(CANCERRESEARCH46, 2665-2669, June 19861
Quantitative Differences in Host Cell Reactivation of Ultraviolet-damaged Virus in
Human Skin Fibroblasts and Epidermal Keratinocytes Cultured from the Same
Foreskin Biopsy'
RexM. Tyrrell andMireille Pidoux
Swiss Institutefor Cancer Research, 1066 Epalinges/Lausanne, Switzerland
ABSTRACT
Repair efficiency of cultured cells may be estimated by measuring the
ability ofa particular cell type to support virus damaged by an appropriate
agent. In this study we have compared the inactivation of ultraviolet (254
nm)-damaged herpes simplex virus in human fibroblast and epidermal
keratinocyte cell lines derived from the same foreskin biopsy and found
the epithelial cells to be a factor of 3 times less efficient in supporting
the damaged virus. The two different cell types show comparable ultra
violet inactivation of clone-forming ability, indicating that the difference
is specific to viral host cell reactivation. This study required the devel
opment of a quantitative infectious centers assay for the measurement of
viral titer in human epithelial cells, a system which may be of more
general application
in studies of potential
human carcinogens.
epidermal cell as a target for solar UV damage, we have
developed a quantitative assayfor measurementof productively
HSV-infected human epidermal keratinocyte lines. In an initial
study, this technique has enabled us to compare various aspects
of the processing of UV-damaged HSV in primary fibroblasts
and epidermal keratinocytes cultured from the skin of the same
donor.
MATERIALS
AND METHODS
Cell Strains and Culture
Human fibroblast strains, 48BR, GMO73OA, and IBR/3 (from the
MRC Cell Mutation Unit, Brighton, England), were obtained from
skin biopsy of normal adult individuals
INTRODUCTION
Since most human cancers are of epithelial origin, cultured
epithelial cells are the logical target material for basic studies
in carcinogenesis. However, although squamous and basal cell
carcinomas account for the majority of all solar UV-induced
skin cancers, most in vitro studies concerning the induction and
processing of UV damage to DNA have, for practical reasons,
usedcells offibroblastic origin. Human epidermal keratinocytes
can be grown efficiently through several passagesfrom a pri
mary explant using either lethally irradiated mouse fibroblasts
(1) or techniques
utilizing
collagen
(e.g., Ref. 2). Using such
quantitative
studies of viral replication
Received 9/9/85; revised 12/1 1/85; accepted 2/11/86.
The costs of 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.
investigation
was
supported
by
grants
from
the
Swiss
National
Science
abbreviations
used
are:
HSV,
herpes
simplex
virus
MEM,
sodium bicarbonate,
glutamine,
and 15% fetal
developed
by Dr. P. H. Gallimore
(Cancer
Research
Campaign
Labo
ratories, Birmingham, England) by transfection of human fetal kerati
nocytes
with the DNA of SV61,
a mutant of SV4O which lacks the
origin of DNA replication (12). They were grown in the same medium
as the primary
fibroblasts,
except
that only 10% fetal calf serum
was
added, and the medium was supplemented with 4 @g
of hydrocortisone
of hydrocortisone
(10 times that used for
the primary keratinocytes, see below) was found to be optimal for this
transformed line. To maintain healthy rapid growth without feeder,
cells were seeded at high density (5 x 10―/cm2plastic) and split twice
perweekata
1:2 ratio.
Swiss 3T3 fibroblasts (Flow) were cultured in Dulbecco's MEM
(Flow) with 10% fetal calf serum, and African green monkey kidney
cells (CV-ls) were cultivated in Earle's MEM supplemented with 1%
glucose and 5% fetal calfserum.
intervals.
Both cell types were passaged at weekly
Preparation
and Cell Lines
of Primary
Primary epidermal
Cultures
cultures were prepared by a slight modification
ofthe technique developed by Rheinwald and Green (13, 14). Foreskins
were collected in PBS containing antibiotics and cut into 2-mm2 pieces
after removal of the s.c. tissue. For further processing, the fragments
were placed on filter paper, epidermal side outward, and incubated in
0.25% trypsinat 4C overnight.After removingthe dermis,the epider
mis was dissociated
in 0.25% trypsin-0.02%
EDTA for 30 mm at room
temperature, the cells were thoroughly disaggregated by pipeting, and
keratinocytes
and 10@lethally
irradiated
(3.5 kilo
rads) 3T3 feeder cells were plated together in a 9-cm Petri dish in
MEM (calcium free) containing 10% fetal calf serum and hydrocorti
sone (0.4 @ig/ml).The calcium concentration of the medium was main
tamed at 0.3 mM (to further slow down terminal differentiation) by
screening for fetal calf serum containing 3 mrs calcium. Cholera toxin
(100 ng/m1 Sigma) and epidermal growth factor (10 ng/ml; Collabo
rative Research, Inc.) were added 3 days later. The dermis was finely
chopped, allowed to attach to Petri dishes containing a small quantity
ofEarle's MEM, and then covered with medium containing 15% fetal
Foundation (3.397.083 and 3.108.085) and the Swiss Cancer League.
2 The
streptomycin,
5 x i0@ epidermal
in cultured epidermal
keratinocytes have only recently been undertaken (1 1). As ex
pected pretreatment of HSV with UV reduced the production
of virus in infected cells. In view of the importance of the
I This
penicillin,
calf serum. Cells were passaged at weekly intervals by trypsinization.
The transformed epidermal keratinocyte line (SV61 BaIn/HFK) was
per ml. This concentration
culture techniques, it has beenshown that repair ofUV damage,
as measured either by removal of pyrimidine dimers (3) or by
repair replication measurements (4, 5), occurs at similar rates
in skin fibroblasts and keratinocytes. However, the ability of
cells to survive UV-induced damage has not beensystematically
compared in the two cell types, presumably becauseof the low
cloning efficiency of epidermal keratinocytes.
Measurements of the ability of damaged virus to survive in
host cells have contributed considerably to our understanding
of the induction and processing of lethal damage in cells (6).
Both fibroblasts(7) and transformedlymphocytes(8) cultured
from patients with a variety of genetic diseases, known or
suspectedto be associated with repair differences, are less able
to support the reproduction of UV-damaged virus. Viral sys
tems are particularly useful for studying the relative roles of
constitutive and induced repair processes,since host cell and
virus can be irradiated separately (9). Although human epider
mal cells can be infected with HSV2 in vitro (e.g., Ref. 10),
aged 46 (female), 45 (female),
and 33 (male), respectively. EK13 and EK4 fibroblasts were derived
from foreskin explants in this laboratory(see below). Fibroblast cultures
were grown in Earle's modified MEM (Gibco) supplemented with
calf serum to allow outgrowth
minimal
essential medium; PBS, phosphate-buffered saline; MOI, multiplicity of infection.
of primary
fibroblasts.
The primary
epidermal cultures were allowed approximately 3 wk to become con
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HOST CELL REACTIVATIONOF UV-DAMAGEDVIRUS
fluent. At weekly intervals the feeder was removed by vigorous pipeting
ing 2 x 10@freshly trypsinized
following a 30-mm incubation with 0.2% EDTA at room temperature
and replaced with new feeder. The keratinocyte populations were pan
infection. To avoid infection of3T3 fibroblasts with herpes virus, feeder
cells were removed from the epidermal cell culture dishes 24 h prior to
the experiment. At the start of the experiment, 2 or 3 plates were
saged by removing
the feeder with EDTA and then treating
the adhering
keratinocytes for 30 mm at 37'C with 0.05% trypsin and 0.01 % EDTA.
The disaggregated cell suspension was plated out as before, except that
all medium components were present from the start. In general a large
seed stock was frozen down at the end of the second passage.To
prepare an experiment, several (approximately 10) 6-cm plates were
seeded with 10@epidermal keratinocytes (passage 3 or 4) and 5 x 1O@
feeder cells and incubated for 2 wk with 2 medium changes and 1 feeder
change per wk. At this time, the dish surface was 70 to 80% covered
with large stratified epidermal keratinocyte colonies (13, 14).
Virus Strains and Growth
The macroplaque strain of herpes simplex virus type I was kindly
supplied
by Dr. D. Lytle (Department
of Health
and Human
Services,
Rockville, MD). A large stock was prepared by infection of several
confluent plates of CV-l cells and frozen in many aliquots.
UV Radiation and Dosimetry
trypsinized and counted to facilitate an accurate MOl. Appropriately
treated virus at an MO! of 0.1 was then infected into the monolayers,
and incubation continued for 1 h at 37'C. The monolayers were then
treated with ‘y-globulin
for 20 mm and then trypsinized for determina
tion of cell number. The microtiter
with
experiment but was in the range of 20 to 50% with a mean of 32% (14
experiments).
Measurement of Clone-forming Ability
was monitored by an IL 700 International Light radiometer in combi
nation with a calibrated
probe incorporating
a 254-nm interference
times. Viral populations
were irradiated
Clone-forming
ability of fibroblast
cell lines was measured
as previ
ously described by plating appropriate cell numbers with rny-irradiated
filter. The rate was adjusted according to the experiment to give
irradiation
plates were then inoculated
HSV-infected epidermal cell suspension appropriately diluted in me
dium (without -y-globulin) to give approximately 1 infected cell per
well. Plates were then incubated at 3'7T for 5 days. Uninfected wells
were scored at x 10 magnification with no staining. The efficiency of
infection was calculated from the relationship, plating efficiency = —
ln Po/n, where Po is the fraction of uninfected wells, and n is the
number of cells inoculated per well. For the transformed epidermal
cells, the efficiency of infection with HSV was close to 100%. For the
primary lines, the efficiency was more variable from experiment to
The fluence rate of UV (254 nm) radiation from a germicidal lamp
appropriate
CV-1 cells 2 days prior to the epidermal
in PBS
feeder layers from the same culture (15). The cloning efficiencies were
in the range of 33 to 40% for the foreskin-derived fibroblasts and 7 to
25% for the adult-derived cells. Epidermal keratinocytes were cloned
by seeding appropriate cell numbers into 6-cm dishes under the same
feeder conditions as for preparation of stock cultures. However, the
and the irradiation was carried out with the cells maintained at 1—'VC. MEM was changed (16) for a mixture of 1 part ofDulbecco's modified
To irradiate in suspension,cells were trypsinised and resuspendedat a Eagle's medium and 3 parts of Ham's F-12 nutrient mixture (both
with constant agitation at a concentration of 1O@'/ml
at ice temperature
in a dish covered with a quartz plate. To irradiate cell monolayers,
medium was removed from the dishes, the cells were washed with PBS,
concentration of l0@/ml in PBS and maintained at l-4C.
No control
inactivation occurred during the irradiation periods used. Monochro
matic radiation at 405 nm for photoreactivation
was obtained and
measured as previously described (15).
Viral Assays
Direct Assay. Freshly confluent
fibroblasts in 9-cm dishes were used
for plaque assays as previously described (15). Cells were infected
directly with appropriate dilutions of HSV (approximately 100/plate)
for 90 mm at 37'C. Plates were incubated for 3 days with medium
containing 0.25% -y-globulin. Direct assay on the freshly confluent (2
to 3 daysafter seeding)transformed keratinocyte line wassimilar except
that only I h of incubation with virus was used before removing
unabsorbed virus with @y-giobulin.Incubation for longer periods led to
a very gradual, microscopically observable, degradation of the cell
culture.
Infectious Centers Assay with Primary Infection in Fibroblasts.
Freshly confluent fibroblast plates were infected for 90 mm with HSV
as for the direct
assay except
that the MOl
was equal
to 0.2. The
monolayer was then exposed to PBS containing 0.25% ‘y-globulin
for
20 mm. This was removed, and the cells were trypsinized, counted
electronically (All 34; Analytical Instruments, Stockholm, Sweden),
and distributed onto plates containing freshly confluent CV-ls in
medium containing 0.25% y-globulin. The infected monolayers were
incubated 65 h before staining for plaques. The efficiency
of infection
was close to 100% (based on plaque-forming units, determined by direct
assay).
Infectious Centers Assay with Primary Infection inEpidermal Cells.
Direct assay on epidermal cell lines is. not practical because of the
limited number of dishes that can be brought to confluence. An infec
tious centers assay protocol such as that described above does not work
because the epidermal cells do not efficiently attach to the CV-l
containing calcium), the serum level was dropped to 5% and the
medium was additionally supplemented with adenine (25 @g/ml)and
insulin (5 sg/ml). Plates were stained for colony formation after 2 wk
with no feeder changes and a single medium change. Under these
conditions we reproducibly obtained cloning efficiencies of 3 to 5% in
passages 4 to 6.
RESULTS
LW Inactivation
ofVirus
Direct Assay on Fibroblasts. The fluence-dependent macti
vation of UVC-damaged HSV-1 asassayedby direct plating in
monolayers of several independently derived fibroblast lines is
shown in Fig. 1. As observed previously in several lines derived
from both human and nonhuman materials, the curves can be
resolved into two components (9), with a faster rate 4 times
[0.049 (J/m2)'J that of the slowerrate [0.012 (J/m2)@1.There
is a remarkable similarity between viral inactivation kinetics in
the 5 independent strains. Of special relevance to this study is
that UV inactivation in the 2 foreskin-derived lines (EK4 and
EK13) is indistinguishable from that observed in 3 strains
derivedfrom punchbiopsiesof adult human skin.
Indirect Assay on Epidermal Keratinocytes and Fibroblasts.
Since direct assay of virus on keratinocytes has not proved
possible, we have compared the survival of UV-damaged virus
in fibroblasts and keratinocytes by an indirect infectious centers
assay (see “Materials
and Methods―).Biphasic curves are not
obtained by this procedure (Fig. 2), and the virus appears to
survive UV damage a factor of 2.5 times better than direct
necessity of contact of the infected cells with the monolayer, we used a
plating on fibroblasts monolayers, as assessedby comparing
the slope of the appropriate curve in Fig. 2 [0.020 (J/m2)'I
with that for the faster component of inactivation in Fig. 1.
However, the most striking factor to emergefrom theseexper
microtiter plate assay. The flat-bottomed wells of a series of 96-well
iments is that UV-damaged
microtiter
2.8 times faster when assayed on 2 independently
monolayers.
Also unsuccessful
were attempts
to absorb
the infected
epidermal cells to the CV-l monolayer using a low volume of liquid
and then fixing the cells with a soft agarose gel (8). To avoid the
plates were each inoculated with 100
@l
of medium contain
virus is inactivated by a factor of
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derived
HOST CELL REACTIVATIONOF UV-DAMAGEDVIRUS
100
0
z
C,
z
0
z
0
at
@
I-
U
(4)
I
10
z
0
I-
.
C.)
0
4
at
U.
_2_____________________
Fig.
3.
UV(254
nm)radiation
inactivatio
ofHSV-1
in
transfo
epid
0
100
FLUENCE
10
0
i
20
FLUENCE
I
40
I
60
UV (254nm)
I
80
U
100
200
UV (254nm)
300
Jm-2
keratinocytes (SV61/Bam) as assayedby infectious centers assay(0) or direct
plating(X).ThemeancurveforinactivationofHSV-1
inprimaryepidermal
keratinocytes(Fig. 2) is shownfor comparison (—).
Jm2
Fig. 1. UV (254 nm) radiation-induced inactivation of HSV-l in primary
human fibroblasts as assayed by direct plating. •,48BRI@
0, GMO73OA;L@,IBR/
3; A, EKI3; 0, EK4. Each curve is the result of a single experiment. Fluence in
this and later figures is expressed as i/rn2 (Jnf!).
virus particles were infected into monolayers ofthe transformed
line prior to infectious centers assay. Under these conditions,
the viral populationsare at least 5 times as resistantto UV
inactivation [slope = 0.011 (J/m2)'J compared with infection
using a nontransformedhost. As observedin Fig. 2 for the
nontransformedcell lines, an exponential curve is obtained
when the infectiousassayis usedrather than the direct assay,
I 0@
and the direct assaygivesa biphasiccurve,the slopeof the fast
and slow componentsbeing 0.028 and 0.0095 ((J/m@], re
0
spectively.
>
Uv Inactivationof aone-fo@nin@
Ability of EpidermalKerati
nocytes and Fibroblasts
U)
z
10
In view of the markeddifferenceof the 2 host cell typesto
0
support UV-damaged virus (Fig. 2), we compared directly the
IU
UV inactivation of clone-forming ability of the keratinocytes
and fibroblasts derived from the same foreskin biopsy. In
contrast to that seen for inactivation of viral populations,
the fluencedependenceof inactivationis similar for the 2 cell
types (Fig. 4), the keratinocytes being marginally more resistant
than the fibroblaststo UV treatment.The cloningefficiencyof
the EK13 keratinocyteline wastoo low to perform this type of
experiment.However, it shouldbe noted(seeFig. 4) that the 2
foreskin-derived fibroblast lines are 2 to 3 times as resistant to
far UV (254 nm) as the adult-derived lines used in this labora
U.
1 o2
tory (although this was not reflected in a modified response of
EK4(+) andEK13(x) fibroblasts.
UV-treated virus; Fig. 1). The resistance was not related to
passage number, and a similar resistance was observed in 2
independentlyderivedforeskinfibroblastlines,EK12 andEKI6
(results not shown). There is no general indication from the
literature that foreskin-derived lines are more resistant to UV
keratinocyte lines (slope = 0.056 (J/m2)@1as compared to a
similar assayusing the corresponding fibroblast lines.
Photoreactivation
FLUENCE
UV
(254nm)Jm2
Fig. 2. UV (254 nm) radiation-induced inactivation of HSV-1 in primary
human fibroblastsand epidermal keratinocytesas assayedby infectiouscenters.
Curves are shown for EK4 (O, 0) and EKI3 (Lx,A) epidermal keratinocytes and
In the initial stagesof theseexperiments,we useda trans
formedepidermalline derivedfrom humanembryonicmaterial
treatment, and it would be interesting to examine additional
linesderivedfrom adult skin biopsymaterial.
and IN Reactivation of UV-damaged Virus
Infected into Epidermal Keratinocytes
in order to save valuable primary material. However, as illus
Several attempts were made to enhance the survival of the
trated in Fig. 3, the transformed line serves as a poor quanti
tative model for the nontransformed cell lines. UV-damaged
UV-damaged virus by pretreatment of the host cells with a
rangeof fluencesof radiation at 254 nm and incubatingthem
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HOST CELL REACTIVATIONOF UV-DAMAGEDVIRUS
close to identical in the 2 cell types (Fig. 3). Furthermore, UV
inactivation of host cell capacity to support unirradiated virus
is close to identical in the 2 cell types when correction is made
for reduced UV transmission through the epidermal cell sheets
(result not shown). These findings suggestthat overall repair of
cellular DNA damage is similar in the 2 cell types and agrees
I 0@
0
z
with findings from comparative
biochemical
repair measure
ments (3, 4). Why repair of biologically effective viral damage
in keratinocytes is less efficient than in fibroblasts remains to
U)
io@
be clarified. One possibilityis that the accessibilityof repair
z
enzymes
to viral DNA is different between the 2 cell types, so
0
that damagemay remain unrepaired long enough for additional
IU
lethal events to arise in virus-infected keratinocytes. Alterna
lively,
the differences may reflect cell type variability in enzymes
U.
(or other factors) limiting for repair of damaged viral DNA.
Severalother findingshaveemergedfrom theseexperiments
which merit further study. (a) The transformed epidermal line
permits recovery of UV-damaged virus a factor of5 times better
1 o2
than the 2 non-transformed lines (Fig. 3). Although capacity to
support damaged virus is known to vary between host cells
0
10
20
30
40
50
from the same species(18), and virus transformation of human
cells
is associated with a loss of ability to repair adenovirus
FLUENCE
UV(254nm)
Jm-2
damaged by alkylating agents (19), we are unaware of any
Fig.4. UV(254 nm)radiation-induced
inactivationofclone-formingefficiency
reportedassociationbetweencell transformationand ability to
of primary human fibroblasts and epidermal keratinocytes. Curves are shown for
EK4keratinocytes(x, is), EK4fibroblasts(0, +), EKI3 fibroblasts(0), andthe support UV-damaged virus. (b) A second observation is that
adult-derived fibroblasts GMO73OA (V), 48BR (s), and IBR/3 (A) irradiated in
assayof damaged virus by an infectious centers technique leads
suspension. In one experiment adult fibroblasts were irradiated on plates
to exponential curves (Figs. 2 and 3) rather than the biphasic
(IBR/3,Y).
curves characteristic
for periods from 16 h to 2 days. In no casewas UV reactivation
observed (results not shown). However, it should be noted that
a direct plating assaycouldnot be usedand that, underparallel
conditions using an infectious centers assay for the primary
fibroblasts, we also failed to seeUV reactivation. Using a direct
assay we obtained small reactivation factors comparable to
those seenby other authors (e.g., Ref. 17). We were also unable
to observe photoreactivation of UV-damaged virus in primary
epidermal keratinocytes. However, for practical reasons, we
limited the photoreactivation period to a maximum of 15 mm
at 20°C(405 nm, 10 W/m2) for these experiments. In similar
preliminaryexperimentswith the transformedkeratinocyteline
where direct plating can be used, we reproducibly observed
small increases in survival of UV-damaged virus as a result of
photoreactivation. Maximum increaseswere always seen after
10-mm photoreactivation. In 2 independent experiments using
virus damaged by 300 J of radiation per m2 at 254 nm (2 log
cycles of inactivation), we observedsurvival increasesof 35 and
41 %, respectively,
after 10-mm treatment of the virus-infected
(2 h) epidermal sheetwith radiation at 405 nm (data not shown).
DISCUSSION
The ability of damaged virus to survive and replicate is
generally believed to reflect the ability of the host cell to repair
the damage.Thus the large differencein survivalof UV-dam
agedvirus between a repair-proficient and an excision-deficient
host is assumed to reflect the capacity of the repair-proficient
cells for host cell reactivation. The major new finding reported
in this study is that lines of human epidermal keratinocytes and
fibroblasts derived from the same skin biopsy show marked
differences in their capacity to support UV-damaged viruses.
According to the data in Fig. 2, the reactivation capacity of
fibroblasts is approximately 3 times as great as that of the
epidermal keratinocytes. However, the resistanceof clone-form
ing ability of the 2 cell types to inactivation by UV (254 nm) is
of the direct assay for viral plaques (Figs.
1 and 3). Since the slopeof the infectiouscenterscurvecorre
sponds more closely to the slower component of the biphasic
curves from the direct assay(e.g., see Fig. 3), this may help in
identifying the factor responsible for the biphasic nature of the
curves. (c) Finally, we have observedthat the 2 foreskin-derived
fibroblast lines, whose UV sensitivity is shown in Fig. 4, and 2
additional lines (results not shown) developed in this laboratory
are markedly more resistant than the 3 lines developed else
where by punch biopsy of human skin. Again, there is no clear
indication from the literature of such a difference, and we are
currently developing additional cell lines from adult biopsy
material in order to understand this observation.
From the practicalviewpoint,we presenta new quantitative
assaywhichallowsthe useof humanepidermalcellsasthe host
cell for infection and initial processingof damagedvirus. In
this initial work, we have used far UV (254 nm) radiation as
the damagingagent. However, this systemis applicableto the
testing of other DNA damaging agents where an epithelial (or
specifically epidermal) cell is required as the host cell. Also,
since host and virus can be treated separately, the effect of
specific damage to the host cell on viral survival may also be
examined. For example, treating monkey cells with various
carcinogens increasesthe survival of UV-irradiated herpes sim
plex virus (20), which led to the suggestionthat sucha system
may provide a potential rapid screening system for testing the
carcinogenic potential of chemical compounds in mammalian
cells. The system presented here would be particularly attractive
for such studies, since human epidermal keratinocytes have
been shown to be much more effective than fibroblasts in
metabolizing carcinogens and to retain the activity through
many population doublings(13). However, sinceUV reactiva
tion was not observed using the infectious centers assay (see
“Results―),
further experiments are required to examine the
feasibility of using such an approach. It is possible that the
enhanced resistance of UV-damaged virus as measured via the
infectious centers assay (see Figs. 1 to 3) is related to the
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HOST CELL REACTIVATION OF UV-DAMAGED VIRUS
trypsinization process which may temporarily inhibit the lytic
infection at an early stage and allow more time for repair. This
enhanced repair could then mask out the relatively small UV
reactivation effects.
In summary we have devised an assay for quantitating survival
of damaged virus in human epidermal keratinocytes and dem
onstrated that the epithelial cells are defective(as compared
with fibroblasts) in their ability to support UV-damaged virus.
The system has wide potential for studying the processing of
damage in a human epithelial cell culture system.
ACKNOWLEDGMENTS
The authors wish to acknowledge the technical assistance of Patricia
Werfelli
in the early stages of this work and Sue Harcourt
and Dr.
Cohn Arlett (Medical Research Council Mutation Unit, Sussex,En
gland) for their gift of the adult primary fibroblast lines. We are also
grateful to Dr. Walser and Dr. Frenck of the Dermatology Department
of the Centre Hospitalier
coordinating
the collection
Universitaire Vaudois for their efforts in
and initial handling ofthe foreskin biopsies.
Dr. Ken Parkinson (Paterson Laboratories, Manchester, England) pro
vided us with valuable advice on culturing
human keratinocytes
and
donated the keratinocyte line developed by Dr. P. H. Gallimore. Dr. C.
D. Lytle (Rockville, MD) kindly provided us with the HSV-l macro
plaque strain.
nocytes.i. Invest. DermatoL,81: 179s—183s,
1983.
6. Sarasin, A. The use of DNA
viruses as probes for studying DNA
repair
pathways in eucaryotic cells. 1n.@
S. Okada, M. Imamura, 1. Terashima, and
H. Yamaguchi(eds.), Proceedingsof the Sixth InternationalCongressof
Radiation Research, pp. 462-470. Tokyo, Japan: Toppan PrintingCo., 1979.
7. Aaronson, S. A., and Lytle, C. D. Decreased host cell reactivation of irradi
ated SV4O in Xeroderma pigmentosuin. Nature (Lond.)228: 359—361,1970.
8. Henderson, E. E., and Long, W. K. Host cell reactivation of UV- and X-ray
damaged herpes simplex virus by Epstein Barr virus (EBV-) transformed
lymphoblastoid cell lines. Virology, 115: 237—248,1981.
9. Bockstahler,L E.Inductionandenhancedreactivationof mammalianviruses
by light. Prog. Nucleic Acid Res. MoL Biol., 26: 303—313,1981.
10. Kitano,Y., and Hats, S. The humanepidermalcells infectedwith herpes
simplexvirusin in vM,.Arch.Dermatol.Forsch.,245:203—210,
1972.
11. Schnipper, L E., Levin, M., Crumpacker, C. S., and Gilchrest, B. A. Virus
replication and induction of interferon in human epidermal keratinocytes
following infection with herpes simplex virus. J. Invest. Dermatol., 82: 94—
96, 1984.
12. Parkinson, E. K., Grabhani, P., and Emmerson, A. A subpopulation of
cultured human keratinocytes which is resistant to the induction of terminal
differentiation-relatedchanges by phorbol, 12-myristate, 13-acetate: evidence
for an increase in the resistant population during transformation. Carcino
genesis (Lond.), 4: 857—861,1985.
13. Parkinson, E. K., and Newbold, R. F. Benzo(a)pyrene metabolism and DNA
adduct formation in seriallycultivatedstrains of human epidermal keratino
cytes. Int. J. Cancer, 26: 289—299,1980.
14. Rheinwald, J. G., and Green, H. Epidermal growth factor and the multipli
cation ofcultured human epidermal keratinocytes. Nature (Lond.), 265: 421—
424, 1977.
15. Tyrrell, R. M., and Amaudruz, F. Alpha polymerase involvement in excision
repair ofdamage inducedbysolar radiation at definedwavelengthsin human
fibroblasts. Photochem. Photobiol., 40: 449-451, 1984.
16. Allen-Hoffmann,B. L, and Rheinwald,J. G. Polycyclicaromatichydrocar
hon mutagenesisof human epidermal keratinocytesin culture. Proc. Nail.
Aced.Ad. USA, 81: 7802-7806, 1984.
17. Lytle, C. D., Day, R. S., III, Hellman, K. B., and Bockstahler, L E. Infection
of UV-irradiated xeroderma pigmentosum fibroblasts by herpes simplex
REFERENCES
virus: study of capacity and Weiglereactivation.Mutat. Res., 36: 257—264,
I . Rheinwald, J. G., and Green, H. Serial cultivation of strains of human
epidermal keratinocytes: the formation of keratinizing colonies from single
cells. Cell, 6: 331—344,1975.
2. Liu, S., and Karasek, M. Isolation and growth of human epidermal kerati
nocytes in cell culture. J. Invest. Dennatol., 71: 157—162,1978.
3. Taichman, L. B., and Setlow, R. B. Repair of ultraviolet light damage to the
DNA of cultured human epidermal keratinocytes. J. Invest. Dermatol., 73:
217—219,
1979.
4. Hannawalt,
P. C.,
Liu,
S., and
Parsons,
C. S. DNA
repair
responses
in
human skin cells. J. Invest Dermatol., 77: 86—90,1981.
5. Liu, S., Parsons, S., and Hannawalt, P. C. DNA repair in cultured kerati
1976.
18. Lytle, C. D. Host cell reactivation in mammalian cells. I. Survival of ultra
violet-irradiated herpes virus in different cell lines. mt. 3. Radiat. BioL, 19:
329—357,1971.
19. Yarosh, D. B., Rice, M., Ziolkowzki, C. H. J., and Day, R. S. O'-methyl
guanine-DNA methyltransferase in human tumor cells. In. E. C. Friedburg
and B. A. Bridges (eds.), Cellular Responses Do DNA Damage, pp. 261270. New York Alan R. Lisa, Inc., 1983.
20. Lytle, C. D., Coppey, 1., and Taylor, W. D. Enhanced survival of ultraviolet
irradiated herpes simplex virus in carcinogen-pretreated cells. Nature
(Lond.),272: 60—62,
1978.
2669
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1986 American Association for Cancer Research.
Quantitative Differences in Host Cell Reactivation of
Ultraviolet-damaged Virus in Human Skin Fibroblasts and
Epidermal Keratinocytes Cultured from the Same Foreskin
Biopsy
Rex M. Tyrrell and Mireille Pidoux
Cancer Res 1986;46:2665-2669.
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1986 American Association for Cancer Research.