Irreversible Inhibitor Of Ornithine Decarboxylase Research Articles

Stress-induced dynamic changes in mouse brain polyamines. Role in behavioral reactivity

Recent findings indicate that rapid and transient changes in polyamine metabolism, termed the polyamine-stress-response, may occur repeatedly in the brain after chronic intermittent stress. Here, we sought to examine the effects of chronic intermittent restraint stress, or of daily intraperitoneal dexamethasone injections on polyamine concentrations in the hippocampus of adult male C57BL/6 mice. Additionally, we studied the effects of α-difluoromethylornithine, an irreversible ornithine decarboxylase inhibitor, on stress-induced changes in polyamines and on behavioral reactivity to novelty stress measured in an open-field arena. As previously observed, following a single stress episode putrescine concentration increased transiently, but the polyamines spermidine and spermine remained unchanged. Following chronic restraint stress, putrescine concentration was increased after each daily stress episode with the largest increase observed after the 4th episode, while spermidine was increased just after the 2nd and 4th episodes and spermine only after the 4th daily episode. In contrast, all polyamine concentrations were increased after 10 injections of either dexamethasone or vehicle (0.9% NaCl). A 14-day course of α-difluoromethylornithine treatment resulted in a complete putrescine depletion and over 50% reduction in polyamines, and led to changes in open field activity indicative of altered emotional behavior. Conclusions: (a) while putrescine concentration increases in the hippocampus after each restraint stress episode, spermidine and spermine undergo a delayed but transient increase; (b) in contrast, chronic dexamethasone treatment may lead to a permanent increase in the concentrations of all polyamines and; (c) chronic α-difluoromethylornithine treatment reduces brain polyamine concentrations and modulates emotional reactivity to novelty stress. The study indicates that the brain polyamine-stress-response is a dynamic process that varies with the type, intensity and length of stressful stimuli, and implicates this response as an adaptive mechanism in the reaction to stressors.

Effects of Difluoromethylornithine on Growth Inhibition and Apoptosis in Human Cervical Epithelial and Cancerous Cell Lines

Difluoromethylornithine(DFMO), an irreversible inhibitor of ornithine decarboxylase and an angiogenesis inhibitor, has been used in phase I cervical intraepithelial neoplasia (CIN) trials, producing a 50% regression of CIN 3 lesions. DFMO is currently in phase II trials. In the experiments reported here, DFMO's growth inhibition and apoptosis induction were explored in an in vitro model to elucidate mechanisms of action. Four immortalized cervical epithelial cell lines, serving as in vitro models of precancerous CIN lesions, and nine cervical carcinoma cell lines were studied. DFMO's growth inhibitory effect was tested in monolayer culture and in semisolid medium, and concentrations required for a 50% growth inhibition (IC(50)) with a 5-day treatment were determined. Apoptosis induction was analyzed using the terminal deoxynucleotidyl transferase assay of DNA fragmentation. DFMO inhibited growth of immortalized cervical epithelial cell lines and cervical cancer cell lines in monolayer culture and in semisolid medium. The immortalized cervical epithelial cell lines were more sensitive than the cervical cancer cell lines to DFMO's growth inhibitory effect. Concentrations required for 50% growth inhibition after a 5-day treatment ranged from 100 microM to >5 mM for cervical carcinoma cell lines and from 100 microM to 1 mM for immortalized cervical epithelial cell lines. DFMO induced apoptosis in precancerous and cancerous cell lines at a concentration of 5 mM, regardless of the cells' human papillomavirus status. DFMO inhibits the growth of cervical precancerous and cancerous cells in vitro in a dose-dependent and time-dependent manner, partially through inducing apoptosis.

Quantitation of polyamines in hypothalamus and pituitary of female and male developing rats

The quantitation of four polyamines in hypothalamus and pituitary is studied in male and female developing rats using an improved high-performance liquid chromatography method. In the hypothalamus, putrescine (PUT) reaches the highest concentration (nmol/mg protein) on day 6. It shows the lowest value in comparison with any other polyamine. Spermidine (SPD) is high during the first postnatal days. Spermine (SPM) fluctuates, and agmatine (AGM) is highest during the first week. SPD, SPM and AGM are lower in females. In the pituitary, PUT, SPD and AGM are high during the first week. SPM remains constant and it is higher in males. AGM is higher in males only on day 1. PUT shows the lowest concentration of all. Concentrations of PUT, SPD and SPM are higher in the pituitary; AGM is higher in the hypothalamus. α-Difluoromethylornithine (a specific and irreversible inhibitor of ornithine decarboxylase) decreases PUT and SPD, increased SPM and AGM remain unchanged in the hypothalamus and pituitary. Thus, each polyamine has its own pattern in hypothalamus and in pituitary during development in males and females; these changes could be related to the hypothalamic control of pituitary secretion of hormones related to reproduction in mammals.

Inhibition of placental ornithine decarboxylase by DL-α-difluoro-methyl ornithine causes fetal growth restriction in rat

The roles of polyamines in intrauterine growth restriction (IUGR) is studied. The DL-α-difluoromethyl ornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC) which is a rate limiting enzyme of polyamine synthesis was administrated to pregnant rats so that we obtained rat fetuses with IUGR. The changes of maternal nutrition, damage of the placenta, and the direct effect of DFMO on the fetus were examined in this IUGR model. Administration of DFMO did not induced changes of maternal nutrition except for triglyceride and the fetal metabolic state. But the placental weight, ODC activity, and DNA in the placenta were decreased significantly. The ODC activity in the total placenta decreased to less than 10 % of that of the control. Depression of ODC activity in the placenta may be the major cause of IUGR induced by DFMO administration, and polyamines play important roles to carry pregnancy.

Activities of arginase I and II are limiting for endothelial cell proliferation.

Polyamines are essential for cell proliferation; therefore, we hypothesized that arginase I or arginase II activities, via production of ornithine for polyamine synthesis, may be limiting for proliferation of endothelial cells (EC). Bovine coronary venular EC stably transfected with a lacZ gene (lacZ-EC, control), rat arginase I cDNA (AI-EC), or mouse arginase II cDNA (AII-EC) were utilized to test this hypothesis. Cell-proliferation assays showed that EC proliferation was markedly increased in AI-EC and AII-EC compared with lacZ-EC. Expression of proliferating cell nuclear antigen was also enhanced in AI-EC and AII-EC. DL-alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, was used to establish that increased polyamine synthesis was involved in mediating the enhanced growth of AI-EC and AII-EC. Addition of 5 mM DFMO to the culture medium completely abolished the differences in cellular putrescine concentrations and reduced the differences in spermidine concentrations among AI-EC, AII-EC, and lacZ-EC. The DFMO treatment also prevented an increase in AI-EC and AII-EC proliferation compared with lacZ-EC. Addition of 10 and 50 microM putrescine dose-dependently increased AI-EC, AII-EC, and lacZ-EC growth to the same extent. These results demonstrate that either arginase isoform can potentially play a role in modulating EC proliferation by regulating polyamine synthesis.

Neonatal polyamine depletion by α-difluoromethylornithine: effects on adenylyl cyclase cell signaling are separable from effects on brain region growth

Ornithine decarboxylase (ODC) and the polyamines play an essential role in brain cell replication and differentiation. We administered α-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, to neonatal rats on postnatal days 5–12, during the mitotic peak of the cerebellum, a treatment regimen that leads to selective growth inhibition and dysmorphology. In adulthood, cell signaling responses mediated through the adenylyl cyclase pathway were evaluated in order to determine if synaptic dysfunction extends to regions that appear to be otherwise unaffected by DFMO. Total adenylyl cyclase catalytic activity, evaluated with the direct enzymatic stimulant, Mn 2+, was significantly elevated in male rats both in the cerebellum and in brain regions showing no growth retardation (cerebral cortex, brainstem); there were no significant effects in females. In contrast, signaling mediated through the G proteins that couple neurotransmitter receptors to adenylyl cyclase showed a deficit in the DFMO group, as evaluated with the response to fluoride; in males, there was no corresponding increase in activity as would have been expected solely from the enhancement of adenylyl cyclase, and in females, there was actually a significant decrease in the response to fluoride. Again, the deficits were not restricted to the cerebellum. Stimulation of adenylyl cyclase by isoproterenol, a β-adrenergic receptor agonist that acts through G s, likewise displayed deficits in both males and females, and without distinction by brain region. These results indicate that the ODC/polyamine pathway plays a role in the development of cell signaling, and hence in neurotransmission, above and beyond its role in cell replication and differentiation. Given the fact that numerous drugs and environmental contaminants have been shown to alter ODC and the polyamines in the developing brain, our findings suggest that changes in brain region growth or structure are inadequate to predict the targeting of specific neurotransmitter or signaling pathways, and that gender-selective functional defects may be present despite the absence of morphological differences.

Sensitivity of Trypanosomatid Protozoa to DFMO and Metabolic Turnover of Ornithine Decarboxylase

α-Difluoromethylornithine (DFMO), the specific and irreversible inhibitor of ornithine decarboxylase (ODC), was able to induce the arrest of proliferation in Leishmania mexicana and ODC-transformed Trypanosoma cruzi cultures grown in a semi-defined medium essentially free of polyamines. Conversely, Crithidia fasciculata and Phytomonas 274 were not affected by the inhibitor. The drug-resistance of Crithidia and Phytomonas was neither caused by an impairment of DFMO uptake nor by a decrease of the enzyme affinity for the inhibitor. We were also able to rule out the possibility of ODC overexpression in the drug-tolerant parasites. The measurements of ODC metabolic turnover indicated that the enzymes from Crithidia and Phytomonas have a short half-life of 20–40 min, while ODC from Leishmania and transgenic Trypanosoma cruzi are rather stable with a half-life longer than 6 hours. Analyses of polyamine internal pools under different growth conditions have shown that DFMO was able to markedly decrease the levels of putrescine and spermidine in all parasites, but the depletion of spermidine was higher in trypanosomatids containing an ODC with slow turnover. Our results suggest that in these parasites cultivated in the presence of the drug, spermidine might decrease below critical levels needed to maintain trypanothione concentrations or other conditions essential for normal proliferation.

α-Difluoromethylornithine induces apoptosis as well as anti-angiogenesis in the inhibition of tumor growth and metastasis in a human gastric cancer model

Polyamines are essential in various biological systems such as cellular proliferation including tumor growth, differentiation and neoplastic transformation including carcinogenesis. alpha-Difluoromethylornithine (DFMO) is a specific irreversible inhibitor of ornithine decarboxylase, a key enzyme in polyamine biosynthesis, and has been used for clinical chemotherapy and chemoprevention trials against several tumors with various effects. The cellular mechanisms of DFMO action are unclear. Because our hypothesis with regard to polyamine-directed chemoprevention includes anti-angiogenesis and apoptosis as essential parts of the cellular mechanism of action of DFMO, we examined these effects in our human gastric cancer model. In our initial experiments, DFMO inhibited the growth of both human umbilical vein endothelial cells (HUVEC), angio-endothelial cells in vitro, and KKLS, a gastric cancer cell line, in culture, and also the growth of KKLS cells transplanted into nude mice. DFMO also inhibited liver metastasis of KKLS orthotransplanted in the stomach of nude mice. The vessel density of DFMO-treated tumors was significantly lower than that of non-treated tumors. The apoptotic index was significantly greater in DFMO-treated tumors than in non-treated tumors. These results suggest that anti-angiogenesis and apoptosis play significant roles in the DFMO inhibition of the growth and metastasis in this human gastric cancer model and provide evidence that DFMO induces apoptosis.

Alpha-Difluoromethylornithine blocks thymocyte apoptosis via a reduction in tyrosine phosphorylation.

The effect of alpha-difluoromethylornithine on cell apoptosis was investigated. Freshly isolated mouse thymocytes were cultured in the medium alone or with dexamethasone, and apoptotic cell death was monitored after 6 h. A correlation was seen between cell apoptosis and a reduction in the polyamine levels of thymocytes. Addition of exogenous polyamines decreased the levels of apoptosis induced spontaneously in the culture medium or by dexamethasone. However, addition of alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, to the cultures did not enhance apoptosis but rather caused inhibition of thymocyte apoptosis. Analysis of the mechanism of alpha-difluoromethylornithine-mediated inhibition of apoptosis indicated that alpha-difluoromethylornithine treatment blocked protein tyrosine phosphorylation, which was elevated drastically during the first hour of thymocyte cultivation. Treatment with the phosphotyrosine phosphatase inhibitor phenylarsine oxide reversed this inhibitory effect of alpha-difluoromethylornithine on apoptotic cell death. Our results provide an alternative mechanism for alpha-difluoromethylornithine showing the inhibition of apoptosis via reduction of protein tyrosine phosphorylation.

Difluoromethylornithine in combination with tamoxifen in female rats: 13-week oral toxicity study.

Cancer chemoprevention is the use of pharmacologic or natural agents to inhibit the development of cancer. Difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase, the rate-limiting enzyme in the biosynthesis of polyamines. DFMO has demonstrated chemopreventive efficacy in animal models of tumorigenesis. Tamoxifen (TAM) is currently used for treatment of estrogen receptor-positive breast carcinoma and has demonstrated efficacy in chemoprevention of breast cancer in women at high risk for the disease. The administration of tamoxifen with DFMO is being considered for development by the National Cancer Institute as a potential drug regimen for the chemoprevention of breast carcinoma. The toxicity of DFMO in combination with TAM was evaluated in female rats following 13 weeks of daily administration by gavage. Dose groups were vehicle control, DFMO (1000 mg/kg per day), low TAM (0.25 mg/kg per day), high TAM (2.5 mg/kg per day), low combination (1000 + 0.25) and high combination (1000 + 2.5). No mortalities occurred in the study. Clinical signs of toxicity were limited to dermal lesions consisting of scab formation and abrasions produced by DFMO. Administration of either DFMO or TAM resulted in decreased body weight gains, with coadministration having an additive effect. Serum albumin, total protein, cholesterol and triglyceride levels were decreased in all drug-treated dose groups, although histologic evidence of liver lesions were not seen. TAM resulted in increased numbers of red blood cells, whereas DFMO produced a slightly anemic response. DFMO produced lesions in the small intestine consisting of necrosis of crypt epithelium and crypt microabscess, which were enhanced by TAM coadministration. Administration of TAM resulted in histologic changes in the ovaries, fallopian tube, vagina, cervix and uterus, indicating that inhibition of ovulation and reproductive cycle arrest in the proestrus stage had occurred. Coadministration with DFMO did not affect the changes to the reproductive system induced by TAM. Coadministration of DFMO with tamoxifen did not result in toxicity unique to the combination drug regimen, but rather toxicity resulted from administration of each drug. Under the conditions of the study, the overall toxicity produced by dual administration of DFMO with tamoxifen was additive with respect to the toxicity associated with each agent alone.

Tumor promoter-induced ornithine decarboxylase gene expression occurs independently of AP-1 activation

Activator protein 1 (AP-1) transactivation and ornithine decarboxylase (ODC) activity have been established as essential downstream effectors of mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). Previous studies have shown that inhibition of either AP-1 transactivation or ODC activity suppressed tumor promoter-induced transformation. By utilizing the JB6 mouse epidermal cell system, the present study determined whether TPA-induced ODC gene expression and activity is independent of AP-1 transactivation. In three independent JB6 (P+) clones, stably expressing dominant negative c-jun, TPA-induced ODC gene expression and activity were similar compared to JB6 P+ cells expressing vector-control alone, while AP-1-dependent transcription was inhibited. Transformation-insensitive JB6 (P-) cells, which lack TPA-inducible c-jun expression, also exhibited similar induction of ODC activity by TPA. alpha-Difluoromethylornithine, an irreversible inhibitor of ODC, attenuated, at an equivalent IC50, both TPA-induced ODC activity and anchorage-independent growth of JB6 P+ cells, despite no inhibition of AP-1 transactivation. Taken together, the results presented indicate that TPA-induced ODC gene expression and activity are independent of AP-1 transactivation. Because inhibition of either AP-1 or ODC precludes TPA-induced transformation, and because ODC is independent of AP-1, we propose that there are at least two pathways to transformation. Each pathway is required but not sufficient for transformation.

D,L-α-DIFLUOROMETHYLORNITHINE, AN IRREVERSIBLE INHIBITOR OF ORNITHINE DECARBOXYLASE, INDUCES DIFFERENTIATION IN MEL CELLS

In the present study, the effect of D,L-α-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), on Friend's murine erythroleukemia (MEL) cell differentiation is investigated. DFMO was able to induce differentiation of MEL cells in culture as determined by haemoglobin (Hb) content and percentage of cells synthesizing Hb detected by benzidine staining. DFMO at a concentration of 2m m resulted in about 70% benzidine-positive cells on the fifth day. There was a time-dependent increase in the percentage of benzidine-positive cells starting from day three. However, only a 24h presence of DFMO in the medium was required to induce differentiation suggesting that DFMO switches on a pathway during this period leading to terminal differentiation of MEL cells. DFMO induced differentiation of MEL cells was sensitive to dexamethasone and 5-bromo-2′-deoxyuridine.

Nitric oxide mediates either proliferation or cell death in cardiomyocytes. Involvement of polyamines.

Nitric oxide (NO) is a molecule involved in several signal transduction pathways leading either to proliferation or to cell death. Induction of ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, represents an early event preceding DNA synthesis. In some cell types increased ODC activity seems to be involved in cytotoxic response. We investigated the role of NO and ODC induction on the events linked to cell proliferation or to cell death in cultured chick embryo cardiomyocytes. Exposure of cardiomyocytes to tumor necrosis factor (TNF) and lipopolysaccharide (LPS) caused NO synthase (NOS) and ODC induction as well as increased incorporation of [3H]-thymidine. This last effect was blocked by a NOS inhibitor and was strongly reduced by difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. Sodium nitroprusside (SNP), an exogenous NO donor, inhibited the increases of NOS and ODC activities and abolished the mitogenic effect of TNF and LPS. Moreover, SNP alone caused cell death in a dose dependent manner. The cytotoxicity of SNP was not affected by DFMO while it was prevented by antioxidants. The results suggest that different pathways would mediate the response of cardiomyocytes to NO: they can lead either to ODC induction and DNA synthesis when NO is formed through NOS induction or to growth inhibition and cell death, when NO is supplied as NO donor. Increased polyamine biosynthesis would mediate the proliferative response of NO, while the cytotoxicity of exogenous NO seems to involve some oxidative reactions and to depend on the balance between NO availability and cellular redox mechanisms.

Early alterations of polyamine metabolism induced after acute administration of clenbuterol in mouse heart

An acute treatment of mice with clenbuterol, a β-adrenergic agonist, produced a marked increase of polyamines levels in heart, particularly during the early phase of administration of the drug. A single dose of 1.5 mg/kg caused as much as a 10 fold induction in activity of ornithine decarboxylase (ODC) and 3 to 4 fold increase in levels of putrescine, spermidine and spermine in mouse heart. Maximum changes were observed 3 to 4 hours post-administration of clenbuterol. This treatment did not produce any change in S-adenosylmethionine decarboxylase activity. The induction of cardiac ODC by clenbuterol was also dose dependent with a peak at about 5 μmol/kg. Co-administration of difluoromethylornithine, an irreversible inhibitor of ODC, or propranolol, a nonspecific β-antagonist, with clenbuterol completely prevented the induction of ODC activity as well as the increase in polyamine levels in heart. However, pretreatment with alprenolol or metoprolol, the specific β 1 and β 2-antagonists, respectively, produced only partial prevention. The cardiac ODC from controls as well as clenbuterol treated mice exhibited similar affinity (Km) for its substrate, ornithine, while maximum enzyme activity (Vmax) was about 14 fold higher in clenbuterol treated mouse heart than in the control. Clenbuterol produced no change in the level of specific ODC mRNA or the protein, but the enzyme from the drug-treated mouse heart was considerably more stable than the control. Pretreatment of mice with either cycloheximide or actinomycin D followed by administration of clenbuterol could not prevent the induction in ODC activity suggesting that de novo biosynthesis of the enzyme protein or ODC mRNA was not responsible for induction of ODC activity. Post-translational changes in ODC may be responsible for an early increase of ODC activity due to clenbuterol treatment.

Thirteen-week oral toxicity study of difluoromethylornithine in combination with tamoxifen citrate in female dogs.

Cancer chemoprevention is the use of pharmacologic or natural agents to inhibit the development of cancer. Difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase, the rate-limiting enzyme in the biosynthesis of polyamines. DFMO has demonstrated chemopreventive efficacy in animal models of tumorigenesis. Tamoxifen (TAM), a nonsteroidal antiestrogen, is approved for use in the treatment of estrogen receptor-positive breast carcinoma and has demonstrated efficacy in chemoprevention of breast cancer in women at high risk for the disease. The administration of TAM with DFMO is being considered for development by the National Cancer Institute as a potential drug regimen for the chemoprevention of breast carcinoma. The toxicity of DFMO in combination with TAM was evaluated in female Beagle dogs following 13 weeks of daily oral administration by capsule. Dose levels in milligrams per kilogram body weight per day were: 0 (vehicle control), 100 DFMO, 0.1 TAM, 1.0 TAM, 0.1 TAM + 100 DFMO and 1.0 TAM + 100 DFMO. No mortalities occurred. Diarrhea was produced by TAM and vaginal discharge, due to reproductive tract lesions, was produced by both DFMO and TAM, either alone or in combination. DFMO decreased reticulocyte counts and TAM increased counts of mature neutrophils. DFMO alone resulted in lesions to the intestines and ovaries, and cornified epithelium of vagina and cervix. TAM produced cornified epithelium of vagina and cervix, and numerous lesions in the ovaries, fallopian tube, uterus, cervix and vagina which were likely due to an estrogen agonist effect. Coadministration of DFMO increased the incidence and/or severity of these reproductive tract lesions. Each compound alone produced ovarian atrophy, and antral follicles and corpora lutea were completely absent in the 1.0 TAM + 100 DFMO group. Coadministration of DFMO and TAM resulted in additive toxicity involving the female reproductive system.

Comparative effects of TGFβ on proliferation of 7‐ and 14‐day‐old chick embryo fibroblasts and lack of involvement of the ODC/PA system in the TGFβ signaling pathway

The growth regulatory activity of transforming growth factor β (TGFβ) on chick embryo skin fibroblasts was compared in two developmental ages, days 7 and 14. The time course of 3H-thymidine incorporation, an S-phase marker of replication, was determined during 36 hr of TGFβ treatment. Seven-day-old cells showed a prereplicative phase of 6 hr, and 14-day-old cells showed a prereplicative phase of 12 hr. DNA synthesis peaked at 24 hr in 7-day-old fibroblasts and was 10 times higher than that in 14-day-old fibroblasts. Ornithine decarboxylase (ODC) activity and content of the natural polyamines spermine (Spm), spermidine (Spd), and putrescine (Put) differed during cell cycle. ODC activity peaked at 12 hr in 7-day-old cells and at 6 hr in 14-day-old cells. Its level was two times higher at day 7 and was associated with a greater content of ODC mRNA. The maximum of polyamine (PA) concentration was determined after 12 hr of treatment in 7-day-old cells and after 36 hr in 14-day-old cells. These findings indicate that the TGFβ proliferative response of embryo fibroblasts changes during development and is associated with activation of the ODC/PA system. Cotreatment with α-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ODC, did not reduced growth rate. Inhibition of ODC resulted in levels of Put and Spd comparable to that of quiescent fibroblasts, whereas Spm concentration remained higher. Because an altered ODC metabolism does not convey the effects of TGFβ on DNA synthesis, the ODC/PA system may not play a role in the pathway of TGFβ signaling. J. Cell. Physiol. 178:304–310, 1999. © 1999 Wiley-Liss, Inc.

Thiols and polyamines in the cytoprotective effect of taurine on carbon tetrachloride-induced hepatotoxicity.

The mechanism by which taurine (2-aminoethanesulfonic acid) protects hepatocytes injury induced by carbon tetrachloride (CCl4) is not fully understood. In a previous study, we reported that cellular polyamines play an important role in this mechanism. The relationship between cellular glutathione (GSH), protein-SH levels, and lactate dehydrogenase (LDH), with respect to the effect of polyamine on the cytoprotective ability of taurine in CCl4-induced toxicity in isolated rat hepatocytes, was examined. CCl4 induced a LDH release and decreased cellular thiols and polyamine levels. Treating with taurine reversed these depletions. The effect of CCl4 was also reversed by the addition of exogenous polyamines. Pretreating with alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, which is a key enzyme in polyamine biosynthesis and therefore used to deplete cellular polyamine, prevented the protective effect of taurine. Adding diethyl maleate, a cellular glutathione-depleting agent, reduced the effect of exogenous polyamines. The role of polyamine in the cytoprotective effect of taurine in CCl4-induced toxicity may therefore be by preventing, among others, GSH and protein-SH depletions.

Effects of polyamine levels on the degradation of short-lived and long-lived proteins in cultured L-132 human lung cells.

Biogenic polyamines have important regulatory functions in various biological processes and it has also been suggested that they could modulate intracellular protein degradation. For an overall assessment of the role of polyamines in this process, we have investigated the effect that the decrease in intracellular polyamine levels caused by inhibitors of polyamine biosynthesis brings about on the degradation of the pools of short- and long-lived proteins in cultured L-132 human lung cells. Treatment of cells with 100 microM (2R,5R)-delta-methyl acetylenic putrescine (MAP), a potent enzyme-activated irreversible inhibitor of ornithine decarboxylase, or with 100 microM MAP plus 50 microM N-butyl 1,3-diaminopropane, a specific inhibitor of spermine synthase, caused a similar decrease (65-70% of control) in the total intracellular levels of polyamines, although they affected the concentrations of spermidine and spermine differently. The effect of the two treatments on protein degradation was essentially the same. In polyamine-depleted cells we observed an inhibition of degradation in long-lived proteins of 16% (P<0.05), with a significant increase in the half-life (t12) of this pool from 100.5 to 120.1 h. This was concomitant with an increase of 26% (P<0. 05) in degradation in short-lived proteins, with a significant decrease in the t12 of this pool from 0.85 to 0.67 h. Recovery of polyamine levels by the addition of 50 microM spermidine to polyamine-depleted cells resulted in a restoration of the degradation rates in both pools of proteins. The way(s) by which polyamines could modulate proteolysis are discussed.

Effects of DFMO on glioma cell proliferation, migration and invasion in vitro.

The polyamine inhibitor DL-alpha-difluoromethylornithine (DFMO) is a specific irreversible inhibitor of ornithine decarboxylase which is a rate-limiting enzyme in the polyamine bio-synthesis pathway. The present study describes the effects of DFMO on glioma cell proliferation, migration and invasion using multicellular spheroids from three glioma cell lines (GaMg, U-251 Mg and U-87 Mg). 10 mM DFMO reduced cell migration in the three cell lines by about 30-50%. 1 mM putrescine, added together with DFMO inhibited the DFMO effect. A stronger effect was observed in the growth assay where 10 mM DFMO reduced the spheroid growth, for all cell lines, by 90%. This effect was also reversed by adding 1 mM of putrescine. In vitro tumor cell invasion experiments indicated after 3 days of confrontation, an extensive invasion also after 10 mM DFMO treatment. The brain aggregate volumes were reduced to about the same extent as in the absence of drug, suggesting essentially no effects of DFMO on the invasive process. It is concluded that the tumor spheroids retained their ability to invade normal brain tissue even after DFMO exposure. However, DFMO inhibited spheroid growth and cell migration which supports the notion that cell growth, migration and invasion are biological properties that are not necessarily related to each other.

Increased expression of c-jun, but not retinoic acid receptor β, is associated with F9 teratocarcinoma stem cell differentiation induced by polyamine depletion

alpha-Difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase, and all-trans-retinoic acid (RA) are known to induce F9 teratocarcinoma stem cell differentiation. Both compounds induce the formation of the same cell type, i.e., parietal endoderm-like cells expressing tissue plasminogen activator and collagen type IV alpha-1. The present study shows that DFMO and RA induce terminal differentiation of F9 cells through different pathways. Thus, retinoic acid receptor (RAR) alpha mRNA is weakly expressed during DFMO treatment, but strongly induced during an early phase of RA treatment. RAR beta mRNA is not detectable in DFMO-treated cells, but very strongly induced by RA and maintained at a high level throughout the differentiative process. RAR gamma mRNA is relatively strongly expressed in untreated control cells and remains at approximately the same level during DFMO-induced differentiation. In RA-treated cells, however, RAR gamma mRNA is rapidly down-regulated and becomes nondetectable during the final course of differentiation. These experiments show that the differentiation of F9 cells into parietal endoderm-like cells does not necessarily involve changes in any of the RAR mRNA subtypes. Even though the steady-state levels of the RAR alpha and RAR gamma transcripts may be sufficient to support the differentiative process, our data clearly show that induction of RAR beta mRNA transcription is neither a prerequisite for F9 cell differentiation, nor an absolute consequence of the elevated c-jun mRNA expression that is consistently observed during the course of parietal endoderm differentiation.