A Novel Role of Telomerase in DNA Repair and Genome Maintenance in Myeloma.

Abstract

Abstract 628We have previously shown that telomerase activity is significantly elevated in multiple myeloma (MM) cell lines and primary cells, and inhibition of telomerase over a period of 3–5 weeks induces telomere shortening and growth arrest in these and other cancer cells. Here, we have further investigated our novel findings, suggesting the role of telomerase in repair of DNA breaks and genome maintenance in MM cells. Double-strand breaks (DSBs) in genome are deleterious because if left unrepaired, they may lead to aberrant DNA recombination, instability, or cell death. We observe that induction of DSB in MM cells with UV (20 J/m2) leads to induction of phosphorylated-H2AX (p-H2AX; a marker for DNA breaks) within 24 hrs and the induction is markedly increased following inhibition of telomerase. We have further evaluated the impact of telomerase inhibition on DNA integrity, utilizing comet assay, a sensitive gel-based technique for detection and assessment of DNA breaks in individual cells. Cells were cultured in the presence or absence of telomerase inhibitor and exposed to UV (20 J/m2) for induction of DSBs and cultured for a short period. Cells were then evaluated by comet assay and the fraction of cells with DNA breaks and the intensity and size distribution of comets were graded as small; medium, large, or very large. Under our experimental conditions, the DNA breaks (with comet size varying from small to medium in individual cells) in untreated myeloma cells and following exposure to UV were detected in 25% and 38% of cells, respectively. However, following treatment with telomerase inhibitor, significantly higher number of comets (58%) were observed in MM cells, indicating a substantial increase in the fraction of cells with DNA breaks.These data are consistent with P-H2AX data and show that telomerase inhibition significantly increases DNA breaks and telomerase may have a role in DNA repair in these cells. To further investigate if telomerase has a role in DNA repair and genome maintenance in cancer, we evaluated the impact of telomerase inhibition on instability at Alu elements, which are 300 base pair repetitive sequences dispersed throughout genome and may serve as substrates for deregulated homologous recombination. Cells were cultured in the presence or absence of telomerase inhibitor for 10 days and Alu copy number was determined by Alu-specific real-time PCR. As a positive control for Alu instability, DNA breaks were induced by transfection of restriction enzyme “Alu I” in myeloma cells. In two independent experiments, the treatment of RPMI 8226 cells with telomerase inhibitor, Alu restriction enzyme, and combination of both resulted in an average of 3.1, 5.3, and 8.7-fold increase in Alu copy number, respectively. Similar observations were made in an additional MM cell line. Interestingly, the inhibition of telomerase or induction of DNA breaks by Alu I restriction enzyme, both induced copy number variation at Alu elements in the genome. We next evaluate whether telomerase contributes to DNA repair and genome stability by adding telomeric sequences at DNA break sites in cancer cells. We initially looked for any telomeric sequences, incorporated within Alu elements. A PCR using a primer annealing at 5’-end of Alu and a telomere-specific primer, gave ∼ 200 bp band in both RPMI 8226 and U266 cells, indicating the presence of telomeric sequence within 300 bp Alu elements in these cells. In summary, our data shows that telomerase contributes to DNA break repair and genome stability in MM cells providing an additional importance to understanding its molecular and cellular role and its therapeutic application in MM. Disclosures:Munshi:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees.