Telomere maintenance by telomerase and by recombination can coexist in human cells

Abstract

Immortal human cells maintain their telomeres by two independent mechanisms, a prevalent one dependent on de novo synthesis of telomeric DNA by telomerase, and a rarer one based on telomere recombination [alternative lengthening of telomeres (ALT)]. Studies with yeast have indicated that expression of telomerase inhibits telomere recombination. In the present study, we have investigated whether expression of telomerase in cells that use ALT would similarly reveal dominance of telomere elongation by telomerase over telomere recombination. Telomerase-negative WI38 VA13/2RA ALT cells were reconstituted for telomerase activity through ectopic expression of the enzyme subunits, hTERT and hTR, and the presence and function of telomerase and ALT were monitored during long term cell growth by enzymatic assays, detection of the ALT-associated PML bodies (APBs) and analysis of telomere dynamics. Our results indicate that telomerase activity and APBs persisted in the cells over at least 90 population doublings. The activity of both pathways on telomeres was determined by analysis of telomere length versus time by gel electrophoresis and in situ hybridization. ALT cells are characterized by very heterogeneous telomeres with a much longer average size than the telomeres of telomerase-positive cells. Telomere dynamics in our cells were compatible with both ALT and telomerase being biologically active since the long telomeres typical of ALT were maintained, while short telomeres, thought to be the preferential substrate of telomerase, were elongated. These findings, indicating that human cells may be capable of concomitantly utilizing both mechanisms of telomere maintenance without effects on their growth and viability, have implications for cancer therapy.

Conditionally replicative adenovirus driven by the human telomerase promoter provides broad-spectrum antitumor activity without liver toxicity

“…No AdhTERTp-E1A-mediated cytotoxicity was detected in these WI38-VA13 tumor cells, in which telomere maintenance has been attributed to the ''ALT'' pathway. 38 Taken together, these data suggest that AdhTERTp-E1A oncolytic virus-mediated cytolysis is largely restricted to human tumor cells that are positive for telomerase expression.…”

“…Virtually no cytotoxicity was detected with either of the normal cell lines tested (Fig 1d). To further confirm that AdhTERTp-E1A-mediated cytotoxicity to tumor cells was directly linked to their hTERT expression, a telomerase negative SV-40 large T antigen transformed cell line, WI38-VA13, 38 was subjected to AdhTERTp-E1A virus infection and subjected to MTS assay for determining cell viability (Fig 1e). No AdhTERTp-E1A-mediated cytotoxicity was detected in these WI38-VA13 tumor cells, in which telomere maintenance has been attributed to the ''ALT'' pathway.…”

Subtelomeric DNA hypomethylation is not required for telomeric sister chromatid exchanges in ALT cells

“…To this end, we ectopically expressed both subunits of telomerase in U2OS osteosarcoma ALT cells. Earlier studies reported distinct outcomes after telomerase introduction into ALT SV40T-immortalized fibroblasts: either ALT was preserved and T-SCEs were unaffected (Cerone et al, 2001) or ALT was abolished and T-SCE frequency was reduced (Ford et al, 2001;Bechter et al, 2004). These apparent discrepancies may possibly be due to distinct telomerase levels in the cells (Henson et al, 2002).…”

Telomerase promotes efficient cell cycle kinetics and confers growth advantage to telomerase-negative transformed human cells

“…The VA13 subtype was created with SV40 transformation, which inactivates the tumor suppressors p53 and Rb (Tokutake et al, 1998), and alters signal transduction pathways regulated by the phosphatase family of PP2A. The recombinant expression replacement of TERT and TER in this cell line does not result in the loss of ALT phenotypes, indicating that ALT and telomerase-dependent telomere maintenance can coexist (Cerone et al, 2001). As telomere length maintenance can be completely uncoupled from telomerase action in ALT cells, we can specifically test the growth-promoting functions of telomerase and compare them with the parental isogenic VA13 cell controls expressing empty retroviral vector.…”