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E J Herbst

Publications and source records attributed to E J Herbst.

At least 19 recordsLinked to original sources
PubMed1992

Deficiencies in DNA replication and cell-cycle progression in polyamine-depleted HeLa cells.

Synchronized HeLa cells depleted of polyamines by alpha-difluoromethylornithine exhibited substantially decreased DNA synthesis, and proliferation ceased after the release of the cells into S phase. Nuclei from these cells synthesized 70-80% less DNA than did nuclei from control cells. Extraction of isolated nuclei with 0.3 M-KCl decreased DNA synthesis by about 60%, which was recovered almost completely in control cell nuclei by reconstitution with the salt extracts of these nuclei. On the other hand, salt extracts of polyamine-depleted nuclei restored only 50% of DNA synthesis in extracted control nuclei. Salt extracts of control cell nuclei contained twice the DNA polymerase alpha activity of polyamine-depleted nuclear extracts. Extracts of cell lysates of both control and polyamine-depleted HeLa cells exhibited similar DNA polymerase alpha activity, suggesting that uptake of the enzyme or its retention by the nuclei of polyamine-depleted cells was decreased. Polyamine-depleted nuclei also showed altered phosphorylation of a 31 kDa protein as compared with control nuclei. Almost normal DNA synthesis, cell proliferation, DNA polymerase alpha activity and nuclear protein phosphorylation were restored in polyamine-depleted cells grown in medium supplemented with 20 microM-spermidine at least 10-12 h before S phase. Cultures in which proliferation was blocked by alpha-difluoromethylornithine did not exhibit synchronous growth after the block was removed. Thus it may be concluded that HeLa cells depleted of polyamines are not inhibited at a single control point in the cell cycle, but are arrested at diverse sites throughout G1 phase.

Cell Cycle↗
PubMed1986

Polyamines and HeLa-cell DNA replication.

HeLa cells were synchronized for S-phase DNA synthesis by the double thymidine-block procedure. A comparison was made of the polyamine content and S-phase DNA synthesis in cells from control cultures and cultures to which an inhibitor of polyamine biosynthesis, alpha-difluoromethylornithine, was added to the synchronization medium. Control cells showed a peak of synchronous DNA synthesis at 3 h and a maximum concentration of polyamines at 6-9 h after release of the second thymidine block. Cells from cultures containing the inhibitor were severely inhibited in the synthesis of DNA and contained no putrescine and only traces of spermidine while the spermine content was lowered by as much as 80%. Supplementation of cultures containing alpha-difluoromethylornithine with a polyamine, at the time of release of the second thymidine block, replenished the intracellular pool of the administered polyamine and partially restored S-phase DNA synthesis, with a lag of 3-6 h. Almost complete restoration of DNA synthesis in cells depleted of polyamines was achieved by the addition of a polyamine to cultures at least 10 h before release of the second thymidine block. The lag in initiation of synchronous S-phase DNA synthesis was eliminated in these cells. It is concluded that reversal by polyamines of the deficiency in S-phase DNA synthesis, in polyamine-depleted HeLa cells, is a time-dependent process indicative of the necessity for the replenishment of replication factors or their organization into an active replication complex.

DNA Replication↗
PubMed1985

Effect of concentration of D,L-2-difluoromethylornithine on murine mammary carcinogenesis.

The appearance of chemically induced mammary gland carcinomas in virgin female Sprague-Dawley rats was blocked by the administration of D,L-2-difluoromethylornithine (DFMO) in drinking water during the stage of tumor promotion. Rats were given injections s.c. at 50 days of age with either 35 mg of 1-methyl-1-nitrosourea (MNU) per kg of body weight or the 0.9% NaCl solution in which the carcinogen was dissolved. At 57 days of age, the rats were each randomly allocated to one of 14 treatment groups. Ten groups (five solvent treated and five MNU treated) were assigned to treatments consisting of 0.00, 0.0625, 0.125, 0.25, or 0.50% (w/v) solution of DFMO in their drinking water; two MNU-treated groups were placed on or removed from DFMO treatment (0.5%; w/v) at 90 days post-carcinogen exposure; and two carcinogen-treated groups received either putrescine (0.5-g/kg diet) or putrescine and DFMO (0.5%; w/v) throughout the experiment. The study was terminated 183 days after carcinogen treatment. All doses of DFMO exerted a protective effect against the induction of mammary cancer; however, only the feeding of the 0.125% and the 0.5% solutions of DFMO resulted in a significant reduction in cancer incidence. The average number of cancers per rat was reduced, and cancer-free time was extended at all concentrations of DFMO. The protective effect of DFMO was sustained following withdrawal of treatment at 90 days post-MNU injection. Feeding putrescine in conjunction with DFMO treatment partially blocked the inhibitory activity of DFMO. DFMO treatment did not affect food or water intake; body weight gain; the weight of ovaries, uterus, adrenal glands, liver, kidney, or spleen; or the periodicity of the estrous cycle. These data provide evidence of an inhibitory effect of DFMO against mammary cancer induced by MNU which cannot be attributed to a systemic toxic effect of this compound.

Animals↗
PubMed1984

Isolation of rat mammary epithelial cells for polyamine analysis.

Mammary epithelial cells were isolated from either abdominal-inguinal glands or mammary tumours of rats, after enzymic digestion of the tissues, and were analysed for polyamine content. Optimum conditions were developed for the isolation of cells in sufficient yield for the analysis of polyamines from 1 g of mammary gland or 0.5 g of tumour tissue. Complete recoveries of the polyamines in the tissues were achieved in the isolated epithelial cells.

Animals↗
PubMed1984

Effect of D,L-alpha-difluoromethylornithine on murine mammary carcinogenesis.

The development of chemically-induced mammary gland carcinomas in rats was dramatically suppressed by provision of a 1% solution of D,L-alpha-difluoromethylornithine (DFMO) in drinking water. Treatment with DFMO significantly reduced cancer incidence and the average size and number of cancers per rat and prolonged the cancer-free time. DFMO appears to be effective in blocking some aspect of the promotion stage of chemically induced mammary carcinogenesis in the rat.

Animals↗
PubMed1981

Role of polyamines in HeLa cell proliferation.

HeLa cells were synchronized for S phase DNA synthesis by the double thymidine block procedure and simultaneously depleted of putrescine and spermidine with the irreversible inhibitor of ornithine decarboxylase, alpha-difluoromethyl ornithine. S phase DNA synthesis was inhibited and cell proliferation was prevented in the cells in which polyamines were depleted by the inhibitor of ornithine decarboxylase. Nuclei prepared from synchronized, polyamine-depleted cells were almost totally inactivated for in vitro DNA synthesis. S phase DNA synthesis was restored and normal cell proliferation occurred if either putrescine, spermidine, or spermine was added to the medium during the synchronization of the cells in the presence of alpha-difluoromethyl ornithine. Nucleic prepared from cells synchronized in media containing the inhibitor but supplemented with a polyamine during the synchronization procedure were also completely active in the synthesis of DNA in vitro. The results indicate that polyamines are required for the replication of DNA in HeLa cells.

Cell Division↗
PubMed1980

Inhibition of ornithine decarboxylase of HeLa cells by diamines and polyamines. Effect on cell proliferation.

1. Ornithine decarboxylase activity is stimulated in high-density HeLa-cell cultures by dilution of or replacement of spent culture medium with fresh medium containing 10% (v/v) horse serum. 2. After stimulation, ornithine decarboxylase activity reaches a peak at 4-6h, then rapidly declines to the low enzyme activity characteristic of quiescent cultures, where it remains during the remainder of the cell cycle. 3. The stimulation of ornithine decarboxylase is eliminated by the addition of 0.5mum-spermine or -spermidine or 10mum-putrescine to the HeLa-cell cultures at the time of re-feeding with fresh medium. Much higher concentrations (1mm) of the non-physiological diamines, 1,3-diamino-propane or 1,3-diamino-2-hydroxypropane, are required to eliminate the stimulation of ornithine decarboxylase in re-fed HeLa-cell cultures. 4. A heat-labile, non-diffusible inhibitor, comparable with the inhibitory protein ornithine decarboxylase antizyme, is induced in HeLa cells by the addition of exogenous diamines or polyamines. 5. Intracellular putrescine is eliminated, intracellular spermidine and spermine are severely decreased and proliferation of HeLa cells is inhibited when cultures are maintained for 48h in the presence of the non-physiological inducer of ornithine decarboxylase antizyme, 1,3-diamino-2-hydroxypropane. Exogenous putrescine, a physiological inducer of the antizyme, does not decrease intracellular polyamines or interfere with proliferation of HeLa cells.

Carboxy-Lyases↗
PubMed1978

Liver ornithine decarboxylase during phenobarbital promotion of nitrosamine carcinogenesis.

The incidence of liver tumors induced in rats by N-diethylnitrosamine was increased by the feeding of phenobarbital. Liver ornithine decarboxylase activity did not increase in animals receiving phenobarbital in the diet, and the concentration of polyamines in the liver was similarly unchanged. No relationship between the promotion of N-nitrosamine-induced liver tumors by phenobarbital and the ornithine decarboxylase activity of the liver was indicated by these experiments.

Animals↗
PubMed1977

Ornithine decarboxylase and polyamines in liver and kidneys of rats on cyclical regimen of protein-free and protein-containing diets. Relationship to deoxyribonucleic acid synthesis in liver.

1. The activity of ornithine decarboxylase in the liver and kidneys of rats maintained on a cyclical regimen of protein-free and protein-containing diets was investigated. There was a daily activation of the enzyme in response to the feeding of protein after 3 days feeding of protein-free diet. 2. The activation of ornithine decarboxylase in the liver and kidneys of rats re-fed on protein was demonstrable throughout 16 cycles of alternating 3-day periods of protein-free and protein-containing diets. The magnitude of the activation in the kidneys diminished from 20-fold stimulation in the first cycle to 5-fold stimulation (compared with animals fed with protein-free diet) in the later cycles of protein re-feeding. The activation of the enzyme in liver was decreased from 20-fold stimulation in the first cycle to approx. 10-fold stimulation in later cycles. 3. The concentration of spermidine was increased by approx. 50% in the liver of animals during cycling from protein-free to protein-containing diets. Spermine was unchanged, and putrescine was maintained at a low concentration approx. one-fifth to one-tenth that of spermidine after protein re-feeding. 4. The incorporation of [(3)H]thymidine into liver DNA was increased 10-fold in animals re-fed with protein compared with animals receiving protein-free diets. 5. The activation of ornithine decarboxylase by re-feeding of protein was inhibited 90% by the injection of propane-1,3-diamine during re-feeding. The stimulation of DNA synthesis was inhibited 60% by multiple injections of propane-1,3-diamine during the re-feeding of protein.

Animals↗
PubMed1976

The effect of polyamines on the synthesis of ribonucleic acid by Drosophila melanogaster larvae.

1. To elucidate further the possible role of polyamines in the synthesis of nuclei acids, a study of the effect of exogenously administered amines on the synthesis of RNA by Drosophila melanogaster larvae was undertaken. This system was chosen because of the previous investigations [Dion, A.S. & Herbst, E.J. (1967) Proc. Natl. Acad. Sci. U.S.A. 58, 2367-2371; Herbst, E.J. & Dion, A.S. (1970) Fed. Proc. Fed. Am. Soc. Exp. Biol. 29, 1563-1567] relating putrescine and spermidine to growth and development of Drosophila.

Animals↗
PubMed1976

Decarboxylases for polyamine biosynthesis in Drosophila melanogaster larvae.

Ornithine decarboxylase (L-ornithine carboxy-lase, EC 4.1.1.17) and S-adenosyl-methionine decarboxylase (S-adenosyl-L-methionine carboxy-lase, EC 4.1.1.50) were assayed in Drosophilia melanogaster larvae. The highest enzyme activities were detected in 24 and 48 h larvae, with diminishing activities in subsequent larval stages. Stimulation of S-adenosylmethionine decarboxylase by putrescine was demonstrable in late but not in early stages of larval development.

Adenosylmethionine Decarboxylase↗