Figure 1.
Telomere size and telomerase activity among pancreatic cancer cell lines.
A) Telomere size measurements. Genomic DNA was digested with restriction enzymes, resolved by electrophoresis in agarose gels and detected by in situ hybridization to [32P]-(CCCTAA)4. B) Quantification of telomere size. Gel in A was scanned and the intensity of each lane was plotted as a function of telomere size, as described in the Material and Methods section. The median telomere size was estimated as the size corresponding to half the cumulative sum of the intensities. C) Detection of telomerase activity by the TRAP assay. Cell extracts containing 300 cells each were assayed using a Cy5-labeled TS oligo substrate. After PCR with the same oligo and a telomeric comeback primer, products were resolved by electrophoresis and detected with a Typhon PhosphoImager. Buffer was lysis buffer only. ITAS, Internal Telomerase Assay Standard. D) Quantification of relative telomerase activity. Relative telomerase activity was calculated as the ratio of the intensity of the telomerase ladder over the intensity of the ITAS. Each measurement is the Mean ± S.D. of triplicate samples (n = 3). Telomerase activity in each line is expressed as a percent of HeLa cells’ activity.
Figure 2.
Short-term responses of pancreatic cancer cell lines to GRN163L.
A) Dose-Response curve of telomerase inhibition by GRN163L. HPAF cells were treated in triplicate with increasing concentrations of GRN163L (50 nM to 4 µM). Twenty-four hours later, telomerase activity was measured using the TRAP assay. Samples treated with no drug were set to 100%. B) IC50 of each line for the inhibition of telomerase by GRN163L. Dose-response curves were performed as in panel A. Non-linear curve fitting allowed calculation of an IC50 for each line. Results are expressed as the IC50 (middle bar) and its 95% interval of confidence (flanking bars).
Figure 3.
Effects of chronic GRN163L on cellular lifespan.
A) Pancreatic cancer cell lines CAPAN1 and CD18 were selected for these studies. Every 2–3 days, each lines was given either no drug (vehicle), GRN163L (1 µM) or the Mismatched oligo (1 µM). Once a week, cells were counted and the results are plotted as the number of population doublings achieved as a function of time. Every other week, samples were put aside for TRF analysis (DNA) or frozen down as backup. B,C) Growth curves of the drug-treated CAPAN1 (B) and CD18 (C) cells are shown.
Figure 4.
Markers of apoptosis, senescence and DNA damage response in the GRN163L-treated CAPAN1 and CD18 cells.
At the end of the growth curves presented in Figure 3, cells treated with no drug (CTR), mismatch oligo (MIS) and GRN163L (GRN) were analyzed for SA-β-galactosidase activity. A) Histological analysis of SA-β-galactosidase activity. Histochemical staining reveals SA-β-galactosidase activity in the GRN163L-treated CAPAN1 and CD18 cells, as evidenced by the deposition of insoluble blue pigments (Right panels). B) Percent of cells in each sample that stained positive for SA-β-galactosidase activity. Measurements were done in triplicates (Mean ± S.D., n = 3). C) Western blot analysis. Samples were probed with antibodies against histone H2AX, phosphorylated H2AX (γ-H2AX), PARP and actin. D) Flow cytometric analysis of DNA content. Cells were stained with propidium iodide and analyzed for DNA content. Measurements were made twice at one week interval for the CAPAN1 (Mean ± S.D., n = 2). In the case of the CD18, one measurement only could be made before the GRN163L-treated cells were lost to crisis (n = 1).
Figure 5.
Effects of chronic GRN163L on telomere maintenance.
Cells were treated every 2–3 days with no drug (CTR), mismatch oligo (MIS) or GRN163L (GRN). A–B) Southern blot analysis of the telomeres. At the indicated population doubling (PD), genomic DNA samples were collected and subsequently processed for telomere size measurements, as described in Figure 1A. C–D) Telomere size measurements. Median telomere sizes are shown for the drug- and control-treated CAPAN1 and CD18 cells. E) Detection of ALT by quantitative PCR. Samples tested included CAPAN1 and CD18 cells treated with no drug (CTR), mismatch oligo (MIS) and GRN163L (GRN), all of which harvested at the end of the growth curves presented in Figure 3. Also included were stocks of CAPAN1, CD18 and VA13 cells. Samples were tested in triplicate with (+) and without (−) the Φ29 DNA polymerase. A representative dot blot is shown.
Figure 6.
Immunofluorescence analysis of telomeres in the GRN163L-treated CD18 cells.
CD18 treated with GRN163L (GRN) or with no drug (CTR) were harvested at the end of the growth curve presented in Figure 3. Cells were fixed and stained with antibodies against TRF2 (green) and γ-H2AX (red) and were counter-stained with DAPI (blue). Images were visualized on a confocal microscope (Zeiss 510 Meta Confocal Laser Scanning Microscope). Three representative images of each sample are shown.
Figure 7.
Recovery from chronic GRN163L exposure after removal of the drug.
After 63 days of continuous GRN163L exposure, CAPAN1 cells were sub-cultivated for 3 weeks in the presence or absence of GRN163L. A, B) Quantification of telomerase activity. Telomerase activity was measured using the TRAP assay at time 0 and after 1, 2, and 3 weeks. Relative telomerase activity was calculated as the ratio of the intensity of the telomerase ladder over the intensity of the ITAS. Each measurement is the Mean ± S.D. of triplicate samples (n = 3). C, D) Telomere size measurements. Telomeres were analyzed at time 0 and after 1, 2, and 3 weeks. Median telomere size was estimated as the size corresponding to half the cumulative sum of the intensities. E) Levels of the γ-H2AX. The indicated samples were Western blotted with antibodies against phosphorylated H2AX (γ-H2AX) and actin. F) Growth rate measurements. Once a week, cells were counted and the number of population doublings done was plotted as a function of time.