Induction of apoptosis by hepatocyte growth factor/scatter factor and its augmentation by phorbol esters in Meth A cells.
Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional cytokine with mitogenic, motogenic, and morphogenic activities. In addition, HGF/SF inhibits the proliferation of some tumor cell lines, but its mechanism remains poorly understood. We determined in this study whether HGF/SF induces cell death of a Meth A mouse sarcoma cell line in vitro, whose proliferation is remarkably suppressed by HGF/SF. Inhibition of Meth A cell growth by HGF/SF was dose-dependent and maximal at a concentration of 30 ng/ml. The percentage of dead cells increased to 22% upon treatment with 30 ng/ml of HGF/SF for 96 h, whereas that in untreated cultures was less than 5%. Staining of these cells nuclei with Hoechst 33342 revealed condensation of the chromatin and nuclear fragmentation. Gel electrophoresis of DNA from HGF/SF-treated cells showed a typical ladder pattern. Cells with a fractional DNA content also increased five-fold in the HGF/SF-treated cultures as analyzed by flow cytometry after propidium iodide staining. These are features typical of apoptosis. Concurrent addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) with HGF/SF augmented the apoptosis induced by the growth factor, while TPA alone caused little death. This enhancement was largely blocked by addition of the specific protein kinase C inhibitor GF 109203X. These results indicate that HGF/SF induced the apoptotic cell death of the Meth A sarcoma cell line and that protein kinase C activation augmented the growth factor-induced apoptosis.
Gohda
E
E
Department of Immunochemistry, Faculty of Pharmaceutical Sciences, Okayama University, Tsushima-naka, Okayama, 700-8530, Japan. gohda@pheasant.pharm.okayama-u.ac.jp
Okauchi
H
H
Iwao
M
M
Yamamoto
I
I
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001562:Benzimidazoles / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism; D017228:Hepatocyte Growth Factor / Q000494:pharmacology*; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D001562:Benzimidazoles; D005456:Fluorescent Dyes; D007211:Indoles; D008301:Maleimides; D017228:Hepatocyte Growth Factor; D011493:Protein Kinase C; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate; C017807:bisbenzimide ethoxide trihydrochloride