[CANCER RESEARCH 48, 4620-4628, August 15. 1988)
Characterization of Normal Human Exocervical Epithelial Cells Immortalized in
Vitro by Papillonâ„¢virusTypes 16 and 18 DNA1
C. D. Woodworth,2 P. E. Bowden, J. Doniger, L. Pirisi,3 W. Barnes, W. D. Lancaster, and J. A. DiPaolo
Laboratory of Biology, Division of Cancer Etiology, National Cancer Institute, Bethesda, Maryland 20892 [C. D. W., P. E. B., J. D., L. P., J. A. D.J, and Department of
Obstetrics and Gynecology, Georgetown University Medical Center, Washington, DC 20007 (W. B., W. D. L.J
ABSTRACT
An In vitro system for studying the interaction between human papillomavirus (HPV) 16 and 18 recombinant DNA and normal human
exocervical epithelial cells is described. Eight HPV-immortalized human
exocervical epithelial cell lines were established; all the lines contained
either integrated HPV16 or 18 sequences and expressed HPV mRNAs.
Thus, integration and expression appear to be required for immortaliza
tion. Immortalized cells (>200 population doublings to date) divided
rapidly (doubling time of 30 to 46 h) and morphologically resembled
primary cultures of normal human exocervical epithelial cells. They
expressed a keratin pattern consistent with their origin from exocervical
epithelium. When cultured at high density or in the presence of serum
they terminally differentiated. Sublines resistant to terminal differentia
tion were selected by growth in serum-supplemented medium. Keratin
pattern changes suggest they have some properties in common with
cervical squamous carcinoma cells. However, HPV-immortalized cell
lines were not tumorgenic in nude mice. Thus, HPV16/18 is not carcin
ogenic by itself. These cell lines represent an appropriate model for
studying factors that regulate HPV gene expression in normal cervical
epithelial cells and examining the influence of cocarcinogens on neoplastic
progression.
INTRODUCTION
Cervical carcinoma is one of the most frequent cancers among
women and a major cause of deaths attributable to cancer (1,
2). The etiological mechanisms contributing to the development
of cervical carcinoma are incompletely understood; however,
circumstantial evidence suggests a causal relationship between
HPV4 and cervical neoplasia (summarized in Refs. 3 to 5). HPV
DNAs have been identified in preneoplastic and neoplastic
lesions of the uterine cervix in the majority of patients (80 to
90%) examined (6, 7). This association is specific as certain
types of HPV (HPV 16 and 18) occur consistently in advanced
cervical intraepithelial neoplasia and in cervical carcinomas,
whereas others such as HPV6 and HPV11 are frequently found
in benign lesions, suggesting a difference in oncogenic potential
(8, 9). A number of different cell lines derived from cervical
tumors retain integrated HPV 16 or HPV 18 DNA and express
HPV RNA after extended passage in culture (10-12), implying
the importance of HPV gene expression for the continued
tumorigenicity of these cells.
Demonstration of the etiological role for HPV in cervical
cancer has not been possible, because no animal model exists
Received 12/16/87; revised 4/12/88, 5/13/88; accepted 5/19/88.
The costs or publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1The investigation was supported in pan by Grant CA32638 awarded to W.
D. L. by the National Cancer Institute, Department of Health and Human
Services, Bethesda, MD 20892.
1To whom requests for reprints should be addressed, at the National Cancer
Institute, Laboratory of Biology, Division of Cancer Etiology, Building 37, Room
2A19. Bethesda, MD 20892.
3 Present address: Department of Chemistry, University of South Carolina.
Columbia. SC 29208.
'The abbreviations used are: HPV, human papillomavirus; HCX, human
exocervical epithelial cells; HK, human foreskin keratinocytes; PD, population
doublings; kbp, kilobase pairs; SDS, sodium dodecyl sulfate; SSC, standard saline
citrate.
for studying the interaction between HPV and cervical cells in
the pathogenesis of cervical neoplasia. Bathing of human cer
vical tissue with condylomata acuminata extracts resulted in
dysplastic growth when grafts were transplanted to the renal
capsule of nude mice (13). However, HPV 16 DNA can trans
form established rodent cell lines to a malignant phenotype
(14-16) and cooperate with an activated c-H-ras oncogene to
fully transform primary rat fibroblasts (17). HPV 16 DNA also
immortalizes primary cultures of foreskin-derived normal HK
(18, 19), thereby providing a model to study the molecular
biology of HPV in normal epithelia of human origin. Expres
sion of HPV genes is regulated in a species- and cell typespecific manner (20-22), and the pathogenesis of HPV-induced
lesions varies in different tissues (23, 24). Because the majority
of cervical tumors originate from cervical epithelium in the
region of the squamocolumnar junction (transformation zone),
it is particularly appropriate to develop a model for studying
the interaction between HPV and HCX. Normal HCX can be
cultured for 30 to 40 PD before they senesce (25, 26). This
study demonstrates that HCX can be transfected and immor
talized with HPV 16 or 18 DNA. The HPV-immortalized cell
lines express HPV genetic information and a keratin pattern
similar to normal cultured exocervical epithelium.
MATERIALS
AND METHODS
Cervical Cell Cultures. Cervical tissue, without gross evidence of
pathology, was obtained from hysterectomies taken from three individ
uals (Table 1). HCX were prepared from the region of the transforma
tion zone (representing the squamocolumnar junction between the
endocervix and exocervix). Tissue fragments (1 cm3) were placed in
MCDB153-LB medium containing 0.25% collagenase (Boehringer
Mannheim, West Germany) at 37°Cfor 24 h. MCDB153-LB is
MCDB1S3 medium (27) in which the basal medium has been modified
by increasing the concentrations of tyrosine, tryptophan, methionine,
histidine, isoleucine, and lysine 3-fold. The concentration of 4-(2hydroxyethyl)-1 -piperazineethanesulfonic acid buffer was reduced to 14
mM, and the osmolarity was adjusted to 300 mOsmol/kg. Other me
dium supplements with the exception of epidermal growth factor (re
duced to 5 ng/ml) were not changed. After 24 h, cell clumps containing
both large squamous cells and small round cells detached from the
underlying tissue and floated in the medium. These cell aggregates were
transferred to a fresh 100 mm culture dish with medium. The remaining
mucosa was gently scraped to dislodge additional clumps of epithelial
cells. HCX were centrifuged (5 min at 800 x g), resuspended in fresh
MCDB153-LB, and transferred to 100-mm dishes. After 24 h, the
medium and debris were removed, and fresh MCDB153-LB was added.
When HCX colonies became larger (0.5 to 1.0 cm2), the primary
cultures were trypsinized and replated into 6-well cluster dishes (Costar,
Cambridge, MA) at a density of approximately 0.5 to 1.0 x IO5 cells/
1.0 cm2. Secondary HCX cultures at 40 to 60% confluence (2 to 5 days)
were used for HPV transfection studies.
Recombinant Plasmids. The plasmid pMHPV16d, a head-to-tail dimer of HPV16 DNA (7) inserted into the BamHl site of the plasmid
pdMMTneo (28), has been described (29). Plasmid pSHPVISm con
tains a single copy of the HPV 18 genome (6) inserted into the EcoRl
site of pSV2neo (30). The physical map of each plasmid is shown in
Fig. 1. For hybridization studies plasmid DNAs were digested with the
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CERVICAL CELL IMMORTALIZATION BY HPV DNA
Table 1 Origin of HPf-immortalized
cervical cell lines
HCX16 and 18 lines were transfected with pMHPVlod
respectively.
lineHCX16-1
Cell
tissue4d
of
and pSHPVISm,
by"Growth
\ r old with endometrial
HCX16-2HCX16-3 leiomyomata34-yr-old
GrowthG4I8
with endometrial
HCX16-4
adenocarcinoma66-yr-old
HCX16-5
HCX16-6HCX16-7
Growth
Growth
G418G418
with benign mature
HCX18-1Source
teratomaSelected
G418
°Ability to proliferate after normal HCX had senesced, or to grow after
treatment with 100 jig/ml of G418 for 48 h.
SV40
polyA LI
Filters were washed twice in 2x SSC (ix SSC is 0.15 M NaCl plus
0.015 M sodium citrate) containing 0.5% SDS for 30 min at room
temperature and then in 0.1 x SSC containing 0.1% SDS at 50'C for
30 min. Filters were exposed to XAR-2 film (Eastman Kodak Co.,
Rochester, NY) with an enhancing screen at -72°C. DNA and RNA
neo
HMT
promoter
molecular weight markers were obtained from Bethesda Research Lab
oratories (Gaithersburg, Ml)).
RNA Analysis. RNA was isolated from subconfluent HCX lines by
lysis in guanidine thiocyanate (32) followed by centrifugation through
cesium trifluoroacetate (Pharmacia Fine Chemicals, Piscataway, NJ).
Polyadenylate-terminated niRNA was enriched using oligodeoxythymidylate cellulose column chromatography. Polyadenylate-terminated
mRNA was separated by electrophoresis in 1.5% agarose gels contain
ing formaldehyde. RNA was transferred to Gene Screen membranes by
electroblotting and hybridized to "P-Iabeled DNA as described.
amp
pMHPViBd
22.5 kbp
PBR322
origin
a modification (18) of the calcium phosphate technique (31). After 2 to
4 h the precipitate was removed, the cells washed twice with MCDB153LB, fresh medium was added, and the cells were incubated an additional
24 to 48 h. The transfected cultures were trypsinized, and cells from a
single well were transferred to one 100-mm dish. Cervical cells that
expressed the transfected DNA were selected by two methods. Cultures
were either treated with G418 (Grand Island Biological Co., Grand
Island, NY) at 100 ¿ig/mlfor 48 h to select drug-resistant cells, or
continually subcultured in the absence of any selective pressure other
than the ability to proliferate after normal HCX had senesced.
DNA Analysis. High-molecular-weight DNA isolated from HCX
lines was digested with restriction endonucleases, and DNA was sepa
rated by electrophoresis in 0.8% to 0.4% agarose gels. DNA was
transferred to Gene Screen membranes (New England Nuclear, Boston,
MA) using an electroblot apparatus (Bio-Rad Laboratories, Richmond,
CA), and filters were hybridized to nick-translated "P-labeled DNA (1
x 10s cpm/ng) under stringent conditions (50% formamide-10% dextran sulfate-5x Denhardt's solution-1% SDS) at 42'C for 16 to 24 h.
Protein Labeling. Total cellular proteins were pulse labeled for 2 h
at 37°Cin methionine-free MCDB153-LB containing 10 mCi/ml of L[35S]methionine (1450 Ci/mmol). Cultures were then rinsed in sterile
phosphate-buffered saline containing 5 HIMethyleneglycol-bis(/3-aminoethylether)-W,N,./V',./V'-tetraacetic acid and 5 mM EDTA and stored
at -40°C until extracted (33, 34).
Keratin Extraction. Cellular cytoskeletons were prepared by treat
ment with Triton X-100 (1%) and high salt (1.4 M NaCl) buffers (34,
35). Total cellular proteins and cytoskeletal pellets (keratin intermedi
ate filament proteins) were extracted with 2% SDS and 2% mercaptoethanol in 0.5 M Tris-HCl (pH 6.80) by heating at 80°Cfor 15 to 20
Eia
min, homogenizing while hot, and centrifuging at room temperature
for 20 min. The supernatants were stored at —20°C.
SV40
poly A
Electrophoresis. Labeled keratins and total cellular proteins were
analyzed by one- and two-dimensional electrophoresis as described (34,
36). Proteins were separated in the first dimension by nonequilibrium
pH gradient (pH 4 to 9) electrophoresis and in the second dimension
by SDS-polyacrylamide gradient (7.5 to 17.5%) slab gel electrophoresis.
The gels were treated with En'Hance (New England Nuclear) and
exposed to Kodak XAR-2 film at -70°C.
Tumor Studies. Cultures of HPV16-immortalized HCX (40 to 60
PD) were trypsinized at confluent density. Trypsin activity was neu
tralized by resuspending cells in MCDB153-LB supplemented with 5%
fetal bovine serum. Cells were washed once in phosphate-buffered
saline, and approximately I x 10' cells were injected s.c. into 6- to 8-
neo
SV40
pSHPVlflm
promoter
PBR322
origin
13.6 kbp
amp
Fig. 1. Molecular structure of the recombinant plasmids pMHPV16d and
pSHPVlSm. The HPV sequences are indicated by bold lines. The arcs outside
the circles indicate the positions of the HPV open reading frames: their distance
from the circle indicates relative reading frames. The /•"/
open reading frame is
wk-old nude mice (athymic/NCR-/ii/; Frederick Cancer Research Fa
cility, Frederick, MD). Approximately 1 x IO6cells also were injected
intracranially (37) into the same animal. Mice were examined for
tumors at weekly intervals and monitored for at least 6 mo.
split into two reading frames due to a frame shift caused by a missing base. .Imp
is the ampicillin resistance gene from pBR322, neo is the neomycin resistance
gene, MMTpromoter is the mouse metallothionine promoter.
RESULTS
appropriate restriction endonucleases, the DNA was separated on agarose gels, and the HPV sequences were purified from gels for use as
probes.
Transaction of HCX. Secondary cultures of normal HCX were
transfected with 2 /¿g
of HPV 16 or HPV 18 DNA per 35-mm dish using
HCX Culture. HCX colonies grew slowly and were composed
predominately of small, closely associated cells of epithelial
morphology (Fig. 2A). Eventually these formed foci of densely
packed cells. Secondary cultures were established from dishes
containing 10 to 20 large colonies (>1 cm2). When DNA
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CERVICAL CELL IMMORTALIZATION BY HPV DNA
Fig. 2. Morphology of normal HCX in culture. A, small outgrowth of HCX from tissue fragment 4 days after plating; B, secondary culture of HCX (approximately
15 PD) illustrating the small size and compact nature of cells; C, HCX that were subcultured 4 times (approximately 40 PD) illustrating the large flattened cell
morphology.
isolated from secondary cultures was analyzed under nonstringent conditions (hybridization at 40°Cbelow maximum tem
perature) for the presence of HPV6, 16, 18, or related se
quences, none was detected. Secondary cultures of HCX grew
rapidly (PD time was approximately 36 h), and cells morpho
logically resembled those in primary cultures (Fig. 2B). After
plating at clonal density (cloning efficiency of approximately
10%), cloned colonies exhibited heterogeneous morphologies.
Most colonies consisted of a mixture of small round cells and
a few large flattened cells; however, others consisted of only
small cells or mainly large differentiated cells. HCX could be
subcultured 3 to 4 times (1:10 split), but their doubling time
increased and they became larger and more flattened (Fig. 2C).
After 30 to 40 PD all HCX underwent terminal differentiation
and could no longer be subcultured.
Immortalization of HCX with HPV16 and 18 DNA. Secondary
cultures of normal HCX were transfected with pMHPV16d or
pSHPVISm DNA and selected for resistance to the antibiotic
G418. Within 7 to 10 days after selection, 1 to 10 resistant
colonies arose per dish. These cells grew rapidly (PD of 36 to
48 h) and could be subcultured repeatedly. The individual cells
in these colonies were similar in morphology to those of normal
HCX secondary cultures (Fig. 3A). Occasionally transfection
with vector DNA only (pdMMTneo) resulted in small G418resistant colonies with limited proliferation capacity (30 to 40
PD). Continuously growing HCX cells were also derived in the
absence of G418 selection by repeatedly subculturing cells
transfected with pMHPV16d. HCX transfected with only vec
tor DNA senesced 1 to 3 subpassages after transfection (30 to
40 PD), whereas HPV-transfected HCX continued to prolifer
ate and overgrew the normal cells. Thus, extension of the life
span of HPV-transfected HCX was not due to pdMMTneo
sequences in the donor recombinant plasmiti. Eight HPVimmortalized HCX lines were established from 3 different
cervical tissues (Table 1). Seven lines were immortalized by
HPV 16 DNA and one by HPV 18 DNA. Cultures are consid
ered immortalized because they have exceeded the life span of
normal HCX by 4- to 6-fold and continue to proliferate at the
same rate as did the original nontransfected controls.
The HPV-immortalized cell lines varied in growth rate and
in morphology of the individual cells (Fig. 3B). At low passage
(40 to 66 PD), cell lines were composed of a mixture of small
undifferentiated cells, large flat multinucleated cells, and
sloughing, differentiating cells. Four lines derived from one
cervical specimen (HCX 16-3, 4, 5, and 6), underwent a period
of crisis in which the majority of the cells differentiated and
sloughed from the substrate (Fig. 3C). When these cultures
were passed at high cell density (1:2 split), a small number of
continuously dividing cells were selected, and these eventually
could be subcultured repeatedly. These postcrisis cell lines
contained more small undifferentiated cells and fewer large
flattened cells than the precrisis cells (Fig. 3D). To date, HPVimmortalized HCX cell lines have been maintained for greater
than 200 PD.
Analysis of Cell Lines for HPV Genetic Information. To
determine whether HPV 16 DNA was present in the HPVI6immortalized HXC lines, high-molecular-weight DNA was ob
tained from 4 lines at low passages (50 to 80 PD). Three of
these cell lines were polyclonal in origin; however, one line,
HCX 16-6, was derived from a single colony after selection with
G418. After BamHl digestion, which releases the HPV 16 se
quences from vector DNA, all lines examined contained an
average of 2 to 20 copies per cell of DNA sequences homologous
to HPV 16 (Fig. 4/1). In addition to the expected 7.9-kbp
complete HPV 16 genome, all lines also contained rearranged
HPV 16 DNA sequences. These rearranged forms of HPV 16
DNA suggest integration. To determine whether any rearranged
sequences were linked to genomic DNA, samples from the same
HPV16-immortalized cell lines were digested with BamHl plus
EcoRV, a noncut enzyme for the plasmid. The expectation that
some rearranged fragments with junctions between cellular and
HPV 16 DNA would be further digested by EcoRV was observed
(Fig. 4A). Thus, at least a portion of the HPV 16 sequences
appear to be integrated into cellular DNA.
To determine
whether
integration
of the plasmid
pMHPV16d occurred by preferential recombination within
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CERVICAL
CELL
IMMORTALIZATION
BY HPV
DNA
Fig. 3. Progressive morphological changes
in HPV 16-immortalized HCX with continued
passage. A, colony of HPV16-immortalized
HCX that arose after transfection with
pMHPVlod and selection with G418. Cells
are small, refractile, and resemble secondary
cultures of normal HCX. In B, after approxi
mately 40 to 60 PD (after normal HCX senesced), HPV16-immortalized cultures con
tain cells with several distinct morphologies.
Many small undifferentiated cells exist along
with large, flat multinucleated cells, and large
round refractile cells presumably in the process
of differentiating. In C, after 50 to 60 PD,
some cell lines [HCX 16-3,4, 5, and 6 (shown)]
entered a period of crisis in which the majority
of cells terminally differentiated and only oc
casional small cells continued to divide. In /».
HPV 16-immortalized HCX after 120 PD were
small and grew rapidly. Few differentiated cells
were present in these cultures.
Fig. 4. Detection of HPV 16 DNA in
HPV16-immortalized HCX lines. Total cellu
lar DNA was extracted from 4 cell lines (2 to
6; HCX 16-2 to HCX 16-6) and 10 ^ of DNA
were digested with restriction enzymes, electrophoresed on 0.8% agarose gels, and electroblotted to Gene Screen filters. In A, DNA was
digested with BamHl (B), which separates vec
tor from HPV 16 sequences, or with /(«mill
plus EcoRV (B/E). EcoRV does not digest
pMHPV16d. Filters were hybridized to the
"P-labeled BamHl fragment of HPV16 DNA.
The last 2 lanes represent a reconstruction
experiment
in which an amount
of
pMHPV16d DNA equal to 2 or 10 copies per
cell was mixed with DNA from nontransfected
cells. In B, Southern blot shown in A was
rehybridized to "P-labeled
vector DNA
(pdMMTneo). The molecular weight markers
are shown at the margins. In C, DNA from the
same cell lines was digested with EcoRV (E)
or Xhol (X), enzymes that do not cut within
pMHPV16d, and blots were hybridized to the
HPV 16 BamHl fragment. The positions of
form I (FI) and II (FII) plasmid DNA are
indicated.
B
a>
il
--Ill
23.1-
-23.1
48.5-
'
-Fil
29.9-9.4
I-6.7
4.4-
-4.4
2.3-
-2.3
19.412.210.1 —
8.3-
-FI
XEXEXEXE
tu
\
m
v
uj
\
111
CQ CD 00
UJ
UJ
UJ
UJ
m ta
HPV16 sequences or by random recombination with the entire
integrated into host cell DNA rather than existing as multimene
plasmid, the same filter was rehybridized to 32P-labeled vector
plasmid episomes. No dominant HPV 16 band comigrated with
DNA. The BamHl digests contained 6.7-kbp pdMMTneo se
Form I or Form II pMHPV16d DNA. Therefore, these four
quences as well as rearranged fragments (Fig. 4/3). Further
cell lines contained little if any monomeric plasmid DNA. The
more, some of the rearranged forms were reduced in size after
lack of a major band indicates that multiple integration events
double digestion with both BamHl plus EcoRV. Thus, integra
have occurred. Because most of the bands were less than 50
tion of plasmid DNA occurred in both vector and HPV 16 kbp in size, integrated HPV 16 DNA was mostly monomeric in
sequences.
form.
Expression of HPV16-specific sequences was measured in 7
The dominant HPV 16 fragment after BamHl digestion was
HPVI 6-immortalized cell lines. All lines contained 1.8- and
7.9 kbp. These fragments derive from either internal parts
integrated into DNA or from episomal pMHPV16d DNA. To 4.2-kbp HPV mRNAs (Fig. 5/1). In addition, each cell line
distinguish between these possibilities, DNA from the cell lines expressed other HPV transcripts that varied in size and abun
was analyzed after digestion with either of two restriction
dance. When the Northern blot in Fig. 5A was rehybridized to
enzymes (EcoRV or Xhol) whose recognition sequences are 32P-labeled vector DNA, several different mRNAs were detected
absent in the transfected plasmid (Fig. 4C). HPV 16 sequences
(Fig. 5B). Transcripts of 2.3 and 6.0 kbp were present in high
were detected in high-molecular-weight DNA after digestion
abundance in 3 cell lines. Small amounts of 1.2-, 3.0-, 3.4-, or
with either enzyme, but the size of the respective HPV 16- 8.2-kbp mRNAs were found in all but one cell line (that was
containing fragments differed. This indicates that HPV 16 DNA
not selected in G418). Several mRNAs of comparable size
4623
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CERVICAL
A
IMMORTALIZATION
B
1234567
6.04.2-
CELL
HK
1
234567
i-I -I:
2.31.81.2-
HK
.
-6.0
-4.2
1.
-2.3
-1.8
-1.2
Fig. 5. Expression of HPV16 RNA in HPV-immortalized HCX lines. North
ern blot containing 10 fig/lane of polyadenylate-containing RNA isolated from 7
HPV16-immortalized cell lines (1 to 7; HCX16-1 to HCX16-7) or from (HK);
(UK were used due to difficulty in obtaining large numbers of normal HCX). In
A, the filter was hybridized to the 7.9-kbp BamHl fragment of HPV16. In B, the
same blot was rehybridized to -"P-labeled vector (pdMMTneo) DNA. Numbers
at the margins indicate the size of the HPV 16 mRNAs (kbp).
B
23.19.4-
_
6.7-*
4.4-
2.31.4-1
2.3-
Fig. 6. Detection and expression of HPV18 sequences in an immortalized
HCX line. In A, cellular DNA was digested with EcoRl, which separates vector
from HPVI8 sequences, and was hybridized to the 3!P-labeled EcoRl fragment
of pSHPVISm DNA. The second lane represents a reconstruction experiment
containing 2 copies of HPV 18 DNA per cell. B, Northern blot containing total
cellular RNA which was hybridized to the EcoRl fragment of pSHPVISm.
Numbers at the margins indicate the size of the HPV 18 RNAs (kbp).
hybridized to both HPV 16 and vector probes. These mRNAs
(1.2, 3.0, and 3.4 kbp) are hybrid transcripts containing infor
mation from both HPV 16 and vector sequences.
After transfection with pSHPVISm, results of analysis for
DNA and for RNA were similar to those obtained with
pMHPV16d. The HPV18-immortalized cell line selected with
G418 contained HPV18 DNA (Fig. 6A). After EcoRl digestion
to separate HPV 18 sequences from vector DNA, the expected
7.9-kbp HPV 18 band was observed along with two rearranged
bands. Approximately two copies of HPV 18 DNA were present
per cell. The HPV18-immortalized cell line expressed HPV 18
RNAs ranging in size from 1.4 to 7.5 kbp (Fig. 6B).
Selection of HCX Cell Lines Resistant to Terminal Differen
tiation. HCX cultured in MCDB153-LB medium underwent
terminal differentiation when the culture medium was supple
mented with 5% fetal bovine serum. Two HPV16-immortalized
HCX lines at low passage (55 to 64 PD) were cultured with
MCDB153-LB medium containing 5% fetal bovine serum.
Initially, most cells flattened, and subseuqently many degener
ated and sloughed off the monolayer. After 10 to 14 days,
occasional foci (approximately 8 to 15 per dish) of small, very
slowly dividing cells were noted. These cells continued to pro
liferate in the presence of serum and were subcultured at high
BY HPV
DNA
cell density (1:2 split). After repeated subculturing, these cells
adapted to growth in serum and proliferated at a rate compa
rable to the original parental cell line (PD time of approximately
36 to 40 h).
Keratin Expression. Keratins are a major differentiation prod
uct of epithelial cells, and their expression differs according to
cell lineage. Keratin synthesis in HPV16-immortalized
HCX
was compared to both normal cultured cervical epithelial cells
and three cervical carcinoma cell lines. Keratins were analyzed
by gel electrophoresis and are referred to numerically (38),
while size and charge data are given in Table 2. HCX secondary
cultures established from endo- or exocervical epithelium dif
fered markedly in keratin expression (Fig. 7). Exocervical cells
expressed keratins 5 and 14 at high level, keratins 6 and 16 at
moderate level, and low levels of simple epithelial keratins
(Nos. 7,8,18,19). In contrast, simple keratins were upregulated
in endocervical cells, keratins 6 and 16 increased slightly, and
keratin 5 decreased. The striking difference was that endocer
vical cells lacked keratin 14.
Immortalization of HCX by HPV 16 had little effect on
keratin expression (Fig. 7). The five lines examined, which
originated from 2 different cervical specimens and had different
HPV 16 DNA copy numbers, were similar in keratin expression
to normal exocervical cells. Keratin 19 was upregulated in all
HCX lines, and other simple epithelial keratins (Nos. 7, 8, 18)
showed minor increases. The serum-selected HPV-immortal
ized lines had a significantly altered pattern of keratin expres
sion. Simple epithelial keratins were more extensively upregu
lated in the 2 lines examined, and keratins 7 and 19 were
expressed at high level.
Keratin expression in three different cervical carcinoma cell
lines, C4-1 (39), QG-U (12), and HeLa (40), was compared to
HPV-immortalized HCX. Carcinoma lines resembled serumselected HCX lines in that the simple keratins were upregulated;
however, all carcinoma lines lacked keratin 14 and, in this
respect, differed from all the immortalized HCX. The tumor
lines also differed from one another (Fig. 7), particularly when
the squamous carcinomas (C4-1, QG-U) and adenocarcinoma
(HeLa) were compared. Cultured squamous carcinoma lines
expressed high levels of keratins 8 and 18, moderate amounts
of 5 and 16, and low levels of 6 and 19. Adenocarcinoma cells
expressed mainly keratins 8 and 16 with smaller amounts of 7
and 18. The largest band in the HeLa extract was not keratin
5 or 6 but vimentin (confirmed by two-dimensional electropho
resis).
Analysis by two-dimensional electrophoresis substantiated
differences in keratin expression (Fig. 8) observed in onedimensional separations. Low levels of vimentin were found in
both exo- and endocervical cultures (Fig. 8, A and fi), and the
absence of keratin 14 in the endocervix was apparent. In addi
tion to confirming the upregulation of keratins 7, 8, 18, and
19, the two-dimensional analysis also showed that keratin 13
was upregulated in serum-selected lines (Fig. 8£>).
The changes
in keratin expression relative to other cellular proteins can be
appreciated in total extracts (Fig. 8, E and F). Relative to actin,
serum selection depressed the synthesis of keratins 5,6,14, and
16 and upregulated synthesis of keratins 7, 8, 13, 18, and 19.
Changes in keratin expression after serum selection can be
explained either by the direct effect of serum components on
keratin gene expression or selection of a minor cell clone
adapted to grow in serum which originally had an altered
keratin pattern. To determine the relationship between the
parental and serum-selected populations, the patterns of
HPV 16 DNA integration were compared (Fig. 9). Both parental
4624
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CERVICAL CELL IMMORTALIZATION BY HPV DNA
Table 2 Relative levels of keratin expression by normal cervical cells, HPV-immortalized HCX, and cervical carcinoma lines
Keratin expression data are from one- and two-dimensional gel analyses: +++, major; ++, moderate; +, minor; (+), trace; —,not detected.
keratins8
II6
CellsNo.°M;
PI"5
607.4Type 588.2keratins7
556.0Type 54
6.213
I
545.414
515.416/17* 49
5.118
445.819
405.2VimentinV595.4
Endo
Exo
HCX 16-5
HCX16-5S
C4-1
QG-U
" Catalogue number of keratin (38).
* Keratins 16 and 17 were not separated by the gel system and were estimated together.
c Molecular mass (x 10~3).
'' Isoelectric point determined in 9 M urea by nonequilibrium pH gradient electrophoresis.
HCX
Endo Exo
Transfected
134
HCX Lines
5
5s
Tumor Lines
2s
C
Q
H
Keratins
Type
II
Type
I
19
Fig. 7. SDS gradient gel electrophoresis of [3!SJmethionine-labeled cytoskeletal extracts of HCX. Lanes 1 and 2, extracts of secondary cultures of endo- and
exocervical cells; Lanes 3 to 7, extracts of HPV16-immortalized HCX (cell lines
HCX 16-1, 3, 4, 2, and 5, respectively); Lanes 8 and 9, extracts of serum-selected
sublines derived from HCX 16-5 and HCX 16-2, respectively. Tumor cell lines
shown in Lanes 10 to 12 (C, C4-1; Q, QG-U; H, HeLa). Keratin intermediate
filament proteins migrate at molecular weights between 60,000 (5) and 40,000
(19) and are numbered (type II, 5 to A, and type I, 14 to 19) as described (38).
Note the absence of keratin 14 in endocervical cells and tumor cell lines, and the
increased expression of keratins 7 and 19 after serum selection.
and serum-selected lines contained the same rearranged bands,
proving their relatedness.
Tumorigenicity. HPV-immortalized HCX lines were tested
for tumorigenicity by inoculating 5 to 10 x IO6 cells s.c. or by
injecting 1 x IO6 cells intracranially into nude mice. Three of
the HPV16-immortalized
HCX lines (55 to 80 PD) and two
cell lines selected for growth in serum were tested. Small
nodules appeared at the site of s.c. injection after 4 to 8 days;
however, these nodules subsequently regressed. Animals have
been observed for 4 to 6 mo, and no tumors have been observed.
As a positive control for tumorigenicity, nude mice were given
injections of a human cervical carcinoma cell line (C4-1). Pro
gressively growing tumors developed after 4 to 6 wk, and these
contained numerous koilocytotic cells and cytologically resem
bled HPV-associated carcinomas.
HPV16- and 18-immortalized
HCX lines derived from three
different cervical specimens. Normal HCX are negative for
HPV DNA and senesce as expected for normal cells (41).
Because HPV-transfected
HCX continue to proliferate
and
express HPV mRNAs, HPV gene expression must be associ
ated with immortalization.
Immortalized
HCX continue to
proliferate and currently are at approximately
200 PD, 5- to 6fold greater than the life span of normal HCX.
Normal HCX and HK differed in that cervical cells senesced
more rapidly. Cervical tissue was obtained from adults and
foreskins from neonates; therefore, differences in culture life
span may reflect the donor's age (41) rather than tissue-specific
differences. HPV-immortalized
HCX and HK also differed in
that HK without exception became immortalized without crisis
(terminal differentiation
of most cells), while in HCX lines
derived from one individual, crisis was observed. Crisis can
occur after transformation
of HK by simian virus 40 (42) but it
is not an obligatory event for establishment
of transformed HK
cell lines (42). All HPV-immortalized
HCX lines (4 of 7) that
entered crisis were derived from the cervix of a 34-yr-old patient
reflecting the variability in proliferative potential in cells iso
lated from adult tissues. The occurrence of crisis might depend
upon the characteristics
of the original stem cell which may
have had a variable number of potential doublings.
HPV gene expression
is regulated in a cell type-specific
manner (20-24). The present model is unique in that cultured
cervical cells are used to investigate the interaction
between
HPV DNA and the specific target cells associated with cervical
cancer.
Normal
HCX
cultured
in serum-supplemented
MCDB153-LB
medium uniformly undergo terminal differen
tiation. Immortalized
HCX express HPV mRNA and retain
the ability to terminally differentiate. However, sublines resist
ant to serum-induced
differentiation
were selected by growth in
serum-supplemented
medium. Therefore, immortalization
by
HPV does inhibit terminal differentiation
in the subpopulation
of cells that survive.
The epithelial origin of cultured HCX was verified by exam
ining keratin synthesis (38, 43). Exocervical cultures expressed
keratins typical of squamous epithelia, Nos. 5, 6, 14, 16/17,
with only a trace of keratins characteristic
of simple epithelia,
Nos. 7, 8, 18, 19, while endocervical cells expressed high levels
of simple keratins, and some other "squamous"
keratins, but
keratin 14 was absent. Similar differences between exo- and
endocervical cells, both in culture and in vivo, have been de
A model for studying the interaction between HPV DNA and tected with antisera specific for keratins 18 and 19 (44).
Complete gel analysis of keratins from cultured HCX makes
human exocervical epithelial cells, the presumed progenitor of
a comparison with in vivo expression possible. Normal uterine
cervical carcinoma, has been developed. One characteristic as
endocervical epithelium expresses simple keratins, but during
sociated with HPV transfection, immortalization, is a repro
squamous metaplasia, keratins 5 and 17 are also present (43).
ducible phenomenon as indicated by the series of unique
4625
DISCUSSION
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1988 American Association for Cancer Research.
CERVICAL CELL IMMORTALIZATION BY HPV DNA
NE«-i
Endocervix
Exocervix
SDS
14
8
16
16
18
19
19
Fig. 8. Two-dimensional electrophoresis of
selected methionine-labeled extracts ¡Ito l>.
cytoskeletal (Csk); E and /'. total cellular pro
tein (Tot)] of normal HCX and HPV16-imnidti,ili/ni lines. Proteins were separated in
the first dimension by nonequilibrium pH gra
dient cleclrophoresis (NE: range shown. pH 9
to 4) followed by SDS-polyacrylamide gradient
gel electrophoresis in the second dimension. A
and B, secondary cultures of exo- and endocervical epithelium; C and E, HPV16-immortalized line HCX 16-5; D and F, serum-selected
sublinc HCX16-5S derived from HCX16-5.
Keratins are numbered (38). and vimentin (v)
is indicated. Endogenous actin (Act) was pre
sent in total extracts and was used as a refer
ence to evaluate relative changes in keratin
expression. Filled triangles (A) in A and C
represent complexes of keratins 5 and 14 (60).
Csk
Csk
HCX16-5
HCX16-5S
B
14
14
» 16
16
18
f
19
19
Csk
Csk
HCX16-5
HCX16-5S
13
4
14
T
Act
Tot
2.3Fig. 9. Comparison of HPVI6 integration patterns in parental and serumselected HCX lines. Cellular DNA was digested with /(»mlII. and the Southern
blots were hybridized to the 7.9-kbp BamHl fragment of pMHPVlod. The
streaking in Lane 2 represents an artifact.
Endocervical cultures synthesized keratins 5, 6, and 16/17, in
addition to simple keratins, suggesting that either the original
endocervical cells were isolated from a metaplastic squamous
epithelium or that altered keratin expression was induced by
culture conditions, as found in HK (45). Cultured exocervical
cells and exocervical epithelium in vivo expressed keratins in
8
18*
16
<
19
E
23.1 -
13
V,
t
. 16
Act
19
Tot
common (Nos. 5, 6, 14, 16/17, and 19), but additional keratins
(Nos. 1,4, 13, and 15) were expressed in vivo.
Keratin expression in HPV16-immortalized HCX cell lines
was similar to nontransfected HCX, indicating that HPV 16
had only a minor effect on keratin expression. HPV16-immortalized cells demonstrated downregulation of keratin 6 and
upregulation of keratin 19. The same changes in keratins 6 and
19 occurred in cervical squamous carcinoma cell lines (C4-1
and QG-U), but the overall keratin profiles of the carcinoma
lines were significantly different than the immortalized HCX.
In comparison, transformation of HK by simian virus 40 has a
dramatic effect on keratin expression (46-48). Squamous ker
atins are dramatically reduced so that the overall profile closely
resembles a simple epithelium (47, 48). Explanted normal and
papillomatous tissues from recurrent laryngeal papillomatosis
also exhibit diverse keratin patterns (49). Sublines of HPVimmortalized HCX resistant to serum-induced terminal differ
entiation demonstrated a more dramatic alteration in keratin
expression. Upregulation of keratins 7, 8, 13, 18, and 19 and
downregulation of keratins 5, 6, 14, and 16/17 are consistent
with a switch to a simple type of epithelial differentiation.
Similar changes have been observed in squamous metaplasia of
the uterine cervix (43, 50) and in cervical neoplasia (51, 52).
The overall keratin profile of serum-selected sublines appears
4626
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CERVICAL CELL IMMORTALIZATION
closer to that of cervical squamous carcinoma lines. In terms
of keratin expression, the upregulation of keratins 7 and 13,
that is also characteristic of the two carcinoma lines examined,
suggests that these subpopulations may have progressed further
towards a transformed phenotype.
Integration of HPV DNA into the host genome may be a
prerequisite for immortalization. All HPV-immortalized HCX
lines had an average of 2 to 20 copies of HPV 16 or 18 DNA
per cell integrated within host sequences. Integration occurred
in both HPV 16 and vector sequences, and there was little or no
tandem duplication of the integrated DNA. There were multiple
sites of integration in the host cell DNA. The HCX 16-6 line,
derived from a single colony after G418 selection, contained
multiple rearranged bands indicating that integration may occur
at many sites within a single cell line. In contrast, Durst et al.
(19) observed oligomeric HPV 16 genomes integrated at a single
site within host DNA of HPV16-transfected HK. No relation
ship existed between the copy number of HPV16 DNA per cell
and the corresponding level of HPV16 mRNA expression. All
HPV16-immortalized lines expressed a 1.8-kbp HPV mRNA.
This mRNA species is present in HPV16-immortalized
HK
(18, 19) and tumorigenic NIH 3T3 cells (29). In the latter the
1.8-kbp species includes the E6 and E7 gene transcripts. Expres
sion of E6/E7 mRNA is usually observed in cervical carcinoma
cell lines containing HPV 16 or 18 DNA (10, 12, 53), implying
the importance of these gene products in carcinogenesis.
Circumstantial evidence suggests that integration of HPV
genomes into cellular DNA is also important in the neoplastic
conversion of premalignant cervical cells (54). HPV 16 or 18
DNA is usually integrated into cellular DNA in cervical tumor
cells but present in episomal form in dysplastic cervical epithe
lium (54). Transfection studies show that HPV16-immortalized
HCX and HK contained integrated HPV 16 DNA (18, 19), but
were not tumorigenic in nude mice. These results are consistent
with epidemiological and clinical studies (reviewed in Refs. 3
to 5), suggesting that HPV alone does not malignantly trans
form HCX because HPV 16 DNA can integrate in mild cervical
intraepithelial neoplasia (55) and in Bowenoid papulosis (56).
Rodent cell lines transfected with HPV16 DNA undergo pro
gressive changes that result in neoplastic transformation and
tumors in nude mice (16, 29, 57). The observed differences
between human and rodent systems probably reflect the relative
genetic stability of human cells (58) and emphasize the impor
tance of using human cells when investigating mechanisms of
cocarcinogenesis (58, 59). HCX were isolated from individuals
ranging in age from 34 to 66 yr. Cell lines derived from the two
older patients did not undergo neoplastic conversion, even
though these cells presumably had been exposed to other cofactors over a number of years.
Immortalization of HCX by HPV DNA may be an important
factor in the sequential development of carcinomas. HPV 16
DNA integrates into the genome of HCX and stimulates con
stitutive cellular proliferation. Because papillomatous lesions
are composed of continuously replicating cells, they might be
particularly susceptible to additional carcinogenic factors. In
addition, the enhanced life span of HPV-immortalized HCX
would be expected to result in a greater cumulative exposure to
carcinogens and increase the likelihood of further neoplastic
progression. It is now possible to study molecular aspects of
HPV genes and the etiological agents and cofactors required
for uncontrolled growth and malignancy using cervical epithelia
that are ordinarily at risk in vivo.
BY HPV DNA
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4628
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Characterization of Normal Human Exocervical Epithelial Cells
Immortalized in Vitro by Papillomavirus Types 16 and 18 DNA
C. D. Woodworth, P. E. Bowden, J. Doniger, et al.
Cancer Res 1988;48:4620-4628.
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