Enhanced Apoptosis in Metallothionein Null Cells YUKIHIRO KONDO, 1 JAMES M. RUSNAK, DALE G. HOYT, CATHERINE E. SETTINERI, BRUCE R. PITT, and JOHN S. LAZO Department of Pharmacology, University of Pittsburgh and Experimental Therapeutics Program, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15261 Received October 29, 1996; Accepted May 2, 1997 SUMMARY Metallothioneins (MTs) are major intracellular, zinc-binding pro- teins with antioxidant properties. Mouse embryonic cells null for MT due to loss of functional MT I and II genes (MT-/-) were more susceptible to apoptotic death after exposure to tert-butyl hydroperoxide or the anti-cancer agents cytosine arabinoside, bleomycin, melphalan, and cis-dichlorodiammineplatinum(II) compared with wild-type mouse embryonic cells (MT+/+). We measured basal levels of the tumor suppressor protein p53 and the death effector protein Bax and found the basal levels of both proteins were higher in MT null cells compared with MT+/+ cells. After treatment with the DNA-damaging agent cis-dichlorodiammineplatinum(II), p53 protein levels were in- duced in both MT+/+ and MT-/- cells with MT null cells always maintaining the highest p53 levels. The elevated sensi- tivity to apoptosis was not restricted to embryonic cells. Pri- mary pulmonary fibroblasts were isolated from distinct litters of MT null, heterozygous, and wild-type mice, and all had unde- tectable basal MT levels. Zinc exposure increased MT levels in the wild-type and heterozygous fibroblasts but not in the MT null fibroblasts. Consistent with the induced MT levels, we found MT+/+ and MT+/- embryonic cells were less sensitive to cis-dichlorodiammineplatinum(II)-induced apoptosis com- pared with MT-/- cells. Our results implicate MT as a stress- responsive factor that can regulate apoptotic engagement. Controlled cell death is essential for the development and survival of multicellular organisms. Several gene products, such as Bax, facilitate the selective and controlled process of death termed apoptosis (1). Apoptosis is inhibited by a vari- ety of proteins including the oncoprotein Bcl-2 and the anti- oxidants superoxide dismutase and glutathione peroxidase (2). Inappropriate apoptosis may precipitate many diseases including Alzheimer’s, Huntington’s, and Parkinson’s dis- eases, neoplasia, autoimmune disorders, immune deficiency, ischemic neurological and cardiovascular damage, and alo- pecia (3). Because apoptosis is a critical homeostatic mecha- nism, it is likely to be finely regulated by both constitutive and inducible proteins. For example, the tumor suppressor p53, which is a zinc-dependent protein, seems to be one inducible stress-responsive protein that facilitates some pathways of apoptosis. The morphological properties of apoptosis, most notably chromatin condensation, are now well accepted (4, 5). The biochemical bases for chromatin condensation and the other morphological aspects of apoptosis remain poorly defined. Some have suggested ROI may participate in apoptotic sig- naling (2, 6 –9), whereas others have disputed this hypothesis (10, 11). DNA cleavage is universally seen in nucleated cells undergoing apoptosis. A Ca 2+ /Mg 2+ -dependent endonuclease activity that produces internucleosomal DNA breaks has been associated with the terminal phases of apoptosis in some cells, and this endonuclease is inhibited by zinc (12). MTs are a family of low molecular weight thiol-rich pro- teins. As one of the major intracellular zinc-binding proteins, they may regulate free zinc levels. The intracellular levels of MT can be readily increased by heavy metals, cytokines, drugs, steroids, and low oxygen via transcriptional activation (13). Because of the nucleophilicity of MT, there has been considerable interest in its ability to protect cells against electrophilic toxins. Indeed, we and others (13–17) previously demonstrated increased MT protects against cytotoxicity from carbon-, oxygen-, and nitrogen-based radicals, including electrophilic mutagens, antineoplastic drugs, nitric oxide, and environmental oxidants. Cells made deficient in MT by homologous recombination and disruption of MT I and II genes are more sensitive to the toxic effect of oxidants, the heavy metal Cd, anti-cancer drugs, and electrophilic muta- gens (18, 19). Zheng et al. (20) found increased hepatic in- duction of p53 in MT I and MT II knockout mice after Cd This research was funded in part by American Institute for Cancer Re- search Grant 95A50), the American Heart Association, American Cancer So- ciety Grant IRG-58 –34 and National Institutes of Health Grants CA61299, DK46935, and HL32154. 1 Current affiliation: Department of Urology, Nippon Medical School, Bunkyo-ku, Tokyo, Japan, 113. ABBREVIATIONS: MT, metallothionein; ROI, reactive oxygen intermediates, ara-C, cytosine arabinoside; tBH, tert-butyl hydroperoxide; CDDP, cis-dichlorodiammineplatinum(II) (cisplatin); QFIGE, quantitative field inversion gel electrophoresis; PBS, phosphate-buffered saline; MEC, mouse embryonic cells; BLM, bleomycin. 0026-895X/97/020195-07$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. 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