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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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Cell survival and MTHFR protein levels after treatment with the antisense oligonucleotide EX5. (A) Colon carcinoma SW620 cells were treated on three successive days with increasing concentration of EX5. Cells were also treated with a control ASO, CTSEX5. The number of surviving cells was determined by SRB staining. Cell survival after transfection with EX5 is expressed as a % of survival after transfection with the control CTSEX5 oligonucleotide. Error bars represent±s.e. of the mean of three experiments, each performed in triplicate. (B) MTHFR protein levels after three rounds of treatments with Lipofectin only (mock transfection), 400 nM of CTSEX5, 200 nM of EX5 or 400 nM of EX5. SW620 cells were harvested after the third treatment and subjected to Western blot analysis. The position of the MTHFR protein and the molecular weight markers are indicated. Protein levels of β-actin were also assayed by Western blotting to verify equal loading of samples.
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fig3: Cell survival and MTHFR protein levels after treatment with the antisense oligonucleotide EX5. (A) Colon carcinoma SW620 cells were treated on three successive days with increasing concentration of EX5. Cells were also treated with a control ASO, CTSEX5. The number of surviving cells was determined by SRB staining. Cell survival after transfection with EX5 is expressed as a % of survival after transfection with the control CTSEX5 oligonucleotide. Error bars represent±s.e. of the mean of three experiments, each performed in triplicate. (B) MTHFR protein levels after three rounds of treatments with Lipofectin only (mock transfection), 400 nM of CTSEX5, 200 nM of EX5 or 400 nM of EX5. SW620 cells were harvested after the third treatment and subjected to Western blot analysis. The position of the MTHFR protein and the molecular weight markers are indicated. Protein levels of β-actin were also assayed by Western blotting to verify equal loading of samples.

Mentions: A BLAST search identified sequences in exon 5 of MTHFR that did not have any homology with other ESTs in the NCBI database. Figure 3aFigure 3


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)

Cell survival and MTHFR protein levels after treatment with the antisense oligonucleotide EX5. (A) Colon carcinoma SW620 cells were treated on three successive days with increasing concentration of EX5. Cells were also treated with a control ASO, CTSEX5. The number of surviving cells was determined by SRB staining. Cell survival after transfection with EX5 is expressed as a % of survival after transfection with the control CTSEX5 oligonucleotide. Error bars represent±s.e. of the mean of three experiments, each performed in triplicate. (B) MTHFR protein levels after three rounds of treatments with Lipofectin only (mock transfection), 400 nM of CTSEX5, 200 nM of EX5 or 400 nM of EX5. SW620 cells were harvested after the third treatment and subjected to Western blot analysis. The position of the MTHFR protein and the molecular weight markers are indicated. Protein levels of β-actin were also assayed by Western blotting to verify equal loading of samples.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Cell survival and MTHFR protein levels after treatment with the antisense oligonucleotide EX5. (A) Colon carcinoma SW620 cells were treated on three successive days with increasing concentration of EX5. Cells were also treated with a control ASO, CTSEX5. The number of surviving cells was determined by SRB staining. Cell survival after transfection with EX5 is expressed as a % of survival after transfection with the control CTSEX5 oligonucleotide. Error bars represent±s.e. of the mean of three experiments, each performed in triplicate. (B) MTHFR protein levels after three rounds of treatments with Lipofectin only (mock transfection), 400 nM of CTSEX5, 200 nM of EX5 or 400 nM of EX5. SW620 cells were harvested after the third treatment and subjected to Western blot analysis. The position of the MTHFR protein and the molecular weight markers are indicated. Protein levels of β-actin were also assayed by Western blotting to verify equal loading of samples.
Mentions: A BLAST search identified sequences in exon 5 of MTHFR that did not have any homology with other ESTs in the NCBI database. Figure 3aFigure 3

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