Phorbol ester (12-O-tetradecanoylphorbol 13-acetate) prevents ornithine decarboxylase inhibition and apoptosis and L1210 leukemic cells exposed to TGF-beta 1.

Previous studies have shown that growth suppression and apoptosis of leukemic cells exposed to TGF-beta 1 is associated with the inhibition of ornithine decarboxylase (ODC)--the key enzyme of polyamine pathway. The aim of the present study was to evaluate the influence of 12-O-tetradecanoylphorbol 13-acetate (TPA)--a potent ODC inducer on antiproliferative and apoptotic effects of TGF-beta 1 in L1210 leukemic cells. Cells were incubated in 2% FCS/RPMI-1640 medium, supplemented with TGF-beta 1 (2 ng/ml). TPA (100 ng/ml) or alpha-difluoromethylornithine (DFMO) (5 mM). Cell proliferation, apoptosis and necrosis were evaluated using [methyl-3H] thymidine, electron microscopy, electrophoresis of DNA and trypan blue exclusion. Expression and activity of ODC were determined by RT-PCR and measurement of 14CO2 release from L-1-14C ornithine, respectively. TGF-beta 1 inhibited proliferation and induced apoptotic and necrotic cell death in L1210 leukemic cells. The above effects were associated with the inhibition of ODC expression and activity, measured 2 and 4 hr after TGF-beta 1 administration, respectively. The presence of DFMO, an irreversible inhibitor of ODC, led to apoptotic fragmentation of DNA, similar to that observed in TGF-beta 1-treated cultures. Administration of TPA simultaneously with TGF-beta 1 significantly reduced antiproliferative, apoptotic and necrotic effects of TGF-beta 1, and prevented its inhibitory action of ODC expression and activity. It is concluded that: down-regulation of ODC expression may be one of the early events associated with TGF-beta 1-evoked suppression of growth and apoptosis; ODC is involved in the mechanism of protective action of TPA on TGF-beta 1-related growth inhibition of L1210 leukemic cells.

Polyamines and pyrimidines in blood plasma and urine of dogs bearing mammary tumours.

The blood plasma and urinary pattern of polyamines and pyrimidines in dogs bearing mammary tumours was examined. A large variability of pyrimidines in blood plasma and spermidine, spermine and pseudouridine in urine of healthy and tumour-bearing dogs was observed. The blood plasma level of uracil and uridine as well as urinary concentration of pseudouridine and spermidine/spermine ratio were significantly elevated in dogs with mammary tumours.

Involvement of polyamines in epidermal growth factor (EGF), transforming growth factor (TGF)-alpha and -beta 1 action on culture of L6 and fetal bovine myoblasts.

1. alpha-Difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase significantly abolished stimulation of protein synthesis evoked by EGF, TGF-alpha or -beta 1 in L6 and fetal bovine myoblasts. 2. The participation of polyamines in early events evoked by growth factors was shown by a significant stimulation of ornithine decarboxylase and S-adenosylmethionine decarboxylase activity as well as increased concentration of spermidine and spermine in L6 cells exposed to TGF-alpha and EGF. 3. TGF-beta 1 at a high concentration (1 ng/ml) increased protein synthesis in L6 myoblasts but inhibited it in fetal bovine myoblasts. Metabolic effects of TGF-beta 1 in L6 cells was associated with an enhancement of decarboxylase activities, however there were no significant changes in cellular polyamine concentrations. Presented data suggest that polyamines are involved in the signal transduction pathway of EGF, TGF-alpha, and -beta 1 in L6 and fetal bovine myoblasts.

TGF-beta 1 inhibits polyamine biosynthesis in K 562 leukemic cells.

The present study proved that TGF-beta 1 significantly inhibited the growth of K 562 cells. The drop in cell numbers after 24 h incubation with increasing concentrations of TGF-beta 1 (0.01, 0.1, 1.0, 10.0 ng/ml) was accompanied by significant suppression of the activity of two key enzymes of polyamine biosynthesis: ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC). In contrast to ODC and SAMDC activity, TGF-beta 1 did not significantly affect the absolute concentration of spermidine and spermine in K 562 cells. We suppose that the lack of an evident drop in concentration of spermidine and spermine in spite of a significant decrease in ODC and SAMDC activity in K 562 cells exposed to TGF-beta 1 resulted from the uptake of polyamines from the extracellular space.

Metabolic effect of orotic acid in calves.

Eight calves (males, Black and White crossbred with Holstein-Fresian) were fed milk and milk replacer without (control group) or with potassium orotate (3 mmol./l.) supplementation for 6 weeks after birth. Orotate depressed the biosynthesis of polyamines in mucosa of the gastrointestinal tract (rumen, omasum, abomasum, colon) by decreasing of ornithine decarboxylase activity with a simultaneous compensatory increase of S-adenosyl-methionine decarboxylase activity. A lower concentration of spermidine and spermine in the mucosa of the colon was also noted. The above changes were accompanied by increased urinary excretion of ornithine and arginine. Calf adaptation to a high OA intake was associated with an increased activity of the OA metabolizing enzyme complex (orotate phosphoribosyl transferase and orotidine monophosphate decarboxylase) in the liver, while urinary OA losses diminished with age. Increased concentrations of uracil and uridine in the liver and higher urinary excretion of pseudouridine in OA-fed calves was also observed. Stimulation of pyrimidine metabolism by OA depressed purine synthesis, which was reflected by a decrease of urate, hypoxanthine, and xanthine concentration in the liver. Interestingly OA enhanced urate excretion by the kidneys. OA strongly affected lipid metabolism in calves because total cholesterol, LDL-, and HDL-cholesterol, and triglycerides in blood plasma decreased while triglycerides accumulated in the liver of OA-fed calves. Milk OA in concentrations characteristic of cows with hereditary orotic aciduria exerts an unfavourable effect on the metabolism of polyamines, purines, and lipids in calf tissues.

Metabolic effect of orotic acid in rat L6 myoblasts.

Orotic acid (OA) is an intermediate in the pyrimidine pathway. The main source of OA in the human and animal diet is bovine milk and its products. OA significantly inhibited the stimulation of protein synthesis by FCS derived growth factors in L6 myoblasts. An increasing OA concentration (0.001 mM, 0.01 mM, and 0.1 mM) in a medium containing 2% FCS decreased the proliferation of L6 myoblasts (as measured by the number of cells) as well as the incorporation of traced thymidine into cellular DNA after 24 h incubation. This mitoinhibitory effect was accompanied by a significant reduction of ornithine decarboxylase (ODC) activity, an enzyme which is believed to be a valuable index of cell proliferation. A drop in the cytosol spermine level of L6 myoblasts treated with OA also occurred. A simultaneous, significant, compensative increase of S-adenosylmethionine decarboxylase (SAMDC) activity was noted. The addition of putrescine (2 microM), a product of ODC activity, abolished the depressional influence of OA on protein synthesis in L6 myoblasts, thus confirming its interference with the polyamine pathway. Dibutyryl-cAMP (0.05-0.2 mM) or adenine (25 microM) supplementation, regardless of OA concentration, significantly attenuated the mitoinhibitory effect and its inhibitory action on protein synthesis. This may suggest the existence of a purine deficiency in OA-treated myoblasts. These experiments indicate that increasing cellular OA concentration inhibits cell growth probably by disturbances in the chain of early events (synthesis of cyclic purine mononucleotides, and activity of the ODC/polyamine system), normally evoked by growth stimulating factors.