Recent Progress with the DNA Repair Mutants of Chinese Hamster Ovary Cells

Thompson, Lawrence H.; Salazar, Edmund P.; Brookman, Kerry W.; Collins, Colin C.; Stewart, S. A.; Busch, David B.; Weber, Christine A.

doi:10.1242/jcs.1984.supplement_6.6

Cited by 72 publications

(25 citation statements)

References 23 publications

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“…Dimers were removed to a much larger extent (-80%) from the transcribed strand in CHO cells than from the nontranscribed strand (-i10%) 24 2). In these cells, we expected to find similar mutation frequencies in both strands because pyrimidine dimers were not removed at all, not even from the actively transcribed hprt gene.…”

Section: Materials and Methods Resultsmentioning

confidence: 99%

DNA strand specificity for UV-induced mutations in mammalian cells.

Vrieling¹,

Rooijen²,

Groen³

et al. 1989

Mol. Cell. Biol.

157

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The influence of DNA repair on the molecular nature of mutations induced by UV light (254 nm) was investigated in UV-induced hprt mutants from UV-sensitive Chinese hamster cells (V-Hi) and the parental line (V79). The nature of point mutations in hprt exon sequences was determined for 19 hprt mutants of V79 and for 17 hprt mutants of V-Hi cells by sequence analysis of in vitro-amplified hprt cDNA. The mutation spectrum in V79 cells consisted of single-and tandem double-base pair changes, while in V-Hi cells three frameshift mutations were also detected. All base pair changes in V-Hi mutants were due to GC --AT transitions. In contrast, in V79 all possible classes of base pair changes except the GC -* CG transversion were present. In this group, 70% of the mutations were transversions. Since all mutations except one did occur at dipyrimidine sites, the assumption was made that they were caused by UV-induced photoproducts at these sites. In V79 cells, 11 out of 17 base pair changes were caused by photoproducts in the nontranscribed strand of the hprt gene.However, in V-Hi cells, which are completely deficient in the removal of pyrimidine dimers from the hprt gene and which show a UV-induced mutation frequency enhanced seven times, 10 out of 11 base pair changes were caused by photoproducts in the transcribed strand of the hprt gene. We hypothesize that this extreme strand specificity in V-Hi cells is due to differences in fidelity of DNA replication of the leading and the lagging strand.Furthermore, we propose that in normal V79 cells two processes determine the strand specificity of UV-induced mutations in the hprt gene, namely preferential repair of the transcribed strand of the hprt gene and a higher fidelity of DNA replication of the nontranscribed strand compared with the transcribed strand.Irradiation of mammalian cells with UV light (254 nm) introduces various lesions into the DNA. Some of these lesions exhibit toxic or mutagenic properties or both and have to be removed from the DNA by repair enzymes to let replication and transcription proceed. The two types of DNA damage which are considered to be primarily responsible for the lethal and mutagenic effects of UV irradiation are the cyclobutane pyrimidine dimer (7,19) and the (6-4) pyrimidine-pyrimidone photoproduct (3, 10). Still, controversy exists over the relative importance of both types of lesions for cell killing and mutagenesis (4,6,13,16).It has been shown for cultured Chinese hamster and human cells that removal of dimers from the transcriptionally active dhfr gene is more rapid than for the genome overall (1, 2, 11) and that possibly even a strand specificity in DNA repair exists (12). Dimers were removed at a faster rate in human cells and to a larger extent in Chinese hamster ovary (CHO) cells from the transcribed strand from the amplified dhfr gene than from the nontranscribed strand. Differential repair of the two DNA strands of a gene after UV irradiation is expected to cause a strand specificity of UV-induced mutations.Several classes of U...

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Section: Materials and Methods Resultsmentioning

confidence: 99%

DNA strand specificity for UV-induced mutations in mammalian cells.

Vrieling¹,

Rooijen²,

Groen³

et al. 1989

Mol. Cell. Biol.

157

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“…The Chinese hamster ovary (CHO) parental cell line CHO-AA8 and the UV-sensitive DNA repair-deficient mutant cell lines CHO-UV23, CHO-UV61 and CHO-UV96 (hereafter named UV23, UV61 and UV96) 11 were kindly provided by Dr. M. Stefanini (CNR IGBE, Pavia, Italy). The UV96 cell line was transfected by the calcium phosphate technique using 10 g of a CMV-ERCC1 plasmid encoding for ERCC1 or with 10 g of CMV empty vector.…”

Section: Cells and Drugsmentioning

confidence: 99%

Unique pattern of ET-743 activity in different cellular systems with defined deficiencies in DNA-repair pathways

et al. 2001

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The cytotoxic activity of ecteinascidin 743 (ET-743), a natural product derived from the marine tunicate Ecteinascidia turbinata that exhibits potent anti-tumor activity in pre-clinical systems and promising activity in phase I and II clinical trials, was investigated in a number of cell systems with well-defined deficiencies in DNA-repair mechanisms. ET-743 binds to N2 of guanine in the minor groove, but its activity does not appear to be related to DNA-topoisomerase I poisoning as the drug is equally active in wild-type yeast and in yeast with a deletion in the DNA-topoisomerase I gene. Defects in the mismatch repair pathway, usually associated with increased resistance to methylating agents and cisplatin, did not affect the cytotoxic activity of ET-743. However, ET-743 did show decreased activity (from 2-to 8-fold) in nucleotide excision repair ( Ecteinascidin 743 (ET-743) is a natural product derived from the marine tunicate Ecteinascidia turbinata, selected for its significant anti-tumor activity in different in vitro and in vivo models. [1][2][3] Currently, ET-743 is undergoing phase II clinical trials after having shown activity in phase I clinical studies with responses observed in different human tumors, including soft tissue sarcoma, osteosarcoma, melanoma and breast cancer. 4,5 The mechanism of action of ET-743 has yet to be fully defined, but DNA appears to be the primary target. Indeed, it has been shown to bind in the minor groove of DNA and to alkylate the N2 position of guanine with some degree of specificity. 6,7 Such ET-743 minor-groove alkylation induces a bend in the DNA helix toward the major groove, a finding not common to the other minor groove-alkylating agents, which bend DNA into the minor groove. 8 No single-strand breaks, double-strand breaks or DNAprotein cross-links could be found by alkaline elution in cells exposed to IC 50 concentrations of ET-743. 9 However, Takebayashi et al. 10 showed that ET-743 was able to induce DNA-topoisomerase I cross-linking but that high concentrations were required to produce the effect.To provide some insight into the mechanism of action of ET-743, we have evaluated its cytotoxic activity using a panel of different cellular systems characterized by well-defined deficiencies in different mammalian repair pathways. Our data indicate that ET-743 has a unique mechanism of interaction with DNA. MATERIAL AND METHODS Cells and drugsYeast strains and the isogenic derivatives CY2823 (⌬rad52) and CY2278 (⌬topI) (kindly provided by Dr. M. Lopes, Instituto F.I.R.C. di Oncologia Molecolare, Milan, Italy) were grown in YPD plates (1% yeast extract, 2% bacto-peptone, 2% glucose) with or without the drugs.The Chinese hamster ovary (CHO) parental cell line CHO-AA8 and the UV-sensitive DNA repair-deficient mutant cell lines CHO-UV23, CHO-UV61 and CHO-UV96 (hereafter named UV23, UV61 and UV96) 11 were kindly provided by Dr. M. Stefanini (CNR IGBE, Pavia, Italy). The UV96 cell line was transfected by the calcium phosphate technique using 10 g of a CMV-ERCC1 plasmid enc...

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“…The first five groups display a similar, high degree of UV sensitivity and are deficient in the incision step of the excision repair process (31)(32)(33). In contrast, the two mutants making up group 6, CHO mutant UV61 (3) and mouse lymphoma mutant US46 (27,37), are only moderately sensitive to UV exposure, and UV61 is partially deficient in the incision of damaged DNA (27,36). Furthermore, mutant UV61 is remarkable in harboring a specific deficiency in the repair of cyclobutane pyrimidine dimers and bulky chemical adducts, but permitting apparently normal repair of (6-4) photoproducts (34).…”

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confidence: 99%

Molecular cloning of the human DNA excision repair gene ERCC-6.

et al. 1990

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The UV-sensitive, nucleotide excision repair-deficient Chinese hamster mutant cell line UV61 was used to identify and clone a correcting human gene, ERCC-6. UV61, belonging to rodent complementation group 6, is only moderately UV sensitive in comparison with mutant lines in groups 1 to 5. It harbors a deficiency in the repair of UV-induced cyclobutane pyrimidine dimers but permits apparently normal repair of (6-4) photoproducts. Genomic (HeLa) DNA transfections of UV61 resulted, with a very low efficiency, in six primary and four secondary UV-resistant transformants having regained wild-type UV survival. Southern blot analysis revealed that five primary and only one secondary transformant retained human sequences. The latter line was used to clone the entire 115-kb human insert. Coinheritance analysis demonstrated that five of the other transformants harbored a 100-kb segment of the cloned human insert. Since it is extremely unlikely that six transformants all retain the same stretch of human DNA by coincidence, we conclude that the ERCC-6 gene resides within this region and probably covers most of it. The large size of the gene explains the extremely low transfection frequency and makes the gene one of the largest cloned by genomic DNA transfection. Four transformants did not retain the correcting ERCC-6 gene and presumably have reverted to the UV-resistant phenotype. One of these appeared to have amplified an endogenous, mutated CHO ERCC-6 allele, indicating that the UV61 mutation is leaky and can be overcome by gene amplification.

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Recent Progress with the DNA Repair Mutants of Chinese Hamster Ovary Cells

Cited by 72 publications

References 23 publications

DNA strand specificity for UV-induced mutations in mammalian cells.

DNA strand specificity for UV-induced mutations in mammalian cells.

Unique pattern of ET-743 activity in different cellular systems with defined deficiencies in DNA-repair pathways

Molecular cloning of the human DNA excision repair gene ERCC-6.

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