GENES. CHROMOSOMES & CANCER 5:150-157 (1992)
Viral Integration and Fragile Sites in Human Papillomavirus-Immortalized Human Keratinocyte Cell Lines Patricia P. Smith, Cynthia L. Friedman, Eileen M. Bryant, and James K. McDougall Fred Hutchinson Cancer Research Center Seattle, Washington Human papillomavirus (HPV) types I 6 and 18 integration sites were mapped in six HPV-immortalized human keratinocyte cell lines by fluorescence in situ hybridization (FISH). Mapping of HPV sequences in these cell lines revealed that HPV integration varied in copy number and location but that integration sites were stable over extended passages in culture. Integration occurred at different sites throughout the genome and did not correspond to the location of specific cellular genes. However, integration sites were consistent with integration near or within known fragile sites in five of the six cell lines. Induction of aphidicolin-sensitive fragile sites in one cell line prior to in situ hybridization revealed that integrated HPV DNA was disrupted by fragile-site expression, suggesting that integration occurred within a fragile site. Genes Chrom Cancer 5: 150-157 ( 1 992). 0
1992 Wiley-Liss. Inc.
INTRODUCTION
Human papillomaviruses (HpVs) are implicated in the etiology of a variety of benign and malignant human epithelial diseases. A subset of the more than 60 specific HPV genotypes that have been identified to date is associated with lesions of the anogenital tract. Of these, HPV types 16, 18, 31, 33, and 35 are associated with premalignant and malignant genital lesions (zur Hausen and Schneider, 1987).HPV DNA is known to persist in plasmid form in benign lesions, whereas, in most tumors and tumor cell lines, single or multiple integrated copies of HPV 16 or 18 have been detected (Durst et al., 1985; Pater and Pater, 1985; Schwarz et al., 1985; McCance, 1986; Spence et al., 1988; zur Hausen, 1989).The presence and expression of these HPV DNA sequences in malignant lesions have been the primary evidence linking HPV and cancers. DNA of HPV types 16 and 18 can transform or immortalize a variety of cell types in vitro, including primary human foreskin keratinocytes (Yasumoto et al., 1986, 1987; Diirst et al., 1987b;Matlashewski et al., 1987; Pirisi et al., 1987; McCance et al., 1988; Schlegel et al., 1988; Kaur et al., 1989 Kaur and McDougall, 1989).This activity has been mapped to the E6 and E7 viral genes (Bedell et al., 1989; Hawley-Nelson et al., 1989; Watanabe et al., 1989). Although these genes clearly have the capacity to immortalize, prolonged passage in culture or cooperation with activated rus has been necessary for full conversion to a malignant phenotype (Matleshewski et al., 1987; Yasumoto et al., 1987; Durst et al., 198713; Hurlin et al., 1991). Cells immortalized by transfection with HPV DNA generally contain a variable number of HPV sequences 0 1992 WILEY-LISS, INC.
integrated at one or more chromosomal sites (Durst et al., 1987b; Kaur and McDougall, 1988; Schlegel et al., 1988; Kaur et al., 1989; Watanabe et al., 1989). Although the copy number and location of HPV integration sites vary among cell lines, preferential integration near fragile sites and/or oncogenes has been reported in HPV-containing tumor cell lines (Diirst et al., 1987a;Popescu et al., 1987,1990;Cannizzaro et al., 1988). Mapping of HPV integration sites may provide clues to the nature of the interaction between cellular and viral sequences leading to altered cell growth and proliferation. Mapping may also address questions regarding the existence of specific chromosomal sites or DNA sequences required for viral integration. The hypothesis that specific sites are involved in HPV integration is supported by evidence that HPV and other DNA viruses preferentially integrate at or near known fragile sites (Cannizzaroet al., 1988;Popescu et al., 1990).However, there has been no direct evidence linking viral integration and fragile sites. We used fluorescence in situ hybridization (FISH) to map the HPV DNA integration sites in six HPVimmortalized human keratinocyte cell lines containing single or multiple integrated copies of HPV (Kaur and McDougall, 1988; Kaur et al., 1989).The cell lines have previously been characterized cytogenetitally, and all contain numerical and structural abnormalities (Smith et a]., 1989).Each cell line showed a unique integration location. No consistent correlation could Received December 13, 1991; accepted February 1, 1992. Address reprint requests to James K Mcnougall, Fred Ilutchinson Cancer Research Center, 1124 Columhia Street, Scattlc, W h 98104.
IS/
HUMAN KERATINOCYTE CELL LINES
TABLE I. Transformed Cell Lines: HPV Integration and Fragile Site Location Cell Line
HPV Type
Copies HPV/ Cella
Integration Siteb
Nearest Fragile Site
lOq23.3 IOq24.2 IOq25.2 I2q24 I2q24. I 3 I2q24.2 lq12 lq21 lp21.2 lp31.2 -c 7p I I.2 7p13
FEP 18-5
18
20-50
IOq23724
FEP18-I I
18
50-100
I2q24
FEA
18
1-5
lq12-qZI
FEH I8L
18
I
I p22-p3 I
FEPE I L8
16
FEPE I L I 3
16
5-10 50-100
Marker
7pl I-p13
aAs previously determined by Southern blot analysis (Kaur and McDougall. 1988; Hurlin e t al., 1991). blntegration site mapping done at the 400 band level of resolution. ‘Unidentified chromosome.
be found between HPV integration sites and the location of specific cellular genes. However, the location of viral sequence integration was correlated with known fragile sites in five of the six cell lines (Table 1). MATERIALS AND METHODS
Cell Lines
The cell lines used in this study have been described previously (Kaur and McDougall, 1988; Smith et al., 1989; Hurlin et al., 1991);they are HPV-immortalized primary human keratinocytes transfected by the calcium phosphate precipitation method. Cell lines FEP18-5, FEP18-11, FEA, and FEH18L were transfected with a plasmid vector containing the entire HPV 18 genome (Kaur and McDougall, 1988). Cell lines FEPElL8 and FEPElL13 were transfected with an 11.4 kb DNA fragment molecularly cloned from a primary cervical carcinoma containing approximately 3.4 kb of integrated HPV 16 DNA, including the E6 and E7 open reading frames (Kaur et al., 1989).All cell lines contained integrated HPV DNA, as determined by Southern blot analysis (Kaur and McDougall, 1988).Cytogenetic analysis showed structural and numerical abnormalities in all cell lines (Smith et al., 1989). Cell Culture and Cytogenetic Analysis
Cell lines were maintained in Keratinocyte SFM supplemented with bovine pituitary extract and human epidermal growth factor (Gibco BRL, Gaithersburg, MD). Cells were routinely subcultured at a dilution of 1:3 every 4-6 days. Cells for fragile-site studies were treated with aphidicolin (APC) (0.2 pM)
for 24-36 hr before chromosome harvest (Murano et al., 1989). Metaphase chromosomes were prepared from subconfluent monolayers of cells incubated in medium containing Colcemid (O.Olpg/ml) for 2 hr before harvest. Cells were removed with trypsin, treated with hypotonic solution, fixed in methanol :acetic acid (3:l),and spread on glass slides. Chromosomes were G-banded as previously described (Smith et al., 1989) or standard stained with Wright’s stain. Metaphases were located and photographed prior to FISH. Slides were destained, air dried, and stored at -20°C. G-banded metaphases were used for identification of chromosomes and mapping of the integration sites. We used Wright’s-stained metaphases to score for fragile-site expression. Locations of HPV integration sites are shown in Table 1. A minimum of ten metaphases for each cell line were relocated and photographed after FISH. Fluorescence microscopy and photography were performed with a Zeiss axioscope equipped with epifluorescence and with Kodak Ektachrome PsoO/lsoO film, respectively. DNA Probes
For detection of HPV 18 sequences, probes consisting of an HPV DNA insert in the plasmid pBR322 and HPV 18 DNA released from the plasmid vector were used. HPV 16 sequences were detected with HPV 16 DNA insert in pBR322 and HPV 16 insert DNA alone (HPV 16 and 18 plasmids were gifts from Drs. M. Durst, H. zur Hausen, and colleagues). HPV 16 and 18 DNA in pBR322 plasmid and HPV 16 and 18 insert released from the vector were nick-translated with
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biotin-l&dATP (Gibco BRL). Nick-translated DNA was lyophilyzed with carrier DNA; was resuspended in 2 x SSC, 10% dextran sulfate, and 50% deionized formamide; and was thermally denatured.
hybridized at various passage levels (p29-p81) in culture, and the characteristic location of integration remained constant throughout all passage levels tested.
FISH
FEPElL8 contained five to ten copies of HPV 16 DNA. Eleven previously G-banded metaphase cells were hybridized, relocated, and photographed. The location of HPV integration was determined by comparison of G-banded and hybridized chromosomes. A single spot of fluorescence signal was present on the long arm of a C-group-size,metacentric marker chromosome. The specific band location of the integration site could not be described because a definitive identification of the chromosome could not be made. This marker was stable, however, with integration at the same location in all metaphase cells. Interphase nuclei also showed a single spot of hybridization.
The procedure used for in situ hybridization was a modification of the methods described by Moyzis et al. (1987). Metaphase chromosomes on glass slides were treated with acetic anhydride, denatured in 70% formamide in 2 x SSC for 2 min at 70°C, dehydrated, and air dried. Approximately 30 ng of denatured probe was applied, and slides were incubated for 12-16 hr at 37°C. The posthybridization wash was carried out for 20 min in 1x SSC, 55% formamide at 42°C. Subsequent washes were done for 10 min in 2 x SSC, 0.03% Triton X at room temperature. For detection of hybridization, fluorescein-conjugated avidin (Vector Laboratories, Burlingame, CA) was bound to the biotin-labeled nucleic acid probe (Pinkel et al., 1986). Signal amplification was accomplished by treatment with biotinylated goat antiavidin (Vector Laboratories),followed by a second layer of fluoresceinated avidin. Slides were counterstained with propidium iodide in phosphate-buffered saline (PBS)and mounted in a DABCO (Eastman Kodak Co.) antifade solution Qohnson et al., 1982). RESULTS
Metaphase chromosomes from four HPV 18- and two HPV 16-transformed cell lines were hybridized with HPV 18 and HPV 16 DNA, respectively,with the use of FISH. Hybridization results were identical with those of HPV DNA in pBR322 plasmid or as a released insert. The fluorescence signal was more intense when HPV DNA in plasmid was used as probe; therefore, mapping and fragile site analysis were conducted with the plasmid probe. The three cell lines with >20 copies of HPV DNA per cell (FEP18-5, FEP18-11, and FEPEIL13) displayed intense signals. The lines with fewer than ten copies of HPV DNA per cell (FEA, FEH18L, and FEPEIL8) showed less intense signals (Fig. 1). A single hybridization signal was detected in four of the six lines (FEP18-5,FEPl811, FEPEIL8, and FEPEIL13).Two hybridization s i g nals were seen in FEA and FEH18L. In each of these cell lines, the two signals were locate on distinct derivative chromosomes and were separated by a region of chromatin with a banding pattern that could not be identified with a specific chromosome. The location of HPV integration was stable in the six cell lines. All metaphase cells examined in each cell line showed integration only at the distinct location characteristic of that line. Cell lines FEPl8-11 and FEP18-5 were
Cell Line FEPEILB
Cell Line FEPE I L I 3
The FEPElL13 cell line contained at least 5C-100 copies of HPV 16 DNA. Twenty-three previously G-banded metaphase cells were hybridized, relocated, and photographed. The hybridization signal was intense and was visualized as a single, large spot at chromosome region 7pllLp13. Classical cytogenetic analysis also demonstrated a structural rearrangement in 7pll-p13. A single large spot of hybridization was also seen in all interphase nuclei. A folic acidinducible fragile site occurs at 7~11.2,and an aphidicolon (APC)-induciblefragile site occurs at 7p13 (Rerger et al., 1985). Cell Line FEA
The FEA cell line, with one to five copies of HPV 18 DNA, showed two sites of hybridization on the long arm of a derivative chromosome 1 by hybridization, relocation, and photography of 19 G-banded metaphase cells. One signal was localized to chromosome region lq12-lq21. A second signal occurred distal to the first; however, the band location of this signal could not be described because all material distal to lq12-lq21 was rearranged. Hybridization occurred only at these two sites. An APC-induciblefragile site is located at lq21 and a 5-azacytidine-induciblefragile site at lq12 (Yunis and Soreng, 1984; Yunis et al., 1987). Cell Line FEH IBL
The FEH18L cell line contained approximately one to five copies of HPV 18. Thirty-nine previously G-banded metaphase cells were hybridized, relocated, and photographed. Hybridization signals were seen at two sites on the short arm of a derivative chromosome
HUMAN KERATINOCWE CELL LINES
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Figure I. Metaphare chromosomes G-banded. followed by hybridization with biotinylated HPV I8 DNA. Hybridized probe was detected with avidin-FITC; the counterstain was propidium iodide. A, 6 The HPV integration site is localized t o two sites on the short arm of a derivative chromosome I in FEH 18L cells. The more proximal slte is at I p22-p3 I, with material of unknown origin intervening between the two integration sites. C , D: HPV sequences are localized near the terminus of a chromosome I 2 long arm (12q24) in the FEP18-I I cell line.
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1 (Fig. 1).As in FEA, chromosome rearrangements occurred distal to the most proximal integration site (lp22-p31), with material of unknown origin located between the first and second signals so that the specific band location of the distal signal could not be determined (Fig. 1).APC-inducible fragile sites occur at lp21.2 and lp31.2 (Yunis and Soreng, 1984). Cell Line FEPI8-I I
Cell line FEP18-11 contained 5&100 copies of HPV 18 DNA. A single fluorescent spot of hybridization was observed in all metaphase cells examined. Nineteen G-banded metaphase cells were hybridized, relocated, and photographed, and the HPV integration site was mapped to one site near the telomere of the long arm of a chromosome 12 (12q24) (Fig. 1).Both chromosome 12 homologs appeared normal by G-banding at the 400 band resolution level. A single spot of hybridization was clearly visible in all interphase nuclei. Three fragile sites occur in the 12q24 region: an APC site at 12q24, a BrdU site at 12q24.2, and a folic acid site at 12q24.13 (Sutherland et al., 1985; Amarose et al., 1987; Yunis et al., 1987). Cell Line FEP18-5
Cell line FEP18-5 contained 2&50 copies of HPV 18 DNA. Hybridization occurred as a single spot on one chromosome in all metaphase cells examined. Thirtynine G-banded metaphase cells were hybridized, relocated, and photographed. HPV integration was mapped to a single site on a derivative chromosome 10 at 10q23-q24 (Fig. 2). This aberrant chromosome 10 appeared to have a normal banding pattern proximal to lOq23q24, with additional material of unknown origin distal to the integration site (Fig. 2). All interphase nuclei also showed a single spot of hybridization. As in cell lines FEA and FEP18L, the HPV integration site in FEP18-5 appeared to map to the site of rearrangement. An APC-inducible fragile site occurs at 10~25.2,and a folic acid-sensitive rare fragile site occurs at 10~23.3or 10q24.2 (Yunis and Soreng, 1984; Berger et al., 1985). APC Fragile Site Induction
To analyze the possible association between HPV integration and fragile sites, we used APC to express fragile sites in the FEP18-5 cell line. The FEP18-5 cell line was chosen for fragile site study because of a single, easily visible integration occurring near a known fragile site on a readily distinguishable chromosome. APC treatment induced fragile site expression on the long arm of the derivative chromosome 10 in the region of HPV integration and at other chromosomal sites. FISH with the HPV 18 probe on meta-
phase chromosomes expressing this fragile site revealed breaks and gaps within the region of hybridization (Fig. 2). In chromosomes in which gaps were induced, the hybridization signal appeared elongated and track-like. This was in contrast to nonAPC-treated cells, in which hybridization showed a discrete spot on each chromatid (Fig. 2). Where breaks were induced, hybridization signals could be seen on both sides of the breakpoints (Fig. 2). In some metaphase cells, faint, threadlike hybridization signals could be seen connecting broken ends of chrornosomes. DISCUSSION
FISH is becoming a widely used technique in cytogenetics. The technique allows rapid and specific hybridization to the single-copy level with minimal background, eliminating ambiguity inherent in hybridization with isotopically labeled probes. We used FISH to map the site of HPV integration in six HI’Vimmortalized human keratinocyte cell lines. Sites of integration were found at unique chromosomal locations in each cell line, with no single preferred site for either HPV 16 or HPV 18 integration. Integration locations were compared with the locations of known oncogenes that have been reported as possible preferential sites of HPV integration (Durst et al., 1987a;Cannizzaro et al., 1988;Popescu et al., 1990). HPV integration near the M C locus (8q24) has been described in cervical carcinoma cell lines and tissues by Diirst et al. (1987a)but was not evident in these cell lines. Integration sites in two of the cell lines did correlate with the G-band location of cellular oncogenes: the 7pll-pl3 integration site in FEPElL13 with ERBB-1 and the lp22-p31 site in FEH18L with /UN(Sakaguchi et al., 1984;Haluska et al., 1988).Both of these locations also contain fragile sites. Fragile sites are candidate sites for integration of viral and other foreign DNA and for action by certain mutagens (Yunis et al., 1987; Popescu and DiPaolo, 1989; Rassool et al., 1991).In this study, a positive correlation was observed between the location of HPV integration and fragile sites. In the five cell lines where HPV integration could be mapped to a chromosome region, the integration sites occurred within the chromosomal regions known to contain fragile sites. Cytogenetically visible rearrangements at these integration sites were also common in these cell lines. In two lines (FEA and FEH18L), the rearrangements appeared to have included HPV DNA that resulted in two hybridization sites separated by rearranged material (Fig. 1).These rearrangements may be evidence for instability during the early evolution of these cell lines, although the lines are now stable. It is possible
HUMAN KERATINOCYTE CELL LINES
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Figure 2. G-banded FEP18-5 meraphase (A) and the same metaphase hybridized with HPV 18 probe (B), showing location of HPV sequences on the long arm of a derivative chromosome I 0 ( I Oq23424). C: FEPl8-5 metaphase APC treated and Wright’s stained. A gap is visible in the derivative chromosome 10 (arrow). D HPV I 8 hybridization of the metaphase in C. showing an elongated track-like pattern of hybridization in the gapped region of the derivative chromosome 10. E APC-treated FEP18-5 chromosomes with a fragile site expressed on the derivative chromosome 10. HPV 18 probe hybridized t o both sides of the breakpoinr at the fragile site.
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SMITH ET A L
to speculate that this is related to the high rate of inter- and intrachromosomal recombination observed at fragile sites in cells cultured under conditions known to cause expression of fragile sites (Yunis et al., 1987; Glover and Stein, 1988). Evidence for integration of HF'V and other viral DNA at fragile sites has been based primarily on mapping at the metaphase chromosome level (Cannizzaro et al., 1988; Popescu et al., 1990).However, chromosome bands represent large amounts of DNA, and it is difficult to determine the exact proximity of DNA integration to fragile sites with these methods. The coincidence of HPV integration and a fragile site in the FEP18-5 cell line provided the opportunity for further study in vitro of fragile site expression. Treatment of FEP18-5 cells with APC resulted in gaps and breaks in several chromosomes, including the derivative chromosome 10 containing integrated HPV DNA. FISH with HPV 18 DNA revealed viral DNA flanking gaps and breaks and lying between gaps in the derivative chromosome 10. We believe that this is the first clear demonstration of HPV integration within a fragile site. Further investigation with these cell lines and in uivo-derived HPV-containing cell lines will be necessary for determining whether this phenomenon is a common feature of HPV DNA integration. The mode of interaction between cellular and HPV DNA at a fragile site in FEP18-5may not be restricted to HPV DNA but may be indicative of a more generalized mode used by many other types of viral and foreign DNAs when they are introduced into cells. Recent in vitro studies by Rassool et al. (1991) have shown that the uptake of DNA at fragile sites may be a very generalized phenomenon and that foreign DNA may preferentially integrate in these regions. The possibility that the HPV DNA, integrated as a tandem array, is itself prone to fragility remains to be explored. In the interpretation of the results of this study and of those of Rassool et al. (I 991),one must consider the fact that DNA was introduced into cells via calcium phosphate precipitation. Conclusions are, therefore, relevant to this in vitro system. The path to in vitro immortalization and tumorigenicity by HPV may or may not be directly related to the action of HPV in vivo. Continued study, particularly with HPV-containing tumor-derived cell lines, will be necessary for further elucidation of the mechanisms of HPV integration in the human host. We believe that the results of this study confirm those of other studies on mapping of HPV integration sites near fragile sites, and that they strengthen the evidence by revealing HPV integration occurring
within a fragile site. HPV DNA stably integrated at a fragile site in the FEP18-5 cell line also provides a target for cloning of the flanking fragile site DNA sequences of human chromosome 10. Other HPV-immortalized cell lines used in this study may provide a means for isolating and characterizing fragile site sequences in other chromosomes. ACKNOWLEDGMENTS
We thank Marci Wright for manuscript preparation. This research was supported by a Public Health Service grant from the National Cancer Institute (CA42792, principal investigator, J.K.M.) REFERENCES Amarose A€',Huttenlocher PR, Sprudzs RM, Laitsch TJ, Pettenati MJ (1987) A heritable fragile 12q24.13 segregating in a family with a fragile X chromosome. Hum Genet 7k4-6. Bedell MA, Jones KH, Grossman SR, Laimins LA (1989) Identification of human papillomavirus type 18 transforming genes in immortalized and primary cells. J Virol 63:1247 -1255. Berger R, Bloomfield CD, Sutherland GR (1985)Report of the committee on chromosome rearrangements in neoplasia and on fragile sites. Cytogenet Cell Genet 40490-535. Cannizzaro LA, Diirst M. Mendez MJ, Hecht BK, Hecht F (1988)Regional chromosome localization of human papillomavirus integration sites near fragile sites, oncogenes and cancer chromosome breakpoints. Cancer Genet Cytogenet 33:93 98. Diirst M, Kleinheinz A, Hotz M, Gissman L (1985)The physical state of human papillomavirus type 16 DNA in benign and malignant genital tumors. J Gen Virol 66:15151522. Diirst M, Croce CM, Gissman L, Schwarz E, Huebner K (1987a) Wpillomavirus sequences integratt near cellular oncogenes in some cervical carcinomas. Proc Natl Acad Sci USA &1:107&1074. Diirst M, Dzarlieva-Petrusevska RT, Boukamp P, Fusenig NE,Gissman L (1987b) Molecular and cytogenetic analysis of immortalized human primary keratinncytes obtained after transfection with human papillomavirus type 16 DNA. Oncogene 1:251-256. Glover TW, Stein CK (1988) Chromosome breakage and recombination at fragile sites. Am J Hum Genet 43264-273. Haluska FG, Huebner K, Isobe M, Nishimura T, Croci CM, Vogt PK (1988)Localization of the human JUN protooncogene to chromosome region lp31-32. Roc Natl Acad Sci USA 85:2215-2218. Hawlcy-Nelson P, Vousden KH, Hubbert NL, Lowy DK, Schiller JT (1989) The HPV E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes. EMBO J 8:3905-3910. Hurlin PJ, Kaur P, Smith PP, Perez-ReyesN, Blanton RA, McDougal! JK (1991) Progression of human papillomavirus type 18-immortalized human keratinocytes to a malignant phenotype. Roc Natl Acad Sci USA 88:57&574. Johnson GD, llavidson RS, McNamee KC, Russell G, Goodwin I), Holborrow EL (1982) Fading of immunofluorescence during microscopy: A study of its phenomenon and its remedy. J Immunol Methods 55:231-242. Kaur P, McDougall JK (1988) Characterization of primary human keratinocytes transformed by human papillomavirus type 18. J Virol 621917-1924. Kaur P, McDougall JK (1989) HF'V-18 immortalization of human keratinocytes. Virology 173302-310. Kaur P, McDougall JK, Cone R (1989)Immortalization of primary human epithelial cells by cloned cervical carcinoma DNA containing human papillomavirus type 16 E6/B7 open reading frames. J Gen Virol 701261- 1266. Matlashewski G, Schneider J, Ranks L, Jones N, Murray A, Crawford L (1987) Human papillomavirus type 16 DNA cooperates with activated 7m in transforming primary cells. EMBO J 6:1741-1746. McCance DJ (1986) Human papillomaviruses and cancer. Riochim Biophys Acta 823:195-205. McCance DJ, Kopan R, Fuchs E, Laimins LA (1988) Human pap& lomavirus type 16 alters human epithelial cell differentiation in vitro. Proc Natl Acad Sci USA 85:7169 7173. Moyzis RK, Albright KL, Bartholdi MF, Cram LS,Deaven LL, Hildebrand CE, Joste NE,LongmireJL, Meyne J, Schwar~acher-Robinson
HUMAN KERATINOCYTE CELL LINES
T (1987) Human chromosome-specific repetitive DNA sequences: Novel markers for genetic analysis. Chromosoma 95:375386. Murano I, Kuwano A, Kajii T (1989) Fibroblast-specific common fragile sites induced by aphidicolin. Hum Genet 8345 48. Pater MM, Pater A (1985) Human papillomavirus types 16 and 18 sequences in carcinoma cell lines of the cervix. Virology 14531% 318. Pinkel D, Straume T, Gray JW (1986) Cytogenetic analysis using quantitative, high sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 832934 2938. Pirisi I,, Yasumoto S,Feller M, Doniger J, DiPaolo JA (1987) Transformation of human fibroblasts and keratinocytes with human papillomavirus type 16 DNA. J Virol 61:1061- 1066. Popescu NC, DiPaolo JA (1989)Preferential sites for viral integration on mammalian genome. Cancer Genet Cytogenet 42157 171. Popescu NC, DiPaolo JA, Amsbaugh SC (1987) Integration sites of human papillomavirus 18 DNA sequences on HeLa cell chromosomes. Cytogenet Cell Genet 4458-42. Popescu NC, Zimonjic D, DiPaolo JA (1W)Viral integration, fragile sites, and proto-oncogenesin human neoplasia. Hum Genet 8438% 386. Rassool FV, McKeithan TW, Neilly ME, van Melle E, Espinosa 111 R, LeBeau MM (1991) Preferential integration of marker DNA into the chromosomal fragile site 3p14 An approach to cloning fragile sites. Proc Natl Acad Sci USA 88:66574661. Sakaguchi AY, Zabel BU, Grzeschik KH, Law ML, Naylor PJ (1984) Human proto-oncogeneassignments. Cytogenet Cell Genet 37572573. Schwarz E, Freese UK, Gissman L, Mayer W, Roggenbuck B, Stremlau A, zur Hausen H (1985)Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature 314111 114.
157
Schlegel R, Phelps WC, Zhang Y-L, Barbosa M (1988) Quantitative keratinocyte assay detects two biological activities of human papillomavirus DNA and identifies viral types associated with cervical carcinoma. EMBO J 7:3181-3187. Smith PP, Bryant EM, Kaur P, McDougall JK (1989) Cytogenetic analysis of eight human papillomavirus immortali7~dhuman keratinocyte cell lines. Int J Cancer 441124 1131. Spence RP, Murray A, Banks L, Kelland LR, Crawford L (1988)Analysis of human papillomavirus sequences in cell lines recently derived from cervical cancers. Cancer Res 48:324--328. Sutherland GR, Parslow MI, Baker E (1985) New classes of common fragile sites induced by 5-azacytidine and bromdeoxyuridine. Hum Genet 69233 237. Watanabe S, Kanda T, Yoshiike K (1989) Human papillomavirus type 16 transformation of primary human embryonic fibroblasts requires expression of open reading frames E6 and E7. J Virol 63:96.5969. Yasumoto S, Burkhardt AL, Doniger J, DiPaolo JA (1986)Human papillomavirus type 16 DNA-induced malignant transformation of NIH3T3 cells. J Virol 57572~577. Yasumoto S, Doniger J, DiPaolo JA (1987) Differential early viral gene expression in two stages of human papillomavirus type 16 DNAinduced malignant transformation. Cell Biol 72165-2172. Yunis JJ, Soreng AL (1984) Constitutive fragile sites and cancer. Science 2261 1%1204. Yunis JJ, Sareng AL,Bowe AE (1987)Fragile sites are targets of diverse mutagens and carcinogens. Oncogene 1:5%69. zur Hausen H (1989) Papillomaviruses in anogenital cancer as a model to understand the role of viruses in human cancers. Cancer Res 494677-4681, zur Hausen H, Schneider A (1987) The role of papillomaviruses in human anogenital cancer. In Salzman N, Lowley PM (eds): Papovaviridae, Vol. 2. New York Plenum Press, pp 245 263.
Viral Integration and Fragile Sites in Human Papillomavirus-Immortalized Human Keratinocyte Cell Lines Patricia P. Smith, Cynthia L. Friedman, Eileen M. Bryant, and James K. McDougall Fred Hutchinson Cancer Research Center Seattle, Washington Human papillomavirus (HPV) types I 6 and 18 integration sites were mapped in six HPV-immortalized human keratinocyte cell lines by fluorescence in situ hybridization (FISH). Mapping of HPV sequences in these cell lines revealed that HPV integration varied in copy number and location but that integration sites were stable over extended passages in culture. Integration occurred at different sites throughout the genome and did not correspond to the location of specific cellular genes. However, integration sites were consistent with integration near or within known fragile sites in five of the six cell lines. Induction of aphidicolin-sensitive fragile sites in one cell line prior to in situ hybridization revealed that integrated HPV DNA was disrupted by fragile-site expression, suggesting that integration occurred within a fragile site. Genes Chrom Cancer 5: 150-157 ( 1 992). 0
1992 Wiley-Liss. Inc.
INTRODUCTION
Human papillomaviruses (HpVs) are implicated in the etiology of a variety of benign and malignant human epithelial diseases. A subset of the more than 60 specific HPV genotypes that have been identified to date is associated with lesions of the anogenital tract. Of these, HPV types 16, 18, 31, 33, and 35 are associated with premalignant and malignant genital lesions (zur Hausen and Schneider, 1987).HPV DNA is known to persist in plasmid form in benign lesions, whereas, in most tumors and tumor cell lines, single or multiple integrated copies of HPV 16 or 18 have been detected (Durst et al., 1985; Pater and Pater, 1985; Schwarz et al., 1985; McCance, 1986; Spence et al., 1988; zur Hausen, 1989).The presence and expression of these HPV DNA sequences in malignant lesions have been the primary evidence linking HPV and cancers. DNA of HPV types 16 and 18 can transform or immortalize a variety of cell types in vitro, including primary human foreskin keratinocytes (Yasumoto et al., 1986, 1987; Diirst et al., 1987b;Matlashewski et al., 1987; Pirisi et al., 1987; McCance et al., 1988; Schlegel et al., 1988; Kaur et al., 1989 Kaur and McDougall, 1989).This activity has been mapped to the E6 and E7 viral genes (Bedell et al., 1989; Hawley-Nelson et al., 1989; Watanabe et al., 1989). Although these genes clearly have the capacity to immortalize, prolonged passage in culture or cooperation with activated rus has been necessary for full conversion to a malignant phenotype (Matleshewski et al., 1987; Yasumoto et al., 1987; Durst et al., 198713; Hurlin et al., 1991). Cells immortalized by transfection with HPV DNA generally contain a variable number of HPV sequences 0 1992 WILEY-LISS, INC.
integrated at one or more chromosomal sites (Durst et al., 1987b; Kaur and McDougall, 1988; Schlegel et al., 1988; Kaur et al., 1989; Watanabe et al., 1989). Although the copy number and location of HPV integration sites vary among cell lines, preferential integration near fragile sites and/or oncogenes has been reported in HPV-containing tumor cell lines (Diirst et al., 1987a;Popescu et al., 1987,1990;Cannizzaro et al., 1988). Mapping of HPV integration sites may provide clues to the nature of the interaction between cellular and viral sequences leading to altered cell growth and proliferation. Mapping may also address questions regarding the existence of specific chromosomal sites or DNA sequences required for viral integration. The hypothesis that specific sites are involved in HPV integration is supported by evidence that HPV and other DNA viruses preferentially integrate at or near known fragile sites (Cannizzaroet al., 1988;Popescu et al., 1990).However, there has been no direct evidence linking viral integration and fragile sites. We used fluorescence in situ hybridization (FISH) to map the HPV DNA integration sites in six HPVimmortalized human keratinocyte cell lines containing single or multiple integrated copies of HPV (Kaur and McDougall, 1988; Kaur et al., 1989).The cell lines have previously been characterized cytogenetitally, and all contain numerical and structural abnormalities (Smith et a]., 1989).Each cell line showed a unique integration location. No consistent correlation could Received December 13, 1991; accepted February 1, 1992. Address reprint requests to James K Mcnougall, Fred Ilutchinson Cancer Research Center, 1124 Columhia Street, Scattlc, W h 98104.
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HUMAN KERATINOCYTE CELL LINES
TABLE I. Transformed Cell Lines: HPV Integration and Fragile Site Location Cell Line
HPV Type
Copies HPV/ Cella
Integration Siteb
Nearest Fragile Site
lOq23.3 IOq24.2 IOq25.2 I2q24 I2q24. I 3 I2q24.2 lq12 lq21 lp21.2 lp31.2 -c 7p I I.2 7p13
FEP 18-5
18
20-50
IOq23724
FEP18-I I
18
50-100
I2q24
FEA
18
1-5
lq12-qZI
FEH I8L
18
I
I p22-p3 I
FEPE I L8
16
FEPE I L I 3
16
5-10 50-100
Marker
7pl I-p13
aAs previously determined by Southern blot analysis (Kaur and McDougall. 1988; Hurlin e t al., 1991). blntegration site mapping done at the 400 band level of resolution. ‘Unidentified chromosome.
be found between HPV integration sites and the location of specific cellular genes. However, the location of viral sequence integration was correlated with known fragile sites in five of the six cell lines (Table 1). MATERIALS AND METHODS
Cell Lines
The cell lines used in this study have been described previously (Kaur and McDougall, 1988; Smith et al., 1989; Hurlin et al., 1991);they are HPV-immortalized primary human keratinocytes transfected by the calcium phosphate precipitation method. Cell lines FEP18-5, FEP18-11, FEA, and FEH18L were transfected with a plasmid vector containing the entire HPV 18 genome (Kaur and McDougall, 1988). Cell lines FEPElL8 and FEPElL13 were transfected with an 11.4 kb DNA fragment molecularly cloned from a primary cervical carcinoma containing approximately 3.4 kb of integrated HPV 16 DNA, including the E6 and E7 open reading frames (Kaur et al., 1989).All cell lines contained integrated HPV DNA, as determined by Southern blot analysis (Kaur and McDougall, 1988).Cytogenetic analysis showed structural and numerical abnormalities in all cell lines (Smith et al., 1989). Cell Culture and Cytogenetic Analysis
Cell lines were maintained in Keratinocyte SFM supplemented with bovine pituitary extract and human epidermal growth factor (Gibco BRL, Gaithersburg, MD). Cells were routinely subcultured at a dilution of 1:3 every 4-6 days. Cells for fragile-site studies were treated with aphidicolin (APC) (0.2 pM)
for 24-36 hr before chromosome harvest (Murano et al., 1989). Metaphase chromosomes were prepared from subconfluent monolayers of cells incubated in medium containing Colcemid (O.Olpg/ml) for 2 hr before harvest. Cells were removed with trypsin, treated with hypotonic solution, fixed in methanol :acetic acid (3:l),and spread on glass slides. Chromosomes were G-banded as previously described (Smith et al., 1989) or standard stained with Wright’s stain. Metaphases were located and photographed prior to FISH. Slides were destained, air dried, and stored at -20°C. G-banded metaphases were used for identification of chromosomes and mapping of the integration sites. We used Wright’s-stained metaphases to score for fragile-site expression. Locations of HPV integration sites are shown in Table 1. A minimum of ten metaphases for each cell line were relocated and photographed after FISH. Fluorescence microscopy and photography were performed with a Zeiss axioscope equipped with epifluorescence and with Kodak Ektachrome PsoO/lsoO film, respectively. DNA Probes
For detection of HPV 18 sequences, probes consisting of an HPV DNA insert in the plasmid pBR322 and HPV 18 DNA released from the plasmid vector were used. HPV 16 sequences were detected with HPV 16 DNA insert in pBR322 and HPV 16 insert DNA alone (HPV 16 and 18 plasmids were gifts from Drs. M. Durst, H. zur Hausen, and colleagues). HPV 16 and 18 DNA in pBR322 plasmid and HPV 16 and 18 insert released from the vector were nick-translated with
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SMITH ET A L
biotin-l&dATP (Gibco BRL). Nick-translated DNA was lyophilyzed with carrier DNA; was resuspended in 2 x SSC, 10% dextran sulfate, and 50% deionized formamide; and was thermally denatured.
hybridized at various passage levels (p29-p81) in culture, and the characteristic location of integration remained constant throughout all passage levels tested.
FISH
FEPElL8 contained five to ten copies of HPV 16 DNA. Eleven previously G-banded metaphase cells were hybridized, relocated, and photographed. The location of HPV integration was determined by comparison of G-banded and hybridized chromosomes. A single spot of fluorescence signal was present on the long arm of a C-group-size,metacentric marker chromosome. The specific band location of the integration site could not be described because a definitive identification of the chromosome could not be made. This marker was stable, however, with integration at the same location in all metaphase cells. Interphase nuclei also showed a single spot of hybridization.
The procedure used for in situ hybridization was a modification of the methods described by Moyzis et al. (1987). Metaphase chromosomes on glass slides were treated with acetic anhydride, denatured in 70% formamide in 2 x SSC for 2 min at 70°C, dehydrated, and air dried. Approximately 30 ng of denatured probe was applied, and slides were incubated for 12-16 hr at 37°C. The posthybridization wash was carried out for 20 min in 1x SSC, 55% formamide at 42°C. Subsequent washes were done for 10 min in 2 x SSC, 0.03% Triton X at room temperature. For detection of hybridization, fluorescein-conjugated avidin (Vector Laboratories, Burlingame, CA) was bound to the biotin-labeled nucleic acid probe (Pinkel et al., 1986). Signal amplification was accomplished by treatment with biotinylated goat antiavidin (Vector Laboratories),followed by a second layer of fluoresceinated avidin. Slides were counterstained with propidium iodide in phosphate-buffered saline (PBS)and mounted in a DABCO (Eastman Kodak Co.) antifade solution Qohnson et al., 1982). RESULTS
Metaphase chromosomes from four HPV 18- and two HPV 16-transformed cell lines were hybridized with HPV 18 and HPV 16 DNA, respectively,with the use of FISH. Hybridization results were identical with those of HPV DNA in pBR322 plasmid or as a released insert. The fluorescence signal was more intense when HPV DNA in plasmid was used as probe; therefore, mapping and fragile site analysis were conducted with the plasmid probe. The three cell lines with >20 copies of HPV DNA per cell (FEP18-5, FEP18-11, and FEPEIL13) displayed intense signals. The lines with fewer than ten copies of HPV DNA per cell (FEA, FEH18L, and FEPEIL8) showed less intense signals (Fig. 1). A single hybridization signal was detected in four of the six lines (FEP18-5,FEPl811, FEPEIL8, and FEPEIL13).Two hybridization s i g nals were seen in FEA and FEH18L. In each of these cell lines, the two signals were locate on distinct derivative chromosomes and were separated by a region of chromatin with a banding pattern that could not be identified with a specific chromosome. The location of HPV integration was stable in the six cell lines. All metaphase cells examined in each cell line showed integration only at the distinct location characteristic of that line. Cell lines FEPl8-11 and FEP18-5 were
Cell Line FEPEILB
Cell Line FEPE I L I 3
The FEPElL13 cell line contained at least 5C-100 copies of HPV 16 DNA. Twenty-three previously G-banded metaphase cells were hybridized, relocated, and photographed. The hybridization signal was intense and was visualized as a single, large spot at chromosome region 7pllLp13. Classical cytogenetic analysis also demonstrated a structural rearrangement in 7pll-p13. A single large spot of hybridization was also seen in all interphase nuclei. A folic acidinducible fragile site occurs at 7~11.2,and an aphidicolon (APC)-induciblefragile site occurs at 7p13 (Rerger et al., 1985). Cell Line FEA
The FEA cell line, with one to five copies of HPV 18 DNA, showed two sites of hybridization on the long arm of a derivative chromosome 1 by hybridization, relocation, and photography of 19 G-banded metaphase cells. One signal was localized to chromosome region lq12-lq21. A second signal occurred distal to the first; however, the band location of this signal could not be described because all material distal to lq12-lq21 was rearranged. Hybridization occurred only at these two sites. An APC-induciblefragile site is located at lq21 and a 5-azacytidine-induciblefragile site at lq12 (Yunis and Soreng, 1984; Yunis et al., 1987). Cell Line FEH IBL
The FEH18L cell line contained approximately one to five copies of HPV 18. Thirty-nine previously G-banded metaphase cells were hybridized, relocated, and photographed. Hybridization signals were seen at two sites on the short arm of a derivative chromosome
HUMAN KERATINOCWE CELL LINES
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Figure I. Metaphare chromosomes G-banded. followed by hybridization with biotinylated HPV I8 DNA. Hybridized probe was detected with avidin-FITC; the counterstain was propidium iodide. A, 6 The HPV integration site is localized t o two sites on the short arm of a derivative chromosome I in FEH 18L cells. The more proximal slte is at I p22-p3 I, with material of unknown origin intervening between the two integration sites. C , D: HPV sequences are localized near the terminus of a chromosome I 2 long arm (12q24) in the FEP18-I I cell line.
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SMITH Er AL.
1 (Fig. 1).As in FEA, chromosome rearrangements occurred distal to the most proximal integration site (lp22-p31), with material of unknown origin located between the first and second signals so that the specific band location of the distal signal could not be determined (Fig. 1).APC-inducible fragile sites occur at lp21.2 and lp31.2 (Yunis and Soreng, 1984). Cell Line FEPI8-I I
Cell line FEP18-11 contained 5&100 copies of HPV 18 DNA. A single fluorescent spot of hybridization was observed in all metaphase cells examined. Nineteen G-banded metaphase cells were hybridized, relocated, and photographed, and the HPV integration site was mapped to one site near the telomere of the long arm of a chromosome 12 (12q24) (Fig. 1).Both chromosome 12 homologs appeared normal by G-banding at the 400 band resolution level. A single spot of hybridization was clearly visible in all interphase nuclei. Three fragile sites occur in the 12q24 region: an APC site at 12q24, a BrdU site at 12q24.2, and a folic acid site at 12q24.13 (Sutherland et al., 1985; Amarose et al., 1987; Yunis et al., 1987). Cell Line FEP18-5
Cell line FEP18-5 contained 2&50 copies of HPV 18 DNA. Hybridization occurred as a single spot on one chromosome in all metaphase cells examined. Thirtynine G-banded metaphase cells were hybridized, relocated, and photographed. HPV integration was mapped to a single site on a derivative chromosome 10 at 10q23-q24 (Fig. 2). This aberrant chromosome 10 appeared to have a normal banding pattern proximal to lOq23q24, with additional material of unknown origin distal to the integration site (Fig. 2). All interphase nuclei also showed a single spot of hybridization. As in cell lines FEA and FEP18L, the HPV integration site in FEP18-5 appeared to map to the site of rearrangement. An APC-inducible fragile site occurs at 10~25.2,and a folic acid-sensitive rare fragile site occurs at 10~23.3or 10q24.2 (Yunis and Soreng, 1984; Berger et al., 1985). APC Fragile Site Induction
To analyze the possible association between HPV integration and fragile sites, we used APC to express fragile sites in the FEP18-5 cell line. The FEP18-5 cell line was chosen for fragile site study because of a single, easily visible integration occurring near a known fragile site on a readily distinguishable chromosome. APC treatment induced fragile site expression on the long arm of the derivative chromosome 10 in the region of HPV integration and at other chromosomal sites. FISH with the HPV 18 probe on meta-
phase chromosomes expressing this fragile site revealed breaks and gaps within the region of hybridization (Fig. 2). In chromosomes in which gaps were induced, the hybridization signal appeared elongated and track-like. This was in contrast to nonAPC-treated cells, in which hybridization showed a discrete spot on each chromatid (Fig. 2). Where breaks were induced, hybridization signals could be seen on both sides of the breakpoints (Fig. 2). In some metaphase cells, faint, threadlike hybridization signals could be seen connecting broken ends of chrornosomes. DISCUSSION
FISH is becoming a widely used technique in cytogenetics. The technique allows rapid and specific hybridization to the single-copy level with minimal background, eliminating ambiguity inherent in hybridization with isotopically labeled probes. We used FISH to map the site of HPV integration in six HI’Vimmortalized human keratinocyte cell lines. Sites of integration were found at unique chromosomal locations in each cell line, with no single preferred site for either HPV 16 or HPV 18 integration. Integration locations were compared with the locations of known oncogenes that have been reported as possible preferential sites of HPV integration (Durst et al., 1987a;Cannizzaro et al., 1988;Popescu et al., 1990). HPV integration near the M C locus (8q24) has been described in cervical carcinoma cell lines and tissues by Diirst et al. (1987a)but was not evident in these cell lines. Integration sites in two of the cell lines did correlate with the G-band location of cellular oncogenes: the 7pll-pl3 integration site in FEPElL13 with ERBB-1 and the lp22-p31 site in FEH18L with /UN(Sakaguchi et al., 1984;Haluska et al., 1988).Both of these locations also contain fragile sites. Fragile sites are candidate sites for integration of viral and other foreign DNA and for action by certain mutagens (Yunis et al., 1987; Popescu and DiPaolo, 1989; Rassool et al., 1991).In this study, a positive correlation was observed between the location of HPV integration and fragile sites. In the five cell lines where HPV integration could be mapped to a chromosome region, the integration sites occurred within the chromosomal regions known to contain fragile sites. Cytogenetically visible rearrangements at these integration sites were also common in these cell lines. In two lines (FEA and FEH18L), the rearrangements appeared to have included HPV DNA that resulted in two hybridization sites separated by rearranged material (Fig. 1).These rearrangements may be evidence for instability during the early evolution of these cell lines, although the lines are now stable. It is possible
HUMAN KERATINOCYTE CELL LINES
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Figure 2. G-banded FEP18-5 meraphase (A) and the same metaphase hybridized with HPV 18 probe (B), showing location of HPV sequences on the long arm of a derivative chromosome I 0 ( I Oq23424). C: FEPl8-5 metaphase APC treated and Wright’s stained. A gap is visible in the derivative chromosome 10 (arrow). D HPV I 8 hybridization of the metaphase in C. showing an elongated track-like pattern of hybridization in the gapped region of the derivative chromosome 10. E APC-treated FEP18-5 chromosomes with a fragile site expressed on the derivative chromosome 10. HPV 18 probe hybridized t o both sides of the breakpoinr at the fragile site.
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SMITH ET A L
to speculate that this is related to the high rate of inter- and intrachromosomal recombination observed at fragile sites in cells cultured under conditions known to cause expression of fragile sites (Yunis et al., 1987; Glover and Stein, 1988). Evidence for integration of HF'V and other viral DNA at fragile sites has been based primarily on mapping at the metaphase chromosome level (Cannizzaro et al., 1988; Popescu et al., 1990).However, chromosome bands represent large amounts of DNA, and it is difficult to determine the exact proximity of DNA integration to fragile sites with these methods. The coincidence of HPV integration and a fragile site in the FEP18-5 cell line provided the opportunity for further study in vitro of fragile site expression. Treatment of FEP18-5 cells with APC resulted in gaps and breaks in several chromosomes, including the derivative chromosome 10 containing integrated HPV DNA. FISH with HPV 18 DNA revealed viral DNA flanking gaps and breaks and lying between gaps in the derivative chromosome 10. We believe that this is the first clear demonstration of HPV integration within a fragile site. Further investigation with these cell lines and in uivo-derived HPV-containing cell lines will be necessary for determining whether this phenomenon is a common feature of HPV DNA integration. The mode of interaction between cellular and HPV DNA at a fragile site in FEP18-5may not be restricted to HPV DNA but may be indicative of a more generalized mode used by many other types of viral and foreign DNAs when they are introduced into cells. Recent in vitro studies by Rassool et al. (1991) have shown that the uptake of DNA at fragile sites may be a very generalized phenomenon and that foreign DNA may preferentially integrate in these regions. The possibility that the HPV DNA, integrated as a tandem array, is itself prone to fragility remains to be explored. In the interpretation of the results of this study and of those of Rassool et al. (I 991),one must consider the fact that DNA was introduced into cells via calcium phosphate precipitation. Conclusions are, therefore, relevant to this in vitro system. The path to in vitro immortalization and tumorigenicity by HPV may or may not be directly related to the action of HPV in vivo. Continued study, particularly with HPV-containing tumor-derived cell lines, will be necessary for further elucidation of the mechanisms of HPV integration in the human host. We believe that the results of this study confirm those of other studies on mapping of HPV integration sites near fragile sites, and that they strengthen the evidence by revealing HPV integration occurring
within a fragile site. HPV DNA stably integrated at a fragile site in the FEP18-5 cell line also provides a target for cloning of the flanking fragile site DNA sequences of human chromosome 10. Other HPV-immortalized cell lines used in this study may provide a means for isolating and characterizing fragile site sequences in other chromosomes. ACKNOWLEDGMENTS
We thank Marci Wright for manuscript preparation. This research was supported by a Public Health Service grant from the National Cancer Institute (CA42792, principal investigator, J.K.M.) REFERENCES Amarose A€',Huttenlocher PR, Sprudzs RM, Laitsch TJ, Pettenati MJ (1987) A heritable fragile 12q24.13 segregating in a family with a fragile X chromosome. Hum Genet 7k4-6. Bedell MA, Jones KH, Grossman SR, Laimins LA (1989) Identification of human papillomavirus type 18 transforming genes in immortalized and primary cells. J Virol 63:1247 -1255. Berger R, Bloomfield CD, Sutherland GR (1985)Report of the committee on chromosome rearrangements in neoplasia and on fragile sites. Cytogenet Cell Genet 40490-535. Cannizzaro LA, Diirst M. Mendez MJ, Hecht BK, Hecht F (1988)Regional chromosome localization of human papillomavirus integration sites near fragile sites, oncogenes and cancer chromosome breakpoints. Cancer Genet Cytogenet 33:93 98. Diirst M, Kleinheinz A, Hotz M, Gissman L (1985)The physical state of human papillomavirus type 16 DNA in benign and malignant genital tumors. J Gen Virol 66:15151522. Diirst M, Croce CM, Gissman L, Schwarz E, Huebner K (1987a) Wpillomavirus sequences integratt near cellular oncogenes in some cervical carcinomas. Proc Natl Acad Sci USA &1:107&1074. Diirst M, Dzarlieva-Petrusevska RT, Boukamp P, Fusenig NE,Gissman L (1987b) Molecular and cytogenetic analysis of immortalized human primary keratinncytes obtained after transfection with human papillomavirus type 16 DNA. Oncogene 1:251-256. Glover TW, Stein CK (1988) Chromosome breakage and recombination at fragile sites. Am J Hum Genet 43264-273. Haluska FG, Huebner K, Isobe M, Nishimura T, Croci CM, Vogt PK (1988)Localization of the human JUN protooncogene to chromosome region lp31-32. Roc Natl Acad Sci USA 85:2215-2218. Hawlcy-Nelson P, Vousden KH, Hubbert NL, Lowy DK, Schiller JT (1989) The HPV E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes. EMBO J 8:3905-3910. Hurlin PJ, Kaur P, Smith PP, Perez-ReyesN, Blanton RA, McDougal! JK (1991) Progression of human papillomavirus type 18-immortalized human keratinocytes to a malignant phenotype. Roc Natl Acad Sci USA 88:57&574. Johnson GD, llavidson RS, McNamee KC, Russell G, Goodwin I), Holborrow EL (1982) Fading of immunofluorescence during microscopy: A study of its phenomenon and its remedy. J Immunol Methods 55:231-242. Kaur P, McDougall JK (1988) Characterization of primary human keratinocytes transformed by human papillomavirus type 18. J Virol 621917-1924. Kaur P, McDougall JK (1989) HF'V-18 immortalization of human keratinocytes. Virology 173302-310. Kaur P, McDougall JK, Cone R (1989)Immortalization of primary human epithelial cells by cloned cervical carcinoma DNA containing human papillomavirus type 16 E6/B7 open reading frames. J Gen Virol 701261- 1266. Matlashewski G, Schneider J, Ranks L, Jones N, Murray A, Crawford L (1987) Human papillomavirus type 16 DNA cooperates with activated 7m in transforming primary cells. EMBO J 6:1741-1746. McCance DJ (1986) Human papillomaviruses and cancer. Riochim Biophys Acta 823:195-205. McCance DJ, Kopan R, Fuchs E, Laimins LA (1988) Human pap& lomavirus type 16 alters human epithelial cell differentiation in vitro. Proc Natl Acad Sci USA 85:7169 7173. Moyzis RK, Albright KL, Bartholdi MF, Cram LS,Deaven LL, Hildebrand CE, Joste NE,LongmireJL, Meyne J, Schwar~acher-Robinson
HUMAN KERATINOCYTE CELL LINES
T (1987) Human chromosome-specific repetitive DNA sequences: Novel markers for genetic analysis. Chromosoma 95:375386. Murano I, Kuwano A, Kajii T (1989) Fibroblast-specific common fragile sites induced by aphidicolin. Hum Genet 8345 48. Pater MM, Pater A (1985) Human papillomavirus types 16 and 18 sequences in carcinoma cell lines of the cervix. Virology 14531% 318. Pinkel D, Straume T, Gray JW (1986) Cytogenetic analysis using quantitative, high sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 832934 2938. Pirisi I,, Yasumoto S,Feller M, Doniger J, DiPaolo JA (1987) Transformation of human fibroblasts and keratinocytes with human papillomavirus type 16 DNA. J Virol 61:1061- 1066. Popescu NC, DiPaolo JA (1989)Preferential sites for viral integration on mammalian genome. Cancer Genet Cytogenet 42157 171. Popescu NC, DiPaolo JA, Amsbaugh SC (1987) Integration sites of human papillomavirus 18 DNA sequences on HeLa cell chromosomes. Cytogenet Cell Genet 4458-42. Popescu NC, Zimonjic D, DiPaolo JA (1W)Viral integration, fragile sites, and proto-oncogenesin human neoplasia. Hum Genet 8438% 386. Rassool FV, McKeithan TW, Neilly ME, van Melle E, Espinosa 111 R, LeBeau MM (1991) Preferential integration of marker DNA into the chromosomal fragile site 3p14 An approach to cloning fragile sites. Proc Natl Acad Sci USA 88:66574661. Sakaguchi AY, Zabel BU, Grzeschik KH, Law ML, Naylor PJ (1984) Human proto-oncogeneassignments. Cytogenet Cell Genet 37572573. Schwarz E, Freese UK, Gissman L, Mayer W, Roggenbuck B, Stremlau A, zur Hausen H (1985)Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature 314111 114.
157
Schlegel R, Phelps WC, Zhang Y-L, Barbosa M (1988) Quantitative keratinocyte assay detects two biological activities of human papillomavirus DNA and identifies viral types associated with cervical carcinoma. EMBO J 7:3181-3187. Smith PP, Bryant EM, Kaur P, McDougall JK (1989) Cytogenetic analysis of eight human papillomavirus immortali7~dhuman keratinocyte cell lines. Int J Cancer 441124 1131. Spence RP, Murray A, Banks L, Kelland LR, Crawford L (1988)Analysis of human papillomavirus sequences in cell lines recently derived from cervical cancers. Cancer Res 48:324--328. Sutherland GR, Parslow MI, Baker E (1985) New classes of common fragile sites induced by 5-azacytidine and bromdeoxyuridine. Hum Genet 69233 237. Watanabe S, Kanda T, Yoshiike K (1989) Human papillomavirus type 16 transformation of primary human embryonic fibroblasts requires expression of open reading frames E6 and E7. J Virol 63:96.5969. Yasumoto S, Burkhardt AL, Doniger J, DiPaolo JA (1986)Human papillomavirus type 16 DNA-induced malignant transformation of NIH3T3 cells. J Virol 57572~577. Yasumoto S, Doniger J, DiPaolo JA (1987) Differential early viral gene expression in two stages of human papillomavirus type 16 DNAinduced malignant transformation. Cell Biol 72165-2172. Yunis JJ, Soreng AL (1984) Constitutive fragile sites and cancer. Science 2261 1%1204. Yunis JJ, Sareng AL,Bowe AE (1987)Fragile sites are targets of diverse mutagens and carcinogens. Oncogene 1:5%69. zur Hausen H (1989) Papillomaviruses in anogenital cancer as a model to understand the role of viruses in human cancers. Cancer Res 494677-4681, zur Hausen H, Schneider A (1987) The role of papillomaviruses in human anogenital cancer. In Salzman N, Lowley PM (eds): Papovaviridae, Vol. 2. New York Plenum Press, pp 245 263.
