Effects of inhibitors of ornithine and S‐adenosylmethionine decarboxylases on L6 myoblast proliferation

Abstract

The role of polyamines in myoblast proliferation was studied by treating cells of Yaffe's L6 line of rat myoblasts with inhibitors of polyamine synthesis. Both an irreversible inhibitor of ornithine decarboxylase--difluoromethyl-ornithine (DFMO)--and a competitive inhibitor of S-adenosyl-methionine decarboxylase--methylglyoxal-bis(guanylhydrazone) (MGBG)--depressed spermidine levels and inhibited myoblast proliferation. Spermine levels were not significantly depressed by either inhibitor and putrescine levels were decreased only by DFMO. Putrescine and spermidine, but not magnesium, prevented inhibition of myoblast proliferation by DFMO and MGBG; determination of 14C-DFMO uptake in the presence and absence of these compounds demonstrated that they did not reduce the rate or extent of inhibitor uptake and thus prevent its inhibition of ornithine decarboxylase. Thus it seems likely that these inhibitors reduce cell proliferation by inhibiting polyamine formation. Addition of spermidine to the cells led to a substantial reduction in the activity of S-adenosyl-methionine-decarboxylase, suggesting that the enzyme is subject to negative regulation by the products of the polyamine biosynthetic pathway. Unexpectedly, addition of spermidine also increased intracellular putrescine levels; this apparently resulted from conversion of spermidine to putrescine. Addition of putrescine or spermidine in the absence of serum did not increase the rate of myoblast proliferation although it did elevate intracellular polyamine levels as expected. We conclude that some threshold level of one or more polyamines (probably spermidine) is necessary but not sufficient for initiation and maintenance of myoblast proliferation in culture.

The role of polyamines in somatomedin‐stimulated differentiation of L6 myoblasts

“…In addition, the cell density of DFMO-treated cultures was essentially the same as that of control cultures (Table 1). These observations are consistent with the results obtained with a closely related subclone (L6-C5) which demonstrated that at least 48 hours of exposure to DFMO is needed before inhibition of cell proliferation becomes manifest (Stoscheck et al, 1982). Therefore, the protocol used in the experiments presented here precludes a n antiproliferative effect of DFMO; thus, the observed antidifferentiative effect of the drug is not secondary to an effect on cell density.…”

“…All three polyamines, including spermine (the levels of which were not decreased by DFMO), prevented the inhibitory effect of DFMO on L6 myoblast differentiation. Previous work has shown that L6 myoblasts are capable of converting spermidine into putrescine (Stoscheck et al, 1982) and contain spermidineispermine-N1acetyltransferase activity (Erwin and Pegg, unpublished observations). This enzyme acetylates the aminopropyl end of either polyamine (Matsui et al, 1981).…”

“…myoblast proliferation (Stoscheck et al, 1982). This observation, plus the demonstration of very low ODC activity in myotubes, suggested that inhibition of ODC, an enzyme associated with cell proliferation, might provide conditions favorable for differentiation rather than proliferation.…”

“…Previously published results indicated that DFMO treatment of L6 myoblasts caused significant inhibition of cell proliferation by 72 hours, but no effect could be seen at 48 hours (Stoscheck et al, 1982). Those experiments were conducted with a slower-fusing clone of L6 MSA effect of stimulating myoblast differentiation (Fig.…”

Differential effect of αdifluoromethyl‐ornithine on the proliferation of balb 3T3 and chemically transformed 3T3 cells

“…This conclusion has been reached through experiments carried out with polyamine-auxotrophic mutants of bacteria (Algranati and Goldemberg, 1977;Abraham and Pihl, 1981) as well as with mammalian cells treated with specific inhibitors which block polyamine biosynthesis (Heby and Janne, 1981). However, some results suggest that the role of polyamines in cell growth is still unclear (Pardee et al, 1978;Stoscheck et al, 1982).…”