塩化コバルトによるチャイニーズハムスター卵巣細胞のアポトーシス誘導とその塩化亜鉛による抑制について

Abstract

Apoptosis is an important and well-controlled form of self-regulated cell death that differs from necrosis. This process has been recognized to be of major importance for embryonic development, tissue homeostasis, neurodegeneration, autoimmune disease, carcinogenesis, cancer progression, and the killing of cancer cells induced by chemotherapeutic drugs. Apoptosis is induced by radiation, heat, chemical agents, anti-cancer drugs, or some kinds of divalent metals. However, the effects of cobalt on cultured cells is still obscure. The role of cobalt in induction of apoptosis in cultured cells also is not known. To determine whether divalent metal compounds can affect apoptosis in Chinese hamster ovary cells (CHO cells), the effects of cobalt chloride and other metal compounds on cultured cells were examined. After reaching subconfluence, the CHO cells were exposed to varying concentrations of cobalt chloride. Cobalt chloride induced cytotoxicity and cell death in CHO cells in a dose-dependent manner up to the concentration of 1.0 mM as determined by WST-1 assay and by phase-contrast microscopy. By using the Hoechst 33342 staining, marked nuclear condensation and fragmentation of chromatin was observed in CHO cells. DNA ladder formation, a hallmark of apoptosis, also was detected in CHO cells by treatment with cobalt chloride. The induced-DNA fragmentation and DNA ladder formation were dose-dependent with maximal effect at concentrations of 0.5 mM cobalt chloride and were timedependent from 12 h to 48 h. Cobalt chloride also induced apoptosis in another human cell lines, MG63 cells, Saos-2 cells, SCC-25 cells, and HSG cells. However, cobalt chloride did not induce apoptosis in mouse cell line, 3T3-L1 cells. At the same concentration, the chloride compound of the other divalent metals including zinc, iron, nickel, and manganese and copper sulfate also induced apoptosis in CHO cells. However, magnesium chloride or calcium chloride did not induce apoptosis in CHO cells. Zinc chloride prevented cobalt chloride-induced apoptosis in CHO cells in a dose-dependent fashion up to 70μM as determined by phase-contrast microscopy, WST-1 assay, Hoechst staining and DNA ladder formation in agarose gel electrophoresis. The same concentrations of zinc chloride alone did not show any apoptotic features in the treated cells. The present results show that the pathway of the apoptosis in the cultured CHO cells is regulated with cobalt chloride and other divalent metals. The cobalt-induced apoptosis is prevented with zinc treatment. These findings indicate that apoptotic steps in CHO cells are related to the production of some kinds of proteases which are regulated by cobalt and zinc.