Upregulation of estradiol C16 alpha-hydroxylation in human breast tissue: a potential biomarker of breast cancer risk.

BACKGROUND: The biotransformation of the natural estrogen 17 beta-estradiol (E2) via the C16 alpha-hydroxylation pathway is elevated in patients with breast cancer, in subjects at increased risk for developing breast cancer, and in c-Ha-ras-initiated mouse mammary epithelial cells. PURPOSE: To determine whether differences in the extent of E2 C16 alpha-hydroxylation are related to the risk of developing breast cancer, we examined the extent of biotransformation of E2 via the C16 alpha-hydroxylation pathway in the mammary terminal duct lobular units (TDLUs), epithelial organoids that are a presumptive target site of human breast carcinogenesis, and in nontarget component mammary fat tissue. METHODS: Noninvolved mammary tissue was obtained from four patients undergoing reduction mammoplasty and from four undergoing mastectomy for breast cancer. A radiometric assay that measures 3H2O formation caused by stoichiometric 3H exchange from [C16 alpha-3H]E2 was utilized to compare the relative extent of C16 alpha-hydroxylation in explant cultures of TDLUs and mammary fat. RESULTS: The extent of E2 C16 alpha-hydroxylation was 1.83-fold higher (95% confidence interval [CI] = 1.71-1.97) in the TDLUs from reduction mammoplasty (i.e., "low-risk") patients and 7.96-fold higher (95% CI = 6.38-10.55) in the TDLUs from mastectomy (i.e., "high-risk") patients than in the corresponding values observed in the mammary fat. In the TDLUs obtained from the patients undergoing mastectomy for cancer, the extent of this metabolism was 4.56-fold higher (95% CI = 3.97-5.33) than that observed in TDLUs obtained from reduction mammoplasty patients who did not have cancer. CONCLUSION: The increase in the extent of C16 alpha-hydroxylation of E2 in the epithelial organoids of the human breast, TDLUs in particular, may be an important factor for breast cancer induction. This upregulation may represent an endocrine biomarker for the risk of developing breast cancer. IMPLICATION: A larger prospective study is required to confirm the clinical significance of this endocrine biomarker.

Induction by estrogen metabolite 16 alpha-hydroxyestrone of genotoxic damage and aberrant proliferation in mouse mammary epithelial cells.

BACKGROUND: Estrogens are potent mammary tumor promoters influencing post-initiation events via epigenetic mechanisms. The upregulation (i.e., induction) of the C16 alpha-hydroxylation pathway during 17 beta-estradiol (E2) biotransformation has been associated with mammary cell transformation. The action of E2 metabolites on tumorigenic transformation, however, is poorly understood. PURPOSE: The newly established mammary epithelial cell line C57/MG, derived from the C57BL mouse strain, was used to examine whether E2 or its metabolites, 16-hydroxyestrone (16 alpha-OHE1) and estriol (E3), function as initiators of mammary cell transformation. METHODS: DNA repair (hydroxyurea-insensitive thymidine uptake), estrogen metabolism (3H exchange to form 3H2O), hyperproliferation (increased cell number), and acquisition of anchorage-independent growth (soft-agar colonies) were used as quantitative end points to measure the relative extent of transformation. RESULTS: Treatment of cells with 200 ng/mL 16 alpha-OHE1 resulted in a 55.2% increase in DNA repair synthesis, a 23.09% increase in proliferative activity, and a 18-fold increase in the number of soft-agar colonies, relative to the solvent controls (P less than .0001). The extent of upregulation of the three end points was similar to that induced by the genotoxic mammary carcinogen 7, 12-dimethylbenz[a]anthracene (DMBA, positive control). DMBA treatment also upregulated the ratio of 16 alpha/C2 hydroxylation of E2 leading to increased formation of 16 alpha-OHE1. E2 and E3 were not effective in upregulating these markers for transformation. CONCLUSION: These results demonstrate that in nontransformed C57/MG cells, 16 alpha-OHE1 may function as an initiator, perturbing the intermediate biomarkers for preneoplastic transformation.

Selective responsiveness of human breast cancer cells to indole-3-carbinol, a chemopreventive agent.

BACKGROUND: Indole-3-carbinol (I3C), a compound found in cabbage, broccoli, and brussels sprouts, inhibits the growth of mammary tumors when fed to certain strains of mice. The chemopreventive and antitumor effects of I3C depend on the species and tissue type. The mechanism of action and specific human cell types that respond to I3C are not known. PURPOSE: Our purpose was to study the mechanism of action of I3C in estrogen-responsive (MCF-7) and estrogen-nonresponsive (MDA-MB-231) human breast cancer cell lines. METHODS: Estrogen responsiveness was determined by the ability of estradiol to stimulate the growth of breast cancer cells deprived of estrogen. The effect of I3C was measured on cell growth, estradiol metabolism, and level of cytochrome P-4501A1. Growth was measured by cell counts and soft-agar assay, estrogen metabolism was examined by a radiometric assay, and the level of cytochrome P-4501A1 was measured by Western blots with a polyclonal antibody. RESULTS: I3C inhibits the growth of estrogen-responsive cell line MCF-7 but has little effect on estrogen-nonresponsive cell line MDA-MB-231. Specific C-2 hydroxylation of estrogen and induction of cytochrome P-4501A1 was enhanced by I3C in the MCF-7 but not in the MDA-MB-231 cells. CONCLUSION: I3C has specific antigrowth effects in human breast cancer cells. The inhibitory effects of I3C may involve selective induction of estradiol metabolism and the related cytochrome P-450 system that may be limited to estrogen-sensitive cells.

Effect of tamoxifen on preneoplastic cell proliferation in N-nitroso-N-methylurea-induced mammary carcinogenesis.

N-Nitroso-N-methylurea (NMU) is an effective carcinogen for the induction of mammary carcinoma in the rat. Tamoxifen (TAM), used as a chemopreventive agent to reduce tumor incidence, has been well studied using this model. We have utilized the rat mammary carcinoma model to assess the effect of TAM on preneoplastic changes. Fifty-day-old virgin female Sprague-Dawley rats were randomized by weight and divided into the following five groups: Group 1, normal controls (n = 24); Group 2, TAM (n = 20); Group 3, NMU-short term (n = 24); Group 4, NMU-short term + TAM (n = 26); and Group 5, NMU-long term (n = 23). Seven weeks after the exposure to NMU, rats in Groups 1, 2, 3, and 4 were given injections of [3H]thymidine and sacrificed 4 h later for autoradiographic determination of thymidine labeling index (TLI). The rats from Group 5 were observed for 30 weeks after NMU exposure to confirm mammary tumor development. TLI in both terminal ducts and terminal end buds was modulated by treatment with TAM. Carcinogen administration induced higher TLI relative to the normal controls [18.3 +/- 1.8% (SD) versus 15.5 +/- 2.1%, P less than 0.001] in terminal end buds. The effect of carcinogen on TLI was also apparent in the terminal ducts (15.8 +/- 1.1% versus 9.5 +/- 1.1%, P less than 0.001). TAM administration was able to suppress both constitutive and NMU-induced TLI increases in terminal end buds (15.5 +/- 2.1% versus 2.8 +/- 1.1% and 18.3 +/- 1.8% versus 6.8 +/- 1.4%, respectively, P less than 0.001). Similar effects were observed in terminal ducts. In addition to its antiproliferative effect on nontransformed mammary tissue, TAM was effective in suppressing NMU-induced mammary tumor incidence and frequency. NMU-induced hyperproliferation is an intermediate stage in NMU carcinogenesis in the rat and is suppressed by TAM. Mammary epithelial hyperproliferation may provide a useful quantitative intermediate end point to evaluate chemopreventive efficacy.

Sensitivity of immunocytochemical detection of breast cancer cells in human bone marrow.

We have previously shown that occult micrometastases can be detected in the bone marrow of breast cancer patients, at the time of initial treatment, using a panel of epithelial specific monoclonal antibodies indirectly labeled with fluorescein. These monoclonal antibodies permit us to detect cancer cells at at concentration of two/million normal bone marrow cells. Immunofluorescence carries the disadvantage that detailed morphological examination of detected cells cannot be accomplished. A modification of the alkaline phosphatase anti-alkaline phosphatase method has been used to detect cancer cells and to observe their morphology in human bone marrow. The sensitivity of this method has been examined using an established human metastatic breast cancer cell line (MCF-7) mixed with normal bone marrow cells at various dilutions from 400 cancer cells/10(6) marrow cells to 10 cancer cells/10(6) marrow cells. The number of immunocytochemically stained MCF-7 cells counted at each concentration was related to the concentration by a simple nonlinear statistical model. At a concentration of 10 cancer cells/10(6) bone marrow cells, the model shows that this method has the sensitivity to detect between four and six MCF-7 cells 95% of the time. Extrapolation, using this model, predicts that at the very low concentration of one cancer cell/10(6) marrow cells, there is a 95% chance of detecting the cancer cell. This assay may be a very sensitive method for detecting cancer cells in the bone marrow of breast cancer patients.

Immunofluorescent monoclonal antibody detection of breast cancer in bone marrow: sensitivity in a model system.

We have previously shown that occult micrometastases can be detected in the bone marrow of breast cancer patients using a panel of epithelial specific monoclonal antibodies in an indirect immunofluorescent assay. The sensitivity of this assay has been examined using cells from an established human breast cancer cell line (MCF-7) mixed with normal bone marrow at various dilutions from 400 cancer cells/10(6) marrow cells (400:10(6] to 10:10(6). MCF-7 cells were detected at the lowest concentration studied, namely 10:10(6). The number of fluorescent labeled MCF-7 cells counted at each concentration was related to the concentration by a simple nonlinear statistical model. At the concentration of 10:10(6), the model shows that this technique has the sensitivity to detect between two and four MCF-7 cells 95% of the time. Moreover, by extrapolation, the model predicts that even at the very low concentration of 2:10(6), there is a 95% chance of detecting one cancer cell. Therefore, this assay may be a very sensitive method for detecting cancer cells in the bone marrow in vivo.

Antisense RNA to the putative tumor-suppressor gene DCC transforms Rat-1 fibroblasts.

Allelic deletions involving chromosomes 18q occur in a significant number of colorectal cancers. Recently, a highly conserved gene called 'deleted in colorectal cancer' (DCC) has been identified on chromosome 18q. DCC has been postulated to be a colorectal tumor-suppressor gene. In order to understand the role of DCC in cell transformation, we have established a stable Rat-1 cell line expressing dexamethasone-inducible DCC antisense RNA. High levels of dexamethasone-inducible DCC antisense RNA were detected in the Rat-1 transfectants. The antisense DCC-expressing Rat-1 cells showed a faster growth rate, anchorage independence and tumorigenicity in nude mice. Exposure of the parental Rat-1 cells to antisense oligodeoxyribonucleotides to DCC resulted in inhibition of cell adhesion to the substratum which could be abrogated by various extracellular matrices. On the other hand, a bone marrow-derived stromal cell line which does not express DCC showed no detachment from the substratum when treated with the antisense oligo to DCC. These results suggest that the DCC gene is involved in cell adhesion and provide the first direct biological evidence for the possible role of DCC as a tumor-suppressor gene.

Prediction of early relapse in patients with operable breast cancer by detection of occult bone marrow micrometastases.

We used monoclonal antibodies to identify occult micrometastases in the bone marrow of 49 patients with operable (stage I and II) breast carcinoma. Follow-up (mean, 29 months; median, 30 months) revealed that 12 patients recurred. The presence of bone marrow micrometastases (BMM) was significantly associated with early recurrence (P less than .04). The estimated 2-year recurrence rate for patients with no BMM detected (BMM-) was 3%; in patients with BMM, the 2-year recurrence rate was 33%. When BMM and axillary lymph node (LN) status were combined, groups of patients at low risk (LN-, BMM-; 2-year recurrence rate, 0%) and high risk (LN+, BMM+; 2-year recurrence rate, 42%) for early recurrence were identified. Bone marrow tumor burden was related to early recurrence. Among patients with BMM, those who did not recur had on average fewer extrinsic cells in their marrow than those who recurred (15 v 43 cells, respectively). Multivariate analysis comparing BMM, LN+ versus LN-, and tumor size (less than or equal to 2 cm v greater than 2 cm) revealed no factor independently associated with early recurrence. Peripheral tumor burden of BMM (0 or less than 10 extrinsic cells v greater than or equal to 10 extrinsic cells) was the only independent predictor of early recurrence (P less than .003). In conjunction with conventional prognostic factors, particularly axillary LN status, evaluation for BMM might be used to stratify patients for adjuvant treatment programs. Because this pilot study involved few patients with short-term follow-up, the results should be interpreted with caution. The examination of bone marrow for micrometastases remains an experimental procedure; the clinical usefulness of the test will be established through larger studies with long-term follow-up.

Breast cancer in patients irradiated for Hodgkin's disease: a clinical and pathologic analysis of 45 events in 37 patients.

PURPOSE: To characterize the clinical and pathologic features of patients who developed breast cancer (BC) after treatment for Hodgkin's disease (HD). Recent epidemiologic studies have shown that women who are cured of HD have an increased risk of developing BC. PATIENTS AND METHODS: The clinical data, mammograms, and pathologic specimens of 37 women who developed 45 BCs (eight bilateral events), and had a prior history of treatment for HD were analyzed. RESULTS: The median age at diagnosis of HD was 27 years (range, 11 to 60). All patients received radiotherapy (RT) to the upper part of their body, and 10 also had chemotherapy for HD. The median interval from the treatment of HD to the diagnosis of BC was 15 years (range, 8 to 34). The median age at diagnosis of BC was 43 years (range, 27 to 75), 41% of patients were 39 years old or younger. Most mammograms (81%) showed abnormal findings of mass and/or microcalcifications. Of the eight patients (22%) with bilateral tumors, four were synchronous and four were metachronous. Involvement of the medial half of the breast occurred more frequently than in patients with primary BC (39% and 21%, respectively; P < .002). But, the histologic types, grades, presence of lymphocytic reaction, and lymphatic invasion were similar to those observed in 935 primary BC patients who were previously analyzed at our center. The 6-year actuarial relapse-free survival (RFS) for node-negative BC after HD was 85%. Node-positive patients had a significantly lower RFS of 33% (P = .002). CONCLUSIONS: In comparison to patients with primary BC, patients who develop BC after HD are more likely to be younger, have bilateral disease, and have their tumors more frequently involve the medial half of the breast. Pathologic characteristics, nodal involvement, and prognosis are similar to those of primary BC. BC in women who were treated for HD is becoming an increasing problem, as more patients cured of HD reach a follow-up time of 10 to 15 years. Breast examination and mammography at an early age should be part of the follow-up program for women who are cured of HD.

Lower esophageal sphincter pressure and serum gastrin levels after mapped antrectomy.

We investigated the effect of mapped antrectomy with gastroduodenostomy on serum gastrin levels in fasting patients and resting lower esophageal sphincter (LES) pressure. Serum gastrin levels in fasting patients were lower in those who had an antrectomy without vagotomy (P less than .05) as compared to control subjects or patients with antrectomy and vagotomy. Resting LES pressure was similar in patients and controls and was not affected by the presence or absence of vagotomy. These findings suggest that (1) mapped antrectomy and gastroduodenostomy without vagotomy are followed by a decrease in serum gastrin and (2) resting LES pressure is not affected by mapped antrectomy and a decrease in serum gastrin level.

Psychological counseling strategies for women at risk of breast cancer.

Women with family histories of breast cancer have a much higher risk of developing the disease than women in the general population. In the absence of primary prevention for breast cancer, secondary prevention in the form of early detection is our best bet against premature morbidity and mortality. This article describes the most salient psychological issues for high-risk women as well as ways for improving screening behaviors. Based on our work and other studies in the literature, we found that there were several key variables related to psychological distress and surveillance behaviors. Barriers to screening were a major reason why women did not engage in any breast cancer prevention behaviors. Cognitive deficits, in terms of lack of knowledge, and breast cancer misbeliefs contributed to poor adherence to screening. Most important, anxiety or emotional distress not only interfered with adherence to screening but also affected quality of life negatively in that many women needed psychological counseling. In developing psychological counseling strategies for high-risk women, we focused on the treatment outcomes of reducing emotional distress, decreasing perceived vulnerability, and improving adherence to screening behaviors. We conducted a preliminary study by piloting a group psychoeducational intervention for 6 consecutive weeks. This intervention was found to significantly reduce perception of risk (P < .02) and to increase adherence to screening behaviors (P < .01). If proven effective in a randomized controlled trial, this intervention can be proposed to other cancer centers and prevention programs for implementation and enhancement of the behaviors among high-risk women that will assure early detection and decrease breast cancer mortality.

Long-term responses of women to indole-3-carbinol or a high fiber diet.

We test the hypothesis that the estrogen metabolite ratio 2-OH-estrone:estriol can be raised via dietary indole-3-carbinol (I3C) and that this higher ratio can be sustained over a 3-month test period. We also explore the possible role of pure fiber on estradiol metabolism. Using a randomized clinical trial with three arms, each containing 20 subjects, arm 1 received 400 mg/day of I3C daily for 3 months, arm 2 received 20 g of alpha-cellulose daily for the same time period as a source of added fiber, and arm 3 received a placebo dose. Blood levels of a variety of biochemical parameters were measured. The urinary 2-OH-estrone:estriol estrogen metabolite ratio was measured monthly at the same time of the menstrual cycle. While no changes were observed in the control and alpha-cellulose-treated arms, a substantial mean increase in the ratio was observed in the I3C-treated arm at month 1; that increase was maintained over the 3-month time period. Three of the 20 subjects in this I3C-treated group differed from the others in that no significant change in the metabolite ratio was observed at any time point. The results suggest that I3C can serve to increase the 2-OH-estrone:estriol metabolite ratio in a sustained manner without detectable side effects and that some individuals may be resistant to such change.

Iridium-192 implants for primary breast cancer: experience with placement at the time of wide local excision.

An Ir-192 boost is a technique frequently used to deliver an additional dose of radiation therapy to the tumor bed following breast conserving surgery and combined with external beam radiation therapy to the entire breast for patients with early breast cancer. Traditionally these catheters are placed following completion of 4500-5000 cGy, as a separate procedure. This paper described a Pilot Study identifying placement of the catheters at the time of primary wide local excision, or re-excision in 52 patients. The key to the success of this technique is the achievement of complete hemostasis in the primary cavity, the presence of the radiation oncologist during the surgical procedure itself, and closure of the wound prior to placement of the catheters. Details of the technique, and preliminary patient results are presented.

Microinvasive carcinoma (T1mic) of the breast: clinicopathologic profile of 21 cases.

Clinicopathologic data on microinvasive carcinoma of the breast (MICB) as defined by the 1997 TNM criteria (T1mic < or = 1 mm) is scarce. Histologic slides of 109 cases from 1993 through 1997, in which microinvasion was either suspected or diagnosed initially, were reviewed. A double immunoenzyme-labeling technique using antismooth muscle actin and anticytokeratin antibody on the same section was used to confirm invasion in equivocal cases. All foci of invasion were measured by ocular micrometer. Twenty-one cases were confirmed to be MICB. The mean age of the patients was 60.9 years. Thirteen patients presented with mammographic abnormalities on routine examination (60.9%). MICB was ductal in 18 patients, including one tubular carcinoma, and was lobular in three patients. The mean number of invasive foci was two per patient (range, one to seven foci). The accompanying duct carcinoma in situ had high-grade nuclei and necrosis in 16 of 18 patients (89%), 13 of which (72%) were comedo-type. Two of the 15 patients had one positive axillary lymph node each (13.3%). Eleven patients underwent mastectomy, nine received radiation therapy, one received chemotherapy, and two underwent lumpectomy only. Median follow up was 28 months (range. 18-63 months). One patient had a chest wall recurrence of infiltrating duct carcinoma and another recurred with duct carcinoma in situ.

Monoclonal antibodies detect occult breast carcinoma metastases in the bone marrow of patients with early stage disease.

Thirty-five to 40% of patients with operable breast carcinoma develop metastases after primary therapy. There is a need for more specific prognostic parameters to identify patients who are most likely to benefit from adjuvant therapy. The success of such treatment stems from its ability to eradicate preclinical microscopic metastases. The bone marrow is an accessible and frequent site of breast carcinoma metastases. Following studies of Redding et al. (16), we used monoclonal antibodies that recognize membrane and cytoskeletal antigens expressed by epithelial cells (C26, T16, AE-1) in an immunohistochemical assay to find cancer cells in bone marrow aspirates. The assay can detect one cancer cell among 50,000-100,000 hematopoietic cells. None of the 44 control bone marrows (from normal individuals and patients with leukemias and lymphomas) contained antigen-positive (extrinsic) cells. We found extrinsic cells in the bone marrow of 35% (18 of 51) of patients with operable breast carcinoma; no extrinsic cells were identified by routine bone marrow cytology in these patients. Twenty-seven percent (six of 22) of patients with negative lymph nodes had antigen-positive cells, while 41% (12 of 29) of patients with lymph node metastases had such cells. Similarly, 23% (three of 13) of patients with TNM stage I disease, 38% (13 of 34) of patients with stage II disease, and 50% (two of four) of patients with stage III disease had extrinsic cells. In those cases where extrinsic cells were identified, stage II patients with negative lymph nodes and patients with stage I disease were found to have fewer such cells in their marrow than patients with lymph node metastases and patients with stage II disease. These trends did not reach the level of statistical significance in this small number of patients. The presence of extrinsic cells did not correlate with tumor size of lymphatic invasion around the tumor. We conclude that the epithelial cells detected in the bone marrow of the patients with breast carcinoma were carcinoma cells based on the following criteria: (a) they expressed both membrane and cytoplasmic epithelia-specific antigens, (b) they possessed the cytologic characteristics of malignant epithelial cells, and (c) these cells were not detected in the bone marrow from normal individuals or patients with nonepithelial neoplasms involving the bone marrow. We have shown that the technique described here can detect occult metastases in bone marrow and that the presence of extrinsic cells correlates with some established predictors of prognosis. Long-term clinical correlative follow-up studies are now underway.

A distinct kinase modulates the expression of IFN-inducible genes in human breast cancer cells.

The biological activity of interferons (IFNs) is presumed to be mediated through the induction of a number of IFN-inducible genes. IFN-mediated gene induction was examined in two human breast cancer cell lines, MCF-7 and BT-20. Both these cell lines were remarkably responsive to IFNs as a number of IFN inducible genes were rapidly induced. We examined the sensitivity of these genes towards 2-aminopurine (2-AP), a known inhibitor of double-stranded (ds) RNA dependent protein kinase. 2-AP has also been reported to inhibit the induction of IFN-beta 1 in response to dsRNA and the genes c-myc and c-fos in fibroblasts. In both MCF-7 and BT-20 cell lines, 2-AP selectively inhibited the IFN-induced gene responses. 2-AP did not affect levels of the oncogene, HER-2/neu. Tamoxifen (TAM), an antiestrogenic drug, which is known to inhibit the activity of protein kinase C at high concentrations, did not affect IFN-mediated gene induction. Our data is consistent with the concept that the 2-AP sensitive kinase is primarily associated with the IFN-induced gene systems and that positive and negative growth regulating stimuli in breast cancer may require the participation of distinct kinases.

Prevention of mammary preneoplastic transformation by naturally-occurring tumor inhibitors.

Aberrant hyperproliferation (AH) is a late occurring post-initiational event that precedes mammary tumorigenesis in vivo. Experiments on the spontaneously immortalized, non-tumorigenic murine mammary epithelial C57/MG and MMEC cells were designed to validate AH as an in vitro cellular marker for preneoplastic transformation. Colony forming efficiency (% CFE) in anchorage-independent conditions of growth represented the quantitative parameter for AH. C57/MG and MMEC cells, upon treatment with chemical carcinogens or transfection with oncogenes, exhibited at least a 60-300-fold increase in AH relative to that seen in appropriate untreated controls. Transplantation of mammary epithelial cells initiated either by chemical carcinogens or by oncogenes into mammary fat pads of syngeneic mice produced rapidly growing tumors at the transplant site within 4-6 weeks. The tumor-derived T1/Pr1 and myc3/Pr1 cell lines (positive controls) exhibited at least an 800-900-fold increase in AH. Treatment of initiated cells with naturally occurring tumor inhibitors eicosapentaenoic acid (EPA), indole-3-carbinol (I3C), (-)epigallocatechin gallate (EGCG), squalene (SQE), and perillyl alcohol (PA) at non-toxic doses, resulted in a 70-99% inhibition of AH, depending on the initiator and the chemopreventive test compound. Upregulation of AH in initiated mammary epithelial cells in vitro prior to tumorigenesis in vivo, and persistent inhibition of AH by diverse naturally occurring tumor inhibitors, provides evidence for AH as a cellular surrogate endpoint for induction and modulation of mammary neoplastic transformation.

Augmentation of cytotoxicity using combinations of interferons (types I and II), tumor necrosis factor-alpha, and tamoxifen in MCF-7 cells.

The cytotoxic effect of a combination of interferons (type I and II) and tumor necrosis factor (TNF), with an antiestrogenic drug, tamoxifen (TAM), was investigated in the estrogen receptor positive human breast carcinoma cell line, MCF-7. Cytotoxicity was measured by the MTT assay. In an attempt to define the molecular basis for the interaction between the interferons (IFNs) and TNF or any one of the cytokines with TAM, the induction characteristics of a number of IFN-induced mRNAs in response to IFNs, TNF, and TAM were studied. We observed an augmentation of the cytotoxic effect of TNF when it was combined with TAM. There appears to be an overlap in signalling mechanisms of IFNs and TNF as two of the IFN-inducible genes, 1-8 and 6-16 are also induced by TNF. mRNA 1-8 was induced by both IFN-alpha (type I) and IFN-gamma (type II). We conclude that TNF potentiates the cytotoxic effects of TAM in MCF-7 cells and that the three cytokines IFN-alpha, IFN-gamma, and TNF share some pathways that lead to specific induction of some cytokine responsive genes.

2-hydroxyestrone: the 'good' estrogen.

The issue of the role of 2-hydroxyestrone (2-OHE1) in breast cancer has been the subject of considerable controversy as to whether it is carcinogenic or anticarcinogenic. The expanding data base outlined below is most consistent with the conclusion that 2-OHE1 is anticarcinogenic. In every experimental model in which 2-hydroxylation was increased, protection against tumors was achieved. Correspondingly, when 2-hydroxylation was decreased, an increase in cancer risk was observed. Even more dramatically, in the case of laryngeal papillomas induction of 2-hydroxylation with indole-3-carbinol (I3C) has resulted in inhibition of tumor growth during the time that the patients continue to take 13C or vegetables rich in this compound.

Estradiol metabolism: an endocrine biomarker for modulation of human mammary carcinogenesis.

The natural estrogen 17beta-estradiol (E2) has a profound influence on proliferation and neoplastic transformation of mammary epithelium. The role of cellular metabolism of E2 in mammary carcinogenesis, however, remains to be elucidated. Explant culture and cell culture models developed from noncancerous human mammary tissue were used to examine modulation of E2 metabolism in response to treatment with prototype rodent mammary carcinogens and the ability of the naturally occurring phytochemical indole-3-carbinol (13C) to influence E2 metabolism and regulate aberrant proliferation. In the two models, treatment with the chemical carcinogens 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene altered the metabolism of E2 as determined from the radiometric (tritium release) and gas chromatography-mass spectrometry (GC-MS) assays. This alteration in E2 metabolism was accompanied by aberrant proliferation and abrogation of apoptosis as determined by the extent of replicative DNA synthesis, S-phase fraction and Sub G0 (apoptotic) peak. Exposure of carcinogen-initiated cultures to 13C resulted in induction of C2-hydroxylation of E2 and of apoptosis and downregulation of hyperproliferation. Determination of altered cellular metabolism of E2 in response to initiators and modulators of carcinogenesis and evaluation of cell cycle related markers for proliferation and apoptosis may provide a mechanism-oriented approach to validate E2 metabolism as an endocrine biomarker for induction and prevention of human mammary carcinogenesis.