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J. Andres Faiña Medín - One of the best experts on this subject based on the ideXlab platform.
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The Engineered Thymidylate Kinase (TMPK)/AZT Enzyme- Prodrug Axis Offers Efficient Bystander Cell Killing for Suicide Gene Therapy of Cancer
2016Co-Authors: Takeya Sato, Anton Neschadim, Teruyuki Yanagisawa, Arnon Lavie, J. Andres Faiña MedínAbstract:We previously described a novel suicide (or ‘Cell fate control’) gene therapy enzyme/prodrug system based on an engineered variant of human thymidylate kinase (TMPK) that potentiates azidothymidine (AZT) activation. Delivery of a suicide gene sequence into tumors by lentiviral transduction embodies a cancer gene therapy that could employ bystander Cell Killing as a mechanism driving significant tumor regression in vivo. Here we present evidence of a significant bystander Cell Killing in vitro and in vivo mediated by the TMPK/AZT suicide gene axis that is reliant on the formation of functional gap-junctional interCellular communications (GJICs). Potentiation of AZT activation by the engineered TMPK expressed in the human prostate cancer Cell line, PC-3, resulted in effective bystander Killing of PC-3 Cells lacking TMPK expression – an effect that could be blocked by the GJIC inhibitor, carbenoxolone. Although GJICs are mainly formed by connexins, a new family of GJIC molecules designated pannexins has been recently identified. PC-3 Cells expressed both connexin43 (Cx43) and Pannexin1 (Panx1), but Panx1 expression predominated at the plasma membrane, whereas Cx43 expression was primarily localized to the cytosol. The contribution of bystander effects to the reduction of solid tumor xenografts established by the PC-3 Cell line was evaluated in an animal model. We demonstrate the contribution of bystander Cell Killing to tumor regression in a xenograft model relying on the delivery of expression of the TMPK suicide gene into tumors via direct intratumora
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evaluation of bystander Cell Killing effects in suicide gene therapy of cancer engineered thymidylate kinase tmpk azt enzyme prodrug axis
Methods of Molecular Biology, 2015Co-Authors: Anton Neschadim, Teruyuki Yanagisawa, Takeya Sato, J. Andres Faiña Medín, Ryo NakagawaAbstract:Suicide gene therapy of cancer (SGTC) entails the introduction of a cDNA sequence into tumor Cells whose polypeptide product is capable of either directly activating apoptotic pathways itself or facilitating the activation of pharmacologic agents that do so. The latter class of SGTC approaches is of the greater utility in cancer therapy owing to the ability of some small, activated cytotoxic compounds to diffuse from their site of activation into neighboring malignant Cells, where they can also mediate destruction. This phenomenon, termed "bystander Killing", can be highly advantageous in driving significant tumor regression in vivo without the requirement of transduction of each and every tumor Cell with the suicide gene. We have developed a robust suicide gene therapy enzyme/prodrug system based on an engineered variant of the human thymidylate kinase (TMPK), which has been endowed with the ability to drive azidothymidine (AZT) activation. Delivery of this suicide gene sequence into tumors by means of recombinant lentivirus-mediated transduction embodies an SGTC strategy that successfully employs bystander Cell Killing as a mechanism to achieve significant ablation of solid tumors in vivo. Thus, this engineered TMPK/AZT suicide gene therapy axis holds great promise for clinical application in the treatment of inoperable solid tumors in the neoadjuvant setting. Here we present detailed procedures for the preparation of recombinant TMPK-based lentivirus, transduction of target Cells, and various approaches for the evaluation of bystander Cell Killing effects in SGCT in both in vitro and in vivo models.
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the engineered thymidylate kinase tmpk azt enzyme prodrug axis offers efficient bystander Cell Killing for suicide gene therapy of cancer
PLOS ONE, 2013Co-Authors: Takeya Sato, Anton Neschadim, Teruyuki Yanagisawa, J. Andres Faiña Medín, Arnon LavieAbstract:We previously described a novel suicide (or ‘Cell fate control’) gene therapy enzyme/prodrug system based on an engineered variant of human thymidylate kinase (TMPK) that potentiates azidothymidine (AZT) activation. Delivery of a suicide gene sequence into tumors by lentiviral transduction embodies a cancer gene therapy that could employ bystander Cell Killing as a mechanism driving significant tumor regression in vivo. Here we present evidence of a significant bystander Cell Killing in vitro and in vivo mediated by the TMPK/AZT suicide gene axis that is reliant on the formation of functional gap-junctional interCellular communications (GJICs). Potentiation of AZT activation by the engineered TMPK expressed in the human prostate cancer Cell line, PC-3, resulted in effective bystander Killing of PC-3 Cells lacking TMPK expression – an effect that could be blocked by the GJIC inhibitor, carbenoxolone. Although GJICs are mainly formed by connexins, a new family of GJIC molecules designated pannexins has been recently identified. PC-3 Cells expressed both connexin43 (Cx43) and Pannexin1 (Panx1), but Panx1 expression predominated at the plasma membrane, whereas Cx43 expression was primarily localized to the cytosol. The contribution of bystander effects to the reduction of solid tumor xenografts established by the PC-3 Cell line was evaluated in an animal model. We demonstrate the contribution of bystander Cell Killing to tumor regression in a xenograft model relying on the delivery of expression of the TMPK suicide gene into tumors via direct intratumoral injection of recombinant therapeutic lentivirus. Taken together, our data underscore that the TMPK/AZT enzyme-prodrug axis can be effectively utilized in suicide gene therapy of solid tumors, wherein significant tumor regression can be achieved via bystander effects mediated by GJICs.
Leona D Samson - One of the best experts on this subject based on the ideXlab platform.
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repair deficient 3 methyladenine dna glycosylase homozygous mutant mouse Cells have increased sensitivity to alkylation induced chromosome damage and Cell Killing
The EMBO Journal, 1996Co-Authors: Bevin P Engelward, A Dreslin, J Christensen, D Huszar, Carole G Kurahara, Leona D SamsonAbstract:Abstract In Escherichia coli, the repair of 3-methyladenine (3MeA) DNA lesions prevents alkylation-induced Cell death because unrepaired 3MeA blocks DNA replication. Whether this lesion is cytotoxic to mammalian Cells has been difficult to establish in the absence of 3MeA repair-deficient Cell lines. We previously isolated and characterized a mouse 3MeA DNA glycosylase cDNA (Aag) that provides resistance to Killing by alkylating agents in E. coli. To determine the in vivo role of Aag, we cloned a large fragment of the Aag gene and used it to create Aag-deficient mouse Cells by targeted homologous recombination. Aag null Cells have no detectable Aag transcripts or 3MeA DNA glycosylase activity. The loss of Aag renders Cells significantly more sensitive to methyl methanesulfonate-induced chromosome damage, and to Cell Killing induced by two methylating agents, one of which produces almost exclusively 3MeAs. Aag null embryonic stem Cells become sensitive to two cancer chemotherapeutic alkylating agents, namely 1,3-bis(2-chloroethyl)-1-nitrosourea and mitomycin C, indicating that Aag status is an important determinant of Cellular resistance to these agents. We conclude that this mammalian 3MeA DNA glycosylase plays a pivotal role in preventing alkylation-induced chromosome damage and cytotoxicity.
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repair deficient 3 methyladenine dna glycosylase homozygous mutant mouse Cells have increased sensitivity to alkylation induced chromosome damage and Cell Killing
The EMBO Journal, 1996Co-Authors: Bevin P Engelward, A Dreslin, J Christensen, D Huszar, Carole G Kurahara, Leona D SamsonAbstract:In Escherichia coli, the repair of 3-methyladenine (3MeA) DNA lesions prevents alkylation-induced Cell death because unrepaired 3MeA blocks DNA replication. Whether this lesion is cytotoxic to mammalian Cells has been difficult to establish in the absence of 3MeA repair-deficient Cell lines. We previously isolated and characterized a mouse 3MeA DNA glycosylase cDNA (Aag) that provides resistance to Killing by alkylating agents in E. coli. To determine the in vivo role of Aag, we cloned a large fragment of the Aag gene and used it to create Aag-deficient mouse Cells by targeted homologous recombination. Aag null Cells have no detectable Aag transcripts or 3MeA DNA glycosylase activity. The loss of Aag renders Cells significantly more sensitive to methyl methanesulfonate-induced chromosome damage, and to Cell Killing induced by two methylating agents, one of which produces almost exclusively 3MeAs. Aag null embryonic stem Cells become sensitive to two cancer chemotherapeutic alkylating agents, namely 1,3-bis(2-chloroethyl)-1-nitrosourea and mitomycin C, indicating that Aag status is an important determinant of Cellular resistance to these agents. We conclude that this mammalian 3MeA DNA glycosylase plays a pivotal role in preventing alkylation-induced chromosome damage and cytotoxicity.
Arnon Lavie - One of the best experts on this subject based on the ideXlab platform.
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The Engineered Thymidylate Kinase (TMPK)/AZT Enzyme- Prodrug Axis Offers Efficient Bystander Cell Killing for Suicide Gene Therapy of Cancer
2016Co-Authors: Takeya Sato, Anton Neschadim, Teruyuki Yanagisawa, Arnon Lavie, J. Andres Faiña MedínAbstract:We previously described a novel suicide (or ‘Cell fate control’) gene therapy enzyme/prodrug system based on an engineered variant of human thymidylate kinase (TMPK) that potentiates azidothymidine (AZT) activation. Delivery of a suicide gene sequence into tumors by lentiviral transduction embodies a cancer gene therapy that could employ bystander Cell Killing as a mechanism driving significant tumor regression in vivo. Here we present evidence of a significant bystander Cell Killing in vitro and in vivo mediated by the TMPK/AZT suicide gene axis that is reliant on the formation of functional gap-junctional interCellular communications (GJICs). Potentiation of AZT activation by the engineered TMPK expressed in the human prostate cancer Cell line, PC-3, resulted in effective bystander Killing of PC-3 Cells lacking TMPK expression – an effect that could be blocked by the GJIC inhibitor, carbenoxolone. Although GJICs are mainly formed by connexins, a new family of GJIC molecules designated pannexins has been recently identified. PC-3 Cells expressed both connexin43 (Cx43) and Pannexin1 (Panx1), but Panx1 expression predominated at the plasma membrane, whereas Cx43 expression was primarily localized to the cytosol. The contribution of bystander effects to the reduction of solid tumor xenografts established by the PC-3 Cell line was evaluated in an animal model. We demonstrate the contribution of bystander Cell Killing to tumor regression in a xenograft model relying on the delivery of expression of the TMPK suicide gene into tumors via direct intratumora
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identification and structural analysis of an l asparaginase enzyme from guinea pig with putative tumor Cell Killing properties
Journal of Biological Chemistry, 2014Co-Authors: Amanda M Schalk, Hien Anh Nguyen, Coraline Rigouin, Arnon LavieAbstract:The initial observation that guinea pig serum kills lymphoma Cells marks the serendipitous discovery of a new class of anti-cancer agents. The serum Cell Killing factor was shown to be an enzyme with l-asparaginase (ASNase) activity. As a direct result of this observation, several bacterial l-asparaginases were developed and are currently approved by the Food and Drug Administration for the treatment of the subset of hematological malignancies that are dependent on the extraCellular pool of the amino acid asparagine. As drugs, these enzymes act to hydrolyze asparagine to aspartate, thereby starving the cancer Cells of this amino acid. Prior to the work presented here, the precise identity of this guinea pig enzyme has not been reported in the peer-reviewed literature. We discovered that the guinea pig enzyme annotated as H0W0T5_CAVPO, which we refer to as gpASNase1, has the required low Km property consistent with that possessed by the Cell-Killing guinea pig serum enzyme. Elucidation of the ligand-free and aspartate complex gpASNase1 crystal structures allows a direct comparison with the bacterial enzymes and serves to explain the lack of l-glutaminase activity in the guinea pig enzyme. The structures were also used to generate a homology model for the human homolog hASNase1 and to help explain its vastly different kinetic properties compared with gpASNase1, despite a 70% sequence identity. Given that the bacterial enzymes frequently present immunogenic and other toxic side effects, this work suggests that gpASNase1 could be a promising alternative to these bacterial enzymes.
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the engineered thymidylate kinase tmpk azt enzyme prodrug axis offers efficient bystander Cell Killing for suicide gene therapy of cancer
PLOS ONE, 2013Co-Authors: Takeya Sato, Anton Neschadim, Teruyuki Yanagisawa, J. Andres Faiña Medín, Arnon LavieAbstract:We previously described a novel suicide (or ‘Cell fate control’) gene therapy enzyme/prodrug system based on an engineered variant of human thymidylate kinase (TMPK) that potentiates azidothymidine (AZT) activation. Delivery of a suicide gene sequence into tumors by lentiviral transduction embodies a cancer gene therapy that could employ bystander Cell Killing as a mechanism driving significant tumor regression in vivo. Here we present evidence of a significant bystander Cell Killing in vitro and in vivo mediated by the TMPK/AZT suicide gene axis that is reliant on the formation of functional gap-junctional interCellular communications (GJICs). Potentiation of AZT activation by the engineered TMPK expressed in the human prostate cancer Cell line, PC-3, resulted in effective bystander Killing of PC-3 Cells lacking TMPK expression – an effect that could be blocked by the GJIC inhibitor, carbenoxolone. Although GJICs are mainly formed by connexins, a new family of GJIC molecules designated pannexins has been recently identified. PC-3 Cells expressed both connexin43 (Cx43) and Pannexin1 (Panx1), but Panx1 expression predominated at the plasma membrane, whereas Cx43 expression was primarily localized to the cytosol. The contribution of bystander effects to the reduction of solid tumor xenografts established by the PC-3 Cell line was evaluated in an animal model. We demonstrate the contribution of bystander Cell Killing to tumor regression in a xenograft model relying on the delivery of expression of the TMPK suicide gene into tumors via direct intratumoral injection of recombinant therapeutic lentivirus. Taken together, our data underscore that the TMPK/AZT enzyme-prodrug axis can be effectively utilized in suicide gene therapy of solid tumors, wherein significant tumor regression can be achieved via bystander effects mediated by GJICs.
Takeya Sato - One of the best experts on this subject based on the ideXlab platform.
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The Engineered Thymidylate Kinase (TMPK)/AZT Enzyme- Prodrug Axis Offers Efficient Bystander Cell Killing for Suicide Gene Therapy of Cancer
2016Co-Authors: Takeya Sato, Anton Neschadim, Teruyuki Yanagisawa, Arnon Lavie, J. Andres Faiña MedínAbstract:We previously described a novel suicide (or ‘Cell fate control’) gene therapy enzyme/prodrug system based on an engineered variant of human thymidylate kinase (TMPK) that potentiates azidothymidine (AZT) activation. Delivery of a suicide gene sequence into tumors by lentiviral transduction embodies a cancer gene therapy that could employ bystander Cell Killing as a mechanism driving significant tumor regression in vivo. Here we present evidence of a significant bystander Cell Killing in vitro and in vivo mediated by the TMPK/AZT suicide gene axis that is reliant on the formation of functional gap-junctional interCellular communications (GJICs). Potentiation of AZT activation by the engineered TMPK expressed in the human prostate cancer Cell line, PC-3, resulted in effective bystander Killing of PC-3 Cells lacking TMPK expression – an effect that could be blocked by the GJIC inhibitor, carbenoxolone. Although GJICs are mainly formed by connexins, a new family of GJIC molecules designated pannexins has been recently identified. PC-3 Cells expressed both connexin43 (Cx43) and Pannexin1 (Panx1), but Panx1 expression predominated at the plasma membrane, whereas Cx43 expression was primarily localized to the cytosol. The contribution of bystander effects to the reduction of solid tumor xenografts established by the PC-3 Cell line was evaluated in an animal model. We demonstrate the contribution of bystander Cell Killing to tumor regression in a xenograft model relying on the delivery of expression of the TMPK suicide gene into tumors via direct intratumora
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evaluation of bystander Cell Killing effects in suicide gene therapy of cancer engineered thymidylate kinase tmpk azt enzyme prodrug axis
Methods of Molecular Biology, 2015Co-Authors: Anton Neschadim, Teruyuki Yanagisawa, Takeya Sato, J. Andres Faiña Medín, Ryo NakagawaAbstract:Suicide gene therapy of cancer (SGTC) entails the introduction of a cDNA sequence into tumor Cells whose polypeptide product is capable of either directly activating apoptotic pathways itself or facilitating the activation of pharmacologic agents that do so. The latter class of SGTC approaches is of the greater utility in cancer therapy owing to the ability of some small, activated cytotoxic compounds to diffuse from their site of activation into neighboring malignant Cells, where they can also mediate destruction. This phenomenon, termed "bystander Killing", can be highly advantageous in driving significant tumor regression in vivo without the requirement of transduction of each and every tumor Cell with the suicide gene. We have developed a robust suicide gene therapy enzyme/prodrug system based on an engineered variant of the human thymidylate kinase (TMPK), which has been endowed with the ability to drive azidothymidine (AZT) activation. Delivery of this suicide gene sequence into tumors by means of recombinant lentivirus-mediated transduction embodies an SGTC strategy that successfully employs bystander Cell Killing as a mechanism to achieve significant ablation of solid tumors in vivo. Thus, this engineered TMPK/AZT suicide gene therapy axis holds great promise for clinical application in the treatment of inoperable solid tumors in the neoadjuvant setting. Here we present detailed procedures for the preparation of recombinant TMPK-based lentivirus, transduction of target Cells, and various approaches for the evaluation of bystander Cell Killing effects in SGCT in both in vitro and in vivo models.
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the engineered thymidylate kinase tmpk azt enzyme prodrug axis offers efficient bystander Cell Killing for suicide gene therapy of cancer
PLOS ONE, 2013Co-Authors: Takeya Sato, Anton Neschadim, Teruyuki Yanagisawa, J. Andres Faiña Medín, Arnon LavieAbstract:We previously described a novel suicide (or ‘Cell fate control’) gene therapy enzyme/prodrug system based on an engineered variant of human thymidylate kinase (TMPK) that potentiates azidothymidine (AZT) activation. Delivery of a suicide gene sequence into tumors by lentiviral transduction embodies a cancer gene therapy that could employ bystander Cell Killing as a mechanism driving significant tumor regression in vivo. Here we present evidence of a significant bystander Cell Killing in vitro and in vivo mediated by the TMPK/AZT suicide gene axis that is reliant on the formation of functional gap-junctional interCellular communications (GJICs). Potentiation of AZT activation by the engineered TMPK expressed in the human prostate cancer Cell line, PC-3, resulted in effective bystander Killing of PC-3 Cells lacking TMPK expression – an effect that could be blocked by the GJIC inhibitor, carbenoxolone. Although GJICs are mainly formed by connexins, a new family of GJIC molecules designated pannexins has been recently identified. PC-3 Cells expressed both connexin43 (Cx43) and Pannexin1 (Panx1), but Panx1 expression predominated at the plasma membrane, whereas Cx43 expression was primarily localized to the cytosol. The contribution of bystander effects to the reduction of solid tumor xenografts established by the PC-3 Cell line was evaluated in an animal model. We demonstrate the contribution of bystander Cell Killing to tumor regression in a xenograft model relying on the delivery of expression of the TMPK suicide gene into tumors via direct intratumoral injection of recombinant therapeutic lentivirus. Taken together, our data underscore that the TMPK/AZT enzyme-prodrug axis can be effectively utilized in suicide gene therapy of solid tumors, wherein significant tumor regression can be achieved via bystander effects mediated by GJICs.
Bevin P Engelward - One of the best experts on this subject based on the ideXlab platform.
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repair deficient 3 methyladenine dna glycosylase homozygous mutant mouse Cells have increased sensitivity to alkylation induced chromosome damage and Cell Killing
The EMBO Journal, 1996Co-Authors: Bevin P Engelward, A Dreslin, J Christensen, D Huszar, Carole G Kurahara, Leona D SamsonAbstract:Abstract In Escherichia coli, the repair of 3-methyladenine (3MeA) DNA lesions prevents alkylation-induced Cell death because unrepaired 3MeA blocks DNA replication. Whether this lesion is cytotoxic to mammalian Cells has been difficult to establish in the absence of 3MeA repair-deficient Cell lines. We previously isolated and characterized a mouse 3MeA DNA glycosylase cDNA (Aag) that provides resistance to Killing by alkylating agents in E. coli. To determine the in vivo role of Aag, we cloned a large fragment of the Aag gene and used it to create Aag-deficient mouse Cells by targeted homologous recombination. Aag null Cells have no detectable Aag transcripts or 3MeA DNA glycosylase activity. The loss of Aag renders Cells significantly more sensitive to methyl methanesulfonate-induced chromosome damage, and to Cell Killing induced by two methylating agents, one of which produces almost exclusively 3MeAs. Aag null embryonic stem Cells become sensitive to two cancer chemotherapeutic alkylating agents, namely 1,3-bis(2-chloroethyl)-1-nitrosourea and mitomycin C, indicating that Aag status is an important determinant of Cellular resistance to these agents. We conclude that this mammalian 3MeA DNA glycosylase plays a pivotal role in preventing alkylation-induced chromosome damage and cytotoxicity.
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repair deficient 3 methyladenine dna glycosylase homozygous mutant mouse Cells have increased sensitivity to alkylation induced chromosome damage and Cell Killing
The EMBO Journal, 1996Co-Authors: Bevin P Engelward, A Dreslin, J Christensen, D Huszar, Carole G Kurahara, Leona D SamsonAbstract:In Escherichia coli, the repair of 3-methyladenine (3MeA) DNA lesions prevents alkylation-induced Cell death because unrepaired 3MeA blocks DNA replication. Whether this lesion is cytotoxic to mammalian Cells has been difficult to establish in the absence of 3MeA repair-deficient Cell lines. We previously isolated and characterized a mouse 3MeA DNA glycosylase cDNA (Aag) that provides resistance to Killing by alkylating agents in E. coli. To determine the in vivo role of Aag, we cloned a large fragment of the Aag gene and used it to create Aag-deficient mouse Cells by targeted homologous recombination. Aag null Cells have no detectable Aag transcripts or 3MeA DNA glycosylase activity. The loss of Aag renders Cells significantly more sensitive to methyl methanesulfonate-induced chromosome damage, and to Cell Killing induced by two methylating agents, one of which produces almost exclusively 3MeAs. Aag null embryonic stem Cells become sensitive to two cancer chemotherapeutic alkylating agents, namely 1,3-bis(2-chloroethyl)-1-nitrosourea and mitomycin C, indicating that Aag status is an important determinant of Cellular resistance to these agents. We conclude that this mammalian 3MeA DNA glycosylase plays a pivotal role in preventing alkylation-induced chromosome damage and cytotoxicity.