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Natalia Y Tretyakova - One of the best experts on this subject based on the ideXlab platform.
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mechlorethamine induced dna protein cross linking in human fibrosarcoma ht1080 cells
Journal of Proteome Research, 2011Co-Authors: Erin D Michaelsonrichie, Simona G Codreanu, Rachel Loeber, Colin R Campbell, Daniel C. Liebler, Xun Ming, Natalia Y TretyakovaAbstract:Antitumor Nitrogen Mustards, such as bis(2-chloroethyl)methylamine (mechlorethamine), are useful chemotherapeutic agents with a long history of clinical application. The antitumor effects of Nitrogen Mustards are attributed to their ability to induce DNA–DNA and DNA–protein cross-links (DPCs) that block DNA replication. In the present work, a mass spectrometry-based methodology was employed to characterize in vivo DNA–protein cross-linking following treatment of human fibrosarcoma (HT1080) cells with cytotoxic concentrations of mechlorethamine. A combination of mass spectrometry-based proteomics and immunological detection was used to identify 38 nuclear proteins that were covalently cross-linked to chromosomal DNA following treatment with mechlorethamine. Isotope dilution HPLC–ESI+–MS/MS analysis of total proteolytic digests revealed a concentration-dependent formation of N-[2-(S-cysteinyl)ethyl]-N-[2-(guan-7-yl)ethyl]methylamine (Cys-N7G-EMA) conjugates, indicating that mechlorethamine cross-links cyste...
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Mechlorethamine-Induced DNA–Protein Cross-Linking in Human Fibrosarcoma (HT1080) Cells
Journal of Proteome Research, 2011Co-Authors: Erin D. Michaelson-richie, Simona G Codreanu, Rachel Loeber, Colin R Campbell, Daniel C. Liebler, Xun Ming, Natalia Y TretyakovaAbstract:Antitumor Nitrogen Mustards, such as bis(2-chloroethyl)methylamine (mechlorethamine), are useful chemotherapeutic agents with a long history of clinical application. The antitumor effects of Nitrogen Mustards are attributed to their ability to induce DNA–DNA and DNA–protein cross-links (DPCs) that block DNA replication. In the present work, a mass spectrometry-based methodology was employed to characterize in vivo DNA–protein cross-linking following treatment of human fibrosarcoma (HT1080) cells with cytotoxic concentrations of mechlorethamine. A combination of mass spectrometry-based proteomics and immunological detection was used to identify 38 nuclear proteins that were covalently cross-linked to chromosomal DNA following treatment with mechlorethamine. Isotope dilution HPLC–ESI+–MS/MS analysis of total proteolytic digests revealed a concentration-dependent formation of N-[2-(S-cysteinyl)ethyl]-N-[2-(guan-7-yl)ethyl]methylamine (Cys-N7G-EMA) conjugates, indicating that mechlorethamine cross-links cyste...
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Proteomic Analysis of DNA-Protein Cross-Linking by Antitumor Nitrogen Mustards
Chemical research in toxicology, 2009Co-Authors: Rachel Loeber, Simona G Codreanu, Erin D. Michaelson-richie, Colin R Campbell, Daniel C. Liebler, Natalia Y TretyakovaAbstract:Nitrogen Mustards are antitumor agents used clinically for the treatment of a variety of neoplastic conditions. The biological activity of these compounds is typically attributed to their ability to induce DNA−DNA cross-links. However, Nitrogen Mustards are able to produce a variety of other lesions, including DNA−protein cross-links (DPCs). DPCs induced by Nitrogen Mustards are not well-characterized because of their structural complexity and the insufficient specificity and sensitivity of previously available experimental methodologies. In the present work, affinity capture methodology in combination with mass spectrometry-based proteomics was employed to identify mammalian proteins that form covalent cross-links to DNA in the presence of a simple Nitrogen mustard, mechlorethamine. Following incubation of 5′-biotinylated DNA duplexes with nuclear protein extracts, DPCs were isolated by affinity capture on streptavidin beads, and the cross-linked proteins were identified by high-performance liquid chroma...
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cross linking of the dna repair protein o6 alkylguanine dna alkyltransferase to dna in the presence of antitumor Nitrogen Mustards
Chemical Research in Toxicology, 2008Co-Authors: Rachel Loeber, Colin R Campbell, Erin Michaelson, Qingming Fang, Anthony E Pegg, Natalia Y TretyakovaAbstract:The antitumor activity of chemotherapeutic Nitrogen Mustards including chlorambucil, cyclophosphamide, and melphalan is commonly attributed to their ability to induce DNA−DNA cross-links by consecutive alkylation of two nucleophilic sites within the DNA duplex. DNA–protein cross-linking by Nitrogen Mustards is not well characterized, probably because of its inherent complexity and the insufficient sensitivity of previous methodologies. If formed, DNA–protein conjugates are likely to contribute to both target and off-target cytotoxicity of Nitrogen mustard drugs. Here, we show that the DNA repair protein, O6-alkylguanine DNA alkyltransferase (AGT), can be readily cross-linked to DNA in the presence of Nitrogen Mustards. Both chlorambucil and mechlorethamine induced the formation of covalent conjugates between 32P-labeled double-stranded oligodeoxynucleotides and recombinant human AGT protein, which were detected by SDS-PAGE. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of AGT ...
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cross linking of the human dna repair protein o6 alkylguanine dna alkyltransferase to dna in the presence of 1 2 3 4 diepoxybutane and antitumor Nitrogen Mustards
Cancer Research, 2006Co-Authors: Rachel Loeber, Anthony E Pegg, Danae Quirk Dorr, Melissa Goggin, Natalia Y TretyakovaAbstract:5244 O6-alkylguanine DNA alkyltransferase (AGT) repairs O6-alkylguanine lesions in DNA by direct transfer of the O6-alkyl group to an active site cysteine (Cys145), restoring normal guanine and preventing mutagenesis. However, previous studies suggested that AGT expression increased the cytotoxic and mutagenic effects of several bis-electrophiles, including 1,2,3,4-diepoxybutane (DEB) and antitumor Nitrogen Mustards (NM). Because these bifunctional alkylating agents produce N7-guanine monoadducts that retain one of the electrophilic groups, they are likely to induce DNA-protein cross-links by interacting with nucleophilic residues within AGT. The purpose of this study was to determine the structures of DEB- and NM-induced AGT-DNA conjugates and to identify specific amino acid residues involved in cross-linking. DNA-protein cross-link formation was detected by SDS-PAGE when 32P-labeled double-stranded oligodeoxynucleotides were exposed to DEB in the presence of either wild-type hAGT or a C145A hAGT mutant lacking the alkyl acceptor residue. Capillary HPLC-ESI+ mass spectrometric analysis of hAGT that had been incubated with N7-(2’-hydroxy-3’,4’-epoxybut-1’-yl)-deoxyguanosine (dG monoepoxide) or N7-guanine-half Mustards of mechlorethamine and chlorambucil revealed the ability of the protein to form either one or two cross-links to dG. The cross-link locations were mapped to Cys145 and Cys150 by HPLC-ESI+-MS/MS analyses of tryptic digests. The same two active site residues participated in cross-linking following the incubation of hAGT with double-stranded DNA in the presence of DEB, mechlorethamine, and chlorambucil. The exact chemical structures of the cross-linked lesions were established as 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol (Cys-Gua-BD), N-(2-[S-cysteinyl]ethyl)-N-(2-[guan-7-yl]ethyl)methylamine (Cys-Gua-EMA), and N-(2-[S-cysteinyl]ethyl)-N-(2-[guan-7-yl]ethyl)-p-aminophenylbuyric acid (Cys-Gua-PBA) by HPLC-ESI+-MS/MS analysis of the amino acids resulting from total digestion of modified proteins analyzed in parallel with synthetic standards. DNA-protein cross-linking is a likely mechanism for cytotoxicity and mutagenicity of DEB and NM in cells expressing AGT protein.
Rachel Loeber - One of the best experts on this subject based on the ideXlab platform.
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mechlorethamine induced dna protein cross linking in human fibrosarcoma ht1080 cells
Journal of Proteome Research, 2011Co-Authors: Erin D Michaelsonrichie, Simona G Codreanu, Rachel Loeber, Colin R Campbell, Daniel C. Liebler, Xun Ming, Natalia Y TretyakovaAbstract:Antitumor Nitrogen Mustards, such as bis(2-chloroethyl)methylamine (mechlorethamine), are useful chemotherapeutic agents with a long history of clinical application. The antitumor effects of Nitrogen Mustards are attributed to their ability to induce DNA–DNA and DNA–protein cross-links (DPCs) that block DNA replication. In the present work, a mass spectrometry-based methodology was employed to characterize in vivo DNA–protein cross-linking following treatment of human fibrosarcoma (HT1080) cells with cytotoxic concentrations of mechlorethamine. A combination of mass spectrometry-based proteomics and immunological detection was used to identify 38 nuclear proteins that were covalently cross-linked to chromosomal DNA following treatment with mechlorethamine. Isotope dilution HPLC–ESI+–MS/MS analysis of total proteolytic digests revealed a concentration-dependent formation of N-[2-(S-cysteinyl)ethyl]-N-[2-(guan-7-yl)ethyl]methylamine (Cys-N7G-EMA) conjugates, indicating that mechlorethamine cross-links cyste...
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Mechlorethamine-Induced DNA–Protein Cross-Linking in Human Fibrosarcoma (HT1080) Cells
Journal of Proteome Research, 2011Co-Authors: Erin D. Michaelson-richie, Simona G Codreanu, Rachel Loeber, Colin R Campbell, Daniel C. Liebler, Xun Ming, Natalia Y TretyakovaAbstract:Antitumor Nitrogen Mustards, such as bis(2-chloroethyl)methylamine (mechlorethamine), are useful chemotherapeutic agents with a long history of clinical application. The antitumor effects of Nitrogen Mustards are attributed to their ability to induce DNA–DNA and DNA–protein cross-links (DPCs) that block DNA replication. In the present work, a mass spectrometry-based methodology was employed to characterize in vivo DNA–protein cross-linking following treatment of human fibrosarcoma (HT1080) cells with cytotoxic concentrations of mechlorethamine. A combination of mass spectrometry-based proteomics and immunological detection was used to identify 38 nuclear proteins that were covalently cross-linked to chromosomal DNA following treatment with mechlorethamine. Isotope dilution HPLC–ESI+–MS/MS analysis of total proteolytic digests revealed a concentration-dependent formation of N-[2-(S-cysteinyl)ethyl]-N-[2-(guan-7-yl)ethyl]methylamine (Cys-N7G-EMA) conjugates, indicating that mechlorethamine cross-links cyste...
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Proteomic Analysis of DNA-Protein Cross-Linking by Antitumor Nitrogen Mustards
Chemical research in toxicology, 2009Co-Authors: Rachel Loeber, Simona G Codreanu, Erin D. Michaelson-richie, Colin R Campbell, Daniel C. Liebler, Natalia Y TretyakovaAbstract:Nitrogen Mustards are antitumor agents used clinically for the treatment of a variety of neoplastic conditions. The biological activity of these compounds is typically attributed to their ability to induce DNA−DNA cross-links. However, Nitrogen Mustards are able to produce a variety of other lesions, including DNA−protein cross-links (DPCs). DPCs induced by Nitrogen Mustards are not well-characterized because of their structural complexity and the insufficient specificity and sensitivity of previously available experimental methodologies. In the present work, affinity capture methodology in combination with mass spectrometry-based proteomics was employed to identify mammalian proteins that form covalent cross-links to DNA in the presence of a simple Nitrogen mustard, mechlorethamine. Following incubation of 5′-biotinylated DNA duplexes with nuclear protein extracts, DPCs were isolated by affinity capture on streptavidin beads, and the cross-linked proteins were identified by high-performance liquid chroma...
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cross linking of the dna repair protein o6 alkylguanine dna alkyltransferase to dna in the presence of antitumor Nitrogen Mustards
Chemical Research in Toxicology, 2008Co-Authors: Rachel Loeber, Colin R Campbell, Erin Michaelson, Qingming Fang, Anthony E Pegg, Natalia Y TretyakovaAbstract:The antitumor activity of chemotherapeutic Nitrogen Mustards including chlorambucil, cyclophosphamide, and melphalan is commonly attributed to their ability to induce DNA−DNA cross-links by consecutive alkylation of two nucleophilic sites within the DNA duplex. DNA–protein cross-linking by Nitrogen Mustards is not well characterized, probably because of its inherent complexity and the insufficient sensitivity of previous methodologies. If formed, DNA–protein conjugates are likely to contribute to both target and off-target cytotoxicity of Nitrogen mustard drugs. Here, we show that the DNA repair protein, O6-alkylguanine DNA alkyltransferase (AGT), can be readily cross-linked to DNA in the presence of Nitrogen Mustards. Both chlorambucil and mechlorethamine induced the formation of covalent conjugates between 32P-labeled double-stranded oligodeoxynucleotides and recombinant human AGT protein, which were detected by SDS-PAGE. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of AGT ...
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cross linking of the human dna repair protein o6 alkylguanine dna alkyltransferase to dna in the presence of 1 2 3 4 diepoxybutane and antitumor Nitrogen Mustards
Cancer Research, 2006Co-Authors: Rachel Loeber, Anthony E Pegg, Danae Quirk Dorr, Melissa Goggin, Natalia Y TretyakovaAbstract:5244 O6-alkylguanine DNA alkyltransferase (AGT) repairs O6-alkylguanine lesions in DNA by direct transfer of the O6-alkyl group to an active site cysteine (Cys145), restoring normal guanine and preventing mutagenesis. However, previous studies suggested that AGT expression increased the cytotoxic and mutagenic effects of several bis-electrophiles, including 1,2,3,4-diepoxybutane (DEB) and antitumor Nitrogen Mustards (NM). Because these bifunctional alkylating agents produce N7-guanine monoadducts that retain one of the electrophilic groups, they are likely to induce DNA-protein cross-links by interacting with nucleophilic residues within AGT. The purpose of this study was to determine the structures of DEB- and NM-induced AGT-DNA conjugates and to identify specific amino acid residues involved in cross-linking. DNA-protein cross-link formation was detected by SDS-PAGE when 32P-labeled double-stranded oligodeoxynucleotides were exposed to DEB in the presence of either wild-type hAGT or a C145A hAGT mutant lacking the alkyl acceptor residue. Capillary HPLC-ESI+ mass spectrometric analysis of hAGT that had been incubated with N7-(2’-hydroxy-3’,4’-epoxybut-1’-yl)-deoxyguanosine (dG monoepoxide) or N7-guanine-half Mustards of mechlorethamine and chlorambucil revealed the ability of the protein to form either one or two cross-links to dG. The cross-link locations were mapped to Cys145 and Cys150 by HPLC-ESI+-MS/MS analyses of tryptic digests. The same two active site residues participated in cross-linking following the incubation of hAGT with double-stranded DNA in the presence of DEB, mechlorethamine, and chlorambucil. The exact chemical structures of the cross-linked lesions were established as 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol (Cys-Gua-BD), N-(2-[S-cysteinyl]ethyl)-N-(2-[guan-7-yl]ethyl)methylamine (Cys-Gua-EMA), and N-(2-[S-cysteinyl]ethyl)-N-(2-[guan-7-yl]ethyl)-p-aminophenylbuyric acid (Cys-Gua-PBA) by HPLC-ESI+-MS/MS analysis of the amino acids resulting from total digestion of modified proteins analyzed in parallel with synthetic standards. DNA-protein cross-linking is a likely mechanism for cytotoxicity and mutagenicity of DEB and NM in cells expressing AGT protein.
Denis Cournoyer - One of the best experts on this subject based on the ideXlab platform.
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increased resistance to Nitrogen Mustards and antifolates following in vitro selection of murine fibroblasts and primary hematopoietic cells transduced with a bicistronic retroviral vector expressing the rat glutathione s transferase a3 and a mutant
Cancer Gene Therapy, 2003Co-Authors: Jeanphilippe Belzile, Antonis Karatzas, Hoiying Shiu, Sylvain Letourneau, Jeansebastien Palerme, Denis CournoyerAbstract:Increased resistance to Nitrogen Mustards and antifolates following in vitro selection of murine fibroblasts and primary hematopoietic cells transduced with a bicistronic retroviral vector expressing the rat glutathione S -transferase A3 and a mutant dihydrofolate reductase
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coexpression of rat glutathione s transferase a3 and human cytidine deaminase by a bicistronic retroviral vector confers in vitro resistance to Nitrogen Mustards and cytosine arabinoside in murine fibroblasts
Cancer Gene Therapy, 2000Co-Authors: Sylvain Letourneau, Jeansebastien Palerme, Jeansebastien Delisle, Christian Beausejour, Richard L Momparler, Denis CournoyerAbstract:The transfer of drug resistance genes into hematopoietic cells is an experimental approach to protect patients from drug-induced myelosuppression. Because anti-cancer drugs are often administered in combination to increase their clinical efficacy, vectors that express two drug resistance genes are being developed to broaden the spectrum of chemoprotection. We have constructed a bicistronic vector, MFG/GST-IRES-CD (MFG/GIC) coexpressing rat glutathione S-transferase (GST) A3 isoform (rGST Yc1) and human cytidine deaminase (CD). Murine NIH 3T3 fibroblast cells transduced with this vector were evaluated for their resistance to Nitrogen Mustards and cytosine nucleoside analogs. GIC-transduced polyclonal cell populations (GIC cells) demonstrated marked increases in selenium-independent glutathione peroxidase (peroxidase) and CD activities, as well as increased resistance to melphalan (2.3-fold), chlorambucil (3.4-fold), and cytosine arabinoside (Ara-C) (8.1-fold). After selection with Ara-C, the peroxidase and CD activities of GIC cells were augmented 2.6- and 2.9-fold, respectively, in comparison with unselected cells, and the resistance to melphalan, chlorambucil, and Ara-C was further increased to 3.7-, 5.9-, and 53-fold, respectively. Melphalan selection of GIC cells likewise augmented their peroxidase (2.3-fold) and CD (1.9-fold) activities. GIC cells proliferated in the simultaneous presence of melphalan and Ara-C at drug concentrations that completely inhibited the growth of untransduced cells. The growth rate of unselected GIC cells exposed to the drug combination averaged 18% that of drug-free cultures. The growth rate of GIC cells exposed to the drug combination increased to 30% of controls after Ara-C selection and to 50% after melphalan selection. Our results suggest that retroviral transfer of MFG/GIC may be useful for chemoprotection against the toxicities of Nitrogen Mustards and cytosine nucleoside analogs.
Colin R Campbell - One of the best experts on this subject based on the ideXlab platform.
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mechlorethamine induced dna protein cross linking in human fibrosarcoma ht1080 cells
Journal of Proteome Research, 2011Co-Authors: Erin D Michaelsonrichie, Simona G Codreanu, Rachel Loeber, Colin R Campbell, Daniel C. Liebler, Xun Ming, Natalia Y TretyakovaAbstract:Antitumor Nitrogen Mustards, such as bis(2-chloroethyl)methylamine (mechlorethamine), are useful chemotherapeutic agents with a long history of clinical application. The antitumor effects of Nitrogen Mustards are attributed to their ability to induce DNA–DNA and DNA–protein cross-links (DPCs) that block DNA replication. In the present work, a mass spectrometry-based methodology was employed to characterize in vivo DNA–protein cross-linking following treatment of human fibrosarcoma (HT1080) cells with cytotoxic concentrations of mechlorethamine. A combination of mass spectrometry-based proteomics and immunological detection was used to identify 38 nuclear proteins that were covalently cross-linked to chromosomal DNA following treatment with mechlorethamine. Isotope dilution HPLC–ESI+–MS/MS analysis of total proteolytic digests revealed a concentration-dependent formation of N-[2-(S-cysteinyl)ethyl]-N-[2-(guan-7-yl)ethyl]methylamine (Cys-N7G-EMA) conjugates, indicating that mechlorethamine cross-links cyste...
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Mechlorethamine-Induced DNA–Protein Cross-Linking in Human Fibrosarcoma (HT1080) Cells
Journal of Proteome Research, 2011Co-Authors: Erin D. Michaelson-richie, Simona G Codreanu, Rachel Loeber, Colin R Campbell, Daniel C. Liebler, Xun Ming, Natalia Y TretyakovaAbstract:Antitumor Nitrogen Mustards, such as bis(2-chloroethyl)methylamine (mechlorethamine), are useful chemotherapeutic agents with a long history of clinical application. The antitumor effects of Nitrogen Mustards are attributed to their ability to induce DNA–DNA and DNA–protein cross-links (DPCs) that block DNA replication. In the present work, a mass spectrometry-based methodology was employed to characterize in vivo DNA–protein cross-linking following treatment of human fibrosarcoma (HT1080) cells with cytotoxic concentrations of mechlorethamine. A combination of mass spectrometry-based proteomics and immunological detection was used to identify 38 nuclear proteins that were covalently cross-linked to chromosomal DNA following treatment with mechlorethamine. Isotope dilution HPLC–ESI+–MS/MS analysis of total proteolytic digests revealed a concentration-dependent formation of N-[2-(S-cysteinyl)ethyl]-N-[2-(guan-7-yl)ethyl]methylamine (Cys-N7G-EMA) conjugates, indicating that mechlorethamine cross-links cyste...
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Proteomic Analysis of DNA-Protein Cross-Linking by Antitumor Nitrogen Mustards
Chemical research in toxicology, 2009Co-Authors: Rachel Loeber, Simona G Codreanu, Erin D. Michaelson-richie, Colin R Campbell, Daniel C. Liebler, Natalia Y TretyakovaAbstract:Nitrogen Mustards are antitumor agents used clinically for the treatment of a variety of neoplastic conditions. The biological activity of these compounds is typically attributed to their ability to induce DNA−DNA cross-links. However, Nitrogen Mustards are able to produce a variety of other lesions, including DNA−protein cross-links (DPCs). DPCs induced by Nitrogen Mustards are not well-characterized because of their structural complexity and the insufficient specificity and sensitivity of previously available experimental methodologies. In the present work, affinity capture methodology in combination with mass spectrometry-based proteomics was employed to identify mammalian proteins that form covalent cross-links to DNA in the presence of a simple Nitrogen mustard, mechlorethamine. Following incubation of 5′-biotinylated DNA duplexes with nuclear protein extracts, DPCs were isolated by affinity capture on streptavidin beads, and the cross-linked proteins were identified by high-performance liquid chroma...
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cross linking of the dna repair protein o6 alkylguanine dna alkyltransferase to dna in the presence of antitumor Nitrogen Mustards
Chemical Research in Toxicology, 2008Co-Authors: Rachel Loeber, Colin R Campbell, Erin Michaelson, Qingming Fang, Anthony E Pegg, Natalia Y TretyakovaAbstract:The antitumor activity of chemotherapeutic Nitrogen Mustards including chlorambucil, cyclophosphamide, and melphalan is commonly attributed to their ability to induce DNA−DNA cross-links by consecutive alkylation of two nucleophilic sites within the DNA duplex. DNA–protein cross-linking by Nitrogen Mustards is not well characterized, probably because of its inherent complexity and the insufficient sensitivity of previous methodologies. If formed, DNA–protein conjugates are likely to contribute to both target and off-target cytotoxicity of Nitrogen mustard drugs. Here, we show that the DNA repair protein, O6-alkylguanine DNA alkyltransferase (AGT), can be readily cross-linked to DNA in the presence of Nitrogen Mustards. Both chlorambucil and mechlorethamine induced the formation of covalent conjugates between 32P-labeled double-stranded oligodeoxynucleotides and recombinant human AGT protein, which were detected by SDS-PAGE. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of AGT ...
Anthony E Pegg - One of the best experts on this subject based on the ideXlab platform.
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cross linking of the dna repair protein o6 alkylguanine dna alkyltransferase to dna in the presence of antitumor Nitrogen Mustards
Chemical Research in Toxicology, 2008Co-Authors: Rachel Loeber, Colin R Campbell, Erin Michaelson, Qingming Fang, Anthony E Pegg, Natalia Y TretyakovaAbstract:The antitumor activity of chemotherapeutic Nitrogen Mustards including chlorambucil, cyclophosphamide, and melphalan is commonly attributed to their ability to induce DNA−DNA cross-links by consecutive alkylation of two nucleophilic sites within the DNA duplex. DNA–protein cross-linking by Nitrogen Mustards is not well characterized, probably because of its inherent complexity and the insufficient sensitivity of previous methodologies. If formed, DNA–protein conjugates are likely to contribute to both target and off-target cytotoxicity of Nitrogen mustard drugs. Here, we show that the DNA repair protein, O6-alkylguanine DNA alkyltransferase (AGT), can be readily cross-linked to DNA in the presence of Nitrogen Mustards. Both chlorambucil and mechlorethamine induced the formation of covalent conjugates between 32P-labeled double-stranded oligodeoxynucleotides and recombinant human AGT protein, which were detected by SDS-PAGE. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of AGT ...
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cross linking of the human dna repair protein o6 alkylguanine dna alkyltransferase to dna in the presence of 1 2 3 4 diepoxybutane and antitumor Nitrogen Mustards
Cancer Research, 2006Co-Authors: Rachel Loeber, Anthony E Pegg, Danae Quirk Dorr, Melissa Goggin, Natalia Y TretyakovaAbstract:5244 O6-alkylguanine DNA alkyltransferase (AGT) repairs O6-alkylguanine lesions in DNA by direct transfer of the O6-alkyl group to an active site cysteine (Cys145), restoring normal guanine and preventing mutagenesis. However, previous studies suggested that AGT expression increased the cytotoxic and mutagenic effects of several bis-electrophiles, including 1,2,3,4-diepoxybutane (DEB) and antitumor Nitrogen Mustards (NM). Because these bifunctional alkylating agents produce N7-guanine monoadducts that retain one of the electrophilic groups, they are likely to induce DNA-protein cross-links by interacting with nucleophilic residues within AGT. The purpose of this study was to determine the structures of DEB- and NM-induced AGT-DNA conjugates and to identify specific amino acid residues involved in cross-linking. DNA-protein cross-link formation was detected by SDS-PAGE when 32P-labeled double-stranded oligodeoxynucleotides were exposed to DEB in the presence of either wild-type hAGT or a C145A hAGT mutant lacking the alkyl acceptor residue. Capillary HPLC-ESI+ mass spectrometric analysis of hAGT that had been incubated with N7-(2’-hydroxy-3’,4’-epoxybut-1’-yl)-deoxyguanosine (dG monoepoxide) or N7-guanine-half Mustards of mechlorethamine and chlorambucil revealed the ability of the protein to form either one or two cross-links to dG. The cross-link locations were mapped to Cys145 and Cys150 by HPLC-ESI+-MS/MS analyses of tryptic digests. The same two active site residues participated in cross-linking following the incubation of hAGT with double-stranded DNA in the presence of DEB, mechlorethamine, and chlorambucil. The exact chemical structures of the cross-linked lesions were established as 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol (Cys-Gua-BD), N-(2-[S-cysteinyl]ethyl)-N-(2-[guan-7-yl]ethyl)methylamine (Cys-Gua-EMA), and N-(2-[S-cysteinyl]ethyl)-N-(2-[guan-7-yl]ethyl)-p-aminophenylbuyric acid (Cys-Gua-PBA) by HPLC-ESI+-MS/MS analysis of the amino acids resulting from total digestion of modified proteins analyzed in parallel with synthetic standards. DNA-protein cross-linking is a likely mechanism for cytotoxicity and mutagenicity of DEB and NM in cells expressing AGT protein.