Nuclear Localization of Catechol-O-Methyltransferase in Neoplastic and Nonneoplastic Mammary Epithelial Cells
2000; Elsevier BV; Volume: 156; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)65057-2
ISSN1525-2191
AutoresJudith Weisz, Gabriella Fritz-Wolz, Shelley A. Gestl, Gary A. Clawson, Cyrus R. Creveling, Joachim G. Liehr, David J. Dabbs,
Tópico(s)Epigenetics and DNA Methylation
ResumoCatechol-O-methyltransferase (COMT) plays both a regulatory and protective role in catechol homeostasis. It contributes to the regulation of tissue levels of catecholamines and catecholestrogens (CEs) and, by blocking oxidative metabolism of catechols, prevents endogenous and exogenous catechols from becoming a source of potentially mutagenic electrophiles. Evidence implicating CEs in carcinogenesis, in particular in the hamster kidney model of estrogen-induced cancer, has focused attention on the protective role of COMT in estrogen target tissues. We have previously reported that treating hamsters with estrogens causes translocation of COMT to nuclei of epithelial cells in the renal cortex, the site of CE biosynthesis and where the cancers arise. This finding suggested that nuclear COMT may be a marker of a threat to the genome by catechols, including CEs. It is postulated that CEs play a role in the genesis of breast cancer by contributing to a state of chronic oxidative stress that is presumed to underlie the high incidence of this disease in the United States. Therefore, here we used immunocytochemistry to re-examine human breast parenchyma for nuclear COMT. In addition to confirming previous reports of cytoplasmic COMT in mammary epithelial cells, we identified nuclear COMT in foci of mammary epithelial cells in histologically normal breast tissue of virtually all control (macromastia) and cancer patients and in breast cancer cells. There was no correlation between tissue histology and the numbers of cells with nuclear COMT, the size of foci containing such cells, or intensity of nuclear COMT immunostaining. The focal nature of the phenomenon suggests that nuclear COMT does not serve a housekeeping function but that it reflects a protective response to an increased local catechol load, presumably of CEs and, as such, that it may be a characteristic of the population of women studied who share the same major risk factor for developing breast cancer, that of living in the industrialized West. Catechol-O-methyltransferase (COMT) plays both a regulatory and protective role in catechol homeostasis. It contributes to the regulation of tissue levels of catecholamines and catecholestrogens (CEs) and, by blocking oxidative metabolism of catechols, prevents endogenous and exogenous catechols from becoming a source of potentially mutagenic electrophiles. Evidence implicating CEs in carcinogenesis, in particular in the hamster kidney model of estrogen-induced cancer, has focused attention on the protective role of COMT in estrogen target tissues. We have previously reported that treating hamsters with estrogens causes translocation of COMT to nuclei of epithelial cells in the renal cortex, the site of CE biosynthesis and where the cancers arise. This finding suggested that nuclear COMT may be a marker of a threat to the genome by catechols, including CEs. It is postulated that CEs play a role in the genesis of breast cancer by contributing to a state of chronic oxidative stress that is presumed to underlie the high incidence of this disease in the United States. Therefore, here we used immunocytochemistry to re-examine human breast parenchyma for nuclear COMT. In addition to confirming previous reports of cytoplasmic COMT in mammary epithelial cells, we identified nuclear COMT in foci of mammary epithelial cells in histologically normal breast tissue of virtually all control (macromastia) and cancer patients and in breast cancer cells. There was no correlation between tissue histology and the numbers of cells with nuclear COMT, the size of foci containing such cells, or intensity of nuclear COMT immunostaining. The focal nature of the phenomenon suggests that nuclear COMT does not serve a housekeeping function but that it reflects a protective response to an increased local catechol load, presumably of CEs and, as such, that it may be a characteristic of the population of women studied who share the same major risk factor for developing breast cancer, that of living in the industrialized West. A link between oxidative stress and carcinogenesis is well documented. Oxidative DNA damage is potentially mutagenic, and promoters used in two-stage models of carcinogenesis are characteristically generators of reactive oxygen species.1Pero RW Roush MD Markowitz MM Miller DG Oxidative stress, DNA repair, and cancer susceptibility.Cancer Detect Prev. 1990; 14: 555-561PubMed Google Scholar, 2Ames BN Shigenaga MK Gold LS DNA lesions inducible by DNA repair and cell division: three key factors in mutagenesis and carcinogenesis.Environ Health Perspect. 1993; 101: S35-S44PubMed Google Scholar, 3Loft S Poulsen HE Cancer risk and oxidative DNA damage in man.J Mol Med. 1996; 74: 297-312Crossref PubMed Scopus (842) Google Scholar, 4Cerutti PA Prooxidant states and tumor promotion.Science. 1985; 227: 375-381Crossref PubMed Scopus (2333) Google Scholar, 5Klaunig JE Xu Y Isenberg JS Bachowski S Kolaja KL Jiang J Stevenson DE Walborg EFJ The role of oxidative stress in chemical carcinogenesis.Environ Health Perspect. 1998; 106: 289-295Crossref PubMed Scopus (474) Google Scholar The findings of Malins and co-workers6Malins DC Polissar NL Nishikida K Holmes EH Gardner HS Gunselman SJ The etiology and prediction of breast cancer. Fourier transform-infrared spectroscopy reveals progressive alterations in breast DNA leading to a cancer-like phenotype in a high proportion of normal women.Cancer. 1995; 75: 503-517Crossref PubMed Scopus (95) Google Scholar indicating extensive oxidative DNA damage in breast tissue of women in the United States suggests that underlying the high rate of breast cancer in the industrialized West is a state of chronic oxidative stress. A mechanism by which primary estrogens (estrone and estradiol), hormones implicated in breast carcinogenesis, may contribute to such a state of oxidative stress has also been identified.7Yager JD Liehr JG Molecular mechanisms of estrogen carcinogenesis.Ann Rev Toxicol Pharmacol. 1996; 36: 203-232Crossref Scopus (615) Google Scholar, 8Cavalieri E Stack D Devanesan P Todorovic R Dwivedy I Higginbotham S Johansson S Patil K Gross M Gooden J Ramanathan R Cerny R Rogan E Molecular origin of cancer: catecholestrogen-3,4-quinones as endogenous tumor initiators.Proc Natl Acad Sci USA. 1997; 94: 10937-10942Crossref PubMed Scopus (689) Google Scholar The mechanism involves two steps of metabolic activation, first, aromatic hydroxylation of primary estrogens leading to the formation of 2- and 4-hydroxylated catechol estrogens (2- and 4-OH-CEs), followed by oxidation of the catecholestrogens (CEs) to their semiquinone and quinone derivatives, the quinone estrogens (QEs). The latter are electrophiles that can form potentially mutagenic DNA adducts.8Cavalieri E Stack D Devanesan P Todorovic R Dwivedy I Higginbotham S Johansson S Patil K Gross M Gooden J Ramanathan R Cerny R Rogan E Molecular origin of cancer: catecholestrogen-3,4-quinones as endogenous tumor initiators.Proc Natl Acad Sci USA. 1997; 94: 10937-10942Crossref PubMed Scopus (689) Google Scholar In addition, CEs can enter into redox cycling and, thereby, become a source of reactive oxygen species.7Yager JD Liehr JG Molecular mechanisms of estrogen carcinogenesis.Ann Rev Toxicol Pharmacol. 1996; 36: 203-232Crossref Scopus (615) Google Scholar Thus, estrogens may participate in carcinogenesis not only via their estrogen receptor-mediated actions as mitogens, but also by contributing to a pro-oxidant state via the CE/QE metabolic pathway. Evidence implicating the CE/QE pathway of estrogen metabolism in carcinogenesis in vivo has been obtained principally from studies of estrogen-induced renal cancers in hamsters.9Weisz J Biogenesis of catecholestrogens: metabolic activation of estrogens by Phase I enzymes.Polycyclic Arom Compd. 1994; 6: 241-251Crossref Scopus (12) Google Scholar, 10Liehr JG Catecholestrogens in the induction of tumors in the kidney of the Syrian hamster.Adv Pharmacol. 1998; 42: 824-828Crossref PubMed Scopus (18) Google Scholar These studies have led to the hypothesis that estrogens may contribute to oxidative stress when local production of CEs exceeds that which can be inactivated by phase II enzymes. The phase II enzyme catechol-O-methyltransferase (COMT) is considered to play a critical role in the regulation of levels of endogenous catechols (catecholamines and CEs). In addition, COMT serves a protective role by blocking oxidative metabolism of endogenous and exogenous catechols and, thereby, the generation of potentially mutagenic electrophiles.11Creveling C Hartman BK Relationship between the cellular localization and physiological function of catechol-O-methyltransferase.in: Usdin E Borchard RT Creveling CR Biochemistry of S-Adenosyl l-Methionine and Related Compounds. Macmillan Press, London1982: 479-486Google Scholar, 12Raftogianis R, Creveling CR, Weinshilboum R, Weisz J: Estrogen metabolism by conjugation. Monogr Natl Cancer Inst 2000 (in press)Google Scholar The effects of estrogens on the expression of COMT in hamster kidney identified by us previously support this postulate.13Weisz J Fritz-Wolz G Clawson GA Benedict CM Abendroth C Creveling CR Induction of nuclear catechol-O-methyltransferase by estrogens in hamster kidney: implications for estrogen-induced renal cancer.Carcinogenesis. 1998; 19: 1307-1312Crossref PubMed Scopus (34) Google Scholar Administration of estrogen to hamsters induced COMT in epithelial cells of proximal tubules, the region of the kidney where the cancers arise. Moreover, it resulted in translocation of COMT to nuclei of these cells. Induction of nuclear COMT in hamster kidney by estrogens, first demonstrated by immunocytochemistry (ICC), was confirmed by immunoblot analysis of subcellular fractions prepared from hamster kidney cortex.13Weisz J Fritz-Wolz G Clawson GA Benedict CM Abendroth C Creveling CR Induction of nuclear catechol-O-methyltransferase by estrogens in hamster kidney: implications for estrogen-induced renal cancer.Carcinogenesis. 1998; 19: 1307-1312Crossref PubMed Scopus (34) Google Scholar Immunoblot analysis also identified nuclear COMT to be the soluble form of the enzyme (S-COMT). The study identified COMT to be a facultative nuclear enzyme and suggested that nuclear localization of COMT may serve as a marker of the threat to the genome posed by excess catechols. In the case of the hamster kidney the catechols responsible are presumably CEs, the putative mediators of actions of estrogens as complete carcinogens in this tissue.13Weisz J Fritz-Wolz G Clawson GA Benedict CM Abendroth C Creveling CR Induction of nuclear catechol-O-methyltransferase by estrogens in hamster kidney: implications for estrogen-induced renal cancer.Carcinogenesis. 1998; 19: 1307-1312Crossref PubMed Scopus (34) Google Scholar. Discussions of COMT's role in physiology and pathophysiology have focused to date predominantly on the loss of reactivity or activity of catechols caused by O-methylation. There is, however, a growing body of evidence that at least in the case of CEs, O-methylation may generate metabolites with distinctive actions.14Zhu BT Conney AH Functional role of estrogen metabolism in target cells.Carcinogenesis. 1998; 19: 1-27Crossref PubMed Scopus (838) Google Scholar Some of these, specifically the anti-angiogenic and anti-mitotic effects of 2,3-O-methyl-estradiol,14Zhu BT Conney AH Functional role of estrogen metabolism in target cells.Carcinogenesis. 1998; 19: 1-27Crossref PubMed Scopus (838) Google Scholar, 15Reiser F Way D Bernas M Witte M Witte C Inhibition of normal and experimental angiotumor endothelial cell proliferation and cell cycle progression by 2-methoxyestradiol.Proc Soc Exp Biol Med. 1998; 219: 211-216Crossref PubMed Scopus (46) Google Scholar, 16Cushman M He HM Katzenellenbogen JA Varma RK Hamel E Lin CM Ram S Sachdeva YP Synthesis of analogs of 2-methoxyestradiol with enhanced inhibitory effects on tubulin polymerization and cancer cell growth.J Med Chem. 1997; 40: 2323-2334Crossref PubMed Scopus (94) Google Scholar, 17Schumacher G Kataoka M Roth JA Mukhopadhyay T Potent antitumor activity of 2-methoxyestradiol in human pancreatic cancer cell lines.Clin Cancer Res. 1999; 5: 493-499PubMed Google Scholar, 18Arbiser JL Panigrathy D Klauber N Rupnick M Flynn E Udagawa T DÆAmato RJ The antiangiogenic agents TNP-470 and 2-methoxyestradiol inhibit the growth of angiosarcoma in mice.J Am Acad Dermatol. 1999; 40: 925-929Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar suggest that O-methylation of CEs by COMT may be protective not only by preventing oxidative metabolism of CEs but also by generating metabolite(s) with tumor suppressor functions. The potential utility in cancer treatment of 2-methylated CEs and their synthetic analogs is now being explored by several groups.16Cushman M He HM Katzenellenbogen JA Varma RK Hamel E Lin CM Ram S Sachdeva YP Synthesis of analogs of 2-methoxyestradiol with enhanced inhibitory effects on tubulin polymerization and cancer cell growth.J Med Chem. 1997; 40: 2323-2334Crossref PubMed Scopus (94) Google Scholar, 17Schumacher G Kataoka M Roth JA Mukhopadhyay T Potent antitumor activity of 2-methoxyestradiol in human pancreatic cancer cell lines.Clin Cancer Res. 1999; 5: 493-499PubMed Google Scholar, 18Arbiser JL Panigrathy D Klauber N Rupnick M Flynn E Udagawa T DÆAmato RJ The antiangiogenic agents TNP-470 and 2-methoxyestradiol inhibit the growth of angiosarcoma in mice.J Am Acad Dermatol. 1999; 40: 925-929Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar Evidence for the expression of COMT in mammary gland of both rodents and humans has been obtained previously by biochemical assays and by ICC and a role for the enzyme in local CE homeostasis in this tissue has been suggested.19Amin AM Creveling CR Lowe MC Immunohistochemical localization of catechol methyltransferase in normal and cancerous breast tissues of mice and rats.J Natl Cancer Inst. 1983; 70: 337-342PubMed Google Scholar, 20Assicot M Contesso G Bohuon C Catechol-O-methyltransferase in human breast cancers.Eur J Cancer. 1977; 13: 961-966Abstract Full Text PDF PubMed Scopus (36) Google Scholar, 21Hoffman AR Paul SM Axelrod J Catecholestrogen synthesis and metabolism by human breast tumors in vitro.Cancer Res. 1979; 39: 4584-4587PubMed Google Scholar Nuclear localization of COMT, however, was not noted in any of these studies. In light of the association of nuclear COMT with estrogen-induced cancer in hamster kidney and the many observations implicating estrogens in breast carcinogenesis it was of interest to re-examine by ICC human breast parenchyma for nuclear COMT. We report here on the presence of nuclear COMT in foci of histologically normal epithelial cells in breast parenchyma from patients without as well as with breast cancer and also in breast cancer cells. The focal and apparently random distribution of cells with nuclear COMT suggests that nuclear COMT does not serve a housekeeping function but reflects a response to a localized threat posed to the genome by excess CEs. Breast tissue was obtained from women undergoing reduction mammoplasty for macromastia or mastectomy for breast cancer. Tissue samples from reduction mammoplasty were obtained from lateral portion of the breast to minimize effects that pressure may have on the tissue. Specimens from breast cancer patients were obtained from sites distant from the cancers and, whenever available, also from the cancers. After removal of excess adipose tissue by blunt dissection, portions of breast parenchyma were flash-frozen (at −70°C in liquid freon) and stored at −80°C. Other portions were fixed in 10% neutral-buffered formalin and embedded in paraffin. Cryostat sections and sections from paraffin-embedded tissue were cut at 8 μm and processed for ICC using standard methodology (see below). Tissue sections from a total of 33 subjects were examined by ICC (19 from patients with macromastia, nine from patients with breast cancer, and five from subjects with extensive fibrocystic disease). The ages of the subjects ranged from 16 to 57 years. Use of the tissue for the purposes of this study was authorized by the Institutional Review Board for Use of Human Tissues. ICC for COMT was carried out as described previously and with reagents from the same sources.13Weisz J Fritz-Wolz G Clawson GA Benedict CM Abendroth C Creveling CR Induction of nuclear catechol-O-methyltransferase by estrogens in hamster kidney: implications for estrogen-induced renal cancer.Carcinogenesis. 1998; 19: 1307-1312Crossref PubMed Scopus (34) Google Scholar Briefly, the rabbit polyclonal antibody developed against purified protein was used at a dilution of 1:2,500. The signal from the biotinylated secondary antibody was amplified using Vectastain ABC reagent (Vector, Burlingame, CA) with alkaline phosphatase as the reporter, 5-bromo-4-chloro-3-indolyl phosphate as substrate and 4-nitroblue tetrazolium (Sigma Chemical Co., St. Louis, MO) as the chromogen. Endogenous alkaline phosphatase was inhibited by preincubating sections with 0.2 N HCl for 5 minutes. Sections incubated with only the secondary antibody served as controls. Representative photomicrographs of immunoreactive COMT in nonneoplastic breast parenchyma are presented in Figure 1 and in breast cancers in Figure 2. In agreement with previous reports immunoreactive COMT with cytoplasmic localization was seen uniformly in mammary epithelial cells throughout the mammary gland, as well as in breast cancer cells.20Assicot M Contesso G Bohuon C Catechol-O-methyltransferase in human breast cancers.Eur J Cancer. 1977; 13: 961-966Abstract Full Text PDF PubMed Scopus (36) Google Scholar There were no consistent or obvious differences in the intensity of staining as a function of the stage of differentiation of the terminal ductal lobular units (type I, II, or III lobules)22Russo J Reina D Fredrick J Russo IH Expression of phenotypic changes by human breast epithelial cells treated with carcinogens in vitro.Cancer Res. 1988; 48: 2837-2857PubMed Google Scholar or the histological characteristics of the cancers. Immunostaining of both intra- and interlobular stromal cells was always of a much lower intensity than that of epithelial cells in the same tissue sections. Cytoplasmic COMT immunostaining was also weaker in some epithelial cells within involuting terminal ductal lobular units and in the attenuated layer of epithelial cells lining some cysts (Figure 1J).Figure 2Localization of immunoreactive COMT in five breast cancers. Immunocytochemistry was carried out as described under Methods with alkaline phosphatase (AlkP) as the reporter, 5-bromo-3-chloro-indolyl phosphate as the substrate, and nitroblue tetrazolium as the chromogen. Sections were not counterstained and were visualized using Nomarski optics. A, B, C, D, and E show cancers in which immunostaining is predominantly cytoplasmic. In A cytoplasmic immunostaining is intense and there is no evidence of nuclear COMT. B: From the same cancer as A, shows a terminal lobular ductal unit being invaded by cancer cells: COMT is cytoplasmic in both the ductal epithelial and in breast cancer cells. However, immunostaining in the cancer cells is much more intense than in ductal epithelial cells. C: From another patient, shows some cancer cells with intense cytoplasmic immunostaining, some with intense nuclear immunostaining (indicated by arrows) and a few appear to have both intense nuclear and cytoplasmic COMT. D: Cancer cells with only weak immunostaining that is predominantly cytoplasmic. E: Higher magnification of part of D (outlined). F–I: Shown are cancers in which immunostaining is predominantly nuclear. F: A cancer with intense nuclear immunostaining in the majority of cells. G: Higher magnification of part of F indicated by arrows. A cell with apparently only cytoplasmic COMT among the many with prominent nuclear COMT is indicated by an arrow. H and I: Sections from another mastectomy specimen. H: Cancer cells with intense nuclear immunostaining interspersed with islands of cells apparently devoid of COMT immunostaining. At higher magnification, in I, the areas of apparently devoid immunostaining, such as seen in H, can be seen to be made up of cells with only minimal amount of reaction product in the rim of cytoplasm surrounding large nuclei devoid of reaction product. Original magnification, ×400 (A–D, F, and H) and ×1000 (E, G, and I).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Nuclear COMT was seen in some mammary epithelial cells in breast parenchyma from virtually all patients whether without or with cancer, as well as in breast cancer cells (Figures 1 and 2). In contrast to cytoplasmic COMT, epithelial cells with nuclear COMT were seen only in some regions of individual sections. The size and number of foci of cells with nuclear COMT varied greatly; in some subjects they encompassed large portions of mammary gland within a section whereas in others nuclear COMT was restricted to a few scattered cells intermingled with cells showing only cytoplasmic COMT (Figure 1) In tissue sections from cancers nuclear COMT was present in some cases in the majority of cancer cells whereas in others there were only a few such cells scattered among cells with only cytoplasmic COMT (Figure 2) Nuclear COMT was most prominent in cells in which immunostaining in the cytoplasm was relatively weak compared to that in the nucleus. Control sections incubated with secondary antibody only were consistently negative (Figure 3) As in hamster kidney, nuclear immunoreactivity for COMT could be visualized only in sections from tissue fixed in formalin and embedded in paraffin, reflecting a need for retrieval of COMT antigen associated with nuclear proteins.13Weisz J Fritz-Wolz G Clawson GA Benedict CM Abendroth C Creveling CR Induction of nuclear catechol-O-methyltransferase by estrogens in hamster kidney: implications for estrogen-induced renal cancer.Carcinogenesis. 1998; 19: 1307-1312Crossref PubMed Scopus (34) Google Scholar The presence of nuclear COMT in noncancerous breast parenchyma could not be correlated with patient's age (Figure 1, A and B) or any histological characteristics, including those associated with an increased risk for developing breast cancer, ie, epithelial hyperplasia without or with atypia.23Page DL Dupont WD Benign breast disease: indicators of increased breast cancer risk.Cancer Detect Prev. 1992; 16: 93-97PubMed Google Scholar, 24Bartow SA Pathak DR Black WC Key CR Teaf SR Prevalence of benign, atypical, and malignant breast lesions in populations at different risk for breast cancer. A forensic autopsy study.Cancer. 1987; 60: 2751-2760Crossref PubMed Scopus (143) Google Scholar Nor was there any consistent association between the histological type of cancer and nuclear COMT. It was absent in some highly undifferentiated cancers but prominent in others. As demonstrated here, nuclear COMT can be seen in both breast cancer cells and in histologically normal mammary epithelial cells in women without as well as with breast cancer. The focal and apparently random distribution of epithelial cells with nuclear COMT suggests that nuclear COMT does not serve a housekeeping function, but is induced in response to an excess catechol load. This notion is supported by the numerous observations of the focal nature of responses to even low doses of potential carcinogens administered to experimental animals.25Clawson GA Schalles SL Wolz G Weisz J Crone TM Miranda GQ Focal altered compartmentation of repetitive B2 (Alu-like) sequences in rat liver following hepatocarcinogen exposure.Cell Growth Differ. 1996; 7: 635-646PubMed Google Scholar We postulate that in mammary epithelial cells, as in kidneys of estrogen-treated hamsters, CEs are the inducers of nuclear COMT. The notion that CEs are the principal substrates in mammary epithelial cells for COMT is supported by several lines of evidence. Oxidation of primary estrogens to CEs by human breast parenchyma in vitro has been demonstrated, forms of P450 that can catalyze the reaction have been shown to expressed in this tissue and significant concentrations of CE have been reported to be present in breast cyst fluid.26Levin M Weisz J Bui QD Santen RJ Peroxidatic catecholestrogen production by human breast cancer tissue in vitro.J Steroid Biochem. 1987; 28: 513-520Crossref PubMed Scopus (24) Google Scholar, 27Hellmold H Rylander T Magnusson M Reihner E Warner M Gustafsson JA Characterization of cytochrome P450 enzymes in human breast tissue from reduction mammoplasties.J Clin Endocrinol Metab. 1998; 83: 886-895Crossref PubMed Scopus (79) Google Scholar, 28Castagnetta LA Granata OM Arcuri FP Polito LM Rosati F Cartoni GP Gas chromatography/mass spectrometry of catechol estrogens.Steroids. 1992; 57: 437-443Crossref PubMed Scopus (47) Google Scholar Substrates for CE synthases are also likely to be available in ample amounts in breast parenchyma of both pre- and postmenopausal women, because estrone and estradiol are maintained in this tissue at a high level by deconjugation and aromatization in situ of circulating conjugated and C19 steroid precursors, respectively.29van Landeghem AAJ Poortman J Nabuurs M Thijssen JHH Endogenous concentrations and subcellular distribution of estrogen in normal and malignant human breast tissue.Cancer Res. 1985; 45: 2900-2906PubMed Google Scholar, 30Pasqualini JR Chetrite G Blacker C Feinstein MC Delalonde L Talbi M Maloche C Concentrations of estrone, estradiol, and estrone sulfate and evaluation of sulfatase and aromatase activities in pre- and postmenopausal breast cancer patients.J Clin Endocrinol Metab. 1996; 81: 1460-1464Crossref PubMed Scopus (414) Google Scholar, 31Pasqualini JR Cortes-Prieto J Chetrite G Talbi M Ruiz A Concentrations of estrone, estradiol and their sulfates, and evaluation of sulfatase and aromatase activities in patients with breast fibroadenoma.Int J Cancer. 1997; 70: 639-643Crossref PubMed Scopus (91) Google Scholar Functions of COMT in mammary epithelial cells are likely to include modulation of levels of CEs generated in situ presumably for physiological purposes. Several scenarios could lead to CE formation in breast parenchyma and, secondarily, to the induction of nuclear COMT. These include induction of CE synthases (P450s and peroxidases) that can catalyze 2- and 4-hydroxylation of primary estrogens9Weisz J Biogenesis of catecholestrogens: metabolic activation of estrogens by Phase I enzymes.Polycyclic Arom Compd. 1994; 6: 241-251Crossref Scopus (12) Google Scholar, 32Bui QD Weisz J Wrighton SA Hepatic catecholestrogen synthases: differential effect of sex, inducers of cytochromes P-450 and of anti- body to the glucocorticoid inducible cytochrome P-450 on NADPH-dependent estrogen-2-hydroxylase and on organic hydroperoxide-dependent estrogen-2/4-hydroxylase activity of rat hepatic microsomes.J Steroid Biochem Mol Biol. 1990; 37: 285-293Crossref PubMed Scopus (15) Google Scholar, 33Weisz J Metabolism of estrogens by target cells. Diversification and amplification of hormone action and the catecholestrogen hypothesis. New Biology of Steroid Hormones.in: Hochberg R Naftolin F Raven Press, New York1991: 201-212Google Scholar, 34Spink DC Hayes CL Young NR Christou M Sutter TR Jefcoate CR Gierthy JF The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on estrogen metabolism in MCF-7 breast cancer cells: evidence for induction of a novel 17 beta-estradiol 4-hydroxylase.J Steroid Biochem Mol Biol. 1994; 51: 251-258Crossref PubMed Scopus (193) Google Scholar and increased availability of primary estrogen that are substrates for CE synthases. Information on concentrations of CEs and their metabolites in breast parenchyma needed to test these postulates await the development of analytical methods with the requisite sensitivity. In renal tubular epithelial cells of hamsters, in contrast to breast epithelial cells, the principal substrate for COMT under normal physiological conditions is likely to be dopamine, which is produced in these cells for the purpose of regulating sodium excretion.35Aperia A Dopamine action and metabolism in the kidney.Curr Opin Nephrol Hypertens. 1994; 3: 39-45Crossref PubMed Scopus (46) Google Scholar CEs are postulated to become major substrates for COMT in these cells only after the administration of doses of estrogens known to cause renal cancers in this species and only then did nuclear COMT become prominent.13Weisz J Fritz-Wolz G Clawson GA Benedict CM Abendroth C Creveling CR Induction of nuclear catechol-O-methyltransferase by estrogens in hamster kidney: implications for estrogen-induced renal cancer.Carcinogenesis. 1998; 19: 1307-1312Crossref PubMed Scopus (34) Google Scholar Induction of nuclear COMT in hamster kidney was evident by 2 weeks after initiation of the treatment with estrogens (the earliest time point examined), many months before the appearance of the cancers.13Weisz J Fritz-Wolz G Clawson GA Benedict CM Abendroth C Creveling CR Induction of nuclear catechol-O-methyltransferase by estrogens in hamster kidney: implications for estrogen-induced renal cancer.Carcinogenesis. 1998; 19: 1307-1312Crossref PubMed Scopus (34) Google Scholar In interpreting the finding presented here it is necessary to consider the characteristics of the tissue donor population. For noncancerous tissue we had to rely on women with macromastia. Whether and how such tissues may differ from the norm is not known. More importantly, however, all of the tissues used were obtained from subjects sharing the same significant risk factor for breast cancer, that of living in an industrialized Western country. It is estimated that one in eight women living in the United States can expect to be diagnosed with breast cancer in her lifetime.36Feuer EJ Wun LM Boring CC Flanders WD Timmel MJ Tong T The lifetime risk of developing breast cancer.J Natl Cancer Inst. 1993; 85: 892-897Crossref PubMed Scopus (421) Google Scholar Environment is recognized to be the major risk factor for breast cancer in this population. Its importance is underscored by the rise in breast cancer incidence in immigrants to the United States from countries such as Japan where the incidence, although rising, is still only one-sixth of that in the United States.37Adams JB Human breast cancer: concerted role of diet, prolactin and adrenal C19-(5-steroid in tumorogensis.Int J Cancer. 1992; 50: 854-858Crossref PubMed Scopus (22) Google Scholar, 38Muir CS Epidemiology of cancer in ethnic groups.Br J Cancer Suppl. 1996; 29: S12-S16PubMed Google Scholar, 39Kelsey KL Horn-Ross PL Breast cancer: magnitude of the problem and descriptive epidemiology.Epidemiol Rev. 1993; 15: 7-16PubMed Google Scholar The finding of a pattern of extensive oxidative DNA damage in breast parenchyma of women in the United States suggests that underlying the high breast cancer incidence in this population is a state of chronic oxidative stress.40Malins DC Polissar NL Schaefer S Su Y Vinson M A unified theory of carcinogenesis based on order-disorder transitions in DNA structure as studied in the human ovary and breast.Proc Natl Acad Sci USA. 1998; 95: 7637-7642Crossref PubMed Scopus (54) Google Scholar Diverse factors could contribute to this state. The findings presented here support the notion that one such factor could be electrophiles generated as a consequence of oxidative metabolism of CEs. To determine whether this interpretation is correct will require examining breast tissue from subjects from a population with a significantly lower breast cancer incidence than that characteristic of our study population. Since completion of this study Tenhunen and co-workers41Tenhunen J Heikkila P Alanko A Heinonen E Akkila J Ulmanen I Soluble and membrane-bound catechol-O-methyltransferase in normal and malignant mammary gland.Cancer Lett. 1999; 144: 75-84Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar have reported their findings on the localization of COMT in breast cancers from 32 women and in histologically normal breast tissue from the same patients in a cohort of Finnish women. Using ICC these investigators did observe nuclear COMT but only in an unspecified number of invasive ductal breast cancers. There is no obvious explanation for the differences in findings. They could reflect a difference in study population. This is of special interest in light of differences in the composition diet of women in the United States and Finland and the effect that this may have on estrogen metabolism.42Adlerkreutz H Diet and sex hormone metabolism. Nutrition, Toxicology and Cancer.in: Rowlands IR CRC Press, Boca Raton, FL1991: 137-195Google Scholar, 43Adlercreutz H Epidemiology of phytoestrogens.Baillieres Clin Endocrinol Metab. 1998; 12: 605-623Abstract Full Text PDF PubMed Scopus (223) Google Scholar An opportunity to test the hypothesis that COMT plays a protective role in breast carcinogenesis in the United States is provided by a well-characterized COMT polymorphism. This polymorphism is responsible for reduced catalytic activity of the enzyme in ∼25% of Caucasians.12Raftogianis R, Creveling CR, Weinshilboum R, Weisz J: Estrogen metabolism by conjugation. Monogr Natl Cancer Inst 2000 (in press)Google Scholar Several epidemiological studies have been initiated to test the hypothesis that the low-activity COMT genotype is associated with an increased breast cancer risk. Overall, the findings from three of the four case control studies reported to date are consistent with the hypothesis.44Huang CS Chern HD Chang KJ Cheng CW Hsu SM Shen CY Breast cancer risk associated with genotype polymorphism of the estrogen-metabolizing genes CYP17, CYP1A1, and COMT: a multigenic study on cancer susceptibility.Cancer Res. 1999; 59: 4870-4875PubMed Google Scholar, 45Lavigne JA Helzlsouer KJ Huang HY Strickland PT Bell DA Selmin O Watson MA Hoffman S Comstock GW Yager JD An association between the allele coding for a low activity variant of catechol-O-methyltransferase and the risk for breast cancer.Cancer Res. 1997; 57: 5493-5497PubMed Google Scholar, 46Thompson PA Shields PG Freudenheim JL Stone A Vena JE Marshall JR Graham S Laughlin R Nemoto T Kadlubar FF Ambrosone CB Genetic polymorphisms in catechol-O-methyltransferase, menopausal status, and breast cancer risk.Cancer Res. 1998; 58: 2107-2110PubMed Google Scholar A significant positive association between breast cancer risk and low-activity COMT genotype was identified in three of these studies. However, the findings differ with respect to the age group in which this association is evident (ie, whether in pre- or postmenopausal women or both). In the fourth study no association was found between low-activity COMT genotype and breast cancer risk.47Millikan RC Pittman GS Tse CK Duell E Newman B Savitz D Moorman PG Boissy RJ Bell DA Catechol-O-methyltransferase and breast cancer risk.Carcinogenesis. 1998; 19: 1943-1947Crossref PubMed Scopus (107) Google Scholar These differences in findings may be resolved by larger prospective studies. Properties of COMT differ not only as a function of the genotype. The soluble (S-COMT) and membrane bound (M-COMT) forms of the enzyme, generated from alternative start sites of the COMT transcript, also differ in their catalytic properties.48Ball P Haupt M Knuppen R Biogenesis and metabolism of catechol estrogens in vitro.in: Merriam GR Lipsett MB Catechol Estrogens. Raven Press, New York1982: 91-103Google Scholar, 49Roth JA Membrane-bound catechol-O-methyltransferase: a reevaluation of its role in the O-methylation of the catecholamine neurotransmitters.Rev Physiol Biochem Pharmacol. 1992; 120: 1-29Crossref PubMed Google Scholar, 50Tenhunen J Salminen M Lundstrom K Kiviluoto T Savolainen R Ulmanen I Genomic organization of the human catechol-O-methyltransferase gene and its expression from two distinct promoters.Eur J Biochem. 1994; 223: 1049-1059Crossref PubMed Scopus (301) Google Scholar The affinity of S-COMT for 2- and 4-OH-CEs is one order of magnitude higher than that for catecholamines.48Ball P Haupt M Knuppen R Biogenesis and metabolism of catechol estrogens in vitro.in: Merriam GR Lipsett MB Catechol Estrogens. Raven Press, New York1982: 91-103Google Scholar The additional 50 amino acids at the N-terminus of M-COMT increase the affinity of the enzyme for catecholamines by an order of magnitude without, however, affecting its affinity for 2-OH-CEs.49Roth JA Membrane-bound catechol-O-methyltransferase: a reevaluation of its role in the O-methylation of the catecholamine neurotransmitters.Rev Physiol Biochem Pharmacol. 1992; 120: 1-29Crossref PubMed Google Scholar Whether S-COMT and M-COMT differ in their catalytic properties with respect to 4-OH-CEs has yet to be determined. Nuclear COMT in kidneys of estrogen-treated hamsters and in transfected cells has been identified to be S-COMT.13Weisz J Fritz-Wolz G Clawson GA Benedict CM Abendroth C Creveling CR Induction of nuclear catechol-O-methyltransferase by estrogens in hamster kidney: implications for estrogen-induced renal cancer.Carcinogenesis. 1998; 19: 1307-1312Crossref PubMed Scopus (34) Google Scholar, 51Ulmanen I Peranen J Tenhunen J Tilgmann C Karhunen T Panula P Bernasconi L Aubry JP Lundstrom K Expression and intracellular localization of catechol-O-methyltransferase in transfected mammalian cells.Eur J Biochem. 1997; 243: 452-459Crossref PubMed Scopus (96) Google Scholar It is, therefore, likely that S-COMT is also the form identified by ICC in nuclei of mammary epithelial cells. It will be of interest to re-examine the catalytic efficiency of the two forms of COMT specifically for 4-OH-CEs because of evidence suggesting that they play a greater role in carcinogenesis than 2-OH-CEs, in particular, as potential mutagens.8Cavalieri E Stack D Devanesan P Todorovic R Dwivedy I Higginbotham S Johansson S Patil K Gross M Gooden J Ramanathan R Cerny R Rogan E Molecular origin of cancer: catecholestrogen-3,4-quinones as endogenous tumor initiators.Proc Natl Acad Sci USA. 1997; 94: 10937-10942Crossref PubMed Scopus (689) Google Scholar With respect to the mechanism of translocation of COMT to the nucleus it is of interest that human COMT sequence contains a classical nuclear localization signal (NLS), as well as a second presumptive NLS (KKGK).13Weisz J Fritz-Wolz G Clawson GA Benedict CM Abendroth C Creveling CR Induction of nuclear catechol-O-methyltransferase by estrogens in hamster kidney: implications for estrogen-induced renal cancer.Carcinogenesis. 1998; 19: 1307-1312Crossref PubMed Scopus (34) Google Scholar Translocation of S-COMT to the nucleus can occur, however, in the absence of a NLS. Nuclear S-COMT was seen in hamster kidney although hamster COMT sequence does not contain a NLS13Weisz J Fritz-Wolz G Clawson GA Benedict CM Abendroth C Creveling CR Induction of nuclear catechol-O-methyltransferase by estrogens in hamster kidney: implications for estrogen-induced renal cancer.Carcinogenesis. 1998; 19: 1307-1312Crossref PubMed Scopus (34) Google Scholar and eliminating the NLS from human COMT by site-directed mutagenesis did not prevent entry of recombinant S-COMT into nuclei of transfected mammalian cells.51Ulmanen I Peranen J Tenhunen J Tilgmann C Karhunen T Panula P Bernasconi L Aubry JP Lundstrom K Expression and intracellular localization of catechol-O-methyltransferase in transfected mammalian cells.Eur J Biochem. 1997; 243: 452-459Crossref PubMed Scopus (96) Google Scholar Conversely, M-COMT has not yet been found in the nucleus although it possesses the same NLS as S-COMT. How the expression of the two forms is regulated and what factors lead to the induction of nuclear S-COMT remains to be determined. In summary, the study establishes the presence of nuclear COMT in histologically normal human mammary epithelial cells as well as in breast cancer cells. The focal and apparently random distribution of cells with nuclear COMT suggests that in these cells, as in epithelial cells of proximal convoluted tubules of kidneys of estrogen treated hamsters, translocation of COMT is a biomarker for an increased catechol load, presumably of CEs. The finding is consistent with the postulate that oxidative metabolism of CEs contributes to the state of chronic oxidative stress that is presumed to exist in breast tissue of the donor population and hence, to the high incidence of breast cancer in this population.40Malins DC Polissar NL Schaefer S Su Y Vinson M A unified theory of carcinogenesis based on order-disorder transitions in DNA structure as studied in the human ovary and breast.Proc Natl Acad Sci USA. 1998; 95: 7637-7642Crossref PubMed Scopus (54) Google Scholar
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