Functional p53 Is Required for Effective Telomerase Inhibition in BCRABL- Positive CML Cells in Vitro

Telomeres consist of repeat structures such as (TTAGGG)n in vertebrates and are localized at the end of chromosomes. Replication-dependent telomere shortening due to the end-replication problem can be counteracted by upregulation of an endogenous reverse transcriptase called telomerase. Increasing evidence suggests that critical telomere shortening results in genetic instability which may promote tumour evolution and telomerase activation during which critically short telomeres are stabilised and ongoing tumour growth is facilitated. In Chronic myeloid leukemia (CML) the high turnover of the malignant clone is driven by the oncogene BCR-ABL and leads to accelerated telomere shortening in chronic phase (CP) compared to telomere length in healthy individuals. Telomere shortening has been demonstrated to be correlated with disease stage, duration, prognosis and response to molecular targeted treatment. Despite of the accelerated telomere shortening observed, telomerase activity is increased in CP CML and further upregulated with progression of the disease to accelerated phase or blast crisis (AP/BC). To investigate the effect of telomerase inhibition on BCR-ABL-positive cells, we expressed a dominant-negative mutant of hTERT (vector pOS DNhTERT-IRES-GFP) in K562 cells. The cells were single sorted and clones in addition to bulk cultures were long term expanded in vitro. The expression of the transgene DNhTERT was monitored by the expression of GFP and function of DNhTERT was analyzed by measurement of telomere length (by flow-FISH) and telomerase activity (TRAP assay). Evaluation of these parameters showed the following patterns of growth kinetics and telomere biology in individual clones: Two clones lost telomere repeats and were transiently delayed in growth kinetics but eventually escaped from crisis without loss of GFP expression (indicated by a re-increase in telomere length and growth rate, group A) Three other clones lost GFP expression after initial and significant telomere reduction indicating loss of the transgene (group B). Finally, telomere length and growth kinetics of two remaining clones and of the bulk culture cells remained unaffected by expression of DN-hTERT (group C). Of note, none of the clones analyzed either died or entered cell cycle arrest. Further analyses of one clone of group A revealed impaired DNA damage response indicated by two fold increase in number of γH2AX foci in comparison to control cells. Moreover, the expression pattern of genes involved in DNA repair was significantly altered (Dual chip®). Network analysis of the altered genes using MetaCore® software confirmed p53 as a key regulator in signaling of DNA damage in these cells. CML blast crisis cell lines such as K562 are typically negative for functional p53 and p16^INK4.

Therefore, we went on and investigate if the presence of functional p53 is required for the induction of telomere-mediated apoptosis or senescence in BCR-ABL-positive cells. For this purpose, we restored p53 in telomerase-negative clones by using an inducible system (vector pBABE p53ER^tam) in two clones from group A and group B. Induction of p53 in cells with critically short telomeres (telomere length 4–5 kb) lead to immediate induction of apoptosis while vector control cells continued to escape from crisis. These results suggest that the success of strategies aimed at telomerase inhibition in CML is dependent on the presence of functional p53 in BCR-ABL-positive cells which argues in favour of applying these strategies preferentially in CP as opposed to BC.