Preferential sites for viral integration on mammalian genome

IOTH ANNIVERSARY ARTICLE Preferential Sites for Viral Integration on Mammalian Genome Nicholas C. Popescu and Joseph A. DiPaolo

ABSTRACT: Chromosomal localization of human papillomavirus (HPV) 16 and 18 on human cervical carcinomas and epithelial cell lines obtained after HPV transfection has uncovered a nonrandora association of viral integration and specific genome sites. Fragile sites appear to be preferential targets for viral integration because of their structural and functional characteristics through which chromosomal anomalies, alterations in protooncogene activity, and gene amplification can occur. Individually or in association, such changes lead ta the acquisition of an unlimited cell growth potential but not tumorigenicity. Genetic instability and uncontrolled cell division resulting from HPV integration increase the cell's susceptibility to other exogenous carcinogenic factors that may complete the process of neoplastic development.

INTRODUCTION DNA- and RNA-containing oncogenic viruses have been implicated in the i n d u c t i o n of certain h u m a n cancers either as causative agents or cofactors [1-5]. The tumors and cells transformed in vitro by oncogenic viruses have integrated viral sequences and express viral antigens [6, 7]. In the case of RNA viruses, complementary DNA resulting from viral RNA transcription [8] is covalently integrated into the cellular DNA [9]. Integration of the viral genome into the cellular DNA can have repercussions on the initial stages of cell transformation and in the perpetuation of the transformed phenotype as well as in tumor progression. The integrated viral genome may lead to the production of proteins with transforming activity that cause cell transformation [7]. A n alternative consequence of viral integration may be the acquisition by the host genome of viral transforming genes with a new regulatory mechan i s m involving either the loss of viral regulatory mechanism or the addition of new ones of cellular origin. In the latter case, an alteration of cellular genes may result from the integrated viral sequences either as a consequence of the viral insertion per se or by the effect of viral regulatory elements on nearby cellular genes. The chromosomal localization of viral integration sites can provide clues for the identification of molecular alterations that have been triggered by the interaction of viral sequences with cellular genes, particularly growth regulatory genes. A central question is whether the viral genome integrates at specific chromosome sites. In early studies, r o d e n t - h u m a n cell hybrids that segregate h u m a n chromosomes were used to assign the viral integration to i n d i v i d u a l chromosomes [6]. Later, analysis of in situ hybridization results led to the recognition that this method was the most direct approach for localizing viral integration in chromosomes [10]. Innovation of chromoFrom the Laboratoryof Biology,NationalCancer Institute, Bethesda, Maryland. Address reprint requests to: Dr. Nicholas Popescu, Department of Health, Education. and Welfare, National Cancer Institute, Bldg. 37, Room 2A13, Bethesda, MD 20892. Received April 14, 1989; accepted May 5, 1989.

157 © 1989 ElsevierScience PublishingCo., Inc. 655 Avenue of the Americas, New York, NY 10010

Cancer Genet Cytogenet42:157-171 (1989) 0165-4608/89/$03.50

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N.C. Popescu and J. A. DiPaolo some h y b r i d i z a t i o n by Harper and Saunders [11], followed by further refinement by b a n d i n g c h r o m o s o m e s through the autoradiographic e m u l s i o n [12, 13], m a d e possible not only the m a p p i n g of single copy genes but also the localization of viral sequences on transformed and tumor cell chromosomes. SV40 has been the most extensively studied of the DNA oncogenic viruses because of its ability to transform rodent cells and induce fibrosarcomas [6]. Chromosomal localization of SV40 has been determined in several transformed lines, and the results obtained by Croce and his coworkers using somatic cell hybrids have been c o m p r e h e n s i v e l y r e v i e w e d [6]. The integration pattern of the E p s t e i n - B a r r virus (EBV), i m p l i c a t e d in the d e v e l o p m e n t of nasopharyngeal carcinoma and certain human l y m p h o m a s and hepatitis B virus (HBV), which is suspected to be causative in h u m a n h e p a t o c e l l u l a r carcinoina [10], has also been determined by in situ hybridization. The data concerning these viruses was reviewed by Henderson several years ago

[10]. In the past few years h u m a n papillomaviruses (HPV), a group of DNA viruses, have received increasing attention because some of them are associated with genital cancer [4, 5]. Certain HPVs persist as extrachromosomal DNA, While DNA of other HPV types such as 16 and 18 usually are integrated into cellular genome of invasive carcinomas [5]. Studies originating from this laboratory were the first p u b l i s h e d accounts using a recombinant technique stating that HPVs associated with invasive cancer could i m m o r t a l i z e normal foreskin-derived epithelial cells, whereas HPVs associated with benign conditions or not associated with cervical cancer were ineffective [14-18]. Only the cell strains in w h i c h the HPV was integrated into the cellular DNA evolved into permanent lines, showing that the genetic alterations associated with the viral integration are necessary for the acquisition and maintenance of c o n t i n u o u s growth [16, 17]. The localization of HPVs in h u m a n cervical cancers and in foreskin keratinocyte and cervical cell lines immortalized by recombinant viral DNA revealed cellular and molecular alterations involving certain genomic regions and genes. Their significance to the process of neoplastic d e v e l o p m e n t will be discussed in an attempt to assert the specificity of viral integration into the h u m a n genome and to evaluate the consequences of HPVs and other viruses integration to carcinogenesis.

CHROMOSOMAL ASSIGNMENT OF HPV-18 INTEGRATION IN CERVICAL CARCINOMA CELL LINES

Schwarz and coworkers e x a m i n e d the structural organization and transcription of HPV-18 in three cervical carcinoma cell lines: HeLa, a w i d e l y used cell line, and two relatively n e w l y isolated lines, C41 and SW756 [19]. All of these lines have HPV-18 DNA integrated into their cellular genome. Whereas C4-1 cells were estimated to contain an average of one copy of HPV-18 DNA per cell, in HeLa and SW756 cells, m u l t i p l e copies of viral DNA were detected. The patterns of blot h y b r i d i z a t i o n of cellular DNA with HPV-18 DNA suggested that viral DNA was integrated into more than one site in HeLa cells and at a single site in SW756 and C4-1 cells [19]. Subsequent m a p p i n g by in situ c h r o m o s o m e hybridization and somatic cell hybrids using viral DNA probes and probes from cellular sequences flanking integrated HPV-18 DNA d e m o n s t r a t e d single and dispersed HPV-18 integration sites in these three cervical carcinoma cell lines [20-25]. In our laboratory, chromosomes derived from HeLa (ATCC CCL-2) (Fig. 1) were h y b r i d i z e d with a radiolabeled HPV-18 probe and, consistent with the blot h y b r i d i z a t i o n pattern, four sites of integration were identified [21]. Two sites were localized on normal chromosomes 8 and 9 at bands q 2 3 - 2 4 and q 3 1 - 3 4 , respectively. The other sites were on an abnormal c h r o m o s o m e 5 at

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p 1 1 - 1 3 and on an abnormal chromosome 22 at q12-13 (Fig. 2). Using the same approach, Minceva et al. identified only one HPV-18 site of integration at 8q24 in HeLa cells, even though the grain distribution on chromosome 9 was suggestive of a second hybridization site (Fig 5c in [23]). Ambros and Karlic utilized a more sensitive nonisotope in situ hybridization technique in conjunction with reflection contrast microscopy and confirmed our localization to 8q24 to an abnormal chromosome 22 and to a third site on an unidentified abnormal chromosome [24]. In contrast, as demonstrated with flanking cellular sequences on cell hybrids [20] and in situ hybridization of viral DNA, SW756 cells had multiple copies of HPV-18 DNA integrated at a single site on chromosome 12 (q11-13) [22]. Similarly, a single HPV-18 integration site was mapped to region 3p21 in C4, a primary cervical carcinoma, and to region 8q21-22.3 in a C4-1 derived cell line. However, it was not indicated whether the hybridization site was on the normal chromosome 8 or on a rearranged 8;12 chromosome characteristic for this line [25]. Durst and collaborators assigned HPV integration sites in two other cervical carcinoma cell lines designated Caski and SiHa, as well as in a primary cervical tumor [20]. This work indicated that Caski cells may have fewer integration sites than had been previously reported [23[, and these were localized on the terminal region of the long arm of chromosome 8, on chromosome 12, and on #20 [20]. The SiHa cells had HPV-16 integrated at 13q14-31, and in the primary cervical carcinoma studied viral sequences were identified on chromosome 20 (pter-q13) and chromosome 3 (p25ter) [20]. These results demonstrate that three of six cervical carcinoma cell lines have viral DNA integrated onto chromosome 8. A similar trend for integration is

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O Figure 2 An HeLa metaphase after in situ hybridization with HPV-18 DNA (A) and G banding (b) exhibiting a label on chromosome 8 at the end of the long arm, on the short arm of an abnormal chromosome 5, and on the long arm of an abnormal chromosome 22 (arrows). The abnormal chromosomes are M6 and M10 in Figure 1.

apparent for HBV, as five hepatomas with HPV integrated on chromosome 11 have been reported, a figure considerably higher than would be expected from r a n d o m distribution [26].

FRAGILE SITES AS PREFERENTIAL TARGETS FOR HPV VIRAL INTEGRATION The current data on viral localization in cervical cancer cells rule out a u n i q u e integration site for HPV and are not sufficient to establish chromosome specificity for integration. However, the search for HPV integration in cervical carcinoma cell lines has led to the realization that certain genomic sites are preferential targets for viral integration. This was first documented in our HPV-18 m a p p i n g of HeLa cells; all of the integration sites coincided with the location of a fragile site [21] (Table 1). This correspondence was not a peculiar case for HeLa cells, as HPV-18 sites of integration in SW756 and C4-1 cell lines were also near fragile sites [22, 25] (Table 1). Furthermore, the location of four other DNA-containing oncogenic viruses relative to fragile sites showed that 65% of the viral integration sites currently mapped are at the same bands as fragile sites, 20% w i t h i n one band, and 15% of the integration sites do not overlap with the position of a fragile site (manuscript at preparation). Recently a new case was added to these statistics. A Burkitt l y m p h o m a (BL) cell line deriving from a North American patient exhibited an 8;22 translocation usually seen at a lower

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Table 1

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oOnly in HeLa, SW756,and C4-1cell lines were HPV integration sites localized by in situ hybridization at specific chromosomebands.

number of BL [27]. On conventionally stained chromosomes an achromatic region indicative of EBV integration [28, 29] was identified on the short arm of chromosome 2 (Fig. 3). The abnormality occurred at region 2p11-14 and coincides with the location of a fragile site at 2p13. Preliminary evidence indicates EBV integration at this site. DNA replication is required for viral integration, as demonstrated several years ago by Varmus and colleagues [30]. Recently, Laird and coworkers, reviewing the data concerning the fragile sites in humans, Drosophila, and Microtus, pointed out that these regions are constitutively or inducibly late replicating [31]. It is conceivable, that, because of their replication pattern, at a certain point in the cell cycle fragile sites may be the only replicating regions available for the entry of viral DNA. The chromatin composition of these regions is, however, also a determining factor for viral integration. Yunis and colleagues demonstrated the capacity of chemicals that induce fragile site to interact with DNase I hypersensitive regions [32, 33]. By taking this further, Le Beau emphasized that hypersensitive sites are generally near the 5' end of the active gene, flanking the promoter and enhancer regions, and she added that like fragile sites, hypersensitive DNAase I regions tend to occur at Giemsa-negative bands [34]. Interestingly, the nonrandomness of chemical carcinogen-induced chromosome damage at Giemsa-negative bands was reported by us in the 1970s [35]. Most importantly, hypersensitive DNase I sites appear to be targets for retroviral DNA integration [36-38]. Structural and functional characteristics of chromatin may, therefore, determine regional specificity for viral integration. In addition to its relevance to the carcinogenesis process, which will also be discussed, the association between oncogenic viruses and specific genomic sites may have implications in the future strategy of human genomic mapping and sequencing. This idea was advanced by Siniscalco, who proposed an experimental approach of rescuing the DNA regions flanking the sites of viral integration for sequence analysis [39].

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Figure 3 A conventionally stained metaphase from a North American Burkitt (NAB-2) lymphoma cell line. An achromatic lesion on the short arm of chromosome 2 is indicated by an arrow.

INTEGRATION OF HPVS NEAR PROTOONCOGENES IN CERVICAL CARCINOMA CELL LINES All four of the HPV-18 integration sites on HeLa cells mapped by in situ hybridization were found to be near the location of a protooncogene. On normal chromosomes 8 and 9, viral sequences were localized near the m y c and abl protooncogenes, respectively [21] (Table 1). On abnormal chromosomes 22 and 5, the viral sequences were localized in the vicinity of sis and MLV12 protooncogenes, respectively [21]. This correspondence was suggestive of an interaction of viral DNA with genes k n o w n to be involved in cell growth regulation and tumorigenesis. Evidence for such an interaction has been provided by Durst and collaborators who localized HPV-18 sequences w i t h i n 40 kb 5' of the m y c gene by somatic cell hybrids and detected a higher level of c - m y c mRNA [20]. Most significantly, increased m y c expression was only detected on those cervical carcinoma ceil lines in which HPV-18 integrated onto chromosome 8 [20]. These results show that HPV integration caused cis-activation of nearby cellular protooncogenes. Integration of other viruses such as HBV have similar consequences on nearby genes, as demonstrated by Dejean and colleagues in a hepatocellular carcinoma [40]. In addition, HBV was found near a liver cell sequence homologous to the v-erb-A gene that becomes inappropriately expressed due to viral integration [40]. Furthermore, insertional activation of protooncogenes by a DNAc o n t a i n i n g virus has recently been documented in two hepatocellular carcinoma i n d u c e d in woodchucks chronically infected with woodchuck hepatitis virus [41].

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Insertional activation of the m y c gene by this hepatitis virus differs from that comm o n l y caused by retroviruses in B- and T-cell l y m p h o m a [41]. The HPV-18 integration site on chromosome 8 of HeLa cells was examined in detail, and concomitant with an increase level of m y c mRNA in the absence of structural gene alterations, amplification of HPV-18 viral transforming genes was observed [42]. The E6, E7, and E1 open reading frames were found amplified fivefold, and the late viral DNA region, viral long control region, and cellular flanking sequences were amplified 15-fold [42]. In addition, a putatively new sequence designated pal-1 (papilloma-associated locus-l) was identified near the m y c locus [421. The biologic significance and the transforming potential of the new sequences remains to be elucidated. Transfection of NIH 3T3 cells with DNA from D98 cells, a HeLa variant, did not induce malignant transformation of murine fibroblasts [43]. In our laboratory, Bloom's cells transfected with DNA from HeLa (ATC-CCL-2) developed foci at a comparable frequency and morphologically indistinguishable from those observed after transfection with Harvey murine sarcoma virus genome 144] ( u n p u b l i s h e d results). The development of foci in these cells after transfection with DNA from HeLa cells and the identification of pal-1 sequences indicate the presence of a transforming gene in this cell line. Nevertheless, viral tagging remains a useful approach for identifying genes involved in neoplastic development [45J. C4-1 and SW756 cell lines are additional examples of cervical malignancies with HPV-18 integrated near protooncogenes [22, 25]. In the C4 primary tumor HPV-18 was integrated near erb A-2 proto-oncogene [25] (Table 1). After we reported the localization of multiple HPV-18 copies at a single site on chromosome 12q11-13 in SW756 cells [22], int-1 protooncogene isolated from a mouse mammary tumor and g/i, a gene isolated from a h u m a n glioblastoma, was mapped within the same region of chromosome 12 [46, 47] (Table 1). In addition, the h u m a n homeobox gene C8 and two type II keratin genes have also been localized to the q11-13 on chromosome 12 [48, 49]. The influence of HPV-18 integration on nearby protooncogenes or other genes k n o w n to be involved in cellular differentiation remains to be examined. There is a distinct possibility that HPV integration into the SW756 genome may have caused a chromosome deletion at 12q11-13. A closer examination of chromosome 12 has revealed a discrete loss of chromatin at the integration site (Fig. 4). In hepatocellular carcinoma deletions, duplications and translocations are frequent alterations associated with HBV integration. Rogler and coworkers identified a deletion on the short arm of chromosome 11 in a hepatocellular carcinoma at the site of HBV integration [50]. Most significantly, Wang and Rogler reported loss of heterozygosity of one or more markers on the short arm of chromosome 11 in six out of 14 hepatocellular carcinomas [51]. Deletion of a DNA segment without its reduplication was considered the most likely m e c h a n i s m of this viral-induced genomic alteration [51]. N o n r a n d o m integration of HBV on chromosome 11 and the resultant deletions are also important

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Figure 4 A chromosome 12 from a SW756 cervical carcinoma cell with a label on the long arm at q12-13, the assigned HPV-18 site of integration (a). Two other chromosomes 12 are shown, one with an apparent interstitial deletion at the HPV-18 integration site (b).

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N.C. Popescu and J. A. DiPaolo for gene balance as this chromosome is believed to contain malignancy suppressor genes [52, 53]. Although alterations associated with viral integration identified in tumors cells could lead to malignancy, the role of viral integration to the neoplastic development can be evaluated only in experimental models, utilizing normal cells in which stages of neoplastic process are distinguishable.

CHROMOSOME CHANGES A N D HPV INTEGRATION IN H U M A N FORESKIN KERATINOCYTES A N D EXOCERVICAL EPITHELIAL CELLS

To define the role of HPV integration in neoplastic development, a model utilizing epithelial cells was required. Such a model has been developed in our laboratory for both foreskin keratinocytes and exocervical epithelial cells, and it has been indispensable in the understanding of the events related to HPV-induced proliferation of human tissue. A recombinant HPV-16 DNA containing a head-to-tail dimer of the full-length HPV-16 genome and the selectable marker [G418) was used to transfect human keratinocytes and exocervical epithelial cells [14-18]. As a result of HPV-16 transfection, several cell lines with an indefinite growth capacity were obtained. The phenomenon was consistently reproducible and occurred with a high frequency independent of the genetic characteristics of the host [16]. Southern blot DNA hybridization with HPV-16 invariably showed that only cell strains having viral sequences integrated into the cellular genome escaped terminal differentiation and exhibited an unlimited growth potential [16, 18]. A variety of chromosome alterations, breakage, pulverization, dicentrics, and multicentrics were observed after HPV-16 DNA transfection and G418 selection of human keratinocytes. The severity of such damage, reflecting an outgoing genomic instability, diminished within several passages as a population with stable structural alterations emerged. This was coincidental with a simplification of the HPV-16 DNA integration pattern as indicated by Southern blot hybridization [16, 54[. Five cell lines examined by G banding at various stages of progression in culture had an abnormal chromosomal constitution. Three lines exhibited complex structural rearrangements involving several chromosomes or chromosome segments, partial or complete duplications, and achromatic lesions [54]. Two lines showed relatively less rearrangement of the karyotype. In terms of numerical deviations, the loss of chromosomes 19 and the gains of chromosomes 9 were observed in three and two lines, respectively. Structural alterations nonrandomly involved chromosomes 1 and 20. Most significantly, all lines had chromosomes with homogeneously staining regions (HSR) and double minutes (drain) [54]. These special classes of alterations, first reported by Biedler and Spengler in Chinese hamster cells [55], reflect gene amplification as discovered by Schimke [56] and are commonly associated with tumor progression or an acquired resistance to chemotherapeutic agents. None of these HPV16-immortalized keratinocyte lines, however, were tumorigenic in nude mice, showing that abnormalities indicative of gene amplification can also occur in nonneoplastic cells at relatively early stages of cell transformation. Until now, a correlation between these types of chromosome changes and nonmalignant cells had only been suggested [57, 58]. The formation of HSR and dmin in nontumorigenic cell lines shows that amplification of cellular genes, including growth regulatory genes, may provide a selective advantage to the cells for continuous multiplication, a requirement not only for tumor progression but also for immortality. Other structural alterations may also be relevant to the process of transformation by HPV. Two cell lines exhibited alterations of the long arm of chromosome 1 resulting in a duplication of the q31-41 region. Sandberg, in a comprehensive and most informative work on cancer cytogenetics, provided several examples of alterations to the long arm of

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c h r o m o s o m e 1, m a i n l y partial d u p l i c a t i o n s in a variety of malignancies [591. In cervical cancer, however, changes in chromosome 1 precede or are coincidental with stromal invasion [60]. Furthermore, in certain cases, duplication of l q may be the only alteration [61]. Nowell and Croce pointed out that an imbalance of protooncogenes due to n o n r a n d o m d u p l i c a t i o n s of certain chromosome segments may be critical for oncogene dosage in some types of neoplasia because it confers selective growth advantage to the abnormal cells [62]. This is particularly relevant for chromosome 1, to w h i c h several protooncogenes have been mapped. Numerical and structural c h r o m o s o m e alteration in HPV16-immortalized keratinocyte lines show that i m m o r t a l i z a t i o n is associated with or mediated by chromosome changes. Other examples of c h r o m o s o m a l l y abnormal nontumorigenic keratinocytes lines have been reported. A keratinocyte line designated HPK-1A obtained after transfection with HPV16 had a h e t e r o p l o i d karyotype with three abnormal chromosomes: an isochromosome deriving from the long arm of #21, and two stable dicentrics originating from telomeric fusion [63]. An isochromosome 21 was also observed in an HPV16transfected keratinocyte line in our laboratory (unpublished results). Another keratinocyte line that escaped terminal differentiation and continued an exponential growth for more than 800 doublings was trisomic for chromosome 8 [64]. Chemical carcinogen treatment of these cells did not result in the acquisition of tumorigenicity [64]. Chromosome changes were also reported in SV40 transformation [65-68]. Chromosomes 8, 11,15, 16, 18, 19, and 21 tend to be frequently affected, and among these, the acrocentric c h r o m o s o m e s 15 and 21 were most often rearranged [68]. These studies demonstrate that in vitro transformation by DNA viruses is associated with a n e u p l o i d y and structural rearrangements. The relatively low number of cell lines analyzed cytogenetically, however, precludes any conclusions on the specificity of c h r o m o s o m e alterations associated with in vitro viral cell transformation. By in situ h y b r i d i z a t i o n of HPV-16, DNA sequences were localized on chromosomes from five i m m o r t a l i z e d keratinocyte lines [54]. Each line had a distinct integration pattern as shown by Southern blot analysis, as well as a different n u m b e r of copies of viral genome [16]. HPV-16 was found exclusively on abnormal chromosomes, at the sites of chromosomal translocations (Fig. 5), at achromatic regions or segments with t a n d e m d u p l i c a t i o n s (Fig. 6) as well as at HSR [54]. The analysis of DNA replication of the c h r o m o s o m e s from these lines by differential staining after 8 hours of incubation in the presence of 5-bromodeoxyuridine demonstrated that all sites of viral integration were late replicating. Incomplete chromatin c o n d e n s a t i o n and r e c o m b i n a t i o n are consequences of the replication junction that flank late-replication DNA [311 and can thus explain the formation of structural alteration due to the viral integration, particularly the formation of achromatic regions and HSR [54]. The localization of viral sequences at aberrant chromosome locations for the first time implicates viral integration in the induction of stable structural alterations [54]. It w o u l d seem most likely that abnormal chromosomes from HeLa cells with integrated HPV-18 occurred as a result of viral integration. Similarly, constricted or achromatic regions, deletions, duplication, and translocations associated with other viruses such as EBV, adenovirus-12, or HBV may also be the consequence of viral integration [69-73]. Consistent with HPV integration in cervical carcinomas, several but not all HPV-16 integration sites in keratinocyte lines were located near fragile sites and protooncogenes [54]. A l t h o u g h c h r o m o s o m a l changes and the integration of HPV-16 show striking similarities with cervical carcinomas, the immortal keratinocyte lines lack tumorigenic potential. The HPV integration can result in uncontrolled transcription of viral genes and can influence the balance of v i r u s - c e l l interaction [74], thus, increasing cell s u s c e p t i b i l i t y to other factors necessary for progression to a malignant pheno-

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F i g u r e 5 A G-banded karyotype from a human keratinocyte line obtained after transfection with HPV-16 DNA. Two abnormal chromosomes placed separately derived from chromosomes 16 and 20, respectively. The terminal segments of these chromosomes may derive from the long arm of chromosome 1. HPV-16 was detected by in situ hybridization on one of the abnormal chromosomes at the site of chromosome translocation (insert). F i g u r e 6 A metaphase with a near-triploid chromosome number from a human keratinocyte line obtained by transfection with HPV-16 DNA. After in situ hybridization with a radiolabeled HPV-16 DNA probe, viral sequences were found integrated on three abnormal chromosomes originating from a complex rearrangement and chromosome duplications (arrows).

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F i g u r e 7 A G-banded karyotype from HCX-16-2HR tumorigenic cell line. These cells had several consistent abnormal chromosomes; a deleted chromosome 17q, a deleted 19q, and three abnormal copies of chromosome 21 are indicated by arrows. One chromosome 21 has an enlarged satellite region, and the two other chromosomes 21 had a homogoneously staining region at the terminus of the long arm. M1 and M2 were partially derived from chromosome 20; their long arms are consistent with chromosome 7 and a deleted 11, respectively. M5, which is from a different cell, is an isochromosome derived from a deleted long arm of chromosome 1. ]Reproduced with permission Oncogene, The MacMillan Press Ltd. (75).]

type. T h i s w a s c o n c l u s i v e l y d e m o n s t r a t e d w i t h h u m a n e x o c e r v i c a l e p i t h e l i a l cells i m m o r t a l i z e d b y H P V - 1 6 D N A t h a t b e c a m e t u m o r i g e n i c o n l y after t r a n s f e c t i o n w i t h a n a c t i v a t e d H a - r a s o n c o g e n e [75]. A t u m o r i g e n i c cell line (HCX16-2H) e x a m i n e d w a s a n e u p l o i d , w i t h s t r u c t u r a l a l t e r a t i o n s f r e q u e n t l y s e e n in s p o n t a n e o u s s o l i d tum o r s (Fig. 7) i n c l u d i n g a n i s o c h r o m o s o m e o r i g i n a t i n g f r o m a d e l e t e d l o n g a r m of c h r o m o s o m e 1, a c h r o m o s o m e 17 w i t h a n i n t e r s t i t i a l d e l e t i o n of t h e l o n g a r m , a n d a c o m p l e x r e a r r a n g e m e n t i n v o l v i n g c h r o m o s o m e s 11 a n d 20 w i t h a n a p p a r e n t loss of t h e s h o r t a r m of c h r o m o s o m e 11. As a l r e a d y m e n t i o n e d , t h e b a l a n c e of g e n e s o n c h r o m o s o m e s I a n d 11 m a y be i m p o r t a n t for t h e e x p r e s s i o n of t u m o r i g e n i c i t y [52, 53, 62, 76, 77]. D e l e t i o n s of t h e s h o r t a r m of c h r o m o s o m e 3 m a y h a v e a s i m i l a r i m p l i c a t i o n i n s e v e r a l f o r m s of c a n c e r [78, 79, 80]. I n t e r e s t i n g l y , a S V 4 0 - t r a n s f o r m e d h u m a n e p i d e r m a l k e r a t i n o c y t e l i n e t h a t b e c a m e t u m o r i g e n i c after 46 i n v i t r o passages e x h i b i t e d a d e l e t e d c h r o m o s o m e 3 o n t h e s h o r t a r m [81]. HCX 1 6 - 2 H l i n e also e x h i b i t a b n o r m a l i t i e s of c h r o m o s o m e 21. T h e s h o r t a r m of c h r o m o s o m e 21 h a d

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an e n l a r g e d satellite region e x h i b i t i n g h e a v y silver nitrate staining specific for 18s and 28s r i b o s o m a l R N A genes i n d i c a t i v e of an increase dosage and activity of these genes, a n d the long arm had a rather large t e r m i n a l HSR (Fig. 7). The HPV-16 integration was e x a m i n e d in this line and, as d e m o n s t r a t e d w i t h k e r a t i n o c y t e s lines, viral s e q u e n c e s w e r e d e t e c t e d at aberrant c h r o m o s o m e locations on both the short and the long arms of c h r o m o s o m e 21. The integration site on the long arm of c h r o m o s o m e 21 was near the ets-2 p r o t o o n c o g e n e , thus p r o v i d i n g a n e w e x a m p l e of HPV integration near a p r o t o o n c o g e n e . S t u d i e s are in progress to d e t e r m i n e the i n f l u e n c e of viral i n t e g r a t i o n on the structure and e x p r e s s i o n of ets-2 p r o t o o n c o g e n e . A l s o u n d e r e x a m i n a t i o n is c h r o m o s o m a l l o c a l i z a t i o n of the transfected H a - m s o n c o g e n e . Specific c h r o m o s o m e changes h a v e been d e m o n s t r a t e d in several forms of c a n c e r [59], but o n l y 1% of the c y t o g e n e t i c data are c o n c e r n e d w i t h p r i m a r y e p i t h e l i a l t u m o r s , w h i c h r e p r e s e n t 80% of all h u m a n c a n c e r [82]. In general, c h r o m o s o m e c h a n g e s in c a r c i n o m a are m o r e c o m p l e x c o m p a r e d to those in h e m a t o l o g i c m a l i g n a n cies or sarcomas. T h i s c o m p l e x i t y and k a r y o t y p i c h e t e r o g e n i c i t y m a y obscure pathol o g i c a l l y r e l e v a n t p r i m a r y changes. C o n s e q u e n t l y , n e w l y d e v e l o p e d in vitro m o d e l s in w h i c h v i r a l l y i m m o r t a l i z e d k e r a t i n o c y t e or cervical e p i t h e l i a l cells can be conv e r t e d to t u m o r i g e n i c cells by o t h e r c a r c i n o g e n i c agents are useful not o n l y for s t u d y i n g c o c a r c i n o g e n e s i s but also for d i s c e r n i n g c h r o m o s o m e changes critical to cell i m m o r t a l i t y and p r o g r e s s i o n to m a l i g n a n c y .

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