scholarly journals Mouse and human ornithine decarboxylase genes. Methylation polymorphism and amplification

1987 ◽  
Vol 242 (1) ◽  
pp. 205-210 ◽  
Author(s):  
L Alhonen-Hongisto ◽  
P Leinonen ◽  
R Sinervirta ◽  
R Laine ◽  
R Winqvist ◽  
...  
Keyword(s):  
Cell Line ◽  
Ornithine Decarboxylase ◽  
Ehrlich Ascites Carcinoma ◽  
Myeloma Cell ◽  
Restriction Pattern ◽  
Restriction Endonucleases ◽  
Cell Leukaemia ◽  
Chromosome 2 ◽  
Ehrlich Ascites ◽  
Mouse Leukaemia

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.

scholarly journals Tumourigenicity, cell-surface glycoprotein changes and ornithine decarboxylase gene pattern in Ehrlich ascites-carcinoma cells

1985 ◽  
Vol 229 (3) ◽  
pp. 711-715 ◽  
Author(s):  
L Alhonen-Hongisto ◽  
A Kallio ◽  
R Sinervirta ◽  
O A Jänne ◽  
C G Gahmberg ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Ornithine Decarboxylase ◽  
Ehrlich Ascites Carcinoma ◽  
Tumour Cells ◽  
Carcinoma Cells ◽  
Surface Glycoprotein ◽  
Cell Surface Glycoprotein ◽  
Phenotypic Changes ◽  
Ehrlich Ascites

We selected a 2-difluoromethylornithine-resistant Ehrlich ascites-carcinoma cell line that grows in the presence of 20 mM-difluoromethylornithine. These cells contain 10-20 times the normal amount of hybridizable sequences for ornithine decarboxylase (EC 4.1.1.17) in their genomic DNA. We used these gene-amplified cells, their revertant counterparts (grown in the absence of the drug after an established gene amplification) and tumour cells grown in the presence of putrescine to investigate the changes of ornithine decarboxylase gene pattern and simultaneously occurring phenotypic changes, such as tumourigenicity and the expression of cell-surface glycoproteins. In the tumour cells reverted back to the normal gene frequency, not only did the amplified sequences disappear, but there were also signs of gene re-arrangements seen as a ‘gene jump’, when a signal evidently moved to a heavier restriction fragment. Similar gene re-arrangement likewise occurred in cells exposed to putrescine. Although the wild-type tumour cells and the gene-amplified cells readily grew in the peritoneal cavity of mice, the revertant cells and the putrescine-treated cells had lost their tumourigenicity in mice. Gene-amplified tumour cells and the revertant cells showed distinct changes in their surface glycoprotein pattern in comparison with the parental cell line. These findings indicate that alterations of ornithine decarboxylase gene pattern/dosage may be associated with phenotypic changes possibly related to the tumourigenicity of these carcinoma cells.

scholarly journals Polyamines in mycoplasmas and in mycoplasma-infected tumour cells

1982 ◽  
Vol 202 (1) ◽  
pp. 267-270 ◽  
Author(s):  
L Alhonen-Hongisto ◽  
P Veijalainen ◽  
C Ek-Kommonen ◽  
J Jänne
Keyword(s):  
Ornithine Decarboxylase ◽  
Ehrlich Ascites Carcinoma ◽  
Carcinoma Cells ◽  
Leukemia Cells ◽  
Animal Cells ◽  
Irreversible Inhibitor ◽  
Ehrlich Ascites ◽  
Infected Cells ◽  
L1210 Mouse Leukemia ◽  
High Concentrations

Three out of four different mycoplasma strains analysed for the polyamine contents contained relatively high concentrations of putrescine, cadaverine, spermidine and spermine. In addition to ornithine decarboxylase (EC 4.1.1.17) activity, the mycoplasmas also exhibited comparable or higher lysine decarboxylase (EC 4.1.1.18) activity fully resistant to the action of 2-difluoromethylornithine, an irreversible inhibitor of eukaryotic ornithine decarboxylase. 2-Difluoromethylornithine did not modify the polyamine pattern of actively growing mycoplasmas. Ehrlich ascites carcinoma cells and L1210 mouse leukemia cells infected with any of the four mycoplasma strains contained, in addition to putrescine, spermidine and spermine, and also easily measurable concentrations of cadaverine; the latter diamine was absent in uninfected cultures. When the infected cells were exposed to difluoromethylornithine, the accumulation of cadaverine was markedly enhanced. The modification of cellular polyamine pattern by mycoplasmas, especially in the presence of inhibitors of eukaryotic ornithine decarboxylase, could conceivably be used as an indicator of mycoplasma infection in cultured animal cells.

scholarly journals Long-term reduction of amplified ornithine decarboxylase sequences in human myeloma cells

1995 ◽  
Vol 310 (1) ◽  
pp. 299-303
Author(s):  
J Wahlfors ◽  
S Myöhänen ◽  
V P Korhonen ◽  
L Alhonen ◽  
J Jänne
Keyword(s):  
Enzyme Activity ◽  
Cell Line ◽  
Ornithine Decarboxylase ◽  
Copy Number ◽  
Methylation Status ◽  
Gene Copy Number ◽  
Myeloma Cell ◽  
Gene Copy ◽  
Myeloma Cell Line ◽  
Myeloma Cells

(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)

Difluoromethylornithine-induced amplification of ornithine decarboxylase genes in Ehrlich ascites carcinoma cells

1985 ◽  
Vol 126 (2) ◽  
pp. 734-740 ◽  
Author(s):  
L. Alhonen-Hongisto ◽  
A. Kallio ◽  
R. Sinervirta ◽  
P/ Seppänen ◽  
K.K. Kontula ◽  
...  
Keyword(s):  
Ornithine Decarboxylase ◽  
Ehrlich Ascites Carcinoma ◽  
Carcinoma Cells ◽  
Ehrlich Ascites

scholarly journals Certain changes in ornithine decarboxylase gene methylation accompany gene amplification

1991 ◽  
Vol 279 (2) ◽  
pp. 435-440 ◽  
Author(s):  
J Wahlfors
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Gene Amplification ◽  
Ornithine Decarboxylase ◽  
Myeloma Cell ◽  
Parental Cell ◽  
Resistant Cell ◽  
Methylation Pattern ◽  
Parental Cell Line ◽  
Flanking Region

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.

scholarly journals Polyamines and their biosynthetic enzymes in Ehrlich ascites-carcinoma cells. Modification of tumour polyamine pattern by diamines

1977 ◽  
Vol 166 (1) ◽  
pp. 89-94 ◽  
Author(s):  
A Kallio ◽  
H Pösö ◽  
S K Guha ◽  
J Jänne
Keyword(s):  
Ornithine Decarboxylase ◽  
Ehrlich Ascites Carcinoma ◽  
Carcinoma Cells ◽  
Molar Ratio ◽  
Decarboxylase Activity ◽  
Complete Disappearance ◽  
Spermidine Synthase ◽  
Spermine Synthase ◽  
Ornithine Decarboxylase Activity ◽  
Ehrlich Ascites

1. Ehrlich ascites-carcinoma cells contained relatively high concentrations of spermidine and spermine, but the putrescine content of the washed cells was less than 10% of that of higher polyamines. 2. Ascites-tumour cells likewise exhibited high activities of L-ornithine decarboxylase (EC 4.1.1.17), S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50), spermidine synthase (EC 2.5.1.16) and spermine synthase. 3. During the first days after the inoculation, the polyamine pattern of the ascites cells was characterized by a high molar ratio of spermidine to spermine, which markedly decreased on aging of the cells. 4. Various diamines injected into mice bearing ascites cells rapidly and powerfully decreased ornithine decarboxylase activity in the carcinoma cells, apparently through a mechanism that was not a direct inhibition of the enzyme in vitro. Cadaverine (1,5-diaminopentane) and 1,6-diaminohexane were the most potent inhibitors of ornithine decarboxylase among the amines tested. 5. Chronic treatment of the mice with diamines resulted in a virtually complete disappearance of ornithine decarboxylase activity, and after 24h a significant decline in spermidine accumulation. 6. Cadaverine appeared to be an especially suitable compound for use as an inhibitor of the synthesis of higher polyamines, at least in Ehrlich ascites cells, since this diamine also acted as a competitive inhibitor for putrescine in the spermidine synthase reaction without being incorporated into the higher polyamines.

Chronic exposure to dexamethasone induces hypomethylation of ornithine decarboxylase genes in a human myeloma cell line

FEBS Letters ◽  
1987 ◽  
Vol 215 (1) ◽  
pp. 68-72 ◽  
Author(s):  
Pekka Leinonen ◽  
Leena Alhonen-Hongisto ◽  
Raija Laine ◽  
Olli A. Jänne ◽  
Juhani Jänne
Keyword(s):  
Cell Line ◽  
Ornithine Decarboxylase ◽  
Chronic Exposure ◽  
Myeloma Cell ◽  
Myeloma Cell Line ◽  
Human Myeloma Cell Line ◽  
Human Myeloma Cell

scholarly journals Mechanism of Antiangiogenic and Antioxidant Activity of Newly Synthesized CAMBA in Ehrlich Ascites Carcinoma-Bearing Mice

2021 ◽  
Vol 33 (10) ◽  
pp. 2465-2471
Author(s):  
Nada I. Abou-Taleb ◽  
Ola A. Elblasy ◽  
Esraa A. Elbesoumy ◽  
Haidy I. Basuny ◽  
Esraa A. Elhamadi ◽  
...  
Keyword(s):  
Cell Line ◽  
Tumor Volume ◽  
Carcinoma Cell ◽  
Ehrlich Ascites Carcinoma ◽  
Liver Carcinoma ◽  
Hep G2 ◽  
Antiangiogenic Activity ◽  
Ehrlich Ascites ◽  
Tnf Α ◽  
Ic50 Value

The aim of present study was to evaluate antiangiogenic activity of newly synthesized caffeic acid methyl benzoate amide (CAMBA) in EAC-bearing mice. The IC50 value of CAMBA against the Hep-G2 liver carcinoma cell line was calculated. Adult albino mice weighing 25 ± 5 g was used to assess the antiangiogenic activity of CAMBA (25 and 50 mg/k.b.w.) in EAC-bearing mice. IC50 CAMBA against the Hep-G2 cell line equals 52.8 μg/mL. The daily oral administration of CAMBA at concentrations of 25 and 50 mg/kg.b.w. for 30 days to EAC-bearing mice resulted in a significant improvement in tumor volume and tumor weight, ALT, AST, ALP, MMP-2 and -9, TNF-α, NOx, TBARs, GSH, CAT, SOD, GPx and VEGF-C gene expression in EAC-bearing mice. Furthermore, CAMBA almost normalized these effects in liver histoarchitecture. The biochemical, histological and ultrasound examinations of our study suggested that CAMBA have antiangiogenic activity in EAC-bearing mice.

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