Mammalian cell polyamine homeostasis is altered by the radioprotector WR1065.

Mammalian cells become more susceptible to radiation-induced death and mutagenesis when restricted in their production of the natural polyamines putrescine, spermidine and spermine. The effects of polyamine deprivation are reversed by N-(2-mercaptoethyl)-1, 3-diaminopropane (WR1065), a simple aminothiol that has been extensively studied for its radioprotectant properties. Because this compound and its oxidized derivative WR33278 bear some resemblance to the polyamines, it was hypothesized that radioprotection by WR1065 or its metabolites is derived, at least in part, from their ability to supplement the natural polyamines. To evaluate the ability of these aminothiol compounds to emulate polyamine function in intact cells, rat liver hepatoma (HTC) cells were treated with radioprotective doses of WR1065; the ability of this compound to affect various aspects of normal polyamine metabolism was monitored. Although cellular WR1065 was maintained at levels exceeding those of the polyamines, this aminothiol did not have any polyamine-like effect on the initial polyamine biosynthetic enzyme, ornithine decarboxylase, or on polyamine degradative reactions. On the contrary, treatment with relatively low levels of WR1065 resulted in an unexpected increase in putrescine and spermidine synthesis. WR1065 treatment enhanced the stability, and consequently the activity, of ornithine decarboxylase. This stabilization seems to result from a WR1065-induced delay in the synthesis of antizyme, a critical regulatory protein required in the feedback modulation of polyamine synthesis and transport. The increase in cellular spermidine induced by WR1065 might explain its antimutagenic properties, but is probably not a factor in protection against cell killing by radiation. This is the first evidence that compounds can be designed to control polyamine levels by targeting the activity of the regulatory protein antizyme.

Osmotic stress induces variation in cellular levels of ornithine decarboxylase-antizyme.

The polyamines, and especially putrescine, play an integral role in the physiological response of cells to varying extracellular osmotic conditions. Ornithine decarboxylase (ODC) synthesis and stability, as well as the activity of the polyamine transporter, had all been reported to be very sensitive to media osmolarity in different cells and tissues, yet the mechanism of this complex, co-ordinated response was not known. In this study we have determined that all these aspects of osmotic-shock response may be mediated by the common regulatory protein, ODC-antizyme. HTC cells were induced for antizyme and then exposed to media of reduced osmotic strength. Both antizyme activity and protein decreased rapidly, under these conditions, to new steady-state levels that depended upon the degree of reduction in media tonicity. This antizyme reduction was found to be due to a rapid increase in antizyme degradation, with a half-life decrease from 75 min down to 45 min occurring immediately upon exchanging media. In complementary experiments, increased media tonicity induced elevated antizyme levels and stability. The sensitivity of antizyme turnover to osmotic conditions was also observed in DH23b cells, which contain elevated levels of more stable antizyme. Interestingly, the two main antizyme proteins, AZ-1 and AZ-2 (presumably products from the first and second translational start sites), differed in their responses to these changing osmotic conditions. Just as feedback regulation of antizyme synthesis provides an effective mechanism for maintaining stable polyamine levels, these studies suggest that alteration in the rate of antizyme degradation may be the mechanism whereby cells adjust steady-state polyamine levels in response to stimulation or stress.

Feedback repression of ornithine decarboxylase synthesis mediated by antizyme.

The induction of antizyme by spermidine and the resulting enhancement of ornithine decarboxylase (ODC) degradation have been well studied; however, little is known about the mechanism whereby elevated spermidine levels decrease synthesis of the polyamine biosynthetic enzyme. To evaluate the relative contribution of inhibited synthesis, as distinct from enhanced degradation of ODC, spermidine levels were manipulated in a variant cell line that overproduces a stable form of ODC. Spermidine did not selectively inhibit ODC synthesis in these variant cells, supporting the concept that spermidine diminishes ODC synthesis in normal cells owing to enhanced degradation of the protein in the presence of elevated antizyme levels. This model was further investigated in vitro by use of rabbit reticulocyte lysate, which catalyses simultaneous ODC mRNA translation and antizyme-stimulated degradation of ODC protein. Antizyme strongly repressed the incorporation of labelled amino acids into normal rat ODC. Unexpectedly it also diminished the apparent translation of ODC mRNA species coding for enzyme forms that are not destabilized by the post-translational addition of antizyme. The effect of antizyme on ODC translation was not observed in wheatgerm extract, in which there is no antizyme-induced degradation. Further, deletion of a short segment of antizyme necessary for the destabilization of ODC (amino acid residues 113-118) resulted in a form that bound ODC but did not diminish its apparent translation. These results suggest that the co-translational addition of antizyme to ODC results in a complex that is different from, and innately less stable than, that formed when antizyme is added post-translationally.

Ornithine decarboxylase stability in HMOA and DH23b cells is not due to post-translational truncation of a C-terminal recognition site.

The normally labile ornithine decarboxylase (ODC) becomes unusually stable when Cys-441 is replaced with Trp in the variant cell lines HMOA and DH23b. This stable ODC is also observed to have higher mobility on SDS/PAGE. Because previous studies have shown that ODC stability can be achieved when as few as five amino acid residues are removed from its C-terminus, it was suggested that the amino acid substitution in the variant ODC might alter its conformation sufficiently to promote a similar proteolytic loss of a C-terminal degradation signal, resulting in a stable yet active ODC. To examine this mechanism, amino acids in the C-terminal regions of both wild-type and stable (Trp-441) ODC proteins were released, by means of carboxypeptidase-Y digestion, and identified by HPLC. The C-terminal ends were found to be the same, and are as predicted from the cDNA sequence. This study proves that stability of the Trp-441 form of ODC is not simply due to proteolytic removal of a C-terminal proteasome-targeting sequence, thereby implying that the stabilization of this mutant ODC form must result directly from a conformational change associated with the loss of Cys-441.

Overproduction of stable ornithine decarboxylase and antizyme in the difluoromethylornithine-resistant cell line DH23b.

DH23b cells, a variant of the HTC line selected for their resistance to difluoromethylornithine, exhibit defective feedback regulation of ornithine decarboxylase (ODC) stability and polyamine transport, and accumulate ODC protein to > 1000 times normal concentrations. The components of the polyamine feedback regulation system have been examined in an attempt to understand these unusual responses. Southern-blot analysis revealed an amplification (approx. 10-fold) in ODC DNA sequence without any concomitant increase in antizyme. Moreover, the amplified ODC sequence contains a single base substitution that results in the conversion of Cys-441 into Trp. This modification has previously been shown to cause ODC stability in HMOA cells. Although antizyme activity has not been noted in DH23b cells, Western-blot analysis revealed the accumulation of antizyme protein to > 50 times that induced in parental HTC cells. This increase is consistent with a 6-9-fold increase in the half-life of antizyme in these cells, a consequence of the inability of the mutant ODC-antizyme complex to be degraded by 26 S proteasome. Associated with the stabilization of antizyme in both DH23b and HMOA cells is the appearance of two additional forms of antizyme protein with apparent molecular masses of 22 and 18.5 kDa. It is suggested that these result from proteolytic removal of discrete fragments from the N-terminal end of antizyme, perhaps an indication of an initial step in rapid antizyme turnover.

Involvement of the polyamine transport system in cellular uptake of the radioprotectants WR-1065 and WR-33278.

High levels of the aminothiol WR-1065 protect cells from ionizing radiation, while much lower levels of this compound or its disulfide, WR-33278, impart anti-mutagenic effects. In view of the structural similarity of these agents to the essential cellular polyamines putrescine, spermidine and spermine we investigated the possibility transport system. WR-33278 appears to be a very close analog of spermidine or spermine in that it not only inhibits spermidine incorporation, but is also transported at the same velocity as spermidine, with a Kt of approximately 0.8 microM compared with 0.4 microM for the polyamine. Further, repression of the activity of the polyamine transporter by antizyme or its elimination by selected mutation affected both transport of WR-33278 and spermidine equally. In contrast, WR-1065 is not a good substrate for the polyamine transporter and appears to enter cells predominantly by non-mediated passive diffusion. There appears to be no uptake of either WR-33278 or the polyamines by this non-mediated diffusion. Thus both the form of the aminothiol and the activity of the polyamine transport system need to be considered in evaluating the efficacy of low exogenous levels of this drug on mutagenesis or carcinogenesis.

Feedback repression of polyamine transport is mediated by antizyme in mammalian tissue-culture cells.

Antizyme, a spermidine-induced protein that binds and stimulates ornithine decarboxylase degradation, is now shown also to mediate the rapid feedback inhibition of polyamine uptake into mammalian cells. Using a cell line (HZ7) transfected with truncated antizyme cDNA, and mutant ornithine decarboxylase cell lines, we demonstrate that this newly discovered action of antizyme is distinct from its role in modulating polyamine biosynthesis.