[CANCER RESEARCH 52. 4254-4260, August I, 1992]
Squamous Metaplasia of Normal and Carcinoma in Situ of HPV 16-Immortalized
Human Endocervical Cells1
Qi Sun, Kouichiro Tsutsumi, M. Brian Kelleher, Alan Pater, and Mary M. Pater2
Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, Canada A1B ÃŒV6
ABSTRACT
The importance of cervical squamous metaplasia and human papillomavirus 16 (HPV 16) infection for cervical carcinoma has been well
established. Nearly 87% of the intraepithelial neoplasia of the cervix
occur in the transformation zone, which is composed of squamous metaplastic cells with unclear origin. HPV DNA, mostly HPV 16, has been
found in 90% of cervical carcinomas, but only limited experimental data
are available to discern the role of HPV 16 in this tissue specific oncogenesis. We have initiated in vivo studies of cultured endocervical cells
as an experimental model system for development of cervical neoplasia.
Using a modified in vivo implantation system, cultured normal endocer
vical epithelial cells formed epithelium resembling squamous metapla
sia, whereas those immortalized by HPV 16 developed into lesions
resembling carcinoma in situ. In contrast, their ectocervical counter
parts formed well differentiated stratified squamous epithelium and a
lesion with mild dysplastic change, respectively. The HPV 16-immortalized cells showed in vivo cytokeratin expression patterns similar to
their respective normal counterparts, confirming their different origins.
Thus, this study provides direct experimental evidence for the transfor
mation of simple epithelial cells of endocervical origin into stratified
squamous metaplasia and indicates the differential susceptibility of endo- and ectocervical epithelial cells for conversion to cancer by HPV 16.
INTRODUCTION
Squamous cell carcinoma of the uterine cervix is one of the
most common neoplasms in the world (1). The normal cervix is
comprised of 2 distinct regions, the ectocervix and endocervix
(2). The former is lined with stratified, nonkeratinizing squa
mous epithelium (native squamous epithelium) and the latter
with simple columnar epithelium. The junction where the 2
regions adjoin, the squamo-columnar junction, undergoes dy
namic modification. Under certain physiological or pathologi
cal conditions, the simple epithelium at the squamo-columnar
junction is replaced by stratified squamous epithelium (or metaplastic squamous epithelium), giving rise to a region referred to
as the transformation zone (2). The transformation zone is the
most common site for development of cervical neoplasia (3).
The origin of cervical squamous metaplastic cells has been
hypothesized to be either endocervical epithelial cells (4) or
subepithelial stromal cells (5).
It has been well established that certain types of HPVs3 are
involved in the oncogenesis
of cervical carcinomas.
HPV
Received 2/21/92; accepted 5/12/92.
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 accord
ance with 18 U.S.C. Section 1734 solely to indicate this fact.
1The investigation was supported in part by grants awarded by the National
Cancer Institute of Canada (with funds from the Canadian Cancer Society) and the
Medical Research Council of Canada to A. P. and M. M. P.
2 To whom requests for reprints should be addressed, at Memorial University of
Newfoundland, Faculty of Medicine, Division of Basic Medical Sciences, St.
John's, Newfoundland, Canada AI B 3V6.
3 The abbreviations used are: HPV, human papillomavirus; CK, cytokeratin;
CIN, cervical intraepithelial neoplasia; HEN, cultured human endocervical epithe
lial cells; HEC, cultured human ectocervical epithelial cells; HEN-16, cell line from
cultured human endocervical epithelial cells immortalized by HPV 16; HEC-16,
cell line from cultured human ectocervical epithelial cells immortalized by HPV 16;
KGM, keratinocyte growth medium.
genomic DNA, most frequently of HPV 16, has been detected
in 90% of the cervical carcinomas and are found to be actively
expressed (6, 7). HPV 16 DNA has been used to transform
human foreskin and ectocervical keratinocytes (8, 9). It immor
talizes human keratinocytes efficiently, producing cell clones
with indefinite life span in culture. Different approaches have
been taken to examine the behavior of these immortalized cell
lines in conditions allowing squamous differentiation (10, 11).
After transplantation in vivo, the HPV 16-immortalized kerat
inocytes retain thépotential for squamous differentiation,
forming abnormal epithelium without dysplastic changes at
early passages and with various dysplastic changes only after
long periods of time in culture (10).
Considering that most cases of cervical neoplasia are located
within the transformation zone and the endocervix (3), it ap
peared logical that cultured endocervical cells should be the
most appropriate system to study cervical oncogenesis by HPV.
Previously we found that HPV 16 DNA can immortalize hu
man endocervical cells in vitro ( 12). We report here that, upon
being implanted into athymic mice, the normal endocervical
cells formed stratified squamous epithelium resembling squa
mous metaplasia, and HPV 16-immortalized endocervical cell
implants displayed a pathological picture analogous to that of
carcinoma in situ.
MATERIALS
AND METHODS
Cell Culture. Endo- and ectocervical epithelial cell cultures were
derived from cervical specimens obtained from hysterectomies per
formed for benign conditions. Primary cultures of endo- and ectocer
vical epithelial cells were initiated and maintained as described (12, 13)
from cervical regions distant from squamo-columnar junctions. All tis
sue culture was performed with KGM (Clonetics, San Diego, CA). Cells
were passaged 1:2 when 60-80% confluent for primary cultures and 1:3
when 90% confluent for immortalized cells.
Transmission Electron Microscopy. Trypsinized cells were seeded
into Millicell-HA cups (Millipore, Bedford, MA). Upon confluence, the
cells together with the cellulose-ester membranes were fixed in 5%
glutaraldehyde and postfixed with 4% OsO.,. Epon 812-embedded thin
sections were stained with uranyl acetate and lead citrate. Thin sections
were examined with a Jeol 1200 electron microscope.
Cell Implantation in Nude Mice and Preparation for Histology.
Trypsinized cells (IO6 to IO7) were seeded into 35-mm Petri dishes in
KGM, in the center of which a 1.5 x 1.5-cm sterile silicone sheet (Dow
Corning, Midland, MI) had been placed. The medium was changed
after 8 h, and after 48 h the silicone sheet with attached cells was
implanted into 2-3-month-old female nude mice (nu/nu; NIH) as de
scribed by Barrandon and Green (14). The implants together with the
skin flaps were recovered after 8 days, fixed with 4% paraformaldehyde
in phosphate buffered saline, embedded in paraffin, sectioned, and
stained with hematoxylin and eosin.
Mucin and Cytokeratin Staining. For detecting mucin, unstained
sections prepared for histology were stained with Alcian blue at pH 2.5
(15).
For cytokeratin staining (16), the unstained sections prepared for
histology were deparaffined and rehydrated. They were treated with
0.25% trypsin (Sigma, St. Louis, MO) at 37°Cfor 30 min for antibodies
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from Sigma or 2 h for antibody from ICN (ICN Biomedicals, Lisle, IL)
and then incubated at 4°Covernight with the mouse anti-cytokeratin
monoclonal antibodies to CK18 (CY-90), CK13 (Ks-lA3) and CK10
(K8.60) (Sigma), and CK19 (Ks.19.1) (ICN) with dilutions recom
mended by the suppliers. The sections were stained with fluorescein
isothiocyanate-labeled goat anti-mouse IgG (Sigma) for l h at room
temperature. Observation was made with a Leitz Diaplan microscope.
RNA Preparation and Northern Blot Analysis. Total cellular RNA
was prepared from cells in culture and analyzed by Northern blot hy
bridization as described previously (17). The probes were labeled with
-12Pusing a random priming kit (BRL, Gaithersburg, MD). All washing
was in 0.1 x sodium saline citrate and 0.1% sodium dodecyl sulfate at
65°C.
RESULTS
HEN-16 and HEC-16 are immortalized cell lines that were
derived from normal human endo- and ectocervical epithelial
cells, respectively, transfected with whole genomic HPV 16
DNA. They both contain integrated HPV 16 genomes and ex
press viral messages (12).
In Vitro Ultrastructure of HPV 16-immortalized Endocervical Cells. Primary cultures of endocervical cells display pleomorphism in culture, which evolves with passage (13). HPV 16
DNA transfection was performed to derive HEN-16, using a
primary parental endocervical cell culture that consisted of 2
cell populations morphologically discernible with the light mi
croscope. Electron microscopy also showed that these endocer
vical cells cultured in KGM have an ultrastructure different
from those of ectocervical cells (13).
To evaluate the effects of immortalization on endocervical
cell morphology, cells were examined with electron micro
scopy. For maintaining intercellular structure, the cells were
grown and fixed in situ on membranes. HEN-16 showed mod
erate amounts of tonofilament bundles, well developed desmosomes, and irregular microvilli (Fig. la), which were compara
ble to those of normal ectocervical cells (13) and HEC-16
(Fig. \b). No glandular elements or cilia were found in the cells.
Squamous Metaplasia of Normal and Carcinoma in Situ of
HPV 16-immortalized Endocervical Cells. The ultrastructure
of HEN-16, together with their in vitro CK expression pattern,
suggested that they possess a keratinocyte-like phenotype that
is similar, but not identical, to that of HPV 16-immortalized
"native" ectocervical epithelial cells (12). Morphologically,
HEN-16 showed no marked difference from HEC-16 in culture
with both light (12) and electron (Fig. 1) microscopes. The
distinction might have been more obvious if their growth had
been under in vivo conditions.
To examine this possibility, HEN-16, HEC-16, and their
normal counterparts, HEN and HEC, were implanted beneath
skin flaps of female nude mice. HEN cells at passage 1, the
passage number at which the parental cells of HEN-16 were
transfected with HPV 16 DNA, developed into mullÃ-la\creel
epithelium (Fig. 2a) resembling squamous metaplasia. The cells
showed clear borders and occasional intercellular bridges. No
atypical nuclei or mature squamous differentiation were ob
served. Endocervical cells from a separate cervix produced the
same morphology in vivo. After 30 weeks in culture following
transfection, HEN-16 also displayed stratified morphology in
vivo (Fig. 2c), and its squamous nature was revealed by the
existence of some intercellular bridges. The entire thickness of
epithelium was occupied with highly disorganized cells that did
not show squamous maturation. The cellularity presented ob-
Fig. 1. Ultrastructure of HPV to-immortalized endo- and ectocervical cells.
Bar (a), 500 nm. Original magnifications x 18,000. D, desmosome; M, microvilli;
T, tonofilament. a. HEN-16; b, HEC-16.
vious nuclear irregularity, and cell division could be observed in
the suprabasal as well as basal layers. Serial sections were ex
amined and no indication of invasion could be found. In all the
features, the morphology of HEN-16 was reminiscent of severe
dysplasia/carcinoma in situ. HEN-16 reproducibly formed in
vivo lesions resembling carcinoma in situ at later passages after
cloning.
In contrast to HEN and HEN-16, at passage 1, HEC cells
formed well differentiated stratified squamous epithelium (Fig.
20), whereas, after 32 weeks in culture following transfection,
HEC-16 formed a multilayered lesion with obvious squamous
differentiation (Fig. Id). The lesion produced by HEC-16
showed cuboid and flattened basal cells and horizontally ar
ranged suprabasal cells. The few atypical nuclei observed in
HEC-16 were mostly in basal and parabasal layers. These fea
tures were analogous to those of mild dysplasia. Similar results
have been also reported by others (10).
Another set of HPV 16-immortalized endo- and ectocervical
cells derived from separate cervices produced similar results in
vivo. CaSki cells, an epidermoid cervical carcinoma cell line,
also formed a stratified lesion in vivo, with morphology resem
bling carcinoma in situ (data not shown).
Mucin and Cytokeratins in in Vivo Implants. Both HEN and
HEN-16 implants showed sporadic cells with vacuolated cyto
plasm suggestive of glandular cell differentiation. Alcian blue
specifically stained these cells (Fig. 3, a and c, respectively),
confirming their mucin content (15,18) and corroborating their
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Fig. 2. Histology of in vivo implants. The sections were stained with hematoxylin-eosin. Black arrowheads, borders of the implant basal cells and the mouse
connective tissue. Black bar (a), 50 urn. Original magnifÃ-cationx 288. a. HEN; b. HEC; r, HEN-16; ti. III ( 16.
origin as simple columnar epithelium (19). HEC was negative
for Alcian blue staining (Fig. 3Z>).While one of the two HPV
16-immortalized ectocervical cell lines examined was negative,
the other one, HEC-16, also was positive for mucin (Fig. 3d).
Stratified squamous, metaplastic, and simple epithelia each
express characteristic patterns of cytokeratins (20, 21 ). Further
more, HPVs are stratified squamous epitheliotropic in nature,
and their gene expression and viral replication are closely as
sociated with cellular differentiation. Since the in vitro immor
talized cervical cells might represent certain early steps in cer
vical carcinogenesis (9), the in vivo CK expression patterns of
HPV 16-immortalized cervical cells should also address issues
such as their cell origin and the relationship between CK ex
pression and cervical precancer associated with HPV. We ex
amined the in vivo CK expression of the 4 types of implants by
indirect immunofluorescent microscopy with CK-specific mon
oclonal antibodies for CK18 and CK19 (markers for simple
epithelium), CK13 (marker for nonkeratinizing
stratified
squamous epithelium), and CK10 (marker for k(.-ratini/al ion)
(20, 21).
Normal endocervical cell implant expressed CK18 uniformly
in the basal layer, with sporadic staining in suprabasal layers
(Fig. 4). CK19 and CK13 antibodies produced staining in the
same implant throughout the layers (Fig. 4), while CK10 anti
body showed negative reaction (Fig. 4). For HEC implant, in
contrast to HEN implant, CK18 staining was negative and
CK10 was positive in the superficial layers. HEC implant was
also stained for CK13, but not in the basal layer. CK19 was
stained in HEC implant uniformly in the basal cells and only
sporadically in suprabasal layers. These data for HEN and HEC
implants are in good agreement with those described by Smedts
et al. (21) for squamous metaplasia and native ectocervical ep
ithelium, respectively. HEN-16 and HEC-16 implants showed
in vivo CK expression patterns similar to those of their normal
counterparts. However, unlike HEN, in which CK18 staining
was mainly limited to the basal cells, HEN-16 was stained for
CK18 in all layers (Fig. 4). The same implant was also stained
for CK13 and CK19 antibodies throughout the layers (Fig. 4).
Similar to HEN, HEN-16 was negative for CK10 staining (Fig.
4). It should be noted that the same staining pattern was ob
served in implants both cloned and not cloned for HEN-16.
Similar to that of HEC, HEC-16 implant showed no staining
for CK 18, but was positive for CK 19, CK 13, and CK 10 (Fig. 4).
Expression of Viral and Cellular Oncogenes in Endocervical
Cells. Expression of E6 and E7 oncoproteins plays important
roles in proliferation of HPV-immortalized cells and tumor
development. There is evidence that the E6/E7 expression level
is correlated with cancer cell growth and tumorigenicity (22).
Furthermore, E5 protein of bovine papillomavirus has been
shown to bind a cytoplasmic H+-ATPase (23), and E5 of HPV
can up-regulate growth factor-controlled signal transduction
(24). Possible mechanisms for higher grade dysplastic change of
HEN-16 cells would involve higher expression levels of these
viral genes. We examined E6/E7 and E5 expression in both
immortalized cell lines. Northern blot analysis showed that
HEN-16 and HEC-16 had a similar expression pattern for
E6/E7 messages (Fig. 5). No conspicuously increased expres
sion of E6/E7 was observed in HEN-16. E5-specific probe de
tected no signal. An important next step will be to use in situ
hybridization to examine expression of HPV in the in vivo
implants, which might be differentially regulated in vivo (6).
Alternatively, since both cell lines are aneuploid (12), it was
possible that, as a result of changes such as gene dosage,
HEN-16 might overexpress certain protooncogene(s). Some
studies have indicated that overexpression of c-myc and H-ras
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IN VIVO IMPLANTS
OF HUMAN
Fig. 3. Alcian blue staining for mucin in in vivo implants. Black bar (a), 25 »mi.
Original magnifications x 590. a, HEN; ft, HEC; c, HEN-16; d, HEC-16.
protooncogenes is associated with oncogenesis of cervical neo
plasia (25, 26). Therefore, Northern blots were performed to
examine expression of H-ras, K-ras, and c-myc in both cell lines
and their normal counterparts (Fig. 5). While both of the HPV
16 immortalized cell lines showed expression levels for H-ras
and K-ras comparable to those of normal primary cells,
HEN-16 and HEC-16 did show an apparently higher expres
sion level of c-myc than their normal counterparts. Neverthe
less, HEN-16 did not show an obviously higher expression level
for any of these three protooncogenes than did HEC-16.
DISCUSSION
Squamous cell carcinoma of the uterine cervix has been rec
ognized as a cancer with well defined, continuously progressive
premalignant lesions referred to as CINs, which can be graded
in severity from CIN I to CIN Ill/carcinoma in situ. It has been
reported that 87 and 10% of CINs occur within the transfor
mation zone and the endocervix, respectively, while only 3%
occur in the native ectocervical epithelium (27). HPV is a family
of DNA viruses consisting of more than 60 types (28), each of
which showing relative specificity for anatomical site and epi
thelial type and inducing characteristic pathological conditions.
HPV 16 is the most prevalent genotype that is associated with
ENDOCERVICAL
CELLS
cervical squamous cell carcinoma (6). The importance of squamous metaplasia and HPV infection for cervical carcinoma has
made it urgent to understand the biology of the metaplastic cells
as well as those infected by HPV 16.
The origin of squamous metaplastic cells of the transforma
tion zone has been hypothesized to be the subcolumnar reserve
cells (3), and the reserve cells might derive from either the
endocervical columnar epithelium (4) or the subepithelial
stroma (5). This study provides direct experimental evidence for
the transformation of simple epithelial cells of endocervical
origin into stratified squamous metaplastic epithelium. Epithe
lial cells from the normal endocervix were used in this study. It
has been shown that only cells with epithelial morphology exist
in primary culture in KGM (13). Furthermore, we monitored
the development of the implant to ensure that the reconstructed
epithelium was indeed derived from proliferation of the im
planted single cell sheet, not from mouse stromal cells. When
the implant was recovered after 4 days, a thin epithelium com
posed of single or double layers of cuboid cells was observed
(data not shown). No cells infiltrating from the distinctive
mouse stroma (Fig. 2) were seen.
The presence of mucin in the squamous metaplastic epithe
lium, which is not detectible in the native ectocervical epithe
lium, led to the suggestion that squamous metaplastic cells are
derived from the endocervical epithelium (19). In accordance
with this, a mucin-like substance was detected in the HEN
implant but not in the HEC implant. The in vivo CK expression
patterns of HEN and HEC implants are in good agreement with
those of squamous metaplasia and the native ectocervical epi
thelium, respectively, reported by Smedts et al. (21). The most
prominent features for squamous metaplasia are the expression
of CK18 and CK13 (the former is not detectible in native ecto
cervical epithelium and the latter is not detectible in endocer
vical simple epithelium). Thus, squamous metaplasia observed
for the morphology of HEN implants was substantiated by both
the mucin and CK expression data. Therefore, the in vivo re
constructed epithelia from the cultured cervical cells, HEN and
HEC, reflected the characteristics of naturally occurring cervi
cal squamous metaplasia and native ectocervical epithelium,
respectively. Our in vivo CK expression data are in partial
agreement with those published by other workers (20, 29-31).
The variation may be due to the different techniques and anti
bodies utilized.
The HPV 16-immortalized endocervical cells exhibited a
more morphologically malignant phenotype in vivo than their
ectocervical counterparts. The most striking in vivo morpho
logical feature for HPV 16-immortalized endocervical cells is
the cellular disorganization. It is possible that this difference
between HPV 16-immortalized endo- and ectocervical cells is
tissue-specific, since HEN-16 and HEC-16 were derived from
the same individual and they had been in culture for a similar
period of time in the same media when they were tested in vivo.
The different origins for HPV 16-immortalized endo- and
ectocervical cells were confirmed by their distinct in vivo cytokeratin expression patterns. Additionally, the up-regulated
CK 18 expression in HEN-16 suggested deregulated CK expres
sion. Correlation between expression of simple epithelial CKs,
such as CK18, and cervical cancer has been reported (21, 32).
Thus, the CK expression pattern of HEN-16 implants not only
confirmed its origin, but also substantiated its pathological
characteristics.
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CO
(D
V
LU
O
LU
O
LU
CK19
Fig. 4. Expression of cytokeratins in in vivo
implants. Short white bars, borders of the im
plants and the mouse connective tissue; long
white bar, upper left corner, 50 urn. Original
magnifications x 288. HEN and HEN-16 were
positive for CK18, CK19, and CK13, but neg
ative for (kid.
Note that CKI8 antibody
stained nearly all the layers in III N 16.
whereas in HEN implant the positive cells
were limited to the basal and parabasal cells.
CKI9, CK13, and CK10 were detectible in
HEC and HEC-16 implants, while CK18 was
not delectible.
CK18
CK13
CK10
The presence of mucin in HEN-16 is in agreement with its
endocervical origin (19). Further investigation is needed to ex
amine the possibility that the native ectocervical epithelial cells
produce glandular elements upon transformation by HPV 16.
Pecoraro et al. (33) also reported that in their HPV 16-immortalized ectocervical cells there exists a secretory component, as
revealed by electron microscopy.
Using a similar in vivo implantation system, Woodworth et
al. (10) reported that their HPV 16-immortalized cells derived
from the transformation zone, like those from foreskin, form
abnormal stratified squamous epithelia with no dysplastic
change at early passages. Possibly cells in the transformation
zone are predominantly mature metaplastic cells, which have
phenotypes similar to the native ectocervical cells. Indeed, the
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O
LU
i
O
(0
I
CELLS
The normal endocervical epithelium contains at least 4 types
of phenotypically different cells (25). From the present study,
the exact origin of the cells giving rise to squamous metaplasia
IB v/vo and the type of endocervical cells immortalized by HPV
16 remain unclear. Our preliminary studies suggest unti iIteren
tiated reserve cell origins (12). The reconstruction model sys
tem described in this report should be of help to address this
issue, as well as to examine the mechanism of metaplastic trans
formation.
The cultured normal endocervical cells display morphologi
cal evolution //; vitro (13) and form squamous metaplasia when
reconstructed HI vivo (Fig. 2a). Therefore, it is an interesting
possibility that the endocervical cells in culture in KGM may
undergo a process similar to or related to naturally occurring
squamous metaplasia. Such cells might present a critical transdifferentiation stage, and the cells arrested by HPV 16 at this
stage may produce clones with a more immature phenotype
than those of native squamous epithelium or mature meta
plasia. As also observed by others (9, 12), HPV-immortalized
human cells at early passage (35) and without the sequential
introduction of another oncogene (36, 37) are not tumorigenic;
obviously, some other cellular events are required for full trans
formation of the HPV 16-immortalized endocervical cells. It is
widely accepted that CINs can progress to cervical carcinoma
and that the probability of malignant conversion is associated
with the severity of the dysplasia (38). Based on this, we spec
ulate that some endocervical cells or their derivatives arrested
by HPV 16 are more prone to develop into full cancer.
UJ
LÜ
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E6/E7
1350
1350
H-RAS
1350
K-RAS
ACKNOWLEDGMENTS
C-MYC
2370
Excellent technical assistance by G. Jin, M. Rahimtula, and M.
Butler is acknowledged. We thank Drs. L. Gissmann and H. zur
Hausen for HPV 16 DNA, Dr. T. Scott and L. Lee for advice on elec
tron microscopy, Dr. C. Ford for use of his nude mice facilities, Dr. T.
Michalak for use of the fluorescence microscope, and Dr. J. Williams,
L. Simms, and the staff of St. Clare's Mercy Hospital for the cervical
2370
ACTIN
tissues.
REFERENCES
1350
Fig. 5. Northern blot analysis for expression of various indicated oncogenes.
Total cellular RNA was prepared from cells in culture and 10 jig were analyzed.
Probes were: E6/E7, 806-base pair Hpa\\-Nco\ fragment of HPV 16 (39); H-ras,
6.6-kilobase BamHl fragment of EJ-ras (40); K-ras, v-K-rai (Oncor, Gaithersburg, MD); c-myc, 1370-base pair Sstl fragment of A-LMC4I (41); actin, 2.2kilobase Hum\\\ fragment of -c-actin cDNA. Size markers are indicated (right) in
bases.
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discrepancy observed between the IB vivo implantation and IB
vitro raft systems might be explained by IB vivo factors regulat
ing the behavior of HPV 16-immortalized cells, as proposed by
zur Hausen (6).
4 Q. Sun, A. Pater, and M. M. Pater, unpublished observations.
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Squamous Metaplasia of Normal and Carcinoma in Situ of HPV
16-Immortalized Human Endocervical Cells
Qi Sun, Kouichiro Tsutsumi, M. Brian Kelleher, et al.
Cancer Res 1992;52:4254-4260.
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