Cancer chemotherapy and pharmacology · 2004
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.
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Bakalova Rumiana, Ohba Hideki, Zhelev Zhivko, Kubo Takanori, Fujii Masayuki, Ishikawa Mitsuru, Shinohara Yasuo, Baba Yoshinobu
Аннотация
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.
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.