Telomere-Driven Karyotypic Complexity Concurs with p16INK4a Inactivation in TP53-Competent Immortal Endothelial Cells

Abstract

Critically short telomeres promote chromosomal fusions, which in TP53-defective cells initiate the formation of cytogenetic aberrations that are typical of human cancer cells. Expression of the enzyme telomerase stabilizes normal and aberrant chromosomes by maintaining telomere length. However, previous investigations, including our own, have shown that overexpression of telomerase reverse transcriptase (hTERT) does not prevent net telomere shortening in human endothelial cells. In the present study, two mass cultures of hTERT-transduced bone marrow endothelial cells (BMhTERT) and 26 clones were employed to further investigate the immortalization process and consequences of telomere shortening. Eighty-five percent (22 of 26) of the clones and both mass cultures were immortalized. However, cytogenetic analyses revealed recurring cytogenetic aberrations in the mass cultures and 12 representative clones. Several of the recurring aberrations, including +5p, +11, À13, +19, and +20, and nonreciprocal translocations involving 17p and 2p were previously implicated in human carcinogenesis. One mass culture and a subset of clones (5 of 12) had complex karyotypes, characterized by cytogenetic heterogeneity and at least five chromosomal abnormalities. p16 INK4a was silenced exclusively in the five clones and mass culture with complex karyotypes, whereas the p53/p21 cip1 pathway was defective in only one clone. Telomere dysfunction was implicated in the evolution of complex karyotypes by the presence of anaphase bridges, telomere associations, and dicentric chromosomes. These results show that complex karyotypes can evolve in TP53-competent cells and provide evidence that p16 INK4a functions as a gatekeeper to prevent telomere-driven cytogenetic evolution. These investigations provide new insight to the role of p16 INK4a as a tumor suppressor. (Cancer Res 2006; 66(22): 10691-700)

Modeling human endothelial cell transformation in vascular neoplasias

“…Telomere dysfunction in pre-malignant and cancerous cells is typically evidenced by dicentric chromosomes, unbalanced translocations, gene amplifications, deletions and ring chromosomes (Ducray et al, 1999; Gisselsson et al, 2001; O’Hagan et al, 2002). These types of chromosomal aberrations are frequently observed in KS and angiosarcoma, and have also been identified in immortalized EC lines, particularly those with defective tumor suppressor pathways (Schuborg et al, 1998; Wong et al, 2001; Wen et al, 2006). Chromosomal instability accelerates the rate and accumulation of molecular changes that facilitate malignant transformation and tumor progression (Rudolph et al, 2001), including activation of a telomere maintenance mechanism, oncogene activation or silencing of tumor suppressor genes.…”

“…Under standard culture conditions, ECs that bypass senescence usually succumb to a proliferative ‘crisis’ that is characterized by an increased rate of cell death and reduced cell expansion (MacKenzie et al, 2002; Gu et al, 2003; Nisato et al, 2004; Wen et al, 2006). Crisis is thought to be initiated by telomere dysfunction, genetic catastrophe and/or fatal oxidative damage.…”

Amplification of Telomerase Reverse Transcriptase Gene in Human Mammary Epithelial Cells with Limiting Telomerase RNA Expression Levels

“…Although immortalization of many types of normal human cells has been achieved by transducing them with exogenous hTERT, for some strains of normal human cells, this is an inefficient process, with periods of poor growth or even crisis being experienced before immortalization (17)(18)(19). Data are accumulating in support of the notion that hTR levels can also be limiting for telomerase activity.…”

Upregulation of survivin during immortalization of nontransformed human fibroblasts transduced with telomerase reverse transcriptase

“…Ectopic expression of hTERT is sufficient to reconstitute telomerase enzyme activity, elongate telomeres and extend the lifespan of BJ foreskin fibroblasts and retinal epithelial cells (Bodnar et al, 1998). However, telomere length independent mechanisms that involve the p16 INK4a /RB and/or p53 tumor suppressor pathways and/or other uncharacterized molecular events restrict the immortalization of many cell types, including human lung fibroblasts, bone marrow endothelial cells, keratinocytes and mammary epithelial cells (Kiyono et al, 1998;MacKenzie et al, 2000MacKenzie et al, , 2002Milyavsky et al, 2003;Taylor et al, 2004;Wen et al, 2006).…”