Inhibition of cell growth by conditional expression of kpm, a human homologue of Drosophila warts/lats tumor suppressor.
kpm is a human serine/threonine kinase that is homologous to Drosophila tumor suppressor warts/lats and its mammalian homologue LATS1. In order to define the biological function of kpm, we generated stable transfectants of wild-type kpm (kpm-wt), a kinase-dead mutant of kpm (kpm-kd), and luciferase in HeLa Tet-Off cells under the tetracycline-responsive promoter. Western blot analysis showed that high levels of expression of kpm-wt as well as kpm-kd with an apparent mass of 150 kDa were induced after the removal of doxycycline. Induction of kpm-wt expression resulted in a marked decline in viable cell number measured by both trypan blue dye exclusion and MTT assay, whereas that of kpm-kd or luciferase had no effect. We then analyzed the cell cycle progression and apoptosis upon induction of kpm expression. 2-3 days after removal of doxycycline, cells underwent G(2)/M arrest, demonstrated by flow cytometric analysis of propidium iodide incorporation and MPM-2 reactivity. In vitro kinase assay showed that induction of kpm-wt led to down-regulation of kinase activity of the Cdc2-cyclin B complex, which was accompanied by an increase in the hyperphosphorylated form of Cdc2 and a change of phosphorylation status of Cdc25C. Furthermore, both DAPI staining and TUNEL assay showed that the proportion of apoptotic cells increased as kpm expression was induced. Taken together, these results indicate that kpm negatively regulates cell growth by inducing G(2)/M arrest and apoptotic cell death through its kinase activity.
Kamikubo
Yasuhiko
Y
Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan.
Takaori-Kondo
Akifumi
A
Uchiyama
Takashi
T
Hori
Toshiyuki
T
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017209:Apoptosis; D015153:Blotting, Western; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D002455:Cell Division; D002470:Cell Survival; D004396:Coloring Agents / Q000494:pharmacology; D019926:Cyclin B / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004318:Doxycycline / Q000494:pharmacology; D029721:Drosophila Proteins; D005434:Flow Cytometry; D016195:G2 Phase; D006367:HeLa Cells; D006801:Humans; D020287:In Situ Nick-End Labeling; D008938:Mitosis; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011419:Propidium / Q000494:pharmacology; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics*; D013752:Tetracycline / Q000494:pharmacology; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014343:Trypan Blue / Q000494:pharmacology; D025521:Tumor Suppressor Proteins; D020687:cdc25 Phosphatases / Q000378:metabolism
D000900:Anti-Bacterial Agents; D018797:Cell Cycle Proteins; D004396:Coloring Agents; C503698:CycB protein, Drosophila; D019926:Cyclin B; D029721:Drosophila Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; D025521:Tumor Suppressor Proteins; D011419:Propidium; D011494:Protein Kinases; C092745:LATS1 protein, human; C093503:wts protein, Drosophila; C406449:LATS2 protein, human; D017346:Protein Serine-Threonine Kinases; C517009:CDC25C protein, human; D020687:cdc25 Phosphatases; C022616:thiazolyl blue; D013752:Tetracycline; D014343:Trypan Blue; D004318:Doxycycline