Implication of the h Topolll alpha in Telomere Maintenance in Leukemia - TOTEKAN

Coordinating Institution: CRP Sante
From: 01/12/2007
To: 30/11/2009
Budget: 152,500.00€
Contact(s): Wenner Thomas

Summary

Telomeres consist of repetitive G-rich DNA sequences that protect the chromosome ends. Two classes of mechanisms allow the maintenance of telomere length in cancer cells. The first involved the telomerase, translated from the hTERT gene, specialized enzyme able to copy the telomeric motif. The second mechanism, observed in about 15% of tumours corresponds to recombination between telomeres. These tumours display a heterogeneous telomere length, maintained by a recombinogenic mechanism involving a complex formed of the Topoisomerase III alpha (TopoIIIa), a RecQ like helicase BLM and a telomeric protein TRF2.

As Topoisomerase I and II are targets of numerous and essential anticancer drugs, the aim of our project is to study the relevance of TopoIIIa as a potential target of anticancer treatment and especially in patients suffering from Chronic Lymphoid Leukaemia (CLL) and Chronic Myeloid Leukaemia (CML). Promoter methylation is thought to play a crucial role during the carcinogenesis process through a tight gene expression regulation. Hence we study the CpG island methylation status of hTERT and hTopoIIIa in cells from CLL and CML patients and will correlate this with the expression level and activity of these genes.

Up to now, the TopoIIIa CpG islands have been mapped and the TopoIIIa promoter methylation status and a part of hTERT promoter have been investigated in 31 patients suffering from CLL, 10 healthy volunteers and 6 cell lines. Moreover the level of expression of 8 genes including telomeric genes as well as TopoIIIa and BLM has been evaluated. We also expressed, purified and raised antibodies against TopoIIIa as no commercial correct antibody was available.

We measured the telomere length of all the samples using a non-radioactive method which has been successfully implemented in our flow cytometry core facility. Concerning the activity of these genes, the hTERT activity results in telomere of homogenous size and will be study using a TRAP assay (Telomere Repeat Amplification Protocol). However, activity of TopoIIIa results in large telomeres of heterogeneous size. Once the TRAP experiments performed, we would then be able to correlate the level of expression/activity/CpG island methylation of the telomerase and TopoIIIa and then implied TopoIIIa as a potential target of anticancer treatments.