Discrimination Factor (DF), defined as the fold increase in ddNTPs or NRTIs concentration required for 50% telomerase inhibition (IC50) above that of each of the respective endogenous competitor dNTP concentrations (DF = IC50NRTI/ ddNTP/[dNTPcompetitor]). A comparison of the DFs for ddTTP, AZT-TP and d4T-TP, under our assay conditions reveals very similar DF values for ddTTP and d4T (3.0 versus 2.4), and a slightly higher value for AZT-TP (4.4; Table 1 and Figure 2C). We concluded that telomerase catalysis is efficiently inhibited by AZT-TP and d4T-TP in vitro, consistent with their roles as chain-terminating thymidine analogs in the context of inhibition of HIV RT.Figure 6. Continuous treatment of HT29 cells with the thymidine analog AZT and d4T causes substantial telomere shortening. A. TRF blots of untreated (left), AZT-treated (right) HT29 cells. PDL at which TRF was analyzed is shown above each lane. Molecular mass markers are shown at left and right of gel images. Each TRF smear was quantified as a weighted average and is shown below each lane. B. Growth curves and telomere maintenance dynamics of HT29 cells treated continuously with AZT. The growth curve and TRF dynamics of untreated HT29 cells (solid blue line) is plotted for comparison. Telomere length maintenance inhibition was observed in both the lower (62.5 mM) and higher (125 mM) AZT doses. C. TRF blots of untreated (left), d4T-treated (right) HT29 cells. D. Growth curves and telomere maintenance dynamics of HT29 cells treated continuously with d4T. The growth curve and TRF dynamics of untreated HT29 cells (solid blue line) is plotted for comparison. Loss of telomere length was observed with the middle (80 mM) and more significant at the higher (160 mM) d4T dose, but less evident in the lowest dose (40 mM) of d4T treatments, suggesting an apparent dose-response relationship. guanosine analog chain terminator (Figure 4B). Non-linear regression analysis of the dose-response relationships (Figure 4C) resulted in a DF of 1.6 for ddGTP and a DF of 18 for CBV-TP (Table 1). Our data indicate that the active form of ABC inhibits telomerase catalysis effectively in vitro, albeit with less potency than ddGTP.
Telomerase Catalysis is not Affected by the NNRTIs Nevirapine and Efavirenz
Unlike NRTIs, which are competitive RT inhibitors, nonnucleoside reverse transcriptase inhibitors (NNRTIs), including nevirapine (NVP) and efavirenz (EFV), inhibit HIV RT noncompetitively via enzyme binding outside the catalytic cleft [30].Table 2. Summary of the impact of NRTIs/NNRTIs on telomere maintenance in HT29 colorectal adenocarcinoma cells.determine whether these agents affect telomerase by binding to its RT domain, like their inhibition of HIV RT. To measure telomerase catalysis in the presence of NVP and EFV, we used an 18 nt telomeric oligonucleotide primer ending in GGG (GGG-primer) and the full complement of three deoxynucleotide triphosphates (dATP, dTTP and dGTP), including radiolabeled dGTP. We tested both NNRTIs at 1 mM (in 5% DMSO) and 4 mM (in 10% DMSO) (Figure 5A). We also conducted dose-response experiments for NVP and EFV, and did not observe any changes in telomerase catalysis, even with concentrations as high as 10 mM (data not shown). We concluded that, under these conditions, telomerase catalysis is not inhibited in vitro by either of the NNRTIs tested.observed 1.9 kb and 2.3 kb losses in mean telomere length, respectively, over 12 population doubling levels (PDL) in continuous culture. At 125 mM AZT, the rate of telomeric DNA loss was 192 bp/PDL, suggesting that telomerase activity was completely inhibited at this dose. Continuous treatment of HT29 cells with d4T also resulted in a dose-dependent loss of telomeric DNA (Figures 6C and 6D). Treatment with 80 mM or 160 mM of d4T resulted in 1.5 kb and 3.7 kb losses in mean TRF signals over 21 and 18 PDL, respectively. Unexpectedly, treatment with tenofovir disoproxil fumarate (TDF, the prodrug of tenofovir) resulted in excessive cell death, and we were unable collect any cell populations beyond 8 PDL for the 50 mM treatment groups and 3 PDL for the 100 mM treatment group (Figures 7A and 7B). This is in contrast with the low cytotoxicity reported with this agent in other cultured cell models [32]. At these early PDLs, the cumulative TRF loss was expected to exceed the inherent variation of our assay. Thus, we cannot deduce the effects of TDF on telomere maintenance from this experiment. Continuous treatment with ddI at 30 and 60 mM showed no observable loss of TRF signals up to 18 PDL (Figures 7C and 7D).
At 120 uM, ddI showed a slight decrease (0.8 kb) in TRF size at 12 PDL. However, treatment cytotoxicity prevented us from collecting cells beyond this time point, and prevented us from confirming the effects of ddI on telomere maintenance. Treatment with a high dose of ABC (100mM) had a profound cytotoxic effect on HT29 cells, and we were not able to collect cells beyond 2 PDL following drug treatment (Figure 8). However, with lower doses of ABC (12.5 mM and 50 mM), we did observe a dosedependent loss of TRF signal (0.8 kb and 2.1 kb, respectively) over 19 PDL. In our experiments, we also included continuous treatment with Lamivudine (3TC), a cytidine analog not expected to affect telomere synthesis by telomerase, since the telomeric repeat lacks cytidine. The inclusion of 3TC as a negative control tested whether inhibition of other DNA polymerases could affect telomere maintenance independently of telomerase. As expected, we did not observe any loss of TRF signals over 19 PDL of continuous treatment with 80 mM of 3TC (Supplementary Figure 2), implying that telomere length changes measured in NRTI-treated cells are specific to telomerase inhibition effects under these conditions. Finally, continuous treatment with the two NNRTIs, EFV and NVP, did not result in significant losses of TRF signal, despite treatment toxicity associated with high doses of EFV (Supplementary Figure 3). This corroborated our in vitro observation that NNRTIs did not affect telomerase catalysis.
Discussion
Using the in vitro primer extension assay for telomerase activity, we demonstrated that all NRTIs in current clinical usage inhibit human telomerase, albeit with different potencies. AZT-TP and d4T-TP were the most potent inhibitors of telomerase relative to their ddNTP counterpart, ddTTP. CBV-TP was the least potent telomerase inhibitor relative to ddGTP, while the TFV-DP/ ddATP comparison showed intermediate inhibitory potency. There was general agreement between our in vitro and cell culture studies of NRTI effects on telomere synthesis, although in some instances, the profound toxicity of high concentrations of several NRTIs likely affected the correlation between the two experiments. In any cell culture system, effects of telomerase inhibition manifest as a gradual loss of telomere length, which is only evident in the TRF assay after sufficient time has passed (10?0 PDL when telomerase is inhibited completely).
