Induction of frameshift mutations in cultured mammalian cells within a transfected sequence containing a poly(dC-dA) · poly(dT-dG) microsatellite

Riedinger, Kara L.; Hanford, Marsha G.; Farber, Rosann A.

doi:10.1002/(sici)1098-2280(1996)28:3<276::aid-em12>3.0.co;2-c

Cited by 3 publications

(2 citation statements)

References 30 publications

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“…The mutation rates of the A 17 and (CA) 17 sequences were similar to each other when compared within each of the MMR + cell lines. Only maximum rate estimates for A 17 in hTERT1604 cells could be determined, since no mutants were recovered in experiments with three independent transfected clones; however, we were able to demonstrate that at least two of these clones were capable of producing neo R revertants after treatment with ICR170 (unpublished data), which is a known frameshift mutagen (16). The mutation rates of the G 17 sequence in the MMR + cell lines were much higher than the rates of the A 17 or (CA) 17 sequences.…”

Section: Resultsmentioning

confidence: 91%

Sequence dependent instability of mononucleotide microsatellites in cultured mismatch repair proficient and deficient mammalian cells

Boyer

Yamada

Roques

et al. 2002

Human Molecular Genetics

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We have measured the mutation rates of G(17) and A(17) repeat sequences in cultured mammalian cells with and without mismatch repair and have compared these rates to those of a (CA)(17) repeat sequence. Plasmids containing microsatellites that disrupt the reading frame of a downstream neomycin-resistance gene were introduced into the cells by transfection and revertants were selected using the neomycin analog G418. Comparison of mutation rates within cell lines showed that the mutation rates of A(17) and (CA)(17) sequences were similar in the mismatch repair proficient cells, but the mutation rate of G(17) was significantly higher than that of either A(17) or (CA)(17). In the mismatch repair deficient cells, the G(17) and (CA)(17) mutation rates were similar and were significantly higher than the A(17) rate. PCR analysis of the mutants showed that 1 bp insertions predominated in both mononucleotide repeats in the mismatch repair proficient cells; in mismatch repair deficient cells, 2 bp deletions were the most common mutation in the A(17) sequence, but 1 bp insertions and 2 bp deletions were equally represented in the G(17) sequence. These results indicate that a G(17) repeat is less stable than an A(17) repeat in both mismatch repair proficient and mismatch repair deficient mammalian cells. This observation implies that the replication fidelity is lower in G(17) repeats.

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Section: Resultsmentioning

confidence: 91%

Sequence dependent instability of mononucleotide microsatellites in cultured mismatch repair proficient and deficient mammalian cells

Boyer

Yamada

Roques

et al. 2002

Human Molecular Genetics

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“…The number of HT-1080 clones is small because the mutation frequency was relatively low (i.e., not all subcultures contained G418-resistant colonies) and because most of the clones that were isolated did not contain frameshifts within the microsatellite region. The latter clones probably experienced frameshift mutations outside the CA-repeat, as has been demonstrated for ICR170-induced mutations in this plasmid sequence in mouse CAK cells (Riedinger et al, 1996).…”

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confidence: 99%

Microsatellite mutation rates in cancer cell lines deficient or proficient in mismatch repair

et al. 1998

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A selectable system has been used to determine mutation rates within a microsatellite sequence in human cancer cell lines with or without defects in mismatch repair. A sequence consisting of 17 repeats of poly (dC-dA).poly(dT-dG) [abbreviated as (Ca) 17 ] was inserted near the 5' end of the bacterial neomycin-resistance gene in a plasmid vector, such that the reading frame of the neo gene is disrupted. This plasmid was introduced into cancer cell lines, where it became integrated into the cellular genome. Clones with insertions or deletions of CA-repeats that restored the normal reading frame of the neo gene were selected in G418, and mutation rates were determined by¯uctuation analysis. The rates of reversion in LoVo cells, which are de®cient for hMSH2, were about one in a thousand per generation, which is approximately two orders of magnitude higher than in the repair-pro®cient HT-1080 human ®brosarcoma cell line. The mutation rates in H6 cells, which are derived from the hMLH1-de®cient HCT116 line, were more heterogeneous than in LoVo, but all were considerably higher than in the repair-pro®cient line. Nearly all of the revertants of the repair-de®cient lines had deletions of a single CA-repeat from the microsatellite sequence, whereas repair-pro®cient cells had a broader spectrum of mutations.

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Mutation frequency analysis of mononucleotide and dinucleotide repeats after oxidative stress

Yamada

Parker

Farber

2003

Environ and Mol Mutagen

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Many tumors exhibit genetic instability at the DNA sequence level in the form of frameshift mutations in simple repeats (microsatellite instability). A high level of microsatellite instability, such as that seen in hereditary nonpolyposis colorectal cancer (HNPCC), arises from defects in the mismatch repair pathway. A low level of microsatellite instability is found in some non-HNPCC-associated cancers, such as those of the breast and lung, and is not attributable to mismatch repair defects. We hypothesized that oxidative DNA damage may be at least partly responsible for the generation of microsatellite mutations in these tumors. We investigated whether oxidative DNA damage can induce microsatellite mutations in mismatch repair-proficient cultured cells. Telomerase-immortalized normal human fibroblasts were stably transfected with a plasmid containing a tk-neo fusion gene, such that the neo coding region was placed out of frame by the presence of an upstream microsatellite sequence. Cells were treated with H(2)O(2) and mutation frequencies were determined for G(17), A(17), and (CA)(17) repeats. Mutation frequencies of mononucleotide repeats in cells with the neo gene in the (+1) reading frame were reduced after treatment. No effect was observed in cells with the mononucleotide repeats in the (-1) reading frame. A small increase in mutation frequency was observed in cells with the (CA)(17) repeat. Our data suggest that diploid human cells may have protective mechanisms that prevent the induction of microsatellite mutations by a short exposure to high levels of oxidative stress.

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Induction of frameshift mutations in cultured mammalian cells within a transfected sequence containing a poly(dC-dA) · poly(dT-dG) microsatellite

Cited by 3 publications

References 30 publications

Sequence dependent instability of mononucleotide microsatellites in cultured mismatch repair proficient and deficient mammalian cells

Sequence dependent instability of mononucleotide microsatellites in cultured mismatch repair proficient and deficient mammalian cells

Microsatellite mutation rates in cancer cell lines deficient or proficient in mismatch repair

Mutation frequency analysis of mononucleotide and dinucleotide repeats after oxidative stress

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