Human myeloma cells acquire resistance to difluoromethylornithine without overproducing ornithine decarboxylase

An exposure of a human myeloma cell line to 2-difluoromethylornithine the mechanism-based inhibitor of ornithine decarboxylase (EC 4.1.1.17), resulted in a selection of tumor cells readily growing in the presence of 4 mM difluoromethylornithine, a concentration that swiftly halted the growth of the parental cells. Determination of the intracellular polyamines revealed that there were measurable amounts of putrescine and spermidine in the resistant cells. Restriction enzyme analyses of genomic DNA isolated from the resistant cells indicated that the gene dosage for ornithine decarboxylase was not increased to any appreciable extent. Similarly, the accumulation of mRNA was unaltered. The resistant myeloma cells, however, displayed arginase (EC 3.5.3.1) activity that was roughly ten times higher than that in the parental cells.     

A mouse lymphoma cell mutant whose major protein product is ornithine decarboxylase

Mutant mouse lymphoma cells that overproduce ornithine decarboxylase have been generated by selection for resistance to difluoromethylornithine, an inhibitor of the enzyme. Starting with wild type S49 mouse lymphoma cells, sensitive to growth inhibition by 10 microM difluoromethylornithine, we obtained the Z.12 line, which is approximately 100 times more resistant to that drug (McConlogue, L., and Coffino, P. (1983) J. Biol. Chem. 258, 8384-8388). Subsequent selection for still higher levels of resistance was applied to the Z.12 cells and resulted in the generation of the D4.1 line, resistant to 10 mM difluoromethylornithine. The relative synthesis of ornithine decarboxylase in wild type, Z.12, and D4.1 cells was assessed by pulse labeling these cells with [35S]methionine and analyzing the radiolabeled proteins directly, or after immunoprecipitation, on sodium dodecyl sulfate-polyacrylamide gels. As shown previously, the rate of ornithine decarboxylase synthesis is augmented in Z.12 as compared to wild type. In D4.1 cells, the rate of synthesis of ornithine decarboxylase exceeds that of any other single protein; about 15% of total protein synthesis is devoted to the enzyme. The relative amounts of translatable ornithine decarboxylase mRNA in each cell line was determined by in vitro translation of extracted RNA. These results showed that the relative rate of synthesis in each cell line is a reflection of the cell's relative content of translatable ornithine decarboxylase mRNA. Examination of the chromosomes of wild type and D4.1 cells revealed that the former are pseudodiploid and the latter tetraploid. Two of the four chromosomes 14 in D4.1 contain large homogeneously staining regions, a finding consistent with the presence of regions of gene amplification.     

Inhibition of phorbol ester-caused synthesis of mouse epidermal ornithine decarboxylase by retinoic acid

The mechanisms by which topically applied retinoic acid to mouse skin inhibits tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced epidermal ornithine decarboxylase activity were analyzed. Retinoic acid inhibition of the induction of epidermal ornithine decarboxylic activity was not the result of nonspecific cytotoxicity, production of a soluble inhibitor of ornithine decarboxylase, or direct effect on its activity. In addition, inhibition of TPA-caused increased ornithine decarboxylase activity does not appear to be due to enhanced degradation and/or post-translational modification of ornithine decarboxylase by transglutaminase-mediated putrescine incorporation. We found that retinoic acid inhibits the synthesis of ornithine decarboxylase caused by TPA. Application of 10 nmol TPA to mouse skin led to a dramatic induction of epidermal ornithine decarboxylase activity which was paralled by increased [3H]difluoromethylornithine binding and an increased incorporation of [35S]methionine into the enzyme. Application of 17 nmol retinoic acid 1 h prior to application of 10 nmol TPA to skin resulted in inhibition of the induction of activity which accompanied inhibition of [3H]difluoromethylornithine binding and [35S]methionine incorporation into ornithine decarboxylase protein as determined by the tube-gel electrophoresis of the enzyme immunoprecipitated with monoclonal antibodies to it. Inhibition of ornithine decarboxylase synthesis was not the result of the inhibitory effect of retinoic acid on general protein synthesis. The results indicate that retinoic acid possibly inhibits TPA-caused synthesis of ornithine decarboxylase protein selectively.     

A human neuroblastoma cell line with an altered ornithine decarboxylase

A human neuroblastoma cell line (Paju) was resistant to 10 mM difluoromethylornithine, a concentration at which the growth of all mammalian cells normally stops. Ornithine decarboxylase from Paju was very resistant to inhibition by difluoromethylornithine in vitro (Ki = 10 microM compared to 0.5 microM for mouse kidney ornithine decarboxylase). After purification, apparently homogeneous Paju ornithine decarboxylase was inactivated with [3H]difluoromethylornithine and analyzed by polyacrylamide gel electrophoresis. Under denaturing conditions it was found to have an altered molecular structure, i.e. two nonidentical subunits of Mr = 55,000 and 60,000. Another unusual feature of Paju ornithine decarboxylase was its long half-life in vivo (T 1/2 = 8 h compared with 36 min in human HL-60 promyelocytic leukemia cells). The disappearance of immunoreactive protein was only slightly slower than the loss of catalytic activity. The long half-life of Paju ornithine decarboxylase was not shared by adenosylmethionine decarboxylase. Despite the altered structure of Paju ornithine decarboxylase, it was recognized by a specific antisera raised in rabbit against mouse kidney ornithine decarboxylase. The Paju karyotype did not contain double minute chromosomes or any large homogeneously staining region such as that seen in a mouse lymphoma cell mutant that is resistant to difluoromethylornithine and overproduces ornithine decarboxylase (McConlogue, L., and Coffino, P. (1983) J. Biol. Chem. 258, 12083-12086).     

A human neuroblastoma cell line with a stable ornithine decarboxylase in vivo and in vitro

A human neuroblastoma cell line (Paju) grew in 10 mM difluoromethyl-ornithine, which at this concentration normally stops the growth of all mammalian cells. Ornithine decarboxylase from Paju was resistant to inhibition in vitro by difluoromethylornithine, and required 10 microM of the compound for 50% inhibition, whereas ornithine decarboxylase from SH-SY5Y cells (another human neuroblastoma) and from rat liver needed only 0.5 microM difluoromethylornithine. Paju ornithine decarboxylase also exhibited a long half-life (over eight hours) in vivo. The half-life of immunoreactive protein was significantly longer than that of the activity. The long half-life of ornithine decarboxylase in Paju cells leads to its accumulation to a specific activity of 2000 nmol/mg of protein per 30 min during rapid growth (the corresponding activity in SH-SY5Y cells was about 2.5). When partially purified ornithine decarboxylase from Paju cells was incubated with rat liver microsomes it was inactivated with a half-life of 75 min. This inactivation was accompanied by a fall in the amount of immunoreactive protein. In the same inactivating system partially purified SH-SY5Y ornithine decarboxylase had a half-life of 38 min and its half-life in vivo was 50 min. The corresponding values for rat liver ornithine decarboxylase were 45 min and 40 min, respectively. Rat liver microsomes also inactivated rat liver adenosylmethionine decarboxylase. These results suggest that Paju ornithine decarboxylase has an altered molecular conformation, rendering it resistant to (i) difluoromethylornithine and (ii) proteolytic degradation both in vivo and in vitro.     

Translational regulation of mammalian ornithine decarboxylase by polyamines

Ornithine decarboxylase, which catalyses the formation of putrescine, is the first and rate-limiting enzyme in the biosynthesis of polyamines in mammalian cells. The enzyme is highly regulated, as indicated by rapid changes in its mRNA and protein during cell growth. Here we report that ornithine decarboxylase is regulated at the translational level by polyamines in difluoromethylornithine-resistant mouse myeloma cells that overproduce the enzyme due to amplification of an ornithine decarboxylase gene. When such cells are exposed to putrescine or other polyamines, there is a rapid and specific decrease in the rate of synthesis of ornithine decarboxylase, assayed by pulse-labeling. Neither the cellular content of ornithine decarboxylase mRNA nor the half-life of ornithine decarboxylase protein is affected. Our results indicate that polyamines negatively regulate the translation of ornithine decarboxylase mRNA, thereby controlling their own synthesis.     

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

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. All 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.     

Gene expression of ornithine decarboxylase in L1210 leukaemia cells exposed to DL-2-difluoromethylornithine in the presence of cadaverine.

Cultured mouse L1210 leukaemia cells treated with DL-2-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase (EC 4.1.1.17), in the presence of micromolar concentrations of cadaverine, started to overproduce ornithine decarboxylase after an exposure of several weeks. The more than 60-fold excess of the enzyme protein in the drug-treated cells apparently resulted from a strikingly enhanced accumulation of mRNA for the enzyme associated with only a modest (about 2-fold) gene amplification.     

Amplification of ornithine decarboxylase gene in response to polyamine deprivation in Chinese hamster ovary cells

We have isolated from an arginase-deficient polyamine-dependent Chinese hamster ovary cell line a new mutant strain that has greatly increased ornithine decarboxylase activity. This enables the cells, in the absence of ornithine, to decarboxylate lysine into cadaverine (diaminopentane) that is further converted into N-(3-aminopropyl)cadaverine and N,N'-bis(3-aminopropyl)cadaverine. These unusual polyamines can support the growth of the cells without added polyamines derived from ornithine. Immunoreactive ornithine decarboxylase-like protein was clearly increased in the mutant cells but could not solely account for the greatly increased enzyme activity. Southern blot analysis of DNA hybridized to a plasmid carrying ornithine decarboxylase-cDNA revealed at least a 32-fold amplification of the ornithine decarboxylase gene. Ornithine decarboxylase-mRNA concentration was also highly increased in the cells. The half-life of the enzyme and the Km for ornithine were not altered from those of the parental cell line.     

Human lung tumor sensitivity to difluoromethylornithine as related to ornithine decarboxylase messenger RNA levels

The differential response to polyamine depletion has been studied in two types of human lung tumor cells. Small cell lung carcinoma cells die following polyamine depletion by difluoromethylornithine treatment while non-small cell lines demonstrate a typical cytostatic response. We now report that a small cell line, NCI H82, has a lower apparent capacity for polyamine biosynthesis than does a representative non-small cell, NCI H157. In subconfluent cultures, the ornithine decarboxylase activity is 25 times lower in the small cell than the non-small cell and by comparison, the polyamines in the small cell line are markedly reduced. Most significantly, levels of mRNA coding for ornithine decarboxylase are approximately 100-fold lower in the small cell than the non-small cell line, and this difference does not appear to be a result of gene rearrangement. These results suggest that differential sensitivity to polyamine depletion is related to different steady-state levels of ornithine decarboxylase mRNA.     

Polyamines in mycoplasmas and in mycoplasma-infected tumour cells.

Three out of four different mycoplasma strains analysed for the polyamine contents contained relatively high concentrations of putrescine, cadaverine, spermidine and spermine. In addition to ornithine decarboxylase (EC 4.1.1.17) activity, the mycoplasmas also exhibited comparable or higher lysine decarboxylase (EC 4.1.1.18) activity fully resistant to the action of 2-difluoromethylornithine, an irreversible inhibitor of eukaryotic ornithine decarboxylase. 2-Difluoromethylornithine did not modify the polyamine pattern of actively growing mycoplasmas. Ehrlich ascites carcinoma cells and L1210 mouse leukemia cells infected with any of the four mycoplasma strains contained, in addition to putrescine, spermidine and spermine, and also easily measurable concentrations of cadaverine; the latter diamine was absent in uninfected cultures. When the infected cells were exposed to difluoromethylornithine, the accumulation of cadaverine was markedly enhanced. The modification of cellular polyamine pattern by mycoplasmas, especially in the presence of inhibitors of eukaryotic ornithine decarboxylase, could conceivably be used as an indicator of mycoplasma infection in cultured animal cells.     

Insensitivity of cardiac phosphofructokinase to adrenergic activation in Zucker rats. A post-receptor defect.

Glyoxal bis(guanylhydrazone), the parent compound of methylglyoxal bis(guanylhydrazone), was synthesized and tested for its ability to inhibit the biosynthesis of polyamines. It was found to be a powerful competitive inhibitor of adenosylmethionine decarboxylase (EC 4.1.1.50), yet the lack of the methyl group at the glyoxal portion increased the apparent Ki value for the enzyme by about 30-fold in comparison with methylglyoxal bis(guanylhydrazone). Glyoxal bis(guanylhydrazone) inhibited diamine oxidase (EC 1.4.3.6) activity as effectively as did methylglyoxal bis(guanylhydrazone). The cellular accumulation curves of glyoxal bis(guanylhydrazone) in L1210 cells were practically superimposable with those of methylglyoxal bis(guanylhydrazone), and the uptake of both compounds was distinctly stimulated by a prior treatment with 2-difluoromethylornithine. The drug decreased the concentration of spermidine in a dose-dependent manner and, in contrast with methylglyoxal bis(guanylhydrazone), without a concomitant accumulation of putrescine. The fact that putrescine concentrations were decreased in cells exposed to glyoxal bis(guanylhydrazone) was, at least in part, attributable to an inhibition of ornithine decarboxylase (EC 4.1.1.17) activity in cells treated with the compound. Under these experimental conditions equivalent concentrations of methylglyoxal bis(guanylhydrazone) [1,1'-[(methylethanediylidine)dinitrilo]diguanidine] elicited large increases in the enzyme activity. When combined with difluoromethylornithine, glyoxal bis(guanylhydrazone) potentiated the growth-inhibitory effect of that drug. Taking into consideration the proven anti-leukaemic activity of glyoxal bis(guanylhydrazone), its effectiveness to inhibit spermidine biosynthesis (without raising the concentration of putrescine) as well as its suitability for combined use with inhibitors of ornithine decarboxylase, this drug is apparently worthy of further testing in tumour-bearing animals, especially in combination with difluoromethylornithine or related inhibitors of ornithine decarboxylase.     

Temperature-dependent and transformation-associated changes in polyamine metabolism in normal and Rous sarcoma virus-infected chick embryo fibroblasts

Tertiary cultures of chick embryo fibroblasts infected and transformed by the wild-type Rous sarcoma virus, when actively growing at 35 degrees C, had higher putrescine levels than the respective uninfected cells. Transformed cells also had much higher specific activity of ornithine decarboxylase (EC 4.1.1.17) than the normal fibroblasts. At 41 degrees C the difference in putrescine levels between the normal and the transformed cells was less marked, and both cell types showed a relative accumulation of spermine. Cultures infected with the NY68 mutant virus, which is temperature-sensitive for transformation, showed at 41 degrees C normal cell morphology and intermediate polyamine patterns, while at 35 degrees C a transformed phenotype was found in both aspects. In shift-down experiments a change towards the permissive temperature pattern of polyamine metabolism was evident within 2-3 h. Difluoromethylornithine, a specific and irreversible inhibitor of ornithine decarboxylase efficiently reduced the enzyme activity as well as the levels of both putrescine and spermidine in all culture types and temperatures. Incubation of Rous sarcoma virus-transformed cells with 3 mM difluoromethylornithine for 36 h did not affect the maintenance of the transformed state. Likewise, when NY68-infected cultures were exposed to difluoromethylornithine at 41 degrees C for 12 h and then shifted down to 35 degrees C, the appearance of the transformed morphology took place concomitantly with that of the control cultures without respective changes in the polyamine levels. This suggests that the transformation-associated pattern of polyamines in chick embryo fibroblasts is not a prerequisite for morphological transformation of these cells.     

Regulation of ornithine decarboxylase activity in rat ovarian cells in vitro.

Incubation of rat ovarian cell suspension with human choriogonadotropin (hCG) caused a marked enhancement of ornithine decarboxylase (EC 4.1.1.17) activity after a lag period of several hours. Even though ovarian ornithine decarboxylase could be induced in minimum essential medium by the hormone alone, supplementation of the medium with various sera greatly enhanced the stimulation of the enzyme activity. All the sera tested (human, fetal calf and horse) were able to stimulate ornithine decarboxylase activity even in the absence of hCG. Maximum stimulation of the enzyme activity by hCG and/or serum occurred in ovarian cell suspensions prepared from 30 to 33-day-old rats. There was a close correlation between the stimulation of ornithine decarboxylase activity and the accumulation fo cyclic AMP in response to the administration of the hormone (in the presence or absence of serum). However, while various sera alone markedly enhanced ovarian ornithine decarboxylase activity in vitro they, if anything, only marginally stimulated the accumulation of cyclic AMP and the secretion of progesterone in ovarian cells in the absence of gonadotropin. A similar dissociation of the stimulation of ornithine decarboxylase activity from the production of cyclic AMP and progesterone was likewise found when the ovarian cells were incubated in an enriched medium (M199) supplemented with albumin and lactalbumin hydrolysate in the absence of the hormone. Under these culture conditions ornithine decarboxylase activity was strikingly enhanced, greatly exceeding the stimulation obtained with various sera, while the accumulation of cyclic AMP and the secretion of progesterone remained virtually unchanged. Specific inhibition (up to 90%) of gonadotropin-induced ornithine decarboxylase activity by difluoromethyl ornithine or 1,3-diamino-2-propanol had little effect on the ability of the ovarian cells to respond to the hormone with increasing production of cyclic AMP and progesterone. While showing that rat ovarian ornithine decarboxylase can be induced in vitro by choriogonadotropin or various sera, our results indicate that the activation of the enzyme involves at least two different mechanisms: (i) One (in response to gonadotropin) involving a prior stimulation of cyclic AMP production, and (ii) another (in response to serum) that is not associated with increases in the accumulation of the cyclic nucleotide.     

Ornithine decarboxylase inhibitors increase the cellular content of the enzyme: implications for translational regulation

Ehrlich ascites tumor cells grown in the presence of inhibitors of ornithine decarboxylase (EC 4.1.1.17) exhibited an elevated content of this enzyme. The increase could not solely be explained by a decrease in the degradation rate of the enzyme. Instead a stimulation of enzyme synthesis, probably mediated via the polyamine-depleting properties of the inhibitors, is suggested. The enhancement of cellular ornithine decarboxylase content was not accompanied by any significant changes in the amount of ornithine decarboxylase mRNA, indicating a regulation at the level of translation.