Clonal analysis of crisis in bone marrow endothelial cells transduced with human telomerase reverse transcriptase (hTERT).
Authors: Victoria W. Wen, Kaida Wu, Sheik Baksh, Malcolm A. S. Moore, Karen L. Mackenzie
Journal: Cancer Research (2004)

Proc Amer Assoc Cancer Res, Volume 45, 2004

2415 

We previously demonstrated that ectopic expression of the catalytic component of telomerase (hTERT) restored telomerase activity and extended the lifespan of normal diploid human bone marrow endothelial cells (BMECs)(1). However, expression of hTERT was not sufficient for immortalization, as 2/3 hTERT-transduced BMEC (BMhTERT) mass cultures were subject to a growth crisis. Crisis was characterized by an increase in cell death and dramatic reduction in growth rate. Immortal cells eventually grew out of one of these crisis cultures. The third BMhTERT culture was immortalized without an obvious crisis. Independent reports on the proliferative effects of hTERT expression in endothelial cells (ECs) have also varied. Our studies showed that culture conditions did not account for the inability of hTERT to immortalize BMECs. To further investigate the effects of hTERT expression in BMECs, 26 post-senescent BMhTERT clones were established by limiting dilution of a mass culture. The BMhTERT mass culture employed in these studies did not exhibit an obvious crisis. However 21/26 (75%) clones derived from this mass culture underwent crisis. There was no significant difference in the level of telomerase activity in the subset of clones that underwent crisis verses the clones that did not exhibit a clear crisis (n=14, p=0.2 Student t-Test). Similarly, telomere length was not significantly different in clones that underwent crisis compared with clones that showed no crisis (n=15, p=0.7). Prior to crisis, the mean telomere length of individual clones ranged from 5Kbp-12Kbp. Dramatic changes in telomere length were observed during the transition through crisis, resulting in a shorter average telomere length in post-crisis clones compared with pre-crisis clones (n=15, p<0.01). It was notable that while non-transduced BMECs senesced with a telomere length of 6-7Kbp, some post-crisis clones continued to proliferate with a mean telomere length less than 3Kbp. Although the duration of crisis varied among the clones, subsets of cells eventually grew out of most crisis cultures (81%). Overall, 22 out of 26 post-senescent clones (85%) were considered immortalized after exhibiting a 3-fold increase in lifespan. These investigations confirm that hTERT-transduced BMECs undergo crisis during immortalization. Furthermore, our results demonstrate that crisis may be masked in mass cultures by clonal variations in proliferation. This may account for discrepant reports on the potential for hTERT to immortalize ECs. Although crisis was found to be independent of telomere length and the level of telomerase activity, there was a trend toward telomere shortening with increasing population doublings. Molecular alterations during crisis may have contributed to immortalization as well as the ability of the cells to proliferate with extremely short telomeres. 1. MacKenzie et al., Oncogene 21; 4200-4211, 2002.