Telomere Shortening Sensitizes Cancer Cells to Selected Cytotoxic Agents: In Vitro and In Vivo Studies and Putative Mechanisms

Background

Telomere/telomerase system has been recently recognized as an attractive target for anticancer therapy. Telomerase inhibition results in tumor regression and increased sensitivity to various cytotoxic drugs. However, it has not been fully established yet whether the mediator of these effects is telomerase inhibition per se or telomere shortening resulting from inhibition of telomerase activity. In addition, the characteristics and mechanisms of sensitization to cytotoxic drugs caused by telomerase inhibition has not been elucidated in a systematic manner.

Methodology/Principal Findings

In this study we characterized the relative importance of telomerase inhibition versus telomere shortening in cancer cells. Sensitization of cancer cells to cytotoxic drugs was achieved by telomere shortening in a length dependent manner and not by telomerase inhibition per se. In our system this sensitization was related to the mechanism of action of the cytotoxic drug. In addition, telomere shortening affected also other cancer cell functions such as migration. Telomere shortening induced DNA damage whose repair was impaired after administration of cisplatinum while doxorubicin or vincristine did not affect the DNA repair. These findings were verified also in in vivo mouse model. The putative explanation underlying the phenotype induced by telomere shortening may be related to changes in expression of various microRNAs triggered by telomere shortening.

Conclusions/Significance

To our best knowledge this is the first study characterizing the relative impact of telomerase inhibition and telomere shortening on several aspects of cancer cell phenotype, especially related to sensitivity to cytotoxic drugs and its putative mechanisms. The microRNA changes in cancer cells upon telomere shortening are novel information. These findings may facilitate the development of telomere based approaches in treatment of cancer.     

Diminished Telomeric 3′ Overhangs Are Associated with Telomere Dysfunction in Hoyeraal-Hreidarsson Syndrome

Background

Eukaryotic chromosomes end with telomeres, which in most organisms are composed of tandem DNA repeats associated with telomeric proteins. These DNA repeats are synthesized by the enzyme telomerase, whose activity in most human tissues is tightly regulated, leading to gradual telomere shortening with cell divisions. Shortening beyond a critical length causes telomere uncapping, manifested by the activation of a DNA damage response (DDR) and consequently cell cycle arrest. Thus, telomere length limits the number of cell divisions and provides a tumor-suppressing mechanism. However, not only telomere shortening, but also damaged telomere structure, can cause telomere uncapping. Dyskeratosis Congenita (DC) and its severe form Hoyeraal-Hreidarsson Syndrome (HHS) are genetic disorders mainly characterized by telomerase deficiency, accelerated telomere shortening, impaired cell proliferation, bone marrow failure, and immunodeficiency.

Methodology/Principal Findings

We studied the telomere phenotypes in a family affected with HHS, in which the genes implicated in other cases of DC and HHS have been excluded, and telomerase expression and activity appears to be normal. Telomeres in blood leukocytes derived from the patients were severely short, but in primary fibroblasts they were normal in length. Nevertheless, a significant fraction of telomeres in these fibroblasts activated DDR, an indication of their uncapped state. In addition, the telomeric 3′ overhangs are diminished in blood cells and fibroblasts derived from the patients, consistent with a defect in telomere structure common to both cell types.

Conclusions/Significance

Altogether, these results suggest that the primary defect in these patients lies in the telomere structure, rather than length. We postulate that this defect hinders the access of telomerase to telomeres, thus causing accelerated telomere shortening in blood cells that rely on telomerase to replenish their telomeres. In addition, it activates the DDR and impairs cell proliferation, even in cells with normal telomere length such as fibroblasts. This work demonstrates a telomere length-independent pathway that contributes to a telomere dysfunction disease.     

Transient introduction of human telomerase mRNA improves hallmarks of progeria cells

Hutchinson–Gilford progeria syndrome (HGPS) is characterized by accelerated senescence due to a de novo mutation in the LMNA gene. The mutation produces an abnormal lamin A protein called progerin that lacks the splice site necessary to remove a farnesylated domain. Subsequently, progerin accumulates in the nuclear envelope, disrupting nuclear architecture, chromatin organization, and gene expression. These alterations are often associated with rapid telomere erosion and cellular aging. Here, we further characterize the cellular and molecular abnormalities in HGPS cells and report a significant reversal of some of these abnormalities by introduction of in vitro transcribed and purified human telomerase (hTERT) mRNA. There is intra‐individual heterogeneity of expression of telomere‐associated proteins DNA PKcs/Ku70/Ku80, with low‐expressing cells having shorter telomeres. In addition, the loss of the heterochromatin marker H3K9me3 in progeria is associated with accelerated telomere erosion. In HGPS cell lines characterized by short telomeres, transient transfections with hTERT mRNA increase telomere length, increase expression of telomere‐associated proteins, increase proliferative capacity and cellular lifespan, and reverse manifestations of cellular senescence as assessed by β‐galactosidase expression and secretion of inflammatory cytokines. Unexpectedly, mRNA hTERT also improves nuclear morphology. In combination with the farnesyltransferase inhibitor (FTI) lonafarnib, hTERT mRNA promotes HGPS cell proliferation. Our findings demonstrate transient expression of human telomerase in combination with FTIs could represent an improved therapeutic approach for HGPS.     

The catalytic and the RNA subunits of human telomerase are required to immortalize equid primary fibroblasts

Many human primary somatic cells can be immortalized by inducing telomerase activity through the exogenous expression of the human telomerase catalytic subunit (hTERT). This approach has been extended to the immortalization of cell lines from several mammals. Here, we show that hTERT expression is not sufficient to immortalize primary fibroblasts from three equid species, namely donkey, Burchelli’s zebra and Grevy’s zebra. In vitro analysis of a reconstituted telomerase composed by hTERT and an equid RNA component of telomerase (TERC) revealed a low activity of this enzyme compared to human telomerase, suggesting a low compatibility of equid and human telomerase subunits. This conclusion was also strengthened by comparison of human and equid TERC sequences, which revealed nucleotide differences in key regions for TERC and TERT interaction. We then succeeded in immortalizing equid fibroblasts by expressing hTERT and hTERC concomitantly. Expression of both human telomerase subunits led to telomerase activity and telomere elongation, indicating that human telomerase is compatible with the other equid telomerase subunits and proteins involved in telomere metabolism. The immortalization procedure described herein could be extended to primary cells from other mammals. The availability of immortal cells from endangered species could be particularly useful for obtaining new information on the organization and function of their genomes, which is relevant for their preservation.     

Activation of Mrc1, a mediator of the replication checkpoint, by telomere erosion

Background information. In budding yeast, the loss of either telomere sequences (in telomerase‐negative cells) or telomere capping (in mutants of two telomere end‐protection proteins, Cdc13 and Yku) lead, by distinct pathways, to telomeric senescence. After DNA damage, activation of Rad53, which together with Chk1 represents a protein kinase central to all checkpoint pathways, normally requires Rad9, a checkpoint adaptor. Results. We report that in telomerase‐negative (tlc1Δ) cells, activation of Rad53, although diminished, could still take place in the absence of Rad9. In contrast, Rad9 was essential for Rad53 activation in cells that entered senescence in the presence of functional telomerase, namely in senescent cells bearing mutations in telomere end‐protection proteins (cdc13‐1 yku70Δ). In telomerase‐negative cells deleted for RAD9, Mrc1, another checkpoint adaptor previously implicated in the DNA replication checkpoint, mediated Rad53 activation. Rad9 and Rad53, as well as other DNA damage checkpoint proteins (Mec1, Mec3, Chk1 and Dun1), were required for complete DNA‐damage‐induced cell‐cycle arrest after loss of telomerase function. However, unexpectedly, given the formation of an active Rad53–Mrc1 complex in tlc1Δ rad9Δ cells, Mrc1 did not mediate the cell‐cycle arrest elicited by telomerase loss. Finally, we report that Rad9, Mrc1, Dun1 and Chk1 are activated by phosphorylation after telomerase inactivation. Conclusions. These results indicate that loss of telomere capping and loss of telomere sequences, both of which provoke telomeric senescence, are perceived as two distinct types of damages. In contrast with the Rad53–Rad9‐mediated cell‐cycle arrest that functions in a similar way in both types of telomeric senescence, activation of Rad53–Mrc1 might represent a specific response to telomerase inactivation and/or telomere shortening, the functional significance of which has yet to be uncovered.     

Thymoquinone Induces Telomere Shortening,DNA Damage and Apoptosis in Human Glioblastoma Cells

Background

A major concern of cancer chemotherapy is the side effects caused by the non-specific targeting of both normal and cancerous cells by therapeutic drugs. Much emphasis has been placed on discovering new compounds that target tumour cells more efficiently and selectively with minimal toxic effects on normal cells.

Methodology/Principal Findings

The cytotoxic effect of thymoquinone, a component derived from the plant Nigella sativa, was tested on human glioblastoma and normal cells. Our findings demonstrated that glioblastoma cells were more sensitive to thymoquinone-induced antiproliferative effects. Thymoquinone induced DNA damage, cell cycle arrest and apoptosis in the glioblastoma cells. It was also observed that thymoquinone facilitated telomere attrition by inhibiting the activity of telomerase. In addition to these, we investigated the role of DNA-PKcs on thymoquinone mediated changes in telomere length. Telomeres in glioblastoma cells with DNA-PKcs were more sensitive to thymoquinone mediated effects as compared to those cells deficient in DNA-PKcs.

Conclusions/Significance

Our results indicate that thymoquinone induces DNA damage, telomere attrition by inhibiting telomerase and cell death in glioblastoma cells. Telomere shortening was found to be dependent on the status of DNA-PKcs. Collectively, these data suggest that thymoquinone could be useful as a potential chemotherapeutic agent in the management for brain tumours.     

Telomerase maintains telomere structure in normal human cells

In normal human cells, telomeres shorten with successive rounds of cell division, and immortalization correlates with stabilization of telomere length. These observations suggest that human cancer cells achieve immortalization in large part through the illegitimate activation of telomerase expression. Here, we demonstrate that the rate-limiting telomerase catalytic subunit hTERT is expressed in cycling primary presenescent human fibroblasts, previously believed to lack hTERT expression and telomerase activity. Disruption of telomerase activity in normal human cells slows cell proliferation, restricts cell lifespan, and alters the maintenance of the 3' single-stranded telomeric overhang without changing the rate of overall telomere shortening. Together, these observations support the view that telomerase and telomere structure are dynamically regulated in normal human cells and that telomere length alone is unlikely to trigger entry into replicative senescence.