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Atypical protein-kinase Cζ, 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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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-Cζ, but not conventional Ca2+-dependent PK-Cα, PK-Cβ or PK-Cγ, 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-Cζ, but not conventional Ca2+-dependent PK-C isoenzymes (α, β, γ) nor the Ca2+/CaM-dependent pathway, in the regulation of telomerase activity in Burkitt’s lymphoma cells.

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Acknowledgements

The technical assistance of Mrs. Kawahara of the National Institute of Advanced Industrial Science and Technology—AIST-Kyushu, Japan, is gratefully acknowledged. This study was supported in part by the JSPS Invitation Fellowship Program for Research in Japan.

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Authors and Affiliations

  1. Single-Molecule Bioanalysis Laboratory, National Institute for Advanced Industrial Science and Technology—AIST-Shikoku, 2217-14 Hayashi-cho, Kagawa-ken, Takamatsu, Japan

    Rumiana Bakalova, Hideki Ohba, Zhivko Zhelev, Mitsuru Ishikawa, Yasuo Shinohara & Yoshinobu Baba

  2. Department of Chemistry, Kyushu School of Engineering, Kinki University, Fukuoka, Japan

    Takanori Kubo & Masayuki Fujii

  3. Faculty of Pharmaceutical Sciences, Tokushima University, Tokushima, Japan

    Yasuo Shinohara & Yoshinobu Baba

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  1. Rumiana Bakalova
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Correspondence to Hideki Ohba.

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Bakalova, R., Ohba, H., Zhelev, Z. et al. Atypical protein-kinase Cζ, but neither conventional Ca2+-dependent protein-kinase C isoenzymes nor Ca2+-calmodulin, participates in regulation of telomerase activity in Burkitt’s lymphoma cells. Cancer Chemother Pharmacol 54, 161–172 (2004). https://doi.org/10.1007/s00280-004-0789-y

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