1. The activation of human peripheral blood lymphocytes by phytohaemagglutinin in vitro was accompanied by striking increases in the concentrations of the natural polyamines putrescine, spermidine and spermine. 2. The enhanced accumulation of polyamines could be almost totally abolished by dl-alpha-difluoromethylornithine, a newly discovered irreversible inhibitor of l-ornithine decarboxylase (EC 4.1.1.17), or by methylglyoxal bis(guanylhydrazone) {1,1'-[(methylethanediylidene)dinitrilo]diguanidine}, an inhibitor of S-adenosyl-l-methionine decarboxylase (EC 4.1.1.50). The inhibition of polyamine accumulation was associated with a marked suppression of DNA synthesis, which was partially or totally reversed by low concentrations of exogenous putrescine, spermidine, spermine and cadaverine and by higher concentrations of 1,3-diaminopropane. 3. In contrast with some earlier studies, we found that methylglyoxal bis(guanylhydrazone), at concentrations that were sufficient to prevent polyamine accumulation, also caused a clear inhibition of protein synthesis in the activated lymphocytes. Similar results were obtained with difluoromethylornithine. The decrease in protein synthesis caused by both compounds preceded the impairment of DNA synthesis. The inhibition of protein synthesis by difluoromethylornithine was fully reversed by exogenous putrescine, spermidine and spermine, and that caused by methylglyoxal bis(guanylhydrazone) by spermidine and spermine. In further support of the idea that the inhibition of protein synthesis by these compounds was related to the polyamine depletion, we found that difluoromethylornithine caused a dose-dependent decrease in the incorporation of [(14)C]leucine into lymphocyte proteins which closely correlated with the decreased concentrations of cellular spermidine. 4. Difluoromethylornithine and methylglyoxal bis(guanylhydrazone) also elicited a variable depression in the incorporation of [(3)H]uridine and [(14)C]adenine into total RNA. The apparent turnover of lymphocyte RNA remained essentially unchanged in spite of severe polyamine depletion brought about by difluoromethylornithine. 5. The present results, as well as confirming the anti-proliferative action of the inhibitors of polyamine biosynthesis, suggest that polyamine depletion may interfere with reactions at different levels of gene expression.
(1) Human myeloma cell line Sultan, resistant to 20 mM difluoro-methylornithine (DFMO) owing to ornithine decarboxylase (ODC) gene amplification, was grown in the absence of DFMO for a period of 10 months. The gene copy number and methylation status of the ODC gene were monitored after withdrawal of DFMO. Moreover, levels of ODC mRNA, immunoreactive ODC protein, ODC activity and polyamine levels were recorded recurrently during the course of the study. (2) The results revealed that ODC gene copy number started to decrease after 4 weeks growth without DFMO, to a final level of less than 30% of the original gene dosage. The methylation status of the ODC gene, however, remained almost unaltered, displaying only a modest increase in methylation after 10 months without DFMO. The amount of ODC message dropped very rapidly to 75% of the original value, then started to decrease in a gene copy-number-dependent manner. The amount of ODC protein closely followed the levels of mRNA during the study, whereas the ODC activity, after a transient increase during the first week, decreased to half of the original level after 4 weeks. Between 6 and 16 weeks ODC activity stabilized to a fifth of the original value and no more changes were detected during the subsequent period of observation. (3) Due to the grossly elevated ODC enzyme activity, levels of putrescine and spermidine first peaked and then stabilized at 6 weeks after DFMO withdrawal. The final spermidine level was comparable with that of the parental Sultan cell line with only one copy of active ODC gene. However, putrescine content was strikingly elevated, being stabilized to a level that was 20 times higher than in parental cells. Spermine concentration was practically unchanged during the study. (4) According to the results obtained in this study, the abnormal level of ODC expression in human myeloma cells is suppressed partially at the level of transcription or post-transcriptionally, but it is not due to increased methylation of the gene. The major regulatory mechanism to compensate for a highly elevated ODC expression was modulation of the enzyme activity. After 10 months without DFMO, the cells still displayed about 20 times higher ODC activity and putrescine concentration than the myeloma cell line with a single copy of the ODC gene. They did not, however, show any signs of growth retardation or other features different from the parental cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Efficient syntheses of metabolically stable alpha-methylspermidine 1, alpha-methylspermine 2, and bis-alpha,alpha'-methylated spermine 3 starting from ethyl 3-aminobutyrate are described. The biological tolerance for these compounds was tested in wild-type mice and transgenic mice carrying the metallothionein promoter-driven spermidine/spermine N(1)-acetyltransferase gene (MT-SSAT). The efficient substitution of natural polyamines by their derivatives was confirmed in vivo with the rats harboring the same MT-SSAT transgene and in vitro with the immortalized fibroblasts derived from these animals. Enantiomers of previously unknown 1-amino-8-acetamido-5-azanonane dihydrochloride 4 were synthesized starting from enantiomerically pure (R)- and (S)-alaninols. The studies with recombinant human polyamine oxidase (PAO) showed that PAO (usually splits achiral substrates) strongly favors the (R)-isomer of 4 that demonstrates for the first time that the enzyme has hidden potency for stereospecificity.
