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

Sekhon J, Pereira P, Sabbaghian N, Schievella AR, Rozen R - Br. J. Cancer (2002)

Bottom Line: We hypothesised that methylenetetrahydrofolate reductase inhibition would affect cell viability through decreased methionine synthesis.Western blotting and enzyme assays confirmed that methylenetetrahydrofolate reductase expression was decreased.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, McGill University Health Centre - Montreal Children's Hospital, 4060 Ste. Catherine West, Room 200, Montreal, Quebec H3Z 2Z3, Canada.

ABSTRACT
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% (P<0.01) and 70% (P<0.0001), 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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Growth of fibroblast cell lines in deficient media. Two fibroblast cell lines (MCH 51, MCH 75) were grown in MEM (▪), M- (×), and M-H+ (○) for 12 days. The number of cells for each line was counted using the SRB assay at three time points. Each point represents the mean of three replicates±s.d.
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fig1: Growth of fibroblast cell lines in deficient media. Two fibroblast cell lines (MCH 51, MCH 75) were grown in MEM (▪), M- (×), and M-H+ (○) for 12 days. The number of cells for each line was counted using the SRB assay at three time points. Each point represents the mean of three replicates±s.d.

Mentions: Two fibroblast strains (Figure 1Figure 1


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

Sekhon J, Pereira P, Sabbaghian N, Schievella AR, Rozen R - Br. J. Cancer (2002)

Growth of fibroblast cell lines in deficient media. Two fibroblast cell lines (MCH 51, MCH 75) were grown in MEM (▪), M- (×), and M-H+ (○) for 12 days. The number of cells for each line was counted using the SRB assay at three time points. Each point represents the mean of three replicates±s.d.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2376111&req=5

fig1: Growth of fibroblast cell lines in deficient media. Two fibroblast cell lines (MCH 51, MCH 75) were grown in MEM (▪), M- (×), and M-H+ (○) for 12 days. The number of cells for each line was counted using the SRB assay at three time points. Each point represents the mean of three replicates±s.d.
Mentions: Two fibroblast strains (Figure 1Figure 1

Bottom Line: We hypothesised that methylenetetrahydrofolate reductase inhibition would affect cell viability through decreased methionine synthesis.Western blotting and enzyme assays confirmed that methylenetetrahydrofolate reductase expression was decreased.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, McGill University Health Centre - Montreal Children's Hospital, 4060 Ste. Catherine West, Room 200, Montreal, Quebec H3Z 2Z3, Canada.

ABSTRACT
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% (P<0.01) and 70% (P<0.0001), 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.

Show MeSH
Related in: MedlinePlus