Telomere shortening sensitizes cancer cells to selected cytotoxic agents: in vitro and in vivo studies and putative mechanisms

Abstract

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.

Figure 1. GRN163 inhibits telomerase activity in a sequence specific manner and shortens telomeres.

a. SK-N-MC, MCF-7 and K562 were exposed to 5uM of GRN163. Telomerase activity was assessed by the TRAP assay after 24hours. C- control untreated cells, M- mismatch nonspecific scrambled oligo, I-telomerase inhibitor GRN163. R8- standard TRAP control, N- negative control with no cell extracts, IC- internal PCR control. The extent of telomerase inhibition is denoted in percentages below the lanes. b. SK-N-MC cells were continuously exposed to 5µM of GRN163. Telomere length was measured by Southern blot. C- control untreated cells, Ia- telomere length of cells exposed to telomerase inhibitor for three months, Ib- telomere length of cells exposed to telomerase inhibitor for 16 months, M- molecular size marker. Mean telomere length is denoted below each lane, and the percent of telomere shortening is shown as well. c. Graphical presentation of the extent of telomere shortening after the exposure of SK-N-MC cells to GRN163 for 1 year as measured by Southern blot.

Figure 2. Sensitivity of SK-N-MC cells to cisplatinum is telomere length dependent.

a. Proliferation of cells with shortened telomeres exposed to cisplatinum. SK-N-MC cells were continuously exposed to GRN163. The sensitivity of the cells to cisplatinum was estimated after three and 16 months of telomerase inhibition. Numbers indicate the IC₅₀ of the drug in these time points (after 22% and 40% reduction in telomere length). P value refers to difference between WT and short tel-16 months. b. Cell cycle status of SK-N-MC cells with shortened telomeres exposed to 0.13µg/ml cisplatinum. SK-N-MC cells with shortened telomeres were exposed to cisplatinum and the cell cycle status was evaluated by FACS. + or − refers to the exposure to cisplatinum. C. The apoptotic index of SK-N-MC cells with shortened telomeres exposed to cisplatinum. The same cells were analyzed by FACS for their apoptotic index, as represented by their preG1 status. The numbers indicate the LD₅₀ of cisplatinum in cells with intact or shortened telomeres.

Figure 3. The migration ability of cells with shortened telomeres.

The migration of cells with shortened telomeres was evaluated by the transwell and the wound healing assays. a. The transwell membrane assay. Cells with intact or shortened telomeres were allowed to migrate through a membrane for 16 hours, Gimza stained and counted. A representative picture is shown. b. Graphical demonstration of the average cell counts of four independent experiments. c. The wound healing assay. Cells with shortened telomeres were plated on Petri dishes, and the culture was “scratched” and followed for 24 hours. Average measurements of the cell free gaps were done. A representative example is shown. d. Graphical demonstration of the average cell counts of four independent experiments.

Figure 4. DNA damage status and repair ability of cells with shortened telomeres- the Comet assay.

a. The principle of the comet assay. Cells nuclei were exposed to electrophoresis and stained with ethidium bromide. Broken DNA migrates out of the nuclei and forms the comet. Three degrees of DNA damage are shown: control nuclei with intact DNA (representing DNA damage in cells with intact telomeres, #1), intermediate state in cells with partially broken DNA (representing cells with mild telomere shortening, #2) and cells with shortened telomeres harboring damaged broken DNA (representing cells with shortened telomeres, #3). b. Quantitation of the extent of the DNA damage status of cells with shortened telomeres, performed by screening 50 images per sample in quadruples. On the left- Tail extent moment parameter, on the right- total comet intensities. c. Quantitation of the extent of the ability of the cells to repair DNA damage applied by doxorubicin (right panel) or cisplatinum (left panel) 2 and 4 hours post drug induced damage. The assay was performed by screening 50 images per sample in quadruples. The DNA damage status was determined by calculating the tail extent moment.

Figure 5. The formation of γH2AX foci in cells with shortened telomeres.

SK-N-MC cells were exposed to cisplatinum or doxorubicin for 1 hour and then resuspended in drug free medium. The formation of γH2AX foci was followed by immunofluorescence at the indicated time points after the releasing from the drugs. Blue color indicates nuclei stained with DAPI, green foci were detected using cy2-labeled anti γH2AX antibody. A- A representative example. B- Quantitation of the number of foci per sample. Averages were calculated for at least 50 nuclei were counted for each duplicated sample and two experiments which yielded similar results.

Figure 6. Tumor dimensions after the inhibition of telomerase.

CRL 1687cells were injected subcutaneously to nude athymic mice, which were treated three times a week with telomerase inhibitor, GRN163L or with PBS as a control. a. Average tumor sizes of the two groups of mice (each contained 18 mice) which were measured every week. b. Each group of mice was subsequently divided into three subgroups: one treated with PBS or GRN163L only, the other treated with doxorubicin and PBS or GRN163L, and a group treated with vincristine and PBS or GRN163L. The sizes of xenografts were measure each week. c. In vivo telomere shortening after GRN163L administration. Nude athymic mice were injected with CRL1687 cells subcutaneously and treated with telomerase inhibitor, GRN163L, or PBS. Telomere length was evaluated by Southern blot. M- molecular size marker (sizes are listed on the left). Numbers below the gel indicate average telomere length in KB and the extent of telomere shortening. 5 samples of each cell type were analyzed. D. Graphical representation of telomere shortening in vivo after telomerase inhibition with GRN163L. TRF- terminal repeats fragment indicated the length of telomeres. The telomeres shortened by ∼40% after telomerase inhibition. The experiment was repeated twice with 5 samples of each cell types.

Figure 7. Histological analyses of tumors treated in vivo with telomerase inhibitor.

Tumors grown in athymic nude mice treated with telomerase inhibitor GRN163L were analyzed histologically after Gimza staining. The upper panel demonstrates less aggressive parameters of tumors treated with telomerase inhibitor, and the lower panel show a more aggressive appearance of tumors treated with PBS only.