Mutant strain of Chinese hamster ovary cells with no detectable ornithine decarboxylase activity.

Abstract

We previously described an arginase-deficient, polyamine-dependent Chinese hamster ovary cell line which grows in serum-free medium. From this strain we isolated a new mutant strain that has no detectable catalytic ornithine decarboxylase activity. The mutant cells contain, however, immunoreactive ornithine decarboxylaselike protein roughly in the same quantity as the parent strain. The mutant and the parent cell line strains also contain similar amounts of ornithine decarboxylase-mRNA hybridizable to a specific cDNA. If polyamines are omitted from the medium, proliferation of the mutant cells is considerably retarded and ceases in 6 to 10 days. Addition of ornithine or a-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, has no effect on these cells. Putrescine and spermidine decreased in the mutant cells to undetectable levels during polyamine starvation, whereas spermine was reduced to 1/5th of that found in the control cultures. Polyamines appear to be indispensable for the mutant strain, but this was obvious only after the amount of polyamines, found as impurities in bovine serum albumin used in the medium, was reduced by dialysis to 10-12 M. Because sera contain polyamines, the ability of the mutant strain to grow in serum-free medium is a great advantage in elucidation of the mechanisms of polyamine function.The polyamines putrescine, spermidine, and spermine are found in all cells, and they appear to be required for optimal growth of both bacteria and eucaryotes. Bacterial mutants that are completely devoid of polyamines have been isolated. These mutants have a considerably retarded rate of growth, but they can replicate indefinitely in the absence of polyamines (4). However, some yeast cell mutants unable to synthesize polyamines will gradually stop replicating (28, 31).We previously isolated a strain of Chinese hamster ovary cells (CHO), designated A7, which grows under serum-free conditions and has deficient arginase activity. Hence, it is unable to synthesize ornithine, the precursor of polyamines. If ornithine and polyamines are omitted from the growth medium, the cells are depleted of polyamines, and their growth is gradually retarded (8,21). At the same time, the cells lose most of their actin filaments and microtubules (21), and they also develop major chromosome aberrations (14,19,20). Thus, polyamine starvation of the A7 cells affects structures characteristic of eucaryotic cells. Steglich and Scheffler later isolated an ornithine decarboxylase-deficient mutant of CHO cells, designated C54, with 3% of the ornithine decarboxylase activity of the wild type (26). These cells, however, showed only a slight retardation of growth when polyamines were omitted from the growth medium based on the otherwise rich nutrient mixture F10 (25).In this communication we report isolation from A7 cells of a mutant strain designated P22 that lacks catalytically active ornithine decarboxylase enzyme and thus cannot synthesize putrescine. When external sources of polyamines were excluded, the P...

Tumor attenuation by combined heparan sulfate and polyamine depletion

“…Unfortunately, the effect of DFMO has not been as effective in vivo, where the antiproliferative activity is substantially diminished. Two major mechanisms are considered to be responsible for this discrepancy: amplification of ornithine decarboxylase, resulting in resistance to DFMO, and upregulation of polyamine import from the extracellular compartment (40)(41)(42). Although other inhibitors are available that target different enzymes in the pathway {e.g., methylglyoxal bis(guanyl hydrazone), CGP 48664, S-(5Ј-deoxy-5Ј-adenosyl)-methylthioethyl-hydroxylamine, 1-aminooxy-3-aminopropane, and ([4-amino-2-butenyl]methylamino)-5Ј-deoxyadenosine}, the enhanced uptake of polyamines caused by decreased de novo synthesis remains a problem.…”

Effects of chronic 5′-{[(Z)-4-amino-2-butenyl]methylamino)-5′-deoxy- adenosine (AbeAdo) treatment on polyamine and eIF-5A metabolism in AbeAdo-sensitive and -resistant L1210 murine leukaemia cells

“…It has long been recognized that the activity of ornithine decarboxylase (ODC), the first enzyme in the polyaminebiosynthetic pathway that forms putrescine, is necessary for mammalian cell growth (Mamont et al, 1978;Pohjanpelto et al, 1981Pohjanpelto et al, , 1985Steglich and Scheffler, 1982). Many studies have now been carried out with inhibitors of this enzyme [reviewed by Pegg and McCann (1982), Danzin and Mamont (1987) and Pegg (1988)].…”

Development of difluoromethylornithine as a chemoprevention agent for the management of colon cancer

“…Subsequent studies by the Merrell group and others, using specific ODC inhibitors (9 -14) or genetic approaches (15,16) to manipulate levels of endogenous polyamines, confirmed that amines derived from ornithine are essential for mammalian cell viability, and high levels are necessary for optimal mammalian cell growth. Corroborative results, demonstrating the importance of the polyamines for viability and growth, were also obtained in nonmammalian systems.…”