Atypical protein-kinase Czeta, but neither conventional Ca2+ -dependent protein-kinase C isoenzymes nor Ca2+ -calmodulin, participates in regulation of telomerase activity in Burkitt's lymphoma cells
Authors: Zhivko Zhelev
Rumiana Bakalova
Hideki Ohba
Yasuo Shinohara
Masayuki Fujii
Takanori Kubo
Mitsuru Ishikawa
Yoshinobu Baba
Journal: Cancer Chemotherapy and Pharmacology (2004)
Discipline: CLINICAL MEDICINE
Keywords: PK-C; Ca2+/calmodulin; telomerase activity; Burkitt's lymphoma cells; inhibitor
Affiliations: Single-Molecule Bioanalysis Laboratory, National Institute for Advanced Industrial Science and Technology-Shikoku, 2217-14 Hayashi-cho, Kagawa-ken, Takamatsu, Japan.

Abstract:  Purpose: To clarify the role of the pathways dependent on protein-kinase C (PK-C) and Ca2+/calmodulin (CaM) in the regulation of telomerase activity in Burkitt's lymphoma cells. Methods: Burkitt's lymphoma cells (Raji and Daudi) were treated with the PK-C inhibitor, bisindolylmaleimide (BIM), or the CaM inhibitor, trifluoperazine (TFPZ), in a dose-dependent manner and in a time-dependent manner. The activities of PK-C isoenzymes were analyzed fluorimetrically using POLARIS assay kits. CaM-kinase II activity was analyzed radiographically, using CaMK-II immunoprecipitation kinase assay kits. Telomerase activity was detected by a conventional telomeric repeat amplification protocol and Stretch PCR. The level of catalytic subunit of telomerase (hTERT) in drug-treated and nontreated cells was analyzed by flow cytometry using anti-hTERT antibody labeled with ZenonAlexa Fluor-488 IgG. Apoptosis was estimated in terms of phosphatidylserine exposure on the cell surface and DNA fragmentation. Results: It was found that BIM inhibited telomerase activity and this process preceded apoptosis. The subsequent addition of exogenous PK-C (mixture of isoenzymes) to the cell lysates restored telomerase activity if incubation of cells with BIM was up to 24 h. Using PK-C isoenzymes, it was established that atypical PK-Czeta, but not conventional Ca2+ -dependent PK-Calpha, PK-Cbeta or PK-Cgamma, is responsible for the reactivation of telomerase in BIM-treated cells. BIM also showed a well-expressed cytotoxicity against intact leukemia cells. In contrast, the CaM inhibitor TFPZ showed the same cytotoxic effect without any influence on telomerase activity during incubation for 24 h with leukemia cells. After incubation for 48 h, TFPZ markedly suppressed telomerase activity. However, the effect followed apoptosis and appeared to be a result of cell death. The addition of exogenous CaMK-II to the cell lysates obtained from TFPZ-treated cells did not reactivate telomerase. Conclusion: The present study confirmed the participation of atypical PK-Czeta, but not conventional Ca2+ -dependent PK-C isoenzymes (alpha, beta, gamma) nor the Ca2+/CaM-dependent pathway, in the regulation of telomerase activity in Burkitt's lymphoma cells. 

Retracted: No

PubPeer comments:
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