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Papers by Martin Hoagland
Review Phytoestrogens and breast cancer: a complex story
Experimental Approaches for the Study of Cytochrome P450 Gene Regulation
Drug Metabolism and Transport, 2005
ABSTRACT This chapter uses cytochrome P450 1A1 (CYP1A1) as a model “xenobiotic inducible” gene an... more ABSTRACT This chapter uses cytochrome P450 1A1 (CYP1A1) as a model “xenobiotic inducible” gene and the aryl hydrocarbon receptor (AHR) as a regulator of this gene product to illustrate how studies may be performed to understand the relationships between the regulator protein and its target genes. The methodology described was developed to study transcriptional activation. Because transcriptional repression occurs nearly as frequently as activation, the assays described may require modifications to accommodate both mechanisms. Approaches typically used to determine whether changes in gene expression induced by a particular drug or xenobiotic are a result of an increase in gene transcription mediated by specific protein/DNA interactions are described. These approaches and assays include the use of inhibitors of transcription and translation, nuclear runoffs, promoter analysis, and reporter assays to determine the role of specific DNA sequences, determination of specific DNA–protein interactions both in vitro and in vivo using electrophoretic gel mobility shift assays, UV-crosslinking, Southwestern blotting, yeast one-hybrid screening, DNA footprinting, site affinity amplification binding, and the chromatin immunoprecipitation (ChIP) assay, and studies to determine functional roles for specific transcription factors. Key WordsAryl hydrocarbon receptor–aryl hydrocarbon receptor nuclear translocator–chromatin immunoprecipitation assay–CYP1A1–cytochrome P450–dioxin responsive element–DNA footprinting–DNA microarrays–electromobility shift assays–gene regulation–luciferase activity–nuclear runoff–promoter analysis–protein–DNA interactions–reporter analysis–reverse transcription-polymerase chain reaction–site-affinity amplification binding–small inhibitory RNA–Southwestern blot, 2,3,7,8-tetrachlorodibenzo-p-dioxin–transfection–transient–transfection–stable–transfection–viral–UV-crosslinking–yeast one-hybrid screening
Lack of the Aryl Hydrocarbon Receptor Leads to Impaired Activation of AKT/Protein Kinase B and Enhanced Sensitivity to Apoptosis Induced via the Intrinsic Pathway
Journal of Pharmacology and Experimental Therapeutics, 2006
The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that is best known... more The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that is best known for its role in mediating the toxicity of many environmental contaminants such as 2,3,7,8 tetrachlorodibenzo-p-dioxin. However, the endogenous role of AHR, especially with respect to the apoptotic process, is largely unknown and contradictory. In this report, we have used a mouse hepatoma cell line (Hepa1c1c7) and its AHR-deficient derivative (LA1) to examine the effect of differing AHR levels on apoptosis susceptibility, in particular, apoptosis regulated by the intrinsic pathway. Toward this end, the cells were subjected to UV irradiation, hydrogen peroxide, and serum starvation. Analyses of a number of different endpoints of apoptosis revealed that the LA1 cells were more sensitive to these stresses than the wild-type cells, indicating that the AHR plays a cytoprotective role in the face of stimuli that initiate the intrinsic apoptotic pathway. A direct role of the AHR in mediating this effect was confirmed using both pharmacological and molecular approaches. Further analyses imply that lack of the AHR leads to an impaired survival response mediated by phosphatidylinositol 3'-OH kinase-Akt/protein kinase B and, to a lesser degree, epidermal growth factor receptor activation. These findings indicate that exploring the use of the AHR antagonist as agents that enhance the proapoptotic actions of cancer therapies may be a valid approach.
The p53 Inhibitor Pifithrin- Is a Potent Agonist of the Aryl Hydrocarbon Receptor
Journal of Pharmacology and Experimental Therapeutics, 2005
The tumor suppressor protein p53 is currently a target of emerging drug therapies directed toward... more The tumor suppressor protein p53 is currently a target of emerging drug therapies directed toward neurodegenerative diseases, such as Alzheimer's and Parkinson's, and side effects associated with cancer treatments. Of this group of drugs, the best characterized is pifithrin-alpha, a small molecule that inhibits p53-dependent apoptosis through an undetermined mechanism. In this study, we have used a number of molecular approaches to test the hypothesis that pifithrin-alpha acts as an aryl hydrocarbon receptor (AhR) agonist and, in this manner, inhibits the actions of p53. Toward this end, we have found that pifithrin-alpha is a potent AhR agonist as determined by its ability to bind the AhR, induce formation of its DNA binding complex, activate reporter activity, and up-regulate the classic AhR target gene CYP1A1. However, examination of its ability to inhibit p53-mediated gene activation and apoptosis revealed that these actions occurred via an AhR-independent manner. The significance of this study is based on the fact that activation of the AhR is typically associated with an increase in phase I and phase II metabolizing enzymes and adverse biological events such as tumor promotion that may contribute to untoward effects of pifithrin-alpha. Hence, this work will aid in the future design of more specific members of this important class of p53 inhibitors for use in a clinical setting.
Genistein is an isoflavone with oestrogenic activity that is present in a variety of soy products... more Genistein is an isoflavone with oestrogenic activity that is present in a variety of soy products as a constituent of complex mixtures of bioactive compounds, whose matrix profiles play an important role in determining the overall oestrogenic bioactivity of genistein. We review data on how the profile of soy bioactive compounds can modulate genistein-stimulated oestrogen-dependent tumour growth. Our research has focused on the effects of dietary genistein on the growth of oestrogen (E)-dependent mammary tumours both in vitro and in vivo. Genistein enhances the proliferation of E-dependent human breast cancer tumour growth. In a similar manner, dietary genistein stimulates tumour growth in the chemically-induced (NMU) mammary cancer rodent model. Genistin, the glycoside of genistein, simulates growth similar to that of genistein and withdrawal of either genistein or genistin results in tumour regression. The extent of soy processing modulates the effects of dietary genistein in vivo as soy protein isolate, a highly purified and widely used source of protein that is processed to contain low, medium, and high amounts of isoflavones, stimulate the growth of the E-dependent mammary tumours in a dose dependent manner. In contrast to the more purified diets, studies with soy flour of equivalent genistein levels did not stimulate the growth of E-dependent breast cancer tumours in vivo. However, the size of these tumours also did not regress as compared with control groups in which oestrogen and genistein have been withdrawn. The expression of the oestrogen-target genes of pS2, progesterone receptor, and cyclin D1 correlates with the growth of E-dependent tumours and has been consistently observed to be induced in response to treatment with dietary genistein. To evaluate whether dietary genistein interacts with current anti-oestrogen breast cancer therapies such as tamoxifen (TAM), we implanted E-dependent tumours into ovariectomized athymic mice and administered oestradiol,
Nature Chemical Biology, 2006
Mutation and aberrant expression of apoptotic proteins are hallmarks of cancer. These changes pre... more Mutation and aberrant expression of apoptotic proteins are hallmarks of cancer. These changes prevent proapoptotic signals from being transmitted to executioner caspases, thereby averting apoptotic death and allowing cellular proliferation. Caspase-3 is the key executioner caspase, and it exists as an inactive zymogen that is activated by upstream signals. Notably, concentrations of procaspase-3 in certain cancerous cells are significantly higher than those in noncancerous controls. Here we report the identification of a small molecule (PAC-1) that directly activates procaspase-3 to caspase-3 in vitro and induces apoptosis in cancerous cells isolated from primary colon tumors in a manner directly proportional to the concentration of procaspase-3 inside these cells. We found that PAC-1 retarded the growth of tumors in three different mouse models of cancer, including two models in which PAC-1 was administered orally. PAC-1 is the first small molecule known to directly activate procaspase-3 to caspase-3, a transformation that allows induction of apoptosis even in cells that have defective apoptotic machinery. The direct activation of executioner caspases is an anticancer strategy that may prove beneficial in treating the many cancers in which procaspase-3 concentrations are elevated.
Review Phytoestrogens and breast cancer: a complex story
Experimental Approaches for the Study of Cytochrome P450 Gene Regulation
Drug Metabolism and Transport, 2005
ABSTRACT This chapter uses cytochrome P450 1A1 (CYP1A1) as a model “xenobiotic inducible” gene an... more ABSTRACT This chapter uses cytochrome P450 1A1 (CYP1A1) as a model “xenobiotic inducible” gene and the aryl hydrocarbon receptor (AHR) as a regulator of this gene product to illustrate how studies may be performed to understand the relationships between the regulator protein and its target genes. The methodology described was developed to study transcriptional activation. Because transcriptional repression occurs nearly as frequently as activation, the assays described may require modifications to accommodate both mechanisms. Approaches typically used to determine whether changes in gene expression induced by a particular drug or xenobiotic are a result of an increase in gene transcription mediated by specific protein/DNA interactions are described. These approaches and assays include the use of inhibitors of transcription and translation, nuclear runoffs, promoter analysis, and reporter assays to determine the role of specific DNA sequences, determination of specific DNA–protein interactions both in vitro and in vivo using electrophoretic gel mobility shift assays, UV-crosslinking, Southwestern blotting, yeast one-hybrid screening, DNA footprinting, site affinity amplification binding, and the chromatin immunoprecipitation (ChIP) assay, and studies to determine functional roles for specific transcription factors. Key WordsAryl hydrocarbon receptor–aryl hydrocarbon receptor nuclear translocator–chromatin immunoprecipitation assay–CYP1A1–cytochrome P450–dioxin responsive element–DNA footprinting–DNA microarrays–electromobility shift assays–gene regulation–luciferase activity–nuclear runoff–promoter analysis–protein–DNA interactions–reporter analysis–reverse transcription-polymerase chain reaction–site-affinity amplification binding–small inhibitory RNA–Southwestern blot, 2,3,7,8-tetrachlorodibenzo-p-dioxin–transfection–transient–transfection–stable–transfection–viral–UV-crosslinking–yeast one-hybrid screening
Lack of the Aryl Hydrocarbon Receptor Leads to Impaired Activation of AKT/Protein Kinase B and Enhanced Sensitivity to Apoptosis Induced via the Intrinsic Pathway
Journal of Pharmacology and Experimental Therapeutics, 2006
The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that is best known... more The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor that is best known for its role in mediating the toxicity of many environmental contaminants such as 2,3,7,8 tetrachlorodibenzo-p-dioxin. However, the endogenous role of AHR, especially with respect to the apoptotic process, is largely unknown and contradictory. In this report, we have used a mouse hepatoma cell line (Hepa1c1c7) and its AHR-deficient derivative (LA1) to examine the effect of differing AHR levels on apoptosis susceptibility, in particular, apoptosis regulated by the intrinsic pathway. Toward this end, the cells were subjected to UV irradiation, hydrogen peroxide, and serum starvation. Analyses of a number of different endpoints of apoptosis revealed that the LA1 cells were more sensitive to these stresses than the wild-type cells, indicating that the AHR plays a cytoprotective role in the face of stimuli that initiate the intrinsic apoptotic pathway. A direct role of the AHR in mediating this effect was confirmed using both pharmacological and molecular approaches. Further analyses imply that lack of the AHR leads to an impaired survival response mediated by phosphatidylinositol 3'-OH kinase-Akt/protein kinase B and, to a lesser degree, epidermal growth factor receptor activation. These findings indicate that exploring the use of the AHR antagonist as agents that enhance the proapoptotic actions of cancer therapies may be a valid approach.
The p53 Inhibitor Pifithrin- Is a Potent Agonist of the Aryl Hydrocarbon Receptor
Journal of Pharmacology and Experimental Therapeutics, 2005
The tumor suppressor protein p53 is currently a target of emerging drug therapies directed toward... more The tumor suppressor protein p53 is currently a target of emerging drug therapies directed toward neurodegenerative diseases, such as Alzheimer's and Parkinson's, and side effects associated with cancer treatments. Of this group of drugs, the best characterized is pifithrin-alpha, a small molecule that inhibits p53-dependent apoptosis through an undetermined mechanism. In this study, we have used a number of molecular approaches to test the hypothesis that pifithrin-alpha acts as an aryl hydrocarbon receptor (AhR) agonist and, in this manner, inhibits the actions of p53. Toward this end, we have found that pifithrin-alpha is a potent AhR agonist as determined by its ability to bind the AhR, induce formation of its DNA binding complex, activate reporter activity, and up-regulate the classic AhR target gene CYP1A1. However, examination of its ability to inhibit p53-mediated gene activation and apoptosis revealed that these actions occurred via an AhR-independent manner. The significance of this study is based on the fact that activation of the AhR is typically associated with an increase in phase I and phase II metabolizing enzymes and adverse biological events such as tumor promotion that may contribute to untoward effects of pifithrin-alpha. Hence, this work will aid in the future design of more specific members of this important class of p53 inhibitors for use in a clinical setting.
Genistein is an isoflavone with oestrogenic activity that is present in a variety of soy products... more Genistein is an isoflavone with oestrogenic activity that is present in a variety of soy products as a constituent of complex mixtures of bioactive compounds, whose matrix profiles play an important role in determining the overall oestrogenic bioactivity of genistein. We review data on how the profile of soy bioactive compounds can modulate genistein-stimulated oestrogen-dependent tumour growth. Our research has focused on the effects of dietary genistein on the growth of oestrogen (E)-dependent mammary tumours both in vitro and in vivo. Genistein enhances the proliferation of E-dependent human breast cancer tumour growth. In a similar manner, dietary genistein stimulates tumour growth in the chemically-induced (NMU) mammary cancer rodent model. Genistin, the glycoside of genistein, simulates growth similar to that of genistein and withdrawal of either genistein or genistin results in tumour regression. The extent of soy processing modulates the effects of dietary genistein in vivo as soy protein isolate, a highly purified and widely used source of protein that is processed to contain low, medium, and high amounts of isoflavones, stimulate the growth of the E-dependent mammary tumours in a dose dependent manner. In contrast to the more purified diets, studies with soy flour of equivalent genistein levels did not stimulate the growth of E-dependent breast cancer tumours in vivo. However, the size of these tumours also did not regress as compared with control groups in which oestrogen and genistein have been withdrawn. The expression of the oestrogen-target genes of pS2, progesterone receptor, and cyclin D1 correlates with the growth of E-dependent tumours and has been consistently observed to be induced in response to treatment with dietary genistein. To evaluate whether dietary genistein interacts with current anti-oestrogen breast cancer therapies such as tamoxifen (TAM), we implanted E-dependent tumours into ovariectomized athymic mice and administered oestradiol,
Nature Chemical Biology, 2006
Mutation and aberrant expression of apoptotic proteins are hallmarks of cancer. These changes pre... more Mutation and aberrant expression of apoptotic proteins are hallmarks of cancer. These changes prevent proapoptotic signals from being transmitted to executioner caspases, thereby averting apoptotic death and allowing cellular proliferation. Caspase-3 is the key executioner caspase, and it exists as an inactive zymogen that is activated by upstream signals. Notably, concentrations of procaspase-3 in certain cancerous cells are significantly higher than those in noncancerous controls. Here we report the identification of a small molecule (PAC-1) that directly activates procaspase-3 to caspase-3 in vitro and induces apoptosis in cancerous cells isolated from primary colon tumors in a manner directly proportional to the concentration of procaspase-3 inside these cells. We found that PAC-1 retarded the growth of tumors in three different mouse models of cancer, including two models in which PAC-1 was administered orally. PAC-1 is the first small molecule known to directly activate procaspase-3 to caspase-3, a transformation that allows induction of apoptosis even in cells that have defective apoptotic machinery. The direct activation of executioner caspases is an anticancer strategy that may prove beneficial in treating the many cancers in which procaspase-3 concentrations are elevated.