Direct Evidence and Functional Significance of DNA Repair by Telomerase in Multiple Myeloma

Abstract 4416

We have 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. We recently reported our novel findings, indicating 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. Using phosphorylated-H2AX staining (a marker for DNA breaks) and the comet assay, a sensitive gel-based technique for detection and assessment of DNA breaks in individual cells, we have shown that telomerase inhibition leads to significantly increased DNA breaks in MM cells. Here we present the direct evidence of repair of DNA breaks by telomerase, in a plasmid substrate. The plasmid (with Kan^r) was linearized in a way that cut-site had limited homology with telomeric sequence. The linearized and a circular plasmid (with Amp^r; used as internal control) were incubated with myeloma cell extracts in the presence or absence of telomerase inhibitor and transferred to e.coli. The ratio of Kan^r and Amp^r colonies indicated the extent of repair and re-circularization of linearized plasmid by telomerase in the cell extracts. In two independent experiments, the inhibition of telomerase led to 60% decrease in the repair of DNA breaks in the plasmid substrate. To investigate if the substrate DNA needs to have a specific homology to telomeric sequence, we replaced the TS substrate in Telomerase Detection kit by various substrates designed by us, ending with TTA, TAG, AGG, and GGG. All sequences were utilized by telomerase with similar efficiency and the products of enzyme activity seen on a gel. To investigate the functional significance of these observations, myeloma cells were cultured in the absence or presence of telomerase inhibitor and cell aliquots were collected weekly for three weeks. An aliquot of cells was saved in the beginning of experiment, to be used as (day 0) control. DNA from cultured and day 0 cells was analyzed by SNP6.0 arrays (Affymetrix) and genomewide changes in copy number in cultured cells were identified, using genome of day 0 cells as baseline. Inhibition of telomerase was associated with 43% and 55% increase in the acquisition of copy number changes throughout genome in U266 and RPMI cells, respectively. In both myeloma cell lines as well as other cancer cells tested by us, the inhibition of telomerase was associated with 40% to 50% decrease in the amplification events, whereas 120% to 320% increase in deletions throughout genome, relative to untreated control cells. These data indicate that telomerase-mediated repair prevents copy number changes, especially the deletion events, associated with genomic instability in myeloma cells. As a positive control, the double-stranded breaks were also induced by restriction enzyme; this led to genomewide increase in copy number changes, mostly the deletions. Thus, DNA breaks, whether produced by telomerase inhibition or restriction enzyme, increase the deletion events throughout genome. This is also consistent with our previous observations showing increased instability of Alu sequences following telomerase inhibition. We conclude that telomerase contributes to survival of myeloma cells, not only by preventing telomere attrition, but also the repair of DNA breaks. Inhibition of telomerase, therefore, may increase the efficacy of chemotherapeutic agents targeting DNA repair.