Published OnlineFirst September 29, 2009; DOI: 10.1158/0008-5472.CAN-09-1003 Molecular Biology, Pathobiology, and Genetics Role of SV40 Integration Site at Chromosomal Interval 1q21.1 in Immortalized CRL2504 Cells Jinglan Liu,1 Gurpreet Kaur,1 Vikramjit K. Zhawar,1 Drazen B. Zimonjic,2 NicholasC. Popescu, 2 Raj P. Kandpal,3 and Raghbir S. Athwal1 1Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania; 2Laboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, Maryland;and 3Department of Basic Medical Sciences, Western University of Health Sciences, Pomona, California Abstract state of crisis that is characterized by massive cell death through apoptosis or necrosis (3). However, a rare variant cell (1 in 107 to We have applied a functional gene transfer strategy to show 8 the importance of viral integration site in cellular immortal- 10 ) survives the crisis state and continues to multiply indefinitely ization. The large tumor antigen of SV40 is capable of (2). These studies suggest that M1 and M2 are regulated through extending the cellular life span by sequestering tumor separate genetic controls, and immortalized cells have either suppressor proteins pRB and p53 in virus-transformed human inactivated or bypassed regulatory pathways involved in both M1 cells. Although SV40 large T antigen is essential, it is not and M2 stages. sufficient for cellular immortalization, suggesting that addi- The presence of SV40 has been reported in some human tumors, tional alterations in cellular genes are required to attain such as mesothelioma, osteosarcoma, ependymomas (4–7), bron- chopulmonary carcinoma, and nonmalignant pulmonary disease infinite proliferation. We show here that the disruption of human chromosomal interval at 1q21.1 by SV40 integration (8). Despite these observations, the role of SV40 in human cancers can be an essential step for cellular immortalization. The remains controversial. On the other hand, HPV has been detected transfer of a 150-kb bacterial artificial chromosome (BAC) in >99% of cervical cancer cases (9). Although the above clone, RP364B14, corresponding to viral integration site in observations suggest viral etiology of these cancers, the contribu- CRL2504 cells, reverted their immortal phenotype. Interest- tion of disrupted host genes due to integration of viral genome has ingly, the BAC transfer clones of CRL2504 cells displayed not been addressed. characteristics of either senescence as shown by B-galactosidase It has been shown that SV40-LT releases the host cell from the activity or apoptosis as revealed by positive staining with G1 checkpoint (M1 stage) and extends its life span by sequestering M30 CytoDEATH antibody. The SV40 integration at 1q21.1, pRB and p53 family of tumor suppressor proteins (10–12). In order in the vicinity of epidermal differentiation complex (EDC) for cell to attain indefinite growth potential, however, additional genes, resulted in the down-regulation of the filaggrin (FLG) mutations in cellular genes are essential to bypass M2 stage (2). gene that is part of the EDC. FLG gene expression was Although SV40 is known to integrate randomly at multiple sites in the host genome, the immortalized cells often show its integration increased in BAC transfer senescent and apoptotic clones. Our results suggest that the disruption of native genomic at a unique site. We propose that the disruption of a specific sequence by SV40 may alter expression of genes involved in genomic site due to the viral integration may contribute to senescence and apoptosis by modulating chromatin struc- malignant transformation of human cells. Furthermore, the ture. These studies imply that identification of genes located integration of SV40 into a specific genomic site may promote in the vicinity of viral integration sites in human cancers may genomic instability and likely confer growth advantage. be helpful in developing new diagnostic and therapeutic We have applied a functional approach to restore senescence in strategies. [Cancer Res 2009;69(19):7819–25] SV40 immortalized cells and investigated the role of the integration site in cellular immortalization. We have characterized the genomic region in the vicinity of the site interrupted by SV40 Introduction integration. The introduction of a genomic clone, corresponding to the genomic site disrupted by SV40 at 1q21.1, restores senescence The mechanisms of neoplastic transformation have been or apoptosis in CRL2504, an immortalized cell line. addressed by using human cells immortalized with DNA tumor viruses or viral sequences, such as SV40 large T antigen (SV40-LT), human papillomavirus (HPV) 16 E6 and E7, and adenovirus E1A Materials and Methods and E1B (1). A two-stage model consisting of M1 (mortality stage 1) Cell Lines and Growth Conditions and M2 (mortality stage 2) has been proposed for SV40-LT– A single-cell subclone of a human bronchial epithelial cell line, À induced immortalization of human cells (2). The cells challenged immortalized with a cloned Ori SV40 (13), was used in these studies. with SV40-LT bypass M1 stage and continue to multiply until they CRL2504 cells contain a single integrated copy of SV40 genome and do not reach M2 stage (2). Subsequently, the entire cell population enters a contain any free virus.4 The cell lines used for comparison included normal human diploid fibroblasts GMO3468A (Genetic Mutant Cell Repository, Camden, NJ) and FS-2 (generated in the lab). The cell lines were routinely Requests for reprints: Raghbir S. Athwal, Fels Institute for Cancer Research cultured at 37jC in a 7.5% CO2 atmosphere in DF/12 medium supplemented and Molecular Biology, Temple University School of Medicine, AHB 202, 3307 North with 10% fetal bovine serum containing 1% penicillin and streptomycin. The Broad Street, Philadelphia, PA 19140. Phone: 215-707-6931;Fax: 215-707-2989;E-mail: [email protected]. I2009 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-09-1003 4 J. Liu and R.S. Athwal, unpublished observations. www.aacrjournals.org 7819 Cancer Res 2009; 69: (19). October 1, 2009 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst September 29, 2009; DOI: 10.1158/0008-5472.CAN-09-1003 Cancer Research gene transfer clones carrying neo marker were isolated and maintained in BrdUrd was carried out for 24 h, and the cells were subsequently fixed for the medium containing G418 (400 Ag/mL). 45 min in a fixative solution (7 volumes of ethanol and 3 volumes of 50 mmol/L glycine). The fixed cells were hybridized with a 1:600 dilution of Isolation of Human DNA Flanking SV40 Insertion Site by mouse anti-BrdUrd monoclonal antibodies (Molecular Probes), and signal Inverse PCR was detected by staining with a 1:200 dilution of the Alexa Fluor The EcoRI-digested CRL2504 DNA (400 ng) was circularized by overnight 488–conjugated donkey anti-mouse IgG. The nuclei were stained with incubation at 16jC in an appropriate reaction buffer containing 2 ALofT4 4¶,6-diamidino-2-phenylindole (DAPI), and cells were visualized under a DNA ligase (New England Biolabs). The circularized DNA was amplified fluorescence microscope. with the primer pair SV40 1726F/796R specific to the SV40 sequences flanking the human genomic DNA. The amplification was performed in a 25 Analysis for Apoptosis CytoDEATH antibody M30 (Roche Diagnostic Corp.), which recognizes a AL reaction mixture containing 5 AL of ligation products, 2 mmol/L MgCl2, 200 Amol/L each of deoxynucleotide triphosphates, 1 Amol/L of each specific caspase cleavage site within CK18, was used to detect apoptosis primer, and 1 unit of Taq polymerase (Promega). The reaction conditions (17). The cells were seeded on eight-chamber slides (Nalgene Nunc À j included initial denaturation at 96jC for 5 min followed by 35 cycles of International) and fixed in ice-cold methanol at 20 C for 30 min. The 1 min at 94jC, 1 min at 58jC, and 2 min at 72jC. The amplified products fixed cells were treated with 0.25% Triton X-100 (Sigma) and then incubated were fractionated by electrophoresis in a 2% agarose gel, visualized by for 2 h with donkey serum (Molecular Probes). The slides were overlaid with A j ethidium bromide staining, and purified from the gel for cloning. a solution (100 L) containing M30 antibody and incubated at 4 C overnight. The signal was detected by staining with a 1:200 dilution of Transfer of Bacterial Artificial Chromosome Clone into donkey anti-mouse IgG linked with Alexa Fluor 488 (Molecular Probes), and CRL2504 Cells cells were photographed using Olympus AHVT3 fluorescence research The bacterial artificial chromosome (BAC) clones RP11-364B14 and photomicroscope system. 152L6 were retrofitted with MJ0X166 to incorporate neo marker using a Sequences of Primers vector exchange procedure (14). Briefly, human DNA inserts from the BAC Sequences of various primers used are as the follows: clones were released by digestion with NotI restriction enzyme (New England Biolabs) and separated by pulsed-field gel electrophoresis (PFGE). SV40-1726F: 5¶-ACA GTT TAC AGA TGA CTC TCC-3¶ The purified inserts were mixed separately with NotI-digested and SV40- 796R: 5¶-TTG CAG TAA AGC TGC AAA TCC -3¶ dephosphorylated retrofitting vector pJMOX166 at a molar ratio of 2:1 or SV40-1745F: 5¶-CCA GAC AAA GAA CAA CTG CC- 3¶ 4:1 and ligated using T4 DNA ligase at 16jC for 16 h. An aliquot (1 AL) of the SV40- 477R: 5¶- CCG TCA ACA GTA TCT TCC CC-3¶ ligated products was transformed into competent DH-5a cells, and cells H-F87: 5¶- GAT CCC AAC TAA AAC ATC ACC- 3¶ were plated on LB agar plates containing chloramphenicol (12.5 Ag/mL) H-R484: 5¶-AGC CCT TAT TGT TTA AAA GAC C-3¶ and kanamycin (30 Ag/mL). The DNA isolated from the retrofitted clones SV2504-IS-F165: 5¶-CTT CTG TTA TAT CAT TTG ACC C-3¶ was digested with NotI, fractionated by PFGE, and blotted onto a SV2504-IS-R453: 5¶-TAC TGT TTC ATT CCT TGA GCC-3¶ membrane.