Life has evolved to function in an environment that contains many harmful chemical agents, whether they be naturallyoccurring toxins or anthropogenic contaminants. Virtually all of these environmental toxicants provoke adaptive responses in the organisms they encounter. Such responses are attempts by the organisms to counteract and/or eliminate the agents, thereby minimizing their abilities to produce harm. In this regard, each organism has evolved a large number of enzymes that enable it to defend itself from its own particular chemical environment. Prominent among these are enzymes that metabolize lipophilic chemicals, which tend to accumulate in the fatty tissues of the organism, to more hydrophilic agents, which are more readily eliminated. These enzymes, especially prominent in the liver, include the so-called phase I enzymes (including the cytochrome P450 superfamily of enzymes) that insert a functional group onto a substrate molecule, phase II enzymes (including the sulfotransferases and glucuronosyltransferases) that add a charged moiety onto the functional group, as well as transporters that actively discharge these metabolites from cells. My research program is directed toward understanding the integrated mechanisms whereby a normal hepatocyte responds to its chemical environment. Much of this research during recent years has focused on understanding the mechanistic basis for the observation that many drugs that interfere with cholesterol metabolism are also modifiers of cytochrome P450 gene expression. At least a portion of these effects are attributable to the alterations in hepatocellular content of biosynthetic intermediates and sterols that accompany metabolic blockade, and it appears likely that these endogenous molecules exert their effects by activating orphan members of the nuclear receptor superfamily.

Selected Publications

Kocarek TA and Mercer-Haines NA. Squalestatin 1-inducible expression of rat CYP2B: Evidence that an endogenous isoprenoid is an activator of the constitutive androstane receptor.Mol Pharmacol 62:1177-1186, 2002.
 
Kocarek TA, Dahn MS, Cai H, Strom SC and Mercer-Haines NA. Regulation of CYP2B6 and CYP3A expression by hydroxymethylglutaryl coenzyme A inhibitors in primary cultured human hepatocytes.Drug Metab Dispos 30:1400-1405, 2002.
 
Shenoy SD, Spencer TA, Mercer-Haines NA, Alipour M, Gargano MD, Runge-Morris M and Kocarek TA. CYP3A induction by liver X receptor ligands in primary cultured rat and mouse hepatocytes is mediated by the pregnane X receptor.Drug Metab Dispos 32:66-71, 2004.
 
Shenoy SD, Spencer TA, Mercer-Haines NA, Abdolalipour M, Wurster WL, Runge-Morris M and Kocarek TA. Induction of CYP3A by 2,3-oxidosqualene:lanosterol cyclase inhibitors is mediated by an endogenous squalene metabolite in primary cultured rat hepatocytes.Mol Pharmacol 65:1302-1312, 2004.
 
Fang HL, Abdolalipour M, Duanmu Z, Smigelski JR, Weckle A, Kocarek TA and Runge-Morris M. Regulation of glucocorticoid-inducible hydroxysteroid sulfotransferase (SULT2A-40/41) gene transcription in primary cultured rat hepatocytes: Role of CCAAT/enhancer-binding protein (C/EBP) liver-enriched transcription factors.Drug Metab Dispos 33:147-156, 2005.
 
Runge-Morris M and Kocarek TA. Regulation of sulfotransferases by xenobiotic receptors.Curr Drug Metab 6:299-307, 2005.
 
Fang HL, Strom SC, Cai H, Falany CN, Kocarek TA and Runge-Morris M. Regulation of human hepatic hydroxysteroid sulfotransferase gene expression by the peroxisome proliferator activated receptor alpha transcription factor.Mol Pharmacol 67:1257-1267, 2005.

Thomas RD, Green MR, Wilson C, Weckle AL, Duanmu Z, Kocarek TA and Runge-Morris M. Cytochrome P450 expression and metabolic activation of cooked food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in MCF10A breast epithelial cells.Chem Biol Interact 160:204-216, 2006.

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The major focus of Dr. Majumdar’s research has been and continues to be the study of the role of growth factor receptor tyrosine kinases, specifically epidermal growth factor receptor (EGFR) and its family members in regulating gastrointestinal mucosal growth  during aging. The primary goal is to delineate the intracellular events responsible for the age-related rise in gastrointestinal malignancies. The insight that EGFR abnormalities might contribute to carcinogenesis in aging has led to study ways in which the EGFR could be regulated.  More recently, Dr. Majumdar’s lab has identified and cloned a novel  EGFR-antagonist called ERRP (EGF-R Related Protein), which has been shown to inhibit cell proliferation and stimulates in a wide variety of malignant epithelial cell lines due to its ability to inhibit EGFR tyrosine kinase activity. ERRP also inhibits growth of colon, pancreatic and breast tumors in experimental animals. ERRP has been patented as a potential therapeutic agent for a wide variety of epithelial cancers.  

In addition to pursuing studies on signal transduction and cloning, Dr. Majumdar’s lab has been investigating the chemopreventive role of certain nutrients, particularly folic acid and curcumin in gastrointestinal cancers.

Selected Publications

Xiao Z-Q, Li J, Majumdar APN: Regulation of TGF-a-induced Activation of AP-1 in the Aging Gastric Mucosa. Am. J. Physiol. Gastrointest. Liver Physiol. 285: G396-G403, 2003.

Xu H, Yu Y, Marciniak DJ, Rishi A, Sarkar FH, Kucuk O, Majumdar APN : EGFR-Related Protein (ERRP) Inhibits Multiple Members of EGFR Family in Colon and Breast Cancer Cells. Mol. Cancer Ther. 4: 435-442, 2005.

Majumdar APN: Therapeutic Potential of EGFR-Related Protein, a Universal EGFR Family Antagonist. Future Oncol. 1: 235-245, 2005.

Majumdar APN, Du J: Phosphatidylinositol 3 Kinase/Akt Signaling Stimulates Colonic Mucosal Cell Survival During Aging. Am. J. Physiol. Gastrointest. Liver Physiol. 290: G49-G55, 2006.

Schmelz EM, Xu H, Sengupta R, Du H, Banerjee S, Sarkar FH,  Rishi AK, Majumdar APN: Regression of Early and Intermediate Stages of Colon Cancer by Targeting Multiple Members of the EGFR Family with EGF-Receptor Related Protein. Cancer Res 67: 5389-5397, 2007.

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Research in our laboratory focuses on examining the effects of hormones, growth factors and environmental agents on intracellular signaling in breast epithelial cells and in primary cultured rat and human hepatocytes. Our research utilizes a series of human breast epithelial cell lines that when implanted in nude/beige mice recapitulates the morphological sequence of changes of human proliferative breast disease, which is associated with an increased risk for breast cancer development. Using these breast epithelial cell lines we have reported differences in their sensitivities to Fas-mediated apoptosis and we have detected differences in several cell cycle checkpoints, including p53, pRB, E2F-1 and several cyclin-dependent kinases. We have also detected differences in the protein levels of various cell adhesion proteins and in their associations following exposure to environmental agents. We have examined global gene expression profiles, pathways and gene ontologies associated with disruption of components in the signaling pathways. In a second research project, we are examining the role of insulin signaling in the altered expression of various forms of cytochromes P450 which metabolize carcinogens, increase oxidative stress and initiate cell transformation and tumorigenesis. We are interested in the role of these processes in hepatocellular carcinoma and have shown that two of these enzymes, CYP2E1, which catalyzes the activation of low molecular weight carcinogens (e.g. nitrosamines, halogenated hydrocarbons, aromatic hydrocarbons), and CYP4A, which metabolizes endogenous fatty acids, are regulated in response to insulin. For CYP2E1 this occurs through the PI 3-kinase, Akt/protein kinase B, mTOR, p70 S6 kinase pathway. Techniques such as immunoprecipitation, immunoblot, Northern blot and RT-PCR analyses, cloning, gene transcription, gel shift assays and transient infections using adenovirus GFP-dominant negative constructs are employed in our research activities. Global gene expression profiles, gene ontologies, pathways, and Principal Components Analysis are accomplished using whole rat and human genome microarrays.

Selected Publications

Woodcroft KJ, Hafner MS, Novak RF. Insulin signaling in the transcriptional and posttranscriptional regulation of CYP2E1 expression. Hepatology. Feb;35(2):263-73, 2002.

Kim SK, Woodcroft KJ, Novak RF. Insulin and glucagon regulation of glutathione S-transferase expression in primary cultured rat hepatocytes. J Pharmacol Exp Ther.  Apr;305(1):353-61, 2003.

Starcevic SL, Diotte NM, Zukowski KL, Cameron MJ, Novak RF. Oxidative DNA damage and repair in a cell lineage model of human proliferative breast disease (PBD). Toxicol Sci. Sep;75(1):74-81, 2003.

Dombkowski AA, Thibodeau BJ, Starcevic SL, Novak RF.  Gene-specific dye bias in microarray reference designs, FEBS Letters. 560(3):120-124, 2004.

Kim SK, Woodcroft KJ, Khodadadeh SS, Novak RF. Insulin signaling regulates {gamma}-glutamylcysteine ligase catalytic subunit expression in primary cultured rat hepatocytes. J Pharmacol Exp Ther. Oct;311(1):99-108, 2004.

Dombkowski AA, Cukovic D, and Novak RF. Potential biomarkers of proliferative breast disease identified through secretome analysis of gene expression data. Secretome analysis of microarray data reveals extracellular events associated with proliferative potential in a cell line model of breast disease. Cancer Lett. Sep 8;241(1):49-58, 2006.

Kim SK, Abdelmegeed MA, Novak RF. Identification of the insulin signaling cascade in the regulation of Alpha-class glutathione S-transferase expression in primary cultured rat hepatocytes. J Pharmacol Exp Ther. Mar;316(3):1255-61, 2006.

S.K. Kim and R.F. Novak. The Role of Intracellular Signaling in Insulin-mediated Regulation of Drug Metabolizing Enzyme Gene and Protein Expression. Pharmacol Ther. Jan;113(1):88-120, 2007.

John J. Reiners, Jr., Ph.D.
Chemical Carcinogenesis and Toxicology
Ph.D., Purdue University, 1977

Selected Publications

Kessel, D., Castelli M. and Reiners, Jr., J.J. Ruthenium red-mediated suppression of Bcl-2 loss and Ca2+ release initiated by photodamage to the endoplasmic reticulum: scavenging of reactive oxygen species. Cell Death Different., 12:502-511, 2005.

Kessel, D., Conley, M., Vicente, M.G.H. and Reiners, Jr., J.J. Studies on the sub-cellular localization of the porphycene CPO. Photochem. Photobiol., 81:569-572, 2005.

Caruso, J.A., Mathieu, P.A. and Reiners, Jr., J.J. Sphingomyelins suppress the targeted disruption of acid organelles and the induction of apoptosis by the lysosomal photosensitizer NPe6. Biochem. J., 392:325-334, 2005.

Mattingly, R.R., Kraniak, J.A., Dilworth, J.T., Mathieu, P., Bealmear B., Benjamins, J.A., Tainsky, M.A. and Reiners, Jr., J.J. The Mitogen-activated protein kinase/Extracelular signal-Regulated kinase kinase inhibitor PD184352/CI-1040 selectively induces apoptosis in malignant schwannoma  cell lines. J. Pharm. Exp. Therap. 316:456-465, 2006.

Caruso, J.A., Mathieu, P.A., Joiakim, A., Zhang, H. and Reiners, Jr., J.J. Aryl hydrocarbon receptor modulation of TNFa-induced apoptosis and lysosomal disruption in a hepatoma model that is caspase-8 independent. J. Biol. Chem., 281:10954-10967, 2006.

Kessel, D., Vicente, G.H., and Reiners, Jr., John J. Initiation of Apoptosis and Autophagy by Photodynamic Therapy. Lasers Surg. Med. 38:482-488, 2006

Caruso J.A. and Reiners, Jr., J.J. Proteolysis of HIP during apoptosis occurs within a region similar to the Bid loop. Apoptosis, 11:1877-1885, 2006.

Dilworth, J.T., Kraniak, J.M., Wojtkowiak, J.W., Gibbs, R.A., Borch, R.F., Tainsky, M.A., Reiners, Jr., J.J. and Mattingly, R.R. Molecular Targets for emerging anti-tumor therapies for neurofibromatosis type 1. Biochem. Pharmacol., 72:1485-1489, 2006.

Kessel, D., Vicente, G.H., and Reiners, Jr., John J. Initiation of apoptosis and autophagy by photodynamic therapy. Autophagy, 2:289-290, 2006.

Kessel, D. and Reiners, Jr. Initiation of apoptosis and autophagy by the Bcl-2 antagonist HA14-1. Cancer Lett., 249:294-299, 2007.

Kessel, D., Reiners, Jr., J.J., Hazeldine S.T., Polin L. and Horwitz, J.P. The drugs XK469 and SH80 induce autophagic death in mouse leukemia L1210 cells. Mol. Cancer Therapeutics., 6:370-379, 2007.

Kessel, D. and Reiners, Jr., J.J. Apoptosis and autophagy after mitochondrial photodamage. Photochem. Photobiol., 83:1024-1028,2007.

Clark, M.K., Scott, S.A., Wojtowiak, J., Chico, R., Mathieu, P., Reiners, Jr., J.J., Mattingly, R.R., Borch R.F. and Gibbs, R.A. Synthesis, biochemical and cellular evaluation of farnesyl monophosphate prodrugs as farnesyltransferase inhibitors. J. Med. Chem., 50:3274-3282, 2007.

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Understanding how carcinogenic DNA adducts compromise accurate DNA replication is an important goal in cancer research.  These lesions can occur by endogenous agents in the cell or by exposure to exogenous agents such as the toxins found in cigarette smoke.  The DNA damage caused by these agents can result in mutations by reducing the accuracy of DNA replication across from or near the damaged site.  A central goal of the studies in our lab is to determine the molecular mechanism that allows a DNA polymerase to incorporate a nucleotide across from and past a bulky adduct in a DNA template, which we anticipate will lead to a better understanding of how the damage leads to mutations.  It is well established that during synthesis DNA polymerases incorporate a nucleotide through a multi-step mechanism that involves a conformational change from an open binary to a catalytically active closed ternary complex.  We have developed a method to detect the formation of this closed complex that forms only in presence of the next correct nucleotide.  Any deviation from correct Watson/Crick (W/C) geometry for the resulting base-pair was shown to have a destabilizing effect on the complex.  The presence of a bulky carcinogenic adduct, such as an N-acetyl-2-aminofluorene-dG adduct (dG-AAF), resulted in two interesting effects.  First, surprisingly, the polymerase binds much more tightly to the modified primer-template than to an unmodified one.  Second, the presence of the adduct completely inhibited the conformational change, explaining why this adduct acts as a strong block to DNA synthesis.  We have recently obtained a crystal structure of a polymerase bound to an AAF-modified primer-template (shown in the figure) and find that this structure provides an explanation for the biochemical effects of the adduct.  We found that the adduct caused the O helix to tilt forward and block the site were the nucleotide must bind prior to incorporation.  We have recently begun to determine how specific amino acid substitutions within the polymerase active site contribute to the properties that affect polymerase mechanism and fidelity.  We have shown that the substitution of a serine for a tyrosine at the base of the O helix result in a significant increase in the relative ability to incorporate the correct nucleotide dC opposite the dG-AAF adduct.  Moreover, we are able to detect a conformational change for the mutant polymerase when it is bound to an AAF-modified template, suggesting that the increased room provided by removing the bulky tyrosine allows for nucleotide binding in the active site.  Finally, our most recent studies involve using FRET at the single molecule level to determine how these types of adducts effect the rate of polymerase binding and synthesis on DNA templates.

Selected Publications

Alekseyev, Y.; Romano, L. J. “Effect of Benzo[a]pyrene Adduct Stereochemistry on Downstream DNA Replication In Vitro: Evidence for Different Adduct Conformations Within the Active Site of DNA Polymerase I (Klenow Fragment)”, Biochemistry, 41, 4467–4479, 2002.

Lone, S., Romano, L. J. “Mechanistic Insights into Replication Across from Bulky DNA Adducts: A Mutant Polymerase I Allows an N-Acetyl-2-aminofluorene Adduct to be Accommodated During DNA Synthesis”, Biochemistry, 42, 3826–3834, 2003.

Dutta, S., Li, Y., Johnson, D., Dzantiev, L., Richardson, C. C., Romano, L. J., and Ellenberger, T. “Crystal Structures of N-2-acetylaminofluorene and N-2-aminofluorene in Complex With T7 DNA Polymerase Reveal Mechanisms of Replication Blockage and Mutagenesis,” Proc. Natl. Acad. Sci. U.S.A., 101, 16186-16191, 2004.

Gill, J. P. and Romano, L. J. “Mechanism for AAF-induced Frameshift Mutagenesis by Escherichia coli DNA Polymerase I (Klenow Fragment),” Biochemistry 44, 15387-15395, 2005.

Lone, S. and Romano, L. J. The Role of Specific Amino Acid Residues in the Active Site of E. coli DNA Polymerase I on Translesion DNA Synthesis Across from and Past an N-2-Aminofluorene Adduct, Biochemistry 47, 2599 – 2607, 2007.

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Selected Publications
H.-L. Fang, M. Abdolalipour, Z. Duanmu, J.R. Smigelski, A. Weckle, T.A. Kocarek and M. Runge-Morris. Regulation of Glucocorticoid-Inducible Hydroxysteroid Sulfotransferase (SULT2A-40/41) Gene Transcription in Primary Cultured Rat Hepatocytes: Role of CCAAT/Enhancer-Binding Protein (C/EBP) Liver-Enriched Transcription Factors. Drug Metab. Dispos. 33: 147-156, 2005.

H.-L. Fang, S. C. Strom, H. Cai, C.N. Falany, T. A. Kocarek and M. Runge-Morris Regulation of Human Hepatic Hydroxysteroid Sulfotransferase Gene Expression by the Peroxisome Proliferator Activated Receptor Alpha Transcription Factor. Mol.  Pharmacol. 67: 1257 – 1267, 2005.

M. Runge-Morris and T.A. Kocarek Regulation of Sulfotransferases by Xenoiotic Receptors. Current Drug Metabolism, (Invited Review), 6: 299-307, 2005.

Z. Duanmu, A. Weckle,  S.B. Koukouritanki, R.N.Hines, J.L. Falany, C.N. Falany, T.A. Kocarek, and M. Runge-Morris Developmental Expression of Aryl- , Estrogen-, and Hydroxysteroid Sulfotransferase in Pre- and Postnatal Human Liver. J. Pharmacol. Exper. Ther., 316: 1310-1317, 2006.

R.D. Thomas, M.R. Green, C. Wilson, A. L. Weckle, Z. Duanmu, T.A. Kocarek, and M. Runge-Morris.  Cytochrome P450 Expression and Metabolic Activation of Cooked Food Mutagen 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in MCF10A Breast Epithelial Cells. Chemico-Biol. Interact.160: 204-216, 2006.

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