Role of SV40 integration site at chromosomal interval 1q21.1 in immortalized CRL2504 cells

Abstract

We have applied a functional gene transfer strategy to show the importance of viral integration site in cellular immortalization. The large tumor antigen of SV40 is capable of extending the cellular life span by sequestering tumor suppressor proteins pRB and p53 in virus-transformed human cells. Although SV40 large T antigen is essential, it is not sufficient for cellular immortalization, suggesting that additional alterations in cellular genes are required to attain infinite proliferation. We show here that the disruption of human chromosomal interval at 1q21.1 by SV40 integration can be an essential step for cellular immortalization. The transfer of a 150-kb bacterial artificial chromosome (BAC) clone, RP364B14, corresponding to viral integration site in CRL2504 cells, reverted their immortal phenotype. Interestingly, the BAC transfer clones of CRL2504 cells displayed characteristics of either senescence as shown by beta-galactosidase activity or apoptosis as revealed by positive staining with M30 CytoDEATH antibody. The SV40 integration at 1q21.1, in the vicinity of epidermal differentiation complex (EDC) genes, resulted in the down-regulation of the filaggrin (FLG) gene that is part of the EDC. FLG gene expression was increased in BAC transfer senescent and apoptotic clones. Our results suggest that the disruption of native genomic sequence by SV40 may alter expression of genes involved in senescence and apoptosis by modulating chromatin structure. These studies imply that identification of genes located in the vicinity of viral integration sites in human cancers may be helpful in developing new diagnostic and therapeutic strategies.

Figure 1

Sequence surrounding SV40 integration site and its chromosomal location. A. The sequence flanking the integration site was isolated by inverse PCR as described in the Methods’ section. The locations of SV40 primers used for amplification are indicated by arrows. B. Primer sequences corresponding to the human DNA region shown in panel A were used to isolate the BAC clone RP11-364B14, and its location as determined by FISH on chromosome 1q21.2 is indicated by arrows.

Figure 2

Morphology of senescent and apoptotic transfer clones. A. Senescent transfer clones of CRL2504/BAC364B14 were grown and photographed after 21 days (panel 1), 28 days (panel 2), 35 days (panel 3) and 42 days (panel 4). B. CRL2504 cells 8 weeks after transfection with the empty vector (panel 1), 8 weeks after transfer of an unrelated BAC 152L6 (panel 2), 23 days after transfer of BAC364B14 (panel 3) and 27 days after transfer of BAC364B14 (panel 4) were photographed as described in Methods’ section.

Figure 3

Visualization of cells after BrdU incorporation and senescence associated β-galactosidase (SA-βgal) assay. A. Native CRL2504 cells (panel 1) and BAC364B14 transfer clone #10 (panel 3) were visualized with anti-BrdU antibody after growing the cells in the presence of BrdU. The nuclei of native cells (panel 2) and the cells from BAC364B14 transfer clone #10 (panel 4) were subsequently stained with DAPI. The cells were photographed as described in Methods’ section. B. The cells from a confluent culture of native CRL2504 (panel 1), GMO3468 cells after 62 passages (panel 2), BAC364B14 transfer clone #10 after 7 weeks (panel 3) and BAC364B14 transfer clone # sen II after 6 weeks (panel 4) were assayed for the presence of SA-βgal activity. The cells were photographed as above.

Figure 4

Staining of apoptotic cells with cytoDEATH M30 antibody. Native CRL2504 cells (A), vector transfer clone (panel B), BAC364B14 transfer clone (panel C) and floating cells from the dish containing BAC364B14 transfer clone (panel D) were stained with cytoDEATH M30 antibody and photographed as described in Methods’ section.

Figure 5

Semi-quantitaive evaluation of filaggrin (FLG) transcript by RT-PCR. A. RT-PCR was performed with FLG specific primers and GAPDH primers on total RNA isolated from CRL2504 parent (lane 1), CRL2504/pJMOX166 vector control (lane 2), CRL-2504/152L16 (lane 3), immortal CRL2504/364B14 Clone 5.1 (lane 4), immortal CRL2504/364B14 Clone 5.3 (lane 5), immortal CRL2504/364B14 Clone 5.7(lane 6), senescent CRL-2504/364B14 clone 5.5 (lane 7), senescent CRL 2504/364B14 clone 5.9 (lane 8) and senescent CRL 2504/364B14 clone 5.17 (lane 9), and the amplified products were electrophoresed on an agarose gel as described. The arrows indicate the amplified products. B. The FLG transcript was normalized by calculating the ratio of FLG and GAPDH products. The ratios are expressed as percentages.