[CANCER RESEARCH 59, 5615–5624, November 1, 1999]
Recurrent Integration of Human Papillomaviruses 16, 45, and 67 Near
Translocation Breakpoints in New Cervical Cancer Cell Lines1
Louise A. Koopman,2,3 Károly Szuhai,3 Jaap D. H. van Eendenburg, Vladimir Bezrookove, Gemma G. Kenter,
Ed Schuuring, Hans Tanke, and Gert Jan Fleuren
Departments of Pathology [L. A. K., J. D. H. v. E., E. S., G. J. F.] and Gynecology [G. G. K], Leiden University Medical Center, and Laboratory for Cytochemistry and Cytometry,
Department of Molecular Cell Biology, Leiden University Medical Center, [K. S., V. B., H. T.], Leiden, The Netherlands
ABSTRACT
Progressive chromosomal changes and integration of human papillomavirus (HPV) sequences mark the development of invasive cervical
cancer. Chromosomal localization of HPV integration is essential to the
study of genomic regions involved in HPV-induced pathogenesis. Yet, the
available information about HPV integration loci is still limited, especially
with respect to different HPV types. We have established cell lines from
five cervical cancers with HPV-16, HPV-45, and HPV-67. We have determined HPV integration sites and karyotype abnormalities by using the
multicolor combined binary ratio-fluorescence in situ hybridization
method (Tanke et al.) with 24 chromosome-specific paints in combination
with full-length HPV DNA probes.
All cell lines were cytogenetically abnormal, and exhibited numerical
and structural chromosomal deviations. HPV sequences were integrated
at various (segments of) chromosomes. Duplicate integration sites were
seen in all multiploid cell lines, suggesting that viral integration had
preceded chromosomal endoreduplication. HPV-16 was found near the
t(3p14.1–14.3;14) breakpoint in cervical squamous cell carcinoma
(CSCC)-7 and mainly in episomal form in CSCC-1. HPV-45 was integrated near 3q26 –29 in cervical (adeno or adenosquamous) carcinoma
(CC)-8 and near 1q21–23 as well as near the t(1q21;22q13) breakpoint in
CC-10A and CC-10B variant lines. HPV-67 was localized near the breakpoint of t(3p23–26;13q22–31) in CC-11. Southern blot analysis showed
that, except for CSCC-1, the physical state of HPV in the cell lines was the
same as in the original tumor lesions.
This set of six cervical cancer cell lines included three lines with
HPV-45, a major non-Western high-risk HPV type, the first reported
HPV-67-positive cell line, and two cell lines with integrated and episomal
HPV-16 DNA, respectively. The novel combined binary ratio-fluorescence
in situ hybridization technique enabled us to simultaneously map chromosomal rearrangements and HPV integration sites, thereby revealing
recurrent integration near translocation junctions for all of these HPV
types in the cell lines from three of the five primary tumors. The detection
of multiple HPV integration sites at rearranged chromosomes at such high
frequency in cervical cancer-derived cells may reflect events that are
relevant to the development of cervical cancer.
INTRODUCTION
Cervical cancer is a common malignancy that affects women
worldwide, especially in developing countries. It is strongly associated with the “high risk” HPVs4, which are detected in practically all
invasive cervical cancers (1). Besides the predominant HPV types,
HPV-16 and HPV-18, other oncogenic HPV types, such as HPV-31,
Received 5/27/99; accepted 9/7/99.
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.
1
Supported in part by Stichting VanderEs (Rotterdam, The Netherlands) and by the
Eureka project Spektrakar (Senter, The Hague, The Netherlands).
2
To whom requests for reprints should be addressed, at Department of Pathology,
Leiden University Medical Center, L1Q/P1– 40, P. O. Box 9600, 2300 RC Leiden, The
Netherlands. Phone: 131-71-5266596; Fax: 131-71-5248158; E-mail: louise_koopman
@yahoo.com.
3
These authors have contributed equally to this study.
4
The abbreviations used are: HPV, human papillomavirus; FISH, fluorescence in situ
hybridization; mAb, monoclonal antibody; DEAC, diethylaminocoumarin; DAPI, 4,6diamino-2-phenylindole; DI, DNA index; CSCC, cervical squamous cell carcinoma; CC,
cervical (adeno or adenosquamous) carcinoma.
HPV-33, HPV-35, and HPV-45 have been detected in cervical malignancies. The prevalence of certain HPV types appears to correlate
with geographical variation; e.g., HPV-45 may represent a major
oncogenic HPV type in Western Africa (2) and Surinam (3).
Infection with HPV is an early event in the multistep cervical
pathogenic process. The oncogenic potential of HPV has been attributed mainly to the continued expression of the early gene products, E6
and E7 (4). These interfere with normal cell cycle regulation through
inactivation of the tumor suppressor proteins p53 and pRB, respectively (5). Although oncogenic HPV types are required for initial
transformation, HPV infection and viral oncogene expression alone
are not sufficient for the completion of the malignant conversion
process. This notion correlates with the facts that (a) a long latency
period precedes tumor occurrence in vivo and that (b) the incidence of
tumors is less frequent than the number of HPV infections (6). In vitro
models established by transfection of primary human keratinocytes
with HPV-16, HPV-18, or HPV-33 have shown that cells could be
immortalized by action of the E6 and E7 genes (7). The full malignant
phenotype, however, was only reached after extensive prolonged
culture in vitro or by cotransfection with the ras oncogene (8, 9).
Cytogenetic studies or loss of heterozygosity analyses in primary
cervical cancers and pre-invasive lesions indicate that secondary
events involving different chromosomes, e.g., chromosomes 1, 3, 4, 5,
6, 11, 15, 17, and 18 are progressively implicated in vivo (10 –12). Yet
the exact nature of cervical cancer-related genes has still to be identified.
The HPV DNA genome persists as an extrachromosomal episome
in premalignant cervical intraepithelial neoplastic lesions, whereas in
invasive cancers and tumor cell lines, multiple copies of the viral
DNA are integrated in the host genome. In contrast with most cervical
tumors, which seem to contain exclusively integrated HPV sequences,
a proportion of HPV-16-positive tumors contains episomal HPV DNA
either alone or in coexistence with integrated HPV sequences (13, 14).
Thus, although differences between the different HPV types may
exist, integration of HPV DNA in the host genome represents an
important event in cervical carcinogenesis. Besides increasing the
transcript stability and protein expression of the viral E6 and E7 genes
(15), integration may cause changes in the transcription and expression of cellular genes targeted by the integration event. The integration of HPV sequences in the host genome might occur randomly or
with a preference in or near fragile sites or oncogenes (16). Various
HPV integration sites have been identified in HPV-16- or HPV-18containing tumor-derived cell lines (16 –18), in a limited number of
primary lesions (19, 20), or in HPV-immortalized primary keratinocytes (21–23). Thus far, these integration sites do not point to preferential chromosome- or HPV type-specific integration. Still, HPV
may integrate at presently unidentified oncogenic loci involved in
cervical carcinogenesis.
In this context, we have mapped HPV DNA integration sites in six
recently established cervical cancer cell lines containing HPV-16,
HPV-45, and HPV-67. In addition, we have characterized the numerical and structural chromosome abnormalities in the cell lines and
compared them with the original tumors with respect to the physical
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HPV INTEGRATION IN CERVICAL CARCINOMA CELL LINES
Table 1 Patient clinical characteristics, histopathology of the original tumor, and HPV types in primary lesions and derivative cell lines
Cell line
d
CSCC-1
CSCC-7f
CC-8f
CC-10A/Bf
CC-11f
Patient age
34
33
30
45
62
Federation Internationale
de Gynecology et Obstetrique
(FIGO) stage
sIIA
IB/IIA
IIA
IB
IIA
HPV typeb
Histopathological diagnosis
NKLCSC
NKLCSC
ASCg
GCCh
ASC
e
LNMa
Survival
Primary tumor
cell linec
1
2
1
2
1
Disease-free
Disease-free
16 mo
Disease-free
4 mo
16
16
45
45
58 1 67
16
16
45
45
67
a
LNM, status of lymph node metastasis, either positive (1) or negative (2).
HPV typing results as determined by GP-PCR with consensus primer sets CPI/II, GP51/61, or MY09/11 followed by sequence analysis. Additional HPV-16-specific PCR
confirmed HPV-16 where listed and was negative in CC-8, CC-10, and CC-11 primary tumor and lines.
c
Typing was done on both early (#p5) and late passages (.20).
d
Caucasian patient.
e
NKLCSC, nonkeratinizing large cell squamous cell carcinoma.
f
Surinamese patient.
g
ASC, adenosquamous carcinoma.
h
GCC, glassy cell carcinoma. It is classified within the group of mixed (adenosquamous) carcinomas (25).
b
status of HPV and the overall DNA content. The multicolor COBRAFISH technique (24), which was extended with HPV hybridization5,
enabled us to map HPVs at unique chromosomal integration locations,
including chromosomal translocation junctions. These sites may reflect events important in HPV-related carcinogenesis.
MATERIALS AND METHODS
Clinical Background. Initially, tumor specimens from 74 FIGO stage
IIA/IB cervical cancer patients who had undergone a Wertheim’s hysterectomy
at Leiden University Medical Center were brought into cell culture. This group
consisted of 19 patients (26%) of multiple ethnicities, such as Creoles, Javanese, and Hindustani, who were all from the former Dutch colony Surinam
(South America) and were referred for treatment in The Netherlands. The other
55 (74%) were Caucasian. The mean patient age was 45.5 years in a range of
23–76 years. All tumors were examined histologically on paraffin-embedded
sections and pathologically diagnosed as having cervical squamous cell carcinoma (55/74), adenocarcinoma (10/74), or adenosquamous carcinoma (9/74).
The cell cultures from five tumor specimens were successfully established as
a continuous cell line. Four of these cell cultures (CSCC-7, CC-8, CC-10, and
CC-11) were from Surinamese patients. CC-10 was split into variants A and B
on morphological grounds at an early passage. Paraffin-embedded original
tumor samples pertaining to the cell lines were revised and further classified on
the basis of keratinization, cell size, and nuclear appearance (25). Staining for
periodic acid-Schiff and Alcian blue confirmed the adeno or adenosquamous
cell features. Clinical characteristics of these five cases are listed in Table 1.
Cell Line Establishment and Cell Culture. Immediately after surgery,
tissue for diagnostic purposes was fixed in formalin, and a piece of the tumor
tissue was snap-frozen in liquid isopentane and stored at 270°C while the
remaining part was minced and used for cell culturing in vitro. Tumor cells
were cultured as described by Hilders et al. (26). In brief, after digestion in a
collagenase/DNase enzyme solution, cells were cultured in complete medium
consisting of DMEM/25 mM Hepes (Life Technologies, Inc., Irvine, Scotland)
supplemented with 2 mM glutamin, 50 mg penicillin-streptomycin/ml, and 10%
FCS (Bio Whittaker, Verviers, Belgium) in a humidified atmosphere containing 5% CO2 at 37°C. After adherence to the flask, the medium was replaced
with keratinocyte serum-free medium (Life Technologies, Inc., Irvine, Scotland), and the flask was transferred to a 37°C CO2/H2O-free incubator.
Medium was replaced 2–3 times a week. This sequence was repeated after each
trypsinization. At each passage number, aliquots of cells were frozen in liquid
nitrogen in 10% DMSO in FCS. Mycoplasma screening was performed regularly on all cell lines. CasKi, SiHa, and HeLa cervical cancer cell lines
(purchased from American Type Culture Collection, Rochester, NY) were
cultured in complete medium and used as HPV-16- and HPV-18-positive
controls.
DNA Extraction. Primary tumor DNA was extracted from deparaffinized
tumor tissues as described previously (27). Tumor cell line DNA was extracted
from washed cell pellets according to the standard proteinase K-SDS procedure.
5
K. Szuhai, V. Bezrookove, J. Wiegant, J. Vrolijk, R. W. Dirks, A. K. Raap, and J.
Tanke. Simultaneous molecular karyotyping and mapping of viral DNA integration sites
by 25-color COBRA-FISH. Genes, Chromosomes & Cancer, in press, 1999.
HPV Detection and Typing. General primer-mediated PCR and subsequent sequencing in combination with type-specific PCR or oligonucleotide
probe-hybridization was used for the detection and typing of HPV DNA. The
b-globin gene was used as an internal control for PCR amplification (28). An
initial general primer-mediated PCR using the HPV consensus primer set
(CPI/CPIIG) to amplify a 188-bp fragment in the highly conserved E1 ORF
region was performed as described previously (29). Inconclusive or negative
samples were subsequently tested with additional HPV consensus primer sets,
i.e., GP51/61 (30) or MY09/MY11 (29). To determine the HPV subtype,
PCR products were subjected to direct sequence analysis. In short, after
purification of the PCR products using the “EasyPrep” kit (Amersham Pharmacia Biotech, Uppsala, Sweden), the products were sequenced directly with
the cycle-sequencing kit (Perkin-Elmer, Norwalk, Connecticut) using the PCR
primers end-labeled with 32P-ATP. For nucleotide sequence analysis and
comparisons, the programs Seqed, Fasta, and MAP of the Wisconsin Genetics
Computer Group (version 8.1) sequence analysis software package were used.
All available sequences of HPV subtypes were retrieved from GenBank, and
they were complemented with sequences stored in a local HPV nucleotide
database. HPV types were identified when nucleotide comparisons revealed an
identity of .95% with a known type. Where indicated, an HPV-16-specific
PCR was done or group-specific typing using a cocktail of specific oligonucleotide probes in an enzyme-immunoassay was performed, followed by
type-specific hybridization (31).
FISH Probes. Probes for all chromosomes were obtained from Cytocell,
United Kingdom. Dr. Nigel Carter (Sanger Institute, Cambridge, United Kingdom) kindly provided additional probes for chromosomes 1– 8, 13–20, 22, and
X. The probe with the best performance was chosen for each chromosome. All
DNA probes were amplified by degenerate oligonucleotide-primed-PCR (32)
to generate a set of 24 human chromosome painting probes for enzymatic
labeling. Full-length DNA probes for HPV-16, HPV-18, and HPV-45 were
kindly provided by Dr. H. Zur Hausen (Heidelberg, Germany), and full-length
DNA probes for HPV-58 and HPV-67 were provided by Dr. T. Matsukura
(Tokyo, Japan).
Multicolor FISH Staining (COBRA). To visualize each separate chromosome as well as the respective HPV genome, we applied the COBRAFISH technique as described in detail elsewhere (24). This method allows
for staining of all 24 human chromosomes in distinguishable colors using
four primary fluorophores versus using five as it was reported thus far for
other multicolor FISH techniques. Thus, the fifth color (in this case, Cy7)
is available for the identification of the integrated HPV. In short, each of
the 24 human chromosomes were fluorescently labeled by incorporating
labeled dUTP-s. This was done by using either PCR or nick translation and
by using the following various combinations of four fluorophores/haptensdUTP according to the COBRA protocol (24): Fluorescein (Boehringer
Mannheim, Germany), Lissamine (NEN Life Science Products, Boston,
MA), Cy5 (Amersham), and Digoxigenin (Roche, Basel, Switzerland). The
HPV probes were labeled with Biotin-dUTP (Sigma) during nick translation. Metaphase chromosomes were prepared according to standard procedures, and they were pretreated with RNase A and pepsin according to
Wiegant et al. (33). After denaturing, 5 ng of the biotin-labeled HPV probe
and a total of 4 mg of a cocktail with each chromosome represented were
hybridized for 120 h at 37°C in a humidified chamber. After washing, a
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HPV INTEGRATION IN CERVICAL CARCINOMA CELL LINES
Table 2 Chromosomal location of HPV and karyotype description in cervical cancer cell lines
HPV status
Cell line
passage
HPV
Probe
Episomal
Integration site
CSCC-1 p26/
p38
HPV-16
Yes
Not found in 37/38 metaphase
spreads
Karyotype abnormalities
,3n.XXXX, 1132, 12, 1332, der(3) del(3p), der(4) t(4;17)32; 15,
der(5) del(5q), der(7) del(7q), 1932, 110, 21132, der(11) t(3;11), 112,
der(12) t(12;15); 113, 114, 115, 11632, 11732, t(17;20), 118,
12032, 121, 122,/idem 32
At normal 14q (34) in one
complex metaphase
Modal
range
Figure
68–79
76–78a
Fig. 2, a, b
and g
(135–154)
Fig. 2, c–f
CSCC-7 p49
HPV-16
No
Near breakpoint of der(14):
t(3;14) (p14.1–14.3;14)32
,4.XX, der(2) t(2;6), 2334, der(3) t(3;7)32, dic (3;7;3), der(3) t(3;5)32,
2432, 25, der(5) t(5;17), der(5) ins(5;6), 2632, der(6) t(3;6;X)32,
2732, der(7) t(2;3;7;)32, der(7) t(4;7), 28, 19, 21132, der(11)
t(6;11)32, 21332, der(13) t(X;13)32, 21434, der(14) t(3;14)32, dic(14;
14)32, 21532, 21732, der(17) t(8;17), 21832, der(20)34, 222, [cp 19]
76–91
87–90a
Fig. 1
CC-8 p21
HPV-45
No
At normal 3: (3q26–29) 3 2
,4n. 2X34, der(X) t(X;1), t(X;2), der(X) t(X;20), 2233 der(3) t(3,4)32,
der(3) t(3;11), der(3) t(3;17), 2433, der(4)iso4q, 1532, der(6)32, der(6)
t(6;14), 932, der(9)32, der(9) t(5;9), 210, 212, 21333, der(14), 21534,
dic(15) t(13;15)32, 216, 217, der(17), 219, 120, der(20) t(7;20), 221X2,
der(21) t(1;21)32, der(21) t(11;21), 222X2, der(22) t(7;15;16), der(22) t(1;
22;4;17), der(22) t(5,22,4,17) [cp 13]
83–93
95–97a
Fig. 2h
CC-10A p25
HPV-45
No
At normal 1: (1q21–23) 3 2;
near breakpoint of der (22):
t(1;22) (q21;q13) 3 2
,5n. XXXXX, 11, der(1) t(1;22), der (2)32, der(2) t(2;3), der(3), der(6),
17, 28, der(8) t(8;20)34, 1932, der(11) (9;11), 213, 21432, 21534,
dic(15;15)32, 218, 120, 221, 22232; der(22) t(1;22)33, [cp 17]
112–119
110–133a
Fig. 2i
CC-10B p25
HPV-45
No
At normal 1:(1q21–23) 3 2;
near breakpoint of der (22):
t(1;22) (q21;q13) 3 2,
,3n.XXX, 1X, der(1) t(1;9), 15, 14, der(7) t(7;8), der(8) t(8;20)32, 19,
der(9) t(9;20), 215, dic(15;15), 119, 120, der(22) t(1;22)32 [cp 13]
65–75
68–69a
Fig. 2j
CC-11 p28
HPV-67
No
Near breakpoint of der(3):
t(3;13)(p23–26;q22–31)
,2n.XX, 21, der(1) t(1;22;13), der(1) t(1;13), der(4) t(4;19), der(5), 27,
der(7) t(7;15), 210, der(10) t(10;22), 213, der(3) t(3;13), 215, 120, 222
46–51
51a
Fig. 2k
a
This value is the modal number as calculated from DI (Table 3) 3 46 chromosomes.
mAb against digoxin followed by a sheep anti-mouse mAb conjugated to
DEAC (Molecular Probes, Eugene, Oregon) was added to detect the
Digoxigenin-labeled probes. The Biotin-labeled HPV probes were detected
using streptavidin-Cy7 conjugates (Amersham) followed by incubations
with a biotinylated mAb against streptavidin (Life Technologies, Inc.) and
streptavidin-Cy7 (Amersham). Chromosomes were counterstained by
DAPI solution. The slides were embedded in Vectashield (Vector) prior to
microscopical evaluation. Digital fluorescence imaging and analysis were
done as described (24, 34). Multicolor FISH karyotyping of the cell lines
was simultaneously done by the same procedure (24).
Analysis of the Physical State of HPV DNA/Southern Blot Analysis.
The restriction enzymes used were EcoRI (Roche), HindIII (Roche), and PstI
(Amersham). With each restriction enzyme, 10 mg of genomic DNA were
digested, electrophoresed on 1% agarose gel, and transferred to nylon filters
(Hybond N1, Amersham) by Southern blotting. Full-length HPV DNA probes
were radiolabeled with 20 mCi of [a32P]dCTP using a random-primed labeling
kit (Amersham Pharmacia Biotech). Southern blots were prehybridized at
65°C in hybridization mix (53 Denhardt’s, 63 SSC, 0.5% SDS) and after 2 h,
they were hybridized with the HPV probes overnight at 67°C. Blots were
washed for 30 min at 65°C in 23 SSC/0.1% SDS, for 30 min in 13 SSC/0.1%
SDS, and finally for 15 min in 0.53 SSC/0.1% SDS. Membranes were
exposed to Kodak X-Omat AR films with intensifying screens at 270°C.
Ploidy Analysis. For paraffin-embedded primary tumor material, the pepsin digestion method of Hedley et al. (35) was used for nuclear isolation from
40-mm sections. Nuclei from the cell lines were isolated using the detergent
trypsin method of Vindeløv et al. (36). Propidium iodide was used as a DNA
stain for both methods. DNA content was measured on a FACScan flow
cytometer (Becton Dickinson, Mountainview, CA) and expressed as a DI (37).
RESULTS
Establishment of Six Novel Human Cell Lines From Five Cervical Carcinomas. In our attempts to establish long-term cell cultures
from cervical carcinoma specimens obtained from 74 patients, we
established six continuous cell lines from five primary tumors. The
reasons for the lack of success in establishing cell lines from the other
69 primary cultures were (a) the lack of adequate numbers of tumor
cells, (b) overgrowth by fibroblasts, (c) senescence of short-term
tumor cell cultures after 3– 6 passages, or (d) microbial contamination.
Long-term cultures, i.e., cultures that could be maintained beyond at
least 20 passages, were established from five primary cultures. Histopathology, patient characteristics, and lymph node status of these
cases are listed in Table 1.
In Vitro Characteristics of the Cervical Cancer Cell Lines. All
cell lines have now undergone .100 population doublings over at
least 20 –50 passages in keratinocyte serum-free medium. Phasecontrast microscopy revealed flat adherent monolayers with epithelial
cell morphology; cells ranged in size and shape and were arranged in
a pavement-like architecture (not shown). One of the cell lines,
CC-10, was split into two subpopulations (A and B) on morphological
grounds at an early passage. At later passages beyond p30, the
resulting variant cell lines, CC-10A and 10B, no longer showed
obvious morphological differences but appeared distinct by other
parameters (Tables 2 and 3).
Ploidy Analysis. To determine whether the cell lines had changed
their overall chromosome content during culturing, we studied DNA
ploidy in the primary tumor and in early and late passage cell lines.
The DIs of the cell lines versus the paraffin-embedded tumor material
are listed in Table 4. Allowing for technical variability due to the
different methods used to obtain and analyze the DNA from the
different sources, the DI of CC-11 could represent the stemline DI
detected in the primary tumor. In the other cell lines, the DNA content
was different from the corresponding primary tumors. This was probably a result of endoreduplication and subsequent DNA loss during
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HPV INTEGRATION IN CERVICAL CARCINOMA CELL LINES
Table 3 Chromosomes involved in genomic rearrangements and HPV integration
#
a
CSCC-1
CSCC-7
CC-8
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
x
Totald
CC-10A
T
T
T/intHPV16(p)
T
T ins 6
T
T
T
T del p
T
del q
del q
T/intHPV45
intHPV45 (q)
T/der
T/der
T
intHPV45(q)
T iso q
T
T der
T
CC-10B
T/intHPV45
intHPV45 (q)
CC-11
T
4
T/intHPV67 (p)
T
der
der
T der
T
T
T
T
T
T
T/intHPV16
T
T der
T
T
T der
T
T
T
T
T
T
T
[intHPV16 (q)]c
T
T/intHPV67 (q)
dic
dic
T
T
T/intHPV45 (q)
T/intHPV45 (q)
T
T
T
T
T
T
13
18
10
7
10
T
T
T
Der
T
T
10
Totalb
3
5
4
4
3
5
4
3
1
4
1
3
2
5
1
3
0
1
5
1
4
2
a
#, chromosome.
b
Total number of cases per chromosome.
c
Only in 1/38 studied metaphases.
d
Total number of chromosomes involved in changes per cell line.
tion of HPV sequences was 55.4% HPV-16 (n 5 41), 20% HPV-18
(n 5 15), 6.8% HPV-45 (n 5 5), 4% HPV-33 (n 5 3), and one each
of HPV-31, HPV-35, HPV-52, HPV-56, and HPV-58 1 HPV-67
(7%). Five (7%) cases remained HPV negative. In CSCC-1 and
CSCC-7, HPV-16 was detected, and in CC-8 and both CC-10 variants,
HPV-45 was found. Identical typing results were found in the corresponding primary tumors (Table 1). In CC-11, HPV-67 was detected
by using all of the CPI/II, GP51/61, or My09/My11 primer sets. In
the primary tumor, however, HPV-58 was detected with the CPI/II
primer set, whereas HPV-67 was found with the GP51/61 and the
My09/11 sets. In consecutive culture passages (p3-p27), CC-11 was
consistently positive for only HPV-67 with all three primer sets.
Additional HPV-58-specific hybridization performed on the primary
tumor and early and late passages of CC-11 were positive on the
primary tumor only. Apparently, the primary tumor contained both
HPV-58 and HPV-67, and the cell line CC-11 was cultured from the
HPV-67-containing tumor cells.
Chromosomal HPV-integration Sites. To analyze the localization of HPV integration, we applied the modified multicolor COBRA-
culturing. In most cases, this was evident already at early passages
(Table 4).
Karyotypes. Description of the COBRA-FISH karyotypes in each
cell line was done according to the standard nomenclature (Table 2).
Modal chromosome numbers varied from the hyperdiploid range
(CC-11) to the triploid (CSCC-1, CC-10B), tetraploid (CSCC-7, CC8), and pentaploid range (CC-10A), and they were in a similar range
as those calculated from the measured DIs (Table 3). In addition to
gross numerical changes, structural chromosomal abnormalities were
abundant in all cell lines. Table 3 lists the chromosomes according to
their involvement in structural changes as studied by multicolor FISH.
Most chromosomes showed structural rearrangements in three or
more of the six cell lines. Chromosomes 3, 7, 15, and 20 were most
frequently affected, but no single chromosome was structurally aberrant in all cell lines. Chromosomes 10, 12, 16, 19, 21, and X were only
occasionally affected, and chromosome 18 appeared unchanged in all
cell lines.
HPV Typing of Primary Tumors and Cell Lines. HPV was
detected in 69 (93%) of the 74 primary tumors. The overall distribu-
Table 4 Cell line growth rates and ploidy in cervical tumor cell lines
Doubling times shown are the mean 6 SE of values estimated on at least three consecutive passages within the range listed in the last column.
DNA index
Early passage (p)b
Cell lineb
Passages studied and number
of measurements (n)
1.0
1.10
1.70
1.78
1.84 (p4)
2.07; (1.06c) (p4)
2.5; (4.94c) (p5)
1.71 (susp)d
;31 6 2
1.78
1.71 (susp)
;62 6 10
1.23; 1.35
1.21 (p4)
1.66 6 0.02
1.89 6 0.02
2.06 6 0.04
2.42 6 0.01; 2.90e 6 0.01
2.45 6 0.02; 2.84e 6 0.01
2.55 6 0.06
1.48 6 0.02
1.49;
2.89
1.12 6 0.01
1.08
p21–26 (5)
p17–20 (6)
p15–19 (3)
p10–16 (4)
p20–30 (4)
p37–43 (10)
p10–13 (3)
p43
p10–14 (5)
p27
Cell line
Doubling time (h)
CSCC-1
CSCC-7
CC-8
CC-10A
;74 6 5
;50 6 4
;55 6 7
;58 6 1
CC-10B
CC-11
a
Primary tumor
a
DIs were determined on primary tumors from paraffin-embedded tissue sections as described in “Materials and Methods.”
DIs were determined on cell line suspensions as described in “Materials and Methods.”
Minor subpopulations with DIs as listed were present beside prominent peaks in the sample. Multiple ploidy analyses were performed at indicated passages to obtain mean
DIs 6 SE. For CC-10, uncultured primary suspension was available for DNA-analysis. For the remaining cell lines, only early passage samples were available.
d
Susp, uncultured primary suspension.
e
The DI 2.9 peak in CC-10A gradually disappeared within the range of passage 20 –30, whereas it emerged at late passage in CC-10B.
b
c
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HPV INTEGRATION IN CERVICAL CARCINOMA CELL LINES
FISH technique (24) in combination with HPV probe hybridization.
Single cross-hybridizations using unique HPV-16, HPV-18, HPV-45,
HPV-58, and HPV-67 probes on all studied cell lines completely
matched the HPV-PCR data (not shown). The set-up for the COBRA
analysis in combination with HPV hybridization is shown in Fig. 1 for
cell line CSCC-7. Combined results for the other cell lines are
depicted as metaphase spread images in Fig. 2.
HPV was integrated in four of the five cell lines, when, by origin,
considering CC-10A and CC-10B as one cell line (Table 2). Both
CC-10 variants contained integrated HPV-45 sequences near the
breakpoint of derivative chromosome 22, t(1q;22q), and at chromosome 1q (Fig. 2, i-j). Despite differences in the modal chromosomal
number and the DI (Table 4), these variant lines showed no differences in the HPV integration pattern, which probably represented that
of the primary tumor. In CSCC-7, the integration of HPV-16 was
detected in the derivative chromosome 14 near the (3p;14) translocation breakpoint (Fig. 1). In CC-8, the integration of HPV-45 was
observed on chromosome 3q26 –29 (Fig. 2h). CC-11 contained integrated HPV-67 sequences near the breakpoint region of derivative
chromosome 3 [t(3p;13q); Fig. 2k]. This cell line was near-diploid
(DI;1.1), and the integration was consistently found in one chromosome copy. In metaphase spreads of the hyperploid cell lines, on the
other hand, the integration sites were seen in multiple chromosome
copies, thereby indicating that the integration of HPV at these sites
had preceded chromosomal endoreduplication (Fig. 2; Table 4). The
pattern observed in the interphase nuclei of CSCC-1 was typical for
the presence of episomal virus particles (Fig. 2, a and b). In 37 of the
38 metaphases studied from this cell line, only scattered signals and
no integration sites were visible (Fig. 1g). In one metaphase spread
however, integration was observed on four copies of chromosome 14q
(Fig. 2, c-d).
Southern Blot Analysis. To investigate whether the physical status of HPV in the cell lines represents the HPV status of the original
tumors, we performed Southern blot analysis (Fig. 3). In cases where
no frozen primary tumor material was available for this method, the
earliest available culture passage was used. The restriction patterns in
late passages of cell lines CSCC-7, CC-8, CC-10, and CC-11 (Fig. 3,
B-E) were in agreement with the presence of integrated HPV DNA as
determined by HPV-FISH (Fig. 1; Fig. 2, h-k). Moreover, the patterns
were identical to those observed in the primary tumor material or early
passages.
In CSCC-1, however, the restriction pattern changed upon culturing
from passage p6 to p20 (Fig. 3A). The digestion of genomic DNA
from CSCC-1 at p20 with HindIII or EcoRI indicated the presence of
episomal HPV forms, as was also found by HPV-FISH analysis at late
passage. HindIII digests of p6 showed several additional bands that
may represent the coexistence of integrated HPV sequences. When
EcoRI was used, the restriction patterns between p6 and p20 also
differed, but both patterns contained a fragment of approximately 7.3
kb, which could have resulted from the disruption of circular episomal
HPV-16 DNA. The difference in restriction patterns observed between early and late passage may have resulted from the selection
for episomal HPV-16 and/or may represent a change in the constitution of the extrachromosomal episomal genomes that may
have occurred during culturing.
DISCUSSION
By using a novel combination of complete chromosome painting by
COBRA (24) and HPV-FISH, we simultaneously mapped sites of
HPV integration and chromosomal rearrangements in cell line metaphase spreads from five cervical cancers. Integrated HPV was found
at unique chromosomal sites in each cell line, with no single preferred
site for either HPV-16, HPV-45, or HPV-67. Also, among multiple
cell lines with the same HPV type, integration sites were different
(Table 2). Interestingly, however, HPV-16, HPV-45, and HPV-67
were integrated near translocation junctions in the cell lines from three
of the five tumors (Fig. 1 and 2; Table 3). Chromosome 3 was
involved in integration in three cases, two of which involved translocation (Table 3). HPV-16 was found at the junction of chromosome
3p14, with a whole chromosome 14 in CSCC-7, whereas HPV-67 was
found near the junction of chromosome 3p23–26, with chromosome
13q in CC-11. HPV-45 was integrated at chromosome 3q in CC-8 and
at 1q21–23 as well at the junction of 1q21 and 22q13 in CC-10A and
CC-10B. In CSCC-1, HPV-16 was present mainly in episomal form,
although integration at chromosome 14q was also observed in one
metaphase.
The chromosomal locations for the HPV integration described here
are different from the integration sites that have been previously
described in a number of cervical cancer derived cell lines, a few
primary cervical cancer cell cultures, and in HPV-immortalized keratinocyte models. Although the latter are fundamentally different
from tumor-derived immortal cells, they provide useful models for
studying transformation in vitro. Integration of HPV-16 DNA has
been found at multiple sites including chromosomes 3, 4, 7, 11, 13,
15, 17, and 20 (19, 21, 38). HPV-18 integration has been reported at
3p21, 8q21–22, and 12q14 –15 (17, 39) and in the HeLa cell line at
multiple sites including normal and rearranged 8q24 (18), a site also
implicated in HPV-16 and HPV-18 integration in two primary cervical cancers (20). HPV-68 integration at 18q21 was recently described
in a cancer cell line (40). In HPV-33-immortalized cell lines, integration was recurrently found at chromosomes 13q33–34 and 9p13 (22,
23). These previous reports and our present findings do not point
toward the existence of a single preferential chromosomal integration
site for HPV. However, recurrent integrations of different HPV types
at chromosomal breakpoint regions in three of the five cervical
cancers as described in this study provide novel perspectives on the
role of HPV in cervical cancer. To our knowledge, HeLa is the only
tumor-derived cell line in which HPV was detected at rearranged sites
(18). Over the past few years, almost 20 new cervical cancer cell lines
with HPV-16, HPV-18, HPV-31, and HPV-33 have been established,
but the chromosomal localization of HPV was not assessed (41, 42).
Thus, chromosomal integration sites involving multiple HPV types in
cervical cancer-derived cells are narrowly explored.
By using a novel combination of HPV detection with simultaneous
multicolor-FISH analysis as applied on our tumor-derived cell lines,
not only the chromosomal HPV integration sites but also the related
breakpoint regions and chromosomes putatively involved in HPVrelated immortalization can be identified (Table 3). The cytogenetically abnormal chromosomes described in this study include those
that are most frequently described in primary cervical cancers, such as
chromosomes 1, 3, 5, 11, and 17 (10). Chromosomes 3, 11, and 17, as
well as other chromosomes such as 18q and 6p have also been noted
in loss of heterozygosity studies (11). Chromosome 3 was involved in
HPV integration at three different sites: 3q (HPV-45), 3p23–26 (HPV67), and 3p14 (HPV-16; Table 3). The 3p14 region contains the
FRA3B fragile site located within the FHIT gene locus, which is
commonly altered in cervical carcinomas (43, 44). Interestingly, this
region coincides with an earlier reported HPV-16 integration site in a
primary cervical cancer (45). To shed light on the involvement of
possible fragile sites or specific cellular sequences targeted by HPV
integration in the present cell lines, further fine mapping of the
chromosomal abnormalities with regard to the exact chromosomal
loci is in progress. Because our (and other) established cell lines from
already invasive cervical cancers constitute the starting point of our
analyses, we cannot provide solid evidence concerning the sequence
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HPV INTEGRATION IN CERVICAL CARCINOMA CELL LINES
Fig. 1. Set-up for the simultaneous COBRA and
HPV FISH analysis. A representative COBRA-FISH
karyogram of cell line CSCC-7 is shown. a, image (12
colors) resulting from the three primary dyes (Fluorescein, Lissamine, and Cy5) used in ratio-labeling. b,
DEAC image (fourth dye, so-called binary-1 image)
that is used to identify the odd and even numbered
chromosomes that carry the same set of three primary
labels (a). For instance, chromosome 1 (odd, binary
1-negative) and 4 (even, binary 1-positive, purple) have
the same color probe (a) but a different binary color
label (b). c, Cy7 image (fifth dye, so-called binary 2
image) shows the HPV-16 hybridization signals (arrows). d, DAPI image (used as a technical reference
image, not for banding purposes). e, Overlay of the
images in a and c reveals the chromosomal HPV integration sites. In this case, the integration of HPV-16 is
near the translocation region of chromosomes 3p (red)
and 14 (blue), as indicated by arrows. In all panels, the
boxed chromosomes represent derivative chromosomes
14 [t(3;14)] involved in translocation and HPV integration.
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HPV INTEGRATION IN CERVICAL CARCINOMA CELL LINES
Fig. 2. COBRA-HPV FISH analysis using relevant full-length HPV probes on cervical cancer cell lines. HPV-16 is present in episomal form in the interphase nuclei of CSCC-1. a, DAPI
image. b, HPV signals. One analyzed metaphase was near-octaploid (c) and contained four chromosome 14q copies with HPV-16 integration signals (inset, d, arrows). e, DEAC image. f, DAPI
image. g, representative metaphase spread with scattered extra-chromosomal HPV signals (arrows). h, HPV-45 integration on two copies of chromosome 3q (arrows) in CC-8. i-j, HPV-45
integration on two copies of chromosome 1q (arrows with p) and on two copies of derivative chromosomes 22 [t(1q;22q)] near the translocation junction (arrows) in CC-10A and CC-10B.
k, HPV-67 integration on one copy of derivative chromosome 3 [t(3p;13q)] near the translocation junction (arrow) in CC-11. An interphase nucleus (below) shows one HPV-67 integration
signal.
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HPV INTEGRATION IN CERVICAL CARCINOMA CELL LINES
Fig. 3. Southern blot analyses. Total genomic
DNAs from cell lines or primary tumors (pt) at
early and late passages were digested with EcoRI
(cuts twice in HPV-16 and not in HPV-45 and
HPV-67), HindIII (cuts once in HPV-45 and
HPV-67 and not in HPV-16) and PstI (multiple
cuts in HPV-16, HPV-45, and HPV-67). Blots were
hybridized with HPV-16 (A and B), HPV-67 (C),
and HPV-45 (D and E). Size markers are indicated
in kilobases. Line with circle, ;8 kb. Dotted line,
;7.3 kb. Restriction patterns in early (or pt) and
late cultures were identical for CSCC-7, CC-11,
CC-8, and CC-10A&B, indicating that HPV integration status, as defined by multicolor FISH, is
unchanged during culturing. The pattern for
CSCC-1 changes from early to late culture passage
(A) in which episomal HPV was found by multicolor HPV FISH.
of events, i.e., the causal relationship between HPV integration and
genomic rearrangements during malignant transformation. For this
purpose, HPV-immortalized keratinocytes might be suitable models
for the study of sequential stages of malignancy with application of
the methodology presented here.
Southern blot results indicated that the HPV integration status in all
of the present cell lines, except for CSCC-1, is representative of the
HPV integration status in the original primary tumors. Thus, although
gross numerical changes occur in most cell lines through endoreduplication and chromosomal losses (Table 4), the physical HPV status
is unchanged. This was also demonstrated by the multiplication of
integration sites seen in the metaphase spreads (Fig. 2).
In CSCC-1, HPV-16 was predominantly present in episomal
form. The changes in the HPV-16 restriction pattern in CSCC-1
from early to late culture passages (Fig. 3A) suggest that the
physical status of HPV sequences changed during culturing. From
these data, it is not clear whether this is caused by a change in
nature and/or relative contributions of episomal and/or integrated
HPV DNA. Integrated HPV-16 was found at chromosome 14q in
only one metaphase spread, which contained about four times as
many chromosomes as the other metaphases analyzed (Fig. 2a).
The DNA index of CSCC-1, which was measured at the same
passage numbers as was used for the preparation of chromosome
spreads, was in the triploid range (Table 4). This suggests that the
octaploid cells in which integration was found probably represent
a negligible subpopulation. The changed restriction pattern may,
however, be explained by an aberrant physical nature of the episomal HPV-16 DNA, as has been reported in a cervical carcinoma
cell line harboring a 10-kb HPV-16 genome that was maintained as
an extrachromosomal episome (46). The presence of extrachromosomal HPV DNA has been reported in cervical cancers and a few
other cervical cancer cell lines (13, 14, 46, 47) and seems restricted
to HPV-16 DNA. This is in contrast with the general idea that
oncogenic HPV types integrate in cervical neoplastic lesions as
they acquire an invasive character. Viral integration enhances the
expression of E6/E7 mRNAs, presumably by disrupting the HPV
E2 gene, which encodes a transcriptional repressor of the E6/E7specific promoter (15). Integration events may thus be required for
the continued expression of the E6/E7 oncogenes and consequent
genomic instability. The presence of high copy numbers of extrachromosomal HPV DNA, however, may lead to lower but sufficient levels of E6/E7 mRNAs in comparison with cells that contain
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HPV INTEGRATION IN CERVICAL CARCINOMA CELL LINES
fewer, but integrated HPV copies (47). It will be interesting to
further investigate these aspects in CSCC-1 at consecutive passages.
Well-known HPV-16- or HPV-18-containing cell lines such as
HeLa, SiHa, and CasKi (48 –50) have been used in many studies
world-wide and have contributed a great deal to our understanding of
HPV-induced pathogenesis. Because of their establishment, a number
of new cell lines, including mostly HPV-16 and HPV-18-positive cell
lines, have been reported. Still, the relative difficulty to propagate
tumor cells in vitro (51, 52) has limited the representation of various
HPV types and histological backgrounds. Although HPV-16 and
HPV-18 are present in the majority of cervical cancers in the Western
world, other HPV types may predominate in non-Western countries,
where cervical cancer is even more abundant (2, 53). It is in this
context that we point out that four of the five primary tumors from
which the present cell lines were derived were from Surinamese
patients, whereas only 26% of the 74 primary tumors were of Surinamese origin. Although we did not systematically assess the variables that may have contributed to the culture success of Surinamese
tumors, one explanation may be the relatively large tumor size observed in these patients (3) and consequently, the increased amount of
tumor material usually brought into initial culture. These patients also
appear to present with non-HPV-16 or HPV-18 at a 2-fold higher
frequency than the Dutch population (3). In the present study, 14% of
the Dutch primary cancers were non-HPV-16/18 versus 35% among
the Surinamese cancers, which may have contributed to the establishment of four non-HPV-16/18-positive cell lines.
To date, only one HPV-45-positive cell line has been described
(54). CC-11 is the first known cell line with HPV-67. This HPV was
only recently cloned from a vaginal intraepithelial neoplasia grade I
and identified as a new HPV type phylogenetically clustered with
HPV-16, HPV-31, HPV-33, HPV-35, HPV-52, and HPV-58 (55).
This new set of cervical carcinoma derived cell lines, with localized
integration of HPV-16, HPV-45, and HPV-67, will be useful tools for
further investigations of HPV-related cervical carcinogenesis, especially to advance the identification of cellular target sequences for
HPV-integration.
ACKNOWLEDGMENTS
We thank Dr. T. Matsukura for kindly supplying the HPV-58 and HPV-67
DNA probes and Dr. H. Zur Hausen for supplying the HPV-16, HPV-18, and
HPV-45 DNA. We are grateful for the technical support of Sandra Uljee in
HPV typing, and of Yvonne Zomerdijk-van Nooyen in the Southern blot
hybridizations. We thank Marcel Jacobs and Jan Walboomers (Free University
Hospital, Amsterdam) for generously providing additional HPV typing. We
further thank Nel Kuipers-Dijkshoorn for technical assistance and Willem
Corver and Cees Cornelisse for advise concerning the ploidy analyses.
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Recurrent Integration of Human Papillomaviruses 16, 45, and
67 Near Translocation Breakpoints in New Cervical Cancer Cell
Lines
Louise A. Koopman, Károly Szuhai, Jaap D. H. van Eendenburg, et al.
Cancer Res 1999;59:5615-5624.
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