Biocloretos de bifenila (PCBs) em tecido mamário de mulheres / Bichlorinated biphenyls in female mammary tissue

Os policloretos de bifenila (PCBs) foram determinados em tecidos humanos e testada a hipótese de maior acúmulo no tecido de glândula mamária aparentemente normal e que, eventualmente, estaria relacionado com o aparecimento de tumores. Os níveis encontrados mostram que a glândula mamária é semelhantes aos demais tecidos.

Ultrastructural and immunocytochemical characterization of an immortalized human breast epithelial cell line, MCF-10.

The human breast epithelial cell line MCF-10 was derived from s.c. mastectomy tissue from a 36-year-old, parous, premenopausal woman with fibrocystic disease. It was initiated as a mortal cell line (MCF-10M), which senesces when transferred serially in 1.05 mM calcium. These cells spontaneously gave origin to two immortal sublines, MCF-10A, or attached cells, and MCF-10F, or floating cells, which have proliferated for more than 4 years in Dulbecco's modified essential medium and Ham's F-12 either with the customary calcium concentration of 1.05 mM (DMEM-H) or in medium containing 0.04 mM calcium or low calcium. Studies reported here indicate that MCF-10 is a mammary epithelial cell line. Electron microscopy showed that both MCF-10A and MCF-10F have characteristics of luminal ductal cells, but not of myoepithelial cells. When grown for more than 1200 days in Dulbecco's modified essential medium-Ham's F-12 and low calcium media, respectively, they maintained their epithelial characteristics, although the concentration of calcium exerted a powerful influence on cell morphology. Cells grown in Dulbecco's modified Eagle's medium-Ham's F12 medium are low cuboidal with numerous desmosomes and short microvilli, whereas those grown in low calcium medium have significantly reduced number of desmosomes, are more spherical, and have greater numbers of microvilli which are longer than those of cells grown in DMEM-H. The breast epithelial orgin of these cells was confirmed by immunocytochemical detection of epithelial sialomucins and keratins. The monoclonal antibodies MFA-breast and MC5 and the polyclonal antibody epithelial membrane antigen were used to detect the epithelial sialomucins. Keratins were characterized by using KA-4, K-14, AE1/AE3, and K-19 specific antibodies. It was concluded that MCF-10A and MCF-10F cells are breast epithelial cells and that they represent an important tool for studies of the basic processes of growth and carcinogenesis.

Characterization, in some human breast cancer cell lines, of gastrin-releasing peptide-like receptors which are absent in normal breast epithelial cells.

The binding of 125I-Tyr4 bombesin was investigated on plasma membranes of 8 human breast cancer cell lines and 2 long-term cultures of normal human breast epithelial cells. Scatchard plots were compatible with high-affinity, single-site class of receptors in 3 cell lines (KD of 0.75 x 10(-9) and 10(-9) M, Bmax of 0.75 x 10(-13) and 9.7 x 10(-13) M/mg protein in MDA-MB231 and in T47D cells, respectively) while no binding was observed in 5 other cell lines and normal epithelial cells. The neuropeptide and its structural analogues (natural or synthetic) inhibited the binding of 125I-Tyr4 bombesin in the following order of potency: gastrin-releasing peptide (GRP, EC50 = 1.7 x 10(-10) M) greater than BIM 26159 greater than bombesin, Tyr4 bombesin greater than BIM 26147 greater than litorin greater than neuromedin C. In contrast, 125I-Tyr4 bombesin binding was not displaced by neuromedin B, somatostatin, bradykinin and insulin. In agreement with our binding data, SDS-PAGE of the complex 125I-Tyr4 bombesin-receptor covalently linked by ethylene glycol-bis succinimidyl succinate (EGS) identified after autoradiography a single band with a molecular weight of 75,000, which disappeared in the presence of bombesin in excess. No transcription of either GRP or neuromedin B mRNA could be shown in tumor or normal cells. Exogenous gastrin-releasing peptide had no effect on growth of the cell lines when a serum-free medium was used, implicating that in breast cancer cell lines this receptor does not mediate growth but has a functional role.

Mammary hypertrophy is not associated with increased estrogen receptors.

Estrogen promotes secondary female sex characteristics, including breast enlargement. Since excessive breast hypertrophy is unrelated to elevated serum estrogen levels, it has been postulated that the enlarged breast is a hypersensitive "target organ." At the cellular level, estrogen crosses the cell membrane, is bound to a cytoplasmic estrogen receptor (ER), and induces the formation of specific anabolic proteins. In breast cancer, this estrogen receptor is regarded as a measure of the sensitivity of the cell to estrogen. To determine if mammary hypertrophy is related to an increase in the number of estrogen receptors, we assayed breast tissue, not fat, from 25 consecutive breast reductions. The median age of patients was 26 years (17 to 77 years), with 752 gm per breast removed on average. Twenty-four percent of the patients were taking estrogens, primarily birth control bills. Cellular estrogen-receptor status was measured by a standardized cytosol extraction radioactive estradiol technique. Estrogen receptors were undetectable (less than 3 fmol/mg cytosol protein) in all patients. We conclude that estrogen receptors alone, and hence estrogen, are not a determinant in mammary hypertrophy. If the enlarged breast is a "target organ," it is by another mechanism.

Cyclic changes in composition and volume of the breast during the menstrual cycle, measured by magnetic resonance imaging.

The volumes and spin-lattice (T1) relaxation times of breast tissues and parenchymal water content were measured non-invasively by magnetic resonance imaging (MRI) in eight healthy women during four to eight consecutive menstrual cycles. Total breast volume, and parenchymal volume, T1 relaxation time and water content were lowest between days 6 and 15. Between days 16 and 28, parenchymal volume, T1 relaxation time and water content rose sharply by 38.9%, 15.1% and 24.5%, respectively, and peaked after day 25. Within 5 days of the onset of menses, parenchymal volume fell sharply by 30.3%, while water content declined by 17.5%. Rising parenchymal volume in the second half of the menstrual cycle is not solely due to increased tissue water content and provides in vivo evidence for both growth and increased tissue fluid at this time.

Thrombospondin forms complexes with single-chain and two-chain forms of urokinase.

Thrombospondin (TSP), an adhesive glycoprotein found in platelets and extracellular matrix, has been shown previously to interact with plasminogen and tissue plasminogen activator, resulting in efficient plasmin generation. We now demonstrate specific complex formation of TSP with both the single-chain and two-chain forms of urokinase (scuPA and uPA). Binding of uPA and scuPA to immobilized TSP was detected and quantified using colorimetric immunoassays and a functional amidolytic assay. Binding was time and concentration dependent with apparent affinity constants of 40-50 nM. Binding was not affected by serine protease inhibitors, EDTA, or epsilon-aminocaproic acid. scUPA and uPA bound to TSP retained functional activity. Using a sensitive amidolytic assay we found that TSP. scuPA complexes were efficiently converted to TSP. uPA by catalytic plasmin concentrations. Additionally, TSP.uPA complexes were found to have plasminogen-activating activity equivalent to fluid-phase uPA and to be protected from inhibition by plasminogen activator inhibitor type 1, the major plasma and matrix plasminogen activator inhibitor. Using immunohistochemical techniques, we also demonstrated co-distribution of TSP and uPA in normal and malignant breast tissue. Complex formation of TSP with uPA may serve to localize, concentrate, and protect these enzymes on cell surfaces and within the extracellular matrix, thereby providing a reservoir of plasminogen activator activity.

neu oncogene protein and epidermal growth factor receptor are independently expressed in benign and malignant breast tissues.

The neu oncogene protein, p185, and epidermal growth factor receptor (EGFR) were localized immunohistochemically in benign and malignant human breast tissues using monoclonal antibodies. Both benign and malignant epithelial cells were positive for these oncogene proteins in acetone-postfixed frozen sections. Stromal cells were negative for p185, but occasionally positive for EGFR. Myoepithelial cells were consistently positive for EGFR, and p185 was localized predominantly in duct-lining cells, where the basolateral plasma membrane was the normal expression site of both substances. Paraformaldehyde-prefixed frozen sections were less sensitive for antigen demonstration. Based on the intensity of immunoreactivity, 11 of 37 acetone-postfixed breast carcinomas (30%) were judged neu overexpressors, while none of 24 benign tissues overexpressed neu. Epidermal growth factor receptor was demonstrated in 18 of 36 acetone-postfixed cancer tissues (50%) and was overexpressed in three (8%). At the cellular level, heterogenous expression of p185 and EGFR was occasionally observed in both benign and malignant tissues, and a single case of cancer overexpressing both neu and EGFR showed reciprocal patterns of staining, indicating their independent expression. In some carcinomas, EGFR was localized only in stromal cells. Our findings confirmed mutually independent expression of the two closely related protooncogenes in benign and malignant breast tissues.

Automated quantitation of immunocytochemically localized estrogen receptors in human breast cancer.

Frozen sections of breast tumor tissue have been stained using an immunoperoxidase [estrogen receptor (ER)-immunocytochemistry] kit incorporating a monoclonal antiserum [H222] to visualize nuclear human ERs. Quantitation of specific staining has been performed by manual procedures using optical microscopy and by a computer-assisted image analysis system (CAS 100). Initial investigations with a test panel of ER-immunocytochemistry-positive tumors revealed a good qualitative agreement between CAS and manual assessments. Reduced variance was, however, observed between quantified ER-immunocytochemistry results from four experienced investigators using the CAS analysis. An extended study confirmed the relationships between CAS and manual methods of assessment. These findings were evident when studies were scored either by assessment of the percentage of positively stained cells (n = 92; r = 0.919; P less than 0.01) or by H-score calculations (n = 92; r = 0.913; P less than 0.01). A good correlation was also found between CAS quantification and the results of an ER enzyme immunoassay of 48 primary breast cancer specimens (r = 0.715; P less than 0.05). In 49 cases it was possible to relate CAS-defined ER status and levels to the subsequent response of patients to endocrine therapy. ER was assessed on specimens obtained prior to commencement of treatments for recurrent breast cancer. Presuming the presence of ER to be a prerequisite for successful therapy, very good correlations between response and both status and levels of positivity were recorded. None of 16 patients with CAS-ER-negative tumors responded to treatment, while 16 of 33 (48.4%) CAS-ER-positive patients achieved an objective response according to International Union Against Cancer criteria. A relationship between response and the degree of CAS-ER positivity was obtained when the CAS score divisions of 0, 1-100, and greater than 100 (response rates, 0, 41, and 64%, respectively) were used. These data demonstrate that automated image analysis offers a reliable, reproducible procedure for quantifying ER in immunocytochemically stained sections. It has potential advantages over manual procedures, providing less opportunity for subjective influences in scoring sections. Future advances in software design should further reduce elements of subjectivity and increase both the speed and reliability of results. We anticipate image analysis becoming a valuable tool in investigations concerning, for example, the influence of heterogeneity of steroid receptor distribution on the rate of recurrence of breast cancer after mastectomy and in the clinical course of the disease.

Collagenous spherulosis. A comment on its histogenesis.

Collagenous spherulosis is a benign breast lesion involving lobular acini and ductules consisting of eosinophilic spherules measuring up to 100 mu in diameter. It is a myoepithelial product. We described similar lesions in salivary gland tumors and a benign lymphoepithelial lesion of the parotid gland.

[The relationship between the receptor status and the mammographic picture in operable breast cancer]. / Relazione tra assetto recettoriale e quadro mammografico nel cancro mammario operabile.

To verify the relationship of mammographic (Mx) patterns of breast cancer to hormone receptor content (ER, PgR) we studied 129 women (59% in postmenopause; average age 55) in the last 3 years. All women had operable breast cancer and were submitted to mammography before surgery. The tumors were classified, according to Mx characteristics, in 5 classes (Broberg, 1983). ER and PgR contents (cut-off for positivity: 10 fmol/mg prot cyt) were analyzed in all patients by means of DCC method. The percentage of ER+ cases was significantly higher in class I than in all other Mx classes (85% vs 57%; p = 0.02), whereas it was lowest in class IV (51% vs 70%; p = 0.03). The percentage of PgR+ cases was significantly different only in class I with respect to class IV (70% vs 41%; p = 0.002). As for ER and PgR mean tumor content, no statistically significant difference was observed between the 5 classes. When the 2 receptors were simultaneously considered the percentage of ER+ PgR+ cases was higher in class I than in all the extant classes (p = 0.01), and the percentage of ER- PgR- was higher in class IV than in the extant ones (p = 0.02). In selected subgroups of patients, Mx classification of breast cancer can help the physician predict the hormone receptor tumor status with sufficient reliability.

Quantitative DNA patterns in human preneoplastic breast lesions.

In 12 cases of human mammary carcinoma in which a preneoplastic atypical ductal hyperplasia was also identified, quantitative DNA (QDNA) measurements of thionein-stained samples from both lesions were performed using the Cell Image Analysis 100 system. The QDNA values in the preneoplastic and neoplastic lesions from each case showed concordance (six as euploid and six as aneuploid/hyperdiploid). Such congruence suggests a stable inheritance of the somatic mutation(s) that is involved in carcinogenesis and that affects ploidy. If this relationship between concurrent preneoplasia and neoplasia in the ipsilateral breast is confirmed, it offers the possibilities of (1) identifying individuals at risk for developing neoplasias with defined biologic characteristics and (2) developing therapeutic regimens more appropriate to the risk assessment of each patient. It may be possible to conceive of a rational preventive regimen for cancer of the breast.

Endothelin messenger RNA and receptors are differentially expressed in cultured human breast epithelial and stromal cells.

Paracrine regulation is implicit in the biosynthesis and secretion of milk in the breast. An important determinant for this regulation in vivo is proximate cellular location as exemplified by stromal and epithelial cells in breast tissue. Cultured human breast epithelial cells exhibited low constitutive expression of mRNA for endothelin which was enhanced 20-fold after prolactin stimulation. Human breast stromal cells did not express measurable levels of endothelin mRNA under similar conditions. In a similar differential manner, the stimulated release of immunoreactive endothelin into medium overlay was observed only for breast epithelial and not stromal cells. Specific cell-surface receptors for endothelin and biochemical responsiveness to the peptide were observed only in the stromal cells.

Distribution of oncofetal fibronectin in human mammary tumors: immunofluorescence study on histological sections.

Two types of human fibronectin have been detected by the reactivity of specific monoclonal antibody FDC-6: (a) those present in normal plasma and adult tissues, which do not react with FDC-6 (normal fibronectin or norFN); and (b) those present in fetal tissues and in tumoral cell lines, which do react with FDC-6 (oncofetal fibronectin or onfFN) (H. Matsuura and S. I. Hakomori, Proc. Natl. Acad. Sci. USA, 82: 6517-6521, 1985). We compared the distribution of norFN and onfFN in normal breast tissue (15 samples), breast fibroadenoma (ten samples), and breast adenocarcinoma (80 samples), using an immunofluorescence technique with monoclonal antibody FDC-6. A polyclonal antiserum against human normal fibronectin was used as a control. While norFN was diffusely present in normal gland and in benign and malignant tumors, onfFN was absent in normal gland and in benign tumors. In carcinomas, however, its presence was frequent, as we found it in 60% of cases. Among classical prognosis factors in breast carcinomas, onfFN distribution was significantly correlated with histological grade. Indeed, the presence of FDC-6 labeling was significantly linked with intermediary and high malignancy grades, while its absence was significantly linked with low malignancy grade. onfFN could be considered a marker of the neoplastic state; its immunohistological detection may represent a new prognosis factor in breast carcinomas.

Distribution of estrogen and progesterone receptors in healthy tissue adjacent to breast lesions at various stages--immunohistochemical study of 107 cases.

The purpose of this study was to determine the distribution of ER+ (estrogen receptor) and PR+ (progesterone receptor) epithelial cells in normal mammary tissue or in tissue in contact with or involved in benign or malignant processes. Three important findings emerged from this study. First, a true dissociation was observed between ER+ and PR+ cells in mammary tissue. In premenopausal women some cells express only progesterone receptors. In premenopausal normal tissue, regardless of the menstrual cycle status, 6% of cells are ER+ and 29% PR+. Second, during the menstrual cycle the percentage of positive cells varies. This finding would indicate a change in cell recruitment rather than in intracellular levels. Finally, specific changes in the proportion of positive cells in normal tissue in contact with epithelial proliferations were noted. This finding suggests the possibility of either a diffusible factor or a cellular pathological process spreading beyond areas displaying morphological changes.

Tenascin distribution in the normal human breast is altered during the menstrual cycle and in carcinoma.

Tenascin is a novel extracellular matrix glycoprotein which appears to have a major role in tissue development. Previous studies have stated that tenascin is absent from the normal human, rat and mouse breast, its distribution being restricted to embryonic and malignant mammary tissues. No previous studies have investigated tenascin distribution as a function of the normal menstrual cycle. Therefore this study addresses the cyclical appearance of tenascin in the normal breast and associated changes in distribution in preinvasive cancer (carcinoma-in-situ) and invasive infiltrating ductal carcinoma. Tenascin is present in the normal human adult mammary gland, principally in the basement membrane, sub-basement-membrane zone and delimiting layer of fibroblasts around the ductules. Both the distribution and quantity of tenascin change during the menstrual cycle. In carcinoma-in-situ (preinvasive cancer) tenascin is present in the attenuated basement membrane/sub-basement-membrane zone around the expanded ductules and in small amounts in the stroma. In infiltrating ductal carcinoma, tenascin is absent from the remnants of the basement membrane and sub-basement-membrane zone but greatly increased in the adjacent intralobular and interlobular stroma. Therefore, if tenascin is used as a basement membrane/sub-basement-membrane marker for distinguishing carcinoma-in-situ from invasive ductal carcinoma, the time of the menstrual cycle is of importance in interpreting the biopsy appearance. This study suggests that the optimal time for biopsy is between weeks 3 and 4 of the cycle, to avoid confusion between the normal low levels of tenascin (due to hormonal status) and those due to microinvasive disease.(ABSTRACT TRUNCATED AT 250 WORDS)

[Correlation with mammography and estrogen receptors in breast neoplasms]. / Correlazione tra aspetti mammografici e recettori estrogenici nelle neoplasie mammarie.

A comparative study of 71 patients with unilateral primary breast cancer was performed to verify a possible correlation between the mammographic findings of the cancer and the content of hormonal receptor. The Nielsen & Poulsen, and Wolfe classification were employed. Hormonal receptor content was determined using the Taylor method. The estrogen content was found to be high in type 1, intermediate in 2 and 4, and low in 3 and 5. Statistically significant difference in the estrogen receptor content was found between type 1 and others groups. Using the Wolfe classification, the hormonal content was found to be high in P1. Statistically significant difference was found between N1 and P2; N1 and DY. No relationship was found between histologic cancer type and hormonal receptor content. The Nielsen & Poulsen classification seems to be most simple and reproducible method respect of the Wolfe's parenchymal patterns. Mammographic findings could be used in selecting patients for endocrine therapy where no estrogen-receptor assay is available.