Rational design of point mutation-selective antisense DNA targeted to codon 12 of Ha-<i>ras</i>mRNA in human cells

Duroux, Isabelle; Godard, Gérard; Boidot-Forget, Marc; Schwab, G.; Hélène, Claude; Saison‐Behmoaras, Tula

doi:10.1093/nar/23.17.3411

Cited by 31 publications

(21 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The differences in cell survival between the 677T and 677C oligos were significantly different for concentrations of 200, 300 and 400 nM. These results are consistent with previous reports demonstrating the specificity for one form of a target gene using phosphorothioate oligonucleotides with single base mismatches as controls (Duroux et al, 1995;Bennet et al, 1996;Basilion et al, 1999). Our observations also suggest that allele-specific targeting may be possible for MTHFR.…”

Section: Discussionsupporting

confidence: 91%

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

et al. 2002

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionsupporting

confidence: 91%

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

et al. 2002

View full text Add to dashboard Cite

show abstract

“…This result indicates that it would be possible to design anti-sense oligonucleotides which inactivate the mutated K-ras gene selectively, without suppressing the normal proto-oncogene that is essential to physiological cells. Several reports demonstrated the selective inhibitory effect of anti-sense oligonucleotides specific to point mutation of Ha-ras mRNA (Monia et al, 1992;Duroux et al, 1995;SaisonBehmoaras et al, 1991;Chang et al, 1991). These anti-sense oligonucleotides preferentially inhibited the mutated Ha-ras mRNA expression, with little effect on the wild type.…”

Section: Discussionmentioning

confidence: 99%

“…They have shown that a maximum (5-fold) selectivity for the mutant was achieved with a 17 mer anti-sense oligonucleotide. Duroux et al(1995) concluded that 12 or 13 mer anti-sense oligonucleotides centered on the mutation had the best selectivity. In either case, they concluded that longer oligomers lost the ability to distinguish the mutated mRNA from the wild one.…”

Section: Discussionmentioning

confidence: 99%

“…These reverse transcriptions and PCR were performed using the Superscript Preamplification System (GIBCO BRL) according to the manufacturer's instructions. As a control, human ␤ 2 -microglobulin (␤ 2 MG) cDNA was simultaneously amplified (Duroux et al, 1995). To ensure linear amplification, the number of cycles was limited to 25 for K-rascDNA (94°C for 60 sec, 50°C for 60 sec, 72°C for 60 sec) and 21 for ␤ 2 MG cDNA (94°C for 50 sec, 57°C for 50 sec, 72°C for 20 sec).…”

Section: Semi-quantitative Rt-pcr Analysis Of K-ras Mrnamentioning

confidence: 99%

“…These oligonucleotides inhibit the expression of normal as well as mutated genes. However, several reports have shown that anti-sense oligonucleotides specific to point mutations could selectively inhibit mutated gene expression (Monia et al, 1992;Duroux et al, 1995).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Growth inhibition of human pancreatic cancer cell lines by anti‐sense oligonucleotides specific to mutated K‐ras genes

et al. 1999

View full text Add to dashboard Cite

About 90% of human pancreatic cancers carry K-ras point mutation, which may play an important role in tumorigenesis. We investigated the inhibitory effects of anti-sense oligonucleotides targeting K-ras point mutation on the growth of cultured human pancreatic cancer cells. Eight human pancreatic cancer cell lines were screened for K-ras codon 12 point mutations by PCR-RFLP analysis and direct sequencing. Then, 3 cell lines with the major types of K-ras point mutation, i.e.,HuP-T1, HuP-T3 and PANC-1, and 1 without mutation, BxPC-3, were used for the experiments. Seventeen mer anti-sense oligonucleotides were designed, targeting the point mutation of K-ras codon 12, and transfected into the cells by the liposome-mediated method. Cell-growth activities were estimated by MTT assay. Levels of K-ras mRNA expression were determined using quantitative RT-PCR, and K-ras p21 protein synthesis was evaluated with Western blotting. Mutation-matched anti-sense oligonucleotides effectively inhibited the growth of these pancreatic cancer cell lines, except for BxPC-3, by suppressing K-ras mRNA expression and K-ras p21 protein synthesis. Moreover, mutation-matched antisense oligonucleotides showed stronger anti-proliferative effects than did mutation-mismatched ones. Our results suggest that anti-sense therapy specific to point mutations of K-ras mRNA is a practical approach to selective suppression of tumor growth, with little effect on normal cells. Int.

show abstract

Growth inhibition of human pancreatic cancer cell lines by anti-sense oligonucleotides specific to mutated K-ras genes

et al. 1999

View full text Add to dashboard Cite

Rational design of point mutation-selective antisense DNA targeted to codon 12 of Ha-rasmRNA in human cells

Cited by 31 publications

References 23 publications

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

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

Growth inhibition of human pancreatic cancer cell lines by anti‐sense oligonucleotides specific to mutated K‐ras genes

Growth inhibition of human pancreatic cancer cell lines by anti-sense oligonucleotides specific to mutated K-ras genes

Contact Info

Product

Resources

About