NON-RANDOM ALLELIC LOSSES AT 3p, 11p AND 13q DURING HPV-MEDIATED IMMORTALIZATION AND CONCOMITANT LOSS OF TERMINAL DIFFERENTIATION OF HUMAN KERATINOCYTES Renske D.M. STEENBERGEN, Mario A.J.A. HERMSEN, Jan M.M. WALBOOMERS, Gerrit A. MEIJER, Jan P.A. BAAK, Chris J.L.M. MEIJER and Peter J.F. SNIJDERS* Units of Molecular and Quantitative Pathology, Department of Pathology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands To obtain a comprehensive overview of chromosomal alterations that may underlie human papillomavirus (HPV)- mediated immortalization, 4 foreskin keratinocyte cell lines generated by transfection with either HPV 16 (cell lines FK16A and FK16B) or HPV 18 (FK18A and FK18B) were subjected to chromosomal analysis using comparative ge- nomic hybridization (CGH). Three cell lines were analyzed both in the mortal state during their extended lifespan and in the subsequent immortal state. From cell line FK18A, only immortal cells were tested. Chromosomal imbalances in- creased in number through the process of immortalization. Subsequent loss of heterozygosity (LOH ) analysis, using a panel of 21 microsatellite markers selected on the basis of CGH losses, revealed no clonal LOHs in cells at the mortal stage. However, in the immortal descendants 67% of under- representations detected by CGH were expressed as clonal LOH at the respective loci. Clonal LOHs at 3p, 11p and 13q were detected in 2 cell lines each and were thus considered non-random. Immortal cells of 1 cell line (FK18B) revealed LOH at all 3 loci. Moreover, all immortal cell lines displaying allelic losses at one or more of these loci shared a severely dysplastic phenotype after organotypic culturing, as shown previously. T herefore, loss-of-function mutations of genes at these loci, eventually in combination, are potentially involved in the process of HPV-mediated immortalization that is attended by a loss of terminal differentiation. Since chromo- somal changes at these loci are also found in H PV-associated carcinomas in vivo, the HPV-transfected cell lines seem to provide a valuable model system for studying HPV-mediated carcinogenesis. Int. J. Cancer 76:412–417, 1998.  1998 Wiley-Liss, Inc. It is currently widely accepted that certain high-risk human papillomavirus (HPV) types, most notably HPV 16 and HPV 18, are etiologically involved in carcinogenesis of the uterine cervix and oropharynx (zur Hausen, 1994; Snijders et al., 1994). One hallmark of these HPV types is their ability to induce immortaliza- tion of primary human epithelial cells in vitro. Moreover, in both anogenital and respiratory tract epithelial cells this process depends on functions encoded by the viral E6 and E7 genes (reviewed by zur Hausen, 1994). The high-risk HPV E6 and E7 gene products have the intrinsic property to inactivate the tumor suppressor gene products p53 and pRb, respectively (reviewed by zur Hausen, 1994). Therefore, it is conceivable that p53 and pRb inactivation are important steps in immortalization and, likewise, in HPV- mediated carcinogenesis in vivo. However, although essential, such HPV functions are not sufficient for immortalization and need to be supplemented with genetic alterations involving the host cell genome. Somatic cell fusion experiments have suggested that recessive changes (i.e., tumor suppressor gene inactivations) are pivotal (Chen et al., 1993; Seagon and Du ¨rst, 1994). Still, little is known about the nature of these changes. In an attempt to unravel host cell alterations that may be involved in HPV-mediated immortalization, we have described a longitudinal in vitro model system using HPV 16- and HPV 18-transfected foreskin keratinocytes (Steenbergen et al., 1996). Comparative analysis of 2 HPV 16- and 2 HPV 18-containing cell lines at different time intervals during monolayer culture revealed an association between strong telomerase activity and the immortal phenotype. Moreover, loss of heterozygosity (LOH) analysis using 10 informative microsatellite markers revealed that LOH at 3p combined with either 11q or 18q, or at 10p was associated with immortalization in 3 of the 4 cell lines. In contrast, in the 4th cell line no alterations were found after immortalization. This may either reflect the restrictions of LOH analysis, the limited number of markers used, or a combination thereof. Therefore, it is unclear to what extent we have mapped all changes potentially important for immortality. In the present study, we tried to obtain a more extensive assessment of the genetic alterations that may underlie immortalization. Comparative genomic hybridization (CGH) al- lows genome-wide mapping of regions with altered copy numbers (i.e., gains and losses) in a single experiment (du Manoir et al., 1995). Consequently, we applied CGH to supplement previous LOH analysis of the cell lines during and subsequent to immortal- ization. Additional LOH analysis, based on losses detected by CGH, finally revealed that clonal allele losses at 3 loci, i.e., 3p, 11p and 13q, were non-randomly associated with immortalization. MATERIAL AND METHODS Cell lines The cell lines FK16A, FK16B, FK18A and FK18B were established by transfection of primary human foreskin keratino- cytes (EK94–2) with the entire HPV 16 and HPV 18 genome, respectively (Steenbergen et al., 1996). Cells were grown in serum- free keratinocyte growth medium (Life Technologies, Breda, The Netherlands) supplemented with bovine pituitary extract (50 μg/ml), epidermal growth factor (5 ng/ml), penicillin (100 U/ml), strepto- mycin (100 μg/ml) and L-glutamine (2 mM; Life Technologies). During culture, cells were regularly harvested at different passages for subsequent DNA isolation. For the same purpose cells from non-transfected donor keratinocytes were harvested at passage 6. Genomic DNA was isolated using the Puregene DNA isolation kit according to the manufacturer’s recommendations (Biozym, Land- graaf, The Netherlands) Comparative genomic hybridization One microgram of each cell line and reference DNA was labeled by nick translation with biotin-16-dUTP and digoxygenin-11- dUTP (Boehringer Mannheim, Germany), respectively. DNA from the primary donor keratinocytes (EK94–2) served as reference DNA for CGH analysis of HPV-transfected cell lines, after the Grant sponsor: Dutch Cancer Society; Grant numbers: VU93–605 and VU96–1151; Grant sponsor: Royal Netherlands Academy of Arts and Sciences. *Correspondence to: Unit of Molecular Pathology, Department of Pathology, University Hospital Vrije Universiteit, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. Fax: (31)20–4442964. E-mail: pjf.snijders@azvu.nl Received 29 September 1997; Revised 5 December 1997 Int. J. Cancer: 76, 412–417 (1998)  1998 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer