University of Iowa
Iowa Research Online
Theses and Dissertations
2007
Mechanisms of human epithelial cell
immortalization and p16NK4a induced telomereindependent sencescence
Benjamin Will Darbro
University of Iowa
Copyright 2007 Benjamin Will Darbro
This dissertation is available at Iowa Research Online: http://ir.uiowa.edu/etd/183
Recommended Citation
Darbro, Benjamin Will. "Mechanisms of human epithelial cell immortalization and p16NK4a induced telomere-independent
sencescence." PhD (Doctor of Philosophy) thesis, University of Iowa, 2007.
http://ir.uiowa.edu/etd/183.
Follow this and additional works at: http://ir.uiowa.edu/etd
Part of the Molecular Biology Commons
MECHANISMS OF HUMAN EPITHELIAL CELL IMMORTALIZATION AND
P16INK4A INDUCED TELOMERE-INDEPENDENT SENESCENCE
by
Benjamin Will Darbro
An Abstract
Of a thesis submitted in partial fulfillment
of the requirements for the Doctor of
Philosophy degree in Molecular Biology
in the Graduate College of
The University of Iowa
May 2007
Thesis Supervisor: Associate Professor Aloysius Klingelhutz
1
ABSTRACT
As human epithelial cells age in culture, protein levels of the tumor suppressor
protein p16INK4a continue to increase resulting in growth arrest independent of telomere
length. Telomere-independent senescence induced by the p16INK4a/Rb tumor suppressor
pathway prevents many epithelial cells from becoming immortalized by telomerase
alone. Differences in culture conditions have been hypothesized to modulate both
p16INK4a expression and replicative capacity of human epithelial cells; however, the
mechanism(s) of p16INK4a regulation under these conditions is unknown.
We have demonstrated that p16INK4a expression is delayed and replicative
capacity increased in human keratinocytes grown in co-culture with post-mitotic,
fibroblast feeder cells as compared to keratinocytes grown on tissue culture plastic alone.
We have found that p16INK4a induction in human keratinocytes cultured on plastic alone is
associated with a migratory phenotype and that p16INK4a expression is selectively induced
in cells possessing markers of keratinocyte migration. Furthermore, we have identified
that tyrosine kinase activity and proper functioning of the urokinase plasminogen
activation system are required for p16INK4a induction during keratinocyte migration
whereas specific signaling through either Src-PTKs or FAK does not appear to regulate
this phenomenon.
We have shown that human keratinocytes possessing telomerase activity and cocultured with feeder cells do become immortal without any apparent cellular crisis. In
contrast to previous reports, however, we demonstrate that telomerase immortalized
keratinocytes co-cultured with feeders do not consistently growth arrest upon transfer to
the plastic culture condition and display an increased frequency of p16INK4a promoter
methylation.
In summary, p16INK4a-induced, telomere-independent senescence is associated
with an epithelial migration response and provides a significant proliferation barrier to
2
epithelial cell immortalization regardless of culture conditions. These results provide
new insights into p16INK4a regulation and have significant implications for the study of
epithelial tumor cell invasion and telomerase reactivation therapies.
Abstract Approved: ____________________________________
Thesis Supervisor
____________________________________
Title and Department
____________________________________
Date
MECHANISMS OF HUMAN EPITHELIAL CELL IMMORTALIZATION AND
P16INK4A INDUCED TELOMERE-INDEPENDENT SENESCENCE
by
Benjamin Will Darbro
A thesis submitted in partial fulfillment
of the requirements for the Doctor of
Philosophy degree in Molecular Biology
in the Graduate College of
The University of Iowa
May 2007
Thesis Supervisor: Associate Professor Aloysius Klingelhutz
Graduate College
The University of Iowa
Iowa City, Iowa
CERTIFICATE OF APPROVAL
_______________________
PH.D. THESIS
_______________
This is to certify that the Ph.D. thesis of
Benjamin Will Darbro
has been approved by the Examining Committee
for the thesis requirement for the Doctor of Philosophy
degree in Molecular Biology at the May 2007 graduation.
Thesis Committee: ___________________________________
Aloysius Klingelhutz, Thesis Supervisor
___________________________________
Beverly Davidson
___________________________________
Frederick Domann
___________________________________
Dwight Look
___________________________________
Dawn Quelle
“You can rest when it’s over.”
Michael L. Darbro
Advice to his only begotten son
ii
ACKNOWLEDGMENTS
As surely as no man is an island, no graduate student survives his or her training
without the assistance of others. First and foremost, I offer my sincerest thanks to the
members of the Klingelhutz lab who have always been more than simply co-workers.
From the cozy days in the basement of Bowen through the time spent in the fishbowl of
MERF, my fellow graduate students Kristi Berger and Michael James have provided not
only assistance with experiments and helpful scientific discussions but invaluable
friendship and moral support. I wish you both the best in all your endeavors. Hello
(meow). I would like to thank the original “Lab Nazi”, Stacia Phillips, without whom I
would never have made it past PCR as well as our current RA, Kim Lee, who contributed
to the bisulfite sequencing and RQ-TRAP assays in this thesis and could have made a
fortune off me if she charged by the miniprep. The Klingelhutz lab has been very
fortunate to attract some great undergraduate students over the years and I would like to
thank Heather Allen, Erin Collins, Erik Peterson, Heather Krueger, and Felicia Barriga
for making my experience that much more enjoyable. I would especially like to thank
Nguyen Nguyen who not only helped me establish a working bisulfite sequencing
protocol, but also rekindled my excitement for research. Throughout it all, I could not
have asked for a better guide than Al Klingelhutz. My graduate school career has not
been without some significant speed bumps and I can’t thank Al enough for his patience,
understanding, and encouragement. The lessons I have learned from Al extend far
beyond the lab and I will forever be grateful for his commitment to my education. I am
greatly looking forward to the day when we can collaborate on an “engaging” project
together.
I have been very fortunate over the years to work with a wide variety of
individuals outside the Klingelhutz lab who have provided both technical assistance and
scientific guidance. I would like to thank the members of the Richard Roller and John
iii
Lee labs for helpful discussions during the early and late years of my graduate training as
well as Dr. Galen Schneider and the members of his lab for their technical assistance and
informative discussions concerning my work with FAK. In my never-ending search for
an immunofluorescent microscope, I would like to thank Drs. James Martin, Martine
Dunnwald, Joseph Zabner, and the members of their labs for assisting with and allowing
me the use of their microscopy equipment. I would also like to thank Dr. Douglas Spitz
for the use of his low oxygen incubator and Dr. Frederick Domann and members of his
lab for technical assistance with bisulfite sequencing protocols and the use of their realtime PCR apparatus. I am also grateful to the following individuals who provided either
experimental advice or laboratory reagents that contributed to this thesis: Drs. Kevin
Ault, Jackie Bickenbach, Keith Burridge (University of North Carolina-Chapel Hill),
William Carter (Fred Hutchinson Cancer Research Center, Seattle, WA), Shiva Patil,
Michael Schaller (University of North Carolina-Chapel Hill), and Richard Seftor
(Children's Memorial Research Center, Chicago, IL).
Much of the success I have experienced during this thesis project can be attributed
to the guidance of my thesis committee and their interest in my work. I would like to
thank Drs. Beverly Davidson, Frederick Domann, Dwight Look, and Dawn Quelle for
their critical analyses of my work and always steering me in the right direction. In
addition, I would like to thank the Medical Scientist Training Program and
Interdisciplinary Graduate Program in Molecular Biology for granting me the opportunity
to be a part of their excellent programs while pursuing my graduate degree.
Outside the scientific realm, several individuals have played a pivotal role in my
reaching this point. I owe a great deal of my success to these individuals and would like
to thank them for their unwavering support and significant contributions to my sanity.
Included in this group are my incredibly supportive parents. I can’t begin to thank my
mother and father enough for all their encouragement over the years. I certainly could
never have made it this far without their love and support. I also would not have gotten
iv
far without the help of Dr. Adam Bell (I’m on fire today!), Julieann Grant (In the words
of Martinez, “You’re so cool”), Jim Kerfien and Dominic Cirillo (All things just keep
getting better), Shinkai Hakimi (Nako! Nako!), Leslie Arnold (One of these days we’ll
solve all the world’s problems), Dr. Scott Temple (I’ll get that fire to the village yet), the
WNG (One day at a time), the staff at Perkins and Steak N Shake (“Looks like you could
use some more coffee”), Gary Brolsma (Numa Numa Forever!), the Pastor Robert Tilton
(Hoobaba Kanda—Isn’t that something?), and last, but certainly not least, The Squirddler
(I’m #*@! Innocent!). To all of you, “Thank you.”
v
ABSTRACT
As human epithelial cells age in culture, protein levels of the tumor suppressor
protein p16INK4a continue to increase resulting in growth arrest independent of telomere
length. Telomere-independent senescence induced by the p16INK4a/Rb tumor suppressor
pathway prevents many epithelial cells from becoming immortalized by telomerase
alone. Differences in culture conditions have been hypothesized to modulate both
p16INK4a expression and replicative capacity of human epithelial cells; however, the
mechanism(s) of p16INK4a regulation under these conditions is unknown.
We have demonstrated that p16INK4a expression is delayed and replicative
capacity increased in human keratinocytes grown in co-culture with post-mitotic,
fibroblast feeder cells as compared to keratinocytes grown on tissue culture plastic alone.
We have found that p16INK4a induction in human keratinocytes cultured on plastic alone is
associated with a migratory phenotype and that p16INK4a expression is selectively induced
in cells possessing markers of keratinocyte migration. Furthermore, we have identified
that tyrosine kinase activity and proper functioning of the urokinase plasminogen
activation system are required for p16INK4a induction during keratinocyte migration
whereas specific signaling through either Src-PTKs or FAK does not appear to regulate
this phenomenon.
We have shown that human keratinocytes possessing telomerase activity and cocultured with feeder cells do become immortal without any apparent cellular crisis. In
contrast to previous reports, however, we demonstrate that telomerase immortalized
keratinocytes co-cultured with feeders do not consistently growth arrest upon transfer to
the plastic culture condition and display an increased frequency of p16INK4a promoter
methylation.
In summary, p16INK4a-induced, telomere-independent senescence is associated
with an epithelial migration response and provides a significant proliferation barrier to
vi
epithelial cell immortalization regardless of culture conditions. These results provide
new insights into p16INK4a regulation and have significant implications for the study of
epithelial tumor cell invasion and telomerase reactivation therapies.
vii
TABLE OF CONTENTS
LIST OF TABLES...............................................................................................................x
LIST OF FIGURES ........................................................................................................... xi
LIST OF ABBREVIATIONS.......................................................................................... xiii
CHAPTER I. INTRODUCTION.........................................................................................1
Senescence........................................................................................................1
Cellular Immortalization...................................................................................3
Telomeres and Telomere-Dependent Senescence .....................................4
Telomere-Independent Senescence .........................................................11
STASIS, p16, and Culture Conditions............................................................17
Significance of the Present Study ...................................................................20
Description of Thesis Content ........................................................................23
CHAPTER II. INDUCTION OF P16 EXPRESSION AND TELOMEREINDEPENDENT SENESCENCE ARE ASSOCIATED WITH A
KERATINOCYTE MIGRATORY RESPONSE ......................................25
Introduction.....................................................................................................25
Methods and Materials ...................................................................................30
Cell Culture .............................................................................................30
RNA and Protein Isolation ......................................................................31
Oligonucleotide Microarray Analysis .....................................................32
Immunoblot Analysis ..............................................................................33
Semi-Quantitative RT-PCR Analysis......................................................34
Immunocytochemistry.............................................................................34
Involucrin .........................................................................................34
Laminin γ2........................................................................................36
β-Catenin ..........................................................................................36
Results.............................................................................................................37
Co-culture with feeder cells alters both human keratinocyte
replicative capacity and p16 expression ..................................................37
Microarray analysis and validation of differential gene expression
and phenotype in different culture conditions .........................................40
Discussion.......................................................................................................48
Growth arrest and p16 expression in different culture conditions ..........49
p16 is co-expressed with keratinocyte migration genes ..........................50
Possible signaling pathways involved in p16 induction..........................52
The role of p16 in keratinocyte migration...............................................53
Summary.........................................................................................................55
CHAPTER III. INHIBITION OF KERATINOCYTE MIGRATION BUT NOT
FAK ACTIVATION REDUCES P16 EXPRESSION ............................56
Introduction.....................................................................................................56
Materials and Methods ...................................................................................60
Immunoblot Analysis ..............................................................................60
Real-Time RT-PCR Analysis ..................................................................60
viii
Immunocytochemistry.............................................................................61
FAK and Vinculin ............................................................................61
FAK, uPAR, and p16 .......................................................................61
Retroviral Constructs and Infection.........................................................62
Migration Assays.....................................................................................63
Results.............................................................................................................63
Induction of p16 expression correlates with markers of
keratinocyte migration and FAK activation ............................................63
Inhibition of tyrosine kinase activity or uPA/uPAR function
reduces p16 expression............................................................................66
Specific inhibition of FAK signaling does not reduce p16
expression ................................................................................................67
Discussion.......................................................................................................71
Induction of p16 expression during keratinocyte migration....................74
Upstream regulators of p16 expression during keratinocyte
migration..................................................................................................76
Summary.........................................................................................................82
CHAPTER IV. P16 PROMOTER METHYLATION IN TELOMERASE
IMMORTALIZED HUMAN KERATINOCYTES COCULTURED WITH FEEDER CELLS ...................................................83
Introduction.....................................................................................................83
Materials and Methods ...................................................................................89
Retroviral Constructs and Infections .......................................................89
Real-Time Quantitative TRAP Analysis and Cytogenetics ....................90
5-Azadeoxycytidine Treatment ...............................................................91
Bisulfite Sequencing................................................................................91
Immunocytochemistry.............................................................................93
Results.............................................................................................................94
TERT immortalization of keratinocytes cultured with feeders
persists upon transfer to plastic with an associated loss of p16
expression ................................................................................................94
Immortal HFKs transferred to plastic retain telomerase activity and
do not exhibit consistent genetic aberrations...........................................98
Inactivation of p16 by promoter methylation in keratinocytes
immortalized by TERT and co-cultured with feeder cells ....................102
Reintroduction of p16 expression into TERT immortalized
keratinocytes on plastic causes growth arrest........................................105
Discussion.....................................................................................................108
Inactivation of p16 during human keratinocyte immortalization ..........110
Safety of telomerase based cell therapies ..............................................113
Summary.......................................................................................................115
CHAPTER V. CONCLUSIONS .....................................................................................116
Mechanisms of Telomere-Independent Senescence.....................................116
Implications of this Work to Human Tumor Cell Metastasis and
Telomerase Reactivation Therapies..............................................................123
REFERENCES ................................................................................................................132
ix
LIST OF TABLES
Table 1: Cellular life extension and immortalization of normal human fibroblasts
and keratinocytes by DNA tumor virus oncogenes ...............................................5
Table 2: Primer sequences and PCR conditions used for semi-quantitative and realtime RT-PCR amplification of the indicated genes .............................................35
Table 3: Functional classification of differentiation-associated genes downregulated
in normal human foreskin keratinocytes grown on tissue culture plastic
alone.....................................................................................................................41
Table 4: Functional classification of migration-associated genes upregulated in
normal human foreskin keratinocytes grown on tissue culture plastic alone ......43
Table 5: Replicative capacity of HFKs transduced with FRNK or LXSN and
cultured on plastic alone ......................................................................................73
Table 6: Chromosomal analyses of TERT transduced cell lines .....................................101
x
LIST OF FIGURES
Figure 1: The end-replication problem ................................................................................7
Figure 2: Human cell mortality stages .................................................................................9
Figure 3: Telomere-Dependent Senescence.......................................................................10
Figure 4: Telomere-Independent Senescence ....................................................................14
Figure 5: Serial passage of human keratinocytes induces p16 expression ........................16
Figure 6: Lifespan extension and delay in p16 induction in keratinocytes cocultured with feeder cells ...................................................................................38
Figure 7: No significant lifespan extension or delay in p16 induction in
keratinocytes cultured on plastic alone in physiologic oxygen conditions........39
Figure 8: Validation of microarray results.........................................................................46
Figure 9: Phenotype Validation: Involucrin ......................................................................47
Figure 10: Phenotype Validation: Laminin γ2...................................................................48
Figure 11: Phenotype Validation: β-Catenin .....................................................................49
Figure 12: FAK signaling pathways ..................................................................................54
Figure 13: Proposed model of p16 induction in human keratinocytes cultured on
plastic alone .....................................................................................................59
Figure 14: Autophosphorylation of FAK in keratinocytes cultured on plastic alone ........64
Figure 15: Immunocytochemical staining for both vinculin and FAK in early
passage (PD ~6-7) Strain 2 HFKs grown on plastic alone ..............................65
Figure 16: Co-staining of autophosphorylated FAK and uPAR with p16.........................65
Figure 17: Herbimycin A treatment alters HFK morphology and migration patterns.......66
Figure 18: Herbimycin A inhibits keratinocyte migration and p16 expression.................68
Figure 19: Amiloride inhibits keratinocyte migration and p16 expression .......................69
Figure 20: PP1 inhibits keratinocyte migration but not p16 expression............................70
Figure 21: FRNK inhibits keratinocyte migration but not p16 expression........................72
Figure 22: β-catenin signaling pathway.............................................................................80
Figure 23: Immortalization of TERT transduced Strain C HFKs co-cultured with
feeder cells .......................................................................................................95
xi
Figure 24: Expression of p16 in tranduced cell lines.........................................................96
Figure 25: Immortalization of TERT transduced Strain A HFKs co-cultured with
feeder cells .......................................................................................................97
Figure 26: Expression of p16 in TERT transduced Strain A HFKs transferred from
co-culture with feeders to plastic alone ...........................................................99
Figure 27: Telomerase activity in transduced Strain C and A HFKs ..............................100
Figure 28: Induction of p16 expression upon treatment with 5-azadeoxycytidine in
immortalized TERT transduced HFKs transferred from the co-culture
environment to plastic alone ..........................................................................103
Figure 29: Induction of p16 expression upon treatment with 5-azadeoxycytidine in
immortalized TERT transduced HFKs maintained in co-culture with
feeder cells .....................................................................................................104
Figure 30: Bisulfite sequencing analysis of the p16 promoter in LXSH and TERT
transduced Strain C HFKs .............................................................................106
Figure 31: Bisulfite sequencing analysis of the p16 promoter in primary and TERT
transduced Strain A HFKs .............................................................................107
Figure 32: Re-introduction of p16 expression causes growth arrest and a senescent
morphology in immortalized Strain C and A TERT transduced HFKs
transferred from co-culture with feeders to plastic ........................................109
Figure 33: The metastatic cascade ...................................................................................125
xii
LIST OF ABBREVIATIONS
PD
Population Doubling
HPV
Human Papillomavirus
SV40
Simian Virus 40
Ad
Adenovirus
Rb
Retinoblastoma
TR
Telomerase RNA Component (human—unless otherwise specified)
TERT
Telomerase Catalytic Component (human—unless otherwise specified)
M1
Mortality Stage One
M2
Mortality Stage Two
CDK
Cyclin Dependent Kinase
ARF
Alternative Reading Frame Protein (p14ARF or p19ARF)
BCC
Basal Cell Carcinoma
SCC
Squamous Cell Carcinoma
DC
Dyskeratosis Congenita
ECM
Extracellular Matrix
ROS
Reactive Oxygen Species
HFK
Human Foreskin Keratinocyte
MMP
Matrix Metalloproteinase
MAPK
Mitogen Activated Protein Kinase
uPA
Urokinase Plasminogen Activator
uPAR
Urokinase Plasminogen Activator Receptor
FAK
Focal Adhesion Kinase
FA
Focal Adhesion
FAT
Focal Adhesion Targeting Domain
FRNK
FAK-Related Non-Kinase
xiii
TGF-β
Tumor Growth Factor Beta
Pyk2
Proline-Rich Tyrosine Kinase Two
Src-PTK
Src-Protein Tyrosine Kinase
RTK
Receptor Tyrosine Kinase
Neo
Neomycin
Hygro
Hygromycin
Puro
Puromycin
5-Aza
5-Azadeoxycytidine
xiv
1
CHAPTER I
INTRODUCTION
Senescence
In the 1950s, improved cell culture methods had been developed for the study of
cancer. However, at this time, an investigator by the name of Leonard Hayflick was
having a great deal of trouble culturing human fibroblasts. The cells he was culturing
would divide nicely for about 50 population doublings (PDs) before their proliferation
eventually ceased. Over lunch, a colleague of Hayflick’s made the off-hand suggestion
that maybe the cells he was culturing were simply growing old. This comment, likely
combined with Hayflick’s own curiosity, is what ultimately gave rise to the aptly named
“Dirty Old Man Experiment”. To exclude inadequate culture conditions as the reason for
the limited cell division he had been observing, Hayflick co-cultured late passage male
cells with early passage female cells. He observed that the “old” male cells stopped
dividing shortly after the start of the experiment, whereas the “young” female cells
continued to proliferate. To further confirm this result, and perhaps to maintain political
correctness, Hayflick also performed the reciprocal “Dirty Old Woman Experiment” and
found the same result. Convinced that human cells did age in culture, Hayflick submitted
his results for peer review. He received the following feedback from the Nobel laureate
Peyton Rous:
“The largest fact to have come from tissue culture in the last fifty
years is that cells inherently capable of multiplying will do so
indefinitely if supplied with the right [culture conditions].” (West,
2003)
Despite this less than glowing review of his work, Hayflick persisted and eventually got
his work published (Hayflick & Moorhead, 1961). Hayflick challenged the current
dogma of cellular aging and was rewarded with authorship of a paper that has since
become one of the most frequently referenced papers in the history of medical research.
2
Also as a result of his discovery, the term “Hayflick Limit” was adopted to describe the
number of cell population doublings a specific cell type can undergo before reaching this
non-proliferative state termed senescence (West, 2003). Following Hayflick’s initial
discovery that human cells did have a finite replicative capacity in vitro, it was found that
the majority of human cells senesce in the G1 phase of the cell cycle. Further studies
concluded that this growth arrest was irreversible and could not be overcome by
stimulation with growth factors (Seshadri & Campisi, 1990). These initial studies laid
the foundation for the controversial hypothesis that cellular senescence may function as
an in vivo mechanism controlling organism aging and maximum lifespan capacity. This
hypothesis continues to be controversial today, as conclusive evidence linking these
phenomena remains elusive. The desire to eliminate the senescence response and
potentially increase one’s maximum lifespan lost some appeal, however, following the
observation that cellular senescence may serve as an important in vivo mechanism of
tumor suppression. One of the hallmarks of cancer cells is the ability to divide
indefinitely. This phenomenon is referred to as immortalization. Immortalization
provides for the necessary extension of cellular lifespan often required to accumulate the
successive mutations needed for cell transformation. In addition, the lack of senescence
in cancer cells prevents steady-state growth within tumors allowing them to reach
magnitudes that are lethal. In a theoretical sense, cellular senescence may act as a type of
fulcrum. Enhanced cellular senescence may result in premature aging but with the
benefit of a cancer free lifespan. Alternately, reduced cellular senescence may cause an
extension of maximum lifespan that would likely never be realized due to the increased
incidence of lethal cancers. Intriguingly, recent experimental evidence suggests that this
relationship may be more than just theoretical. Studies involving transgenic mice have
shown that when the activity of senescence inducing genes is enhanced mice exhibit
reduced longevity and develop organ pathologies consistent with premature aging;
however, these mice also display a greatly reduced incidence of spontaneous tumor
3
formation (Maier et al., 2004; Tyner et al., 2002). Conversely, transgenic mice
engineered to express anti-senescence genes have been shown to have a higher incidence
of both induced and spontaneous tumor formation, but those few mice that do not
succumb to cancer have slightly extended lifespans and display fewer signs of age-related
degenerative diseases (Gonzalez-Suarez et al., 2005). These studies offer compelling
evidence that cellular senescence plays an intricate role in both aging and cancer.
Cellular Immortalization
Approximately ten years before Hayflick performed his “Dirty Old Man
Experiment”, a young woman named Henrietta Lacks made a visit to Johns Hopkins
Hospital following some unusual menstrual bleeding. The gynecologist that examined
her found an unusual cervical growth that he suspected was cancer. Before the end of the
day Henrietta was diagnosed with cervical cancer. As was typical for such cases,
Henrietta returned to the hospital eight days later for removal of the tumor and radiation
treatment. Unfortunately, Henrietta’s treatment was not successful and she died nearly
eight months later. On the very same day that Henrietta lost her battle with cervical
cancer, a researcher from Johns Hopkins University, Dr. George Gey, appeared on
national television with a vial of cancer cells. Dr. Gey held these cells up for the camera
saying that someday it may be possible to wipe cancer out completely through the
fundamental study of cells like these. The cells he was holding up, which he called HeLa
cells, had come from Henrietta Lacks’ cervical tumor and were among the very first
immortalized human cells extensively characterized (West, 2003). After years of
investigation, these cells were found to have been infected with a sexually transmitted
tumor virus called human papillomavirus (HPV). It was the extensive use of DNA tumor
virus, like HPV, that began to define the cellular events needed to achieve human cell
immortalization. DNA viruses such as HPV, simian virus 40 (SV40), and adenovirus
(Ad) have all been shown to be capable of immortalizing certain human cell types under
4
specific conditions (Table 1). Viral oncogenes that inactivated p53 and retinoblastoma
(Rb) identified these tumor suppressor proteins as important regulators of cellular
lifespan in culture. Inhibition of both tumor suppressor pathways consistently resulted in
an extension of cellular lifespan but only rarely did disruption of these pathways alone
lead to immortalization. In situations where immortalization did occur, there was
typically a prolonged period of cellular “crisis” during which additional genetic
mutations were allowed to accumulate until one eventually provided the necessary gene
activation event that would allow for continued proliferation. In the overwhelming
number of cases, this gene activation event was the upregulation of the catalytic
component of an enzyme called telomerase.
Telomeres and Telomere-Dependent Senescence
Nearly a decade after the dogma pertaining to human cell immortality reached its
“Hayflick Limit”, Alexy Olovnikov, a young Russian scientist, was waiting to catch a
train. Having just come from a seminar at which Hayflick’s work had been discussed,
Olovnikov entered a Moscow subway station pondering what mechanism might account
for normal cells having a limited replicative capacity. As the train approached the station
and came to a stop, Olovnikov had an epiphany. He saw that the tracks resembled a
double-stranded DNA molecule and that the train may be mimicking the action of some
type of cellular copying machinery, manufacturing the new DNA strand while riding
along the template strand. When the train stopped, he noticed that none of the would-be
passengers boarded the train at the very end, but rather near the end. He reasoned that
perhaps the nucleotides, like the individuals needing to board the train, also got on the
copying machine near the end of the DNA strand. Thus, the DNA would not be able to
copy itself at the terminal ends. By this analogy, he surmised that the DNA track would
become shorter each time the train made a stop at the station. Olovnikov realized that
this repeated shortening of the DNA molecule during each round of replication might be
Table 1: Cellular life extension and immortalization of normal human fibroblasts and keratinocytes by DNA tumor virus oncogenes
Viral Oncogene/Cell Type
Rb Status
p53 Status
Extended Lifespan
Immortal
+
+
-
-
HPV E6
+
-
+/-
-
HPV E7
-
+
-
-
(Ide et al., 1998; Kiyono et al., 1998;
Shay et al., 1991a; White et al., 1994)
(Kiyono et al., 1998; Shay et al., 1991a;
White et al., 1994)
SV40 Large T
-
-
+
+1
(Shay et al., 1993)
Ad E1A
-
+
-
-
(Shay et al., 1991a)
Ad E1B
+
-
-
-
(Shay et al., 1991a)
HPV E6+E7
-
-
+
+1
(Kiyono et al., 1998; Shay et al., 1991b;
White et al., 1994)
+
+
-
-
HPV E6
+
-
-
-
HPV E7
-
+/-2
+
-
(Kiyono et al., 1998; Klingelhutz et al.,
1996)
(Helt & Galloway, 2001; Kiyono et al.,
1998)
SV40 Large T
-
-
+
-
(Yuan et al., 2002)
HPV E6+E7
-
-
+
+
(Kiyono et al., 1998)
SV40 Large + Small T
-
-
+
+
(Yuan et al., 2002)
Human Fibroblast
Human Keratinocytes
References
1
: Immortalization occurred only after a period of crisis
2
: E7 has been found to disrupt p21 function in human keratinocytes (p21 is a downstream, p53-inducible gene) (Helt et al., 2002)
5
6
the reason for Hayflick’s observation that normal human cells can undergo only a finite
number of cell divisions. This conclusion was remarkable by itself, however, Olovnikov
went even further and proposed that the specialized structures at the ends of the
chromosomes, called telomeres, must consist of repeat nucleotide sequences that did not
contain genetic information. Instead, they acted as a buffer, preventing DNA shortening
from disrupting any crucial downstream genes. He postulated that the length of the
repeated sequence at the telomere would determine the number of cell divisions that
could occur. Olovnikov even proposed that a yet-to-be-discovered DNA copying
machine existed that was specifically designed to replicate the terminal ends of DNA
strands (Olovnikov, 1971; Olovnikov, 1973; West, 2003). Unfortunately, as
extraordinary as Olovnikov’s theories were, they did not elicit much attention until nearly
a decade later.
At approximately the same time that Olovnikov published his theories, James
Watson, co-discoverer of the structure of DNA, was describing what he called “The End
Replication Problem” (Figure 1). Watson realized that because DNA polymerase
required a free 3’ end, synthesis of the lagging strand would be incomplete because it
would be unable to replicate the 5’ ends on new strands of DNA (Watson, 1972). Like
Olovnikov, Watson’s theory pointed to the ends of DNA, the telomeres, as potential
replicometers; shortening every time the cell divided and providing a type of mitotic
clock. It was clear from experiments performed by Woodring Wright, a graduate student
in Leonard Hayflick’s lab, that at least one mechanism controlling replicative senescence
existed in the nucleus of the cell (Wright & Hayflick, 1975a; Wright & Hayflick, 1975b),
but exactly how telomeres were involved in cellular aging, if at all, was still unclear.
Knowledge of the existence of telomeres dated back to the work of Hermann Muller and
Barbara McClintock who showed that the ends of chromosomes were capped and these
structures functioned to prevent end-to-end chromosomal fusions (McClintock, 1941;
Muller, 1962). It was a series of experiments performed in the immortal, unicellular
7
Figure 1: The end-replication problem. A) Synthesis of the 3’-5’ leading strand occurs
continuously to the end of the template DNA strand whereas synthesis of the 5’-3’
lagging strand occurs discontinuously by way of Okazaki fragments, initiated by labile
RNA fragments (black boxes). Following DNA strand synthesis, RNA primers are
degraded and gaps are filled by DNA polymerase in a 3’-5’ manner. Okazaki fragments
are then ligated together to form the completed DNA strand. B) The terminal gap of the
lagging strand is not filled due to the lack of a free 3’ end required for initiation of DNA
replication. This results in a loss of terminal DNA sequences with each replication.
Source: Figure modified from (Rubin, 2002).
organism Tetrahymena themophila that eventually brought telomeres to the forefront of
cellular aging research.
Tetrahymena have thousands of DNA strands and were therefore considered to be
an ideal species in which to examine telomeres. It was reasoned that these ciliated
protozoa would have thousands of telomeres across their many DNA strands, and
potentially multiple copies of any cellular machinery required to maintain them.
Olovnikov’s theories concerning telomeres were ultimately confirmed when experiments
performed in Tetrahymena displayed that telomeres were composed of hexameric repeats
of nucleotides and there existed a telomere terminal transferase capable of maintaining
8
them (Blackburn & Gall, 1978; Greider & Blackburn, 1985). Following the discovery of
the telomere sequence in humans, TTAGGG, studies aimed at measuring both their
shortening and elongation could be conducted (Moyzis et al., 1988). It was subsequently
found that telomere lengths were not the same in all human tissues and that telomeres did
shorten as normal human fibroblasts divided in culture (Cooke & Smith, 1986; Harley et
al., 1990). Furthermore, it was observed that immortal cells, such as HeLa cells and other
human tumors, possessed telomere terminal transferase activity (Kim et al., 1994; Morin,
1989). This telomere terminal transferase would later be called telomerase. The
telomerase enzyme complex was found to be composed of both an RNA template (TR)
and a catalytic reverse transcriptase component (TERT) (Collins, 1996; Feng et al., 1995;
Lingner et al., 1997; Nakamura et al., 1997). And in a landmark study conducted in the
lab of Jerry Shay and Woodring Wright, introduction of the telomerase catalytic
component into normal human fibroblasts was found to result in both telomerase activity
and cellular immortalization (Bodnar et al., 1998). Thus, the telomere-dependent
mechanism of senescence was established.
The question still remained as to how telomere shortening was leading to growth
arrest. Utilization of DNA tumor viruses demonstrated that the inhibition of specific
tumor suppressor proteins, namely p53 and Rb, could lead to extensions of cellular
lifespan without subsequent immortalization (Table 1). This led to the definition of
several “stages of mortality”. These stages were called mortality stage 1 (M1) and
mortality stage 2 (M2) (Figure 2). M1 was recognized as the stage at which normal
human cells senesced. Following the abrogation of tumor suppressor pathways, cells are
allowed to continue proliferation until they reach mortality stage M2. At this point, gross
genetic abnormalities occur, such as end-to-end chromosomal fusions, and lead to a state
of cellular “crisis”. Following reactivation of telomerase expression, and maintenance of
telomere length, cells escape M2 mortality and become immortal.
9
Figure 2: Human cell mortality stages. Continued passage of human cells in vitro leads
to a senescent state termed mortality stage 1 (M1). Following the inactivation of tumor
suppressor pathways, namely Rb and p53, cells are allowed to continue proliferating until
telomere lengths reach a critical point and chromosomal instability induces a state of
crisis termed mortality stage 2 (M2). Reactivation of mechanisms allowing for telomere
maintenance allow for continued proliferation and subsequent immortality.
Source: Figure modified from (Shay & Wright, 2005).
Telomere shortening is clearly involved in the M2 mortality barrier; however, there is
significant evidence suggesting that progressive telomere shortening also contributes to
M1.
Several studies have suggested that telomere shortening induces DNA damage
signals (d'Adda di Fagagna et al., 2003; Gire et al., 2004; Takai et al., 2003). DNA
damage signals have been found to activate the tumor suppressor protein p53, that in turn
leads to the induction of p21 expression. The p21 protein is capable of inhibiting the
function of cyclin dependent kinase (CDK) 4 and CDK6-cyclin D complexes, as well as,
CDK2-cyclin E complexes. Inhibition of these CDK-cyclin complexes prevents the
phosphorylation and inactivation of the tumor suppressor protein Rb (Figure 3). In it’s
active, hypophosphorylated state, Rb binds E2F transcription factors positioned at the
promoters of S-phase specific genes and recruits histone deacetylase enzymes (HDACs)
to these loci. Recruited HDACs then deacetylate nearby histones causing the
surrounding chromatin to adopt a transcriptionally repressive conformation. This results
in growth arrest in the G1 phase of the cell cycle. In the absence of p53 function, some
cell types, including human fibroblasts, continue to divide beyond M1 suggesting that
10
Figure 3: Telomere-Dependent Senescence. Continued shortening of telomeres leads to
the generation of DNA damage signals and the consequent activation of p53. Activated
p53 induces p21 expression. Increased levels of p21 cause the inhibition of CDK4/6cyclin D and CDK2-cyclin E complexes. Inhibition of CDK complexes prevents
hyperphosphorylation of Rb and transition to S phase. Hypophosphorylated Rb is free to
bind E2F transcription factors and recruit HDACs to the promoter regions of S phase
specific genes. Histone deactylation induces a transcriptionally repressive chromatin
state around S phase specific genes and cell cycle arrest in G1.
Source: Figure modified from (Piepkorn, 2000).
p53 expression is intricately involved in this proliferation barrier (Bond et al., 1994; Ide
et al., 1998). Further evidence supporting this point is the observation that DNA repair
foci form at shortened telomeres and the subsequent growth arrest experienced by these
cells is dependent on the p53/p21 signaling pathway (Herbig et al., 2004). These studies
11
have led to a model of telomere-dependent senescence in which the shortening of
telomeres induces DNA damage signals capable of activating the p53/p21 pathway.
Activation of p53 and the consequent induction of p21 expression lead to a senescent
growth arrest in the G1 phase of the cell cycle presumably by hypophosphorylated Rb.
While this model is supported by experiments in human fibroblasts, which are
immortalized by telomerase activity alone, studies in additional cell types, specifically
human epithelial cells, would soon show that this mechanism of growth arrest is
employed by more than just telomeres.
Telomere-Independent Senescence
The immortalization of human fibroblasts with exogenous TERT expression
sparked a flurry of debate and speculation concerning the implications this study would
have for human aging. When the results of this experiment were prematurely leaked,
Geron, the biotechnology company that had patented the human telomerase gene, noticed
a sudden rush of potential stock buyers and had to call Nasdaq to order a halt to the
trading. Press releases of the result contained phrases such as “telomerase rewinds the
clock of cell aging” and “by all accounts these cells had found their cellular fountain of
youth” (West, 2003). The Los Angeles Times did a story on the result, part of which
read,
“Breaking the biological barrier once thought out of reach,
scientists have for the first time endowed healthy human cells
growing in a dish with a quality that alchemists, explorers and
mystics have vainly sought for ages: immortality.” (West, 2003)
Clearly, this result was remarkable. However, as is the case with most sensationalized
scientific discoveries, on-going research would point out the premature nature of such
conclusions, and in the case of cellular senescence, reveal that telomeres are not the
whole story.
12
In the years following this seminal observation, the results were confirmed in
several laboratories around the world and additional cell types were added to the list of
those that could be immortalized by TERT expression alone. These cell types included
human fibroblasts, retinal pigment epithelial cells, vascular endothelial cells, and
mesothelial cells (Bodnar et al., 1998; Dickson et al., 2000; Yang et al., 1999). Even
fibroblasts carved from the calf of Leonard Hayflick himself had been immortalized by
telomerase (West, 2003). It is perhaps uniquely fitting that the first challenge to this
rapidly emerging new dogma of human cell aging came from investigators at the Fred
Hutchinson Cancer Research Center who, like Hayflick in his time, were having trouble
culturing supposedly immortal cells.
Tohru Kiyono and Aloysius Klingelhutz, post-doctoral students in the lab of
Denise Galloway and James McDougall, were attempting to immortalize human
keratinocytes and mammary epithelial cells with different combinations of TERT and the
HPV oncogenes E6 and E7. Earlier work in their lab had shown that the E6 viral
oncogene of HPV, which was previously known to cause p53 degradation, was capable
of inducing telomerase expression in human epithelial cells (Klingelhutz et al., 1996).
Seemingly inconsistent with the new data suggesting telomerase activity alone could
immortalize human cells, E6 expression alone was incapable of immortalizing epithelial
cells in vitro. It was postulated that perhaps E6 was not inducing as high of levels of
telomerase as exogenous expression of TERT itself and thus, not adequately preventing
telomere loss. This theory was dispelled, however, when E6 expressing cells were found
to still undergo senescence despite having high levels of TERT expression, high levels of
telomerase activity, and maintenance of telomeres (Kiyono et al., 1998). Further
evidence that human epithelial cell senescence was not controlled solely by telomere loss
came from the direct observation that exogenous expression of TERT alone rarely
resulted in immortalization of these cells and only after a significant period of “crisis”
during which additional gene activation and/or silencing events were likely occurring
13
(Kiyono et al., 1998). Immortalization of human keratinocytes and mammary epithelial
cells was possible with E6 if another HPV oncogene, E7, was also exogenously
expressed. E7 is known to bind, and subsequently inactivate, the tumor suppressor
protein Rb (Gage et al., 1990; Imai et al., 1991). Exogenous expression of E7 alone in
human epithelial cells was capable of extending in vitro lifespan but did not result in
immortalization (Kiyono et al., 1998). However, the exogenous expression of E7 in
combination with either TERT or E6 mutants that could not cause the degradation of p53,
but remained able to induce telomerase activity, was found to consistently immortalize
human epithelial cells (Kiyono et al., 1998). These results suggested that immortalization
of human keratinocytes and mammary epithelial cells required not only an escape from
telomere-dependent senescence but also specific inhibition of Rb function. This
conclusion was further supported by examination of the rare TERT immortalized cell
lines that emerged from crisis. These cells consistently exhibited loss of a tumor
suppressor named p16INK4a (Kiyono et al., 1998).
The locus harboring p16INK4a is referred to as the INK4a/ARF locus. The
INK4a/ARF locus encodes two proteins, p16INK4a (from this point on referred to as p16)
and the alternative reading frame protein ARF (p14ARF in humans and p19ARF in mice)
(Quelle et al., 1995). ARF has been shown to negatively regulate an inhibitor of p53 and
has been shown to induce growth arrest through the p53 pathway (Sherr & Weber, 2000).
In human cancers, loss of the entire INK4a/ARF locus is common, however, in the TERT
immortalized epithelial cells examined by Kiyono and Klingelhutz, ARF expression
remained high, suggesting that the specific loss of p16 expression was the event allowing
these cells to become immortal (Kiyono et al., 1998). The p16 protein is similar to the
p53-inducible protein p21 in that it is capable of inhibiting cyclin dependent kinases
(Figure 4). Specifically, increased levels of p16 disrupt CDK4 and CDK6-cyclin D
complexes. The inhibition of cyclin D complexes by p16 has been shown to release other
sequestered cyclin dependent kinase inhibitors, such as p21, so that they can exert their
14
Figure 4: Telomere-Independent Senescence. Increased levels of p16 lead to the
inactivation of CDK4/6-cyclin D complexes. Inhibition of these complexes by p16
allows for the release of p21 that can then exert inhibitory action upon CDK2-cyclin E
complexes. The inhibition of these CDK complexes allows for the maintenance of Rb in
the hypophosphorylated state and cell cycle arrest in G1. It has been hypothesized that
p16 expression is induced in response to cellular stresses.
Source: Figure modified from (Piepkorn, 2000).
inhibitory action at CDK2-cyclin E complexes (McConnell et al., 1999; Mitra et al.,
1999). The combined inhibition of CDK4/6-cyclin D and CDK2-cyclin E complexes
results in the maintenance of Rb in an active, hypophosphorylated form and cell cycle
arrest in the G1 phase. The consistent finding that p16 was inactivated in TERT
immortalized human keratinocytes and mammary epithelial cells suggested to Kiyono
and Klingelhutz that a p16/Rb-mediated mechanism may be enforcing an additional
proliferation barrier in these cell types that needed to be overcome to achieve
immortalization. Their conclusion was consistent with previous work done by others that
had also found p16 function commonly lost in both human tumors and human cells
immortalized in vitro (Loughran et al., 1996; Noble et al., 1996; Okamoto et al., 1994;
15
Sharpless, 2005; Tsutsui et al., 2002; Whitaker et al., 1995). The work of Kiyono and
Klingelhutz laid the foundation for what would eventually be called p16-induced,
telomere-independent senescence.
As exogenous expression of TERT became more common, several epithelial cell
types were found to possess telomere-independent mechanisms of senescence. Human
keratinocytes, mammary epithelial cells, bladder urothelial cells, and prostatic epithelial
cells were all found to undergo senescence despite increased telomerase activity (Brenner
et al., 1998; Dickson et al., 2000; Foster & Galloway, 1996; Jarrard et al., 1999; Kiyono
et al., 1998; Puthenveettil et al., 1999; Sandhu et al., 2000; Stampfer & Yaswen, 2003).
Several lines of evidence pointed to p16 as being the driving force behind this telomereindependent senescence mechanism. Ectopically expressed p16 can induce premature
growth arrest in a variety of cell types (Calbo et al., 2001; Dai & Enders, 2000; Schwarze
et al., 2001; Timmermann et al., 1998; Uhrbom et al., 1997), and in those cell types that
growth arrested despite TERT expression, elevated levels of p16 protein were commonly
observed during senescence (Brenner et al., 1998; Hara et al., 1996; Jarrard et al., 1999;
Kiyono et al., 1998; Reznikoff et al., 1996). In human keratinocytes, a progressive
accumulation of p16 protein can be seen during serial passage in vitro, reaching a
maximal point at the onset of senescence (Figure 5A). Accumulation of p16 protein as
human epithelial cells are serially passaged is likely the result of increased transcription
of the p16 gene (Fu et al., 2003) (Figure 5B), however, increased mRNA stability has
also been suggested (Wang et al., 2005). Furthermore, specific inhibition of p16
function, by antisense constructs, siRNA, overexpression of CDK4, or expression of a
CDK4 mutant that is insensitive to p16 inhibition, has been shown to extend cellular
lifespan in culture without immortalization (Bond et al., 2004; Duan et al., 2001; Ramirez
et al., 2001; Rheinwald et al., 2002). Induction of p16 expression maintains Rb in an
active, hypophosphorylated state capable of shutting down the cell cycle in G1.
16
Figure 5: Serial passage of human keratinocytes induces p16 expression. A) Immunoblot
of p16 protein levels during serial passage of human keratinocytes cultured on tissue
culture plastic alone. Protein levels of actin are included as a loading control. B) Semiquantitative RT-PCR of p16 mRNA levels during serial passage of human keratinocytes
cultured on tissue culture plastic alone. GAPDH, a housekeeping gene, is included as an
internal control. Approximate population doublings are represented below each lane.
Conversely, active Rb has been found to negatively regulate p16 expression (Hara et al.,
1996). In the context of Rb inhibition (induced by E7 or overexpression of CDK4), p16
expression is increased but cells do not immediately growth arrest (Kiyono et al., 1998;
Ramirez et al., 2003; Rheinwald et al., 2002) suggesting that Rb is responsible for
enforcing any p16-induced mechanism of senescence. Cumulatively, these results
suggest that p16 induction, and consequent Rb hypophosphorylation, is responsible for a
telomere-independent mechanism of growth arrest. In addition, levels of p16 protein
have been found elevated in adult tissues compared to infants suggesting that this
mechanism of senescence may be limiting human cell replicative capacity in vivo
(Nielsen et al., 1999). However, the question still remained as to what cellular event or
stimulus was causing p16 induction in aging human cells.
17
STASIS, p16, and Culture Conditions
News of a telomere-independent mechanism of senescence may not have been
well received by telomerase enthusiasts. Soon after the term “telomere-independent
senescence” was coined and started showing up in journal articles there appeared to be a
rapid effort to rename this new mechanism of growth arrest. Terms such as “premature
senescence”, “stress-induced senescence”, and STASIS (STress or Aberrant Signaling
Induced Senescence) were developed to represent this phenomenon (Drayton & Peters,
2002; Serrano & Blasco, 2001; Stampfer & Yaswen, 2003). Of the various terms used to
redefine telomere-independent senescence one was particularly rich with irony: “Culture
Shock” (Sherr & DePinho, 2000). It appeared that history had now come full circle.
Once again, cell culture conditions were cited to account for a mechanism of senescence
that did not fit the current dogma.
In a report entitled “Putative telomere-independent mechanisms of replicative
aging reflect inadequate growth conditions” investigators in the lab of Jerry Shay and
Woodring Wright described how p16-induced, telomere-independent senescence may
simply be an artifact of the way in which some epithelial cells are cultured (Ramirez et
al., 2001). In the vast majority of studies that observed either late passage p16 induction
or a p16/Rb mediated barrier to immortalization, human epithelial cells had been cultured
on tissue culture plastic alone in a serum-free, defined medium. This culture system
allows for a wide dispersal of the epithelial cells across the surface of the tissue culture
plate and the defined medium, which generally contains a very low concentration of
calcium (< 0.1 mM), reduces the degree of terminal differentiation experienced by these
cells during serial passage. In this new study, human keratinocytes and mammary
epithelial cells were co-cultured with post-mitotic, fibroblast feeder cells in a serum
containing medium that also had much higher levels of calcium (>1.2 mM). The coculture environment caused the epithelial cells to grow in tightly packed, confluent
colonies that enlarged and compressed the surrounding fibroblasts as the epithelial cells
18
continued to proliferate. Data presented in this new report showed that co-culture of
human keratinocytes or mammary epithelial cells with feeder cells extends replicative
lifespan in vitro and “avoids the growth arrest associated with increased p16 expression”
(Ramirez et al., 2001). Exogenous expression of TERT alone was sufficient to
immortalize these epithelial cell types when they were grown in the co-culture
environment (Ramirez et al., 2001). Furthermore, p16 induction and subsequent growth
arrest was observed upon transferring TERT immortalized cells from the co-culture
environment to tissue culture plastic alone, suggesting that these cells had not inactivated
the p16/Rb pathway. These results prompted the authors to conclude that “..the level of
p16 may be a general indicator of stress,” caused by inadequate culture conditions
(Ramirez et al., 2001). Another report to come out of this lab displayed data suggesting
that human mammary epithelial cells do not require p16 inactivation to be immortalized
by TERT alone when co-cultured with feeder cells (Herbert et al., 2002). In addition, an
independent study in human esophageal keratinocytes also showed that TERT alone
could immortalize epithelial cells co-cultured with feeders, and that TERT
immortalization did not lead to p16 inactivation (Harada et al., 2003). While these
reports presented some evidence for culture conditions as the origin of p16-induced,
telomere-independent senescence there were several issues the authors failed to address.
1) Replicate experiments with additional, independent primary keratinocyte and
mammary epithelial cell strains were not regularly performed; 2) No explanation was
given for the repeated observation of inflection points (periods of slowed growth
potentially indicating minor crisis) in the growth curves of TERT immortalized epithelial
cells; 3) In one of these reports, authors failed to address the decrease in p16 expression
observed between early and late passage human mammary epithelial cells co-cultured
with feeder cells; 4) Functional integrity of the p16 gene in TERT immortalized epithelial
cells was not examined in all cases and was incomplete in others; 5) No experiments
were performed to address what “stress” inadequate culture conditions were generating or
19
how this stress translated into p16 induction. These reasons alone are enough to question
whether telomere-independent senescence is purely a “culture shock” phenomenon;
however, in contrast to these studies, there was also published data suggesting that p16induced, telomere-independent senescence was still occurring in human keratinocytes cocultured with feeder cells. James Rheinwald and colleagues had observed that whereas
human keratinocytes did have an extended lifespan when co-cultured with feeder cells,
they still induced p16 expression in later passages at the time of growth arrest (Rheinwald
et al., 2002). Their results led them to conclude, “…that there is not an apparent
qualitative difference in the activation of the p16-dependent senescence arrest mechanism
and immortalization barrier between cells cultured in the two systems” (Rheinwald et al.,
2002). As had been observed previously, Rheinwald also noted that inflection points
occurred in the growth curves of TERT-expressing keratinocytes and noted that this
period of slowed growth correlated with the point in time that control primary cells
induced p16 expression and underwent senescence. Thus, the “culture shock” theory of
p16-induced, telomere-independent senescence was far from complete.
One reproducible observation that was seen in all of the above studies was a
reduction or delay of p16 induction in human epithelial cells co-cultured with feeder
cells. This observation suggests that p16 expression may be regulated differently in the
two culture conditions. There is limited data on what specific genes are induced in
human epithelial cells cultured in the presence or absence of feeder cells. The first in
vitro cultures of human keratinocytes were in the presence of feeder cells (Rheinwald &
Green, 1975). In this co-culture system, a fraction of keratinocytes was found to undergo
terminal differentiation and form an envelope resistant to detergents and reducing agents
(Sun & Green, 1976). This suggests that human keratinocytes co-cultured with feeder
cells may be engaged in the normal process of differentiation. Consistent with this
conclusion is the observation that human keratinocytes induce several genes known to be
involved in differentiation upon the addition of both serum and calcium to serum-free
20
medium (Olsen et al., 1995). Co-culture of human keratinocytes with fibroblast feeder
cells has also been shown to induce enhanced expression of keratinocyte growth factor
(KGF) and the interleukin-1 receptor in the fibroblasts (Maas-Szabowski et al., 2000).
This in turn creates a positive feedback loop with keratinocytes that are expressing the
KGF receptor at high levels as well as releasing interleukin-1α and interleukin-1β into
the medium. It has been hypothesized that this interaction represents a basic mechanism
for keratinocyte growth regulation in the co-culture system. If human keratinocytes are
engaged in the normal process of differentiation in the co-culture environment it would
be of significant importance to determine what gene expression program keratinocytes
engage when cultured on plastic alone. Since p16 expression is induced to a greater
extent in human keratinocytes cultured in the absence of feeder cells this information
would suggest candidate signal transduction pathways involved in upregulation of p16
during telomere-independent senescence. This information may help determine whether
p16-induced, telomere-independent senescence is merely a culture artifact or an authentic
aging mechanism limiting the replicative capacity of human cells.
Significance of the Present Study
Study of the p16/Rb-mediated barrier to human epithelial cell immortalization is
of extreme significance as p16 is one of the most frequently lost tumor suppressor
proteins in human cancers. Several human cancers have been found to inactivate p16
expression. A brief list of those cancers that have a high frequency of p16 loss include
breast (21% of total cases exhibit p16 inactivation), nonsmall cell lung carcinoma (64%),
colorectal (27%), bladder (59%), nonhodgkin’s lymphoma (33%), head and neck (68%),
melanoma (65%), leukemias (61%), pancreas (85%), ovary (25%), stomach (44%),
esophagus (70%), multiple myeloma (60%), and glioma (84%) (Liggett & Sidransky,
1998; Sharpless, 2005). In all, p16 inactivation is observed in approximately 1/3 of all
human cancers, a figure rivaled only by p53. Better understanding of upstream signaling
21
pathways that lead to p16 induction could lead to the development of more efficacious,
senescence-inducing chemotherapeutic medications for the treatment of human cancers.
At present, few upstream regulators of p16 are known. Oncogenic expression of Ras, and
subsequent signaling through the Ras/mitogen activated protein kinase (MAPK) pathway,
has been shown to induce p16 expression in human fibroblasts with some consistency
(Lin et al., 1998; Ohtani et al., 2001; Serrano et al., 1997), however, little data exists to
suggest the same pathway induces p16 expression in human epithelial cells. Limited
reports have shown p16 expression to be inducible by DNA damaging agents such as UV
radiation, γ-irradiation, topoisomerase I and II inhibitors, and cisplatin (Ahmed et al.,
1999; Chazal et al., 2002; Shapiro et al., 1998). Various other observations have shown a
potential role for 14-3-3σ and interferon-JAK/STAT signaling in the upregulation of p16
expression in human epithelial cells (Chaturvedi et al., 2003; Dellambra et al., 2000). A
more refined understanding of what mechanisms cause p16 induction and consequent
telomere-independent senescence in human epithelial cells will help narrow the field of
possible therapeutic targets.
Human keratinocytes are an appropriate cell type in which to examine p16induced, telomere-independent senescence given both the apparent epithelial cell typespecific nature of this phenomenon (discussed above) and the known differences between
mouse and human cell senescence. Laboratory mice are known to possess extremely
long telomeres and express telomerase in somatic tissues (Kipling & Cooke, 1990;
Prowse & Greider, 1995). Thus, telomere maintenance is not a limiting factor for cellular
immortalization. Despite the fact that murine cells would then appear to exhibit only a
p16-induced, telomere-independent senescence during continuous culture, several studies
have suggested that the p53 pathway is the principal mediator of this growth arrest. Cells
derived from p53 or ARF null mice do not senesce in culture and can be propagated
indefinitely (Kamijo et al., 1997). In contrast, cells derived from mice in which the p16
gene has been specifically knocked-out do not exhibit an extension of replicative capacity
22
in vitro and still require a loss in some component of the p53 pathway to become
immortal (Sharpless et al., 2001). Thus, p16-induced, telomere-independent senescence
is primarily a human cell occurrence and as such should be examined in human epithelial
cells. Human keratinocytes also serve as a model epithelial cell type for this study as
they are known to give rise to human cancers such as squamous and basal cell
carcinomas. Basal cell carcinoma (BCC) is the most common malignancy in humans.
BCC rarely metastasizes but can cause significant morbidity if allowed to progress.
Untreated BCCs are known to cause significant local destruction and disfigurement if not
treated in a timely fashion. Squamous cell carcinoma (SCC) is the second most common
form of skin cancer behind BCC. Whereas most SCCs are readily identified and
removed, high-risk SCC has a considerable metastatic rate and can be life-threatening.
SCC is capable of metastatic spread through regional lymph nodes resulting in distant
metastasis. Over the last three decades, the incidence of SCC has risen steadily likely
due to increased sun exposure of the general population. Study of the mechanism of p16induced, telomere-independent senescence in human keratinocytes is appropriate given
that inactivation of p16 expression has been found in both BCC and SCC (Green &
Khavari, 2004; Saridaki et al., 2003; Soufir et al., 1999). Furthermore, human urothelial,
mammary, and prostatic epithelial cells all exhibit increased p16 expression at senescence
and experience telomere-independent senescence despite exogenous TERT expression
(Brenner et al., 1998; Jarrard et al., 1999; Reznikoff et al., 1996). Thus, it is likely that
information concerning the mechanism controlling p16-induced, telomere-independent
senescence in human keratinocytes will be applicable to these cell types, as well as those
malignancies that predominantly arise from other epithelial tissues.
The demonstration that exogenous expression of TERT alone can immortalize
several different cell types, with or without feeder cells present, has led to the
development of strategies aimed at reactivating telomerase expression in human cells as a
means to treat both genetic disorders and age-related tissue degenerative diseases
23
(Harley, 2005). Cell types and disorders such as fibroblasts/keratinocytes and chronic
skin ulcers, retinal pigmented epithelial cells and macular degeneration, and certain
immune cells and AIDS have all been suggested as potential targets for telomerase
reactivation therapy (Harley, 2002; Harley, 2005). Additionally, patients suffering from
the genetic disorder Dyskeratosis congenita (DC), often caused by a mutation in the RNA
component of telomerase, have been cited as potential benefactors of telomerase
reactivation therapies (Harley, 2002; Mason et al., 2005). Before such therapies should
be attempted, however, the safety of such cells must be rigorously examined. Whereas
some reports have indicated that p16 mechanisms of growth arrest are maintained in
TERT immortalized cells, there is considerable evidence to suggest these cell lines
undergo a brief period of crisis prior to immortalization which may compromise p16
function in some way (Rheinwald et al., 2002). Furthermore, recent data suggests that
TERT may have additional functions independent of the ability to maintain telomere
length that may contribute to carcinogenesis (Chang & DePinho, 2002; Stewart et al.,
2002). Thus, preliminary investigations into the status of tumor suppressor genes, such
as p16, in TERT immortalized human cells must be performed as the first step in
evaluating telomerase reactivation as a potential therapeutic modality.
Description of Thesis Content
This thesis is divided into three data chapters (Chapters II, III, and IV) and a final
unified conclusions chapter that will also provide direction for future research goals in
this field (Chapter V). The three data chapters will address the points of significance
above. Specifically, Chapters II and III will attempt to answer the question of what
upstream events cause the induction of p16 expression in culture conditions that promote
telomere-independent senescence, and Chapter IV will attempt to address the potential
p16 inactivation and subsequent safety of TERT immortalized human keratinocytes cocultured with feeder cells. The overall goal of this thesis project is to elucidate in part or
24
whole the mechanism of human keratinocyte p16-induced, telomere-independent
senescence and whether this proliferation barrier is inactivated in TERT immortalized
human keratinocytes co-cultured with feeder cells.
25
CHAPTER II
INDUCTION OF P16 EXPRESSION AND TELOMEREINDEPENDENT SENESCENCE ARE ASSOCIATED
WITH A KERATINOCYTE MIGRATORY RESPONSE
Introduction
It is unknown what upstream signaling events cause the progressive increase in
p16 expression that precedes keratinocyte telomere-independent senescence. Increased
cellular stress induced by inadequate culture conditions has been hypothesized to induce
p16 expression and consequent telomere-independent senescence (Ramirez et al., 2001),
however, the identity of this stress and the mechanism through which it translates into
upregulation of p16 in keratinocytes is unclear. It has been reported that co-culture of
human keratinocytes with post-mitotic, fibroblast feeder cells, as opposed to culture on a
plastic substrate alone, extends keratinocyte replicative capacity, delays the induction of
p16 expression, and allows for immortalization with TERT alone (Harada et al., 2003;
Ramirez et al., 2001). Previous studies have suggested that the co-culture environment
can lead to keratinocyte differentiation (Sun & Green, 1976), however, little is known as
to what gene expression program keratinocytes engage in the absence of feeder cells or to
what additional stresses they may be subjected.
Multiple stresses have been associated with cell culture, including high oxygen
tension, lack of interactions with neighboring cells, as well as, growth factor and nutrient
deficiencies (Ben-Porath & Weinberg, 2004; Ben-Porath & Weinberg, 2005; Sherr &
DePinho, 2000). Of these various potential stresses, several studies have suggested that
elevated levels of reactive oxygen species (ROS) may be responsible for a cultureinduced increase in p16 expression. In human fibroblasts, expression of p16 was
elevated during the induction of premature senescence by hydrogen peroxide or
oncogenic Ras, two conditions known to produce increased levels of ROS (Chen et al.,
26
2001b; Lee et al., 1999; von Zglinicki et al., 1995). In addition, ultraviolet radiation,
known to generate increased levels of ROS, has been shown to induce p16 expression in
human keratinocytes cultured in vitro (Ahmed et al., 1999; Chazal et al., 2002; Peus &
Pittelkow, 2001). In the co-culture environment, feeder cells may be reducing the level
of oxidative stress experienced by human keratinocytes, thereby delaying the onset of
p16 induction and telomere-independent senescence. Culture of human keratinocytes in a
reduced oxygen environment, more consistent with physiologic oxygen conditions, may
produce a similar effect and result in an extension of replicative capacity and a reduction
in p16 expression. However, it is also possible that a non-stress mediated mechanism
may be responsible for p16 induction and consequent telomere-independent senescence.
Keratinocytes are the primary cell type composing the human epidermis. In this
tissue context, keratinocytes engage in two very important processes: differentiation and
wound healing. The normal process of keratinocyte differentiation provides for the
specialized protective barrier attributed to skin. Keratinocyte differentiation within the
epidermis creates histologically distinguishable basal, spinous, granular, and cornified
layers. The result of keratinocyte differentiation is the progression of cells from the
replicative basal layer to the mechanically rigid cornified layer. Terminally differentiated
keratinocytes present in the cornified layer (corneocytes) are no longer viable and are
encapsulated within a highly specialized structure known as the cornified envelope
(Kalinin et al., 2002). The layer of dead corneocytes and the lipid rich cornified envelope
surrounding them is essential for the effective physical and water barrier function of the
epidermis. As basal layer keratinocytes differentiate upwards, changes in gene
expression result in the loss of proliferative capacity and the emergence of various
protein markers that distinguish different layers of the epidermis. Changes in keratin
intermediate filament expression are particularly useful in identifying cells committed to
differentiation (Olsen et al., 1995; Steinert & Roop, 1988). Keratins 5 and 14 are
expressed in the proliferating basal layer while keratins 1 and 10 are characteristic of
27
suprabasal, differentiating keratinocytes (Dale et al., 1990; Fuchs et al., 1987).
Profilaggrin, a component of the keratohyalin granules, is found primarily in the granular
layer while proteins such as involucrin, loricrin, and cystain A are found mainly in the
cornifed layers where they contribute to the formation of the cornified envelope (Kalinin
et al., 2001; Kalinin et al., 2002). Human keratinocyte differentiation requires a complex
program of gene expression that is responsible for producing the above-mentioned
proteins at specific points in time during a cell’s movement from the basal to cornified
layer. Previous studies have suggested that when in co-culture with feeder cells some
human keratinocytes undergo the normal process of differentiation and form a cornifiedenvelope-like structure (Sun & Green, 1976). Although there is some evidence to
suggest that p16 may be involved in keratinocyte differentiation (Fuchs & Byrne, 1994;
Harvat et al., 1998; Lee et al., 2000) it would appear that p16 is induced to a greater
extent in the absence of feeders, suggesting that p16-induced, telomere-independent
senescence may be caused by another program of keratinocyte gene expression.
When the epidermis is compromised, keratinocytes must undergo a
reprogramming of gene expression to facilitate healing of the wound and restoration of
the protective barrier. To achieve epidermal closure, keratinocytes must acquire both the
ability to migrate across the open wound and increase proliferation to fill the cellular
vacancy. This process, characterized by increased cellular proliferation that occurs
behind a leading edge of migrating keratinocytes, is referred to as reepithelialization
(Garrett, 1997). The change in keratinocyte phenotype required to perform the function
of reepithelialization has been termed “activation” (Grinnell, 1992). As is the case for
differentiation, keratinocyte activation has several recognizable features including
hyperproliferation, migration, cytoskeletal changes, augmentation of cell surface
receptors (such as integrins and other extracellular matrix receptors), and the production
of basement membrane components that serve as a provisional extracellular matrix
(ECM) (Coulombe, 1997; Freedberg et al., 2001). For migration to occur, keratinocytes
28
must first undergo detachment from neighboring cells by removal of intracellular
desmosomes (Singer & Clark, 1999; Yates & Rayner, 2002). This loss of cell contacts
can result in the redistribution of cell-cell adhesion proteins such as β-catenin (Dietrich et
al., 2002). Migrating cells at the leading edge of the wound also undergo rearrangements
of the actin cytoskeleton to produce lamellipodia and filopodia, structures necessary for
adhesion to and movement along the provisional extracellular matrix (Yates & Rayner,
2002). The provisional ECM, which serves to both assist in keratinocyte migration and
restore a proper basement membrane, is produced by migrating keratinocytes that have
increased the expression and secretion of ECM proteins, such as fibronectin, collegen,
and laminin (Liggett & Sidransky, 1998; O'Toole, 2001). These changes in cellular
morphology, protein localization and secretion, as well as proliferative index, have all
served to help distinguish activated/wound healing keratinocytes from their
differentiating counterparts.
It is not difficult to hypothesize that differences in culture conditions may tip the
balance of which gene expression program keratinocytes engage. In culture conditions
that better mimic the in vivo environment of epidermal keratinocytes, it would be
expected that cells would express markers characteristic of terminal differentiation.
However, culture conditions that do not provide the necessary factors that are present in
the epidermis may stimulate keratinocytes to activate the signaling pathways necessary
for wound healing and migration. Intriguingly, recent studies in both normal human
keratinocytes and invasive epithelial cancers have suggested that p16 expression may be
upregulated during the process of migration. Co-expression of p16 and laminin-γ2 (a
component of laminin 5) has been observed at the leading edge of in vitro wounded
keratinocytes (Natarajan et al., 2003) suggesting that p16 expression is activated in
keratinocytes induced to migrate during wound healing. Additionally, greater levels of
p16 expression have been found at the infiltrative front of squamous and basal cell
carcinomas migrating across the basement membrane (Nilsson et al., 2004; Svensson et
29
al., 2003). As with differentiation, human keratinocyte migration is controlled by a
complex program of gene expression responsible for the coordinated co-expression of
several genes. Thus, experimental techniques capable of assaying global gene expression
patterns would be extremely useful in determining whether culture of human
keratinocytes on plastic alone is inducing a normal cellular response or an artificial stress
response.
With the advent of oligonucleotide microarrays, investigators have been enabled
to measure the expression level of thousands of genes in parallel. This technology has
proved quite useful in examining global gene expression patterns in various cellular
contexts. In recent years, gene expression studies using microarrays have revealed new
information about specific signaling pathways involved in skin related pathologies such
as melanoma and psoriasis (Bittner et al., 2000; Bowcock et al., 2001), viral infections
such as HPV (Chang & Laimins, 2000; Duffy et al., 2003), and the growth inhibitory
effects of tumor suppressor proteins such as p53 (Zhao et al., 2000). Microarrays have
also been used to gain insight into the mechanism of senescence (Zhang et al., 2004).
This technology is uniquely suited for the analysis of signaling pathways and potentially
co-regulated genes involved in p16-induced, telomere-independent senescence; the study
of which is of extreme significance given the fact that many epithelial cancers bypass p16
mediated tumor suppression.
In an effort to identify the proposed culture-induced “stress” leading to p16induced, telomere-independent senescence, we have cultured human keratinocytes on
plastic alone in both atmospheric (~20%) and physiologic (~4%) oxygen conditions. We
found no difference in either replicative capacity or p16 induction between the two
oxygen conditions. To characterize the phenotype associated with culture-induced p16
upregulation we have used DNA oligonucleotide microarrays to assess the global gene
expression patterns between human keratinocytes grown on tissue culture plastic alone or
in co-culture with post-mitotic, gamma irradiated fibroblast feeder cells. These two
30
culture conditions were selected for microarray analysis since we found them to
differentially modulate both p16 expression and keratinocyte replicative capacity.
Analysis of the microarray data indicated that, compared to keratinocytes co-cultured
with feeder cells, keratinocytes grown on plastic alone increase the expression of several
genes involved in migration and decrease the expression of genes associated with
differentiation. These findings suggest that the increased “stress” experienced by
keratinocytes grown on plastic alone is likely the result of a culture-induced migratory
response that is absent or reduced when keratinocytes are grown in co-culture with feeder
cells.
Methods and Materials
Cell Culture
The following cell culture protocols are adhered to in each subsequent chapter of
this thesis. As such, they will only be described once. Human foreskin keratinocytes
(HFKs) were isolated as previously described (Blanton et al., 1991). Foreskin tissue was
obtained using a protocol approved by the University of Iowa Institutional Review Board
in accordance with HIPAA guidelines and the Declaration of Helsinki Principles. HFKs
were grown under two different culture conditions. In the “plastic alone” condition,
HFKs were grown on tissue culture plastic in keratinocyte-serum free media (KSFM;
Invitrogen, Carlsbad, CA: 10724-011) supplemented with 0.2 ng/ml human recombinant
epidermal growth factor (EGF), 30 µg/ml bovine pituitary extract (BPE), and 1%
Penicillin-Streptomycin (Invitrogen: 15140-122). In the “feeder cell” condition, HFKs
were grown on tissue culture plastic in co-culture with post-mitotic, gamma irradiated J2
3T3 fibroblasts in E-media. E-media consists of a base of Dulbecco’s modified Eagle’s
media (Invitrogen: 11965-092) and HAM F-12 nutrient mixture media (Invitrogen:
11765-054) at a 3:1 ratio, respectively, supplemented with 10% fetal bovine serum (FBS)
(Invitrogen: 26140-079), 1% Penicillin-Streptomycin (Invitrogen: 15140-122), 1.36
31
ng/ml tri-iodo-thyronine (Sigma, St. Louis, MO: T5516), 0.5 µg/ml hydrocortisone
(Sigma: H0396), 8.4 ng/ml cholera toxin (Sigma: C3012), 5.0 µg/ml transferrin (Sigma:
T1147), 5.0 µg/ml insulin (Sigma: I6634), and 4.5 ng/ml EGF (Invitrogen: 13247-051).
The final calcium concentrations in both KSFM and E-media were 0.09 mM and 1.28
mM, respectively. In the co-culture condition, irradiated fibroblasts were removed by
incubation with a solution of 0.05% trypsin (Invitrogen: 15400-054), 50 mM hepes buffer
solution (Invitrogen: 15630-080) in Hanks balanced salt solution (Invitrogen: 14170112). This protocol effectively removed the fibroblast feeder cells while leaving the
keratinocytes attached. HFKs were removed by further incubation in 0.05%
trypsin/EDTA solution (Invitrogen: 25300-054). Trypsinized HFKs were pelleted by
centrifugation and replated onto plastic alone or into co-culture with a new population of
irradiated J2 3T3 fibroblasts. All cells were passaged at a 1:4 or 1:6 split ratio when 7080% confluent. The majority of cells were maintained at 37˚C with 5% CO2 at
atmospheric (~20%) oxygen levels, however, in some experiments, primary HFKs were
cultured on plastic alone in a Nuaire Autoflow incubator (Nuaire, Plymouth, MN: NU4950) maintained at 37˚C with 4% O2 and 5% CO2. Population doublings (PDs) were
calculated using the following equation: PDn = PD(n-1) + log10[split ratio]/log10[2]
(Reznikoff et al., 1987).
RNA and Protein Isolation
The following RNA and protein isolation protocols are adhered to in each
subsequent chapter of this thesis. As such, they will only be described once. Both RNA
and protein were collected at selected PDs when HFKs reached 70-80% confluence.
RNA and protein were collected from HFKs grown with feeders after the removal of the
irradiated fibroblasts. Total RNA was collected using TriReagent (Molecular Research
Center, Cincinnati, OH) and purified according to the manufacturer’s instructions. All
RNA samples were quantified by spectrophotometry and quality was analyzed by
32
resolution of the 18S and 28S rRNA bands. Total protein was collected using WE16
lysis buffer, as previously described (Foster & Galloway, 1996). Lysates were sonicated
and centrifuged at 15,000g for 30 minutes at 4˚C. Supernatants were collected and
protein concentration was quantified using the Bio-Rad Protein Assay (Bio-Rad
Laboratories, Hercules, CA) according to the manufacturer’s instructions.
Oligonucleotide Microarray Analysis
RNA samples were prepared for hybridization as previously described (Duffy et
al., 2003). Affymetrix HG-U133A GeneChips were hybridized, washed, and stained by
the University of Iowa DNA Facility according to Affymetrix protocols (Affymetrix,
Inc., Santa Clara, CA). Oligonucleotide arrays were scanned by the University of Iowa
DNA Facility using a confocal scanner manufactured for Affymetrix by Molecular
Dynamics (Sunnyvale, CA). Data analysis was performed using the GeneChip software
(Version 5.0) supplied with the Affymetrix instrumentation system. RNA was collected
from two independent strains of primary HFKs (Strain 1 and Strain 2) serially
subcultured on plastic alone or in co-culture with feeder cells to approximately half of
their maximum lifespan (Strain 1: PDs ~8 and Strain 2: PDs ~14). For each condition
one GeneChip was analyzed and the following comparisons were made: Strain 1 on
plastic vs. Strain 1 on feeders, Strain 1 on plastic vs. Strain 2 on feeders, Strain 2 on
plastic vs. Strain 1 on feeders, and Strain 2 on plastic vs. Strain 2 on feeders. For each
comparison the co-culture condition was used as a baseline. For each gene, across all
four GeneChip comparisons, the average fold change was calculated from the signal log
ratio value. The change in gene expression level was considered significant if (1) the
average fold change was greater than or equal to twofold, and (2) the change in transcript
level was considered significant by the Affymetrix difference call metric (only values of I
= Increase and D = Decrease were accepted) in each comparison performed. Functional
classification of a subset of these genes was performed by referencing public databases
33
such as Netaffx (http://www.affymetrix.com/analysis/index.affx) and utilizing microarray
analysis software including D-Chip (Harvard, Boston, MA) and GeneSpring (Silicon
Genetics, Redwood City, CA).
Immunoblot Analysis
The following immunoblot analysis protocol is adhered to in each subsequent
chapter of this thesis. As such, it will only be described once. Subsequent Methods and
Materials sections will only address additional primary antibodies not used in this
chapter. Immunoblot analyses of cellular protein levels were performed as previously
described (Foster et al., 1994) using 20µg of protein lysate, run on polyacrylamide gels of
the appropriate concentration, and transferred onto Immobilon-P membranes (Millipore,
Billerica, MA). Membranes were stripped by incubation and agitation at 50˚C in a
solution of 100 mM 2-mercaptoethanol, 2% (w/v) SDS, 62.5 mM Tris-HCl at pH 6.7.
Stripped membranes were then reblocked and reprobed with different antibodies. The
following antibodies were used in this study: p16 (Pharmingen, San Diego, CA: G175405), Fibronectin (BD Transduction Laboratories, San Jose, CA: 10), Laminin γ2
(Chemicon International, Temecula, CA: D4B5), Vimentin (Santa Cruz Biotechnology
Inc., Santa Cruz, CA: V9), Keratin 19 (ICN Biomedicals, Inc., Irvine, CA: KS19.1),
Keratin 14 (Cymbus Biotechnology Ltd, United Kingdom: LL002), Keratin 13 (Santa
Cruz Biotechnology Inc.: DE-K13), Keratin 8 (Sigma, St. Louis, MO: K8.60), and Actin
(Santa Cruz Biotechnology Inc.: I-19). Blots were probed with the appropriate HRPconjugated secondary antibody: goat anti-mouse IgG (Jackson ImmunoResearch
Laboratories, West Grove, PA), goat anti-rabbit IgG (Santa Cruz Biotechnology Inc.), or
donkey anti-goat IgG (Santa Cruz Biotechnology Inc.). Detection was performed using
the Western Lightning chemiluminescence kit (Perkin Elmer Life Sciences, Boston,
MA).
34
Semi-Quantitative RT-PCR Analysis
The following semi-quantitative RT-PCR protocol is adhered to in each
subsequent chapter of this thesis. As such, it will only be described once. cDNA
synthesis reactions were carried out using the RETROscript kit (Ambion, Austin, TX).
RNA samples (2µg) were used as the template for single-stranded cDNA synthesis
reactions primed with the oligo dT primers included in the RETROscript kit. PCR
analysis was carried out using the SuperTaq kit (Ambion) according to the manufacture’s
instructions. RT-PCR reaction products were separated by agarose gel electrophoresis,
stained with ethidium bromide, and visualized by UV light. Primer sequences and PCR
conditions used for semi-quantitative RT-PCR amplification of the indicated genes (in all
chapters of this thesis) are provided in Table 2.
Immunocytochemistry
Involucrin
Early passage HFKs (PD ~5-7) were grown on 1 well permanox Lab-Tek chamber slides
(Nalge Nunc International, Naperville, IL) with or without feeder cells in either E-media
or KSFM, respectively. Chamber slides were fixed with 20% DMSO in methanol for 20
minutes at 4˚C. Slides were blocked with 5% BSA in PBS for 30 minutes. Slides were
incubated for 1 hour with involucrin primary antibody (Sigma: SY5) at a 1:400 dilution
in blocking buffer. Donkey anti-mouse rhodamine conjugated secondary antibody was
diluted 1:25 in blocking buffer and incubated with chamber slides for 45 minutes.
Images were collected on an Olympus BX60 fluorescence microscope (Olympus
Corporation, Tokyo, Japan). PBS washes were performed between each step in all
immunocytochemistry protocols.
Table 2: Primer sequences and PCR conditions used for semi-quantitative and real-time RT-PCR amplification of the indicated genes
Gene
Amplified
Forward Primer
Reverse Primer
Temp
Cycles
36B4
5’-GGCCAGCTGGAAGTCCAACT-3’
5’-CCATCAGCACCACAAGCCTTC-3’
60˚C
22
GAPDH1
5’-TGAAGGTCGGAGTCAACGGATTTGGT -3’
5’-CATGTGGGCCATGACCTCCACCAC -3’
60˚C
30
18S
5’-CCTTGGATGTGGTAGCCGTTT -3’
5’-AACTTTCGATGGTAGTCGCCG -3’
60˚C
40
p162
5’-CAACGCACCGAATAGTTACGG-3’
5’-TGCCCATCATCATGACCTG-3’
62˚C
28
SPRR1A
5’-ACACAGCCCATTCTGCTCCG-3’
5’-TGCAAAGGAGCGATTATGATT-3’
54˚C
27
SPRR2B
5’-TGAGCACTGATCTGCTTTGG-3’
5’-CTGGGAACTGACACTGCTGA-3’
60˚C
27
uPAR
5’-CTGCGGTGCATGCAGTGTAAG-3’
5’-GGTCCAGAGGAGAGTGCCTCC-3’
63˚C
22
Integrin α2
5’-TGGTCTCATCAATCTCATCT-3’
5’-TGACATCAGTTGTAATGCAG-3’
52˚C
27
Kallikrein 7
5’-AATGAGTACACCGTGCACCTG-3’
5’-AGACTCCTGGGTCATTGGGTT-3’
60˚C
30
MMP-10
5’-GTCACTTCAGCTCCTTTCCT-3’
5’-ATCTTGCGAAAGGCGGAACT-3’
60˚C
30
Envoplakin
5’-TGAATTCCAGCTGCAGGAGGAGTCG-3’
5’-AGTCGACGGTCTCCCCAGCTACAAGC-3’
57˚C
30
MMP-9
5’-GACCTGGGCAGATTCCAAAC-3’
5’-CACGCGCAGTGAAGGTGAGC-3’
60˚C
30
1
: cDNA was diluted 1:100 for GAPDH amplification
2
: Primers specific for p16 transcript variant 1
Reference
(Fu et al.,
2003)
(Montuori et
al., 2001)
(Kita et al.,
2001)
(Komatsu et
al., 2003)
(Liu et al.,
2001)
(Kazerounian
et al., 2002)
(Hsieh et al.,
2005)
35
36
Laminin γ2
Early passage HFKs (PD ~5-7) were cultured on glass coverslips with or without
feeder cells in either E-media or KSFM, respectively. Cells were fixed with 3.7%
formaldehyde in PBS for 10 minutes at room temperature. Cells were permeabilized with
0.5% Triton X-100 in PBS for 7 minutes. Coverslips were blocked in 10% normal goat
serum, 1% BSA, and 0.1% Tween-20 in PBS for 45 minutes. Coverslips were incubated
for 1 hour with laminin γ2 primary antibody (antibody B4-6, gift from W Carter, Fred
Hutchinson Cancer Research Center, Seattle, WA) at a 1:25 dilution in blocking buffer.
Donkey anti-mouse Texas-red conjugated secondary antibody (Jackson ImmunoResearch
Laboratories) was diluted 1:100 in blocking buffer and incubated with the coverslips for
45 minutes. The coverslips were mounted on glass slides using a 60% glycerol solution.
Images were collected on a Nikon Eclipse E800 fluorescence microscope (Nikon
Corporation, Tokyo, Japan).
β-Catenin
Early passage HFKs (PD ~5-7) were cultured on glass coverslips with or without
feeder cells in either E-media or KSFM, respectively. Cells were fixed with 3.7%
formaldehyde in PBS for 10 minutes at room temperature. Cells were permeabilized with
0.5% Triton X-100 in PBS for 7 minutes. Coverslips were blocked in 10% normal goat
serum, 1% BSA, and 0.1% Tween-20 in PBS for 45 minutes. Coverslips were incubated
for 1 hour with β-catenin primary antibody (Sigma: 6F9) at a 1:100 dilution in blocking
buffer. Donkey anti-mouse Texas-red conjugated secondary antibody (Jackson
ImmunoResearch Laboratories) was diluted 1:100 in blocking buffer and incubated with
the coverslips for 45 minutes. The coverslips were mounted on glass slides using a 60%
glycerol solution. Images were collected on a Nikon Eclipse E800 fluorescence
microscope (Nikon Corporation).
37
Results
Co-culture with feeder cells alters both human keratinocyte
replicative capacity and p16 expression
Variable results have been reported concerning both p16 expression and
replicative capacity of human keratinocytes cultured on tissue culture plastic alone or in
co-culture with post-mitotic fibroblast feeder cells (Baek et al., 2003; Fu et al., 2003;
Kang et al., 2003; Kang et al., 2004; Ramirez et al., 2001; Rheinwald et al., 2002). Using
three primary strains of HFKs derived from different donors, we found that HFKs serially
subcultured on plastic alone growth arrested at approximately 16.6 +/- 3.9 PDs. In
contrast, HFKs grown in co-culture with feeder cells reached approximately 28.1 +/- 2.3
PDs before proliferation ceased. Statistical analysis using the paired student’s t-test
found this difference to be significant (p = 0.030). Comparing replicative capacities of
each HFK strain, we found co-culture with feeder cells extended HFK replicative
capacity by an average 11.4 +/- 3.5 PDs. Representative growth curves from the three
primary HFK strains are shown in Figure 6A and 6B. Protein levels of p16 were also
altered in HFKs grown in the two culture conditions. We observed a steady
accumulation of p16 protein in HFKs cultured on plastic alone that reached a maximal
level at growth arrest (Figure 6C). In contrast, HFKs grown in the presence of feeder
cells exhibited delayed p16 accumulation that ultimately increased in later passages
correlating with growth cessation (Figure 6C). At late passage, an increase in p16
expression was seen in all primary HFKs co-cultured with feeders (data not shown).
We also tested whether culture of human keratinocytes on plastic alone at
different oxygen levels modulated replicative capacity or p16 expression. Using two
primary strains of HFKs derived from different donors, we found that HFKs serially
passaged on plastic alone in either atmospheric (~20%) or physiologic (~4%) oxygen
conditions did not exhibit a significant difference in replicative capacity (Figure 7A).
38
Figure 6: Lifespan extension and delay in p16 induction in keratinocytes co-cultured with
feeder cells. A) Replicative capacities of three primary HFK cell strains grown on plastic
alone (Strain 1: 12.9 PDs, Strain 2: 20.7 PDs, and Strain 3: 16.3 PDs). B) Replicative
capacities of three primary HFK cell strains grown in co-culture with post-mitotic
fibroblast feeder cells (Strain 1: 25.8 PDs, Strain 2: 28.1 PDs, and Strain 3: 30.3 PDs).
C) Immunoblot of p16 protein levels in Strain 1 HFKs cultured on plastic alone (lanes 14) and in co-culture with feeder cells (lanes 5-8). Protein levels of actin are included as a
loading control. Approximate population doublings are represented below each lane.
39
Figure 7: No significant lifespan extension or delay in p16 induction in keratinocytes
cultured on plastic alone in physiologic oxygen conditions. A) Replicative capacities of
two primary HFK cell strains grown on plastic alone in both atmospheric (~20%) and
physiologic (~4%) oxygen (Strain 4: 20.1 PDs in both oxygen conditions, Strain 5: 15.2
PDs in both oxygen conditions). B) Immunoblot of p16 protein levels in Strain 4 HFKs
cultured on plastic alone in either atmospheric oxygen (odd lanes) and physiologic
oxygen (even lanes). Protein levels of actin are included as a loading control.
Approximate population doublings are represented below each lane.
Furthermore, we did not observe a difference in p16 induction in HFKs cultured under
both oxygen conditions (Figure 7B). Thus, co-culture of human keratinocytes with
feeder cells delays p16 induction and keratinocyte telomere-independent senescence but
differences in oxygen levels during culture appear to have no effect on these phenomena.
40
Microarray analysis and validation of differential gene
expression and phenotype in different culture conditions
Differences in oxygen conditions did not significantly alter either replicative
capacity or p16 expression to the same extent that culture in the presence or absence of
feeders was capable. Thus, to determine which genes and signaling pathways are
activated or repressed in association with culture-induced p16 expression, we used
Affymetrix oligonucleotide microarrays to assay global gene expression patterns in HFKs
grown on plastic alone compared to those in co-culture with feeder cells. RNA was
collected from two independent, midpassage strains of HFKs serially subcultured under
both culture conditions. Following hybridization and detection of transcript abundance
on Affymetrix HG-U133A GeneChips, we compared gene expression in HFKs cultured
on plastic to that of HFKs in co-culture with feeder cells. The change in gene expression
level was considered significant if (1) the average fold change across all comparisons was
greater than or equal to twofold, and (2) the change in transcript level was considered
significantly increased or decreased by the Affymetrix difference call metric in each
microarray comparison performed. When the microarray data between the two culture
conditions were compared, we found 183 gene transcripts significantly decreased and 255
significantly increased in HFKs cultured on plastic alone as compared to HFKs cocultured with feeder cells. Functional classification of a subset of these genes was
performed as described in the Materials and Methods.
Many of the genes expressed at lower levels in HFKs cultured on plastic alone as
compared to HFKs in co-culture with feeder cells are characteristically expressed and
involved in keratinocyte differentiation. Many of these genes are involved in formation
of the cornified envelope (Kalinin et al., 2002) (Table 3). We also found several known
components of keratinocyte desmosomes downregulated in HFKs cultured on plastic
alone (Kitajima, 2002). In addition, several other genes which code for proteins
41
Table 3: Functional classification of differentiation-associated genes downregulated in
normal human foreskin keratinocytes grown on tissue culture plastic alone
Gene Description
Gene ID
UniGene ID
Fold Reduction
Small proline-rich protein 3
NM_005416
Hs.139322
18.0
Kallikrein 7 (Stratum corneum
chymotryptic enzyme)
NM_005046
Hs.151254
17.7
Transglutaminase 1
(Keratinocyte
Transglutaminase)
NM_000359
Hs.22
14.6
Small proline-rich protein 1A
NM_005987
Hs.211913
9.1
Sciellin
NM_003843
Hs.115166
8.6
Involucrin
NM_005547
Hs.157091
8.4
Envoplakin
NM_001988
Hs.25482
8.4
S100 calcium-binding
protein A9
NM_002965
Hs.112405
5.4
Elafin/SKALP
NM_002638
Hs.112341
5.2
Periplakin
NM_002705
Hs.74304
4.6
Small proline-rich protein 1B
NM_003125
Hs.1076
4.5
Small proline-rich protein 2B
NM_006945
Hs.231622
4.0
Cystatin A
NM_005213
Hs.2621
2.7
Desmoglein 1
NM_001942
Hs.2633
32.0
Plakophilin 1
NM_000299
Hs.198382
4.8
Desmocollin 1
NM_004948
Hs.69752
3.5
Plakophilin 3
NM_007183
Hs.26557
2.2
X07695
Hs.3235
>100
Cornified Envelope Formation
Desmosomal Proteins
Keratins
Keratin 4
42
Table 3 Continued
Keratin 13
NM_002274
Hs.74070
58.9
Keratin 1
NM_006121
Hs.80828
6.4
Keratin 10
X14487
Hs.99936
4.2
Serine protease inhibitor,
Kazal type, 5 (SPINK5)
NM_006846
Hs.331555
24.0
Lymphocyte antigen 6
complex, locus D (E48)
NM_003695
Hs.3185
23.2
AF216693
Hs.207224
6.7
NM_001444
Hs.153179
6.0
Other Genes Downregulated
Interleukin-1 receptor
antagonist homolog 1
Fatty acid binding protein 5
specifically expressed in differentiating keratinocytes were found to be downregulated in
HFKs cultured on plastic alone, including SPINK5, lymphocyte antigen 6 complex (E48),
interleukin-1 receptor antagonist, and fatty acid binding protein 5 (Brakenhoff et al.,
1995; Corradi et al., 1995; Komatsu et al., 2002; Olsen et al., 1995). Expression of
keratin intermediate filaments is known to be altered during the process of differentiation.
Keratins such as 1, 10, 4, and 13 are preferentially expressed in suprabasal,
differentiating keratinocytes in an epidermis (Dale et al., 1990) and were also found
downregulated in HFKs cultured on plastic alone compared to HFKs co-cultured with
feeder cells.
HFKs cultured on plastic alone upregulated genes that play a role in keratinocyte
migration. Several genes involved in formation of the provisional extracellular matrix
were upregulated, as well as, specific integrins needed for migration (Table 4). In
addition to the ECM genes themselves, several genes that code for ECM modifying
proteins were also upregulated, including matrix metalloproteinases, regulators of
plasmin activation, transglutaminase 2 (tissue transglutaminase), and thrombospondin 1.
43
Table 4: Functional classification of migration-associated genes upregulated in normal
human foreskin keratinocytes grown on tissue culture plastic alone
Gene Description
Gene ID
UniGene ID
Fold Induction
Fibronectin 1
X02761
Hs.287820
38.1
Chondroitin sulfate proteoglycan 2
(Versican)
D32039
Hs.81800
9.4
Collagen, type VIII, alpha 1
NM_001850
Hs.114599
6.4
Laminin, gamma 2
NM_005562
Hs.54451
4.6
Laminin, alpha 3
NM_000227
Hs.83450
4.3
Laminin, beta 3
NM_000228
Hs.75517
3.5
Integrin, beta 6
NM_000888
Hs.123125
3.3
Transforming growth factor, betainduced, 68kD
NM_000358
Hs.118787
2.9
Integrin, alpha 2
NM_002203
Hs.271986
2.4
Integrin, alpha V
NM_002210
Hs.295726
2.2
Hexabrachion (Tenascin C)
NM_002160
Hs.289114
2.1
Collagen, type XVI, alpha 1
NM_001856
Hs.26208
2.1
Matrix metalloproteinase 10
(stromelysin 2)
NM_002425
Hs.2258
7.1
Matrix metalloproteinase 1
(interstitial collagenase)
NM_002421
Hs.83169
3.9
Matrix metalloproteinase 2
(gelatinase A)
NM_004530
Hs.111301
3.2
Matrix metalloproteinase 9
(gelatinase B)
NM_004994
Hs.151738
2.3
Extracellular Matrix Proteins
Matrix Metalloproteinases (MMPs)
44
Table 4 Continued
Plasminogen Activation Components
Plasminogen activator, urokinase
(uPA)
NM_002658
Hs.77274
7.9
U08839
Hs.179657
3.1
NM_000602
Hs.82085
2.8
Keratin 19
NM_002276
Hs.182265
5.6
Keratin 8
NM_002273
Hs.242463
3.9
Keratin 18
NM_000224
Hs.65114
3.6
Inhibin, beta A
M13436
Hs.727
59.5
Transglutaminase 2 (Tissue
Transglutaminase)
M98478
Hs.8265
24.7
Vimentin
NM_003380
Hs.297753
6.9
Thrombospondin 1
NM_003246
Hs.87409
6.3
M92934
Hs.75511
6.0
Cysteine-rich, angiogenic
inducer, 61
NM_001554
Hs.8867
3.5
Follistatin
NM_013409
Hs.9914
2.4
Urokinase-type plasminogen
activator receptor (uPAR)
Plasminogen activator inhibitor
type 1
Keratins
Other Genes Upregulated
Connective tissue growth factor
Since keratinocyte migration is induced during the wound healing response it was not
surprising to see several genes involved in wound healing upregulated in HFKs cultured
on plastic alone compared to in co-culture with feeder cells. The antagonistic TGF-β
family proteins activin βA and follistatin are known to be expressed by keratinocytes in
wounded epithelium in vivo and were upregulated in HFKs grown on plastic alone, as
were two members of the CCN family of growth and angiogenic regulators, connective
45
tissue growth factor and cysteine-rich, angiogenic inducer 61 (Chen et al., 2001a;
Igarashi et al., 1993; Wankell et al., 2003). Also consistent with the observation that
HFKs cultured on plastic alone altered their normal program of differentiation was the
increase in expression of keratins typically found in simple, non-stratified epithelia
(keratins 8, 18, and 19).
Differential gene expression and phenotypic differences (differentiation in the coculture environment versus migration in the plastic alone culture condition), as
ascertained by microarray analysis, were validated by semi-quantitative RT-PCR,
immunoblot analysis, and immunocytochemistry. Early passage HFKs were cultured
under both conditions and transcript levels of uPAR, MMP-10, integrin α2, SPRR2B and
1A, kallikrein 7 and envoplakin were analyzed. We found the expression patterns seen in
the RT-PCR analysis were consistent with the microarray data (Figure 8A).
Protein levels of fibronectin, laminin γ2 (a component of laminin 5), vimentin,
and keratins 8, 13, and 19 were examined by immunoblot analysis. Keratin 14 was
examined as an additional control since, like actin, it was not found to be differentially
expressed by microarray analysis. The protein expression patterns of these genes were in
agreement with the microarray data (Figure 8B).
Immunocytochemical staining was performed for involucrin, laminin γ2, and βcatenin to validate the microarray analysis and the differences in phenotype that can be
inferred from the differential gene expression in the two culture conditions. Involucrin is
a vital structural component of the cornified envelope and was found to be expressed
primarily in keratinocytes co-cultured with feeder cells. Involucrin staining in
keratinocytes cultured with feeder cells exhibited a sheet-like morphology consistent with
the formation of a cornified envelope-like structure (Figure 9) (Okumura et al., 1991). In
contrast, the little involucrin staining seen in keratinocytes cultured on plastic alone was
sporadic and had no recognizable pattern of expression. Laminin γ2 staining in
46
Figure 8: Validation of microarray results. A) Semi-quantitative RT-PCR analysis of
differentially expressed gene transcripts in early passage (PD ~6-7) Strain 1 HFKs grown
under both culture conditions. The acidic ribosomal protein P0 gene, 36B4, a
housekeeping gene, is included as an internal control. Transcript levels of p16 in both
culture conditions are included for comparison. B) Immunoblot analysis of differentially
expressed genes in Strain 1 HFKs cultured on plastic alone (lanes 1-4) and in co-culture
with feeder cells (lanes 5-8). Protein levels of actin are included as a loading control.
Approximate population doublings are represented below each lane.
keratinocytes cultured on plastic alone was consistent with initial attachment of the cells
to the tissue culture plate followed by the deposition of a “laminin track” as the cells
migrated across the empty surface (Figure 10) (Kirfel et al., 2003; Nguyen et al., 2000a).
In contrast, the only laminin γ2 staining seen in the co-culture environment is found in
the regions occupied by the feeder cells. This result is consistent with the formation of a
primitive basement membrane-like structure between the keratinocytes and feeder cells.
Components of laminin 5 are a few of the several ECM proteins that are incorporated into
the basement membrane at the dermal-epidermal border (Nishiyama et al., 2000; Ryan et
al., 1996).
47
Figure 9: Phenotype Validation: Involucrin. Immunocytochemical staining for the
differentiation marker involucrin in early passage (PD ~6-7) Strain 1 HFKs grown under
both culture conditions. Arrows are pointing out the location of involucrin positive
staining HFKs in both culture conditions.
In keratinocytes cultured on plastic alone, β-catenin is located sporadically at the cell
periphery, can be seen throughout the cytoplasm, and may be present in the nucleus
(Figure 11). This staining pattern is consistent with the disruption of cell-cell contacts
and the redistribution of β-catenin (Dietrich et al., 2002). In contrast, β-catenin is located
exclusively at the cell periphery in keratinocytes cultured with feeder cells consistent
with the preservation of cell-cell contacts (Dietrich et al., 2002).
Overall, we found a high degree of correlation between our microarray results and
alternative expression assays, indicating that HFKs cultured in the absence of feeder cells
alter their normal gene expression program and phenotype of differentiation to one
characteristic of keratinocyte migration.
48
Figure 10: Phenotype Validation: Laminin γ2. Immunocytochemical staining for the
migratory track and basement membrane marker laminin γ2 in early passage (PD ~6-7)
Strain 2 HFKs grown under both culture conditions. Arrows in the top panel are pointing
out the location of laminin γ2 positive staining feeder cells. Arrow in the bottom panel is
pointing out the location of laminin γ2 positive staining HFKs cultured on plastic alone.
*: The laminin γ2 migration track.
Thus, culture conditions that promote p16-induced, telomere-independent senescence
also activate signaling pathways involved in keratinocyte migration.
Discussion
It has been suggested that human keratinocytes cultured under different conditions
exhibit differences in both replicative capacity and p16 expression. In this study, we
have shown that HFKs cultured on plastic alone exhibit a phenotype characteristic of a
keratinocyte migration response. This phenotypic change in keratinocyte behavior and
gene expression suggests an identity for the culture-induced “stress” experienced when
keratinocytes are grown in the absence of post-mitotic feeder cells and poses new
questions as to a possible role for p16 in keratinocyte migration.
49
Figure 11: Phenotype Validation: β-Catenin. Immunocytochemical staining for the cellcell contact marker β-catenin in early passage (PD ~6-7) Strain 2 HFKs grown under
both culture conditions. Arrows are pointing out the location of β-catenin positive
staining HFKs in both culture conditions.
Growth arrest and p16 expression in different culture
conditions
Similar to what has been observed previously, we found that both replicative
capacity and p16 expression are altered in human keratinocytes grown with or without
feeder cells. Our results are consistent with previous reports that have shown a decrease
in p16 expression and increase in replicative capacity in the co-culture system, however,
unlike previous reports that characterized the increase in cellular lifespan to be
approximately 20-30 PDs, we found it to be more modest (Fu et al., 2003; Kang et al.,
2003; Kang et al., 2004; Ramirez et al., 2001; Rheinwald et al., 2002). What accounts for
the variation seen between co-culture results remains to be identified, however,
50
possibilities include differences in precise media constituents, passage protocols, feeder
cell number and/or the method of induction of the feeder cell post-mitotic state.
Increased levels of oxidative stress have been found to regulate p16 expression in
certain cell types under specific conditions (Ahmed et al., 1999; Chazal et al., 2002; Chen
et al., 2001b; Lee et al., 1999; Peus & Pittelkow, 2001; von Zglinicki et al., 1995).
Culture of human fibroblasts in a low oxygen environment, a setting more akin to the
level of oxygen exposure in vivo, has often been employed to determine whether cultureinduced oxidative stress influences cellular senescence (Chen et al., 1995; Packer &
Fuehr, 1977; Saito et al., 1995). However, very little data exists linking increased levels
of ROS and epithelial senescence. In this study, we found that neither human
keratinocyte replicative capacity nor induction of p16 expression was influenced by
differences in oxygen conditions when the cells were cultured on plastic alone. In our
microarray analysis comparing HFKs cultured with or without feeder cells, we did not
find genes such as superoxide dismutase, catalase, thioredoxin peroxidase, or glutathione
peroxidase differentially expressed, suggesting that oxidative stress is not responsible for
modulating p16 expression in these two culture conditions. In addition, previous
attempts in our lab to modulate oxidative stress with either N-acetyl-cysteine or hydrogen
peroxide have failed to show any significant difference in either p16 expression or
replicative capacity of human keratinocytes (data not shown). Thus, whereas studies in
transgenic mice have shown that cellular responses to oxidative stress may be involved in
organism aging (Migliaccio et al., 1999), it would appear from our experiments that
culture-induced oxidative stress is not involved in p16-induced, telomere-independent
senescence in human keratinocytes.
p16 is co-expressed with keratinocyte migration genes
The stimulus for p16 induction under certain culture conditions has not been fully
elucidated, however, several theories have suggested that a cellular stress, as opposed to a
51
predetermined program of gene expression, is responsible for telomere-independent
senescence (Ben-Porath & Weinberg, 2004; Lowe & Sherr, 2003; Shay & Wright, 2001;
Sherr & DePinho, 2000). During the wound healing process, keratinocytes undergo a
complex reprogramming of gene expression that allows them to alter their normal
behavior of terminal differentiation to one of migration and proliferation. This change
induces a switching of keratinocyte integrin profiles from one that favors attachment to
the basement membrane (mediated by α6β4 integrins) to one necessary for migration over
a provisional matrix (α5β1, αVβ6, αVβ5, α3β1, and α2β1) (Martin, 1997; Nguyen et al.,
2000a). To facilitate migration, keratinocytes must also acquire the ability to remodel
extracellular matrix proteins. Activation of plasmin, the main fibrinolytic enzyme
necessary to dissolve a fibrin clot, is achieved by either tissue-type plasminogen activator
(tPA) or urokinase-type plasminogen activator (uPA). To increase the likelihood of
plasmin activation, the receptor for uPA (uPAR) is also known to be upregulated during
the wound healing process (Blasi & Carmeliet, 2002). Keratinocyte migration is also
aided by upregulation of matrix metalloproteinases 1, 9, and 10 (Martin, 1997).
Additionally, in an effort to both restore the basement membrane and aid migration,
keratinocytes engaged in the wound healing response produce extracellular matrix
proteins including laminin, fibronectin, and collagen (Kirfel et al., 2003; O'Toole, 2001).
Our microarray results and subsequent validation indicate that, in contrast to
differentiating HFKs in co-culture with feeder cells, HFKs cultured on plastic alone are
engaged in a migratory response that displays upregulation of several genes also involved
in the wound healing process. Most of the genes mentioned above were found
upregulated in HFKs grown on plastic alone as were components of the TGF-β signaling
pathway, also thought to play a role in migration during the keratinocyte wound healing
response (Decline et al., 2003; Martin, 1997; Wankell et al., 2003). From the noticeable
differences in culture conditions, it is probable that this change in phenotype is induced
by a lack of specific cell-cell contacts between keratinocytes and/or fibroblasts. In an
52
effort to exclude differences in culture media as the reason for the phenotypic switch we
attempted to culture HFKs in E-media without feeder cells, however, HFKs cultured in
this fashion did not survive beyond one passage (data not shown). It should be noted that
the concentration of calcium in E-media is much higher than in KSFM and calcium has
been shown to be an important determinant in causing keratinocyte differentiation (Boyce
& Ham, 1983). We attempted to determine whether the increased calcium content of Emedia was responsible for inhibition of p16 expression in the co-culture environment but
when KFSM was supplemented with a similar level of calcium (1.28mM) we actually
saw an increase in p16 expression (data not shown). Thus, it appears that the feeder cells
are a primary factor responsible for inhibition of p16 expression in the co-culture
environment.
Possible signaling pathways involved in p16 induction
Previous studies combined with our microarray results suggest a number of
different signaling pathways that may be involved in p16 induced telomere-independent
senescence when human keratinocytes are grown on tissue culture plastic alone and
engaged in the process of migration. As discussed above, activated keratinocytes
undergo several changes in an effort to adopt a migratory phenotype. Changes in integrin
profiles and the secretion of ECM proteins provides for the development of a
microenvironment where cell surface EMC receptors bind their respective ligands and
form adhesion structures known as focal adhesions. Focal adhesion formation causes the
clustering of integrins and the recruitment of focal adhesion kinase (FAK), a major
signaling protein involved in transducing extracellular signals from the ECM to the
nucleus (Schlaepfer et al., 1999). FAK activity has been shown to be induced during the
wound healing response and may serve to activate the necessary signal transduction
pathways responsible for acquiring the activated/migratory phenotype (Kim et al., 2000;
Kim et al., 2001). Upon binding to focal adhesions, FAK is activated through an
53
autophosphorylation event that leads to the formation of a signaling complex including
proteins such as c-Src, Shc, and Grb2 (Figure 12). The association of activated FAK with
Grb2, and subsequently SOS, has been shown to increase signaling through the
Ras/MAPK pathway (Blasi & Carmeliet, 2002; Schlaepfer et al., 1999). This finding is
relevant to p16 expression since overexpression of oncogenic Ras has been shown to be
capable of upregulating p16 expression in human fibroblasts (Lin et al., 1998; Ohtani et
al., 2001; Serrano et al., 1997). One proposed mechanism for the upregulation of p16 by
Ras signaling identifies the Ets family members, Ets1 and Ets2, as transcriptional
activators of p16 expression (Ohtani et al., 2001). Whether a functional relationship
between FAK and Ets factors exists is still speculative (Naito et al., 2002); however, the
relationship between ECM regulation and Ets transcription factors has been well
established (Trojanowska, 2000) suggesting that p16 regulation during keratinocyte
migration may be modulated by differences in ECM composition. This hypothesis is
consistent with findings that p16 expression is increased in dysplastic or transformed
keratinocytes invading through the basement membrane, which may be providing
migrating keratinocytes with a unique composition of ECM ligands (Natarajan et al.,
2003; Nilsson et al., 2004; Svensson et al., 2003). Overexpression of FAK has also been
found in several types of epithelial derived cancers (Cance et al., 2000; Glukhova et al.,
1995; Han et al., 1997; Kornberg, 1998; Owens et al., 1996; Tremblay et al., 1996) and
has specifically been linked to increased invasion (Schneider et al., 2002; Weiner et al.,
1993). These results imply that a relationship between FAK and p16 may exist in the
context of keratinocyte migration. This hypothesis will be tested in the next chapter.
The role of p16 in keratinocyte migration
Our results imply that p16 is co-expressed with several genes known to be
involved in keratinocyte migration. Our results are consistent with other findings that
54
Figure 12: FAK signaling pathways. Integrin receptor engagement with ligands such as
fibronectin, laminin, and collagen causes the formation of focal adhesions and can
stimulate FAK autophophorylation at Tyr-397. In several studies, uPA bound to uPAR is
also present in focal adhesions via a uPA-dependent uPAR-integrin interaction.
Autophosphorylation of Tyr-397 creates a SH2 binding domain which leads to the
subsequent recruitment of Src-family protein tyrosine kinases (PTKs). Src-mediated
phosphorylation of FAK at Tyr-925 creates a binding site for the Grb2 adaptor protein.
Binding of Grb2 to FAK, or FAK/Src tyrosine phosphorylated Shc, can lead to the
translocation of the GDP/GTP exchange protein SOS to the plasma membrane and
enhanced GTP exchange on Ras. This event activates the Ras/MAPK signaling pathway
that can then potentially influence both cellular gene expression and migration.
Source: Figure modified from (Schlaepfer et al., 1999).
p16 expression is not induced as a feature of normal differentiation or epithelial renewal
(Keating et al., 2001; Klaes et al., 2001; Nielsen et al., 1999). Also in agreement with our
finding that p16 induction is associated with a migratory response, is the observation that
p16 expression is induced in human keratinocytes subjected to scraping in vitro
(Natarajan et al., 2003). Furthermore, it was shown by Natarajah and colleagues that p16
and laminin γ2 were co-expressed under these conditions. This result is consistent with
our finding that laminin γ2, as well as laminins α3 and β3, was upregulated in HFKs
55
cultured on plastic alone compared to in co-culture with feeder cells. It has been
proposed that p16 induction during migration may have evolved to facilitate wound
closure by halting proliferation in favor of increased cell motility (Natarajan et al., 2003).
However, in several contexts, p16 has been shown to inhibit migration of various cell
types (Adachi et al., 2001; Fahraeus & Lane, 1999) and may play a role in contactmediated growth inhibition (Wieser et al., 1999). An alternative hypothesis is that
migration-induced p16 expression evolved to prevent constitutive activation of the genes
necessary for migration. Thus, loss of p16 in cancer cells may allow for continued
activation of migratory genes and an increased incidence of invasion. If this is the case,
future therapies designed to modulate p16 expression in invasive carcinomas could
prevent both tumor proliferation and metastasis.
Summary
In summary, we have shown that human keratinocytes, grown in the absence of
feeder cells, are subjected to an additional culture-induced stimulus that alters
keratinocyte gene expression and phenotype. Furthermore, we have shown that p16induced, telomere independent senescence occurs in culture conditions that induce a
keratinocyte migratory response. These results provide new insight into keratinocyte
migration as well as p16 regulation and may ultimately provide a mechanism for the
telomere-independent senescence experienced when primary keratinocytes are cultured in
the absence of feeder cells.
56
CHAPTER III
INHIBITION OF KERATINOCYTE MIGRATION BUT
NOT FAK ACTIVATION REDUCES P16 EXPRESSION
Introduction
In the previous chapter, we have shown that p16 expression in human
keratinocytes cultured in the absence of feeder cells, a culture condition that promotes
telomere-independent senescence, correlates with the reprogramming of keratinocyte
gene expression and phenotype (Darbro et al., 2005). Keratinocytes grown on tissue
culture plastic alone exhibited a phenotype characteristic of “activated” migrating cells
and induced the expression of several genes known to be involved in the wound healing
process. Upon wounding, keratinocytes acquire an activated phenotype and switch their
profile of cell surface integrins from one favoring cell attachment to one necessary for
migration (Martin, 1997). Migratory integrins come into contact with provisional ECM
proteins and form focal adhesions (FAs). FAK is then recruited to FAs through a Cterminal focal adhesion targeting domain (FAT) (Hildebrand et al., 1993), and upon
proper localization and binding to integrin-associated proteins, undergoes an
autophosphorylation event at Tyr-397 that leads to the formation of a signaling complex
(Schlaepfer et al., 1999). FAK associates with several different signaling proteins and
affects a wide variety of cellular events, including cellular spreading and migration
(Gates et al., 1994; Kim et al., 2000; Schlaepfer et al., 1999) (Figure 12). Based on our
previous findings that both migratory integrins and their respective ECM protein ligands
are upregulated in human keratinocytes cultured in the absence of feeder cells, as well as
the observation that FAK activity is known to be induced in “activated” keratinocytes
(Kim et al., 2001), we hypothesized that FAK may be directly involved in the
upregulation of p16 during keratinocyte migration and telomere-independent senescence.
57
FAK signaling has been shown to transduce extracellular signals to the nucleus
through a series of tyrosine phosphorylation events involving signaling proteins such as
Src, p130Cas, and Shc (Schlaepfer et al., 1999). Binding of ECM proteins fibronectin and
laminin to their respective integrin receptors has been shown to cause recruitment of
FAK to FAs and induce the autophosphorylation of FAK at Tyr-397 (Kim et al., 2005;
Schlaepfer et al., 1999). FAK autophosphorylation of Tyr-397 forms a SH2 binding
domain for Src family protein tyrosine kinases (Src-PTKs) that can then induce
subsequent phosphorylation of FAK at Tyr-925 (Schlaepfer & Hunter, 1996). Src-PTK
phosphorylation of FAK at Tyr-925 serves as a binding site for Grb2 and allows for the
activation of the Ras/MAPK pathway, a known regulator of p16 expression in human
fibroblasts (Lin et al., 1998; Ohtani et al., 2001; Schlaepfer et al., 1994; Serrano et al.,
1997). FAK has also been found to directly regulate the expression of MMPs-2 and 9, as
well as uPA (Hauck et al., 2001; Irigoyen & Nagamine, 1999; Sein et al., 2000; Shibata
et al., 1998). These genes are involved in remodeling ECM and, like fibronectin and
components of laminin 5, were found upregulated in keratinocytes cultured in the
absence of feeder cells. It is also significant that these genes, like FAK itself, all exhibit
increased expression in cancers that have acquired the ability to invade through the
basement membrane, a phenomenon that has also been shown to induce p16 expression
(Jung et al., 2001; Natarajan et al., 2003; Nilsson et al., 2004; Svensson et al., 2003).
These observations suggest that FAK signaling should be active in human keratinocytes
cultured in the absence of feeder cells and may be an upstream regulator of p16
expression.
Another protein that cooperates with FAK in transducing extracellular signals to
the nucleus is the urokinase plasminogen activator receptor (uPAR). Binding of pro-uPA
to uPAR activates uPA, facilitating the generation of plasmin from plasminogen,
resulting in proteolysis of the ECM and increased mobility of migrating cells (Collen,
1999). However, in addition to this proteolytic mechanism of migration, uPA/uPAR
58
interactions have also been shown to be connected with a variety of intracellular
signaling pathways that involve both tyrosine and serine protein kinases such as Src,
FAK, and the Ras/MAPK pathway (Blasi & Carmeliet, 2002). uPAR, which lacks a
cytosolic domain, transmits extracellular signaling through associations with integrins,
namely α3β1, α5β1, αVβ1, and αVβ3 (Blasi & Carmeliet, 2002). Binding of uPA to uPAR
has been shown to stimulate the Ras/MAPK pathway through a mechanism that requires
FAK, Src and Shc (Nguyen et al., 2000b). There is also the potential for a positive
feedback loop to emerge during the wound healing process as both constitutively active
Ras and FAK/Src/Ras signaling activated by cytoskeletal reorganization lead to the
induction of uPAR and uPA, respectively (Irigoyen & Nagamine, 1999; Muller et al.,
2000). As shown in the previous chapter, both uPA and uPAR transcript levels were
found to be increased in human keratinocytes cultured on plastic alone suggesting that
these proteins could be participating with FAK in regulating p16 expression.
In both FAK and uPA/uPAR signaling, Src-PTKs play a significant role. c-Src is
involved in both FAK and uPA/uPAR induced activation of the Ras/MAPK pathway
(Irigoyen & Nagamine, 1999; Nguyen et al., 2000b; Schlaepfer et al., 1994; Schlaepfer et
al., 1999; Schlaepfer & Hunter, 1996), and has been found to be activated and associated
with microtubules in human keratinocytes subjected to scrape-wounding in vitro
(Yamada et al., 2000). Consistent with a role in cellular migration, c-Src is known to
tyrosine phosphorylate β-catenin leading to disruption of E-cadherin/β-catenin cell-cell
contacts and a loss of cell adhesion (Behrens et al., 1993; Lilien & Balsamo, 2005;
Matsuyoshi et al., 1992). This is relevant to keratinocytes cultured on plastic alone since
β-catenin was found to have been redistributed from the cell periphery to the cytoplasm
(Figure 11). The reliance of FAK and uPA/uPAR signaling on Src-PTKs, combined with
evidence of c-Src activation in migrating epithelial cells, suggests that Src-PTKs may be
involved in migration-mediated induction of p16 expression.
Based on these observations we constructed a model for p16 induction in human
59
Figure 13: Proposed model of p16 induction in human keratinocytes cultured on plastic
alone. Secretion and subsequent ligation of ECM proteins with their pro-migratory
integrin receptors causes the formation of focal adhesions. Focal adhesions, which may
very well contain uPA/uPAR receptor-ligand complexes, facilitate the recruitment of
FAK to these sites and subsequent autophosphorylation of Tyr-397. Activated FAK then
initiates a signal transduction cascade involving Src and the Ras/MAPK pathway.
Increased FAK signaling induces the expression of MMPs-2, 9 and uPA, and may
potentially be responsible for inducing p16 expression and consequent telomereindependent senescence.
keratinocytes cultured on plastic alone (Figure 13). Based on this model, we focused our
studies on FAK, uPA/uPAR, and Src-PTKs as potential upstream regulators of p16
expression in activated keratinocytes cultured in the absence of feeder cells. We found
that both the localization and phosphorylation status of FAK were modulated by
differences in culture conditions and, in human keratinocytes cultured on plastic alone,
the induction of p16 expression was tightly correlated with increased cellular levels of
either autophosphorylated FAK or uPAR. When uPA/uPAR function or the cascade of
tyrosine phosphorylation downstream of FAK, Src, and uPA/uPAR signaling were
inhibited by various methods, we saw a reduction in both keratinocyte migration and p16
60
expression; however, specific inhibition of Src-PTKs or FAK autophosphorylation
reduced only keratinocyte migration without a concomitant suppression of p16 induction.
These findings suggest that p16 induction during keratinocyte migration is regulated by
tyrosine phosphorylation events and proper functioning of the urokinase plasminogen
activation system, however is not directly dependent on either FAK or Src-PTK activity.
Materials and Methods
Immunoblot Analysis
The following primary antibodies were used in this study: pY397-FAK (BD
Transduction Laboratories: 14), FAK (Upstate Cell Signaling Solutions, Charlottesville,
VA: 06-543), Phospho-MEK1/2 (Ser217/221) (Cell Signaling Technology, Danvers,
MA: 9121), and Phospho-ERK1/2 (Thr202/Tyr204) (Cell Signaling Technology: 9101).
Real-Time RT-PCR Analysis
cDNA synthesis reactions were carried out as described in Chapter II using the
RETROscript kit. Real-time PCR was performed on 100ng of cDNA using the Sybr
Green system (Applied Biosystems, Foster City, CA, Product Number: 4309155) and a
7000 Sequence Detection System real-time thermalcycler (Applied Biosystems). Realtime PCR reactions were carried out for p16, MMP-9, and 18S (a ribosomal gene
analyzed as a control) with an annealing temperature of 60˚C for 40 cycles. Analysis was
performed using the ABI Prism 7000 SDS software supplied with the 7000 Sequence
Detection instrumentation system. Levels of p16 were standardized against 18S levels
and normalized by relative comparison. All real-time reactions were carried out in
triplicate and error bars created using standard deviation of the relative mean.
61
Immunocytochemistry
FAK and Vinculin
Early passage HFKs (PD ~5-7) were cultured on glass coverslips without feeder
cells in KSFM. Cells were fixed with 3.7% formaldehyde in PBS for 10 minutes at room
temperature. Cells were permeabilized with 0.5% Triton X-100 in PBS for 7 minutes.
Coverslips were blocked in 5% normal donkey serum, 5% normal goat serum, 1% BSA,
and 0.1% Tween-20 in PBS for 45 minutes. Coverslips were incubated for 1 hour with a
mixture of both vinculin (antibody 7F9, gift from K Burridge, University of North
Carolina) and FAK (Upstate Cell Signaling Solutions, Charlottesville, VA: 06-543)
primary antibodies. FAK primary antibody was diluted to a ratio of 1:100 in the provided
solution of vinculin antibody. A mixture of donkey anti-mouse Texas-red conjugated
(Jackson ImmunoResearch Laboratories) and goat anti-rabbit fluorescein conjugated
(Chemicon International) secondary antibodies, both diluted 1:100 in blocking buffer,
was incubated with the coverslips for 1 hour. The coverslips were mounted on glass
slides using a 60% glycerol solution. Images were collected on a Bio-Rad Radiance 2100
MP Confocal/Multiphoton microscope (Bio-Rad Laboratories), at the University of Iowa
Central Microscopy Core Facility. Images were analyzed and merged using ImageJ
software (National Institutes of Health). PBS washes were performed between each step
in all immunocytochemistry protocols.
FAK, uPAR, and p16
Mid to late passage HFKs (PD ~12-17) were grown on permanox chamber slides
without feeder cells in KSFM. Cells were fixed with 3.7% formaldehyde in PBS for 10
minutes at room temperature. Cells were permeabilized with 0.5% Triton X-100 in PBS
for 7 minutes. Slides were blocked in 10% normal donkey serum, 1% BSA in PBS for 45
minutes. Slides were incubated with pTyr-397 FAK (BD Transduction Laboratories: 14)
or uPAR (American Diagnostica Inc., Greenwich, CT: 3936) primary antibodies both
62
diluted in blocking buffer at a ratio of 1:100 for 1 hour. Texas-red conjugated donkey
anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories), diluted 1:100
in blocking buffer, was incubated with the chamber slides for 1 hour. Slides were then
incubated with an Oregon-green conjugated p16 primary antibody diluted 1:40 in a
blocking buffer consisting of 10% normal goat serum, 1% BSA in PBS for 1 hour. The
Oregon-green conjugated p16 antibody was created using a primary antibody to p16
(Pharmingen: G175-405) and a Zenon Oregon-green labeling kit (Molecular Probes,
Eugene, OR) according to the manufacturer’s instructions. Chamber slides were
mounted with VECTASHIELD mounting medium with DAPI (Vector Laboratories,
Burlingame, CA) to provide for visualization of HFK nuclei. Images were collected on a
Nikon Eclipse E800 fluorescence microscope (Nikon Corporation). To ascertain pTyr397 FAK/p16 and uPAR/p16 co-staining, HFKs staining positive for the different
antibodies were visualized and quantified in four separate microscopic fields.
Retroviral Constructs and Infection
The FRNK retroviral construct was made by PCR cloning avian FRNK cDNA out
of an avian FRNK-pcDNA3 vector (gift from M Schaller, University of North CarolinaChapel Hill) (Lin et al., 1997) and inserting it into the retroviral vector pLXSN.
Generation of virus with Phoenix amphotropic packaging lines has been previously
described (Der et al., 1986; Whitehead et al., 1995; Zohn et al., 1998). Exponentially
growing HFKs at approximately PD 4 were infected overnight in 4 µg/ml polybrene.
Infected cells were washed in regular media the following morning. Cells were passed
the following day, and selective media containing 50 µg/ml neomycin (Geneticin)
(Invitrogen: 11811-031) was added on the next day. Control experiments were
conducted by infecting parallel cultures of HFKs with the pLXSN vector alone. All
infections were performed in the plastic culture condition and selection was carried out
63
over 7 days. Clonal populations were generated by splitting infected HFKs 1:100 prior to
selection and performing ring cloning.
Migration Assays
Outgrowth migration assays were performed as previously described by Ura and
colleagues (Ura et al., 2004). Briefly, early passage HFKs (PDs ~5-7) were suspended in
KSFM or KSFM containing different concentrations of either Herbimycin A (Sigma:
H6649), Amiloride (Sigma: A7410), or PP1 (BIOMOL Int., Plymouth Meeting, PA:
EI275). Approximately 4x104 HFKs were then plated inside a 5mm cloning ring placed
on a noncoated, plastic tissue culture plate and allowed to attach for 4 hours. After the
HFKs had attached, the cloning ring was removed to yield a circular monolayer sheet.
Migration outward was measured for 7 days with media changes every other day. In
treatment groups, Herbimycin A, Amiloride, or PP1 were maintained in the media for the
duration of the experiment. After 7 days, protein and RNA were collected as described
above. For each treatment group, triplicate monolayer sheets were plated, measured for
migration, and collected for protein and RNA. Statistical significance was measured by a
two-sample student’s t-test.
Results
Induction of p16 expression correlates with markers of
keratinocyte migration and FAK activation
To determine the activation status of FAK in both culture conditions, FAK
phosphorylation at Tyr-397 was assayed by immunoblotting. FAK was
autophosphorylated at Tyr-397 almost exclusively in HFKs grown on plastic alone
(Figure 14). Immunocytochemistry was performed to establish if FAK was being
recruited to focal adhesions, the site at which FAK becomes active and
64
Figure 14: Autophosphorylation of FAK in keratinocytes cultured on plastic alone.
Immunoblot of pTyr397-FAK, total FAK, and the activated Ras/MAPK signaling
intermediates phospho-MEK1/2 (Ser217/221) and phospho-ERK1/2 (Thr202/Tyr204) in
Strain 3 HFKs cultured on plastic alone (lanes 1-4) and in co-culture with feeder cells
(lanes 5-9). Protein levels of actin are included as a loading control. Approximate
population doublings are represented below each lane.
autophosphorylates Tyr-397. As expected, FAK was found to co-localize with vinculin,
a marker of focal adhesions, in HFKs cultured on plastic alone (Figure 15). In contrast,
FAK staining in HFKs cultured with feeder cells was diffuse throughout the cytoplasm
(data not show). We also confirmed that HFKs with increased levels of Tyr-397
autophosphorylated FAK and uPAR were selectively expressing p16. We found the
overall correlation for both pTyr-397 FAK/p16 and uPAR/p16 co-staining to be 90-98%
(Figure 16). HFKs staining negative for FAK autophosphorylation or uPAR showed
little or no increase in p16 protein. Interestingly, we did not observe an increase in the
active, phosphorylated forms of the Ras/MAPK pathway signaling intermediates
MEK1/2 and ERK1/2 in HFKs cultured on plastic alone (Figure 14). In fact, greater
levels of phosphorylated MEK1/2 and ERK1/2 were present in HFKs co-cultured with
feeder cells. Thus, the induction of p16 expression in HFKs cultured on plastic alone is
65
Figure 15: Immunocytochemical staining for both vinculin and FAK in early passage (PD
~6-7) Strain 2 HFKs grown on plastic alone. Arrows indicate positive staining for
vinculin and FAK in focal adhesions.
Figure 16: Co-staining of autophosphorylated FAK and uPAR with p16. Mid to late
passage (PD ~12-17) Strain 1 HFKs cultured on plastic alone were stained for uPAR and
p16 or pTyr-397 phosphorylated FAK and p16. DAPI staining was performed for
visualization of HFK nuclei.
66
tightly correlated with both FAK autophosphorylation and increased levels of uPAR
protein but not increased activation of the Ras/MAPK signaling pathway.
Inhibition of tyrosine kinase activity or uPA/uPAR function
reduces p16 expression
Tyrosine phosphorylation is a common event in both FAK activation and
downstream signal transduction initiated by uPA/uPAR or Src-PTKs (Blasi & Carmeliet,
2002; Danen et al., 1998; Longhurst & Jennings, 1998; Parsons & Parsons, 2004;
Schlaepfer et al., 1999). Herbimycin A, a nonspecific inhibitor of tyrosine kinases,
Amiloride, a competitive inhibitor of uPA, and PP1, a specific inhibitor of Src family
kinases, have all been shown to inhibit keratinocyte migration in vitro (Daniel & Groves,
2002; Kansra et al., 2005; Kim et al., 2001) and were used in our studies to inhibit
migration of HFKs cultured on plastic alone. Using an outgrowth migration assay, we
treated primary HFKs cultured on plastic alone with different concentrations of
Figure 17: Herbimycin A treatment alters HFK morphology and migration patterns.
Early passage (PD ~5-7) Strain 3 HFKs were grown in an outgrowth migration assay on
tissue culture plastic alone and treated with 875nM Herbimycin A (Herb A) for 7 days.
Control HFKs were treated with an equivalent amount of DMSO. The morphology and
migration pattern exhibited by HFKs treated with Herbimycin A is representative of that
observed with both Amiloride and PP1.
67
Herbimycin A, Amiloride, or PP1 and assayed for keratinocyte migration and p16
expression. Morphologically, HFKs treated with Herbimycin A, Amiloride, or PP1
displayed more sheet-like growth with cells closely packed together. In contrast,
untreated, control cultures displayed many cells individually migrating outward from the
pre-formed circular keratinocyte sheet (Figure 17). We found that Herbimycin A,
Amiloride, and PP1 all significantly inhibited HFK migration in the outgrowth assay (p =
0.003 for 219nM Herbimycin A, p = 0.0001 for 875nM Herbimycin A, p = 0.001 for
50µM Amiloride, p = .047 for 250nM PP1, p = .001 for 500nM PP1) (Figures 18A, 19A,
and 20A). Furthermore, it was observed that both Herbimycin A and Amiloride
treatment reduced p16 expression at both the mRNA and protein levels (Figures 18B and
C as well as Figure 19B and C). Treatment with PP1, however, did not reduce p16
expression at either the mRNA or protein levels (Figures 20B and C). Real-time RTPCR analysis confirmed that p16 mRNA levels were significantly different between
untreated HFKs and those treated with either Herbimycin A or Amiloride, although there
was not a significant difference in p16 mRNA levels between the two Herbimycin
concentrations examined (Figure 18D and 19D). Real-time analysis confirmed that there
was no difference in p16 expression in HFKs treated with PP1 (Figure 20D). Thus,
inhibition of tyrosine kinase activity, uPA/uPAR function, or Src-PTK activity is
sufficient to reduce keratinocyte migration, but only the disruption of tyrosine
phosphorylation or uPA/uPAR function is capable of suppressing p16 expression in
HFKs cultured on plastic alone.
Specific inhibition of FAK signaling does not reduce p16
expression
To specifically test the involvement of FAK signaling in p16 regulation we
infected primary HFKs with a retroviral vector encoding the C-terminal end of FAK.
68
Figure 18: Herbimycin A inhibits keratinocyte migration and p16 expression. A) Early
passage (PD ~5-7) Strain 3 HFKs were grown in an outgrowth migration assay on plastic
alone for 7 days with or without Herbimycin A. Error bars represent standard deviation
of at least three separate circular monolayer sheets. B) Immunoblot of pTyr397-FAK,
total FAK, and p16 in Strain 3 HFKs treated with Herbimycin A. Consistent with its role
as a tyrosine kinase inhibitor, Herbimycin A reduced the levels of autophosphorylated
FAK. Protein levels of actin are included as a loading control. Herbimycin A
concentrations used are represented below each lane. C) Semi-quantitative RT-PCR
analysis of p16 in Strain 3 HFKs treated with Herbimycin A. GAPDH, a housekeeping
gene, is included as an internal control. Herbimycin A concentrations used are
represented below each lane. D) Real-time RT-PCR analysis of p16 in Strain 3 HFKs
treated with Herbimycin A. Levels of p16 mRNA were standardized against expression
levels of the 18S rRNA gene transcript and then normalized relative to the untreated,
control group. Error bars represent standard deviation of the relative mean.
69
Figure 19: Amiloride inhibits keratinocyte migration and p16 expression. A) Early
passage (PD ~5-7) Strain 3 HFKs were grown in an outgrowth migration assay on plastic
alone for 7 days with or without Amiloride. Error bars represent standard deviation of at
least three separate circular monolayer sheets. Amiloride concentrations 250 µM and
above were found to be cytotoxic to HFKs and cells treated with these concentrations
were not used for further analysis. B) Immunoblot of p16 in Strain 3 HFKs treated with
Amiloride. Protein levels of actin are included as a loading control. The Amiloride
concentration used is represented below lane 2. C) Semi-quantitative RT-PCR analysis
of p16 and uPAR in Strain 3 HFKs treated with Amiloride. Consistent with the results of
previous studies (Wang et al., 1994), Amiloride treatment reduced uPAR expression.
GAPDH, a housekeeping gene, is included as an internal control. The Amiloride
concentration used is represented below lane 2. D) Real-time RT-PCR analysis of p16 in
Strain 3 HFKs treated with Amiloride. Levels of p16 mRNA were standardized against
expression levels of the 18S rRNA gene transcript and then normalized relative to the
untreated, control group. Error bars represent standard deviation of the relative mean.
70
Figure 20: PP1 inhibits keratinocyte migration but not p16 expression. A) Early passage
(PD ~5-7) Strain 6 HFKs were grown in an outgrowth migration assay on plastic alone
for 7 days with or without PP1. Error bars represent standard deviation of at least three
separate circular monolayer sheets. B) Immunoblot of laminin γ2 and p16 in Strain 6
HFKs treated with PP1. Consistent with being a downstream Src-inducible gene (Hlubek
et al., 2001; Lilien & Balsamo, 2005), protein levels of laminin γ2 were reduced in PP1
treated cells. Protein levels of actin are included as a loading control. PP1
concentrations used are represented below each lane. C) Semi-quantitative RT-PCR
analysis of p16 in Strain 6 HFKs treated with PP1. GAPDH, a housekeeping gene, is
included as an internal control. PP1 concentrations used are represented below each lane.
D) Real-time RT-PCR analysis of p16 in Strain 6 HFKs treated with PP1. Levels of p16
mRNA were standardized against expression levels of the 18S rRNA gene transcript and
then normalized relative to the untreated, control group. Error bars represent standard
deviation of the relative mean.
71
This region of FAK contains the focal adhesion targeting domain but does not possess the
kinase domain necessary for autophosphorylation of Tyr-397. This FAK mutant has been
termed FAK-related non-kinase (FRNK). FRNK acts as a dominant negative inhibitor of
FAK signaling by competing with endogenous FAK for localization at focal adhesions
(Richardson & Parsons, 1996). Similar to treatment with Herbimycin A, Amiloride, or
PP1, FRNK infected HFKs exhibited a significant reduction in migration (p = .012)
(Figure 21A). Expression of FRNK reduced FAK autophosphorylation, consistent with
its role as a dominant negative inhibitor of FAK signaling (Figure 21B). Levels of MMP9 mRNA were also measured as expression of this pro-migratory gene is induced by FAK
signaling (Hauck et al., 2001; Sein et al., 2000; Shibata et al., 1998). We found that in
addition to a reduction in FAK autophosphorylation, MMP-9 expression was also
suppressed in FRNK transduced HFKs cultured on plastic alone (Figure 21C) (Hauck et
al., 2001). FRNK expression, however, did not reduce p16 expression at either the
protein or mRNA levels (Figure 21B and 21C). Real-time RT-PCR analysis confirmed
that p16 mRNA levels were not significantly reduced in FRNK transduced HFKs
compared to those infected with LXSN vector alone, in fact, there appeared to be an
increase in p16 mRNA associated with FRNK transduction (Figure 21D). Real-time RTPCR also confirmed that there was a significant reduction in MMP-9 expression between
FRNK transduced HFKs and those infected with LXSN vector alone (Figure 21D).
Furthermore, FRNK expression did not significantly extend HFK replicative capacity
(Table 5). Thus, inhibition of FAK signaling is not sufficient to reduce p16 expression or
increase replicative capacity in human keratinocytes cultured on plastic alone.
Discussion
We have shown previously that human keratinocytes cultured in the absence of
feeder cells induce the expression of several genes known to be involved in migration. In
72
Figure 21: FRNK inhibits keratinocyte migration but not p16 expression. A) Mid
passage (PD ~8-12) Strain 6 HFKs transduced with either FRNK or LXSN were grown in
an outgrowth migration assay on plastic alone for 7 days. Error bars represent standard
deviation of at least three separate circular monolayer sheets. B) Immunoblot of
pTyr397-FAK, total FAK, FRNK, and p16 in Strain 6 HFKs transduced with either
FRNK or LXSN. Protein levels of actin are included as a loading control. Transduced
construct and approximate population doublings are represented below each lane. C)
Semi-quantitative RT-PCR analysis of MMP-9 and p16 in Strain 6 HFKs transduced with
either FRNK or LXSN. GAPDH, a housekeeping gene, is included as an internal control.
Transduced construct and approximate population doublings are represented below each
lane. D) Real-time RT-PCR analysis of MMP-9 (shaded bars) and p16 (clear bars) in
Strain 6 HFKs transduced with either FRNK or LXSN. Levels of MMP-9 mRNA were
standardized against expression levels of the 18S rRNA gene transcript and then
normalized relative to the PD12 LXSN sample. Levels of p16 mRNA were standardized
against expression levels of the 18S rRNA gene transcript and then normalized relative to
the PD12 FRNK sample. Error bars represent standard deviation of the relative mean.
73
Table 5: Replicative capacity of HFKs transduced with FRNK or LXSN and cultured on
plastic alone
HFK Strain/
Transduced Gene
PD 8
PD 10
PD 12
PD 16
PD 20
PD 22
PD 24
LXSN
(15 Clones)
15/15
12/15
6/15
2/15
0/15
FRNK
(15 Clones)
15/15
4/15
2/15
0/15
LXSN (Pooled)
+
+
+
+
FRNK (Pooled)
+
+
+
-
LXSN (Pooled)
+
+
+
+
+
+
-
FRNK (Pooled)
+
+
+
+
+
-
Strain 5 HFKs1
-
Strain 6 HFKs2
1
: Strain 5 HFKs were transduced with either FRNK or LXSN vectors. Ring cloning
isolated 15 individual clones from each the FRNK and LXSN infections. In addition to
the isolated clones, one plate of infected HFKs transduced with either FRNK or LXSN
was pooled following selection. Transduced clones and pooled populations were serially
passaged on plastic alone until they reached senescence. The number of clones (or the
pooled population of cells) surviving to each population doubling (PD) is indicated
above.
2
: Strain 6 HFKs were transduced with either FRNK or LXSN vectors. Ring cloning was
not performed. Only pooled populations of cells surviving selection were serially
passaged on plastic alone. The number of PDs reached by the pooled populations is
indicated above.
74
this study, we have shown that p16 induction in this setting is dependent on the activity
of tyrosine kinases and the proper functioning of the uPA/uPAR system. We have also
provided evidence that p16 expression during keratinocyte migration is not directly
associated with either Src-PTK activity or FAK signaling. Our results strengthen the
connection between p16 expression and keratinocyte migration and suggest that tyrosine
phosphorylation events and uPA/uPAR signaling may be important upstream regulators
of p16 induction in this context.
Induction of p16 expression during keratinocyte migration
Previously we have shown that p16 expression in human keratinocytes cultured in
the absence of feeder cells is correlated with the induction of migration associated genes
(Darbro et al., 2005). In this study, we have shown that FAK, a principal regulator of cell
spreading and migration, is preferentially activated in keratinocytes cultured on plastic
alone. Both FAK localization and autophosphorylation status were consistent with
keratinocyte migration in the absence of feeder cells. Furthermore, p16 expression was
selectively induced in HFKs with increased amounts of both uPAR and
autophosphorylated FAK, two well established markers of keratinocyte migration. These
results contribute to the growing evidence for p16 induction during keratinocyte
migration and continue to pose new questions as to what role p16 plays in this process.
The negative feedback role of p16 expression in response to oncogenic Ras
signaling is well documented in human fibroblasts and has provided a cellular mechanism
to explain how p16 serves a tumor-suppressing role in the setting of enhanced
proliferation stimuli (Lin et al., 1998; Ohtani et al., 2001; Serrano et al., 1997). However,
little has been reported as to the inducibility of p16 to Ras in human epithelial cells. In
this study, we observed reduced levels of Ras/MAPK phosphorylated intermediates
(MEK1/2 and ERK1/2) in keratinocytes cultured on plastic alone compared to the coculture environment. This result suggests that p16 expression in the context of human
75
keratinocyte telomere-independent senescence is not associated with enhanced
proliferation stimuli transduced by the Ras/MAPK pathway. In retrospect, this result
may have been expected. Previous studies in our lab have never observed a prominent
induction of p16 expression prior to or during growth arrest in response to transduction of
HFKs with oncogenic Ras (data not shown). Further evidence suggesting that
Ras/MAPK signaling in human epithelial cells does not induce p16 expression is the
observation that Raf-1 induced growth arrest in human mammary epithelial cells appears
to be p16-independent (Raf-1 is immediately downstream of Ras in the Ras/MAPK
signaling pathway) (Olsen et al., 2002). Taking this data into consideration, it would
appear that there are cell type specific differences with regard to p16 expression as a
negative feedback, tumor-suppressing mechanism in response to increased proliferation
signals.
One of the primary functions of human epithelial cells is migration. Human
keratinocytes migrate across open wound beds in an effort to restore the protective barrier
our skin provides. In several settings, enhanced or abnormal migratory stimuli can lead
to invasion across the basement membrane, potentially disrupting the function of
surrounding tissues. Thus, it is possible that human epithelial cells evolved a negative
feedback mechanism to prevent cells that had acquired constitutive activation of
migratory genes from surviving long enough to cause local tissue dysfunction. The
induction of p16 in response to migratory stimuli may serve this function.
The finding that p16 expression is induced at the leading edge of keratinocytes
scrape-wounded in vitro, led to the hypothesis that p16 induction during keratinocyte
migration may have evolved to facilitate directional motility of keratinocytes during the
wound healing process by preventing proliferation from being a competitive cellular
process (Natarajan et al., 2003). This hypothesis suggests that p16 competent
keratinocytes would facilitate wound healing but an absence of p16 expression in these
cells would presumably lead to less efficient wound closure. However, we have observed
76
that p16 expression is not necessary for in vitro wound closure since p16 negative,
immortalized HFKs can also accomplish this task and with greater speed than primary,
p16 positive keratinocytes (data not shown). In addition, one study of chronic venous leg
ulcers, a condition stemming from dysfunction of the wound healing processes, showed
positive p16 expression at the leading edge of the wound in nearly half of the biopsies
taken (Impola et al., 2005). It was only when keratinocytes made the transition to
invasive squamous cell carcinoma that p16 expression was consistently absent (Impola et
al., 2005). These observations, combined with data from several studies that have shown
p16 expression can inhibit migration in different epithelial cell types (Adachi et al., 2001;
Fahraeus & Lane, 1999; Wieser et al., 1999) are consistent with the hypothesis that p16
expression during migration may have evolved as a negative feedback mechanism to
prevent cells that acquire constitutive activation of migratory genes from invading
through the basement membrane. This hypothesis is consistent with data that shows
induction of p16 at the invasive front of several epithelial cancers (Jung et al., 2001;
Natarajan et al., 2003; Nilsson et al., 2004; Svensson et al., 2003) and the high incidence
of p16 silencing in human cancers known to have a high rate of metastasis (Liggett &
Sidransky, 1998; Sharpless, 2005). Future studies designed to evaluate whether the
specific loss of p16 function predisposes transformed cells to metastasis are clearly
required to test this hypothesis and may provide innovative therapeutic options in clinical
cases of metastatic cancer.
Upstream regulators of p16 expression during keratinocyte
migration
Several genes have been found to regulate p16 expression in both human
fibroblasts and keratinocytes. A partial list of these genes includes Ets1 and Ets2, Id-1,
Bmi-1, 14-3-3σ, and Egr-1 (Alani et al., 2001; Chaturvedi et al., 2003; Dellambra et al.,
2000; Itahana et al., 2003; Jacobs et al., 1999; Nickoloff et al., 2000; Ohtani et al., 2001).
77
In this study, we attempted to answer the question of whether FAK, uPA/uPAR, or SrcPTKs were involved in the regulation of p16 expression during keratinocyte migration.
Our results suggest that whereas tyrosine kinase activity and proper uPA/uPAR
functioning are necessary for p16 expression during keratinocyte migration, neither FAK
nor Src-PTK signaling appear to be involved in this event.
Although we did observe a decrease in both FAK autophosphorylation and the
expression of MMP-9 in HFKs transduced with FRNK, suggesting FAK signaling
through MAPK pathways had been inhibited (Hauck et al., 2001; Sein et al., 2000;
Shibata et al., 1998), we cannot completely rule out FAK signaling as a potential
regulator of p16 expression. It is possible that the reduction in FAK activity seen in our
experiments was simply not complete enough to exert an inhibitory effect on p16
expression. We attempted to inhibit FAK expression with the use of several different
siRNA constructs, however, we did not observe FAK knockdown with any of them (data
not shown). Inhibition of FAK signaling is a fine line in that a complete lack of FAK
signaling has been shown to induce apoptosis in some cell types in a manner consistent
with activation of signaling pathways involved in anoikis (induction of apoptosis upon
loss of cell anchorage) (Frisch & Screaton, 2001; Frisch et al., 1996). Future experiments
designed to inactivate FAK expression and prevent any resultant anoikis (perhaps with
co-expression of a constitutively active mutant of p130Cas) (Wei et al., 2004) may be
better able to address this question of whether reduced FAK signaling causes p16
repression. Since we did not examine the expression of p16 in the context of
constitutively active FAK it is impossible to know whether or not redundancy in FAK
function was why a reduction in p16 expression was not seen. Proline-rich tyrosine
kinase 2 (Pyk2), also known as FAK2 tyrosine kinase, is a non-receptor tyrosine kinase
that shares a high degree of sequence conservation with FAK, specifically surrounding
the SH2 and SH3 binding sites (Schlaepfer & Hunter, 1998). Pyk2 has been shown to
compensate for FAK function in FAK-/- fibroblasts but does not fully restore cell
78
migration ability (Sieg et al., 1998). If Pyk2 is compensating for specific FAK functions
in FRNK transduced HFKs this might account for the decreased migration seen in our
experiments but the lack of reduction in p16 expression. Pyk2 expression is upregulated
in FAK deficient fibroblasts and exhibits tyrosine phosphorylation in response to
fibronectin stimulation, it does not however colocalize with vinculin at focal adhesions
(Schaller & Sasaki, 1997; Schlaepfer & Hunter, 1998). It has been proposed that binding
interactions of the N-terminal domain of Pyk2 might override those of the C-terminal
domain (where the focal adhesion targeting domain is located), thereby differentiating
Pyk2 localization and activation from FAK (Xiong & Parsons, 1997). Thus, competition
for localization at focal adhesions produced by FRNK may not have the same inhibitory
effect on Pyk2 as FAK. Future studies aimed at reducing both FAK and Pyk2 activity in
human keratinocytes would better elucidate any role FAK signaling may play in p16
regulation.
Based on our results concerning both tyrosine kinase and uPA/uPAR inhibition
there are several candidate signaling pathways that may be inducing p16 expression
during keratinocyte migration. uPAR, a GPI-anchored protein, has been found to
associate with integrins and various receptor tyrosine kinases (RTKs) in lipid rafts
(Simons & Toomre, 2000). Several well-established signaling pathways originating at
RTKs have been described and there is growing evidence that suggests uPAR can
modulate signaling through many of these pathways (Blasi & Carmeliet, 2002). One
example is the interferon signaling pathway in which uPAR has been found to associate
with gp130 and contribute to activation of Janus kinases (JAK) (Koshelnick et al., 1997).
JAKs have been found to be important in interferon-induced activation of signal
transducer and activator of transcription (STAT) proteins. The potential for uPAR to
activate or modulate the same pathway as interferon cytokines is relevant since Egr-1, an
IFN-γ inducible transcription factor, has been implicated as a potential regulator of p16
expression in human keratinocytes (Chaturvedi et al., 2003). Also consistent with a role
79
for interferon signaling in p16 regulation is the observation that STAT-1 protein levels
increase in a passage dependent manner in human keratinocytes cultured on plastic alone
(data not shown). uPAR signaling has been shown to cross-talk with signaling pathways
involved in both keratinocyte migration and proliferation and as such provides a very
attractive candidate for the regulation of p16 expression during migration. Studies
designed to specifically knockdown or increase the expression levels of uPA, uPAR, or
both will assist in clarifying what role uPA/uPAR signaling may have in p16 regulation.
In addition to uPA/uPAR signaling, signaling through either β-catenin or TGF-β
pathways could be involved in p16 induction during keratinocyte migration. We have
previously shown that β-catenin localization is modulated by differences in culture
conditions. β-catenin is known to be tyrosine phosphorylated by several proteins
including both c-Src and Pyk2 (Behrens et al., 1993; Lilien & Balsamo, 2005;
Matsuyoshi et al., 1992; van Buul et al., 2005). Our results suggest that Src-PTK activity
is not associated with p16 induction in keratinocytes cultured in the absence of feeders,
but Pyk2 cannot be ruled out as a potential upstream regulator of p16 expression
(especially in light of our results concerning inhibition of FAK signaling). Pyk2 tyrosine
phosphorylation of β-catenin results in the loss of cell-cell contacts and relocalization of
β-catenin (van Buul et al., 2005). Under normal circumstances, cytoplasmic β-catenin is
rapidly phosphorylaed by the serine/threonine kinases casein kinase I (CKI) and glycogen
synthase kinase-3β (GSK-3β) bound to a scaffolding complex of axin and adenomatous
polyposis coli (APC) proteins (Nelson & Nusse, 2004) (Figure 22). This complex can be
inhibited, however, by signaling through the Wnt/Frizzled/Dishevelled pathway.
Increased Wnt signaling leads to the inhibition of the GSK-3β complex and the
accumulation of both cytoplasmic and nuclear β-catenin (Harris & Peifer, 2005). In our
previous microarray experiments, we found that both Wnt 5b and 7a were upregulated in
HFKs cultured on plastic alone. Thus, another possible mechanism of p16 induction in
migrating keratinocytes is through tyrosine phosphorylation of β-catenin, inhibition of
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Figure 22: β-catenin signaling pathway. β-catenin (β) is part of a complex at cell-cell
contacts that includes plakoglobin (Pg), α-catenin (α), and cadherins. Upon
disassociation of cell-cell contacts or phosphorylation by tyrosine kinases (such as Pyk2),
β-catenin relocalizes from the cell periphery to the cytoplasm and nucleus. When the
Wnt signaling pathway is inactive, the cytoplamsic pool of β-catenin is degraded by a
complex including casein kinase 1 (CK1), glycogen synthase kinase-3β (GSK), APC and
Axin. The resultant serine/threonine phosphorylated β-catenin is ultimately degraded by
the 26S proteasome. The binding of Wnt to Frizzled (Frz) receptors activates Wnt
signaling, that induces a disheveled (Dsh)-mediated inhibition of β-catenin
phosphorylation by GSK. This results in β-catenin accumulation and its interaction with
T-cell factor (TCF) transcription factors in the nucleus.
Source: Figure modified from the website of Professor Avri Ben-Ze’ev, Weizmann
Institute of Science, Rehovot, Israel
(http://www.weizmann.ac.il/mcb/avri/images/newfig1l.jpg).
81
GSK-3β complexes by Wnt signaling, and consequent β-catenin induced p16 expression.
This potential mechanism of p16 induction may be at work in invasive human colorectal
adenocarcinomas that show co-localization of nuclear β-catenin and p16 in regions of
low proliferation (Jung et al., 2001). Future experiments aimed at examining p16
expression in the context of inhibited or enhanced β-catenin signaling could prove
enlightening. Also seen in our previous microarray study was the upregulation of several
genes known to be involved in TGF-β signaling. Despite the fact that most TGF-β
signaling involves serine/threonine kinases, there is growing evidence that tyrosine
kinases are capable of modulating this well-defined pathway. A family of transcription
factors, known as the Smad family, primarily carries out TGF-β signaling. Smad1, which
mediates TGF-β signaling through the binding of bone morphogenetic proteins to their
respective receptors, has been found to be negatively regulated by receptor tyrosine
kinase pathways invoked by epidermal growth factor (EGF) and hepatocyte growth factor
(HGF) (Kretzschmar et al., 1997). Similarly, JAK/STAT signaling in response to
treatment with IFN-γ has been found to prevent TGF-β induced phosphorylation and
nuclear translocation of Smad3 (Ulloa et al., 1999). These results suggest that inhibition
of tyrosine kinase activity would lead to derepression of both Smad1 and Smad3 induced
gene expression. In contrast to these studies, it has also been found that the
phosphorylation and subsequent nuclear translocation of Smad2 is enhanced by the
activation of receptor tyrosine kinases by HGF (de Caestecker et al., 1998). Lastly, one
study links both Smad proteins and tyrosine kinases to the β-catenin signaling pathway.
In this study, treatment of human proximal tubular epithelial cells with TGF-β induced a
loss of cell-cell contacts that was associated with the tyrosine phosphorylation and
nuclear localization of β-catenin (Tian & Phillips, 2002). Furthermore, after treatment
with TGF-β, β-catenin was found to associate with both Smad3 and Smad4 suggesting a
cooperative function for these two pathways (Letamendia et al., 2001; Tian & Phillips,
2002). Thus, inhibition of tyrosine kinase activity could have various effects on both
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TGF-β and β-catenin signaling and may be one reason for the decrease in p16 expression
seen in human keratinocytes treated with Herbimycin A.
Summary
In summary, we have shown that p16 expression in human keratinocytes cultured
on plastic alone is selectively induced in cells possessing markers of keratinocyte
migration. Furthermore, we have shown that inhibition of tyrosine kinase activity or
uPA/uPAR function, but not Src-PTKs or FAK signaling, is sufficient to reduce p16
expression in migrating keratinocytes. These results continue to suggest that p16
induction is associated with keratinocyte migration and provide new targets for study as
potential upstream regulators of p16 induction during both keratinocyte migration and
telomere-independent senescence.
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CHAPTER IV
P16 PROMOTER METHYLATION IN TELOMERASE
IMMORTALIZED HUMAN KERATINOCYTES COCULTURED WITH FEEDER CELLS
Introduction
Previous reports have shown that exogenous expression of the catalytic
component of telomerase (TERT) is capable of immortalizing many different human cell
types (fibroblasts, retinal pigmented epithelial cells, vascular endothelial cells, and
mesothelial cells) without the need for additional genetic alterations (Bodnar et al., 1998;
Dickson et al., 2000; Yang et al., 1999). Several human epithelial cell types
(keratinocytes, mammary epithelial cells, bladder urothelial cells, and prostatic epithelial
cells), however, have been shown to require an abrogation of the p16/Rb pathway, in
addition to telomerase activity, to acquire an immortal phenotype (Brenner et al., 1998;
Dickson et al., 2000; Foster & Galloway, 1996; Jarrard et al., 1999; Kiyono et al., 1998;
Puthenveettil et al., 1999; Sandhu et al., 2000; Stampfer & Yaswen, 2003). The p16/Rb
pathway enforces a telomere-independent mechanism of growth arrest that has been
shown to be modulated by differences in culture conditions. When grown in co-culture
with post-mitotic fibroblast feeder cells, many epithelial cell types, including human
keratinocytes, exhibit a delay in passage dependent p16 expression and have an extended
lifespan in culture (Baek et al., 2003; Darbro et al., 2005; Fu et al., 2003; Herbert et al.,
2002; Kang et al., 2003; Ramirez et al., 2001; Rheinwald et al., 2002). There is evidence
that suggests some epithelial cell types can be immortalized by TERT expression alone
when cultured in the presence of feeder cells (Harada et al., 2003; Herbert et al., 2002;
Ramirez et al., 2001). Furthermore, it has been suggested that the p16/Rb pathway is still
intact in these immortalized epithelial cells and remains responsive to both UV irradiation
and transfer to the plastic culture condition. These observations are relevant when
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considering telomerase based tissue therapies and implies that telomerase expression
alone, and immortalization in vitro, does not require the permanent inactivation of the
p16/Rb tumor suppressor mechanism.
There has been considerable debate as to whether or not controlled telomerase
activation should be considered for the treatment of human disease and age-related loss
of function disorders (Chang & DePinho, 2002; Harley, 2002; Harley, 2005). In any
tissue supported by proliferative cells the continued loss of telomeres and consequent
cellular senescence would be expected to decrease overall function and conceivably lead
to the development of age-related disorders (Harley, 2005; Rowe-Rendleman &
Glickman, 2004; Wright & Shay, 2005). Restoration of telomerase activity in this
context would be expected to extend the functional lifespan of these tissues and decrease
the incidence of tissue dysfunction or degeneration. Telomerase based therapies are also
being examined for use in patients suffering from dyskeratosis congenita (DC). DC is an
inherited condition in humans characterized by abnormalities in skin, nails, hair and bone
marrow (Mason et al., 2005; Rubin, 2002). In some DC families, a mutation in the RNA
component of telomerase is sufficient to cause the disease phenotype and affected
individuals possess significantly shorter telomeres than the general population (Vulliamy
et al., 2001a; Vulliamy et al., 2001b). It has been proposed that re-introduction of
telomerase activity into the cells of DC patients might restore telomere length and reduce
the severity of the disease phenotype. DC patients have also been found to have an
increased incidence of cancer, a finding that has been proposed to result from genetic
instability caused by dysfunctional telomeres (Collins & Mitchell, 2002; Marciniak &
Guarente, 2001). In this context, telomerase therapy may decrease the rate of cancer in
DC patients by reducing the level of genetic instability at chromosomal ends. The link
between telomerase insufficiency, genetic instability and cancer is also supported by 5th
generation telomerase knockout mice in which genetic instability caused by telomere
dysfunction has been found to contribute to tumor initiation (Artandi & DePinho, 2000;
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Blasco et al., 1997; Hande et al., 1999; Rudolph et al., 1999). These telomerase knockout
mice experience end-to-end chromosomal translocations and exhibit pathologies in
several tissues similar to what is observed in DC patients (Herrera et al., 2000; Herrera et
al., 1999a; Herrera et al., 1999b; Lee et al., 1998; Samper et al., 2002). Encouraging is
the result that telomerase re-introduction into late generation telomerase deficient mice is
able to elongate critically short telomeres preventing end-to-end chromosomal fusions
and the pathologies associated with telomere dysfunction (Samper et al., 2001).
Furthermore, recent data from transgenic mice engineered to overexpress telomerase
have shown a lower incidence of certain age-related degenerative diseases, specifically
those related to kidney function and germline integrity (Gonzalez-Suarez et al., 2005).
These results suggest that therapeutic telomerase activation could be extremely beneficial
in both restoring the functional capacity of proliferative tissues and reducing genetic
instability at chromosomal ends. However, reactivation of telomerase expression in
somatic tissues has also been found associated with over 90% of human cancers (Hiyama
& Hiyama, 2002) suggesting that there may be significant risk in telomerase based
therapies.
Introduction of exogenous TERT, or derepression of the endogenous TERT gene,
in humans would very likely result in the maintenance of telomere length in vivo.
Whereas this event could potentially increase cellular lifespan by doing so would also be
circumventing a generally agreed upon tumor suppressor mechanism, replicative
senescence. Exogenous expression of TERT alone has not proven to be sufficient to
induce transformation in various human cell types (Drayton & Peters, 2002; Jiang et al.,
1999). However, immortalization is one of several abilities cells must acquire prior to
transformation. Immortalization of human cells by TERT expression could be viewed as
providing one “hit” in a multistage progression to malignancy. In the backdrop of an
immortal cell few further genetic aberrations are required to achieve a transformed
phenotype. Namely, loss-of-function mutations in tumor suppressors and gain-of-
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function alterations in growth promoting, proto-oncogenes is all that would be required to
convert ageless, therapeutic cells into life threatening, malignant tumors. This theoretical
consideration is undoubtedly concerning but recent studies in both tissue culture systems
and animal models provides evidence that this concern is more than just theoretical.
Tissue culture studies have provided evidence that expression of telomerase can
both enhance cellular growth and proliferation as well as lead to the inhibition of several
tumor suppressor genes. Long-term culture of TERT transduced human mammary
epithelial cells has been reported to be associated with increased expression of the protooncogene c-myc and resistance to growth inhibition by TGF-β (Stampfer et al., 2001;
Wang et al., 1998). In an immortal telomerase-negative fibroblast cell line, one in which
telomere length is maintained by the ALT (alternative lengthening of telomeres)
mechanism, forced expression of a telomerase mutant defective in telomere maintenance
was found to cooperate with Ras to produce highly tumorigenic cells (Stewart et al.,
2002). Studies in human lens epithelial cells immortalized by SV40 Large T antigen
have shown that exogenous expression of TERT accelerates growth, decreases p53
protein levels, p21 mRNA levels, as well as mRNA and protein levels of the cyclin-CDK
inhibitor Grap2 cyclin D interacting protein (GCIP) (Xiang et al., 2002). Furthermore,
two studies examining human fibroblasts immortalized by telomerase alone found a
significant repression of both p16 and p53 expression as cells continued to be passaged in
vitro. In one study, p16 and p53 expression were lost due to gene deletion events and
allelic mutations, respectively (Noble et al., 2004); in the other, p16 expression was
found to be significantly repressed in TERT immortalized human fibroblasts and not
inducible by expression of oncogenic Ras (Taylor et al., 2004). Ironically, several of the
same transgenic mouse studies that have shown a beneficial role for telomerase in the
prevention of age-related tissue degenerative disorders have also exposed telomerase as a
possible proto-oncogene and provided more evidence for non-telomere lengthening
functions. Laboratory mice are known to possess extremely long telomeres and express
87
telomerase in somatic tissues (Kipling & Cooke, 1990; Prowse & Greider, 1995). Thus,
telomere maintenance is not a limiting factor for cellular transformation. Yet, transgenic
mice engineered to constitutively express the mouse form of TERT specifically in basal
layer keratinocytes or in all cell types have an increased incidence of chemically induced
and spontaneous tumor formation, respectively (Artandi et al., 2002; Gonzalez-Suarez et
al., 2005). These combined results strongly suggest additional, telomere-independent
functions for telomerase that when combined with it’s ability to maintain telomere length
could lead to the development of tumorigenic cells at increased frequency.
It is clear that there are many potential benefits and dangers associated with
telomerase based cell therapies. Studies in human fibroblasts have shown that
immortalization with TERT can lead to the repression of p16 expression, however, no
data exists on whether p16 expression is eventually inactivated in TERT immortalized
epithelial cells co-cultured with feeder cells. The examination of TERT immortalized
keratinocytes is significant to the therapeutic telomerase field since the function of human
skin is often compromised in an age-related fashion leading to pathologies such as
chronic skin ulcers. Epidermal keratinocytes also provide an easily assessable target
tissue for preliminary clinical trials. These features make keratinocytes an ideal cell type
to examine the efficacy of telomerase activation and as such every effort must be made
prior to human trials to determine whether TERT immortalized keratinocytes provide a
significant risk to potential recipients.
We chose to focus our study primarily on the tumor suppressor p16. The p16/Rb
pathway has been shown to enforce a telomere-independent mechanism of growth arrest
in human epithelial cells cultured in the absence of feeder cells, and must be inactivated
in this setting for TERT immortalization (Brenner et al., 1998; Dickson et al., 2000;
Jarrard et al., 1999; Kiyono et al., 1998; Reznikoff et al., 1996; Rheinwald et al., 2002;
Tsutsui et al., 2002). As shown in Chapter II, co-culture of keratinocytes with feeder
cells delays the accumulation of p16 protein but does not prevent the eventual increase of
88
p16 expression in late passage keratinocytes cultured with feeder cells. This observation
suggests that p16 expression may still be an important growth regulatory mechanism in
keratinocytes despite co-culture with feeders. Previous reports that have shown
immortalization of co-cultured human epithelial cells with TERT alone imply that
telomere-dependent mechanisms are the sole barrier preventing continued proliferation in
this culture system. However, the presence of inflection points in the growth curves of
TERT immortalized epithelial cells co-cultured with feeders (Herbert et al., 2002;
Ramirez et al., 2003; Rheinwald et al., 2002), combined with the observations that
telomeres experience only limited shortening in co-cultured human keratinocytes (Kang
et al., 2004), suggest that telomere-independent mechanisms may still be involved in
limiting the replicative capacity of these cells. Thus, examination of p16 expression in
TERT transduced human keratinocytes co-cultured with feeder cells should provide vital
information concerning not only the potential safety of telomerase based cell therapies
but also the mechanism of epithelial cell senescence in the co-culture environment.
In this study, we demonstrate that telomerase activity alone in human
keratinocytes co-cultured with feeder cells results in immortalization of these cells and
that switching these cells from the co-culture environment to plastic leads to a brief
induction of p16 expression followed by continued proliferation and maintenance of the
immortal phenotype. Examination of telomerase immortalized cell lines by 5azadeoxycytidine treatment and bisulfite sequencing demonstrated p16 promoter
methylation. Reintroduction of p16 into TERT immortalized cell lines resulted in growth
arrest and a senescent phenotype. Our results suggest that immortalization of human
keratinocytes with TERT is associated with a high incidence of p16 inactivation
regardless of culture conditions and emphasizes the need for caution in the use of such
cells for potential telomerase-based therapies.
89
Materials and Methods
Retroviral Constructs and Infections
The TERT-neo, TERT-hygro, and pLXSH vectors have all been described
previously (Dickson et al., 2000; Farwell et al., 2000; Kiyono et al., 1998). The p16
retroviral construct was made by excising p16 cDNA out of a p16-pBluescript construct
(gift from J Martin, University of Iowa) with EcoRI and cloning it directly into pBABEpuro. DNA sequencing performed by the University of Iowa DNA Sequencing Facility
confirmed proper orientation and sequence of the p16 gene. Generation of virus with
Phoenix amphotropic packaging lines has been previously described (Der et al., 1986;
Whitehead et al., 1995; Zohn et al., 1998).
For TERT-neo, TERT-hygro, and pLXSH infections, exponentially growing
HFKs were infected overnight in 4 µg/ml polybrene. Infected cells were washed in
regular media the following morning. Cells were passed the following day, and selective
media containing either 50 µg/ml neomycin (G418) (Invitrogen: 11811-031) or 10 µg/ml
hygromycin (Invitrogen: 10687-010) was added on the next day. All infections were
performed in the plastic culture condition and selection was carried out over 7 days.
Following selection, clonal populations were pooled and split into either the feeder coculture environment or left on tissue culture plastic in the absence of feeder cells. We
performed two separate series of infections on two independent HFK cell strains (Strain
A and Strain C). In the first infection, Strain C primary HFKs cultured on plastic alone
were infected with TERT-hygro or pLXSH at approximately 4 PDs. Following selection
on plastic, clones were pooled and divided between the two culture conditions. When
Strain C TERT transduced HFKs on feeders reached PD 22 they were split normally and
half the cells were replated into the co-culture environment and the other half were plated
onto tissue culture plastic alone. In the second infection, Strain A primary HFKs cultured
on plastic alone were infected with TERT-neo at approximately 4 PDs. Following
90
selection on plastic, clones were pooled and divided between the two culture conditions.
When Strain A TERT transduced HFKs on feeders reached PDs 25, 29, 35, 41, and 51
they were split normally and half the cells were replated into the co-culture environment
and the other half were plated onto tissue culture plastic alone.
For p16 and pBABE infections, late passage (PD > 100) exponentially growing
TERT transduced HFKs, that had been switched from the feeder co-culture environment
to plastic, were infected overnight in 4 µg/ml polybrene. Infected cells were washed in
regular media the following morning. Cells were passed the following day onto glass
coverslips, and selective media containing 1 µg/ml puromycin (Sigma: P8833) was added
on the next day. Control experiments were conducted by infecting parallel cultures of
HFKs with the pBABE vector alone. All infections were performed in the plastic culture
condition and selection was carried out over 4 days. Prior to the end of the selection
period, cultures were incubated with 100 µM BrdU (Sigma: B5002) for 12 hours.
Following incubation with BrdU, cells were fixed in 3.7% formaldehyde for future
immunocytochemical staining.
Real-Time Quantitative TRAP Analysis and Cytogenetics
Real-time quantitative TRAP analysis (RQ-TRAP) was performed as previously
described by Wege and colleagues (Wege et al., 2003). Briefly, extracts from ~1000
cells were mixed with 0.1µg of telomerase primer TS (5’-AATCCGTCGAGCAGATGG3’), 0.05µg of anchored return primer ACX (5’-GCGCGG(CTTACC)3CTAACC-3’), and
Syber green PCR master mix (Applied Biosystems: 4309155). Reaction samples were
incubated at 25˚C for 20 minutes prior to amplification to allow telomerase to elongate
the TS primer by adding TTAGGG repeat sequences. Real-time PCR amplification was
carried out in a 7700 Sequence Detection System real-time thermalcycler (Applied
Biosystems) with the following reaction conditions: Cycles at 95˚C for 10 minutes, 95˚C
for 15 seconds, and 60˚C for 1 minute. Threshold cycle values were determined from
91
amplification plots and compared with standard curves generated from serial dilutions
(1000, 100, 10, 1 cell) of a telomerase-positive cell line (HPV16 E6/E7 HFKs).
Telomerase activity is reported as standard cell equivalents. All RQ-TRAP reactions
were carried out in triplicate and error bars created using standard deviation of the mean.
Chromosomal analyses were performed as previously described (Klingelhutz et
al., 2005) on late passage (PD > 100) Strain A and C TERT transduced HFKs
continuously cultured with feeders or transferred to plastic. Briefly, cells growing on
coverslips were arrested in metaphase by adding ethidium bromide followed by colcemid.
Cell were incubated at room temperature with hypotonic solution (3:1 mixture of 0.8%
sodium citrate and 0.075 M potassium chloride) and then fixed three times with a 3:1
methanol/acetic acid solution. Ten to twenty G-banded metaphases were analyzed for
each cell type.
5-Azadeoxycytidine Treatment
Strain A and C TERT transduced HFKs continuously co-cultured with feeders or
transferred to plastic were treated with 1.5 µM 5-azadeoxycytidine (Sigma: A3656) at
subconfluency for approximately 7 days before cells were collected for both protein and
RNA. Untreated, parallel cultures were also collected for both protein and RNA at the
same passage.
Bisulfite Sequencing
DNA was collected at selected time points from either primary or transduced
Strain A and C HFKs at 70-80% confluence using the DNeasy Kit (Qiagen, Valencia,
CA: 69504) according to the manufacturer’s instructions. Following DNA isolation, 5µg
of genomic DNA was digested with EcoRI overnight at 37˚C. Digested DNA was
diluted to 54µl in ddH2O and denatured by adding 6µl of freshly prepared 3.0 M NaOH
followed by incubation at 37˚C for 15 minutes. Following denaturation of the DNA,
431µl of 3.6 M sodium bisulfite/1 mM hydroquinone was added and the sample was
92
incubated at 55˚C for 14 hours with 5 minute spikes in temperature to 95˚C every 2
hours. Bisulfite treated DNA was subsequently desalted using the Wizard DNA
Purification System (Promega, Madison, WI: A7100) according to the manufacturer’s
instructions. Bisulfite treated DNA was eluted in 54µl of ddH2O and desulphonated by
adding 6µl of freshly prepared 3.0 M NaOH followed by incubation at 37˚C for 15
minutes. Bisulfite treated DNA was then precipitated by adding 40µl of 7.5 M NH4OAc
and 300µl 95% EtOH and incubating at –20˚C for at least 1 hour. Precipitated DNA was
centrifuged at 4˚C for 30 minutes at max speed in a table top centrifuge. Following
centrifugation, the supernatant was removed, the DNA pellet was allowed to air dry for 510 minutes at room temperature, and was ultimately resuspended in 50µl of ddH2O.
PCR amplification of the p16 promoter region from –351 to –73 was performed
with the sense primer 5’-GTGGGGAGGAGTTTAGTTTTTTTTTTTTG-3’ and antisense
primer 5’-TCTAATAACCAACCAACCCCTCCTCTTTC-3’ (Cody et al., 1999).
Reaction conditions were 40 cycles at 95˚C for 30 seconds, 60˚C for 30 seconds, and
72˚C for 30 seconds. PCR products were isolated by first running the PCR reactions on a
2% agrose gel and then extracting the desired PCR product using a gel extraction kit
(Qiagen: 28704). Isolated PCR products were cloned into the pGEM-T cloning vector
(Promega: A3600) according to the manufacturer’s instructions. Ligation products were
transformed into DH5α max efficiency competent cells (Invitrogen: 18258-012) and
grown on LB+Amp plates that had been supplemented with both X-Gal (Fisher
Scientific, Hampton, NH: BP1615) and IPTG (Research Products International
Corporation, Mt. Prospect, IL: 367-93-1). Selection of bacterial clones was performed by
blue/white colony screening. At least 15 bacterial clones were selected and subjected to
plasmid minipreps (Qiagen: 27106) to ensure the recovery of at least 10 epigenotypes per
sample. Plasmid DNA was then submitted to the Uiniversity of Iowa DNA Sequencing
Facility for sequence analysis. Analysis of non-CpG cytosines indicated the efficiency of
bisulfite conversion at ~99%.
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Immunocytochemistry
Strain A and C TERT transduced HFKs switched from feeders to plastic and
infected with either the p16 or pBABE retroviruses were fixed with 3.7% formaldehyde
in PBS for 10 minutes at room temperature. Cells were permeabilized with 0.5% Triton
X-100 in PBS for 7 minutes. Infected cells were treated with 100 µM BrdU for 12 hours
prior to fixation to label cells in the S phase of the cell cycle. After fixation and
permeabilization, cultures that had received BrdU were treated for 1 hour with 0.2 M HCl
to make DNA containing BrdU accessible to the antibody. HCl-treated cultures were
then neutralized with 0.1 M borate buffer (pH 8.5) and washed with 1X PBS before
proceeding with immunostaining. Coverslips were blocked in 10% normal goat serum,
1% BSA, and 0.1% Tween-20 in PBS for 45 minutes. Coverslips were incubated for 1
hour with a mixture of both BrdU (Becton Dickinson, Franklin Lakes, NJ: 347580) and
p16 (Santa Cruz Biotechnology Inc., Santa Cruz, CA: C-20) primary antibodies. BrdU
and p16 primary antibodies were diluted to a ratio of 1:20 and 1:100 in blocking buffer,
respectively. A mixture of donkey anti-mouse Texas-red conjugated (Jackson
ImmunoResearch Laboratories) and goat anti-rabbit fluorescein conjugated (Chemicon
International) secondary antibodies, both diluted 1:100 in blocking buffer, was incubated
with the coverslips for 45 minutes. The coverslips were mounted onto glass slides with
VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA)
to provide for visualization of HFK nuclei. Images were collected on a Nikon Eclipse
E800 fluorescence microscope (Nikon Corporation). PBS washes were performed
between each step of this immunocytochemical staining protocol.
94
Results
TERT immortalization of keratinocytes cultured with
feeders persists upon transfer to plastic with an associated
loss of p16 expression
Previous reports have suggested that human keratinocytes can be immortalized by
telomerase activity alone when grown in co-culture with fibroblast feeder cells (Harada et
al., 2003; Ramirez et al., 2001). Furthermore, these reports suggest that p16 expression
in TERT immortalized keratinocytes is not inactivated and remains inducible by either
transfer to plastic culture conditions or exposure to UV irradiation. Recent evidence has
shown that p16 expression is permanently inactivated in TERT immortalized fibroblasts
(Noble et al., 2004; Taylor et al., 2004), thus we sought to determine if a similar result
could be seen in TERT immortalized human keratinocytes co-cultured with feeder cells.
We observed that Strain C human keratinocytes transduced with TERT and co-cultured
with feeder cells do acquire an immortal phenotype without an apparent crisis (Figure
23). Similar to previous reports (Kiyono et al., 1998; Rheinwald et al., 2002), we also
observed that TERT transduced HFKs cultured on plastic alone did not become immortal
and growth arrested at approximately PD 26. HFKs transduced with pLXSH expectedly
did not become immortal on either plastic or in co-culture with feeders and displayed
cellular lifespans similar to previous observations (Darbro et al., 2005). At PD 22, Strain
C TERT transduced HFKs were transferred from the co-culture environment to tissue
culture plastic alone. In contrast to previous reports, transferred HFKs did not undergo
rapid growth arrest. In fact, TERT HFKs transferred to the plastic culture condition
mirrored the proliferation rate of TERT HFKs remaining in the co-culture environment
over PDs 24-48, and after a period of slowed growth spanning PDs 48-54, resumed a
rapid proliferation rate and then continued to proliferate indefinitely.
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Figure 23: Immortalization of TERT transduced Strain C HFKs co-cultured with feeder
cells. Replicative capacities of Strain C HFKs transduced with either LXSH or TERT
and cultured on plastic alone (Plastic), in the presence of feeder cells (Feeders), or
transferred from the co-culture environment to plastic alone (Feeders to Plastic).
TERT/Feeders to Plastic cells were transferred form the co-culture environment to plastic
at PD 26. TERT/Feeders and TERT/Feeders to Plastic HFKs were found to be immortal.
The following transduced cell lines senesced at the specified PDs: LXSH/Plastic (~16
PDs), LXSH/Feeders (~32 PDs), TERT/Plastic (~26 PDs).
Expression levels of p16 were examined in each of the transduced cell lines and were
found to be consistent with both growth rate and arrest (Figure 24A). In both pLXSH
and TERT transduced HFKs cultured on plastic, p16 levels were increased prior to and at
the point of growth arrest, suggesting that these cells succumbed to p16 enforced
telomere-independent senescence. Protein levels of p16 were increased but to a lesser
extent in pLXSH cells co-cultured with feeder cells. TERT transduced HFKs co-cultured
with feeder cells maintained a relatively low level of p16 expression throughout the
duration of the experiment. Soon after the transfer of TERT transduced HFKs from the
co-culture environment to plastic, p16 protein levels began to rise and reached a maximal
96
Figure 24: Expression of p16 in tranduced cell lines. A) Immunoblots of p16 protein
levels in Strain C HFKs transduced with either LXSH or TERT and cultured on plastic
alone (Plastic: lanes 1, 2, and 10-12), in the presence of feeder cells (Feeders: lanes 3-6
and 13-15), or transferred from the co-culture environment to plastic alone
(Feeders/Plastic: lanes 7-9). B) Immunoblot of p16 protein levels in Strain C HFKs
transduced with TERT and transferred from the co-culture environment to plastic alone
(TERT Feeders to Plastic) at PD 26. Protein levels of actin are included as a loading
control. Approximate population doublings are represented below each lane.
level during the period of slow growth experienced by these cells between PDs 48-54
(Figure 24B). As transferred TERT HFKs began to reacquire a rapid growth rate p16
protein levels began to decline. No further p16 expression was seen in the transferred
TERT HFKs for the duration of this experiment (data not shown). Thus, p16 expression
was lost in TERT HFKs transferred from the co-culture environment to plastic alone.
To determine whether the loss of p16 expression seen in Strain C TERT
transduced HFKs transferred from the co-culture environment to plastic was reproducible
and dependent on the number of PDs cells had experienced we repeated TERT retroviral
infections in another HFK cell strain derived from a different donor (Strain A). Strain A
TERT transduced HFKs were transferred from the feeder culture system to plastic at
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various points in their lifespan (Figure 25). Consistent with our previous result, Strain A
HFKs co-cultured with feeder cells were immortalized by transduction with TERT
without an apparent crisis. TERT transduced HFKs were transferred from the co-culture
environment to plastic at PDs 25, 29, 35, 41, and 51 to examine their viability in the
absence of feeder cells. TERT transduced HFKs transferred at PDs 25, 29, and 35 all
underwent growth arrest within 7 passages after transfer. Cells transferred after PD 35,
however, became immortal in the plastic culture condition.
Figure 25: Immortalization of TERT transduced Strain A HFKs co-cultured with feeder
cells. Replicative capacities of Strain A HFKs transduced with TERT and transferred
from the co-culture environment to plastic alone (Feeders to Plastic) at various PDs.
TERT/Feeders to Plastic cells were transferred from the co-culture environment to plastic
at the following PDs: #1 (PD 25), #2 (PD 29), #3 (PD 35), #4 (PD 41), #5 (PD 51).
TERT/Feeders, TERT/Feeders to Plastic #4, and TERT/Feeders to Plastic #5 HFKs were
found to be immortal. The following TERT/Feeders to Plastic cell lines senesced at the
specified PDs: #1 (~31 PDs), #2 (~33 PDs), #3 (~49 PDs).
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TERT transduced HFKs transferred at PD 41 experienced a slowed growth phase
between PDs 61-69 before resuming a regular proliferation rate. TERT transduced HFKs
transferred at PD 51 experienced a slight growth inhibition between PDs 57-65 before
acquiring a proliferation rate that exceeded that of TERT transduced HFKs remaining in
the co-culture environment. Protein levels of p16 were elevated above the levels seen in
parallel cultures of TERT HFKs left on feeders during the period of slowed growth
exhibited by Strain A TERT HFKs transferred from the co-culture environment to plastic
at PD 51 (Figure 26A). Loss of p16 expression in these cells correlated with the
resumption of a rapid proliferation rate (Figure 26B). Expression levels of p16 were also
examined in TERT transduced HFKs that growth arrested after being transferred to
plastic at PD 35. Unexpectedly, we found that whereas p16 expression was increased
immediately following transfer to the plastic, p16 protein levels began to decrease with
subsequent passage (Figure 26C). At the point of growth arrest (PD 49), the level of p16
protein had decreased to levels similar to equivalent passage TERT transduced HFKs
remaining in co-culture with feeder cells. This result suggests that the growth arrest seen
in these cells was mediated by p16-independent mechanisms. Thus, the loss of p16
expression appears to be a common event upon transfer of TERT immortalized
keratinocytes from the co-culture environment to plastic. Furthermore, maintenance of
TERT induced HFK immortality upon transfer from the co-culture environment to plastic
appears to be dependent on the length of time the cells are co-cultured with feeders.
Immortal HFKs transferred to plastic retain telomerase
activity and do not exhibit consistent genetic aberrations
Analysis of telomerase activity in all HFK cell lines was performed to determine
whether TERT transduced HFKs that remained immortal upon transfer from the coculture environment to plastic retained telomerase activity. As expected, all HFK cell
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Figure 26: Expression of p16 in TERT transduced Strain A HFKs transferred from coculture with feeders to plastic alone. A) Immunoblot of p16 protein levels in both TERT
transduced Strain A HFKs retained on feeders (TERT Feeders: odd lanes) and
TERT/Feeders to Plastic #5 cells transferred to plastic from the co-culture environment at
PD 51 (TERT Feeders to Plastic: even lanes). B) Immunoblot of p16 protein levels in
TERT/Feeders to Plastic #5 cells. C) Immunoblot of p16 protein levels in both TERT
transduced Strain A HFKs retained on feeders (TERT Feeders: lanes 1, 2, 4, 6, and 8) and
TERT/Feeders to Plastic #3 cells transferred to plastic from the co-culture environment at
PD 35 (TERT Feeders to Plastic: lanes 3, 5, 7, and 9). Protein levels of actin are included
as a loading control. Approximate population doublings are represented below each lane.
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lines that had been transduced with TERT exhibited telomerase activity (Figure 27).
Cytogenetic analysis was also performed to determine if any gross chromosomal
alterations had occurred that may have contributed to maintenance of the immortal
phenotype in TERT HFKs transferred from the co-culture environment to plastic. In
Strain C TERT HFKs we found no gross genetic abnormalities associated with transfer
from the co-culture system to plastic (Table 6). In fact, the only genetic abnormalities in
Figure 27: Telomerase activity in transduced Strain C and A HFKs. Real-time
quantitative TRAP assays were performed on cell extracts from Strain C and A HFKs
transduced with either LXSH or TERT and cultured on plastic alone (P), in the presence
of feeder cells (F), or transferred from the co-culture environment to plastic alone (F to
P). Strain A TERT/Feeders to Plastic #5 cells, transferred form the co-culture
environment to plastic at PD 51, are represented above. Telomerase activity is measured
in standard cell equivalents. Error bars represent standard deviation of the mean.
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Table 6: Chromosomal analyses of TERT transduced cell lines
Transduced Cell Line
Karyotype
Strain C HFKs
TERT Feeders
46, XY, iso (8)(q10)1
TERT Feeders to Plastic
46, XY
Strain A HFKs
TERT Feeders
TERT Feeders to Plastic #5 (transferred at PD 51)
46, XY
48, XY, +7, +20
1
: These cells contain one copy of the short arm of chromosome 8 and 3 copies of the
long arm of chromosome 8.
Strain C TERT HFKs were found in the cells that remained in co-culture with feeder
cells. This result suggests that there were no gross genetic abnormalities (such as
chromosomal amplification, duplication, deletion, or translocations) acquired by TERT
transduced HFKs transferred to plastic that allowed them to remain immortal. In Strain
A TERT HFKs transferred from the co-culture environment to plastic we observed
duplication of chromosomes 7 and 20. In contrast, Strain A TERT HFKs continuously
co-cultured with feeder cells exhibited no gross genetic abnormalities. These results
suggest that it is unlikely that chromosomal loss or gain is responsible for p16
inactivation in TERT transduced HFKs that remain immortal upon transfer from the coculture environment to plastic. Thus, the maintenance of immortality in TERT
transduced HFKs transferred from the co-culture environment to plastic is likely not the
result of gross genetic abnormalities. This result, combined with the increased incidence
of p16 promoter methylation as a mechanism of p16 silencing in many tumor types
(Liggett & Sidransky, 1998; Rocco & Sidransky, 2001; Sharpless, 2005), directed our
investigations toward epigenetic mechanisms of p16 inactivation.
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Inactivation of p16 by promoter methylation in
keratinocytes immortalized by TERT and co-cultured with
feeder cells
Growing evidence has suggested that hypermethylation of CpG islands is a
common mechanism employed by cancer to permanently silence expression of tumor
suppressor genes (Curtis & Goggins, 2005; Das & Singal, 2004; Herman & Baylin, 2003;
Momparler, 2003; Paz et al., 2003). Furthermore, in vitro immortalized human cell lines
have been found to methylate a wide array of genes involved in cell cycle regulation (Liu
et al., 2005). The promoter region of the p16 gene contains a 5’ CpG island that has been
found to exhibit increased levels of methylation in various cancers (Liggett & Sidransky,
1998; Rocco & Sidransky, 2001; Sharpless, 2005). In addition, methylation has been
found to be responsible for p16 inactivation in several human keratinocyte cell lines
immortalized by telomerase alone in the absence of feeder cells (Farwell et al., 2000).
We hypothesized that p16 inactivation in TERT transduced HFKs that had been
transferred from the co-culture environment to plastic may be the result of promoter
methylation. To examine the status of p16 promoter methylation in immortal TERT
transduced HFKs we examined the inducibility of p16 by the DNA methyltransferase
inhibitor 5-azadeoxycytidine and the methylation status of individual CpG sites in the
p16 promoter by bisulfite sequencing.
Following treatment with 5-azadeoxycytidine, re-expression of p16 at both the
mRNA and protein levels was observed in TERT transduced HFKs that had lost p16
expression upon being transferred from the co-culture environment to plastic (Figure 28A
and B). This result is highly suggestive of p16 promoter methylation in these cells.
Inducibility of p16 expression to 5-azadeoxycytidine was also examined in TERT
transduced HFKs that remained in the co-culture environment. We also observed an
increase in p16 expression at both the mRNA and protein levels when these cells were
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Figure 28: Induction of p16 expression upon treatment with 5-azadeoxycytidine in
immortalized TERT transduced HFKs transferred from the co-culture environment to
plastic alone. A) Immunoblot of p16 protein levels in immortalized Strain C (lanes 1 and
2) and A (3 and 4) TERT Feeders to Plastic cells treated with or without 5azadeoxycytidine (5-AZA). Protein levels of actin are included as a loading control. B)
Semi-quantitative RT-PCR of p16 mRNA levels in immortalized Strain C (lanes 1 and 2)
and A (3 and 4) TERT Feeders to Plastic cells treated with or without 5-azadeoxycytidine
(5-AZA). GAPDH, a housekeeping gene, is included as an internal control. In both A)
and B) above, Strain A TERT/Feeders to Plastic #5 cells are represented.
treated with the DNA methyltransferase inhibitor (Figure 29A and B). This result
suggests p16 promoter methylation may be occurring in TERT transduced keratinocytes
co-cultured with feeder cells and not simply an effect of transfer to the plastic culture
condition. To confirm that p16 promoter methylation had occurred in both TERT
transduced HFKs co-cultured with feeders and those transferred to plastic we performed
bisulfite sequencing on the p16 promoter region spanning from –351 to –73. This region
of the p16 promoter has been shown previously to be important for p16 downregulation
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Figure 29: Induction of p16 expression upon treatment with 5-azadeoxycytidine in
immortalized TERT transduced HFKs maintained in co-culture with feeder cells. A)
Immunoblot of p16 protein levels in immortalized Strain C (lanes 1 and 2) and A (3 and
4) TERT Feeders cells treated with or without 5-azadeoxycytidine. Protein levels of actin
are included as a loading control. B) Semi-quantitative RT-PCR of p16 mRNA levels in
immortalized Strain C (lanes 1 and 2) and A (3 and 4) TERT Feeders cells treated with or
without 5-azadeoxycytidine. GAPDH, a housekeeping gene, is included as an internal
control.
by DNA methylation (Cody et al., 1999; Farwell et al., 2000; Wong et al., 1997).
Bisulfite sequencing relies on the ability of bisulfite to induce modifications to genomic
DNA, specifically, the conversion of unmethylated cytosines to uracils. Following
treatment of genomic DNA with bisulfite, the specific region of the p16 promoter was
amplified and PCR products were cloned into pGEM vectors and transformed into
competent bacterial cells. Subsequent sequencing revealed which specific cytosines had
been methylated in the HFK cell lines tested. As controls, bisulfite sequencing was
performed on Strain C HFKs transduced with pLXSH and grown in both culture
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conditions, Strain C HFKs transduced with TERT and cultured on plastic alone, and
Strain A primary HFKs grown in both culture conditions. Furthermore, bisulfite
sequencing was performed on both early and late passage TERT transduced HFKs cocultured with feeder cells to determine if promoter methylation occurred as a result of
time in culture. We observed little, if any, p16 promoter methylation in Strain C HFKs
transduced with pLXSN (in either culture condition), Strain C TERT HFKs cultured on
plastic alone, or Strain A primary HFKs (in either culture condition) (Figures 30 and 31).
Little p16 promoter methylation was detected in early passage Strain A or C TERT HFKs
co-cultured with feeder cells. However, a significant increase in p16 promoter
methylation was observed in late passage Strain A and C TERT HFKs co-cultured with
feeders. The degree of methylation was also increased in both Strain A and C TERT
HFKs that had lost p16 expression upon transfer from the co-culture environment to
plastic. Thus, the loss of p16 expression in TERT transduced HFKs transferred from coculture with feeders to plastic is most likely attributed to promoter methylation.
Furthermore, the process of p16 promoter methylation begins before TERT transduced
HFKs are transferred from the co-culture environment to plastic suggesting that during
prolonged culture TERT transduced HFKs co-cultured with feeders progressively
inactivate p16 expression.
Reintroduction of p16 expression into TERT immortalized
keratinocytes on plastic causes growth arrest
To determine whether reintroduction of p16 expression is sufficient to induce
growth arrest in TERT transduced HFKs transferred from the co-culture environment to
plastic these immortalized cells were infected with a retrovirus containing p16. We
hypothesized that p16 expression would lead to rapid senescence of immortal
keratinocytes. Following retroviral infection, cell cultures were exposed to a brief period
of selection with puromycin. This period of selection provided for the vast majority of
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Figure 30: Bisulfite sequencing analysis of the p16 promoter in LXSH and TERT
transduced Strain C HFKs. Numbers on the x axis represent CpG sites in the p16
promoter region spanning from –351 to –73. Methylation status of CpG sites in each
individual epigenotype is indicated as either unmethylated (open circle) or methylated
(closed circle). DNA from LXSH on Plastic, LXSH on Feeders, and TERT on Plastic
Strain A HFKs was collected from cells at PDs ~12-16. Early passage TERT on Feeders
cells were at PD 14, and both late passage TERT on Feeders and TERT Feeders to Plastic
cells were at PD >100.
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Figure 31: Bisulfite sequencing analysis of the p16 promoter in primary and TERT
transduced Strain A HFKs. Numbers on the x axis represent CpG sites in the p16
promoter region spanning from –351 to –73. Methylation status of CpG sites in each
individual epigenotype is indicated as either unmethylated (open circle) or methylated
(closed circle). DNA from primary Strain A HFKs cultured under both conditions was
collected from cells at PD ~10, early passage TERT on Feeders cells at PD 20, and both
late passage TERT on Feeders and TERT Feeders to Plastic cells at PD >100. Strain A
TERT/Feeders to Plastic #5 cells are represented above.
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nontransduced HFKs to be selected against but allowed for a small population of
uninfected cells to remain viable. Following the brief selection period we fixed infected
cultures and subjected them to immunocytochemical staining for both p16 and BrdU. We
observed that in both Strain A and C TERT HFKs that remained immortal following
transfer to plastic, p16 expression caused both growth arrest and morphological changes
consistent with senescence (Figure 32). We detected a lack of BrdU incorporation in
cells expressing p16 consistent with p16-induced growth arrest. Cells that did not
express p16 exhibited positive BrdU staining, suggesting they had entered the S phase of
the cell cycle during the 12 hour BrdU treatment period prior to fixation.
Morphologically, HFKs expressing p16 were also much larger than those cells in which
p16 expression was absent. This observation is consistent with the morphology of HFKs
that have undergone p16-induced, telomere-independent senescence following serial
passage in the plastic culture condition. Control cultures infected with the pBABE vector
alone also exhibited BrdU staining with a lack of any significant p16 expression. Thus,
reintroduction of p16 expression into immortal TERT transduced HFKs transferred from
the co-culture environment to plastic is sufficient to cause both growth arrest and
acquisition of a senescent morphology.
Discussion
It has been shown previously that human keratinocytes can apparently be
immortalized by exogenous expression of TERT alone if they are co-cultured with
fibroblast feeder cells. Furthermore, it has been suggested that these immortalized
keratinocytes retain inducibility and function of the p16 tumor suppressor gene. In this
study, we have shown that human keratinocytes can be immortalized by TERT
expression alone when co-cultured with feeder cells, however, loss of p16 expression by
promoter methylation is a frequent event in these cell lines. These results suggest that the
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Figure 32: Re-introduction of p16 expression causes growth arrest and a senescent
morphology in immortalized Strain C and A TERT transduced HFKs transferred from coculture with feeders to plastic. Late passage (PD > 100) Strain C and A TERT Feeders to
Plastic cells were infected with retrovirus carrying either pBABE-p16 or pBABE alone.
Cells were incubated with BrdU prior to fixation and stained with antibodies to both p16
and BrdU. DAPI staining was performed for visualization of all HFK nuclei in the
microscopic field. Strain A TERT/Feeders to Plastic #5 cells are represented above.
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p16/Rb pathway is inactivated in telomerase immortalized human keratinocytes
regardless of culture conditions and provides more evidence that telomerase based cell
therapies may lead to the transformation of human cells.
Inactivation of p16 during human keratinocyte
immortalization
Several studies have suggested that p16 inactivation is required for human
epithelial cell immortalization in vitro (Brenner et al., 1998; Dickson et al., 2000; Jarrard
et al., 1999; Kiyono et al., 1998; Reznikoff et al., 1996; Rheinwald et al., 2002; Tsutsui et
al., 2002). Most of these studies, however, have involved human epithelial cells cultured
in the absence of feeder cells. In the absence of feeder cells, cultures of human epithelial
cells accumulate p16 protein in a passage dependent manner. This increase in p16
expression eventually leads to growth arrest by telomere-independent mechanisms.
There is a growing body of evidence that suggests induction of epithelial cell migration is
associated with upregulation of p16 expression (Darbro et al., 2005; Jung et al., 2001;
Natarajan et al., 2003; Nilsson et al., 2004; Svensson et al., 2003). Previously, we have
shown that human keratinocytes cultured in the absence of feeder cells acquire a
phenotype consistent with a migratory response and that this migratory stimulus may be
responsible for inducing p16-mediated, telomere-independent senescence in this culture
condition (Darbro et al., 2005). Co-culture with post-mitotic fibroblast feeder cells has
been shown to increase human epithelial cell lifespan in culture but appears to only delay
or reduce the upregulation of p16 that precedes growth arrest. In the co-culture
environment, we have shown that human keratinocytes downregulate the expression of
genes involved in migration, suggesting that p16 expression is regulated by different
mechanisms in this culture system. Whereas it has been suggested that p16 expression
can be induced by telomere dysfunction (Jacobs & de Lange, 2004), it has also been
shown that human keratinocytes experienced very limited shortening of telomeres in the
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co-culture environment (Kang et al., 2004). These observations suggest that a telomereindependent mechanism, separate from a migratory stimulus, may be responsible for
upregulation of p16 expression in human keratinocytes co-cultured with feeder cells. If
telomere-independent mechanisms are responsible for p16 induction in human
keratinocytes co-cultured with feeder cells one would expect to observe immortalization
of these cells by exogenous expression of TERT alone if one of two conditions were true
1) p16 expression is inactivated or 2) induction of p16 expression is not responsible for
the growth arrest seen in human keratinocytes co-cultured with feeder cells.
Interestingly, the results of this study would seem to suggest that neither condition is
entirely true.
We found that human foreskin keratinocytes immortalized by exogenous TERT
expression and co-cultured with feeder cells exhibited p16 promoter methylation. At first
glance, this result would suggest that p16 inactivation is required to immortalize human
keratinocytes regardless of culture conditions. However, the presence of several
unmethylated p16 promoter sequences in late passage TERT transduced HFKs cocultured with feeders and the observation that p16 expression is induced when these cells
are transferred from the co-culture environment to plastic suggest this might not be the
case. Whereas it is possible that other epigenetic mechanisms of gene regulation may be
responsible for p16 silencing in the absence of any detectable DNA methylation, such as
DNA methylation-independent histone modification (Lewis et al., 2004; Thiagalingam et
al., 2003; Zhao et al., 2005), the consistent induction of p16 expression following transfer
from the co-culture environment to plastic is highly suggestive of a mixed population of
both p16 positive and negative TERT transduced HFKs. It is reasonable to conclude that
upon transfer to the plastic culture condition, those keratinocytes that had retained
functional p16 immediately upregulated p16 expression and senesced. Following the
senescence of p16 positive cells, those keratinocytes that had inactivated p16 expression
in the co-culture environment continued to proliferate and remained immortal. We can
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be reasonably confident that methylation of the p16 promoter occurred as a result of
continued co-culture of TERT transduced HFKs, and was not the result of a pre-existing
epigenetic mutation, since TERT HFKs cultured only on plastic alone induced p16
expression and failed to become immortal. The question then becomes what is the
significance of a mixed population of both p16 positive and negative TERT immortalized
keratinocytes.
The observation that a p16 negative population of cells is frequently selected for
during continuous co-culture of TERT transduced HFKs suggests that p16 expression
does limit proliferation of human keratinocytes in this culture environment. If p16
expression were not exerting a growth inhibitory effect cells would acquire no selective
growth advantage by inactivating p16. Thus, the inactivation of p16 expression in a
subpopulation of TERT immortalized keratinocytes co-cultured with feeder cells suggests
that a telomere-independent induction of p16 expression in late passage co-cultured
keratinocytes does contribute to their growth arrest in this culture system. However, the
retention of p16 expression in a subpopulation of TERT immortalized keratinocytes cocultured with feeder cells suggests that p16 inactivation is not required to immortalize
human keratinocytes in this culture system. That being said, we cannot exclude the
possibility that p16 negative HFKs may in some way be contributing to the survival of
their p16 competent counterparts. Experiments that strictly require the presence of
functional p16 in all cultured cells could prove technically difficult; however,
experiments in which primary keratinocytes are co-cultured with both feeder cells and
p16 negative, TERT immortalized keratinocytes could provide useful information as to
whether a p16 negative population of keratinocytes enhances the replicative capacity of
p16 positive ones.
The ability of TERT transduced keratinocytes to maintain the immortal phenotype
upon transfer from the co-culture environment to plastic appears to be dependent on the
length of time they spend in co-culture. Previous work has shown that inactivation of the
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p16/Rb pathway is required for immortalization of human keratinocytes in the plastic
culture condition and our results are consistent with this observation. However, in at
least one TERT transduced HFK cell line transferred from the co-culture environment to
plastic (Strain A TERT HFKs transferred at PD 35), it appeared that p16 expression had
already been compromised yet these cells still growth arrested in the plastic culture
system. The proliferation barrier observed in these cells was presumably not the result of
p16-induced, telomere-independent senescence as the levels of p16 protein were in a
steady state of decline as these cells underwent growth arrest. This observation suggests
that additional telomere-independent mechanisms of senescence exist in human
keratinocytes that prevent immortalization with TERT alone in the plastic culture
condition. Previous studies performed by Rheinwald and colleagues have suggested that
p53 is responsible for enforcing a telomere-independent mechanism of senescence in
human keratinocytes separate from that induced by p16 (Rheinwald et al., 2002). As p16
was the focus of our study we did not examine the status of p53 in our TERT transduced
HFK cell lines, however, our results would appear consistent with this theory. If p53 is
responsible for inducing a separate telomere-independent mechanism of senescence we
would expect to find inactivation of p53 in those cell lines that maintained the immortal
phenotype upon transfer to the plastic culture condition and induction of p53 signaling in
those cells that under went growth arrest following transfer. Since forced expression of
p16 induced rapid growth arrest and a senescent morphology in TERT immortalized
keratinocytes it is likely that Rb function is retained in these cells. Functional Rb may be
cooperating with p53/p21 signaling in the growth arrest of cells that have already
inactivated p16 expression. Future studies aimed at determining the status of p53 and
p21 signaling in our TERT transduced cell lines may provide further evidence of a p53induced, telomere-independent mechanism of senescence in human keratinocytes.
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Safety of telomerase based cell therapies
Whether p16 and/or p53 inactivation are strict requirements for human
keratinocyte immortalization in vitro remains to be determined, however, the increased
frequency of p16 promoter methylation in TERT transduced keratinocytes is of extreme
significance when considering telomerase based cell therapies. The loss of p16
expression has been observed in a myriad of human malignancies and is a negative
prognostic marker in many of them (Chakravarti et al., 2003; Esteller et al., 2001;
Groeger et al., 1999; Korkolopoulou et al., 2001; Partridge et al., 2005; Tsihlias et al.,
1999; Weinberger et al., 2004). Thus, p16 inactivation in TERT engineered cells is an
event that needs to be addressed prior to any therapeutic interventions.
It is conceivable that reduced periods in culture might be sufficient to reduce the
incidence of tumor suppressor gene inactivation in TERT transduced human cells as our
results suggest that early passage TERT transduced HFKs co-cultured with feeders did
not possess a significant number of p16 negative clones. Furthermore, transfer of early
passage TERT transduced HFKs from the co-culture environment to plastic did not result
in subsequent immortalization in the new culture environment. Even though we observed
a reduction in p16 expression in some of these cultures it is clear that additional tumor
suppressor pathways were still intact as these cells growth arrested within a few passages.
From our results, it would be advisable to culture TERT transduced keratinocytes no
longer than 40 PDs before their potential use as therapeutic agents. This strategy,
however, is not without risk, as it appeared that p16 inactivation might have already taken
place in some TERT transduced keratinocytes prior to this point. Culture of TERT
transduced human cells in more physiologic oxygen conditions may reduce the incidence
of epigenetic mutations in tumor suppressor genes. This strategy may be particularly
relevant to p16 mutations since several studies have found an association between
increased levels of ROS and p16 methylation during tumor progression (Govindarajan et
al., 2002; Jarmalaite et al., 2003; Romanenko et al., 2002). We have begun preliminary
115
investigations designed to address the question of whether or not p16 inactivation occurs
at the same frequency observed in this study in TERT transduced human keratinocytes
co-cultured with feeder cells in more physiologic oxygen conditions. Thus far we have
observed that co-culture of TERT transduced HFKs with feeder cells in physiologic
oxygen conditions does not decrease the frequency of immortalization but does appear to
decrease the frequency of p16 promoter methylation in later passages (data not shown).
Further analysis of these cells is required to determine if additional tumor suppressor
genes have been inactivated (such as Rb) but our initial observations are encouraging.
Summary
In summary, we have shown that exogenous expression of TERT in human
keratinocytes co-cultured with feeder cells is sufficient to induce immortalization,
however, these cells have an increased frequency of p16 promoter methylation and
subsequent inactivation. These results suggest that inactivation of the p16/Rb tumor
suppressor pathway occurs in TERT immortalized human keratinocytes regardless of
culture conditions and emphasizes the need for further experimentation prior to
telomerase based therapeutic interventions.
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CHAPTER V
CONCLUSIONS
Mechanisms of Telomere-Independent Senescence
One of the goals of this thesis project was to elucidate in part or whole the
mechanism of p16-induced, telomere-independent senescence. Previous work had
suggested that an ill-defined “stress” mechanism, caused by inadequate growth
conditions, was the cause of p16 induction and consequent growth arrest in human
epithelial cells continuously passaged in vitro. This hypothesis stemmed from the
observations that co-culture of human epithelial cells with post-mitotic, fibroblast feeder
cells allowed for an extension of replicative lifespan and immortalization with TERT
alone (Ramirez et al., 2001). It was also shown that the level of p16 expression was
reduced in human epithelial cells co-cultured with feeders. Following this report,
additional studies both affirmed and questioned that p16-induced, telomere-independent
senescence was merely a stress-mediated culture artifact (Baek et al., 2003; Fu et al.,
2003; Harada et al., 2003; Herbert et al., 2002; Kang et al., 2003; Rheinwald et al., 2002).
In this thesis, we have shown that co-culture of human keratinocytes with post-mitotic,
fibroblast feeder cells does significantly extend their replicative capacity in vitro and
delays the induction of p16 expression. Furthermore, we have shown that a similar
extension of lifespan and delay in p16 expression cannot be elicited by culture of human
keratinocytes in more physiologic oxygen conditions, suggesting that increased oxidative
stress generated by culture in the absence of feeder cells is not the cause of p16-induced,
telomere-independent senescence. Whereas it remains unknown what mechanism
accounts for the eventual p16 induction and growth arrest of human keratinocyte cocultured with feeder cells it is clear that culture on plastic alone promotes p16 expression
and senescence by telomere-independent mechanisms. As such, culture of human
keratinocytes on plastic alone provided the best model system for us to determine the
117
mechanism of p16-induced, telomere-independent senescence. Having come to this
conclusion, we performed a series of microarray experiments to determine which genes
in addition to p16 were being preferentially induced in keratinocytes grown on plastic
alone compared to those in the co-culture environment. We found that human
keratinocytes cultured on plastic alone, as compared to those in co-culture with feeder
cells, induced the expression of several genes known to be involved in keratinocyte
migration. Following validation of the microarray results by several methods it was clear
that the two culture systems differed in their ability to induce either a differentiating or
migratory phenotype in human keratinocytes. The observation that human keratinocytes
were differentiating in the co-culture condition was consistent with previous finding (Sun
& Green, 1976). However, the observation that p16 induction occurred preferentially in a
culture system that “activated” human keratinocytes suggested that perhaps a migratory
response, as opposed to a stress mechanism, was responsible for increased p16 expression
and consequent telomere-independent senescence. Further analysis confirmed that p16
expression was selectively induced in human keratinocytes possessing markers of a
migratory phenotype (pTyr-397 FAK and uPAR), and that inhibition of keratinocyte
migration, by impairment of either tyrosine kinase activity or uPA/uPAR function, was
sufficient to reduce p16 expression in human keratinocytes cultured on plastic alone.
Thus, our data would suggest that p16 expression in human keratinocytes cultured on
plastic alone is indeed the result of a “specific” culture condition that induces a
phenotypic change in human keratinocytes. The phrase “inadequate culture conditions”
is clearly inappropriate as culture of human keratinocytes without feeder cells induces a
normal, if not potentially enhanced, cellular response (migration). Our conclusion that
p16 expression is associated with a migratory stimulus in human epithelial cells is
supported by several recent reports (Jung et al., 2001; Natarajan et al., 2003; Nilsson et
al., 2004; Svensson et al., 2003) and, as reviewed below, has significant implications for
the study of human tumor cell metastasis.
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Future studies are required to delineate the precise signal transduction pathway(s)
that are responsible for p16 induction by migratory stimuli. Our results suggest that p16
induction in this setting is not the result of increased signaling through FAK, Src-PTKs,
or the Ras/MAPK pathways. Unlike in human fibroblasts, p16 expression in human
keratinocytes does not appear to be induced by oncogenic growth signals (such as
increased Ras activation), however, it remains likely that p16 induction during
keratinocyte migration is the result of a feedback mechanism designed to induce
senescence in cells that have acquired enhanced or abnormal migratory stimuli. It is
possible that instead of being inducible by increased proliferation signals, such as
oncogenic Ras, p16 expression is enhanced in the setting of increased migratory stimuli,
possibly originating from signaling pathways such as uPA/uPAR, β-catenin, or TGF-β.
Our results clearly implicate both tyrosine kinase activity and the uPA/uPAR
plasminogen activation system as regulators of p16 expression during keratinocyte
migration, and suggest that signaling through the β-catenin and TGF-β pathways may be
increased in human keratinocytes cultured on plastic alone. Delineation of the individual
contributions each of these pathways offer to p16 regulation could prove difficult as there
is considerable overlap of these pathways in terms of both gene expression and signaling
intermediates. Activation of latent TGF-β, and subsequent signaling, by uPA/uPAR
interactions (Blasi & Carmeliet, 2002), cooperation of Smads and β-catenin in gene
expression (Letamendia et al., 2001; Tian & Phillips, 2002), and the induction of uPAR
expression by β-catenin signaling (Wong & Pignatelli, 2002) are all previously described
points of interaction between these pathways. Thus, experiments designed to test the
involvement of these signaling pathways in p16-induced, telomere-independent
senescence may have to inhibit or activate more than just one. A complete mechanism
describing p16-induced, telomere-independent senescence in human epithelial cells
cultured on plastic alone is of extreme significance. Such knowledge would provide not
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only information pertinent to epithelial cell transformation and invasion but also help in
establishing better culture methods for the study of human epithelial cell immortalization.
Our results in human keratinocytes cultured on plastic alone led us to conclude
that p16-induced, telomere-independent senescence in this setting was likely caused by a
migratory stimulus induced by specific features of this culture environment (Darbro et al.,
2005); however, it remained unclear what mechanism controlled p16 expression in
human epithelial cells co-cultured with feeders. In the co-culture environment, increased
levels of p16 are still observed prior to growth arrest (Darbro et al., 2005; Fu et al., 2003;
Kang et al., 2003; Rheinwald et al., 2002). However, the induction of p16 expression is
greatly delayed in this co-culture setting and human keratinocytes appear to downregulate
the expression of several genes involved in migration. It is also unclear whether this
increase in p16 expression preceding growth arrest is what causes the proliferation barrier
experienced by human epithelial cells co-cultured with feeders. The original experiments
of Kiyono and Klingelhutz clearly established that p16 was involved in the proliferation
barrier experienced by human epithelial cells cultured on plastic alone since loss of either
p16 or Rb was required for lifespan extension and immortalization of these cells with
TERT (Kiyono et al., 1998). In the co-culture environment, however, there was data
suggesting that p16 loss was not required for immortalization of human epithelial cells
with TERT alone implying that telomere-dependent mechanisms, not p16 induction, were
responsible for growth arrest in this culture condition (Harada et al., 2003; Herbert et al.,
2002; Ramirez et al., 2001). The regular appearance of inflection points in the growth
curves of TERT immortalized epithelial cells co-cultured with feeders led this conclusion
to be questioned as it appeared that these periods of slowed growth occurred at the same
time control cells were undergoing growth arrest with high levels of p16 expression
(Rheinwald et al., 2002). Furthermore, it had also been observed that telomeres exhibit
only limited shortening in human keratinocytes co-cultured with feeder cells suggesting
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that a telomere-independent mechanism of senescence was in fact leading to growth
arrest in these cells (Kang et al., 2004).
We hypothesized that if p16 expression was exerting a growth inhibitory effect,
limiting the proliferative potential of human keratinocytes co-cultured with feeders, that
some form of functional p16 inactivation would be present in TERT immortalized cells.
If p16 expression were not limiting the replicative capacity of human epithelial cells cocultured with feeders then loss of p16 would presumably provide no selective growth
advantage to these cells suggesting that p16 function would not be selected against in
TERT containing cells. The results presented in this thesis confirmed that exogenous
expression of TERT alone in human keratinocytes co-cultured with feeder cells leads to
immortalization. We further confirmed that loss of p16 expression was required for
continued proliferation of TERT transduced human keratinocytes transferred from the coculture environment to plastic alone. We observed that maintenance of the immortal
phenotype following transfer from the co-culture environment to plastic appeared to be
dependent on the length of time TERT transduced keratinocytes spent in co-culture with
feeders. Having found evidence that p16 promoter methylation had occurred in
immortalized transferred cells we sought to determine whether some level of p16
promoter methylation was occurring in TERT transduced cells co-cultured with feeders.
Bisulfite sequencing clearly demonstrated that a subpopulation of cells had undergone
p16 promoter methylation suggesting that p16 inactivation is selected for in TERT
immortalized human keratinocytes co-cultured with feeder cells. Forced expression of
p16 caused rapid growth arrest in immortal keratinocytes transferred from the co-culture
environment to plastic implying that the specific inactivation of p16 is significant since
the cellular machinery required for it’s growth inhibitory effect, namely Rb, was not
altered. These results led us to conclude that p16 expression does contribute a growth
inhibitory effect to human keratinocytes co-cultured with feeder cells and as such may
very well be the cause of growth arrest in the absence of TERT expression. Our
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conclusion that p16 inactivation occurs in TERT immortalized human keratinocytes cocultured with feeder cells is supported by several recent studies that have shown similar
results for TERT immortalized human fibroblasts (Noble et al., 2004; Taylor et al., 2004)
and, as reviewed below, has significant implications for the use of telomerase activation
as a therapeutic modality.
Whereas our results confirm that p16 promoter methylation is an epigenetic event
selected for by TERT transduced human keratinocytes co-cultured with feeders it is
unclear at this point in time why this occurs in only a subpopulation of cells. Results
obtained from transferring TERT transduced human keratinocytes from the co-culture
environment to plastic alone confirmed that some cells maintain functional p16
expression that is induced and presumably causes their growth arrest after transfer.
Whether p16 negative cells contribute to the continued proliferation of their p16 positive
counterparts is an area of interest that requires further experimentation. As mentioned
above, it is likely that telomere-independent mechanisms are responsible for the eventual
induction of p16 expression in human keratinocytes co-cultured with feeder cells. Since
our microarray results suggest that keratinocytes co-cultured with feeder cells are not
subjected to a migratory stimulus, which appears to be associated with p16 induction in
the absence of feeder cells, it is likely that other factors are involved in inducing
telomere-independent expression of p16 in the co-culture environment. One factor
unique to the co-culture environment that may be responsible for p16 induction in this
setting is increased calcium concentration. In an attempt to exclude calcium
concentration as a reason for p16 repression in the co-culture environment, we added an
amount of calcium to KSFM medium that was equivalent to that present in E-media
(1.28mM). To our surprise, we found p16 expression was increased in keratinocytes
treated with calcium. This result clearly excludes calcium as being the reason for p16
downregulation in the co-culture environment but suggests that it may be involved in the
eventual induction of p16 expression in the co-culture system. Increased levels of
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calcium in culture medium have been shown to induce human keratinocyte differentiation
in vitro (Lee et al., 2000). Thus, despite the fact that p16 expression is not induced as a
feature of normal differentiation or epithelial renewal (Keating et al., 2001; Klaes et al.,
2001; Nielsen et al., 1999), it may be playing a role in calcium-induced keratinocyte
differentiation in vitro. In fact, it has been shown that TERT immortalized human
keratinocytes that have lost p16 expression undergo abnormal differentiation in
oganotypic raft cultures (Farwell et al., 2000). Additionally, treatment of human
keratinocytes with corticotropin-releasing hormone (CRH), which induces keratinocyte
differentiation through the release of intracellular calcium stores, has been shown to
cause p16 upregulation and subsequent growth arrest (Zbytek & Slominski, 2005). The
possibility that telomere-independent p16 expression is increased in the co-culture
environment by calcium-induced differentiation provides a potential explanation for how
p16 negative keratinocytes may facilitate the continued proliferation of p16 positive cells.
The loss of p16 expression in a fraction of keratinocytes co-cultured with feeders may be
altering the calcium-induced differentiation of their p16 positive counterparts. This
altered state of differentiation may reduce the stimulus for p16 induction in those cells
that have not inactivated p16, thus, reducing the selective pressure to lose p16 expression.
This situation would provide for the indefinite proliferation of both p16 negative and
positive TERT transduced keratinocytes in the co-culture system. To test this hypothesis,
one could perform a study similar to Hayflick’s original “Dirty Old Man Experiment”.
The co-culture of early passage (“young”) primary female keratinocytes with both feeder
cells and immortal (“extremely old”) p16-negative, TERT transduced male keratinocytes
would provide the proper context to examine this hypothesis. Following the senescence
of parallel control cultures, in which primary female keratinocytes are co-cultured with
feeder cells alone, any primary female cells continuing to proliferate in the “immortal”
co-culture environment would constitute an increase in replicative capacity conveyed by
their immortal, p16-negative neighbors. Characterization of differentiation markers in
123
these cells may further identify whether keratinocyte differentiation is involved in any
resultant increase in replicative capacity.
At present, we cannot confidently conclude that inactivation of the p16/Rb
pathway is required for TERT immortalization of human keratinocytes co-cultured with
feeder cells. Our results clearly indicate that loss of this cell cycle checkpoint is a
frequent event in a fraction of co-cultured keratinocytes immortalized by TERT but
whether this phenomenon is required for the indefinite proliferation of those cells that
have not lost p16 expression remains to be determined. Future experiments designed to
test the specific contribution of p16 expression to growth arrest in human keratinocytes
co-cultured with feeder cells are also needed as it is still unclear whether replicative
senescence in this culture system is enforced by p16. Furthermore, if p16 induction does
prove to be responsible for the eventual proliferation barrier reached by primary human
epithelial cells co-cultured with feeder cells it will become important to determine how
p16 expression is regulated differently in this culture system compared to the plastic
alone condition. Since co-culture with feeder cells induces human keratinocytes to
acquire a phenotype more akin to their normal state of terminal differentiation,
elucidation of the mechanism controlling p16-induced senescence in this culture system
may provide important information concerning the process of epithelial cell
transformation in vivo.
Implications of this Work to Human Tumor Cell Metastasis
and Telomerase Reactivation Therapies
Epithelial derived cancers account for over 90% of all human malignancies.
Cancers of epithelial origin, such as breast, colon, prostate, and bladder, also have a high
frequency of metastasis. The inactivation of p16 expression in epithelial derived cancers
is common (Liggett & Sidransky, 1998; Rocco & Sidransky, 2001; Sharpless, 2005).
Human mammary, prostatic, and uroepithelial cells have all been found to exhibit p16-
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induced, telomere-independent senescence and, in most experimental settings, require
inactivation of the p16/Rb pathway for immortalization in vitro (to date, immortalization
of normal human colonic epithelial cells with telomerase expression alone has not been
attempted) (Brenner et al., 1998; Foster & Galloway, 1996; Jarrard et al., 1999;
Puthenveettil et al., 1999; Reznikoff et al., 1996; Sandhu et al., 2000). Human
keratinocytes have been shown to share these characteristics and are also known to give
rise to metastatic cancers (Dickson et al., 2000; Kiyono et al., 1998). As such, results
obtained from the study of p16-induced, telomere-independent senescence in human
keratinocytes should prove very applicable to other epithelial cell types. The results
presented in this thesis strongly suggest that p16 induction and consequent telomereindependent senescence in human keratinocytes is associated with cellular migration.
Combined with the observations of others, these results may have significant implications
for defining p16 as a metastasis-suppressing gene in epithelial derived human cancers.
Metastasis of human tumor cells occurs in several well-defined steps, known as
the metastatic cascade (Figure 33). Following growth of the primary tumor, cells must
invade locally (in the case of epithelial cells invasion across some type of basement
membrane is usually required) and intravasate into the bloodstream or lymphatic
channels. Once in circulation, the tumor cells must survive long enough to reach a
secondary site. Following extravasation into the tissue at the secondary site, tumor cells
complete the cascade by surviving and proliferating into a micrometastatic lesion.
Evidence presented in this thesis, combined with the observations of others, suggests that
p16 may play a role in metastasis suppression at two stages in the human metastatic
cascade: tumor cell invasion and survival in circulation.
In an effort to determine upstream regulators of p16 expression in the context of
human epithelial cell telomere-independent senescence, we discovered that p16
expression is associated with a keratinocyte migratory response. In culture conditions
125
Figure 33: The metastatic cascade. Metastasis of epithelial tumors typically begins with
an in situ cancer juxtaposed to an intact basement membrane. Following the loss of cellcell contacts and the upregulation of pro-migratory genes, tumor cells acquire a migratory
phenotype and invade past the basement membrane. There is evidence to suggest that
p16 expression is induced directly preceding invasion and is subsequently lost in cells
that successfully invade past the basement membrane. After passing through the
basement membrane, metastasizing cells can be disseminated throughout the body by
either the lymphatics or the circulatory system. To survive transit to distant sites within
the body, metastasizing cells must overcome anoikis. There is evidence that suggests
loss of p16 function aids in this process. Following extravasation of the circulatory
system, micrometastases begin to form from single cells. Metastatic colonization at
distant sites can eventually develop into angiogenic metastases.
Source: Figure modified from (Steeg, 2003).
126
that promote p16-induced, telomere-independent senescence we found p16 is coexpressed with several genes known to facilitate both cellular migration and tumor cell
invasion. Pro-migratory integrins, MMPs, uPA, uPAR and several other genes we found
to be upregulated in human keratinocytes cultured on plastic alone have all been
implicated in tumor cell invasion and the metastasis of human epithelial cancers (Aguirre
Ghiso et al., 1999; Blasi & Carmeliet, 2002; Cairns et al., 2003; Kramer et al., 2005;
Varner & Cheresh, 1996; Wittekind & Neid, 2005; Yoon et al., 2003). These results
suggest that p16 expression may be regulated by the same cellular mechanisms that lead
to human epithelial cell migration and invasion. As discussed in Chapter III, our results
have led us to believe that p16 expression in this context evolved as a negative feedback
mechanism to induce growth arrest in epithelial cells that had acquired enhanced or
abnormal cellular signals to migrate. Consistent with this hypothesis, several studies
have reported that epithelial tumor cells induce p16 expression directly preceding or at
the onset of invasion (Jung et al., 2001; Natarajan et al., 2003; Nilsson et al., 2004;
Svensson et al., 2003). In these studies that examined cellular markers of proliferation,
p16 expression was found to correlate with the induction of growth arrest (Jung et al.,
2001; Svensson et al., 2003). Furthermore, loss of p16 expression was commonly
observed in those cells that successfully invaded past the basement membrane (Natarajan
et al., 2003; Nilsson et al., 2004) suggesting p16 inactivation facilitates the process of
invasion. Based on these results, one can envision a model of epithelial tumor cell
invasion in which p16 expression is induced as tumor cells come into contact with the
basement membrane. At this point, invading cells either senesce via p16-induced growth
arrest or acquire p16-inactivating mutations that allow for continued invasion into the
surrounding tissue. Further study is required to test this model of p16-induced growth
arrest upon tumor cell invasion and may contribute more evidence for a potential role of
p16 in suppressing epithelial tumor cell invasion.
The model above predicts that the loss of p16 expression may facilitate the
127
process of tumor cell invasion. However, there is additional evidence to suggest that p16
loss during this event may also contribute to the continued survival of metastasizing cells
while in transit within the circulatory system. The study of pancreatic cancer cells is
extremely relevant to the study of p16 loss and metastasis, as they have been found to
have the highest frequency of p16 loss of all human malignancies (Liggett & Sidransky,
1998; Rocco & Sidransky, 2001; Schutte et al., 1997; Sharpless, 2005) and are known to
exhibit early and aggressive local growth and metastasis (Rosewicz & Wiedenmann,
1997). The correlation between p16 loss and increased metastatic potential is extremely
high in pancreatic cancers and there is evidence that suggests the phenomenon of anoikis
is involved. Anoikis is the term given to the induction of apoptosis experienced by cells
upon loss of cell anchorage (Frisch & Francis, 1994; Frisch & Ruoslahti, 1997). Anoikis
has long been thought a safety mechanism in that it prevents survival and reattachment of
metastatic cells at new locations. Following cellular transformation, many tumor cells
become resistant to anoikis thereby allowing them to spread and reattach at distant sites.
Studies in metastatic human pancreatic tumor cells have shown that re-expression of p16
is found to restore anoikis suggesting p16 inactivation may have contributed to the loss of
anoikis and the enhanced metastatic spread of these cells (Plath et al., 2000). Consistent
with this finding are several in vitro studies that have shown loss of p16 increases soft
agar colonization of human cells and that re-expression of p16 in human tumor cell lines
reduces anchorage independent growth (Higashi et al., 1997; Wang et al., 1999; Wang et
al., 2001; Wei et al., 2003). These findings suggest that p16 may have functions
independent of its ability to induce growth arrest, which is particularly relevant to a rather
unexpected result presented in Chapter III. In Chapter III, we examined p16 expression
in the setting of reduced FAK signaling. In human keratinocytes transduced with FRNK,
a dominant negative inhibitor of FAK, we observed a decrease in both FAK
autophosphorylation and expression of the FAK-inducible gene MMP-9. We
hypothesized p16 expression would also be downregulated in these cells but were
128
surprised when we observed what appeared to be an increase in p16 mRNA levels by
real-time RT-PCR. As we did not observe a concomitant increase in p16 protein levels in
these cells further experiments are needed to determine if this association is real;
however, such an association may explain how decreased signaling through FAK, upon
loss of cell anchorage, can lead to induction of anoikis (Frisch & Screaton, 2001; Frisch
et al., 1996). As induction of anoikis occurs most prominently in epithelial and
endothelial cells, FAK-p16-induced anoikis may be yet another mechanism through
which p16 can limit the metastatic spread of epithelial derived tumors.
The current definition of a metastasis suppressor gene is that when said gene is reexpressed in a tumor line it is capable of inhibiting metastasis without a significant
reduction in tumorigenicity (Steeg, 2003). Much like the gene maspin, defining p16 as a
metastasis suppressor gene would be complicated since it can also function as a tumor
suppressor (Zou et al., 1994). One condition that may facilitate defining p16 as a
metastasis suppressor gene is the discovery of unknown functions of p16 that are
independent of its ability to induce growth arrest. The finding that p16 is associated with
anoikis is one such discovery. In addition, there is also substantial evidence to suggest
p16 may be capable of directly inhibiting the process of migration. In our study and
several others (Natarajan et al., 2003; Nilsson et al., 2004; Svensson et al., 2003)
immunocytochemical staining for p16 has shown it to be distributed not only in the
nucleus but throughout the cytoplasm as well suggesting a potential role for p16 in this
cellular compartment. Consistent with this possibility is the finding that expression of
full-length p16 protein in a human melanoma cell line inhibited spreading on vitronectin
by a mechanism involving CDK6 localized to the spreading edge of cells (Fahraeus &
Lane, 1999). Additionally, a recent report has shown that p16, cyclin D1, CDK4 and
CDK6 were immuno-colocalized with Ezrin, Rac, Vinculin, αV-integrin, and FAK
proteins in the ruffles and lamellipodia of migratory cells (Alhaja et al., 2004). These
studies suggest that p16 may inhibit metastatic spread of tumor cells through the specific
129
inhibition of migration and not simply by inducing senescence of cells that have acquired
abnormal migratory stimuli. Studies employing the use of keratinocytes with inactivated
Rb could prove useful in determining whether p16 inhibition of CDK4/6-cyclin D
complexes at the cell membrane leads to the inhibition of keratinocyte migration
independent of Rb function.
The role p16 plays in suppressing metastasis, if any, remains to be elucidated.
Studies in transgenic knockout mice have suggested that p16 inactivation can confer
increased metastatic potential to tumor cells but the associated loss of ARF in many of
these experiments has made interpretation of the individual role of p16 loss complicated
(Aguirre et al., 2003; Krimpenfort et al., 2001). If p16 loss does increase metastatic
spread of epithelial cancer cells, therapies designed to re-activate p16 expression in tumor
cells may prove efficacious in the treatment of these malignancies.
The loss of p16 expression is clearly associated with an increased risk of tumor
development and, as discussed above, may be implicated in the metastatic spread of
epithelial tumor cells. As such, our finding that p16 promoter methylation occurs in
TERT immortalized human keratinocytes co-cultured with feeder cells is of extreme
significance given that any such cells used for therapeutic purposes may have an
increased risk of both tumor development and subsequent metastatic spread.
The field of therapeutic telomerase reactivation is still in its infancy, as such it is
extremely important for investigators to rigorously examine telomerase immortalized
human cells for the existence of potential cellular “side effects” associated with
telomerase expression. The potential for telomerase based cell therapies to lessen the
effects of degenerative aging disorders and possibly rescue the disease phenotype of
dyskeratosis congenita is offset by recent studies suggesting that telomerase immortalized
cells often exhibit deregulation of both proto-oncogenes and tumor suppressor pathways
(Artandi et al., 2002; Chang & DePinho, 2002; Gonzalez-Suarez et al., 2005; Noble et al.,
2004; Stampfer et al., 2001; Stewart et al., 2002; Taylor et al., 2004; Wang et al., 1998;
130
Xiang et al., 2002). In this thesis, we demonstrate that p16 promoter methylation is a
frequent event in TERT immortalized human keratinocytes co-cultured with feeder cells
suggesting that the p16/Rb tumor suppressor pathway has been compromised in these
cells. Our results are consistent with studies reporting p16 inactivation in TERT
immortalized human fibroblasts and suggest that such cells may pose an increased risk of
cancer if used for therapeutic purposes.
In our study, we found that the incidence of p16 promoter methylation was
increased in late passage TERT immortalized keratinocytes suggesting that prolonged
culture of these cells likely contributes to tumor suppressor inactivation. Further
evidence to this point is the observation that additional telomere-independent
mechanisms of growth arrest, which are likely responsible for growth arrest in the
absence of p16 expression (as seen in Strain A TERT HFKs transferred at PD 35), are
also compromised as a function of in vitro culture duration. Based on this result, one can
conclude that shorter periods of culture may decrease the frequency of additional genetic
and epigenetic mutations. Shorter periods of in vitro culture, culture in low oxygen
environments, and more regulated expression of TERT have all been proposed as
potential methods to decrease the occurrence of genetic aberrations in telomerase
immortalized human cells. However, it may not be possible to completely eliminate the
risk of generating tumor suppressor compromised, telomerase positive cells. In this case,
strategies designed to eliminate transgenic cells once transferred to a human recipient are
needed. It is possible that telomerase engineered cells might also be fitted with a Herpes
simplex virus thymidine kinase gene that in effect becomes a suicide gene when the
antiviral drug ganciclovir is applied (Painter et al., 2005). In addition to suicide genes,
telomerase expressing cells may also be engineered with additional tumor suppressor
gene constructs that have been designed to be induced upon stimulation with a particular
hormone, such as estrogen inducible p53 or p16 expression constructs (Sengupta et al.,
2000). Unfortunately, this approach is also not devoid of risk. Like any cellular gene
131
that functions to restrict growth, transgenic suicide or growth arrest genes would also be
subject to the same random process of inactivation either by gene deletion or epigenetic
effects. The inducible nature of these transgenic constructs would be expected to lessen
the frequency of this event as cells will not be under constant selective pressure to
eliminate their expression. One method that might decrease the risk of “safety” gene
inactivation is the placement of such a gene next to a cellular gene that is absolutely
required for cell viability. Experiments in which a viral thymidine kinase gene is inserted
in close proximity to DNA polymerase I may produce this effect. This type of strategy
appears to hold the most promise for telomerase engineered cell therapies but as with any
medical breakthrough is not without some risk to the patient. In the end, telomerase
based cell therapies will come down to a risk/benefit analysis. Considerations such as
potential benefits and therapeutic alternatives will have to be determined on a patient-topatient basis. The selective targeting of specific tissues in which tumor progression is
rare could also reduce the potential risk of telomerase therapies. Although a great deal
more experimentation is needed before such therapies should be considered for use in
humans, the potential for telomerase therapies to correct genetic defects, such as in DC
patients, and age-related tissue dysfunction disorders is well worth the additional effort.
132
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