Expression of mTert in primary murine cells links the growth-promoting effects of telomerase to transforming growth factor-beta signaling.

Here, we show that ectopic expression of the catalytic subunit of mouse telomerase (mTert) confers a growth advantage to primary murine embryonic fibroblasts (MEFs), which have very long telomeres, as well as facilitates their spontaneous immortalization and increases their colony-forming capacity upon activation of oncogenes. We demonstrate that these telomere length-independent growth-promoting effects of mTert overexpression require catalytically active mTert, as well as the formation of mTert/Terc complexes. The gene expression profile of mTert-overexpressing MEFs indicates that telomerase enhances growth in these cells through the repression of growth-inhibiting genes of the transforming growth factor-beta (TGF-beta) signaling network. We functionally validate this result by showing that mTert abrogates the growth-inhibitory effect of TGF-beta in MEFs, thus demonstrating that telomerase increments the proliferative potential of primary mouse embryonic fibroblasts by targeting the TGF-beta pathway.

New androgen response elements in the murine pem promoter mediate selective transactivation.

The Pem homeobox transcription factor is expressed under androgen control in the testis and epididymis. It is also transcribed in the ovary, muscle, and placenta. The mouse Pem gene promoter was cloned and sequenced. It was analyzed in transactivation tests using CV-1 and PC-3 cells expressing the AR and found to be strongly stimulated by androgens. EMSAs and mutational analysis of the Pem promoter allowed the identification of two functional androgen response elements named ARE-1 and ARE-2. They both differed from the consensus semipalindromic steroid response element and exhibited characteristics of direct repeats of the TGTTCT half-site. Unlike the steroid response element, both Pem androgen response elements were selectively responsive to androgen stimulation. Specific mutations in the left half-site of Pem ARE-1 and ARE-2, but not of the steroid response element, were still compatible with AR binding in the EMSA. In addition, Pem ARE-1, but not ARE-2 or the steroid response element, showed some flexibility with regard to spacing between half-sites. These results strongly suggest that the AR interacts differently with direct repeats than with inverted repeats, potentially leading to cis element-driven selective properties. Thus, the existence of several classes of DNA response elements might be an essential feature of differential androgen regulation.

Enhanced productivity during controlled proliferation of BHK cells in continuously perfused bioreactors.

A perfused cell-culture process was developed to investigate the stability of IRF-1-mediated proliferation control in BHK cells and to evaluate the efficacy of a novel promoter in these cells. The cell density of proliferation-controlled producer cells was effectively regulated for over 7 weeks in a microcarrier-based continuously perfused bioreactor. An IRF-1-inducible promoter was employed to express a heterodimeric IgG antibody as a relevant model protein. Basal expression levels were equivalent to that of a highly active viral promoter, while productivity increased up to sixfold during growth arrest. However, no stably expressing clone was isolated in this study. Protein expression decreased gradually with time and could not be induced further in subsequent growth-repression cycles. The results demonstrate that the regulatory system is sufficiently stable to allow controlled growth in a continuous scalable reactor system and that productivity increases can be achieved in a proliferation controlled microcarrier culture.