Ras Transformation Overrides a Proliferation Defect Induced by Tpm3.1 Knockout.

Extensive re-organisation of the actin cytoskeleton and changes in the expression of its binding proteins is a characteristic feature of cancer cells. Previously we have shown that the tropomyosin isoform Tpm3.1, an integral component of the actin cytoskeleton in tumor cells, is required for tumor cell survival. Our objective was to determine whether cancer cells devoid of Tpm3.1 would evade the tumorgenic effects induced by H-Ras transformation. The tropomyosin isoform (Tpm) expression profile of a range of cancer cell lines (21) demonstrates that Tpm3.1 is one of the most broadly expressed Tpm isoform. Consequently, the contribution of Tpm3.1 to the transformation process was functionally evaluated. Primary embryonic fibroblasts isolated from wild type (WT) and Tpm3.1 knockout (KO) mice were transduced with retroviral vectors expressing SV40 large T antigen and an oncogenic allele of the H-Ras gene, H-RasV12, to generate immortalized and transformed WT and KO MEFs respectively. We show that Tpm3.1 is required for growth factor-independent proliferation in the SV40 large T antigen immortalized MEFs, but this requirement is overcome by H-Ras transformation. Consistent with those findings, we found that Tpm3.1 was not required for anchorage independent growth or growth of H-Ras-driven tumors in a mouse model. Finally, we show that pERK and Importin 7 protein interactions are significantly decreased in the SV40 large T antigen immortalized KO MEFs but not in the H-Ras transformed KO cells, relative to control MEFs. The data demonstrate that H-Ras transformation overrides a requirement for Tpm3.1 in growth factor-independent proliferation of immortalized MEFs. We propose that in the SV40 large T antigen immortalized MEFs, Tpm3.1 is partly responsible for the efficient interaction between pERK and Imp7 resulting in cell proliferation, but this is overidden by Ras transformation.

Up-regulation of survivin during immortalization of human myofibroblasts is linked to repression of tumor suppressor p16(INK4a) protein and confers resistance to oxidative stress.

Survivin is an essential component of the chromosomal passenger complex and a member of the inhibitor of apoptosis family. It is expressed at high levels in a large variety of malignancies, where it has been implicated in drug resistance. It was also shown previously that survivin is up-regulated during telomerase-mediated immortalization, which occurs at a relatively early stage during carcinogenesis. This study shows that up-regulation of survivin during immortalization of human myofibroblasts is an indirect consequence of the repression of p16(INK4a). Survivin and p16(INK4a) were functionally linked by assays that showed that either the up-regulation of survivin or repression of p16(INK4a) rendered telomerase-transduced MRC-5 myofibroblasts resistant to oxidative stress. Conversely, siRNA-mediated down-regulation of survivin activated caspases and enhanced the sensitivity of immortal MRC-5 cells to oxidative stress. The E2F1 transcription factor, which is negatively regulated by the pRB/p16(INK4a) tumor suppressor pathway, was implicated in the up-regulation of survivin. Using the ChIP assay, it was shown that E2F1 directly interacted with the survivin gene (BIRC5) promoter in cells that spontaneously silenced p16(INK4a) during telomerase-mediated immortalization. E2F1 binding to the BIRC5 was also enhanced in telomerase-transduced cells subjected to shRNA-mediated repression of p16(INK4a). Together, these data show that repression of p16(INK4a) contributes to the up-regulation of survivin and thereby provides a survival advantage to cells exposed to oxidative stress during immortalization. The up-regulation of survivin during immortalization likely contributes to the vulnerability of immortal cells to transformation by oncogenes that alter intracellular redox state.

Mild hyperoxia limits hTR levels, telomerase activity, and telomere length maintenance in hTERT-transduced bone marrow endothelial cells.

Reactivation of telomerase in endothelial cells (ECs) may be an effective approach to the treatment of vascular disorders associated with telomere attrition and EC senescence. However, overexpression of human telomerase reverse transcriptase (hTERT) does not prevent net telomere loss in ECs grown in standard culture medium with exposure to atmospheric oxygen (21% O(2)). Since these culture conditions are hyperoxic relative to normal tissue in vivo, where oxygen tension is estimated to be 1%-6%, we examined the effects of reduced exposure to oxidative stress (OS) on telomere length maintenance in hTERT-transduced bone marrow endothelial (BMhTERT) cells. Propagation of BMhTERT cells in the free radical scavenger, tert-butylhydroxylamine (tBN), and/or in 5% O(2) increased telomerase enzyme activity and facilitated telomere length maintenance. The enhancement of telomerase activity correlated with higher levels of the telomerase RNA component (hTR). We also investigated the role of the telomere binding protein, TRF1, in telomere length regulation under alternate OS conditions. Inhibition of TRF1 function had no effect on telomere length in BMhTERT cells grown under standard culture conditions. However, alleviation of OS by growth in tBN plus 5% O(2), elevated hTR levels, enhanced telomerase enzyme activity, and enabled progressive telomere lengthening. The direct impact of hTR levels on telomerase-mediated telomere lengthening was demonstrated by overexpression of hTR. BMhTERT cells transduced with hTR exhibited very high telomerase enzyme activity and underwent dramatic telomere lengthening under standard culture conditions. Overall, these results demonstrate that hTR levels are reduced by mild hyperoxia and limit telomerase-mediated telomere lengthening in hTERT-transduced ECs.