Figure 1. (A) Homologous recombination substrate plasmid (luminescence based assay). The plasmid contains two incomplete fragments of a firefly luciferase gene, indicated as LUC-N and LUC-C, sharing a small region of homology (shown in blue). The luciferase gene fragments are separated by an AmpR gene, serving as spacer. HR between homologous sequences of these two fragments generates a functional luciferase gene, resulting in the excision of the AmpR gene. The plasmid also has a Gaussia luciferase gene which serves as an internal control (IC) and is not affected by recombination. (B-C) TRF2-suppression by siRNAs. BAC (FLO-1) cells, transfected with siRNAs (CS, control; TR, TRF2-targeting), were evaluated for TRF2 expression by Western blotting. Western blot (B) and quantitation of TRF2 expression, following normalization with -Actin (C) are shown. (D) Telomere length in OE33. DNA from OE33 and four cell lines, for which telomere length (in kbp) has been analyzed by southern blotting, was evaluated for telomere length by quantitative PCR, as described in Methods. Relative T/S ratio of four cell lines was plotted against their median telomere length (in kbp) to make a standard curve, which was then used to determine telomere length in OE33 cells. Figure 2. Impact of HR inhibition on efficacy of telomerase inhibitor in OE19 and FLO-1 cells. (A-C) Impact of chemically-suppressed HR on efficacy of GRN163L. (A) Impact on growth. The BAC (OE19) cells were treated with mismatch control oligonucleotide (C), telomerase inhibitor GRN163L (G; 2 µM), nilotinib (N; 5 M) or combination (NG), and viable cell number determined at different time points as indicated. The growth curve shows the mean of triplicate values, with S.E.M. (B) Impact on telomere length. OE19 cells, treated as described above for 10 days, were harvested, genomic DNA purified and telomere length determined by real time PCR as described in Methods; error bars represent SEMs of triplicate assays. (C) Impact on apoptosis. OE19 cells, treated as described above for 10 days, were examined for apoptosis by evaluating their ability to bind lactadherin; extent of green fluorescence indicates early or late apoptosis. (D-F) Impact of transgenically-suppressed HR on efficacy of GRN163L. FLO-1 cells, transduced with lentivirus particles producing control (CS) or RAD51-specific (R) shRNAs, were cultured in the presence or absence of telomerase inhibitor GRN163L (G; 2.0 µM) and evaluated for impact on growth, telomere maintenance, and apoptosis. (D) Impact on growth. The transduced cells were cultured in the presence or absence of GRN163L and cell viability determined at different time points as indicated by counting the substrate attached cell number, and confirmed by trypan blue exclusion; the growth curve shows the mean of triplicate assays, with S.E.M. (E) Impact on telomere length. The transduced cells were cultured in the presence or absence of GRN163L for 10 days and telomere length determined by real time PCR as described; error bars represent SEMs of triplicate assays. (F) Impact on apoptosis. Transduced FLO-1 cells, treated as above for 10 days, were examined for apoptosis by evaluating their ability to bind lactadherin; extent of green fluorescence indicates early or late apoptosis. CS, control shRNA transduced cells, R, RAD51targeting shRNA transduced cells; CG, CS cells treated with GRN163L (G; 2 µM) for 10 days; RG, R cells treated with GRN163L (G; 2 µM) for 10 days. Figure 3. Suppression of HR by nilotinib prevents the acquisition of copy number changes in BAC cells. DNA from OE33 cells cultured in the presence or absence of nilotinib (5 M) for three weeks and baseline control (day 0) cells was purified. Accrual of copy number changes in cultured vs. baseline control cells was monitored by investigating SNP and CNV loci throughout genome, using SNP6.0 arrays (Affymetrix). (A) Representative copy-number changes detected on chromosomes 13 and 19 in cultured compared to baseline (day 0) cells are shown; Day 0, Cells harvested at beginning of experiment; C, untreated control cells cultured for three weeks; N, nilotinib-treated cells cultured for three weeks. (B) Bar graphs summarize copy-number change events (where an event is defined as the observed change in five or more consecutive SNP/CNV loci) at SNP (Panel I) and CNV loci (Panel II), throughout genome.