Expression of mutant telomerase in immortal telomerase-negative human cells results in cell cycle deregulation, nuclear and chromosomal abnormalities and rapid loss of viability

Abstract

We have reconstituted wild type or mutant telomerase activity in two human cell lines that lack constitutive expression of both core subunits of the enzyme and maintain telomeres by a telomerase-independent mechanism (ALT cells). Wild type telomerase RNA and four telomerase RNAs with single point mutations in their template domain were used to express enzymes specifying di erent telomeric DNA sequences. Expression of wild type telomerase for up to 32 days had no detectable e ect on cell growth or viability. In contrast, cells expressing mutant telomerases had slower growth rate, abnormal cell cycle and reduced viability. Dramatically aberrant nuclei, typical of cells undergoing mitotic catastrophe, and large numbers of fused chromosomes were also characteristic of these populations. Notably, all phenotypes were apparent within the ®rst few cell divisions after expression of the enzymes. Unlike wild type, mutant telomerase activity was progressively selected against with cell culturing, and this correlated with the disappearance of cells with aberrant phenotypes. Our results suggest that even very limited synthesis of mutated sequences can a ect telomere structure in human cells, and that the toxicity of mutant telomerases is due to telomere malfunction. Oncogene (2001) 20, 714 ± 725.

Mutated telomeres sensitize tumor cells to anticancer drugs independently of telomere shortening and mechanisms of telomere maintenance

“…These changes are indicative of cell death by mitotic catastrophe. Previous studies in human ALT cells and in lower eucaryotes have shown that mutated telomeres have deleterious effects on cell viability and result in cell cycle abnormalities and cell death by mitotic catastrophe (Yu et al, 1990;Kirk et al, 1997;Smith and Blackburn, 1999;Guiducci et al, 2001;Lin et al, 2004). Thus, although the expression of MuA-hTR does not affect cell viability in normal growth conditions, it may enhance the antiproliferative effects of doxorubicin, resulting in higher drug sensitivity compared to control cells.…”

“…phTR and phTR-MuA containing, respectively, wild-type hTR and a mutant hTR specifying TTTGGG telomeric sequences (Marusic et al, 1997;Guiducci et al, 2001) driven by the endogenous hTR promoter, and phTERT containing wild-type hTERT and the puromycin resistance gene were obtained from Dr Silvia Bacchetti. phTERT/hTR-MuA was obtained by subcloning hTR-MuA into the phTERT plasmid.…”

“…This RNA reconstitutes a mutant holoenzyme that adds mutant repeats (T 3 G 3 ) onto the telomeres (Marusic et al, 1997;Guiducci et al, 2001;Kim et al, 2001). YCC-B1, MCF-7 and YCC-B2 cells were transfected with the vector alone or a plasmid encoding MuA-hTR and clonal populations were selected.…”

“…However, as previously reported, cells expressing the mutant RNA formed smaller colonies than the control cells when plated at low Figure 1SE and data not shown). The mutant hTR-dependent effects require the presence of a biologically active telomerase enzyme (Guiducci et al, 2001). To confirm the dependence of MuA-hTR action on a functional telomerase, we used ALT VA13 cells that do not express hTERT, transfected them with MuA-hTR and selected clonal populations (Supplementary Figure 3SA).…”

“…These effects are most likely owing to the inability of telomeric proteins to bind to the mutated repeats and do not require telomere shortening (Marusic et al, 1997;Kim et al, 2001). However, unless the mutant hTR is highly overexpressed or the endogenous hTR is absent (Guiducci et al, 2001;Li et al, 2004), the endogenous telomerase complex adds wild-type sequences to mutant ones, partially restoring telomere function and mitigating the deleterious effects of the mutant telomerase. Hence, the expression of mutant template RNAs may only be partially effective in inducing cell death of telomerase-positive cancer cells and human tumors.…”