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Molecular analysis of 5-azacytidine-induced variants in mammalian cells
Z Kelecsényi1, D L Spencer, W J Caspary
1Cancer Genetics Group, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Abstract:
5-azacytidine induces 6-thioguanine resistance in AS52 cells. To characterize these resistant clones, we isolated 148 of them from 50 independently treated flasks. Less than nine (6%) of the 148 variants were spontaneous. PCR amplification of the DNA primers flanking the gpt gene produced no product in 15 clones (10%). Of the 133 remaining clones, 52 showed sequence alterations in the gpt structural gene. Of these 52, 34 (65%) were GC-->CG transversions. Only seven were located in CpG sequences. Thus, methyltransferase complexes are not major contributors to 5-azacytidine-induced point mutations in AS52 cells. The remaining 81 clones had no sequence alterations within the coding region of the gpt gene. Southern blot analysis of a sample of these variants (37/81) indicated that the 6-thioguanine-resistant phenotype was not due to local rearrangements or deletions (resolution 50 bp). Sequence analysis of the early promoter region of another sample of these variants (24/81) indicated that lesions in the promoter could not be responsible for the 6-thioguanine resistance observed. Thus, a majority of these variants were formed via a mechanism other than small genomic rearrangements, point mutations or deletions of the gpt structural gene or its promoter. Neither the mechanisms leading to these variants nor the biological and morphological consequences of these variants are known.
Insights
5-azacytidine treatment generated 6-thioguanine resistant AS52 cell clones. Most resistant clones arose from mechanisms beyond direct gpt gene mutations or rearrangements.
Area of Science:
- Molecular Biology
- Genetics
- Drug Resistance
Background:
- 5-azacytidine is a DNA methyltransferase inhibitor.
- AS52 cells are a mammalian cell line used for mutagenicity studies.
- 6-thioguanine resistance can arise from mutations in the gpt gene.
Purpose of the Study:
- To investigate the mechanisms of 5-azacytidine-induced 6-thioguanine resistance in AS52 cells.
- To characterize genetic alterations in resistant clones.
Main Methods:
- Isolation and characterization of 148 resistant clones.
- Polymerase Chain Reaction (PCR) for gpt gene amplification.
- DNA sequencing of the gpt structural gene and promoter region.
- Southern blot analysis for genomic rearrangements.
Main Results:
- Less than 9% of resistant clones were spontaneous.
- 10% of clones showed no PCR product for the gpt gene.
- 52 clones had sequence alterations in the gpt gene, with 34 GC-->CG transversions, few at CpG sites.
- 81 clones had no coding region alterations; Southern blots and promoter sequencing ruled out deletions, rearrangements, or promoter lesions.
Conclusions:
- Methyltransferase complexes are not major contributors to 5-azacytidine-induced point mutations in AS52 cells.
- A majority of resistant variants arose through mechanisms other than small genomic rearrangements, point mutations, or deletions of the gpt gene or its promoter.
- The mechanisms and consequences of these variants remain unknown.