Disregulation of mitotic checkpoints and regulatory proteins following acute expression of SV40 large T antigen in diploid human cells

Abstract

SV40 large T antigen (T) inactivates the tumor suppressor proteins p53 and pRb, and can induce cells to enter DNA replication at inappropriate times. We show here that T also compromises three cell cycle checkpoints that regulate the entry into and exit from mitosis. Human diploid ®broblasts infected with a retrovirus expressing T displayed an attenuated radiation-induced mitotic delay, were more susceptible to chemical-induced uncoupling of mitosis from the completion of DNA replication, and were more likely to exit mitosis and rereplicate their DNA when mitotic spindle assembly was inhibited. Consistent with altered mitotic checkpoint control, cells expressing T displayed elevated protein levels and/or associated activities of the mitotic regulatory proteins cyclin A, cyclin B, Cdc25C and p34 cdc2 . These changes in mitotic control were evident within 5 ± 10 population doublings after retroviral infection, indicating a direct e ect of T expression. Cells acutely infected with the T-expressing retrovirus su ered numerical and structural chromosome aberrations, including increases in aneuploidy, dicentric chromosomes, chromatid exchanges and chromosome breaks and gaps. These ®ndings indicate that T rapidly disrupts mitotic checkpoints that help maintain genomic stability, and suggest mechanisms by which T induces chromosome aberrations and promotes the immortalization and neoplastic transformation of human cells.

The SV40 large T oncoprotein disrupts DNA-binding of the cell cycle-regulated transcriptional repressor CDF

“…Taken together, our observations therefore demonstrate that SV-LT abrogates repression of the cdc25C promoter which is fully consistent with the genomic footprinting data showing a loss of CDF-DNA interaction. Our observations are also in agreement with published data showing that SV-LT expressing cells contain elevated levels of Cdc25C (Chang et al, 1997).…”

“…The transcriptional deregulation of cell cycle genes appears to be a major strategy employed by the oncoproteins of DNA tumor viruses to override growth control mechanisms in mammalian cells (Buchou et al, 1993;Oshima et al, 1993;Spitkovsky et al, 1994;Cheng et al, 1995;Schulze et al, 1995;Chang et al, 1997). Oncoproteins, including the adenovirus E1a gene product, the SV40 large T antigen (SV-LT), and the human papilloma virus E7 protein, bind pocket proteins through regions which are necessary for transformation (DeCaprio et al, 1989;Dyson et al, 1989).…”

The Werner syndrome protein has separable recombination and survival functions☆

“…The apparent copy number independence of WRN function in our experiments may reflect the comparatively high level of WRN protein in fibroblast cell lines [27] and/or attenuated DNA damage checkpoint functioning in SV40-transformed fibroblast cell lines (see, e.g. [28]). Fig.…”

The mitogen-activated protein (MAP) kinase cascade can either stimulate or inhibit DNA synthesis in primary cultures of rat hepatocytes depending upon whether its activation is acute/phasic or chronic

“…Since both oncogenic Ras and oncogenic Raf will cause potent activation of the MAP kinase cascade, these data would initially appear to be in conflict with our results. However, because these hepatocyte cell lines were immortalized with SV40 virus the function of p53 may have been compromised, and p53 function has been demonstrated to be one mechanism by which cells can induce expression of p21 Cip-" [40][41][42]. Thus it is probable that SV40 transformation of these hepatocyte cell lines may have made these cells incompetent to induce p21 Cip-" via chronic activation of the MAP kinase cascade.…”