title: 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.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Single-Molecule Bioanalysis Laboratory, National Institute for Advanced Industrial Science and Technology-Shikoku, 2217-14 Hayashi-cho, Kagawa-ken, Takamatsu, Japan.
authors.forename: Rumiana
authors.initials: R
authors.lastname: Bakalova
authors.forename: Hideki
authors.initials: H
authors.lastname: Ohba
authors.forename: Zhivko
authors.initials: Z
authors.lastname: Zhelev
authors.forename: Takanori
authors.initials: T
authors.lastname: Kubo
authors.forename: Masayuki
authors.initials: M
authors.lastname: Fujii
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Ishikawa
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Shinohara
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Baba
mesh_terms: D017209:Apoptosis; D002051:Burkitt Lymphoma / Q000201:enzymology*; D002147:Calmodulin / Q000494:pharmacology*; D018492:Dopamine Antagonists / Q000494:pharmacology; D004789:Enzyme Activation; D005434:Flow Cytometry; D005470:Fluorometry; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes; D008301:Maleimides / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011493:Protein Kinase C / Q000494:pharmacology*; D011494:Protein Kinases / Q000494:pharmacology*; D019098:Telomerase / Q000494:pharmacology*; D014268:Trifluoperazine / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000099221:Protein Kinase C zeta
chemical_list: D002147:Calmodulin; D018492:Dopamine Antagonists; D007211:Indoles; D007527:Isoenzymes; D008301:Maleimides; D011493:Protein Kinase C; D011494:Protein Kinases; D019098:Telomerase; D014268:Trifluoperazine; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000099221:Protein Kinase C zeta; C088060:bisindolylmaleimide