A highly selective telomerase inhibitor limiting human cancer cell proliferation

Abstract

Telomerase, the ribonucleoprotein enzyme maintaining the telomeres of eukaryotic chromosomes, is active in most human cancers and in germline cells but, with few exceptions, not in normal human somatic tissues. Telomere maintenance is essential to the replicative potential of malignant cells and the inhibition of telomerase can lead to telomere shortening and cessation of unrestrained proliferation. We describe novel chemical compounds which selectively inhibit telomerase in vitro and in vivo. Treatment of cancer cells with these inhibitors leads to progressive telomere shortening, with no acute cytotoxicity, but a proliferation arrest after a characteristic lag period with hallmarks of senescence, including morphological, mitotic and chromosomal aberrations and altered patterns of gene expression. Telomerase inhibition and telomere shortening also result in a marked reduction of the tumorigenic potential of drug-treated tumour cells in a mouse xenograft model. This model was also used to demonstrate in vivo efficacy with no adverse side effects and uncomplicated oral administration of the inhibitor. These findings indicate that potent and selective, non-nucleosidic telomerase inhibitors can be designed as novel cancer treatment modalities.

Fig. 1. Specific and selective telomerase inhibitors. (A) Chemical structure of the BIBR compound class of inhibitors. BIBR1532, R = H; BIBR1591, R = morpholin-4-yl. (B) Dosis-dependent inhibition of telomerase activity by BIBR1532 (solid squares) and BIBR1591 (open circles). Assays were performed and quantified using a PCR-based protocol followed by a TCA precipitation step. The incorporated activity of samples with inhibitor was normalized to the control and plotted against the inhibitor concentration. (C) Selectivity profile of BIBR1532. Enzymatic activity was assayed in the presence of 0–50 µM BIBR1532 as described in Materials and methods. –, no effect at 50 µM. (D) Direct assay of telomerase activity. Telomerase was reconstituted with insect cell expressed hTERT and in vitro transcribed RNA, affinity purified and incubated in the presence of different concentrations of BIBR1532. Telomerase products were separated on a sequencing gel.

Fig. 2. Telomerase inhibitors induce telomere shortening and limit cell proliferation. (A) Total genomic DNA prepared from untreated (lane 1), solvent- (lane 2) or inhibitor-treated (lanes 3 and 4) NCI-H460, HT1080, MDA-MB231 or DU145 cells was assessed for telomere restriction fragment size by Southern blot analysis with a telomeric probe. PD, population doubling; –, absence and +, presence of BIBR1532 or BIBR1591. (B) NCI-H460, HT1080, MDA-MB231 and DU145 cells were plated in 24-well plates in duplicate in the presence of 10 µM BIBR1532 or 50 µM BIBR1591 dissolved in 0.1% DMSO (closed symbols). Control cells were untreated (open triangles) or treated with corresponding solvent concentrations (open circles). Cultures were replated every 2–3 days to maintain log-phase growth and to calculate the growth rate.

Fig. 3. Reversibility of inhibition. (A) NCI-H460 cells were cultivated in 24-well plates in the absence (open circles) or presence of 50 µM BIBR1591 (closed triangles). After 130 days, compound-treated cells were replated only when the culture dishes reached subconfluence. At day 220, these cells were washed, replated in medium without compound and the growth rate monitored for additional 50 days (open squares). (B) The median TRF size of inhibitor-treated NCI-H460 cells at day 220 corresponds to only 1.5 kb (lanes 1 and 2). Removal of the inhibitor and cultivation of these cells for 40 PD in absence of inhibitor leads to a significant telomere elongation (lane 3). Untreated control cells at day 260 are also shown for comparison (lane 4).

Fig. 4. Induction of a senescence phenotype. Phase contrast micrographs show the cellular morphology of (A) untreated and (B) inhibitor-treated NCI-H460 cells at PD162 and PD130, respectively. (C) Inhibitor-treated NCI-H460 cells from a separate inhibition experiment were stained for β-galactosidase activity at pH 6.0. Phenotypic changes in this experiment were already observed at PD72. (D–G) Flow cytometric analysis of NCI-H460 cells shown in (C). Dot density maps of PI-staining (x-axis) and 90-degree light scatter (y-axis) are shown for control (D), and inhibitor-treated cells (PD72) (E). DNA content analysis of control (F), and inhibitor-treated cells (PD72) (G).

Fig. 5. Telomere analysis of inhibitor-treated NCI-H460 cells. (A) Q-FISH analysis of metaphase chromosomes from inhibitor-treated NCI-H460 cells. DAPI-stained chromosomes and Cy-3-labeled telomeres are shown in blue and yellow, respectively. The NCI-H460 cell line is hypotriploid with seven marker chromosomes (modal chromosome number 57) and exhibits a characteristic chromosome with interchromosomal telomere signals. Arrowhead denotes missing telomeres; arrow denotes fused chromosomes; dashed arrow denotes interchromosomal telomere signals. (B) Details of end-to-end fusions from another metaphase with telomere signals present at the fusion site. (C) Histograms express the fluorescence intensity and frequency of all individual telomere spots from NCI-H460 derived metaphases. Twenty metaphases (n = 3740) were derived from control cultures (PD68) and eight metaphases (n = 1366) were derived from inhibitor treated cells (PD120). n is the number of individual telomere signals. The differences in mean fluorescence intensity (arbitrary fluorescence units ± SD) between the control cells (321 ± 160) and the treated cells (217 ± 101) were highly significant (p <0.0001).

Fig. 6. Tumorigenicity assay. Nude mice were each injected with 1.5 × 10⁶ HT1080 control or pre-senescent, telomere shortened cells. Tumour size was measured in regular intervals. (A) Telomere length of untreated control (–) and inhibitor-treated (+) HT1080 cells used to inject nude mice. (B) Mean tumour size of animals bearing control (n = 24) (open circles) and pre-senescent cells in the absence (n = 27) (open triangles) or presence (n = 19) (closed triangles) of BIBR1532. (C) The number of animals (as a percentage) with a tumour >1000 mm³ for untreated control (black) and pre-treated cells in the absence (thin stripes) or presence (bold stripes) of BIBR1532 at the indicated days after injection. Statistical analysis was performed using Fisher’s exact test, with significant differences (p <0.05) between untreated control and pre-treated cells (*; p <0.0002) as well as between pre-treated cells in the presence or absence of BIBR1532 (**; p = 0.01034) at the indicated time points.