Methylenetetrahy drofolate Reductase in Cultured Human Cells. I. Growth and Metabolic Studies

Rosenblatt, David S.; Erbe, Richard W.

doi:10.1203/00006450-197711000-00004

Cited by 39 publications

(21 citation statements)

References 10 publications

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“…Their patient showed a relatively mild clinical phenotype, with a high residual enzyme activity (20%) in cultured fibroblasts. Based on the absence of flavin adenine dinucleotide (FAD) stabilisation of the MTHFR protein in their patient during heat inactivation, 26 it was postulated that this region of the protein is critical in FAD binding. Our patient showed a more severe clinical phenotype with no residual enzyme activity in cultured fibroblasts, regardless of FAD addition.…”

Section: Discussionmentioning

confidence: 99%

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Identification of four novel mutations in severe methylenetetrahydrofolate reductase deficiency

Wendel

et al. 1998

Eur J Hum Genet

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Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is an inborn error of folate metabolism, and is inherited as an autosomal recessive trait. MTHFR is a key enzyme in folate-dependent remethylation of homocysteine, and reduces 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. Patients with this severe enzymatic deficiency are biochemically characterised by homocystinuria and hypomethioninaemia, and may suffer from neurological abnormalities, mental retardation and premature vascular disease. Here we report the molecular basis of severe MTHFR deficiency in four unrelated families from Turkish/Greek ancestry. By use of reverse-transcriptase (RT)-PCR, subsequently followed by direct sequencing analysis, we were able to identify four novel mutations in the MTHFR gene: two missense (983A®G; 1027T®G) and two nonsense (1084C®T; 1711C®T) mutations. Furthermore, a splice variant containing a premature termination codon, was observed in one patient, probably as a secondary effect of the 1027T®G missense mutation. The ongoing identification and characterisation of mutations in the MTHFR gene will provide further insight into the heterogeneity of the clinical phenotype in severe MTHFR deficiency.

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Section: Discussionmentioning

confidence: 99%

“…26 In the assay 14 C Me-THF served as a cosubstrate in the presence of menadione as electron acceptor. Fibroblasts were resuspended in 50 µM potassium phosphate buffer (pH 7.2), sonicated on ice, and subsequently centrifuged for 40 min at 15 800 g at 4°C.…”

Section: Mthfr Assaymentioning

confidence: 99%

Identification of four novel mutations in severe methylenetetrahydrofolate reductase deficiency

Wendel

et al. 1998

Eur J Hum Genet

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“…Methylenetetrahydrofolate reductase (MTHFR) activity was measured in isolated lymphocytes (16) in the absence and presence of 75 or 400 M AdoMet, and in cultured fibroblasts (17). The specific MTHFR activity is expressed in nmol formaldehyde formed per mg protein per hour.…”

Section: Methodsmentioning

confidence: 99%

Defective cystathionine beta-synthase regulation by S-adenosylmethionine in a partially pyridoxine responsive homocystinuria patient.

Kluijtmans¹,

Boers²,

Stevens³

et al. 1996

J. Clin. Invest.

101

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We determined the molecular basis of cystathionine ␤ -synthase (CBS) deficiency in a partially pyridoxine-responsive homocystinuria patient. Direct sequencing of the entire CBS cDNA revealed the presence of a homozygous G 1330 A transition. This mutation causes an amino acid change from aspartic acid to asparagine (D444N) in the regulatory domain of the protein and abolishes a TaqI restriction site at DNA level. Despite the homozygous mutation, CBS activities in extracts of cultured fibroblasts of this patient were not in the homozygous but in the heterozygous range. Furthermore, we observed no stimulation of CBS activity by S-adenosylmethionine, contrary to a threefold stimulation in control fibroblast extract. The mutation was introduced in an E. coli expression system and CBS activities were

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“…Dialysed serum was used for all experiments with deficient media. These conditions were based on similar studies in fibroblasts and in transformed lines (Hoffman and Erbe, 1976;Rosenblatt and Erbe, 1977).…”

Section: Deficient Culture Mediamentioning

confidence: 99%

Antisense inhibition of methylenetetrahydrofolate reductase reduces survival of methionine-dependent tumour lines

et al. 2002

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Transformed cells have been documented to be methionine-dependent, suggesting that inhibition of methionine synthesis might be useful for cancer therapy. Methylenetetrahydrofolate reductase synthesises 5-methyltetrahydrofolate, the methyl donor utilised in methionine synthesis from homocysteine by vitamin B 12 -dependent methionine synthase. We hypothesised that methylenetetrahydrofolate reductase inhibition would affect cell viability through decreased methionine synthesis. Using medium lacking methionine, but containing homocysteine and vitamin B 12 (M-H+), we found that nontransformed human fibroblasts could maintain growth. In contrast, four transformed cell lines (one colon carcinoma, two neuroblastoma and one breast carcinoma) increased proliferation only slightly in the M-H+ medium. To downregulate methylenetetrahydrofolate reductase expression, two phosphorothioate antisense oligonucleotides, EX5 and 677T, were used to target methylenetetrahydrofolate reductase in the colon carcinoma line SW620; 400 nM of each antisense oligonucleotide decreased cell survival by approximately 80% (P50.01) and 70% (P50.0001), respectively, compared to cell survival after the respective control mismatched oligonucleotide. Western blotting and enzyme assays confirmed that methylenetetrahydrofolate reductase expression was decreased. Two neuroblastoma and two breast carcinoma lines also demonstrated decreased survival following EX5 treatment whereas nontransformed human fibroblasts were not affected. This study suggests that methylenetetrahydrofolate reductase may be required for tumour cell survival and that methylenetetrahydrofolate reductase inhibition should be considered for anti-tumour therapy.

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Methylenetetrahy drofolate Reductase in Cultured Human Cells. I. Growth and Metabolic Studies

Cited by 39 publications

References 10 publications

Identification of four novel mutations in severe methylenetetrahydrofolate reductase deficiency

Identification of four novel mutations in severe methylenetetrahydrofolate reductase deficiency

Defective cystathionine beta-synthase regulation by S-adenosylmethionine in a partially pyridoxine responsive homocystinuria patient.

Antisense inhibition of methylenetetrahydrofolate reductase reduces survival of methionine-dependent tumour lines

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