Document Detail


Expression of mammalian O6-alkylguanine-DNA alkyltransferase in a cell line sensitive to alkylating agents.
MedLine Citation:
PMID:  2766456     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Chinese hamster ovary cells (CHO) were co-transfected with pSV2neo and sheared DNA from either a human cell line (HT29) expressing high levels of O6-alkylguanine-DNA alkyltransferase (AGT) or from a cell line (BE) deficient in this activity. Cells expressing the selectable marker were obtained by exposure to G418 and colonies resistant to alkylation damage isolated by growth in the presence of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU). The number of colonies of cells expressing AGT activity arising after transfection with DNA from BE cells was similar to the number arising from cells exposed to HT29 DNA. Although the amount of AGT repair protein expressed in the transfectant colonies from this experiment was relatively low, these results indicate that repair of alkylation damage can be restored in AGT-deficient cells by transfection of human DNA from both repair-deficient and proficient cells. A separate transfection of CHOMG cells [a mutant of CHO cells resistant to the drug, methylglyoxal bis(guanylhydrazone) (MGBG)] with HT29 DNA and pSV2neo followed by selection of G418 and 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) resulted in three colonies with high AGT levels. These transfectants had different growth rates and expressed levels of the AGT protein between 230 and 300 fmol/mg protein. The transfectants were as resistant to the cytotoxic effects of BCNU, Clomesone, methylnitrosourea (MNU) and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) as HT29 cells which were much more resistant than the parental CHOMG cells. Pretreatment of transfectant cells with 0.4 mM O6-methylguanine for 24 h reduced AGT activity to 14% basal levels, which upon removal of the base increased to approximately 74% basal level within 8 h. The sensitivity to the cytotoxic effects of both the chloroethylating and methylating agents was enhanced by treatment with O6-methylguanine. In the same manner, the number of BCNU-induced DNA interstrand cross-links increased in transfectant cells pretreated with O6-methylguanine. These results provide further evidence that the formation of methyl or chloroethyl adducts at the O6-position contribute significantly to cell lethality.
Authors:
M E Dolan; L Norbeck; C Clyde; N K Hora; L C Erickson; A E Pegg
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Carcinogenesis     Volume:  10     ISSN:  0143-3334     ISO Abbreviation:  Carcinogenesis     Publication Date:  1989 Sep 
Date Detail:
Created Date:  1989-10-12     Completed Date:  1989-10-12     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  8008055     Medline TA:  Carcinogenesis     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  1613-9     Citation Subset:  IM    
Affiliation:
Department of Physiology, Milton S. Hershey Medical Center, Pennsylvania State University, College of Medicine, Hershey 17033.
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MeSH Terms
Descriptor/Qualifier:
Alkylating Agents / pharmacology*
Animals
Carmustine / pharmacology
Cell Division / drug effects
Cell Line
Cricetinae
Cricetulus
Female
Guanine / analogs & derivatives*,  pharmacology
Kinetics
Lomustine / pharmacology
Methylnitronitrosoguanidine / pharmacology
Methylnitrosourea / pharmacology
Methyltransferases / biosynthesis,  genetics*
Mitoguazone / pharmacology
O(6)-Methylguanine-DNA Methyltransferase
Ovary
Transfection
Grant Support
ID/Acronym/Agency:
CA 18137/CA/NCI NIH HHS; CA 45628/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
0/Alkylating Agents; 13010-47-4/Lomustine; 154-93-8/Carmustine; 20535-83-5/O-(6)-methylguanine; 459-86-9/Mitoguazone; 684-93-5/Methylnitrosourea; 70-25-7/Methylnitronitrosoguanidine; 73-40-5/Guanine; EC 2.1.1.-/Methyltransferases; EC 2.1.1.63/O(6)-Methylguanine-DNA Methyltransferase

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