William F. Greenlee

Expression of cytochrome P450 CYP1B1 in breast cancer

The expression of CYP1B1 has been identified in breast cancer using the reverse transcriptase‐polymerase chain reaction and immunoblotting. CYP1B1 mRNA was expressed in the majority of breast tumours and immunoblotting of breast tumours identified a single protein band of molecular weight 60 kDa corresponding to the predicted molecular weight of human CYP1B1. This is the first study to identify CYP1B1 expression in a tumour where it may represent a previously unknown pathway for the metabolism of oestradiol and chemotherapeutic drugs.

Negative selection in hepatic tumor promotion in relation to cancer risk assessment

Mechanistic studies with phenobarbital (PB), 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) and other liver tumor promoters support a general model of promotion involving negative selection where specifically-mutated cells derive a growth advantage in the presence of persistent mitosuppression. Exposure to these liver tumor promoters appears to transiently enhance hepatocyte replication, presumably via transcriptional activation of growth regulatory genes, leading to a homeostatic increase in mitoinhibitory growth factors in the liver to constrain proliferation. Transforming growth factor beta 1 (TGF-beta), a potent mitoinhibitory growth factor for hepatocytes, has been associated with the mitosuppression caused by PB and certain peroxisomal proliferators. Escape from TGF-beta mitosuppression may involve loss or alteration of function of the mannose 6-phosphate/insulin-like growth factor II (M6P/IGFII) receptor, which is required for TGF-beta 1 activation, or alterations of the TGF-beta types I, II and III signal transduction receptors. A risk assessment based on a negative selection mechanism could be conducted for tumor promotion endpoints with TCDD and compared with current approaches that implicitly regard TCDD as an initiator. Benchmark dose calculation using centrilobular induction of cytochromes P450 1A1 and 1A2 as a surrogate for periportal growth stimulation would provide a rational starting point for application of conventional safety factor approaches, similar to those used with non-cancer effects. In the future, tissue and plasma concentrations of specific growth factors, e.g. TGF-beta or hepatocyte growth factor, HGF, might be considered as more direct dose surrogates for tumor-promoting effects of xenobiotics. Uncertainty factor adjustments to a TCDD benchmark dose calculation should eventually rely on direct knowledge of regulation of specific growth regulatory genes and their receptors in relevant species and on species differences in TCDD pharmacokinetics, instead of application of default animal-to-human and interindividual uncertainty factors.

Functional analysis of the promoter for the human CYP1B1 gene.

Our laboratory has cloned the cDNA (Sutter, T. R., Tang, Y. M., Hayes, C. L., Wo, Y.-Y. P., Jabs, E. W., Li, X., Yin, H., Cody, C. W., and Greenlee, W. F. (1994) J. Biol. Chem. 269, 13092–13099) and gene (Tang, Y. M., Wo, Y.-Y. P., Jabs, E. W., Stewart, J. C., Sutter, T. R., and Greenlee, W. F. (1996) J. Biol. Chem. 271, 28324–28330) for humanCYP1B1, a new member of the cytochrome P450 superfamily. Here, we report on the mapping and function of the CYP1B1promoter. The CYP1B1 promoter is fully functional, when it is uncoupled from upstream enhancer elements. Deletion analysis and site-directed mutagenesis identified four regulatory elements required for maximum promoter activity: two antisense Sp1 sites (−84 to −89 and −68 to −73), a TATA-like box (−34 to −29), and an initiator motif (−5 to +3). The initiator and the TATA-like elements are both required for basal promoter activity, with enhanced activity mediated by the two antisense Sp1 elements. The CYP1B1 initiator was demonstrated by in vitro transcription analysis to be a positioning element that maintained fidelity of transcription from a single site. Specific binding to a CYP1B1 initiator probe by human nuclear extract proteins was competed either by the highly homologous murine terminal deoxynucleotidyl transferase initiator or, to a lesser extent, by the adenovirus major late initiator. Taken together, these results indicate that the structure and function of theCYP1B1 promoter confers constitutive expression of the gene and assures fidelity of transcription initiation from a single site. The CYP1B1 promoter is distinct from the promoters of the closely related cytochrome P450s CYP1A1 andCYP1A2 and is structurally and functionally similar to the promoters of constitutively expressed genes and at least two viruses.

2,3,7,8-tetrachlorodibenzo-p-dioxin inhibits DNA synthesis in rat primary hepatocytes

Treatment of Sprague-Dawley (SD) rats with a dosing regimen of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) maintaining a steady-state liver concentration of 150 ng/g results in enhanced hepatocyte proliferation in the periportal region, but reduced proliferation in the remainder of the hepatic lobule (Fox et al. (1993) Cancer Res., 53, 2265-2271). Here, we report an initial characterization of the actions of TCDD on hepatocyte proliferation by monitoring DNA synthesis in primary hepatocytes isolated from SD rats. TCDD caused a dose-dependent inhibition (EC50 = 10 pM) of DNA synthesis in primary hepatocytes isolated from either male or female SD rats in the presence or absence of known hepatocyte mitogens (epidermal growth factor, hepatocyte growth factor, and transforming growth factor alpha). No change in DNA synthesis was observed at TCDD concentrations less than 1 pM. Initial characterization of the EGF response system in these cells revealed that TCDD did not alter the specific binding of EGF, or the levels of EGF receptor protein measured in intact cells or cell lysates. TCDD-dependent inhibition of DNA synthesis occurred independently of the suppression observed with transforming growth factor-beta 1. Estradiol did not alter DNA synthesis in the presence or absence of TCDD. Taken together, these findings indicate that TCDD suppresses DNA synthesis via a novel pathway that is non-responsive to estradiol, independent of TGF-beta, and does not involve a decreased ability of hepatocytes to recognize (bind) EGF, a prototype mitogen.

Molecular basis of dioxin actions: evidence supporting chemoprotection.

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD or dioxin), a highly publicized environmental contaminant, was shown to be chemoprotective against breast cancer in both rats and mice in bioassays conducted in the late 1970s. This fi nding went largely unnoticed as investigators focused on animal tumors that were increased by dioxin. The position that dioxin causes human tumors remains a subject for debate; however, recent epidemiological studies of a population highly exposed to dioxin in 1976 as a result of an industrial accident suggest that women with higher dioxin body burdens may have a lower incidence of breast cancer. With the growth of new knowledge about the molecular basis of dioxin actions in humans and animals, it is clear that most of the responses produced by this agent are initiated by a specifi c recognition protein (designated the Ah receptor) found in almost all animal and human tissues and organs. The recognition event between the Ah receptor and environmental agents like dioxin is due to the formation of a complex. We have observed that in the presence of dioxin, the Ah receptor turns off proliferation in tumor cells and suppresses the ability of these cells to invade normal tissue. We believe that these fi ndings provide a molecular and biochemical basis for understanding the chemoprotective mechanisms suggested by the fi ndings of rodent bioassays and could lead to the development of novel therapeutic agents targeting the Ah receptor.

The Ah Receptor: A Biochemical and Biologic Perspective

Publisher Summary The identification of the induction receptor (Ah receptor) for the polycyclic aromatic hydrocarbon-inducible microsomal mono-oxygenases is a significant event in the maturing of toxicology as a scientific discipline. Subsequent studies on the role of the Ah receptor in mediating the toxicity of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), the prototype ligand, reveal that this protein regulates the expression of a gene battery that includes the structural genes for cytochrome(s) PI-450 and, in certain tissues, interacts with the product of at least one other regulatory locus to control a broader pleiotropic response, resulting in altered patterns of cell proliferation and differentiation. Thus, the disciplines of toxicology and receptor biology find a common focus. The intensive study of the cell and molecular biology of the Ah receptor is providing fundamental insight into understanding the mechanisms of toxicity of TCDD and related halogenated aromatic compounds. The use of TCDD as a molecular probe in defining the biology of the Ah receptor as a regulatory protein is adding to the knowledge of basic cellular and biochemical processes in eukaryotic cells that function, at least in part, to control the programmed patterns of cell proliferation and differentiation. The chapter explains the potential value provided by the understanding of cellular mechanisms to broader issues relevant to TCDD as a major public health problem.