[CANCER RESEARCH 50. 1316-1323, Februar} 15. 1990]
Integration of Human Papillomavirus 16 DNA and Genomic Rearrangements in
Immortalized Human Keratinocyte Lines
Nicholas C. Popescu1 and Joseph A. DiPaolo
Laboratory of Biology, National Cancer Institute, Bethesda, Maryland 20892
ABSTRACT
Five foreskin-derived keratinocyte lines, immortalized by transfection
of human papillomavirus (HPV16) DNA, were cytogenetically abnormal,
exhibiting numerical deviations and altered chromosomes due to translocations, deletions, achromatic lesions, or partial duplications. Further
more, all lines had cells with either homogeneously staining regions or
double minute chromosomes, alterations associated with malignancy or
drug resistance. None of these lines were tumorigenic in nude mice,
showing that such alterations which are a manifestation of DNA ampli
fication also occur in nonneoplastic cells. By in situ chromosome hybrid
ization, viral sequences were identified on abnormal chromosomes at the
junction of chromosome translocations, at achromatic lesions and within
homogeneously staining regions and duplicated chromosome segments.
Thus, for the first time in an experimental system, HPV16 integration
into the cellular genome was associated with the induction of a subset of
chromosome alterations. HPV16 integration that frequently occurred at
fragile sites and near protooncogenes may be a critical alteration which
confers a selective growth advantage and an indefinite proliferative po
tential to HPV-transfected cells.
INTRODUCTION
The development of models for studying the transformation
of the human epithelial cells from which most human adult
cancers arise permits the identification of relevant cellular and
molecular alterations in the neoplastic process. In our labora
tory immortalization of human keratinocytes after transfection
with HPV16 has recently been demonstrated to be as a repro
ducible phenomenon that occurs with a high frequency inde
pendent of the genetic characteristics of the host cells (1, 2).
Significantly only cell strains having HPV162 sequences inte
grated into the cellular DNA become permanent lines, showing
that genetic alterations caused by the viral DNA integration are
necessary for the acquisition and maintenance of continuous
growth (2). In identifying molecular alterations triggered by the
interaction of viral DNA with the cellular genome, chromo
somal localization of viral sequences is essential. Integration
sites were localized by in situ hybridization of a radiolabeled
HPV16 DNA probe to the chromosomes derived from human
keratinocyte lines immortalized by HPV16 DNA transfection.
The cytogenetic and in situ hybridization analysis revealed
that the five lines examined have a stable abnormal karyotype
with chromosome rearrangements and numerical deviations;
most importantly all lines had chromosomes with tandem du
plications, achromatic lesion, and either HSR or cells with
double minutes. The latter two types of abnormalities are
associated with malignancy or drug resistance and reflect am
plification of cellular genes (3-5). Single or multiple HPV16
DNA sequences were found at chromosome translocations,
within HSRs and duplicated segments or achromatic lesions,
showing that a portion of chromosome rearrangements includ-
ing HSR are associated with HPV16 integration into the cel
lular genome. Such genetic alterations may play an essential
role in the induction of immortality.
MATERIALS
AND METHODS
Cell Culture. HKc isolated from newborn foreskins by a collagenous
float technique were cultured in serum-free medium MCDB153-LB (6),
a modification of one described by Boyce and Ham (7).
Transfection and Selection of Transfected Cell. The recombinant
plasmili (pMPHVlód) used has the neomycin antibiotic resistance gene
for selection in mammalian cells and two head-to-tail HPV-16 copies
cloned at the BamHl site. In this double HPV16 construction, the LI
gene is intact. Recombinant HPV16 was transfected into HKc cells
using a modified calcium phosphate technique (6). Neomycin-resistant
colonies were selected with 100 ^g of G418/ml medium (1). Morpho
logical growth characteristics, the pattern of viral integration, and
keratins of the established keratinocyte lines have been reported (2).
Chromosome Banding of HKc HPV16 Immortalized Cell Lines. Con
fluent cultures were subcultured 1:5 and used 24 h later for chromosome
preparation. Colcemid (2 x 107M)was added for 4 h. Thirty min before
harvest the medium was removed and cells were incubated in 0.075 M
KCI. The cells were mechanically detached from the surface of dishes,
centrifuged, and fixed with methanol:acetic acid (3:1); the fixative was
changed three times before making slides. To obtain G-bands, chro
mosome preparations were stained with Giemsa or Wright stain after
trypsin pretreatment (8). Other slides were processed for C-banding or
nucleolar organizer silver staining. Chromosome number was deter
mined on 50 conventionally stained metaphases and 20 karyotypes
were examined for identification of structural or numerical chromo
some changes in each cell line.
In Situ Chromosome Hybridization. A HPV16 DNA probe was used
to assign the integration sites in Hkc HPV16 immortalized cell lines.
The DNA was labeled with all four ['Hjdeoxynucleoside triphosphates
to a high specific activity (2.7 x IO7cpm/^g) by using a nick translation
kit (Amersham Corp., Arlington Heights, IL). Chromosomes obtained
from exponentially growing cultures were treated with ethidium bro
mide (7.5 Mg/m') which reduces chromosome contration for the last 2
h of the total 4 h treatment with Colcemid before harvesting. The in
situ hybridization procedure for mapping single-copy genes was carried
out under stringent conditions of 50% formamide-2x standard salinecitrate (1 X SCC = 0.15 M NaCl, 0.015 M sodium citrate) at 42°Cfor
both hybridization and subsequent washings (9). Autoradiography and
G-banding for chromosome identification have been described previ
ously (9). The HPV16 DNA integration was determined after the
examination of a minimum of 30 labeled chromosome spreads for each
cell line.
RESULTS
Cytogenetic Constitution of Keratinocyte Lines. Two nontransfected epithelial foreskin cell strains, sister strains of those
used for HPV16 DNA transfection, had a normal karyotype at
the second and third passage after initiation of the cultures. In
contrast five immortalized lines examined 9-15 passages (100150 population doublings) (2) after HPV16 DNA transfection
Receivcd 5/22/89; revised 9/19/89; accepted 11/15/89.
and antibiotic selection had 60-90% of the cells with an abnor
The costs of publication of this article were defrayed in part by the payment
mal chromosome number and structural alterations (Table 1).
of page charges. This article must therefore be hereby marked advertisement in
The remaining 10-30% of the cells were aneuploid without
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1To whom requests for reprints should be addressed.
structural abnormalities. All cells with a diploid number had
2The abbreviations used are: HPV16, human papillomavirus type 16 DNA;
HSR, homogeneously staining regions; HKc, human keratinocytes.
structural changes. Three lines contained a mixture of cells with
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HPV INTEGRATION,
Table
1 Cytogenetic
CHROMOSOMAL
ALTERATIONS.
dala of human keratinocyte
AND CELL IMMORTALIZATION
lines obtained
by HPV16 DNA transfection
Chromosome''Cell
with
of
abnormal
abnormal
chromosomes
(markers)t(6?;8)(dup22-35;?;p25)%of
chromosomes80No.chromosomes7Abnormal
line"HKS/HPV16d-2Passage*14(30,
102)2N:4N60:40Gain8Loss11,
cells with
double minutes3
19,6»ofcells
t(22;?)(ql3;?)
t(4;?)(qter;?)
t(8;10)(p;q)
t(l;18;?)(dup q31-41;q23;?)
t(l;19)(dupq31-41;pl3.3)
t(l;HSR;16)(q31-41;HSR;pl3.3)
HKS/HPVI6d-4
10(26,70)
60:40
11,19
60
t(l;?;20)(q41-44;?;ql3.3)
HSR(14)(q?)
HKS/HPV16d-5
15(85)
HSR(20)(ql3.3)
0:100
12
19
80
dic(3;3)(3;q21)
HSR?(12)(q22-24)
HKS/HPV16d-6
9(13,65)
0:100
9
22
70
del(6)(q32)
°Cell lines obtained after transfection with HPV16 DNA that escaped cell senescence and exhibit continuous growth potential.
"Passage numbers after HPV16 DNA transfection and selection when lines were examined cytogenetically. Numbers in parentheses, additional intervals of
examination: data not shown.
c Ploidy level was determined on approximate counts of 100 spreads.
d Chromosome gain and loss relative to the ploidy level, chromosomes involved in rearrangements.
HKS/HPV16d-3
15(40)
70:30
90
a near diploid and a near-tetraploid chromosome number and
two lines had a single population with a tetraploid number
(Table 1). The most prodominant numerical deviations were
the loss of chromosome 11s and 19s in two and three lines,
respectively, and gain of chromosome 9s in two (Table 1). Each
line was reexamined either once or twice at more advanced
passages (Table 1). These analyses showed a stability of the
original abnormalities and an elimination of the cells without
structural changes (data not shown). The number of structurally
altered chromosomes and the complexity of rearrangements
varied; three lines (HKc/HPV16d-2-4) had several highly rear
ranged chromosomes that involved an entire chromosome, seg
ments of duplicated bands and a homogeneously staining region
or an achromatic lesion (Figs. 1-4; Table 1). The remaining
two lines (HKc/HPVd-5 and -6) exhibited fewer and less com
plex rearrangements (Figs. 5 and 6; Table 1). Alterations of
chromosomes 1 and 20 were identified most frequently and
nonrandomly involved regions Iq31-41 and 20q,21, respec
tively (Table 1). As a result of alterations involving chromosome
1, lines HKc/HPV16d-3 and -4 both had 3 or 4 copies of the
segment Iq31-41 (Table 1; Figs. 2 and 3). A common feature
in all lines was the presence of HSR or double minutes (Table
1).
UVI'16 Integration Site on Chromosomes of Keratinocyte
Lines. Each line had a unique integration pattern as revealed
by Southern blot analysis, that remained stable after prolonged
subculturing (2). Furthermore, the number of copies of viral
genome integrated into the cellular DNA varied between indi
vidual lines (2). Viral integration sites were localized by in situ
hybridization of HPV 16 DNA to chromosomes derived from
all five lines at approximately the same passages after HPV 16
DNA transfection and G418 selection (Table 1). Viral se-
H U
Fig. l. G-band karyotype with a near diploid
chromosome number from HKs HPV16d-2:
44,XY,-6.t(6?;8)(dupq22-35:?p;25),t(ll:?:14)
(ql3;?:q32).-19.
Ml and M2 originate from
translocations at the end of the short arm of
chromosome 8 and the end of the long arm of
chromosome 14, respectively.
1 » il II li I! ü
8
II
13
14
10
li16
IE
15
12
11
U
at
17
18
Ã
*
.f-••
I
19
9
20
M1
M2
1317
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HPV INTEGRATION.
CHROMOSOMAL
AND CELL IMMORTALIZATION
{) ï H
ft\K
Ã->H i) Ut
«
1
Fig. 2. G-band karyotype representative of
the near diploid population from Hks/
HPV16d-3
line. 46.XY,
t(4;?) (q35;?),
t(8;IO)(p;q). +9. t(l:18;?)(dup q31-41;q23;?)
t(l;19)(dup q31-41; p 13.3) Ml and M2 were
partially derived from chromosomes 18 and
19, respectively. The midsegment of Ml and
most of the long arm of M2 exhibit a banding
pattern consistent with a tandem duplication
of a segment from the long arm of chromo
some 1.
ALTERATIONS,
M
13
2
4*
"14
3
11
il
li
15
î*'$
20
M1
M2
>
17
n
it
19
II
16
22
21
18
i.
X
Y
K li
ffll
l K U Ml
Fig. 3. G-band karyotype of a hypodiploid
cell from HkcHPV16d-4 line. 43.XY.-7.-11,
t(l;HSR;16)(q31-41;HSR;pl3.3),
14(HSR),
I(l;?;20)(q41-44;?;ql3.3),
+22 Ml derived
from chromosome 16, translocated to a large
segment the only terminal part of which is
identifiable as lq31-44 (also see Fig. 4A for
comparison). The upper part of M 2 is a chro
mosome 20 while the long arm has a series of
uniformly spaced bands. An acrocentric has a
HSR longer than one-half of its length.
8
II
13
i
11
a
I
it
14
fiv.-.-
I
19
10
20
15
16
17
ili
22
u
18
X Y
M1
quences were integrated exclusively on abnormal chromosomes
letter M on the karyotypes from Figs. 1-6, whereas other
(Table 2; Fig. 7). The abnormal chromosomes having integrated
abnormal chromosomes are shown by arrows. HKcHPV16d-4
HPV16DNA sequences (indicated by dots) are designated with was the only line with a single grain at the site of hybridization;
1318
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HPV INTEGRATION. CHROMOSOMAL ALTERATIONS. AND CELL IMMORTALIZATION
Fig. 4. A, comparison of the G-banding of M l
(middle), chromosomes 16 (left) and 1 (right), re
spectively. The homology of the long arm of MI
and the long arm of chromosome 1 is limited to
the region lq31-44. The breakpoint of chromo
some 1 is a Iq25. In B, the C-banding of Ml
clearly confirnms the origin of the paracentromeric
region as deriving from chromosome 16 and also
shows that the long arm does not have heterochromatin below the centromere. Therefore, only the
distal end of Ml derives from the long arm of
chromosome 1; the remaining midpart may be a
homogeneously staining region (arrow).
i
«9
M1 1
B
ïf! i!
Ili II
fi
t\
um m un m
10
8
13
IM!
iti
14
lui
15
IX
19
Fig. 5. A G-band karyotype from Hkc/HPV16d-5
region of the long arm. q 13.3.
il
rt u
11
fi
mi
16
12
17
AMA
»I
21
22
18
line with a near tetraploid chromosome number. Two copies of chromosome 20 (Ml) have HSR at the telomeric
in the other lines several grains indicative of multiple copies of segments tentatively designated as parts of the long arm of
chromosomes 6q22-27 and llql3-24,
respectively (Table 2;
viral DNA clustered at one site or were dispersed over a longer
region.
Figs. 1 and la). Grains at the terminus of 14q32 were close to
In HKcHPV16d-2 specific hybridization was found on two the location of the atkl protooncogene (10) and a fragile site
at 14q32. Line HKcHPV16d-3 also had specific HPV 16 DNA
abnormal chromosomes (Ml and M2) at achromatic regions
connecting the telomere of chromosomes 8p25 and 14q32 with hybridization on two abnormal chromosomes (Fig. 7, b and c).
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HPV INTEGRATION,
J/Av
!/5
W> II*
CHROMOSOMAL
ALTERATIONS.
AND CELL IMMORTALIZATION
1
m\
Vyvv y/r¿
??R5 lilj
9
8
lili
lili
13
14
10
é«*f
12
1 1
M1
itti
15
16
18
USI
19
21
20
22
Y
Fig. 6. Modal G-band karyotype from line Hkc/HPV16d-6 with a near tetraploid chromosome number. The complement have three chromosome I2s with an
HSR or an achromatic lesion on the long arm below the positive band q21 and a stable dicentric resulting from an entire chromosome 3 and a deleted chromosome
3(delq2l).
Table 2 Integration sites of HPl'16 DNA in human keratinocyte lines
of grains
grains/site)Ml*22
(av.
lineHKc/HPV16d-2
Cell
grains524
Remarks44
(4.8)
(2.4)
HKc/HPV16d-3HKc/HPV16d-4
456175 36(2.8)33(1.2)
30 (2.3)
20 labeled metaphases
4NTwo
were
HKc/HPV16d-5HKc/HPV16d-6Total"
360400%63
(3.5)50
copies of M 1
were labeled
(5.4)M2
Two or more copies
of M I were labeled
a The number of grains was determined from a minimum of 30 hybridized
metaphases prior to and after banding for chromosome identification.
* The abnormal chromosomes (Ml and M2) with specific labeling are shown
in Fig. 7.
The origin of Ml and M2 was partially identified as deriving
from chromosomes 18 and 19. respectively (Fig. 2). The midsegment of Ml and most of the long arm of M2 exhibited a
banding pattern consistent with a duplication of Iq31-41 (Fig.
2). Grains were scattered throughout this region (Fig. 7, b and
c} which contains arg and trk protooncogenes as well as two
fragile sites (11, 12).1 In contrast to these lines, HKcHPVlod4 had HPV 16 sequences integrated at a single site on chromo
some Ml (Fig. Id). This marker was derived from the whole
3 M. Zanca et al., personal communication.
chromosome 16 and a large translocated segment the only
terminal part of which is identifiable as lq31-44 with the
breakpoint at Iq25 (Figs. 3 and 4). No positive heterochromatin
staining was observed by C-banding, showing that the midpart
of this long arm may be a HSR-like region (Fig. 4). Hybridi
zation consisting of single grains was detected at the site of
translocation between chromosome 16pl3.3 and the HSR-like
region (Fig. Id).
In lines HKcHPV 16d-5 and -6, multiple grains were observed
on a HSR at the terminus of the long arm of chromosome 20
and on the long arm of chromosome 12 at an achromatic and
a diffusively staining region, respectively (Fig. 7, e and/). The
HPV 16 integration site and HSR on chromosome 20 at band
ql3.3 are near the location of the ire gene (13). The integration
site at the achromatic lesion on chromosome 12 overlaps with
the location of a fragile site 12q24.
DISCUSSION
Transfection of normal human foreskin-derived keratinocytes
with recombinant HPV 16 DNA reproducibly leads to cell im
mortality, provided that the transcriptionally active viral se
quences are integrated into the cellular genome (2, 14). Vector
sequences alone were ineffective in extending the life span of
keratinocytes. Five HPV 16 DNA immortalized keratinocytes
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HPV INTEGRATION. CHROMOSOMAL ALTERATIONS. AND CELL IMMORTALIZATION
l *
•¿Â»
<
vJ
*
V
•¿Â¿
Fig. 7. Complete and partial metaphases from HKc/HPV16d lines after in xilu hybridi/ation with HPV16 DNA. Arrowheads, integration site of HPV16 on two
abnormal chromosomes from HKc/HPV16d-2 (a). The integration sites of HPV16 on the remaining cell lines (A-/) is shown on hybridized and Ci-banded
chromosomes. To facilitate the comparison HPV 16 integration sites are indicated by black solid dois on Figs. 1, 2, 3. 5. and 6.
lines exhibited numerical and structural cytogenetic alterations,
structural changes. Significantly,
HPV sequences of cervical
demonstrating
that immortalization
is associated with or me
carcinoma cell lines integrated on normal and abnormal chro
mosomes near fragile sites and protooncogene
locations (9, 16diated by chromosome changes.
DNA and RNA viruses, like chemical and physical carcino
19), causing activation of cellular oncogenes (19. 20). However,
gens, are clastogenic (15). Chromosome
and chromatid break
it cannot be ruled out that expression of HPV gene products is
age, chromosome
pulverization,
and dicentric or multicentric
also a contributing
factor leading to chromosomal
anomalies
chromosomes
as well as endoreduplications
were observed at
or affecting protooncogene
expression. Certain integration sites
the first subpassage after HPV 16 DNA transfection and G418
mapped here are also close to fragile sites and protooncogenes.
selection of human keratinocytes (data not shown). The number
Fragile sites are induced by chemicals that interact with DNase
I-hypersensitive
chromatin regions (12). These regions are se
of cells with such damage reflecting an ongoing genomic insta
bility decreased, and cultures eventually stabilized as popula
lective sites for retroviral integration (21, 22). Common fragile
tions of aneuploid cells with or without structural changes
sites are constitutively,
or are induced to be, late replicating;
rare fragile sites are considered by some investigators to be cisemerged. This transition was also reflected by a simplification
of HPV 16 DNA integration pattern (2).
acting alterations to DNA leading to late replication (23). The
In situ localization of HPV16 DNA in these cell lines impli
staining pattern of the HPV 16 integration sites after 8 h of
incubation in the presence of 5-bromodeoxyuridine
invariably
cates the integration of viral DNA to the formation of persistent
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HPV INTEGRATION.
CHROMOSOMAL
ALTERATIONS,
showed that these regions replicate late together with the heterochromatic regions of chromosomes 1, 9, and 16. Incomplete
chromatin condensation and recombination are consequences
of the replication junction that flank late replication DNA (23)
and can explain the origin of the structural alterations observed
here, particularly the formation of diffuse staining regions and
HSR. The only consistent alteration associated with EpsteinBarr virus in Burkitt lymphomas and adenovirus 12-infected
cells was a constricted and/or achromatic region (24, 25). In
hepatocellular carcinoma, deletions, duplications, and translo
cations are frequent alterations associated with hepatitis B virus
integration (26-29). Structural alterations in HPV 16 immor
talized lines were diverse and may have a distinctive role in the
transformation process. For example, partial trisomy of the
long arm of chromosome 1 (Iq31-41) was observed in lines
HKcHPV16d-3 and -4. Alterations of the long arm of chro
mosome 1, mainly partial duplications of Iq, have been identi
fied in a variety of malignancies and occur nonrandomly in
cervical cancer preceding or coincidental with stromal invasion
(30, 31). Furthermore, in certain cases Iq duplication may be
the only alteration (32). Several protooncogenes are located on
chromosome 1 and an imbalance of this chromosome material
may be critical for oncogene dosage in certain types of neoplasia
that may confer selective growth advantage to the abnormal
cells (33). The significance of the predominant alterations oc
curring near the atk-\ (HKcHPV16d-2-4) (Figs. 1 and 3) and
the src (HKcHPV16d-5 and -6) (Figs. 3 and 5) protooncogenes,
respectively, or on chromosome 12, where no protooncogenes
are located at the site of the achromatic lesion (HKcHPV16d6) (Fig. 6) is unknown.
The abnormalities common to all of the lines studied were
the HSR and the double minutes both of which reflect DNA
amplification. Amplification of DNA, first reported in leukemic
cells developing resistance to methotrexate (34), has been
shown, however, to occur in untreated cancers as well as in cells
possessing some properties of neoplastic growth (35, 36). This
is conclusively demonstrated here because none of these lines
were tumorigenic in nude mice. Several models implicating the
inhibition and overreplication of DNA, the induction of acentric
fragments, and the formation of inverted duplications have
been proposed as initial steps in gene amplification (37-44).
Isolation and characterization of the flanking sequences of
HPV 16 integrated amplified regions in keratinocyte lines may
provide new insight to the mechanism of DNA amplification.
The presence of amplified DNA regions in HPV16 immortal
ized lines is consistent with the concept that amplification of
cellular genes including protooncogenes provides a selective
advantage for continuous cell division, a requirement not only
for tumor progression but also for immortality.
10.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
ACKNOWLEDGMENTS
26.
The authors thank Drs. June Biedler and Barbara Spengler for their
most valuable opinions on the chromosome abnormalities in these
lines.
27.
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Integration of Human Papillomavirus 16 DNA and Genomic
Rearrangements in Immortalized Human Keratinocyte Lines
Nicholas C. Popescu and Joseph A. DiPaolo
Cancer Res 1990;50:1316-1323.
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