Expression of mammalian (O6-alkylguanine-DNA alkyltransferase in a cell line sensitive to alkylating agents

M. Eileen Dolan, Lauri Norbeck, Christopher Clyde, Nancy K. Hora, Leonard C. Erickson, Anthony Pegg

Research output: Contribution to journalArticle

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Abstract

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-alkylguanlne-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 l-(2-chloroethyl)-3-cyclohexyl-l-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 trans-fectant colonies from this experiment was relatively low, these results indicate that repair of alkylation damage can be restored in AGT-defkient 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-chloroethyD-l-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 cytotoxk effects of BCNU, Clomesone, methylnitrosourea (MNU) and 1-methyl-3-nitro-1-nitroso-guanidine (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 ̃ 74% basal level within 8 h. The sensitivity to the cytotoxk effects of both the chloro-ethylating 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.

Original languageEnglish (US)
Pages (from-to)1613-1619
Number of pages7
JournalCarcinogenesis
Volume10
Issue number9
DOIs
StatePublished - Sep 1 1989

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Alkylating Agents
Cell Line
DNA
Transfection
clomesone
Alkylation
Cricetulus
DNA alkyltransferase
Ovary
Mitoguazone
Cell Count
DNA Repair-Deficiency Disorders
Methylnitrosourea
HT29 Cells
Proteins
Guanidine
Growth

All Science Journal Classification (ASJC) codes

  • Cancer Research

Cite this

Dolan, M. Eileen ; Norbeck, Lauri ; Clyde, Christopher ; Hora, Nancy K. ; Erickson, Leonard C. ; Pegg, Anthony. / Expression of mammalian (O6-alkylguanine-DNA alkyltransferase in a cell line sensitive to alkylating agents. In: Carcinogenesis. 1989 ; Vol. 10, No. 9. pp. 1613-1619.
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abstract = "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-alkylguanlne-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 l-(2-chloroethyl)-3-cyclohexyl-l-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 trans-fectant colonies from this experiment was relatively low, these results indicate that repair of alkylation damage can be restored in AGT-defkient 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-chloroethyD-l-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 cytotoxk effects of BCNU, Clomesone, methylnitrosourea (MNU) and 1-methyl-3-nitro-1-nitroso-guanidine (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 ̃ 74{\%} basal level within 8 h. The sensitivity to the cytotoxk effects of both the chloro-ethylating 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.",
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Expression of mammalian (O6-alkylguanine-DNA alkyltransferase in a cell line sensitive to alkylating agents. / Dolan, M. Eileen; Norbeck, Lauri; Clyde, Christopher; Hora, Nancy K.; Erickson, Leonard C.; Pegg, Anthony.

In: Carcinogenesis, Vol. 10, No. 9, 01.09.1989, p. 1613-1619.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Expression of mammalian (O6-alkylguanine-DNA alkyltransferase in a cell line sensitive to alkylating agents

AU - Dolan, M. Eileen

AU - Norbeck, Lauri

AU - Clyde, Christopher

AU - Hora, Nancy K.

AU - Erickson, Leonard C.

AU - Pegg, Anthony

PY - 1989/9/1

Y1 - 1989/9/1

N2 - 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-alkylguanlne-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 l-(2-chloroethyl)-3-cyclohexyl-l-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 trans-fectant colonies from this experiment was relatively low, these results indicate that repair of alkylation damage can be restored in AGT-defkient 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-chloroethyD-l-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 cytotoxk effects of BCNU, Clomesone, methylnitrosourea (MNU) and 1-methyl-3-nitro-1-nitroso-guanidine (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 ̃ 74% basal level within 8 h. The sensitivity to the cytotoxk effects of both the chloro-ethylating 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.

AB - 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-alkylguanlne-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 l-(2-chloroethyl)-3-cyclohexyl-l-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 trans-fectant colonies from this experiment was relatively low, these results indicate that repair of alkylation damage can be restored in AGT-defkient 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-chloroethyD-l-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 cytotoxk effects of BCNU, Clomesone, methylnitrosourea (MNU) and 1-methyl-3-nitro-1-nitroso-guanidine (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 ̃ 74% basal level within 8 h. The sensitivity to the cytotoxk effects of both the chloro-ethylating 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.

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