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Biomedical subjects

T L Boehm

Publications and source records attributed to T L Boehm.

At least 37 records · Page 2Linked to original sources
PubMed1983

Alteration of enzymatic DNA methylation by chemical carcinogens.

It is generally accepted that the DNA methylation pattern in a particular cell type is stable over the cell generations and is clonally inherited by a semiconservative mechanism in which the methylation of the progeny strand is determined by the 5-methylcytosine residue present in the parental strand. In our experiments, we have been able to show that the chemical carcinogens MNU, MNNG, and L-ethionine affect the methylation process either by modification of the DNA substrate or by interference at the cofactor level. The changes introduced into the cellular methylation pattern persist and are inherited in the progeny cells regardless of whether the carcinogens or their adducts in the DNA persist in the cell or not. The carcinogen-induced hypomethylation correlates with the increased transcriptional complexity of the nRNA of treated cells, indicating the initiation of transcription at sites previously inactive. This means that chemical carcinogens can cause, by interference with the process of DNA methylation, stable changes in the program of genetic expression; the possibility that such changes are related to the process of initiation of carcinogenesis should not be overlooked.

Animals↗
PubMed1983

Aberrant de novo methylation of DNA after treatment of murine cells with N-acetoxy-N-2-acetylaminofluorene.

The ultimate chemical carcinogen N-acetoxy-N-2-acetylaminofluorene inhibits the enzymatic methylation of newly replicated DNA in cultured mouse P815 cells in a dose-dependent manner. After removal of the carcinogen, a significant de novo methylation of newly replicated DNA takes place, the level of methylation being higher than in control cultures. This aberrant methylation persists in the absence of N-acetoxy-N-2-acetylaminofluorene in subsequent cell cycles. Cellular cloning experiments suggest that N-acetoxy-N-2-acetylaminofluorene treatment leads to two distinct sets of cells, one with a higher and another with a lower extent of enzymatic methylation of DNA, contrasting to the apparent uniform methylation pattern in control clones.

2-Acetylaminofluorene↗
PubMed1983

Enzymatic methylation of inverted DNA repeats in the vicinity of the mouse beta-major globin gene.

This paper examines the extent of enzymatic methylation in 5'-CCGG sequences of inverted repeats in DNA isolated from adult liver and bone marrow of DBA/2 mice, with special attention to the methylation of such sequences in the vicinity of the beta-major globin gene. Two thirds of inverted repeats contain 5'-AGCT and 5'-CCGG sequences, as found by a method based on the capability of inverted repeats of forming intramolecular duplexes under the conditions of "zero-time" reassociation. Methylation in internal cytosines of 5'-CCGG sequences of inverted DNA repeats differs between bone marrow and liver tissues. The beta-major globin gene was found in DNA covalently linked to inverted repeats. The enzymatic methylation of inverted repeats neighbouring the beta-major globin gene differs at HpaII recognition sites; the DNA of bone marrow tissue, in which this gene is expressed, is less methylated at such sites as compared to liver DNA.

Animals↗
PubMed1982

Inhibition of thymidylate synthase by hydroxyurea in rapidly proliferating P815 mastocytoma cells.

The incorporation of [14C]deoxycytidine, [3H]deoxyuridine, and [3H]thymidine, respectively into pyrimidine bases of DNA has been measured in rapidly proliferating P815 mouse mastocytoma cells in the presence of hydroxyurea. The incorporation of [14C]deoxycytidine-derived radioactivity into DNA cytosines is increased when compared to the incorporation into DNA thymines. The [3H]deoxyuridine-derived radioactivity is incorporated solely into DNA thymines and this incorporation is inhibited by hydroxyurea in a dose-dependent manner. This suggests an inhibitory effect of hydroxyurea on the thymidylate synthase which was proved in experiments in which the conversion of deoxyuridine monophosphate into deoxythymidine monophosphate catalysed by a crude enzyme preparation from P815 cells was inhibited in the presence of hydroxyurea. Enzymatic DNA methylation as measured by the conversion of incorporated [14C]deoxycytidine into 5-methylcytosines was not affected by hydroxyurea.

Animals↗
PubMed1982

Elevated level of enzymatic DNA methylation in cells treated with 1-beta-D-arabinofuranosylcytosine.

When mouse P815 mastocytoma cells are cultured in the presence of 1-beta-D-arabinofuranosylcytosine (ara-C), a dose-dependent inhibition of cell proliferation and DNA synthesis can be observed. The DNA synthesized in the presence of ara-C is more highly enzymatically methylated than that of controls as measured by conversion of incorporated [14C]deoxycytidine-derived radioactivity into 5-methylcytosines. Analysis of DNAs by means of the bacterial restriction enzyme HpaII and its isoschizomer MspI revealed an aberrant DNA methylation pattern after ara-C treatment. The hypermethylation of DNA persisted also in the cell cycle following the treatment in the absence of ara-C. No such changes in DNA methylation patterns following ara-C treatment were observed in P815/ara-C, a subclone of P815 cells resistant to ara-C.

Animals↗
PubMed1981

Hypomethylation of DNA in Raji cells after treatment with N-methyl-N-nitrosourea.

Human Raji lymphoblast-like cells were propagated in the presence of various concentrations of N-methyl-N-nitrosourea (MNU) and the degree of enzymatic methylation of newly synthesized DNA was analysed by two independent methods. The overall extent of enzymatic DNA methylation was measured on the basis of [14C]deoxycytidine derived radioactivity incorporated into DNA 5-methylcytosine and cytosine residues. Enzymatic methylation of internal cytosines at 5'-CCGG-3' sequences of Raji DNA was analysed by use of the bacterial restriction enzyme HpaII and its isoschizomer MspI. The data obtained by both methods indicate that the treatment with MNU causes a lower level of enzymatic methylation of newly synthesized DNA. This lower extent of DNA methylation persists in the absence of the carcinogen in the cell cycles following the treatment.

Burkitt Lymphoma↗
PubMed1981

Elevated transcriptional complexity and decrease in enzymatic DNA methylation in cells treated with L-ethionine.

This paper analyzes the extent of enzymatic methylation of DNA in P815 cells cultured for several cell cycles in the presence of 1 mM L-ethionine by use of the bacterial restriction enzymes HpaII, MspI, HhaI, HaeII, and AvaI. The results obtained suggest that the extent of methylation of DNA is decreased during L-ethionine treatment and that the degree of this inhibition varies among different methylation sites. The nucleotide sequence complexity of nuclear RNA in P815 cells was analyzed by excess hybridization with tracer amounts of nick-translated nonrepetitive P815 [3H]DNA sequences. About 6.2% of the nonrepetitive sequences form hybrids with nuclear RNA of controls cells, whereas 8.8% are complementary to nuclear RNA from L-ethionine-treated cells. Thus, the decrease in enzymatic DNA methylation correlates with the presence of new transcriptional products suggesting that this modification process inversely correlates with the initiation of transcription.

Animals↗
PubMed1980

Enzymatic hypermethylation of DNA in mouse-mouse somatic cell hybrids.

Somatic cell hybrids between mouse L fibroblasts (A9 cells) and Ehrlich ascites tumour cells were constructed by use of poly(ethyleneglycol). The hybrids were selected in hypoxanthine/aminopterin/thymidine medium and morphologically different syncaryons were isolated by a micromanipulation. They were simultaneously analysed for their tumourogeneity, a release of certain proteins into the culture medium, the activity of pyrimidine salvage pathways, and the extent of enzymatic DNA methylation. None of these hybrids gave a rise of tumours if transplanted into DBA/2 mice. Two of the hybrids released a protein immunologically cross-reacting with antibody against C-peptide of human proinsulin. Activities of pyrimidine salvage pathways as measured by incorporation of [14C]deoxycytidine into DNA cytosine and thymine, respectively, are different in the analyzed cells. Enzymatic DNA methylation in somatic cell hybrids was significantly elevated as compared to the parental cell lines.

Animals↗
PubMed1980

Impaired restriction endonuclease cleavage of DNA modified with N-methyl-N-nitrosourea.

This paper examines the ability of several restriction enzymes to cleave lambda DNA treated in vitro with N-methyl-N-nitrosourea. An impaired digestion was observed with all tested enzymes suggesting that the sequence specific restriction endonucleases are either unable to recognize carcinogen-modified sequences or to insert endonucleolytic cuts at such sites.

Bacteriophage lambda↗
PubMed1979

Distribution pattern and enzymic hypermethylation of inverted repetitive DNA sequences in P815 mastocytoma cells.

A specific class of DNA sequences, the inverted repetitive sequences, forms a double-stranded structure within a single linear polynucleotide chain in denatured DNA. The reassociation process is unimolecular and occurs very fast. Quantitative analyses have shown that in mouse P815 cells these sequences comprise about 4% of the nuclear DNA and are interspersed within sequences of other degrees of repetitiveness. After labeling the cells with L-[Me-3H]methionine and [14C]deoxycytidine, relative rates of enzymic DNA methylation were computed on the basis of radioactivities found in pyrimidine residues of the nuclear DNA. The results indicate that in P815 cells, DNA of inverted repetitive sequences is methylated to a level about 50% higher than the normal repetitive DNA sequences and to about 300% higher than the unique and intermediary intermediatry sequences. The biological function of the inverted repetitive sequences, as well as of the role of enzymic methylation of DNA remains unknown.

Animals↗