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Cadaverine supplementation during a chronic exposure to difluoromethylornithine allows an overexpression, but prevents gene amplification, of ornithine decarboxylase in L1210 mouse leukaemia cells.

ABSTRACT: We recently selected a variant mouse L1210 leukaemia-cell line overproducing ornithine decarboxylase (ODC) (EC 4.1.1.17) as a result of chronic exposure to 2-difluoromethylornithine (DFMO) in the presence of micromolar concentrations of cadaverine. These cells, now grown for more than 2 years in the presence of DFMO and cadaverine, continued to accumulate ODC-specific mRNA in an amount 30-50 times higher than that in the parental cells, yet showing practically no changes in the gene dosage for the enzyme. However, analysis of the genomic DNA with the isoschizomeric restriction enzymes HpaII and MspI revealed that the ODC sequences in the overproducer cells were hypomethylated in comparison with the parental cells. The natural polyamines (putrescine, spermidine and spermine) were almost totally replaced by cadaverine and aminopropylcadaverine. Omission of cadaverine from the culture medium, but leaving 10 mM-DFMO, resulted in an about 10-fold ODC gene amplification within a few weeks. The accumulation of ODC mRNA was enhanced by the same factor. Concomitantly, the content of the natural polyamines was almost normalized, representing about 65% of that found in the parental cells. The present results suggest that, under a given selection pressure, an overproduction of the target gene product may be primarily based on an enhanced transcriptional activity, possibly associated with hypomethylation and, if not sufficient, a secondary amplification of the active gene occurs.

SUBMITTER: Alhonen-Hongisto L

PROVIDER: S-EPMC1148461 | biostudies-other | 1987 Nov

REPOSITORIES: biostudies-other

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Project description:Racemic difluoromethylornithine (D/L-DFMO) is an inhibitor of ODC (ornithine decarboxylase), the first enzyme in eukaryotic polyamine biosynthesis. D/L-DFMO is an effective anti-parasitic agent and inhibitor of mammalian cell growth and development. Purified human ODC-catalysed ornithine decarboxylation is highly stereospecific. However, both DFMO enantiomers suppressed ODC activity in a time- and concentration-dependent manner. ODC activity failed to recover after treatment with either L- or D-DFMO and dialysis to remove free inhibitor. The inhibitor dissociation constant (K(D)) values for the formation of enzyme-inhibitor complexes were 28.3+/-3.4, 1.3+/-0.3 and 2.2+/-0.4 microM respectively for D-, L- and D/L-DFMO. The differences in these K(D) values were statistically significant ( P <0.05). The inhibitor inactivation constants (K(inact)) for the irreversible step were 0.25+/-0.03, 0.15+/-0.03 and 0.15+/-0.03 min(-1) respectively for D-, L- and D/L-DFMO. These latter values were not statistically significantly different ( P >0.1). D-DFMO was a more potent inhibitor (IC50 approximately 7.5 microM) when compared with D-ornithine (IC50 approximately 1.5 mM) of ODC-catalysed L-ornithine decarboxylation. Treatment of human colon tumour-derived HCT116 cells with either L- or D-DFMO decreased the cellular polyamine contents in a concentration-dependent manner. These results show that both enantiomers of DFMO irreversibly inactivate ODC and suggest that this inactivation occurs by a common mechanism. Both enantiomers form enzyme-inhibitor complexes with ODC, but the probability of formation of these complexes is 20 times greater for L-DFMO when compared with D-DFMO. The rate of the irreversible reaction in ODC inactivation is similar for the L- and D-enantiomer. This unexpected similarity between DFMO enantiomers, in contrast with the high degree of stereospecificity of the substrate ornithine, appears to be due to the alpha-substituent of the inhibitor. The D-enantiomer may have advantages, such as decreased normal tissue toxicity, over L- or D/L-DFMO in some clinical applications.

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Cadaverine supplementation during a chronic exposure to difluoromethylornithine allows an overexpression, but prevents gene amplification, of ornithine decarboxylase in L1210 mouse leukaemia cells.

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