Overproduction of the ornithine decarboxylase (ODC) regulatory protein ODC-antizyme has been shown to correlate with cell growth inhibition in a variety of different cell types. Although the exact mechanism of this growth inhibition is not known, it has been attributed to the effect of antizyme on polyamine metabolism. Antizyme binds directly to ODC, targeting ODC for ubiquitin-independent degradation by the 26 S proteasome. We now show that antizyme induction also leads to degradation of the cell cycle regulatory protein cyclin D1. We demonstrate that antizyme is capable of specific, noncovalent association with cyclin D1 and that this interaction accelerates cyclin D1 degradation in vitro in the presence of only antizyme, cyclin D1, purified 26 S proteasomes, and ATP. In vivo, antizyme up-regulation induced either by the polyamine spermine or by antizyme overexpression causes reduction of intracellular cyclin D1 levels. The antizyme-mediated pathway for cyclin D1 degradation is independent of the previously characterized phosphorylation-and ubiquitination-dependent pathway, because antizyme up-regulation induces the degradation of a cyclin D1 mutant (T286A) that abrogates its ubiquitination. We propose that antizyme-mediated degradation of cyclin D1 by the proteasome may provide an explanation for the repression of cell growth following antizyme up-regulation. The regulatory protein antizyme (AZ)1 has been studied primarily in the context of its role in facilitating degradation of the enzyme ornithine decarboxylase (ODC), which catalyzes the rate-limiting step in polyamine synthesis (reviewed in Refs. 1-3). AZ is therefore thought to be dedicated principally to the feedback regulation of polyamine levels. AZ synthesis is controlled via an unusual mechanism of translational frameshifting. The ribosomal frameshift required for the translation of full-length AZ is directly induced by polyamines when their levels rise (4, 5). Polyamines can thus inhibit their own synthesis via AZ-mediated down-regulation of ODC. This mechanism serves to prevent extreme fluctuations in polyamine levels, which are thought to be toxic (6 -8). Despite this homeostatic mechanism, the levels of polyamines and ODC vary markedly during the cell cycle, indicating that additional factors control polyamine levels and suggesting a role for this pathway in the regulation of cell proliferation (9).Overproduction of AZ in a variety of cell types, including malignant oral keratinocytes, hepatoma cell lines, and prostate cancer cells, coincides with growth inhibition (10 -12) and cell cycle arrest in the G 1 phase (13,14). Furthermore, overexpression of antizyme in both mouse skin cancer and gastric epithelia models has been shown to result in tumor suppression (15,16). These and other observations of antiproliferative effects of antizyme prompted us to test whether antizyme may have a specific role in cell cycle regulation, thereby accounting for its potential role as a tumor suppressor.The cell cycle arrest previously seen in prostate carcinoma ...

To examine the role of multidrug resistance protein 1 (MRP1) and glutathione S-transferases (GSTs) in cellular resistance to antineoplastic drugs, derivatives of MCF7 breast carcinoma cells were developed that express MRP1 in combination with one of three human cytosolic isozymes of GST. Expression of MRP1 alone confers resistance to several drugs representing the multidrug resistance phenotype, drugs including doxorubicin, vincristine, etoposide, and mitoxantrone. However, co-expression with MRP1 of any of the human GST isozymes A1-1, M1-1, or P1-1 failed to augment MRP1-associated resistance to these drugs. In contrast, combined expression of MRP1 and GST A1-1 conferred ϳ4-fold resistance to the anticancer drug chlorambucil. Expression of MRP1 alone failed to confer resistance to chlorambucil, showing that the observed protection from chlorambucil cytotoxicity was absolutely dependent upon GST A1-1 protein. Moreover, using inhibitors of GST (dicumarol) or MRP1 (sulfinpyrazone), it was shown that in MCF7 cells resistance to chlorambucil requires both intact MRP1-dependent efflux pump activity and, for full protection, GST A1-1 catalytic activity. These results are the first demonstration that GST A1-1 and MRP1 can act in synergy to protect cells from the cytotoxicity of a nitrogen mustard, chlorambucil.

Model cell lines developed from MCF7 breast carcinoma cells were used to examine the roles of glutathione S-transferase P1-1 (GSTP1-1) and multidrug resistance protein (MRP) in the protection of cells from 4-nitroquinoline 1-oxide (4NQO) toxicities. Increased expression of GSTP1-1 alone in MCF7 cells results in limited protection from the formation of 4NQO-derived covalent adducts of nucleic acids but affords no protection from 4NQO-mediated cytotoxicity. Increased expression of MRP alone conferred modest protection while co-expression of GSTP1-1 with MRP produced high-level protection from both 4NQO-derived adduct formation and 4NQO cytotoxicity. This synergistic resistance to 4NQO toxicities (both nucleic acid adduct formation and cytotoxicity) is associated with a GSTP1-1-dependent increase in 4NQO-glutathione (QO-SG) conjugate formation and a MRP-dependent increase in QO-SG efflux. These data indicate that MRP is an important export transporter for the glutathione conjugate of the carcinogen, 4NQO. Moreover, this MRP-dependent efflux activity is necessary to achieve the full protection from 4NQO toxicity-protection that is potentiated by GSTP1-1-mediated QO-SG formation.

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