Ornithine Decarboxylase Activity In Cells Research Articles

The effect of N-acetylcysteine in combination with vitamin C on the activity of ornithine decarboxylase of lung carcinoma cells — In vitro

Ornithine decarboxylase (ODC) is a marker of lung cancer and is a key enzyme in the synthesis of polyamines, which are necessary for the promotion of the growth of malignant cells. This study assesses the dose-dependent effect of N-acetylcysteine (NAC), a chemopreventive agent, in combination with vitamin C (VC) on the activity of ODC in lung carcinoma cell line, NCI-H82. The cells were subjected to supplementation of NAC and VC both individually and in combination at different dosages for 24 h as well as 48 h. The cells were incubated with radiolabeled l-ornithine ( 14C) after the supplementation of NAC and VC individually as well as in combination. A microprocedure was carried out to estimate the activity of ODC in cells after 24 and 48 h of incubation. The activity which was found to be elevated in control cells was decreased significantly on drug supplementation in dose-dependent fashion. The content of nucleic acids also exhibited similar result and [ 3H]-thymidine incorporation was also affected by the supplementation.

Ornithine decarboxylase activity is inhibited by the polyamine precursor amino acids at the protein stability level in Caco-2 cells

High concentrations of certain amino acids are known to affect hormonal secretion, immune function, electrolyte balance or metabolic functions. However, there is a lack of knowledge regarding the molecular mechanisms responsible for these effects. We showed that, as well as spermidine transport, the activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine biosynthesis, is decreased in human colon adenocarcinoma cells, Caco-2, following a 4-h supplementation with one of the two polyamine precursor amino acids, L-arginine or L-methionine. Dose-response assays indicated that the inhibitory effect of supplemental L-methionine was stronger than that of supplemental L-arginine. However, it was transient, being even replaced by ODC induction after 8 h, whereas the inhibitory effect of L-arginine lasted for at least 8 h. Unlike L-cysteine, neither L-methionine nor L-arginine could inhibit ODC activity in a crude acellular preparation of the enzyme. The inhibition of ODC activity in cells exposed to L-methionine or L-arginine was due to a decreased abundance of ODC protein without change at the mRNA level and each of these amino acids could counteract ODC induction by a glycine supplement. Contrary to the latter, supplemental L-methionine or L-arginine induced a marked decrease in ODC half-life, concomitantly with an increase in the activity of antizyme, an ODC inhibitory protein. Thus, depending on their nature, amino acids can up- or downregulate ODC activity at the protein stability level.

Interaction of asparagine and EGF in the regulation of ornithine decarboxylase in IEC-6 cells.

Our laboratory has shown that asparagine (ASN) stimulates both ornithine decarboxylase (ODC) activity and gene expression in an intestinal epithelial cell line (IEC-6). The effect of ASN is specific, and other A- and N-system amino acids are almost as effective as ASN when added alone. In the present study, epidermal growth factor (EGF) was unable to increase ODC activity in cells maintained in a salt-glucose solution (Earle's balanced salt solution). However, the addition of ASN (10 mM) in the presence of EGF (30 ng/ml) increased the activity of ODC 0.5- to 4-fold over that stimulated by ASN alone. EGF also showed induction of ODC with glutamine and alpha-aminoisobutyric acid, but ODC induction was maximum with ASN and EGF. Thus the mechanism of the interaction between ASN and EGF is important for understanding the regulation of ODC under physiological conditions. Therefore, we examined the expression of the ODC gene and those for several protooncogenes under the same conditions. Increased expression of the genes for c-Jun and c-Fos but not for ODC occurred with EGF alone. The addition of ASN did not further increase the expression of the protooncogenes, but the combination of EGF and ASN further increased the expression of ODC over that of ASN alone. Western analysis showed no significant difference in the level of ODC protein in Earle's balanced salt solution, ASN, EGF, or EGF plus ASN. Addition of cycloheximide during ASN and ASN plus EGF treatment completely inhibited ODC activity without affecting the level of ODC protein. These results indicated that 1) the increased expression of protooncogenes in response to EGF is independent of increases in ODC activity and 2) potentiation between EGF and ASN on ODC activity may not be due to increased gene transcription but to posttranslational regulation and the requirement of ongoing protein synthesis involving a specific factor dependent on ASN.

Synergistic induction of ornithine decarboxylase by asparagine and gut peptides in intestinal crypt cells.

The objective of this study was to determine whether the amino acid asparagine stimulated the activity of ornithine decarboxylase (ODC) synergistically with epidermal growth factor (EGF) or gastrin in IEC-6 cells, a line of normal rat small intestinal crypt cells. Cells were grown in DMEM containing 5% dialyzed fetal bovine serum, and serum was deprived for 24 h before experiments. Exposure to EGF or gastrin alone increased ODC activity 4.5- to 6-fold. Asparagine alone increased the enzyme activity 10- to 13-fold in IEC-6 cells. Simultaneous addition of asparagine and EGF or gastrin, however, increased ODC activity more than 40-fold. In contrast, there was no synergistic induction of ODC activity when gastrin and EGF were added together. Increased ODC activity in cells treated with asparagine and EGF or gastrin was associated with an increase in ODC mRNA and protein levels. The rate of transcription of the ODC gene was significantly increased by exposure to EGF or gastrin. Asparagine alone had little or no effect on the rate of transcription of the ODC gene. When given together with EGF or gastrin, asparagine also had no additional effect on the transcription rate of the ODC gene. The half-life of mRNA for ODC in unstimulated IEC-6 cells was approximately 30 min and increased to more than 2 h in cells exposed to asparagine, although neither gastrin nor EGF prolonged the stability of ODC mRNA. The half-life of mRNA for ODC after combined addition of asparagine and EGF or gastrin was extended to approximately 2 h, similar to asparagine alone. Combined addition of asparagine and EGF or gastrin also significantly increased DNA synthesis compared with cells exposed to each of the three agents alone. In conclusion, 1) simultaneous addition of asparagine and EGF or gastrin increases ODC activity in a synergistic manner and 2) asparagine increases ODC mRNA levels through completely distinct mechanisms from EGF or gastrin. EGF or gastrin specifically stimulates transcription of the ODC gene, whereas asparagine affects a posttranscriptional process.

Role of polyamines in glucocorticoid effects on pancreatic acinar AR42J cell growth and differentiation.

We previously found that glucocorticoids inhibit growth and increase differentiation in rat pancreatic acinar AR42J cells. In the current study, we examined the role of polyamines in these effects. Treatment of AR42J cells with the ornithine decarboxylase (ODC) inhibitor difluoromethylornithine (DFMO) inhibited DNA synthesis. Thus polyamines are required for AR42J cell growth. However, we have previously shown that dexamethasone (Dex) increased AR42J cell ODC activity and mRNA levels. In the current study, we found that Dex treatment increased cellular putrescine levels. These increases in ODC and putrescine occurred during Dex-induced inhibition of DNA synthesis. Therefore, in AR42J cells, ODC activity and polyamine levels are not strictly growth related. To examine the requirement for glucocorticoid induction of ODC activity in glucocorticoid stimulation of differentiation, we examined the effects of DFMO on amylase gene expression and cholecystokinin binding. DFMO reduced cell amylase content while having little effect on mRNA levels in both Dex-treated and untreated cells. In contrast, DFMO had little effect on control CCK binding but inhibited the Dex-induced increase. Thus polyamines are necessary for growth and glucocorticoid-induced differentiation of AR42J cells; however, effects of glucocorticoids on AR42J cell growth and differentiation are not mediated by effects on ODC.

Effects of Tumor Promoters and Cocarcinogens on Growth and Differentiation of Cultured Human Esophageal Epithelial Cells

The acute effects of 12-O-tetradecanoylphorbol-13-acetate [(TPA) CAS: 56937-68-9], T-2 toxin (CAS: 21259-20-1), capsaicin (CAS: 404-86-4), cigarette smoke condensate (CSC), and ethanol (CAS: 3807-77-0) were examined in secondary cultured human esophageal epithelial cells in serum-free LHC-8 medium. Effects were evaluated by morphology and measurement of clonal growth rate (population doublings per day), cross-linked envelope (CLE) formation, and the enzymatic activities of ornithine decarboxylase (ODC) and plasminogen activator (PA). All compounds tested were inhibitory to clonal growth; concentrations causing 50% growth inhibition were estimated as 10 nM TPA, 6 nM T-2 toxin, 40 microM capsaicin, 8 micrograms CSC/ml, 540 mM ethanol, and 0.8 microgram CSC/ml with 220 mM ethanol. None of the compounds tested induced CLE formation, although calcium ionophore (A23187) could induce CLE in at least 60% of the cells. TPA (10 and 100 nM) decreased the ODC activity of cells, and capsaicin (100 microM) induced ODC by 220%. TPA (1-100 nM) and capsaicin (100 microM) also induced PA activity. Slight increases in ODC activity by CSC (10 micrograms/ml), CSC (1 microgram/ml) with ethanol, and T-2 toxin (1 nM) were observed, but PA activity was not affected by these compounds. The results indicated that the response of human esophageal epithelial cells to TPA is both similar to and different from that reported for human epidermal and bronchial cells in vitro. Enhancement of PA activity and decrease in ODC by TPA are found in all three human epithelial cell types. However, these changes are not associated in esophageal cells with increased CLE formation as reported in studies with the use of bronchial and epidermal epithelial cells. The results from these acute studies provide the basis for designing in vitro carcinogenesis investigations using these agents.

Stimulation of ornithine decarboxylase activity by luteinizing hormone in rat testicular interstitial cells in vitro is age dependent

The developmental pattern of ornithine decarboxylase (ODC) responsiveness to luteinizing hormone (LH) in isolated rat testicular interstitial cells in vitro was examined and correlated with testosterone production by the same cells. LH caused a 60-100% stimulation of ODC activity in cells from 60-day-old rats but produced no response in cells from 30, 37, 41, 50 and 55-day-old animals. Interstitial cells from 25-day-old rats responded with a moderate (40%) but statistically significant enhancement of ODC activity to the highest LH dose (100.0 ng/ml) only. Testosterone production by control cells was low until day 41 (0.15-0.30 ng/10(6) cells per 4 h), and then markedly increased to adult levels (2.12 +/- 0.03 10(6) cells per 4 h). LH in all concentrations (0.1 - 100.0 ng/ml) employed caused a consistent 4 to 7-fold stimulation of testosterone production in interstitial cells at all ages studied. This study shows age-dependent stimulation by LH of ODC activity in rat testicular interstitial cells in vitro and no apparent correlation with testosterone production by the same cells.

227A DILANTIN AND NEUROBLASTOMA GROWTH

Recently, a link between Fetal Dilantin Syndrome and the occurrence of neuroblastoma in the affected fetus has been suggested by four cases. Our laboratory has previously presented data on the suppression of normal fibroblast polyamine biosynthesis by Dilantin in vitro. Because polyamine production has been implicated in the pathogenesis of neoplasia, the activity of the key polyamine biosynthetic enzyme, Ornithine Decarboxylase (ODC), was investigated in human neuroblastoma cells. The levels of Ornithine Decarboxylase in human neuroblastoma cells in vitro is 5-10 times greater than in normal fibroblasts. Acute exposure of neuroblastoma cells to Dilantin showed significant enhancement of ODC activity at concentrations of 1-3 ug/ml, while higher concentrations caused suppression of ODC activity. This enhancement effect could not be demonstrated in normal fibroblasts. Dilantin exposure of growing neuroblastoma cells showed enhancement of cell number at the 1-3 ug/ml concentration range, whereas cell growth was inhibited by higher concentrations of Dilantin. The stimulation of neuroblastoma cell ODC activity and growth in vitro by low therapeutic doses of Dilantin may be important in the pathogenesis of neuroblastoma cell tumors in fetuses exposed to Dilantin in utero.

Ornithine Decarboxylase Activity in Cells Acutely and Chronically Transformed by Murine Sarcoma Virus

SummaryOrnithine decarboxyase (ODC) activity increases when cells are acutely transformed with murine sarcoma virus (MSV). Three contact inhibited or MSV transformed clones of Balb/3T3 were transformed or supertransformed by MSV or its accompanying non-transforming ‘helper’ virus (MuLV), and the relationships between ODC activity, morphology, virus production and growth rates were examined. Clones isolated from these lines were also studied. All of the virus infected lines released both MSV and MuLV. ODC activities could not be correlated with differences in growth rates. The only consistent relationship was between elevated ODC activity and acute morphological transformation, suggesting that polyamine metabolism plays a crucial role in the transformation process. With time, the elevated ODC activities returned towards baseline levels. Thus ODC activity does not appear to be a useful marker for chronic infection or transformation by type C viruses.The authors thank John Minna, Harold Stull, Herbert Oie, E...