Human myeloma cells acquire resistance to difluoromethylornithine by amplification of ornithine decarboxylase gene.
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ABSTRACT:
SUBMITTER: Leinonen P
PROVIDER: S-EPMC1147683 | biostudies-other | 1987 Feb
REPOSITORIES: biostudies-other
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Project description:α-Difluoromethylornithine (DFMO) is currently used in chemopreventive regimens primarily for its conventional direct anticarcinogenesic activity. However, little is known about the effect of ornithine decarboxylase (ODC) inhibition by DFMO on antitumor immune responses. We showed in this study that pharmacologic blockade of ODC by DFMO inhibited tumor growth in intact immunocompetent mice, but abrogated in the immunodeficient Rag1(-/-) mice, suggesting that antitumor effect of DFMO is dependent on the induction of adaptive antitumor T cell immune responses. Depletion of CD8(+) T cells impeded the tumor-inhibiting advantage of DFMO. Moreover, DFMO treatment enhanced antitumor CD8(+) T cell infiltration and IFN-γ production and augmented the efficacy of adoptive T cell therapy. Importantly, DFMO impaired Gr1(+)CD11b(+) myeloid-derived suppressor cells (MDSCs) suppressive activity through at least two mechanisms, including reducing arginase expression and activity and inhibiting the CD39/CD73-mediated pathway. MDSCs were one primary cellular target of DFMO as indicated by both adoptive transfer and MDSC-depletion analyses. Our findings establish a new role of ODC inhibition by DFMO as a viable and effective immunological adjunct in effective cancer treatment, thereby adding to the growing list of chemoimmunotherapeutic applications of these agents.
| S-EPMC4707077 | biostudies-literature
Project description:We previously isolated and characterized a human myeloma cell line overproducing ornithine decarboxylase (ODC) due to gene amplification [Leinonen, Alhonen-Hongisto, Laine, Jänne & Jänne (1987) Biochem. J. 242, 199-203]. We have now employed the PCR combined with reverse transcription to determine semiquantitatively ODC gene dosage and the amounts of heterogeneous nuclear (hn) RNA and of mature mRNA of the enzyme in parental and alpha-difluoromethylornithine-resistant human myeloma cells. Experiments with dilution series revealed that the ODC gene copy number and the amount of both hnRNA and mRNA were increased to the same extent (about 100-fold) in the resistant cells. Similar dot-blot analyses of ODC-specific genomic DNA and total RNA indicated that the ODC gene copy number was increased by a factor of 380 and the amount of ODC mRNA by a factor of 700. Our results indicate that the PCR combined with reverse transcription is at least as useful as blot analyses to give semiquantitative assessment of the amounts of specific DNA or RNA sequences. In addition, the use of the PCR enables the analysis of minute sample amounts in extremely short time.
| S-EPMC1151427 | biostudies-other
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.
| S-EPMC1223689 | biostudies-other
Project description:The mechanism of inactivation of rodent ornithine decarboxylase by alpha-difluoromethylornithine (DFMO) was studied using the inhibitor labelled with 14C in both the 1 and the 5 positions. [1-14C]DFMO was a substrate and was decarboxylated by the enzyme yielding 14CO2. A radioactive metabolite derived from [5-14C]DFMO was bound to the enzyme, and the extent of binding paralleled the irreversible inactivation of ornithine decarboxylase. The partition ratio of decarboxylation to binding was approx. 3.3. These results provide support for the postulated mechanism of action of DFMO [Metcalf, Bey, Danzin, Jung, Casera & Vevert (1978) J. Am. Chem. Soc. 100, 2551-2553], in which enzymic decarboxylation of the inhibitor leads to the generation of a conjugated imine, which then alkylates a nucleophilic residue on the enzyme.
| S-EPMC1147558 | biostudies-other
Project description: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.
| S-EPMC1148461 | biostudies-other
Project description:(1) Human myeloma cell line Sultan, resistant to 20 mM difluoro-methylornithine (DFMO) owing to ornithine decarboxylase (ODC) gene amplification, was grown in the absence of DFMO for a period of 10 months. The gene copy number and methylation status of the ODC gene were monitored after withdrawal of DFMO. Moreover, levels of ODC mRNA, immunoreactive ODC protein, ODC activity and polyamine levels were recorded recurrently during the course of the study. (2) The results revealed that ODC gene copy number started to decrease after 4 weeks growth without DFMO, to a final level of less than 30% of the original gene dosage. The methylation status of the ODC gene, however, remained almost unaltered, displaying only a modest increase in methylation after 10 months without DFMO. The amount of ODC message dropped very rapidly to 75% of the original value, then started to decrease in a gene copy-number-dependent manner. The amount of ODC protein closely followed the levels of mRNA during the study, whereas the ODC activity, after a transient increase during the first week, decreased to half of the original level after 4 weeks. Between 6 and 16 weeks ODC activity stabilized to a fifth of the original value and no more changes were detected during the subsequent period of observation. (3) Due to the grossly elevated ODC enzyme activity, levels of putrescine and spermidine first peaked and then stabilized at 6 weeks after DFMO withdrawal. The final spermidine level was comparable with that of the parental Sultan cell line with only one copy of active ODC gene. However, putrescine content was strikingly elevated, being stabilized to a level that was 20 times higher than in parental cells. Spermine concentration was practically unchanged during the study. (4) According to the results obtained in this study, the abnormal level of ODC expression in human myeloma cells is suppressed partially at the level of transcription or post-transcriptionally, but it is not due to increased methylation of the gene. The major regulatory mechanism to compensate for a highly elevated ODC expression was modulation of the enzyme activity. After 10 months without DFMO, the cells still displayed about 20 times higher ODC activity and putrescine concentration than the myeloma cell line with a single copy of the ODC gene. They did not, however, show any signs of growth retardation or other features different from the parental cells.(ABSTRACT TRUNCATED AT 400 WORDS)
| S-EPMC1135887 | biostudies-other
Project description:The ornithine decarboxylase (ODC; EC 4.1.1.17) gene in parental, dexamethasone-resistant and 2-difluoromethylornithine (DFMO)-resistant human IgG-myeloma-cell lines was studied with the aid of methylation-sensitive restriction endonucleases and probes recognizing different parts of the gene. In all cell lines the promoter region of the ODC gene appeared to be heavily methylated, whereas the first long intron was unmethylated. Methylation analyses of several clones from the parental cell line revealed that these cells are heterogeneous with respect to the methylation status of the ODC gene, whereas all clones from DFMO-resistant cell lines displayed the same methylation pattern. Two of the parental clones represented a hypomethylated type very close to that exclusively found among the DFMO-resistant clones with ODC gene amplification. This typical methylation pattern was due to decreased methylation of a few CCGG sequences in the 3'-flanking region of the gene. It is possible that this kind of hypomethylation favours the initiation of the gene-amplification process in certain individual cells. This hypothesis was supported by the finding that no hypomethylation was present in the ODC gene of another human myeloma cell line that had acquired resistance to DFMO without gene amplification. In a dexamethasone-resistant cell line that overproduced ODC mRNA at normal gene dosage there were some minor differences between the methylation pattern of the ODC gene of different clones, but no such hypomethylation could be found in clones from the parental cell line. In dexamethasone-resistant cells the ODC gene was hypomethylated around the two HpaII sites and three CfoI sites in the coding region and also, as well as in cells with amplified ODC sequences, in the 3'-flanking region of the gene. Some hypomethylation in the distant 5'-flanking region was also observed.
| S-EPMC1151623 | biostudies-other
Project description:With the use of the isoschizomeric restriction endonucleases HpaII and MspI, we found that mouse tumour ornithine decarboxylase (ODC; EC 4.1.1.17) genes are extensively methylated. ODC genes in L1210 mouse leukaemia cells were apparently more methylated than in Ehrlich ascites carcinoma, as revealed by the use of HpaII endonuclease, yet the digestion of genomic DNA isolated from these two murine tumour cell lines with MspI, which cleaves at a CCGG sequence, also with internally methylated cytosine, resulted in an apparently identical restriction pattern. It is possible that the amplification of ODC genes in Ehrlich ascites-carcinoma cells in response to 2-difluoromethylornithine (DFMO) was associated with hypomethylation, or that less-methylated genes were amplified. A human myeloma (Sultan) cell line only revealed three separate hybridization signals when cleaved with HpaII. One of these signals was amplified under the pressure of DFMO. When cleaved with MspI, these three HpaII fragments disappeared and were replaced by a double signal of 2.3-2.4 kilobase-pairs (kbp) in size. The amplified ODC sequences in the Sultan myeloma cell line apparently originated from chromosome 2, as indicated by a unique hybridization signal in a 5.8 kbp HindIII fragment specific for the human ODC locus on chromosome 2. A comparison of different human cells, the Sultan myeloma, a lymphocytic B-cell leukaemia (Ball), normal mononuclear leucocytes and leucocytes obtained from leukaemia patients, revealed interesting differences in the methylation of ODC genes. The use of two restriction endonucleases (HpaII and CfoI), the cleavage site for both of which contains a CG sequence and which only cleave when cytosine is unmethylated, indicated that ODC genes in the lymphocytic leukaemia cells were much less methylated than those in the normal leucocytes or in the Sultan cells.
| S-EPMC1147684 | biostudies-other
Project description:Cultured mouse L1210 leukaemia cells treated with DL-2-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase (EC 4.1.1.17), in the presence of micromolar concentrations of cadaverine, started to overproduce ornithine decarboxylase after an exposure of several weeks. The more than 60-fold excess of the enzyme protein in the drug-treated cells apparently resulted from a strikingly enhanced accumulation of mRNA for the enzyme associated with only a modest (about 2-fold) gene amplification.
| S-EPMC1152922 | biostudies-other
Project description:We have studied the regulation of the expression of ornithine decarboxylase with the aid of transgenic mice harbouring either functional human ornithine decarboxylase genes or the mouse ornithine decarboxylase promoter-driven chloramphenicol acetyltransferase fusion gene in their genome. We used three different stimuli which are well known to enhance ornithine decarboxylase activity in their appropriate target tissues: (i) testosterone in female kidney, (ii) a phorbol ester in epidermis and (iii) partial hepatectomy in liver. Endogenous mouse ornithine decarboxylase activity was strikingly stimulated in response to these treatments. Even though containing the 5' flanking region of the mouse ornithine decarboxylase gene, known to possess full promoter activity, the chloramphenicol acetyltransferase reporter gene was entirely insensitive to any of these stimuli. The human transgene-derived ornithine decarboxylase activity in kidney was unaffected by testosterone treatment, but responded in skin to application of the phorbol ester and likewise was clearly enhanced in regenerating liver. Although mouse endogenous ornithine decarboxylase mRNA levels were distinctly elevated after testosterone, this treatment did not influence the accumulation of the human transgene-derived mRNA. The phorbol ester enhanced the accumulation of mouse endogenous ornithine decarboxylase mRNA and also that derived from the human transgene; however, the enzyme activity was stimulated in regenerating liver without appreciable changes in the levels of endogenous or transgene-derived message. Our present results strongly emphasize the central role of the coding sequence or ornithine decarboxylase gene in the induction of the enzyme activity.
| S-EPMC1134203 | biostudies-other