Multiple mechanisms are responsible for altered expression of ornithine decarboxylase in overproducing variant cells.

Abstract

We selected and characterized a series of mouse S49 cell variants that overproduce ornithine decarboxylase (ODC). Previously, we described variants that have an amplified ODC gene and produce about 500-fold more ODC than the wild-type cells of origin (L. McConlogue and P. Coffino, J. Biol. Chem. 258:12083-12086, 1983). We examined a series of independent variants that overproduce ODC to a lesser degree and found that a number of mechanisms other than gene amplification are responsible for the increased ODC activity. Variants were selected for resistance to 0.1 mM difluoromethylornithine, an inhibitor of ODC, by either a single or a multistep process. AU showed increased ODC activity and increased ODC mRNA steady-state levels. The half-life of the enzyme was not increased in any of the variants. In one class of variant the increase of ODC mRNA was sufficient to account for ODC overproduction. In a second class, the rate of synthesis of ODC polypeptide per ODC mRNA was at least four-to eightfold higher than that in wild-type cells. Therefore, these variants were altered in the translatability of ODC mRNA. Southern analysis showed that gene amplification does not account for the increased ODC mRNA levels in any of the variants. In both variant and wild-type cells, ODC activity was responsive to changes in polyamine pools; activity was reduced following augmentation of pool size. This change in activity was associated with modification of the rate of synthesis and degradation of ODC but no change in the level of ODC mRNA.In animal cells ornithine decarboxylase (ODC) is an essential enzyme, the activity of which is regulated by a wide variety of hormonal, developmental, and cell growth-related stimuli (35). It is the initial and rate-limiting enzyme in the synthesis of polyamines; its product, putrescine, is the precursor of the polyamines spermidine and spermine. In general, cells that are proliferating exhibit more activity than their nonproliferating counterparts. There is evidence for multiple forms of regulation of ODC activity, including mRNA steady-state level, polypeptide synthesis, and polypeptide turnover.In S49 mouse T-lymphoma cells cyclic AMP modulates cell growth and ODC activity (7,10,22). ODC activity is also changed by treatments that modulate cellular polyamine levels, such as exposure to putrescine, the direct product of ODC. We generated ODC-overproducing variants of S49 cells by selecting for cells resistant to the ODC inhibitor difluoromethylornithine (DFMO) (19,20). These variants allowed us to identify the ODC polypeptide and to clone the ODC cDNA (21) DFMO. Clone Z.12 was selected from an unmutagenized wild-type (clone 24.3.2) S49 cell population by serially increasing the concentration of DFMO as described previously (20). The D2 clones were selected from a mutagenized population in a single step as follows. Wild-type cells were mutagenized with 2.5 ,g of N-methyl-N'-nitro-N-nitrosoguanidine per ml for 4.5 h. This treatment resulted in 30% cell survival and induction of resistance to 6-thio...

Regulation of mouse ornithine decarboxylase activity by cell growth, serum and tetradecanoyl phorbol acetate is governed primarily by sequences within the coding region of the gene

“…Peak levels of enzymatic activity were similar in both cell types, but initial levels were higher in the transfectants expressing stable that the sequences flanking the coding region participate in regulation of ODC activity when cells are treated with serum or TPA or undergo cell-density-dependent changes in growth. We and other workers have shown that post-transcriptional and post-translational mechanisms as well as transcriptional changes determine ODC steady-state enzymatic activity [3,4,7,31,32]. (There is good evidence that indicates that enzymatic activity is exclusively determined by the number of ODC protein molecules [6,33]).…”

“…The ODC assay quantitates the release of ['4C]CO2 during ODC-catalyzed conversion of ornithine to putrescine and CO2 [20]. The band marked by an asterisk is of a size consistent with that for a hamster ODC fragment as predicted by sequence comparison [46,47].…”

“…Schimke [29] noted that a polypeptide with a short half-life could rapidly and transiently rise in abundance simply as the result of an increase in the overall rate of protein synthesis. The turnover of ODC is among the most rapid known for an enzyme, with a half-life of less than one hour [30]. Mitogenic stimulation increases the rate of protein synthesis.…”

“…Reactions were incubated at 37°C for 2 hours and terminated by heating at 85°C for 5 minutes. ['4C]CO2 was collected and counted as described [20].…”

“…CHO wild type (clone 10001, from Michael Gottesman, National Cancer Institute) and stably transfected cells were grown in the same medium but without putrescine. Quiescent cells were obtained by growing cells to confluence and then incubating them for 30 hours in fresh DMEM supplemented with 0.5% FCS. Stimulation of quiescent or confluent cells was with 30% FCS, 1.6 /AM TPA or 100 ,Ag/ml cycloheximide.…”