title: Antisense inhibition of methylenetetrahydrofolate reductase reduces survival of methionine-dependent tumour lines.
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.
authors.affiliation: Department of Human Genetics, McGill University Health Centre - Montreal Children's Hospital, 4060 Ste. Catherine West, Room 200, Montreal, Quebec H3Z 2Z3, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Sekhon
authors.forename: P
authors.initials: P
authors.lastname: Pereira
authors.forename: N
authors.initials: N
authors.lastname: Sabbaghian
authors.forename: A R
authors.initials: AR
authors.lastname: Schievella
authors.forename: R
authors.initials: R
authors.lastname: Rozen
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000201:enzymology / Q000473:pathology; D002277:Carcinoma / Q000201:enzymology / Q000473:pathology; D002460:Cell Line / Q000187:drug effects; D003110:Colonic Neoplasms / Q000201:enzymology / Q000473:pathology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005091:Exons / Q000235:genetics; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D015316:Genetic Therapy; D006710:Homocysteine / Q000494:pharmacology; D006801:Humans; D008715:Methionine / Q000502:physiology*; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D009447:Neuroblastoma / Q000201:enzymology / Q000473:pathology; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology*; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000235:genetics; D012334:RNA, Neoplasm / Q000037:antagonists & inhibitors / Q000235:genetics; D013873:Thionucleotides / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000201:enzymology; D014805:Vitamin B 12 / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D009363:Neoplasm Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D013873:Thionucleotides; D006710:Homocysteine; D008715:Methionine; D000587:Oxidoreductases Acting on CH-NH Group Donors; D042965:Methylenetetrahydrofolate Reductase (NADPH2); D014805:Vitamin B 12