Telomerase activity in primary cultures of normal adrenocortical cells

Abstract

Telomerase activity was measured in isolated cells from bovine and human adrenal cortex, in cells in primary culture, in cells in later passages in culture, and in cells genetically modified by expression of hTERT (human telomerase reverse transcriptase). Telomerase activity in freshly isolated bovine adrenocortical cells and in human adrenal cells from donors of various ages (6-79 years) was very low or undetected. However, primary bovine adrenocortical cell cultures were strongly positive for telomerase activity, and primary human adrenocortical cell cultures were weakly positive. Both cell types proliferate in primary culture but proliferation of bovine cells is much more vigorous. When primary bovine cells were subcultured to make successively secondary and tertiary cultures, telomerase activity declined strongly, and was undetected by the third passage. There was only a slight decrease in growth rate over this period. Levels of the telomerase RNA component did not change with passage number when assessed by semi-quantitative competitive RT-PCR. When both bovine and human cells were infected with a retrovirus encoding hTERT, telomerase activity in the cells was very high. We conclude that in the adrenal cortex, as in some other tissues, TERT expression is regulated and upregulation of telomerase activity is associated with rapid proliferation in primary culture. Telomerase activity is not maintained, and introduction of TERT is required for stable telomerase activity and for immortalization.

Progressive Loss of Malignant Behavior in Telomerase-Negative Tumorigenic Adrenocortical Cells and Restoration of Tumorigenicity by Human Telomerase Reverse Transcriptase

“…Consistent with this scenario, Ras G12V /SV40 TAg-expressing human and bovine adrenocortical cells had very low levels of telomerase activity (ref. 36; Fig. 4).…”

“…Antibodies used were as follows: SV40 TAg, mouse monoclonal antibody PAb108 (Santa Cruz Biotechnology, Santa Cruz, CA); and Ras, mouse monoclonal antibody (R02120, Transduction Laboratories, Lexington, KY). Telomerase activity in detergent extracts of cells was assessed by the telomerase repeat amplification protocol (TRAP) assay as described previously (36). Telomere restriction fragment (TRF) analysis was performed as described previously (27), except that DNA fragments were separated by pulse-field gel electrophoresis.…”

The level of oncogene H-Ras correlates with tumorigenicity and malignancy

“…Similar to normal human cells, normal bovine cells lack telomerase activity and although the telomeres of bovine cells are relatively long and continue to shorten with each cell division, they eventually become non functional and cell divisions ceases. 25 It has been shown that H-Ras and SV40T can transform normal bovine adrenocortical cells into malignant tumors, indicating that telomerase is not essential for tumorigenesis. Therefore, it is more accurate and physiologically relevant to study the roles of oncogenes such as H-Ras, P53 and pRb in the transformation of human and bovine cells in a telomerase deficient background.…”

Improving cell therapy—experiments using transplanted telomerase-immortalized cells in immunodeficient mice

“…We have generated hTERT-modified bovine and human adrenocortical cells (Thomas et al, 2000;Suwa et al, 2001;Yang et al, 2001;Hornsby, 2004). For both bovine and human adrenocortical cells, we find that when culture conditions are optimized, ectopic expression of hTERT appears to be the only requirement for immortalization (Thomas et al, 2000;Yang et al, 2001;Hornsby, 2004).…”