An early enlargement of the putrescine pool is required for growth in L1210 mouse leukemia cells under hypoosmotic stress.
Hypoosmotic stress is a potent inducer of ornithine decarboxylase (ODC) activity in a variety of mammalian cells, but the physiological relevance of this response has not been determined. To test whether an increased putrescine content confers a growth advantage at lower osmolarities, we compared the ability of L1210 mouse leukemia cells and of ODC-overproducing variants obtained from this cell line (D-R cells) to proliferate after a hypotonic shock (325----130 mosmol/kg). The growth rate of D-R cells at 130 mosmol/kg was greater than or equal to 5-fold higher than in L1210 cells; and unlike the ODC-overproducing strain, L1210 cells underwent up to a 90% loss of viability over time as seen after restoration of normosmotic growth conditions and by trypan blue exclusion tests. The addition of putrescine or L-ornithine stimulated the proliferation of both cell sublines up to 5-fold in a concentration-dependent manner, with a maximal effect observed at about 10 and 100 microM, respectively. Putrescine restored virtually normal growth rates in both sublines at osmolarities as low as 190 mosmol/kg. No other alpha,omega-diamine was active in that respect whereas spermidine was markedly inhibitory. Furthermore, D-R cells incubated at 130 mosmol/kg showed a marked growth inhibition by 1-aminooxy-3-aminopropane (potent ODC inhibitor to which they are resistant in isotonic media) as a result of putrescine but not spermidine depletion. Whereas ODC was strongly and rapidly induced by hypotonic shock there was a precipitous decline in S-adenosylmethionine decarboxylase activity. Putrescine synthesis and accumulation were nevertheless reduced in D-R cells incubated at 130 mosmol/kg because of a decreased availability of L-ornithine. When either putrescine or L-ornithine was added to hypotonic media, D-R cells accumulated putrescine massively for extended periods together with a reduction in spermidine and spermine contents. Putrescine transport patterns were altered by hypotonic shock, net excretion of the diamine being reduced by about 80%, with a concurrent enlargement of the intracellular pool. Finally, parental L1210 cells incubated with an irreversible inhibitor of S-adenosylmethionine decarboxylase for 24 h until hypotonic shock and supplemented with putrescine in the presence of the drug thereafter exhibited a greatly exaggerated growth stimulation by the diamine. These results demonstrate an essential role for an early increase in putrescine content in the growth adaptation of a mammalian cell line to a lower osmolarity.
Poulin
R
R
Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033.
Wechter
R S
RS
Pegg
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AE
D012437:Adenosylmethionine Decarboxylase / Q000494:pharmacology; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D004790:Enzyme Induction; D007939:Leukemia L1210 / Q000201:enzymology / Q000473:pathology*; D051379:Mice; D009952:Ornithine / Q000494:pharmacology; D009955:Ornithine Decarboxylase / Q000096:biosynthesis; D065108:Ornithine Decarboxylase Inhibitors; D009994:Osmolar Concentration; D011073:Polyamines / Q000494:pharmacology; D011700:Putrescine / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D065108:Ornithine Decarboxylase Inhibitors; D011073:Polyamines; D009952:Ornithine; D009955:Ornithine Decarboxylase; D012437:Adenosylmethionine Decarboxylase; D011700:Putrescine