Abstract
Disease progression in CML is associated with accelerated telomere shortening caused by increased turnover of Bcr-Abl positive cells. We have recently proposed a model in which continuous telomere shortening is correlated with increasing genetic instability in Bcr-Abl-positive cells thus facilitating the acquisition of secondary genetic abnormalities and, as a consequence clonal succession and eventually clinical progression of the disease. Based on this hypothesis, telomerase upregulation in late stage disease is a prerequisite for prevention of replication-induced senescence in Bcr-Abl positive cells. As a consequence, treatment of CML with Telomerase inhibitors (TI) represents an attractive strategy aiming at the potential eradication of cycling CML stem cells. Therefore, we have exploited both pharmacological (small molecule inhibitor) and genetic strategies (dominant negative hTERT mutants, DN-hTERT) of telomerase inhibition in CML cells in vitro We first treated K562 cells with the pharmacological telomerase inhibitor BIBR1532 in vitro. After around 400 population doublings (PD), no differences in growth kinetics nor signs of senescence or apoptosis were observed in BIBR1532 treated cells despite of significant telomere shortening (22 base pairs (bp) per PD) compared to control cells. Furthermore, neither significant differences in mRNA expression of telomere/telomerase-associated proteins, nor accumulation of double strand breaks (DSBs) under irradiation was observed in treated cells with short telomeres as opposed to untreated control cells. The very slow shortening rate of 22bp/PD plus the lack of stigmata pointing to induction of senescence in K562 cells lead to the assumption that telomerase activity is not complete inhibited by the compound. In order to verify the potency of telomerase directed treatments in CML, we therefore expressed DN-hTERT in K562 cells. Integration of DN-hTERT led to a significant decrease in telomerase activity (measured by RQ-TRAP). Furthermore, DN-hTERT expressing cells underwent accelerated telomere shortening at a substantially higher rate (>100 bp/PD) from 15kb to around 4kb within 110 days of culture. In contrast to BIBR1532-treated cells, DN-hTERT expressing K562 cells slowed down growth kinetic in comparison to control cells after 80 days of culture. By using Annexin 5 staining, 25% of apoptotic cells could be detected in cells with critically short telomeres as compared to control cells (<3%). Finally, a significantly increased accumulation of double strand breaks (DSBs) detected by gammaH2AX foci after exposure to irradiation was observed in DN-hTERT K562 cells as compared to control cells pointing to an impaired DNA repair machinery in Bcr-Abl positive cells with disrupted telomere maintenance. In summary, the data suggest that pharmacological telomerase inhibition by BIBR1532 is insufficient to induce telomere-mediated senescence in Bcr-Abl-positive cells. However, accelerated telomere shortening, slowing down of growth kinetics, elevated apoptosis and increased radiosensitivity induced by expression of DN-hTERT indicate a therapeutic potential for telomerase-directed treatment strategies in CML.
Targeting Assay To Study the cis Functions of Human Telomeric Proteins: Evidence for Inhibition of Telomerase by TRF1 and for Activation of Telomere Degradation by TRF2
