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Kinetics of polyamine synthesis and turnover in mouse fibroblasts.

ABSTRACT: Kinetics of polyamine synthesis and degradation were studied in mouse fibroblasts growing in suspension culture. The approach was to prelabel cells with radioactive polyamines and to observe the rate of loss of radioactivity and the rate of decrease in specific activity of these compounds in cells. Radioactive putrescine declined with a half-life of 1.5-2h, whether derived directly from exogenous putrescine or indirectly from ornithine. Much of this turnover was due to excretion, the kinetics of which suggested that a steady-state was being established between putrescine inside and outside the cells. Within 5h of medium change, cells growing at a density of 5x10(5)cells/ml had supplied putrescine to the medium to a concentration of about 1mum. When cells were prelabelled with either putrescine or spermidine, radioactivity in cell spermidine declined with a half-life of 60h. This rate of turnover is sufficient to provide all the spermine required by the cell. Spermine synthesis was the only observed reaction of spermidine, although some excretion into the growth medium was detected. Spermine was not degraded at a detectable rate as long as cells were growing exponentially; in stationary phase, degradation to spermidine, which was excreted, became significant. The half-lives of the specific activities of spermine, spermidine and putrescine were 24, 15 and 1.5h respectively. From these values, the rate of synthesis of each was calculated. Spermidine was synthesized at 6.8 times the rate of spermine, and putrescine was synthesized at 0.46nmol/10(6)cells per h, twice the rate of spermidine. The significance of these kinetic parameters is discussed.

SUBMITTER: McCormick F

PROVIDER: S-EPMC1185931 | biostudies-other | 1978 Aug

REPOSITORIES: biostudies-other

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Project description:1. A number of compounds known to inhibit polyamine biosynthesis at various steps in the biosynthetic pathway were tested for their ability to inhibit growth and decrease polyamine concentrations in virally transformed mouse fibroblasts (SV-3T3 cells). 2. Virtually complete inhibition of growth was produced by the inhibitors of ornithine decarboxylase alpha-methylornithine and alpha-difluoromethylornithine and by the inhibitors of S-adenosylmethionine decarboxylase 1,1'-[(methylethanediylidene)dinitrilo]diguanidine and 1,1'-[(methylethanediylidene)dinitrilo]bis-(3-aminoguanidine). The former inhibitors decreased putrescine and spermidine contents in the cells to very low values, whereas the latter substantially increased putrescine but decreased spermidine concentrations. The inhibitory effects of all of these inhibitors on cell growth could be prevented by the addition of spermidine, suggesting that spermidine depletion is the underlying cause of their inhibition of growth. 3. alpha-Difluoromethylornithine, which is an irreversible inhibitor of ornithine decarboxylase, was a more potent inhibitor of growth and polyamine production (depleting spermidine almost completely and spermine significantly) than alpha-methylornithine, which is a competitive inhibitor. This was not the case with the inhibitors of S-adenosylmethionine decarboxylase where 1,1'-[(methylethanediylidene)dinitrilo]diguanidine, a reversible inhibitor, was more active than 1,1'-[(methylethanediylidene)dinitrilo]bis-(3-aminoguanidine), an irreversible inhibitor. It is suggested that this effect may be due to the lesser uptake and/or greater chemical reactivity of the latter compound. 4. Various nucleoside derivatives of S-adenosylhomocysteine that inhibited spermidine synthase in vitro did not have significant inhibitory action against polyamine accumulation in the cell. These compounds, which included S-adenosylhomocysteine sulphone, decarboxylated S-adenosylhomocysteine sulphone, decarboxylated S-adenosylhomocysteine sulphoxide and S-adenosyl-4-thio-butyric acid sulphone did not inhibit cell growth or polyamine content until cytotoxic concentrations were added. 5. 5'-Methylthioadenosine, 5'-isobutylthioadenosine and 5'-methylthiotubercidin, which inhibit aminopropyltransferase activity in vitro, all inhibited cell growth and decreased spermidine content. Although these compounds were most active against spermine synthase in vitro, they acted in the cell primarily to decrease spermidine content. Cell growth could not be restored to normal values by addition of spermidine, suggesting that these nucleosides have another inhibitory action towards cellular proliferation. 6. 5'-Methylthioadenosine and 5'-isobutylthioadenosine are degraded by a phosphorylase present in SV3T3 cells, yielding 5-methylthioribose-1-phosphate and 5-isobutylthioribose-1-phosphate respectively, and adenine. This degradation appears to decrease the inhibitory action towards cell growth, suggesting that the nucleosides themselves are exerting the inhibitory action. 5'-Methylthiotubercidin, which is not a substrate for the phosphorylase and is a competitive inhibitor of it, was the most active of these nucleosides in inhibiting cell growth and spermidine content. 5'-Methylthiotubercidin and alpha-difluoromethylornithine had additive effects on retarding cell growth, but not on cellular spermine accumulation, also suggesting that the primary growth-inhibiting action of the nucleoside was not on polyamine production. 7. These results support the concept that 5'-methylthioadenosine phosphorylase plays an important role in permitting cell growth to continue by preventing the build-up of inhibitory intracellular concentrations of 5'-methylthioadenosine.

Project description:Noncycling and terminally differentiated (TD) cells display differences in radiosensitivity and DNA damage response. Unlike other TD cells, Sertoli cells express a mixture of proliferation inducers and inhibitors in vivo and can reenter the cell cycle. Being in a G1-like cell cycle stage, TD Sertoli cells are expected to repair DSBs by the error-prone nonhomologous end-joining pathway (NHEJ). Recently, we have provided evidence for the involvement of Ku-dependent NHEJ in protecting testis cells from DNA damage as indicated by persistent foci of the DNA double-strand break (DSB) repair proteins phospho-H2AX, 53BP1, and phospho-ATM in TD Sertoli cells of Ku70-deficient mice. Here, we analyzed the kinetics of 53BP1 foci induction and decay up to 12 h after 0.5 Gy gamma irradiation in DNA-PKcs-deficient (Prkdc scid ) and wild-type Sertoli cells. In nonirradiated mice and Prkdc scid Sertoli cells displayed persistent DSBs foci in around 12 % of cells and a fivefold increase in numbers of these DSB DNA damage-related foci relative to the wild type. In irradiated mice, Prkdc scid Sertoli cells showed elevated levels of DSB-indicating foci in 82 % of cells 12 h after ionizing radiation (IR) exposure, relative to 52 % of irradiated wild-type Sertoli cells. These data indicate that Sertoli cells respond to and repair IR-induced DSBs in vivo, with repair kinetics being slow in the wild type and inefficient in Prkdc scid . Applying the same dose of IR to Prdkc -/- and Ku -/- mouse embryonic fibroblast (MEF) cells revealed a delayed induction of 53BP1 DSB-indicating foci 5 min post-IR in Prdkc -/- cells. Inefficient DSB repair was evident 7 h post-IR in DNA-PKcs-deficient cells, but not in Ku -/- MEFs. Our data show that quiescent Sertoli cells repair genotoxic DSBs by DNA-PKcs-dependent NEHJ in vivo with a slower kinetics relative to somatic DNA-PKcs-deficient cells in vitro, while DNA-PKcs deficiency caused inefficient DSB repair at later time points post-IR in both conditions. These observations suggest that DNA-PKcs contributes to the fast and slow repair of DSBs by NHEJ.

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Kinetics of polyamine synthesis and turnover in mouse fibroblasts.

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