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PubMed1999

[Study on the methylation of p15 gene CpG islands in acute leukemia: using methylation-specific PCR method].

OBJECTIVE: To explore the feasibility of the methylation of p15 gene CpG islands as a common gene marker for all types of acute leukemias(AL). METHODS: The methylation of p15 gene CpG islands in bone marrow from 40 cases of newly diagnosed or relapsed AL, 5 cases of AL in CR and 8 of normal subjects was analyzed by using methylation-specific PCR methods(MSP), and the product of MSP was sequenced. RESULTS: p15 gene CpG islands were methylated in 80% (32/40) of the newly diagnosed or relapsed AL, no difference between AML and ALL was observed. One positive result was found in the 5 AL patients in CR and this patient soon relapsed. The bone marrow cells from 8 normal subjects did not have p15 gene CpG islands methylation, which suggested that such methylation was peculiar to AL cells. DNA sequencing confirmed the right expected sequence. The sensitivity of MSP was 10(-3). CONCLUSION: The methylation of p15 gene CpG islands occurs very commonly in every types of AL. It can be used as a gene marker for ALs and for minimal residual disease in CR. MSP needs neither special methylation-sensitive restricted sites, nor high volume of DNA. There is no radioactive pollution, and the sample need not be fresh. MSP is a very sensitive, simple and specific method for the detection of DNA methylation.

Acute Disease↗
PubMed2006

Transitional cell carcinomas and nonurothelial carcinomas of the urinary bladder differ in the promoter methylation status of the caveolin-1, hDAB2IP and p53 genes, but not in the global methylation of Alu elements.

Tumor suppressor genes play a prominent role in the modification and progression of urinary bladder carcinogenesis as a result of classic genetic alterations. Little is known about the potential significance of epigenetic events, mediated by DNA hypermethylation. This prompted our investigation to explore the global Alu methylation and the promoter methylation of the novel putative tumor suppressor genes caveolin-1 and hDAB2IP, and of p53 in transitional cell carcinomas (TCC), squamous cell carcinomas and undifferentiated small cell carcinomas of the urinary bladder. Quantitative GeneScan analysis revealed that the various histopathological tumor entities showed considerable interindividual variations in the global methylation, but the overall rate did not significantly differ between the various cancer subtypes. With methylation-specific PCR, a high frequency of methylation of the promoter region of the caveolin-1 gene was detected in undifferentiated small cell carcinomas (50%) and in squamous cell carcinomas (25.9%), while TCC were found not to be methylated. By immunohistochemistry, all squamous cell carcinomas showed a strong diffuse overexpression of caveolin-1, whereas undifferentiated small cell cancers lacked any expression. High-grade, high-stage TCC disclosed a higher incidence (60%) and a substantially stronger expression than low-grade, low-stage TCC (42.9%). Our findings suggest that hypermethylation of the caveolin-1 gene and an abnormal protein expression play a crucial role in cell differentiation, and in the phenotypical conversion of TCC into nonurothelial carcinomas. Promoter methylation of the hDAB2IP gene occurred more frequently in advanced muscle invasive (72.7%) than in superficial noninvasive (50%) TCC. DNA hypermethylation of p53 was detected in a quarter of the low-grade, low-stage TCC and undifferentiated small cell carcinomas, but only sporadically in squamous cell carcinomas, and was absent in high-grade, high-stage TCC. In conclusion, aberrant methylation and abnormal protein expression of the caveolin-1-gene is involved in the formation of nonurothelial carcinomas of the urinary bladder and promoter methylation of the hDAB2IP gene in the progression of TCC from a low to a high malignant potential.

Alu Elements↗
PubMed1989

Methylation of microinjected isoaspartyl peptides in Xenopus oocytes. Competition with protein carboxyl methylation reactions.

Xenopus oocytes possess a highly conserved protein carboxyl methyltransferase postulated to function in the repair or metabolism of age-damaged protein aspartyl residues (O'Connor, C. M. (1987) J. Biol. Chem. 262, 10398-10403). Three hexapeptides of the general sequence Val-Tyr-Pro-isoAsp-X-Ala, in which isoAsp represents an L-isoaspartyl residue and X represents Gly, Ser, or Ala, are methylated with the same order of preference following their microinjection into oocytes as in a purified system containing bovine brain protein carboxyl methyltransferase and S-adenosyl-L-[methyl-3H]methionine. The affinities of the enzyme for the glycyl, seryl, and alanyl variants of the peptides in vitro are 4.25, 3.04, and 1.67 microM, respectively. A nonapeptide of the sequence Lys-Ala-Ser-Ala-isoAsp-Leu-Ala-Lys-Tyr is a higher affinity substrate for the methyltransferase in vitro, characterized by a Km of 0.88 microM, but it is modified to a lesser extent in oocytes, partially because of its reduced stability in cytoplasm. The hexapeptide Val-Tyr-Pro-Asp-Gly-Ala, which contains an aspartyl residue in the usual stereoconfiguration, is not methylated either in vitro or in intact oocytes. Microinjection of any of the four isoaspartyl-containing peptides greatly stimulates total carboxyl methylation in oocytes, with rate increases ranging from 19- to 51-fold after the injection of 30 pmol of peptide. The protein ovalbumin is also modified following its microinjection into oocytes to near its calculated methyl-accepting capacity. Each of the isoaspartyl peptides can act as a competitive inhibitor of ovalbumin methylation both in vitro and in microinjected oocytes. The inhibitory potencies of the peptides parallel their specific methyl-accepting activities. The results demonstrate that the oocyte may be a useful model for studying the significance of protein carboxyl methylation because of the large functional excess of methylation capacity and the fidelity of the reactions compared to those observed in purified systems. This excess capability may have physiological significance when structurally abnormal proteins accumulate as a result of cellular stress and or aging.

Amino Acid Sequence↗
PubMedDate not supplied

[Methylation of ribonuclease and albumin by methyl cobalamin].

The capacity of methyl cobalamine (14CH3-B12) to methylate RNase and albumin in in vitro systems was studied. Under the experimental conditions, 14CH3-B12 methylated proteins about 100--1000 times more actively than S-adenosyl-methionine, a universal donor of methyl groups. The nature of buffer and pH 2 to 8 had no noticeable effect on the methylating capacity of 14CH3-B12. However, at higher pH rate of incorporation of methyl groups slightly increased. The 14CH3-groups incorporated into RNase amino acids remained stable upon illumination, in the presence of KCN in the incubation medium, and when heated in 20% HCl for 24--72 hr at 105 degrees. Methylation did not influence the enzymic properties of RNase. The automatic amino acid analyzer showed three peaks of the label of modified amino acids in the hydrolzates of methylated RNase, one of which corresponded to methylated methionine.

Albumins↗
PubMed1983

Differential turnover of methyl groups on methyl-accepting membrane proteins of irreversibly sickled erythrocytes.

Methyl group turnover rates for specific methyl-accepting membrane proteins in intact irreversibly sickled cells (ISCs) have been determined. The turnover of methyl groups on all methyl acceptor membrane proteins carboxylmethylated in ISCs is not concerted but proceeds in an ordered sequence which is markedly different from that exhibited by unfractionated normal erythrocytes (AA). In ISCs methyl group turnover based on initial demethylation rate constants is most rapid for membrane polypeptides migrating in sodium dodecyl sulfate at 30,000-39,000 Da, 40,000-55,000 Da, polypeptides comigrating with cytoskeletal component band 4.1, and band 4.5. In contrast, initial methyl group turnover rates obtained after less than 20% of the total methyl groups are turned over in (AA) cells show most rapid demethylation rates for membrane polypeptides migrating at 40,000-55,000 Da, polypeptides comigrating with band 4.5, cytoskeletal components bands 2.1 and 4.1. Results also show significant differences between ISCs and most dense fractions from normal (AA) and nonsickle hemolytic anemias in the demethylation of cytoskeletal proteins, bands 2.1 and 4.1. These findings indicate qualitative differences in accessibility of methyl acceptor substrates to the methylating-demethylating enzyme activity in the cytosol of irreversibly sickled cells compared to discocytic control erythrocytes.

Anemia, Sickle Cell↗
PubMedDate not supplied

[Intragenomic specificity of DNA methylation in animals. Qualitative differences in tissues and changes in methylation of repeating sequences during aging, carcinogenesis and hormonal induction].

5-methylcytosine (m5C) is nonrandomly distributed in mammalian genome. The unique sequences in DNA of all species studied (mouse, rat, cow) methylated to a similar extent and are characterized by a minimal m5C content (0.8--0.9 mole%). The very renaturating sequences (including satellite DNA) possess a maximal m5C amount. The rarely repeated (10 to 1000 folds) sequences which are known to contain genes for rRNA and tRNA as well as for histones are characterized by an elevated level of methylation. It is established that tissue differences in m5C content in DNA as well as decrease in the DNA methylation level with age and on spontaneous lympholeukosis in cattle and, on the contrary, increase in DNA methylation degree in rat liver as a result of induction with hydrocortisone, are due to tissue specific changes in the methylation level of repeated but not unique sequences. The methylation level of sequences differing in the reiteration degree seems to be mainly due to CG-dublet frequencies. In the unique sequences this particular dinucleotide is almost completely methylated. Nevertheless, the multiplicity of DNA methylases in the animal nucleus is not ruled out. Methylation of the CG sequence is supposed to protect animal DNA's, especially structural genes, against restriction endonucleases. The decrease in DNA methylation in animals with age and on lympholeukosis detected by us is considered to be one of the possible reasons for chromosome lesions and for distortions in DNA replication and transcription.

5-Methylcytosine↗
PubMed1980

[On the problems with respect to the formation of N-nitroso compound precursors when using alkylating agents in the fumigation of foodstuffs. II. N-methylation of various amino acids by the action of methyl bromide (author's transl)].

The aim of this investigation was to determine whether the insecticide, methyl bromide, used in the treatment of foodstuffs reacts with the amino group of various amino acids in the sense of an N-methylation, thus generating substrates which can be converted to nitroso compounds, i.e. those which are the precursors of cancerogenic compounds. Cooled liquid methyl bromide was added to the aqueous solutions of five amino acids at different pH values. Samples for thin layer chromatographic separation of the derivatives formed were taken after 1, 2, 3, 4, and 8 hours. The method of proving used was at ninhydrine reaction. The results show that methyl bromide reacts with the amino acids glycine, alanine, valine, leucine and glutamic acid to degrees depending upon the pH value and the length of the reaction. The corresponding N-methyl derivatives are formed, namely sarcosine, N-methyl alanine, N-methyl valine, N-methyl leucine, and N-methyl glutamic acid.

Alanine↗
PubMed1977

Inhibition of DNA methylation by S-adenosylethionine with the production of methyl-deficient DNA in regenerating rat liver.

Ethionine, a liver carcinogen, was administered p.o. (300 mg/kg) to rats 17 hr after partial hepatectomy. At 6 hr after administration of the ethionine, hepatic S-adenosylethionine levels were 30- to 40-fold greater than the hepatic level of S-adenosylmethionine. A 10-fold ratio of S-adenosylethionine to S-adenosylmethionine still persited at 24 hr after ethionine administration. When given at 17 hr after partial hepatectomy, ethionine produced a 30% inhibition of DNA synthesis, measured by the incorporation of [methyl-3H]thymidine at 23 to 24 hr after partial hepatectomy (6 to 7 hr after ethionine administration). DNA synthesized during this interval was methyl deficient as judged by the reduced incorporation of radioactivity from L-[methyl-3H]methionine into 5-methylcytosine residues of DNA. In an assay for DNA methylation in vitro using whole nuclei, the methyl-deficient DNA was methylated by S-adenosylmethionine 8 times more than was control DNA; the DNA methylation was competitively inhibited by S-adenosylethionine. These data suggest that S-adenosylethionine, formed in vivo from ethionine, competitively inhibits the methylation of DNA in vivo by S-adenosylmethionine, resulting in the production of methyl-deficient DNA.

Animals↗