Pierre S. Mamont

Anti-proliferative properties of DL-α-difluoromethyl ornithine in cultured cells. A consequence of the irreversible inhibition of ornithine decarboxylase

Abstract Both DL-α-Methyl orinithine (α-MeOrn), a competitive inhibitor of ornithine decarboxylase (ODC) (L-orinithine carboxy-lyase EC 4.1.1.17) and DL-α-difluoromethyl orinithine (α-DF MeOrn), a catalytic irreversible inhibitor of this enzyme, decrease the concentrations of putrescine and spermidine but not of spermine in rat hepatoma (HTC) cells and in mouse leukemia cells cultured in vitro . The depletion of the two amines is followed by a striking decrease in the rate of cell replication in both cell lines. Growth of human prostate adenoma cells is inhibited by α-DF MeOrn but not by α-MeOrn, illustrating the greater effectiveness of the irreversible inhibitor. These findings again support an essential function for putrescine and spermidine in cell division processes.

Biphasic induction of ornithine decarboxylase and putrescine levels in growing HTC cells.

Abstract A biphasic induction of ornithine decarboxylase with concomitant increase of intracellular putrescine was seen in growing rat hepatoma cells during each generation period. In non-growing HTC cells no coordinate accumulation of intracellular putrescine followed the unique induction of DDC by dilution into fresh serum-deprived medium. The data together suggest that the biphasic increases of ODC activity occur just before and after DNA synthesis and that a growing HTC cell has a finely regulated cycle of ODC activity. Finally, ODC activity may not always correlate with the intracellular putrescine levels.

Regulation of ornithine decarboxylase by ODC-antizyme in HTC cells.

Abstract Low concentrations of putrescine (10 −5 M) blocked ornithine decarboxylase (ODC) in rat hepatoma (HTC) cells in culture, but the lower homologue of putrescine, 1, 3 diaminopropane, had no effect on ornithine decarboxylase at 10 −5 M. Higher concentrations of both putrescine and 1, 3 diaminopropane induced approximately the same amount of soluble ODC antizyme type inhibitor. When concentrated dialyzed supernatants of cells grown in 10 −5 M putrescine were treated with 250 mM NaCl and chromatographed on a superfine Sephadex G-75 column, both ODC and inhibitor were recovered. Spermidine, spermine and cadaverine also induced the inhibitor suggesting a low specificity of induction by amines.

Effect of α-methyl ornithine on ornithine decarboxylase activity of rat hepatoma cells in culture

Abstract DL-α-methyl ornithine (α-MeOrn), a potent competitive inhibitor of ornithine decarboxylase (L-ornithine carboxy-lyase EC 4.1.1.7) (ODC), caused an increase of ODC activity of rat hepatoma tissue culture cells cultured in the presence of the compound. The effect was seen only when there was newly synthesized ODC present. The α-MeOrn mediated increase of ODC activity was independent of RNA synthesis and was expressed at a post-transcriptional level. ODC half-life, measured after cycloheximide treatment, was increased by a factor of three in the presence of α-MeOrn, suggesting the compound slowed enzyme degradation perhaps by its ability to enter the active site.

The pleiotypic response in mammalian cells: Search for an intracellular mediator

Abstract The reactions included in the definition of this regulatory program, called the “pleiotypic response”, are very similar to those under “stringent” control in bacteria. The latter appears to be mediated by a novel nucleotide, ppGpp, and we therefore assayed for the presence of this nucleotide in cultured fibroblasts under a variety of conditions of growth restriction which elicit the pleiotypic response. We were unable to detect it in any of these circumstances and we conclude that ppGpp is not the mediator of the pleiotypic response, at least in the cell lines studied.

Marked and prolonged inhibition of mammalian ornithine decarboxylase in vivo by esters of (E)-2-(fluoromethyl)dehydroornithine

(E)-2-(fluoromethyl)dehydroornithine, a new enzyme-activated irreversible inhibitor of ornithine decarboxylase (ODC) is no more effective than alpha-difluoromethylornithine (DFMO) at inhibiting polyamine biosynthesis in rat hepatoma tissue culture (HTC) cells and in rat organs even though its potency is over 15 times higher than that of DFMO in vitro. The methyl, ethyl, octyl and benzyl esters of (E)-2-(fluoromethyl)dehydroornithine were synthesized as potential prodrugs of the amino acid. When tested at concentration equivalent to the Ki value of the amino acid, they are devoid of ODC-inhibitory property. When measured 6 hr after its addition to the HTC cell culture medium, the absorption of methyl ester was 20 times higher than that of the parent amino acid or that of DFMO, and was accompanied by a more marked intracellular accumulation of (E)-2-(fluoromethyl)dehydroornithine than that achieved by the addition of the parent amino acid. The methyl ester used at 10 times lower concentrations is as effective as its parent amino acid or as DFMO at inhibiting polyamine biosynthesis in HTC cells. Similarly, the methyl and the ethyl esters of (E)-2-(fluoromethyl)dehydroornithine used at 10 times lower doses are as effective as the parent amino acid and as DFMO at inhibiting ODC in the ventral prostate of rat, 6 hr after oral administration. All the esters of (E)-2-(fluoromethyl)dehydroornithine produce a particularly long duration of ODC inhibition in the ventral prostate and in the testes. Repeated administration (25 mg/kg given once a day by gavage) of the methyl ester of (E)-2-(fluoromethyl)dehydroornithine for 8 days to rats results in a constant 80% inhibition of ODC over a 24-hr period, accompanied by a 90% decrease of putrescine and spermidine concentrations in the ventral prostate.

Fluorine-Containing Polyamines: Biochemistry and Potential Applications

Investigations with the fluorinated spermidine analogues show clearly that these compounds have significant potential for studying the metabolism and functions of the polyamines. However, the biochemical and biological properties of these analogues are dissimilar. This is due to the influence of the fluorine substituent(s) on the basicity of the amine function proximal to the fluoromethylene group, this effect being amplified by geminal disubstitution. The monofluorinated spermidine analogues compare well with the natural amine in their ability to regulate the expression of the decarboxylase enzymes, to be substrates of spermine synthase and to support growth of polyamine-deficient cells. It is also likely that 6-monofluorospermine, formed biochemically in situ, shares with spermine similar functions. These findings raise the possibility of using these spermidine analogues to study the metabolism and pharmacology of polyamines in vivo but also to provide more insight into the regulatory role of spermidine in ODC and SAM-DC expression. Another potential application may be the use of these analogues as probes in tumor imaging and therapy control. This indication has been inferred by studies in tumor-bearing animals, using 19F-NMR spectroscopy determination of tissue fluorospermidine and fluorospermine, formed biochemically from the precursors 2-fluoro or 2,2-difluoroputrescine, and which demonstrate preferential accumulation in tumor versus normal tissue. Finally, these monofluorinated spermidine analogues may exert beneficial effects in pathological states associated with polyamine deficiency. These diseases remain however to be identified. Among the difluorinated spermidine analogues, 7,7-difluorospermidine possesses the most interesting properties. This spermidine analogue still possesses ODC and SAM-DC repressing activities although at much higher concentration than spermidine. More importantly it is a potent inhibitor of spermine synthesis both in cultured cells and in vivo due to its efficient competition with spermidine in the spermine synthase reaction. This compound not only depletes tumor cell of its spermine content but, in addition, appears to exert by itself and/or via 6,6-difluorospermine, the product of its metabolism, polyamine antagonist effects. Combined with MAP but also with DFMO, two potent irreversible inhibitors of ODC which block the synthesis of the natural endogenous polyamines, 7,7-difluorospermidine causes an immediate decrease of viability in cultured HTC cells and promotes tumor regression and stabilization in hepatoma-bearing rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Aspects of polyamine metabolism in relationship to S-adenosylmethionine metabolism revealed by the use of α-difluoromethylornithine, an effective inhibitor of putrescine biosynthesis

The polyamines, spermidine and spermine, and their diamine precursor, putrescine, play important but not yet precisely defined regulatory functions in normal and abnormal cellular growth and cell differentiation (For recent reviews, see Heby, 1981; Williams-Ashman and Canellakis, 1979). This fact has recently received extensive support from studies on the biochemical consequences of polyamine deficiency in prokaryotes and eukaryotes. This deficiency has been achieved either by mutation (Tabor et al., 1980; Cohn et al., 1980; Morris, 1981) or by specific inhibition of the biosynthetic enzymes (For reviews, see Mamont et al., 1978; Heby and Janne, 1981). Among inhibitors of polyamine biosynthesis, DL-α-difluoromethylornithine (DFMeOrn), an enzyme-activated irreversible inhibitor of L-ornithine decarboxylase (ODC) (Metcalf et al., 1978), the rate-limiting enzyme of the polyamine biosynthetic pathway, has attracted much attention. This is due to its specific mode of inhibition, its organ-selectivity in vivo (Danzin et al., 1979a,b) and absence of toxicity. DFMeOrn has proved to be valuable for demonstrating that continuous polyamine synthesis is required in order to maintain maximum rates of abnormal and normal mammalian cell proliferation both in vitro (Mamont et al., 1978; Heby and Janne, 1981) and in vivo (Prakash et al., 1980).