Characterization of Bcr-Abl Positive Leukemic Cells under Long-Term In Vitro Treatment with Telomerase Inhibitor BIBR1532.

Abstract

In normal somatic cells telomeres shorten with each cell division because of the end-replication problem. The ribonucleoprotein enzyme telomerase is able to prevent replicative telomere shortening and to maintain or elongate telomere length. In 90 % of tumour cells the enzyme telomerase is found to be upregulated. Chronic myeloid leukemia is a disorder characterized by a reciprocal translocation between Chromosome 9 and 22, leading to the so called Philadelphia chromosome harbouring the BCR-ABL translocation. BCR-ABL positive leukemic stem cells are characterized by increased turnover leading to accelerated telomere shortening as opposed to their normal counterparts. It is unclear to date whether accelerated telomere shortening in Bcr-Abl-positive cells is linked to genetic instability eventually leading to the acquisition of secondary clonal events that might propagate acceleration of the disease to blast crisis. Therefore we aimed to characterize Bcr-Abl positive chronic myeloid leukemia cell line K562 with or without inhibition of telomerase activity under long-term culture conditions. K652 cells were expanded for 400 populations doublings (PD) with or without treatment with the small molecule telomerase inhibitor BIBR1532 in vitro. While telomeres in untreated control cells remained relatively constant, telomeres in BIBR1532 treated cells underwent replicative shortening from 10 kb to 3 kb (as measured by flow FISH), reflecting a rate of 22 base pairs (bp) lost per PD. No difference in growth kinetics were observed until that stage. We next characterized treated K562 with short telomeres (K562-S) in contrast to control cells with long telomeres (K562-L) for the expression of telomere and telomerase-binding proteins. No difference in mRNA expression for any of the candidate proteins were observed by RT-PCR. Comparative analysis of global protein expression was performed by 2D gel electrophoresis. Taken together, 23 protein spots were found to be differentially expressed between treated and untreated cells, fifteen of which were already identified by mass spectometry. Additionally, we analysed the cells for the acquisition of additional cytogenetic abnormalities by M-FISH. Interestingly, in this ongoing study, we consistently found acquisition of genetic material on chromosome 7 in treated as compared to untreated cells. To study radiation sensitivity under BIBR1532 treatment, K562 cells were exposed to increasing doses of irradiation. Interestingly, despite of a dose-dependent increase in the fraction of apoptotic cells in the pre-treated as opposed to control cells, no accumulation in the number of double strand breaks or lethal aberrations were detected. Interestingly, telomere shortening after telomerase inhibition translated to increased sensitivity to Imatinib (IC50 0.6 μM vs. IC50 1.2 μM). Taken together, telomerase inhibition represent a attractive new therapeutic strategy in Bcr-Abl positive leukemias. However, careful evaluation of side effects need to be studied on the proteomics and cytogenetic level.