Normal Human Breast Tissue Research Articles

Differential expression of high-affinity melatonin receptors (MT1) in normal and malignant human breast tissue.

Melatonin is a pineal hormone that strongly inhibits the growth of breast cancer cells in vitro and in vivo. We report thefirst use of immunohistochemical analysis to determine the distribution of the high-affinity melatonin receptor subtype, MTI, in human breast tissue, the hypothalamic suprachiasmatic nucleus, and skin. The MT1 antibody, which is specific for the cytoplasmic portion of the receptor, produced cytoplasmic staining in normal-appearing breast epithelial cells and ductal carcinoma cells; stromal cells, myoepithelial cells, and adipocytes were nonreactive. The majority of nonneoplastic samples (13/19 [68%]) were negative to weakly positive, while moderate to strong reactivity was seen in most cancer samples (49/65 [75%]). Thus, although MT1 receptors were detectable in normal and malignant breast epithelium, high receptor levels occurred more frequently in tumor cells (P < .001), and tumors with moderate or strong reactivity were more likely to be high nuclear grade (P < .045). These findings may have implications for the use of melatonin in breast cancer therapy.

Basal Activation of Transcription Factor Signal Transducer and Activator of Transcription (Stat5) in Nonpregnant Mouse and Human Breast Epithelium

Transcription factor Stat5 (signal transducer and activator of transcription) is essential for PRL-induced terminal differentiation of mouse mammary epithelial cells during pregnancy and lactation and has been implicated in mammary tumorigenesis. A new and sensitive immunological method to detect active, tyrosine phosphorylated Stat5 in situ revealed that Stat5 is continuously activated in luminal epithelial cells of mouse and human breast, not only during pregnancy and lactation, but also outside of pregnancy. Examination of virgin Stat5a or Stat5b null mice suggested that Stat5a was the primary isoform activated in mammary epithelial cells. Basal activation of Stat5 in mammary epithelium of virgin wild-type mice was continuous throughout estrous cycle and was also detected in 17 of 17 normal human breast tissue specimens analyzed. PRL was identified as the principal factor maintaining basal activation of Stat5 in mammary epithelium of nonpregnant mice based on several lines of evidence. First, administration of PRL, but not GH or epidermal growth factor, uniformly enhanced basal activation of Stat5 in luminal mammary epithelial cells. Second, hypophysectomy disrupted basal activation of Stat5, an effect that was completely reversed by administration of PRL, but only partially by GH. Third, knock-out of the PRL receptor gene markedly reduced basal activation of Stat5, an effect that was maintained in a normalized endocrine environment after transplanting PRL receptor null mammary epithelium into wild-type mice. Continuous activation of Stat5 indicates a role of this transcription factor in normal, nonpregnant breast epithelial cells, and may shed new light on Stat5 involvement in breast tumor promotion.

Basal Activation of Transcription Factor Signal Transducer and Activator of Transcription (Stat5) in Nonpregnant Mouse and Human Breast Epithelium

Transcription factor Stat5 (signal transducer and activator of transcription) is essential for PRL-induced terminal differentiation of mouse mammary epithelial cells during pregnancy and lactation and has been implicated in mammary tumorigenesis. A new and sensitive immunological method to detect active, tyrosine phosphorylated Stat5 in situ revealed that Stat5 is continuously activated in luminal epithelial cells of mouse and human breast, not only during pregnancy and lactation, but also outside of pregnancy. Examination of virgin Stat5a or Stat5b null mice suggested that Stat5a was the primary isoform activated in mammary epithelial cells. Basal activation of Stat5 in mammary epithelium of virgin wild-type mice was continuous throughout estrous cycle and was also detected in 17 of 17 normal human breast tissue specimens analyzed. PRL was identified as the principal factor maintaining basal activation of Stat5 in mammary epithelium of nonpregnant mice based on several lines of evidence. First, administration of PRL, but not GH or epidermal growth factor, uniformly enhanced basal activation of Stat5 in luminal mammary epithelial cells. Second, hypophysectomy disrupted basal activation of Stat5, an effect that was completely reversed by administration of PRL, but only partially by GH. Third, knock-out of the PRL receptor gene markedly reduced basal activation of Stat5, an effect that was maintained in a normalized endocrine environment after transplanting PRL receptor null mammary epithelium into wild-type mice. Continuous activation of Stat5 indicates a role of this transcription factor in normal, nonpregnant breast epithelial cells, and may shed new light on Stat5 involvement in breast tumor promotion.

Human mammaglobin RT-PCR assay for detection of occult breast cancer cells in hematopoietic products

BackgroundThe aim of this study was: (i) to evaluate the sensitivity and specificity of mammaglobin as a reverse transcriptase polymerase chain reaction (RT-PCR) marker of breast cancer cells; (ii) to determine the incidence of tumor cell contamination of hematopoietic samples from patients with breast cancer. Materials and methodsA nested RT-PCR assay for mammaglobin was developed. Sensitivity was determined by serial dilution assays with breast cancer cell lines, human breast cancers and normal breast tissue. Specificity was evaluated in hematopoietic samples from healthy volunteers and patients with hematological malignancies or solid tumors other than breast cancer. ResultsThe mammaglobin transcript was detected in all 15 breast cancers, one benign breast tumor and five normal breast tissues studied, as well as in three breast cancer cell lines, in dilutions as low as 10–8. The transcript was not detected in any of 47 peripheral blood samples, 15 bone marrow aspirates and 28 peripheral blood progenitor cell samples from the three control populations. Mammaglobin mRNA was detected in 19 of 78 peripheral blood samples from patients with breast cancer starting systemic chemotherapy, as well as in five of 30 repeat samples collected before the fourth cycle of treatment. The transcript was also present in six of seven bone marrow aspirates from patients with metastatic disease, two of five with loco-regional disease, but not in the aspirate of two patients with thrombocytopenia and a previous history of breast cancer. ConclusionsHuman mammaglobin mRNA is a sensitive and specific marker of breast cancer cells and should be further studied as a molecular marker of tumor cell contamination of hematopoietic tissues.

Transcription factor EGR-1 inhibits growth of hepatocellular carcinoma and esophageal carcinoma cell lines.

The transcription factor EGR-1 (early growth response gene-1) plays an important role in cell growth, differentiation and development. It has identified that EGR-1 has significant transformation suppression activity in some neoplasms, such as fibrosarcoma, breast carcinoma. This experiment was designed to investigate the role of egr-1 in the cancerous process of hepatocellular carcinoma (HCC) and esophageal carcinoma (EC), and then to appraise the effects of EGR-1 on the growth of these tumor cells. Firstly, the transcription and expression of egr-1 in HCC and EC, paracancerous tissues and their normal counterpart parts were detected by in situ hybridization and immunohistochemistry, with normal human breast and mouse brain tissues as positive controls. Egr-1 gene was then transfected into HCC (HHCC, SMMC7721) and EC (ECa109) cell lines in which no egr-1 transcription and expression were present. The cell growth speed, FCM cell cycle, plate clone formation and tumorigenicity in nude mice were observed and the controls were the cell lines transfected with vector only. Little or no egr-1 transcription and expression were detected in HCC, EC and normal liver tissues. The expression of egr-1 were found higher in hepatocellular paracancerous tissue (transcription level P=0.000; expression level P=0.143, probably because fewer in number of cases) and dysplastic tissue of esophageal cancer (transcription level P=0.000; expression level P=0.001). The growth rate of egr-1-transfected HHCC (HCC cell line) cells and ECa109 (EC cell line) cells was much slower than that of the controls. The proportion of S phase cell, clone formation and tumorigenicity were significantly lower than these of the controls' (decreased 45.5% in HHCC cells and 34.1% in ECa109 cells; 46.6% and 41.8%; 80.4% and 72.6% respectively). There were no obvious differences between SMMC7721 (HCC) egr-1-transfected cells and the controls with regard to the above items. The decreased expression of egr-1 might play a role in the dysregulation of normal growth in the cancerous process of HCC and EC. Egr-1 gene of transfected HHCC and ECa109 cells showed obvious suppression of the cell growth and malignant phenotypes, but no suppression in SMMC7721 (HCC cell line) cells.

The expression of cytochrome P450 enzymes in human breast tumours and normal breast tissue.

Environmental chemicals are one of the risk factors in breast cancer genesis. Cytochrome P450 (CYP) enzymes play a major role in the activation of these chemicals. Using highly specific and sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. the expression profile of all major xenobiotic metabolizing CYP forms was screened in breast tumour and surrounding tumour free (control) breast tissue in a series of 20 sample pairs obtained from females with infiltrating ductal carcinoma. The levels of CYPIAI mRNA were very low in both tumour and normal tissue. CYP1B1, CYP2B6, CYP2C, CYP2D6, CYP2E1, CYP4B1, and CYP11A1 expressions were positive in both tumours and control tissue. CYP2A6, CYP2A7, CYP2A13, CYP2F1, CYP3A4, CYP3A5. and CYP3A7 mRNAs were expressed neither in tumours nor in control tissue. These results show that several CYPs. responsible for the activation of a quite large number of procarcinogens and genotoxic estrogen metabolites. are expressed in breast tissue with a lack of qualitative differences in CYP expression at mRNA level between breast tumours and surrounding normal breast.

Functional Analysis of Csk and CHK Kinases in Breast Cancer Cells

In this report, we analyzed the expression and kinase activities of Csk and CHK kinases in normal breast tissues and breast tumors and their involvement in HRG-mediated signaling in breast cancer cells. Csk expression and kinase activity were abundant in normal human breast tissues, breast carcinomas, and breast cancer cell lines, whereas CHK expression was negative in normal breast tissues and low in some breast tumors and in the MCF-7 breast cancer cell line. CHK kinase activity was not detected in human breast carcinoma tissues (12 of 12) or in the MCF-7 breast cancer cell line (due to the low level of CHK protein expression), but was significantly induced upon heregulin (HRG) stimulation. We have previously shown that CHK associates with the ErbB-2/neu receptor upon HRG stimulation via its SH2 domain and that it down-regulates the ErbB-2/neu-activated Src kinases. Our new findings demonstrate that Csk has no effect on ErbB-2/neu-activated Src kinases upon HRG treatment and that its kinase activity is not modulated by HRG. CHK significantly inhibited in vitro cell growth, transformation, and invasion induced upon HRG stimulation. In addition, tumor growth of wt CHK-transfected MCF-7 cells was significantly inhibited in nude mice. Furthermore, CHK down-regulated c-Src and Lyn protein expression and kinase activity, and the entry into mitosis was delayed in the wt CHK-transfected MCF-7 cells upon HRG treatment. These results indicate that CHK, but not Csk, is involved in HRG-mediated signaling pathways, down-regulates ErbB-2/neu-activated Src kinases, and inhibits invasion and transformation of breast cancer cells upon HRG stimulation. These findings strongly suggest that CHK is a novel negative growth regulator of HRG-mediated ErbB-2/neu and Src family kinase signaling pathways in breast cancer cells.

Ovarian hormone receptors in human mammary stromal cells

Mesenchymal cells of the rodent breast express both estrogen and progesterone receptors. Searches for these molecules in the human breast have yielded conflicting results. Following immunohistochemical staining of samples of normal human breast tissue, the authors detected estrogen receptor α protein and progesterone receptor protein in extralobular (non-specialized) fibroblasts and estrogen receptor α protein in adipocytes. Tissues from young teenage girls and pregnant women contained the greatest number of receptor positive fibroblasts. These observations confirm prior reports of the presence of ovarian hormone receptors in mammary fibroblasts. The findings also illustrate similarities in the organization of the rodent and human breasts and thereby suggest that regulation of the gland by ovarian hormones involves similar mechanisms in both species.

P1, a high mobility group-like protein is depressed in human breast adenocarcinoma

Protein P1, which is a nuclear protein resembling high mobility group proteins, has been studied in human breast adenocarcinomas and compared to those from control tissue. The presence of the protein was confirmed by in vitro phosphorylation by casein kinase II and immunoblotting, using antibodies raised in rabbits against rat liver P1. The protein has been isolated by reverse phase HPLC chromatography which provides a more rapid method of purification requiring smaller amounts of material. The levels of P1 expression were investigated and it was found that there was a three-fold increase in the ratio of P1/histone H1 in normal breast tissue as compared to the neoplastic tissue. In two other malignant and non-malignant tissues studied, the level of P1 was also decreased in the malignant tissues. Thermolytic phosphopeptides of P1 from normal and malignant human breast tissues exhibited the same pattern, though when compared to the phosphopeptide pattern from rat tissue, differences were observed.

Monoclonal antibody identifies a distinctive epitope expressed by human multiple myeloma cells.

Employing a technology called differential immunization for antigen and antibody discovery (DIAAD), we aimed to generate monoclonal antibodies (mAbs) specific to human multiple myeloma (MM) cells. The fundamental principles of DIAAD rely on the induction of high zone tolerance to the "wild type" (normal) antigen. followed by immunization with the modified (diseased) antigen. Because chronic myelogenic leukemia (CML) cells are derived from a lineage closely related to MM, we immunized mice by contrasting a pool of MM cells with CML cells. Monoclonal antibody VAC69 reacted exclusively with MM cells, identifying a membrane molecule composed of a single-chain glycoprotein with a molecular weight of 78-120 kd. This antigen exhibited narrow tissue specificity and was not found on human cancers such as prostate, breast, or cervical carcinoma; leukemia; or lymphoma, nor was it seen on normal human peripheral lymphocytes or on Epstein-Barr virus-transformed B-cell lines. By immunohistochemistry, mAb VAC69 showed no binding to antigens expressed on normal human ovary, breast, prostate, lung or colon tissue, nor did it bind to human breast or prostate cancer. Conversely, mAb VAC69 bound strongly to human MM, although showing only slight binding to histiocytes or inflamed cells in human lymph nodes and human tumors of the colon, lung, and ovary. Monoclonal antibody VAC69 also triggered cancer-specific cytotoxicity in vitro (in the presence of complement) as well as in vivo using a sever combined immunodeficiency model transplanted with human MM. Further studies showed the ability of mAb VAC69 to be specifically internalized by human MM cells, indicating its potential use for therapeutic intervention in MM by delivering drugs into cancer cells.

Characterization of bipotent mammary epithelial progenitor cells in normal adult human breast tissue.

The purpose of the present study was to characterize primitive epithelial progenitor populations present in adult normal human mammary tissue using a combination of flow cytometry and in vitro colony assay procedures. Three types of human breast epithelial cell (HBEC) progenitors were identified: luminal-restricted, myoepithelial-restricted and bipotent progenitors. The first type expressed epithelial cell adhesion molecule (EpCAM), alpha6 integrin and MUC1 and generated colonies composed exclusively of cells positive for the luminal-associated markers keratin 8/18, keratin 19, EpCAM and MUC1. Bipotent progenitors produced colonies containing a central core of cells expressing luminal markers surrounded by keratin 14+ myoepithelial-like cells. Single cell cultures confirmed the bipotentiality of these progenitors. Their high expression of alpha6 integrin and low expression of MUC1 suggests a basal position of these cells in the mammary epithelium in vivo. Serial passage in vitro of an enriched population of bipotent progenitors demonstrated that only myoepithelial-restricted progenitors could be readily generated under the culture conditions used. These results support a hierarchical branching model of HBEC progenitor differentiation from a primitive uncommitted cell to luminal- and myoepithelial-restricted progenitors.

Depolarization of autofluorescence from malignant and normal human breast tissues

We report on steady-state measurements on the anisotropy of autofluorescence from malignant and normal breast tissue as a function of tissue thickness. For thin tissue sections the anisotropy from normal tissue was found to be smaller compared with that from malignant tissue. However, the opposite result was obtained for thicker tissues. A phenomenological model was also developed to simulate the dependence of anisotropy on tissue thickness.

UV reflectance spectroscopy probes DNA and protein changes in human breast tissues.

The absorption spectrum obtained using diffuse reflectance measurements of malignant, fibroadenoma, and normal human breast tissues were studied. The spectral features in the spectrum were assigned to molecular components in the tissues. Over the past decade, the methods of fluorescence, excitation, and Raman spectroscopy have been studied as potential noninvasive diagnostic tools. Useful spectroscopic information may be obtained from absorption spectra of tissues as well. However, direct measurement of absorption spectra of tissues by conventional transmission means is complicated by multiple photon scattering in tissues. Diffuse reflectance spectrum offers an indirect way to obtain absorption spectrum. Excised malignant, fibroadenoma, and normal breast tissue samples without any treatment were obtained from pathology. Samples were placed in a quartz cuvette. The diffuse reflectance measurements between 250 nm to 650 nm were performed using an automated dual lamp spectrophotometer. The absorption spectra of breast tissues were obtained from the diffuse reflectance measurement. Twenty-one invasive carcinoma, 20 mixed in situ and invasive carcinoma, 14 fibroadenoma, and 39 normal breast tissue samples were studied. The absorption spectra of breast tissues were obtained from diffuse reflectance spectra. Spectral features were assigned to DNA and proteins in human breast tissue. Amplitude of changes averaged over 275 nm to 285 nm and 255 nm to 265 nm and were found to be different for malignant, fibroadenoma, and normal breast tissues. These changes arise from differences in content of protein and DNA. The peaks of absorption spectrum derived from diffuse reflectance measurements in the UV region revealed fingerprints from proteins and DNA components. The absorbance in the wavelength ranges of 275-285 nm and 255-265 nm were found to be different for malignant, fibroadenoma, and normal breast tissues. These differences provide a criterion to distinguish malignant from fibroadenoma and normal breast tissues.

Measurement of optical transport properties of normal and malignant human breast tissue

We report measurement of optical transport parameters of normal and malignant (ductal carcinoma) human breast tissue. A spatially resolved steady-state diffuse reflectance technique was used for measurement of the reduced scattering coefficient (mu(s)?) and the absorption coefficient (mu(a)) of the tissue. The anisotropy parameter of scattering (g) was estimated by goniophotometric measurements of the scattering phase function. The values of mu(s)? and mu(a) for malignant breast tissue were observed to be larger than those for normal breast tissue over the wavelength region investigated (450-650 nm). Further, by using both the diffuse reflectance and the goniophotometric measurements, we estimated the Mie equivalent average radius of tissue scatterers to be larger in malignant tissue than in normal tissue.

Technical aspects of microdialysis of human breast

In this study a technique for insertion of microdialysis catheters and the influence of the position of the catheters within normal human breast tissue were evaluated by measuring amino acids. Moreover, to assess variability over time, the levels of amino acids were measured during a period of 3 h. In nine healthy women two parallel microdialysis catheters were implanted, guided by a catheter for intravenous use, into the breast tissue. All insertions were successful and there were no complications. The levels of amino acids were equal in the two parallel catheters and varied less than 10% over a period of 3 h. Insertion of the microdialysis catheter via an intravenous catheter is suitable for the dense breast tissue. The position of the microdialysis catheter within the same breast seems to be of minor importance for measurements of amino acids. Thus, the described technique is a safe and reproducible way of investigating the human breast in vivo.

Relaxin‐like factor (RLF) is differentially expressed in the normal and neoplastic human mammary gland

BACKGROUND Expression of relaxin-like factor (RLF), a member of the relaxin family, was studied in normal, benign, and malignant neoplastic human breast tissue. METHODS Reverse transcription polymerase chain reaction (RT-PCR) and nonradioactive in situ hybridization were employed to detect RLF transcripts. RLF epitopes were detected with a rabbit polyclonal antiserum generated against the putative receptor binding domain of human RLF. The RLF antiserum was characterized by Western blot analysis on human testicular and placental tissues, recombinant glutathione S-transferase-RLF fusion protein, and baculovirus-derived recombinant marmoset-RLF and marmoset-relaxin. RESULTS RT-PCR analysis revealed RLF amplicons in a cDNA library of normal human breast tissue and in malignant neoplastic breast tissue. RLF hybridization signals were localized exclusively in the tubuloalveolar and ductal breast epithelium but were absent in stromal cells. Benign breast disease displayed weaker RLF hybridization signals compared with normal tubuloalveolar breast tissue. Malignant transformation of breast epithelial tissues resulted in down-regulation of RLF gene expression. The weakest expression of RLF mRNA was observed in lymph node metastases of corresponding primary ductal carcinomas. Immunoreactive RLF was exclusively expressed in breast epithelial cells. Despite strong RLF hybridization signals, the tubuloalveolar epithelial cells of normal breast tissue displayed only very weak immunoreactive RLF. Benign breast disease showed clearly detectable levels of both RLF mRNA and immunoreactive protein. In contrast, epithelial cells in breast carcinoma and lymph node metastases displayed strong expression of immunoreactive RLF, although expression of RLF transcripts was weak. CONCLUSIONS Results demonstrated that transcriptional and posttranscriptional mechanisms affected human RLF gene expression in normal and neoplastic epithelial breast cells. Cancer 2000;89:2161–8. © 2000 American Cancer Society.

Tenascin—A Marker for the Malignant Potential of Preinvasive Breast Cancers

Objective. Up to now, the mechanisms responsible for progression from noninvasive to invasive breast cancer have remained obscure. Tenascin is an extracellular matrix glycoprotein, present in embryonal and fetal tissues, which is also found in the stroma of various benign and malignant pathologies. We studied the expression and immunohistochemical behavior of tenascin in specimens of invasive and preinvasive breast cancer in order to assess its potential role as a marker for tumor invasion.Materials and methods. Sixty-eight specimens including 29 noninvasive ductal cancers, 12 invasive ductal cancers, 5 adenoses, 7 fibroadenomas, and 15 samples of normal human breast tissue were evaluated. An immunofluorescent microscopic technique was used for analysis of the localization and distribution of tenascin. Paraffin-embedded biopsies were incubated with primary monoclonal anti-tenascin antibodies (1:25, Dako-tenascin, TN2). Subsequently, trimethylrhodamine-isothiocyanate-conjugated secondary antibodies (rabbit anti-mouse immunglobulins (Dakopatts, Denmark) were added to visualize the protein.Results. A significant tenascin expression was observed around the ducts in all samples of patients with preinvasive breast cancers. Intensive staining was also found in the periductal stroma of all specimens of patients with invasive breast cancers. Benign breast lesions showed weaker reactivity. No tenascin expression was detectable in normal human breasts, while tissue samples of in situ cancers presented variable staining intensities positively correlating with the degree of differentiation.Conclusion. Tenascin immunofluorescence may prove a suitable and helpful adjunct for diagnosing malignant disease and for predicting the invasive potential of premalignant breast lesions.

Differences in oestrogen receptor α variant messenger RNAs between normal human breast tissue and primary breast carcinomas

The presence of high levels of human oestrogen receptor alpha (hERα) is an important predictor of a favourable response of breast cancer patients to tamoxifen therapy. However, approximately 40% of all hERα-positive tumours do not respond to tamoxifen therapy. hERα variant mRNAs have been described extensively for breast cancer patients and are hypothesised to contribute to tamoxifen resistance. However, some studies have shown that the presence of hERα variants is not limited to malignant breast tissue.

P27 KIP1 expression indicates that steroid receptor-positive cells are a non-proliferating, differentiated subpopulation of the normal human breast epithelium

To test the hypothesis that steroid receptor-expressing cells are derived from the proliferative population, we examined expression of the p27 KIP1 inhibitor of cyclin-dependent kinase activity (a differentiation marker) while tracking the fate of proliferating cells in normal human breast tissue implanted into athymic nude mice using tritiated thymidine [ 3H]-dT. We identified a small number of cells that appeared to have divided just once before switching on p27 KIP1 expression. p27 KIP1+ve cells also expressed steroid receptors, but not the Ki67 proliferation-associated antigen. These data support the hypothesis that steroid receptor-expressing cells are a differentiated population within the normal human breast epithelium.

The Syk tyrosine kinase suppresses malignant growth of human breast cancer cells.

Syk is a protein tyrosine kinase that is widely expressed in haematopoietic cells. It is involved in coupling activated immunoreceptors to downstream signalling events that mediate diverse cellular responses including proliferation, differentiation and phagocytosis. Syk expression has been reported in cell lines of epithelial origin, but its function in these cells remains unknown. Here we show that Syk is commonly expressed in normal human breast tissue, benign breast lesions and low-tumorigenic breast cancer cell lines. Syk messenger RNA and protein, however, are low or undetectable in invasive breast carcinoma tissue and cell lines. Transfection of wild-type Syk into a Syk-negative breast cancer cell line markedly inhibited its tumour growth and metastasis formation in athymic mice. Conversely, overexpression of a kinase-deficient Syk in a Syk-positive breast cancer cell line significantly increased its tumour incidence and growth. Suppression of tumour growth by the reintroduction of Syk appeared to be the result of aberrant mitosis and cytokinesis. We propose that Syk is a potent modulator of epithelial cell growth and a potential tumour suppressor in human breast carcinomas.