There are large increases in cellular levels of the polyamines spermidine and spermine in lymphocytes induced to transform by concanavalin A. The anti-leukemic agent methylglyoxal bis(guanylhydrazone) (MGBG) that in the absence of increased cellular levels of polylmines, lymphocytes progress normally from Go through G1 and into S-phase. Furthermore, these experiments suggest that the increased levels of spermidine and spermine generally seen in rapidly proliferating eukaryotic systems are necessary for enhanced rates of DNA replication. Stimulation of the rate of biosynthesis of the aliphatic polyamines spermidine and spermine appears to be an invariable correlate of increased cell proliferation. Striking increases in the levels of the enzymes of polyamine biosynthesis and, to a lesser extent, enhanced cellular levels of these compounds have been observed in a variety of systems (1-5). Although the general phenomenon has been well described, and a variety of hypotheses have been advanced to explain the role of increased polyamine levels, few experiments have been designed to directly test the role of these compounds. Recently, with the discovery of specific inhibitors of polyamine synthesis, a new avenue of experimentation has become accessible. Williams-Ashman and Schenone demonstrated methylglyoxal bis(guanylhydrazone) (MGBG) to be a potent inhibitor of S-adenosylmethionine decarboxylase (6). More recently, Snyder and his coworkers have shown that a-hydrazinoornithine inhibits the conversion of ornithine to putrescine, the biosynthetic precursor of spermidine and spermine, both in vitro and in tnvo (7,8). If these inhibitors are specific for polyamine biosynthesis, and exhibit no other pharmacological actions, they will be powerful tools to examine the cellular function of the polyamines.We have used MGBG to study the role-of polyamines in lymphocytes which have been stimulated by concanavalin A (Con A) to grow and divide. MGBG rapidly brings about complete inhibition of the biosynthesis of spermidine and spermine when added to activated lymphocytes (9, 10). From the studies to date, it is clear that normal transcription and translation continue after polyamine accumulation is blocked with MGBG. We have therefore concluded that the increased cellular levels of polyamines seen in proliferating cells are not involved in protein synthesis or in the synthesis, processing, and accumulation of RNA (10). Hence, in defining a role for the enhanced polyamine levels, our attention has been directed to later events in the process of lymphocyte activation. Specifically, we have been examining the effects of MGBG on DNA replication and cell division (11). We demonstrate in this paper that blocking polyamine accumulation with MGBG leads to decreased rates both of [methyl-3H]thymidine incorporation into DNA and of cell division. Evidence is presented which strongly argues that these effects of MGBG are due directly to inhibition of spermidine and spermine accumulation and not to a pharmacological action of MGBG unr...

Mutants defective in the conversion of arginine to putrescine were found by screening clones from mutagenized cultures for inability to produce urea during growth in arginine-supplemented media. Two partially blocked mutants were isolated; one was deficient in arginine decarboxylase and the other was deficient in agmatine ureohydrolase. As predicted from the pattern of putrescine synthesis in Escherichia coli, these mutants were conditionally putrescine-deficient. When grown in either minimal or ornithine-supplemented media, conditions which lead to preferential utilization of the ornithine to putrescine pathway, the mutants had normal intracellular polyamine levels. However, when the mutants were placed in arginine-supplemented media, the level of intracellular putrescine was lowered markedly. Under conditions where intracellular putrescine was 1 % of normal, the doubling time of the mutants was increased approximately 10%. The putrescinedeficient mutants had wild-type morphology, normal levels of protein and ribonucleic acid (RNA), and stringent amino acid control of RNA synthesis.

A mutant of Escherichia coli with reduced levels of biosynthetic arginine decarboxylase was isolated which required putrescine or spermidine for optimal growth. The stimulation of growth by putrescine was 1.5to 3-fold depending upon the culture medium. Specificity studies supported the concept that the requirement was for spermidine or closely related polyamines, or for diamines which could be converted enzymatically to these compounds. The behavior of the macromolecular composition of the polyamine-starved cells appeared abnormal. The ribonucleic acid to protein and deoxyribonucleic acid to cell ratios in the starved cells were both higher than expected on the basis of their growth rate. The stable ribonucleic acid in the polyamine-limited cells appeared to be normal as judged from size distribution and degree of methylation. The relationship of these results to mechanisms for regulation of nucleic acid and protein synthesis in E. coli is discussed.Although the polyamines putrescine, spermi-

A homologous series of spermidine analogs was synthesized with the general structure NH3+(CH2),NH2+(CH2)3NH3+, where spermidine has n = 4. The influence ofthese compounds on growth and on the syntheses of protein and messenger ribonucleic acid was examined in a spermidine auxotroph of Escherichia coli. All of the homologs tested were taken up by the cells to an intracellular level equivalent to the level of spermidine which gives optimal growth. With increasing chain length of the homologs, there was reduced ability to stimulate growth. The homologs with n = 7 and n = 8 were essentially inactive. A similar specificity was observed when the ability of the homologs to restore the rates of protein and messenger ribonucleic acid chain elongation was compared to that of spermidine. These results suggest that a definite spatial arrangement of the amino groups of spermidine is required for productive interaction at its intracellular site(s) of action.Wild-type Escherichia coli growing in minimal medium contains milimolar intracellular concentrations of spermidine [NH3+(CH2)4-

The rate of polypeptide chain elongation during steady-state, polyaminelimited growth of a mutant of Escherichia coli was measured by two independent techniques. Analysis of polysome patterns gave values of 17.5 and 9.5 amino acids per s at 37 C in unstarved and polyamine-limited cells, respectively. From the kinetics of entry of labeled amino acids into polypeptides of defined molecular weights, values at 30 C of 10.1 and 5.8 amino acids per s were obtained for unstarved and polyamine-limited cultures, respectively. Correction of these values to 37 C resulted in rates of 15.0 and 8.7 amino acids per s. These results support the previous conclusion, based on the kinetics of ,B-galactosidase induction, that polyamine starvation decreases the rate of protein synthesis by limiting the velocity of polypeptide chain elongation.