metallothionein and Breast-Neoplasms

Human metallothioneins are a superfamily of low molecular weight intracellular proteins, whose synthesis can be induced by essential elements (primarily Zn and Cu), toxic elements and chemical agents, and stress-producing conditions. Of the four known isoforms in the human body MT2 is the most common. The expression of metallothioneins is encoded by a multigene family of linked genes and can be influenced by single nucleotide polymorphisms (SNPs) in these genes. To date, 24 SNPs in the MT2A gene have been identified with the incidence of about 1 % in various population groups, and three of them were shown to affect physiological and pathophysiological processes. This review summarises current knowledge about these three SNPs in the MT2A gene and their associations with element concentrations in the body of healthy and diseased persons. The most investigated SNP is rs28366003 (MT2A -5 A/G). Reports associate it with longevity, cancer (breast, prostate, laryngeal, and in paranasal sinuses), and chronic renal disease. The second most investigated SNP, rs10636 (MT2A +838G/C), is associated with breast cancer, cardiovascular disease, and type 2 diabetes. Both are also associated with several metal/metalloid concentrations in the organism. The third SNP, rs1610216 (MT2A -209A/G), has been studied for association with type 2 diabetes, cardiomyopathy, hyperglycaemia, and Zn concentrations. Metallothionein concentrations and MT2A polymorphisms have a potential to be used as biomarkers of metal exposure and clinical markers of a number of chronic diseases. This potential needs to be studied and verified in a large number of well-defined groups of participants (several hundreds and thousands) with a focus on particular physiological or pathological condition and taking into consideration other contributing factors, such as environmental exposure and individual genetic and epigenetic makeup.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Breast Neoplasms; Diabetes Mellitus, Type 2; Female; Genetic Predisposition to Disease; Humans; Male; Metallothionein; Middle Aged; Polymorphism, Single Nucleotide; Trace Elements

Breast cancer is a disease that has plagued many women globally. The rapid rise in the incidence rate has prompted the rigorous search to understand its etiology and find better management strategies for this disease. Metallothionein (MT) belongs to a family of metal-binding proteins where dysregulated expression of this protein has been observed in invasive breast ductal carcinomas. Since its discovery in equine kidney, functions of MT have extended beyond the initial role of heavy metal detoxification to promoting tumorigenesis. MT, which was reported to be highly expressed in many tumors including breast cancers, is known to regulate key processes such as cell proliferation, apoptosis and even chemoresistance. In this patent review, we shall evaluate the roles of 10 functional isoforms of MT in breast neoplasia and discuss the utility of MT isoforms as biomarkers for prognosis. Strategies targeting MT for treatment could provide alternatives to overcome this dreaded disease which has claimed the lives of many women.

Topics: Animals; Antineoplastic Agents; Biomarkers, Tumor; Breast Neoplasms; Drug Delivery Systems; Drug Resistance, Neoplasm; Female; Humans; Metallothionein; Patents as Topic; Prognosis; Protein Isoforms

Zinc(II) ions contribute to a number of biological processes e.g. DNA synthesis, gene expression, enzymatic catalysis, neurotransmission, and apoptosis. Zinc(II) dysregulation, deficiency and over-supply are connected with various diseases, particularly cancer. 98 % of human body zinc(II) is localized in the intracellular compartment, where zinc(II) is bound with low affinity to metallothionein (MT). Zinc transporters ZIP and ZnT maintain transmembrane transport from/to cells or organelles. Imbalance of their regulation is described in cancers, particularly prostate (down-regulated zinc transporters ZIP1, 2, 3 and ZnT-2) and breast, notably its high-risk variant (up-regulated ZIP6, 7, 10). As a result, intracellular and even blood plasma zinc(II) levels are altered. MT protects cells against oxidative stress, because it cooperates with reduced glutathione (GSH). Recent studies indicate elevated serum level of MT in a number of malignancies, among others in breast, and prostate. MT together with zinc(II) affect apoptosis and proliferation, thus together with its antioxidative effects it may affect cancer. To date, only little is known about the influence of zinc(II) and MT on cancer, while these compounds may play an important role in pathogenesis. This review concludes current data regarding the impact of zinc(II) on the pathogenesis of breast and prostate cancers with potential outlines of new, targeted therapy and prevention. Moreover, blood plasma zinc(II) and MT levels and dietary zinc(II) intake are discussed in relation to breast and prostate cancer risk.

Topics: Breast Neoplasms; Carrier Proteins; Female; Humans; Male; Metallothionein; Prostatic Neoplasms; Sulfhydryl Compounds; Zinc

Metallothioneins (MTs) are a family of metal binding proteins that play an important role in maintaining transition metal ion homoeostasis, redox balance in the cell and fundamental cellular processes such as proliferation and apoptosis. In humans, there are 4 groups of MT proteins which are encoded by 10 functional MT isoforms. In breast tissues, MT is primarily expressed in myoepithelial and malignant epithelial cells. Immunohistochemical studies have revealed that 26% to 100% of invasive ductal breast cancers express the MT protein. The MT-1F and MT-2A isoforms have been reported to be associated with higher histological grade in breast cancer, whereas higher MT-1E mRNA expression was found in estrogen receptor-negative tumors compared to their estrogen receptor-positive counterparts. A number of studies have shown that MT expression in breast cancer is associated with poorer prognosis. In addition, metallothionein expression may have a potential role in protecting the breast cancer cell from chemotherapeutic threats to survival.

Topics: Animals; Biomarkers, Tumor; Breast Neoplasms; Humans; Metallothionein; Neoplasm Proteins

Myoepithelial cells participate in the development of mammary glands and have been suggested to play a role in the biology of mammary cancer. Recent studies demonstrated in the cells expression and overexpression of metallothionein (MT): a low molecular weight, cystein-rich protein which participates, i.a., in the multidrug resistance to cvtostatic drugs. The present study was aimed at examining the relation between results of immunocytochemical (ICC) detection of MT expression in myoepithelial cells, present in structures of a ductal mammary carcinoma, and survival of the patients. In sections originating from 43 patients with ductal mammary carcinoma ICC reactions were performed to detect MT and to confirm the presence of myoepithelial cells (using antibodies to smooth muscle actin). Survival of the patients was also determined in the course of 7-year observations. Statistical analysis using the Coxe's model did not detect relations between MT expression intensity, in the myoepithelium on one hand and patient survival on the other (chi2=0.003 p=0.96).

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Female; Humans; Immunohistochemistry; Metallothionein; Middle Aged; Myoepithelioma; Paraffin Embedding; Survival Analysis

Zn2+ dyshomeostasis is an intriguing phenomenon in breast and prostate cancers, with breast cancer cells exhibiting higher intracellular Zn2+ level compared to their corresponding normal epithelial cells, in contrast to the low Zn2+ level in prostate cancer cells. In order to gain molecular insights into the zinc homeostasis of breast and prostate cancer cells, this study profiled the expression of 28 genes, including 14 zinc importer genes (SLC39A1-14) that encode Zrt/Irt-like proteins 1-14 to transport Zn2+ into the cytoplasm, 10 zinc exporter genes (SLC30A1-10) which encode Zn2+ transporters 1-10 to transport Zn2+ out of the cytoplasm, and 4 metallothionein genes (MT1B, MT1F, MT1X, MT2A) in breast (MCF10A, MCF-7, MDA-MB-231) and prostate (RWPE-1, PC3, DU145) cell lines in response to extracellular zinc exposures at a mild cytotoxic dosage and a benign dosage. The RNA samples were prepared at 0 min (T0), 30 min (T30), and 120 min (T120) in a time course with or without zinc exposure, which were used for profiling the baseline and dynamic gene expression. The up-regulation of MT genes was observed across the breast and prostate cancer cell lines. The expression landscape of SLC39A and SLC30A was revealed by the quantitative reverse transcription polymerase chain reaction data of this study, which sheds light on the divergence of intracellular Zn2+ levels for breast and prostate cancer cells. Taken together, the findings are valuable in unraveling the molecular intricacy of zinc homeostasis in breast and prostate cancer cells.

Topics: Breast Neoplasms; Homeostasis; Humans; Male; Metallothionein; Prostate; Prostatic Neoplasms; Zinc

The overexpression of Metallothionein-1 (MT-1) may play an important role in breast cancer; however, few studies have compared MT-1 expression between breast cancer and fibroadenoma. A cross-sectional controlled study was performed in 66 premenopausal women, aged 20-49 years, who had been histologically diagnosed with breast fibroadenoma or breast cancer. The patients were divided into two groups: group A, control (fibroadenoma, n = 36) and group B, study (breast cancer, n = 30). Immunohistochemistry was performed on tissue samples of fibroadenoma and breast cancer patients to evaluate the expression of metallothionein using an anti-MT-1 polyclonal antibody (rabbit polyclonal anti-metallothionein-Catalog Number biorbyt-orb11042) at a dilution of 1:100. The data were analyzed using NOVA (p < 0.05). Microscopic analysis showed a higher concentration of anti-MT-1-stained nuclei in breast cancer tissues than in fibroadenoma tissues. The mean proportion of cells with anti-MT-1-stained nuclei was 26.93% and 9.10%, respectively, in the study and control groups (p < 0.001). Histological grade 3 tumors showed a significantly higher MT-1 expression than hitological grade 1 (p < 0.05), while breast tumors negative for estrogen-, progesterone- and HER2- receptors had a significantly higher MT-1 expression than positive breast tumors positive for these parameters (p < 0.05). MT-1 protein in women of reproductive age was significantly higher in breast cancer than in fibroadenoma in this study. Furthermore, there was higher MT-1 immunoreactivity in more aggressive tumors.

Topics: Adult; Biomarkers, Tumor; Breast; Breast Neoplasms; Case-Control Studies; Cross-Sectional Studies; Female; Fibroadenoma; Humans; Metallothionein; Middle Aged; Young Adult

The present study suggests and describes the application of a delivery system for antisense oligonucleotides against mRNA encoding estrogen receptor proteins α and β. The delivery system is composed of a cationic liposome envelope containing 17β-estradiol (E2) in its structure. Cationic liposomes protect cargo against the extracellular matrix, and E2 can increase its shuttling efficiency into cells. Using MCF-7 cells derived from estrogen receptor-positive ductal carcinoma, treatment with liposomes against ERα was found to decrease MCF-7 proliferation, and importantly the application of both the antisense against ERα and β exhibited an antiproliferative effect expressed as cell viability. Using qRT-PCR, it was shown that MT1A, NF-κB1 and K-ras genes, but not TFF1, were downregulated using E2-based liposomes (evaluated at P=0.05). Further indicators of oxidative stress were employed to assess the effect on treatment efficiency. Glutathione (GSH/GSSG redox ratio), metallothionein (MT) and malondialdehyde (MDA) confirmed a positive effect of antisense therapy resulting in their decreased levels in the MCF-7 cells. Based on these data, we suggest that E2-based liposomes offer sufficient transfer efficiency and moreover, due to the effect on NF-κB1, MT and GSH, tumor cells can be chemosensitized to increase treatment effectiveness.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromatography, High Pressure Liquid; Drug Delivery Systems; Estradiol; Female; Gene Expression Regulation; Genetic Therapy; Glutathione; Humans; Hydrogen-Ion Concentration; Liposomes; Malondialdehyde; MCF-7 Cells; Metallothionein; Microscopy, Phase-Contrast; Oxidation-Reduction; Oxidative Stress; Oxygen; Receptors, Estrogen

Breast cancer patients negative for the nuclear oestrogen receptor α have a particularly poor prognosis. Therefore, the 4T1 cell line (considered as a triple-negative model) was chosen to induce malignancy in mice. The aim of the present study was to assess if zinc ions, provided in excess, may significantly modify the process of mammary oncogenesis. Zn(II) ions were chosen because of their documented antitumour effects. Zn(II) is also known to induce the expression of metallothioneins (MT) and glutathion (GSH). A total dose of zinc sulphate per one gram of mouse weight used in the experiment was 0.15 mg. We studied the expression of MT1, MT2, TP53 and MTF-1 genes and also examined the effect of the tumour on antioxidant capacity. Tumour-free mice had significantly higher expression levels of the studied genes (p<0.003). Significant differences were also revealed in the gene expression between the tissues (p<0.001). The highest expression levels were observed in the liver. As compared to brain, lung and liver, significantly lower concentrations of MT protein were found in the primary tumour; an inverse trend was observed in the concentration of Zinc(II). In non-tumour mice, the amount of hepatic hydrosulphuryl groups significantly increased by the exposure to Zn(II), but the animals with tumour induction showed no similar trend. The primary tumour size of zinc-treated animals was 20% smaller (p=0.002); however, no significant effect on metastasis progression due to the zinc treatment was discovered. In conclusion, Zn(II) itself may mute the growth of primary breast tumours especially at their early stages.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; DNA-Binding Proteins; Female; Humans; Liver; Metallothionein; Mice; Transcription Factor MTF-1; Transcription Factors; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays; Zinc Sulfate

To assess the relation of blood MT-2A expression, serum zinc, copper, Cu/Zn ratio, total antioxidant status (TAS), total oxidant status (TOS) and oxidant status index (OSI) with benign and malignant breast tumors, also, their relation to different clinical stages and grades of breast cancer.. Unrelated 199 female patients with breast tumor and 120 healthy controls were enrolled in this study. Metallothionein-2A (MT-2A) expression was assessed by quantitative real-time polymerase chain reaction (RT-PCR). Serum MT-2A levels were measured by ELISA. Serum copper (Cu) and Zinc (Zn) concentrations were determined by atomic absorption spectrophotometry. Serum TOS and TAS levels were measured colorimetrically.. Our study demonstrated that blood metallothionein-2A mRNA level, serum MT-2A, copper, Cu/Zn ratio, total oxidant status and oxidant status index were significantly increased, while, serum zinc level and total antioxidant status were significantly decreased in patients with breast cancer and benign breast disease as compared to controls and in breast cancer group as compared to the benign one.. Blood metallothionein-2A expression and serum MT-2A levels could be important prognostic indices of less differentiated, more aggressive breast cancer phenotype. Disturbance of copper, zinc and oxidative stress status might contribute to the pathogenesis of breast tumor and could be useful biomarkers for diagnosing and monitoring such disease.

Topics: Analysis of Variance; Antioxidants; Biomarkers, Tumor; Breast Neoplasms; Calorimetry; Copper; Egypt; Female; Gene Expression Regulation, Neoplastic; Humans; Metallothionein; Middle Aged; Neoplasm Grading; Neoplasm Staging; Oxidants; Phenotype; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; Spectrophotometry, Atomic; Zinc

Human metallothioneins (MTs) are important regulators of metal homeostasis and protectors against oxidative damage. Their altered mRNA expression has been correlated with metal toxicity and a variety of cancers. Current immunodetection methods lack the specificity to distinguish all 12 human isoforms. Each, however, can be distinguished by the mass of its acetylated, cysteine-rich, hydrophilic N-terminal tryptic peptides. These properties were exploited to develop a bottom-up MALDI-TOF/TOF-MS-based method for their simultaneous quantitation. Key features included enrichment of N-terminal acetylated peptides by strong cation exchange chromatography, optimization of C18 reversed-phase chromatography, and control of methionine oxidation. Combinations of nine isoforms were identified in seven cell lines and two tissues. Relative quantitation was accomplished by comparing peak intensities of peptides generated from pooled cytosolic proteins alkylated with ¹⁴N- or ¹⁵N-iodoacetamide. Absolute quantitation was achieved using ¹⁵N-iodoacetamide-labeled synthetic peptides as internal standards. The method was applied to the cadmium induction of MTs in human kidney HK-2 epithelial cells expressing recombinant MT-3. Seven isoforms were detected with abundances spanning almost 2 orders of magnitude and inductions up to 12-fold. The protein-to-mRNA ratio for MT-1E was one-tenth that of other MTs, suggesting isoform-specific differences in protein expression efficiency. Differential expression of MT-1G1 and MT-1G2 suggested tissue- and cell-specific alternative splicing for the MT-1G isoform. Protein expression of MT isoforms was also evaluated in human breast epithelial cancer cell lines. Estrogen-receptor-positive cell lines expressed only MT-2 and MT-1X, whereas estrogen-receptor-negative cell lines additionally expressed MT-1E. The combined expression of MT isoforms was 38-fold greater in estrogen-receptor-negative cell lines than in estrogen-receptor-positive cells. These findings demonstrate that individual human MT isoforms can be accurately quantified in cells and tissues at the protein level, complementing and expanding mRNA measurement as a means for evaluating MTs as potential biomarkers for cancers or heavy metal toxicity.

Topics: Alternative Splicing; Breast Neoplasms; Cadmium; Cells, Cultured; Cerebrum; Cysteine; Cytosol; Epithelial Cells; Female; Humans; Kidney; MCF-7 Cells; Metallothionein; Methionine; Organ Specificity; Peptides; Protein Isoforms; Proteomics; Reproducibility of Results; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Metallothioneins (MTs) are a family of metal binding proteins that play an important role in cellular processes such as proliferation and apoptosis. Metallothionein 2A is the most expressed MT isoform in the breast cells. A number of studies have demonstrated increased MT2A expression in various human tumors, including breast cancer. We carried out an association study to examine whether MT2A gene polymorphisms are associated with risk of breast cancer. Information on lifestyle risk factors was collected via a self-administered questionnaire. Genotyping was conducted using polymerase chain reaction-restriction fragment length polymorphism technique. Three single nucleotide polymorphisms (SNP) rs28366003, rs1610216 and rs10636 were genotyped in 534 breast cancer cases and 556 population controls. One SNP in MT2A (rs28366003) showed a positive association with breast cancer. Compared with homozygous common allele carriers, heterozygous for the G variant [odds ratio (OR) = 1.92, 95 % confidence interval (CI):1.28-2.81, p trend <0.01; the OR assuming a dominant model 1.93 (95 % CI: 1.29-2.89, (p dominant) <0.02) after adjustment for age, family history, smoking status, BMI, menarche, parity, menopausal status and use of contraceptive and menopausal hormones] had a significantly increased risk of breast cancer in Polish population, as well as women with haplotypes, including variant allele of rs28366003 SNP (OR = 1.58, CI: 0.41-6.33, p global = 0.03). Our data suggest that the rs28366003 SNP in MT2A is associated with risk of breast cancer in Polish population.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Carcinoma, Ductal; Case-Control Studies; Female; Genetic Association Studies; Genetic Predisposition to Disease; Genotyping Techniques; Humans; Life Style; Metallothionein; Middle Aged; Poland; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Risk Assessment; Risk Factors; Surveys and Questionnaires

This study was designed to evaluate the changes in promoter CpG islands hypermethylation during breast cancer progression from pre-invasive lesions [flat epithelial atypia (FEA), atypical ductal hyperplasia (ADH), and ductal carcinoma in situ (DCIS)] to invasive ductal carcinoma (IDC). We performed MethyLight analysis for the methylation status of 57 promoter CpG island loci in 20 IDCs and their paired normal breast tissues. After selecting 15 CpG island loci showing breast cancer-specific DNA methylation, another set of normal breast tissue (n = 10), ADH/FEA (n = 30), DCIS (n = 35), and IDC (n = 30) of the breast were analyzed for these loci. We found six new methylation markers of breast cancer, namely DLEC1, GRIN2B, HOXA1, MT1G, SFRP4, and TMEFF2, in addition to APC, GSTP1, HOXA10, IGF2, RARB, RASSF1A, RUNX3, SCGB3A1 (HIN-1), and SFRP1. The number of methylated genes increased stepwise from normal breast to ADH/FEA and DCIS, while IDC did not differ from DCIS. Methylation levels and frequencies of APC, DLEC1, HOXA1, and RASSF1A promoter CpG islands were significantly higher in ADH/FEA than in normal breast tissue. GRIN2B, GSTP1, HOXA1, RARB, RUNX3, SFRP1, and TMEFF2 showed higher methylation levels and frequencies in DCIS than in ADH/FEA. DICS and IDC did not differ in the methylation levels or frequencies for most CpG island loci except SFRP1 and HOXA10. Our findings showed that promoter CpG island methylation changed significantly in pre-invasive lesions, and was similar in IDC and DCIS, suggesting that CpG island methylation of tumor-related genes is an early event in breast cancer progression.

Topics: Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; CpG Islands; Disease Progression; DNA Methylation; DNA, Neoplasm; Female; Homeodomain Proteins; Humans; Hyperplasia; Membrane Proteins; Metallothionein; Middle Aged; Neoplasm Proteins; Promoter Regions, Genetic; Proto-Oncogene Proteins; Receptors, N-Methyl-D-Aspartate; Retrospective Studies; Transcription Factors; Tumor Suppressor Proteins

The overexpression of metallothionein-2A (MT-2A) is frequently observed in invasive human breast tumors and has been linked with more aggressive breast cancers. MT-2A overexpression led to the induction of MDA-MB-231 breast cancer cell migratory and invasive abilities. The reduction of MT-2A expression through small interfering RNA (siRNA) targeting MT-2A in invasive MDA-MB-231 cells completely inhibited both cell invasion and migration. In addition, the expression of matrix metalloproteinase-9 (MMP-9) and the transcriptional activity of AP-1 and NF-κB were upregulated by MT-2A overexpression. Collectively, our results provide the first demonstration that MT-2A promotes breast cancer cell invasion by upregulating MMP-9 via AP-1 and NF-κB activation. Furthermore, we found that MT-2A silencing can inhibit breast cancer invasiveness.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; Female; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 9; Metallothionein; Neoplasm Invasiveness; NF-kappa B; RNA, Small Interfering; Transcription Factor AP-1

Human breast tumors accumulate abnormally high levels of zinc (Zn). As a result, numerous studies have implicated Zn hyper-accumulation in the etiology of breast cancer. Zinc accumulation can be cytotoxic, therefore cells have Zn-buffering mechanisms, such as metallothioneins (MT) and vesicular sequestration, which tightly regulate Zn homeostasis. The Zn transporter ZnT2 sequesters Zn into intracellular vesicles and thus can protect cells from Zn cytotoxicity. Herein, we report that malignant breast tumor (T47D) cells do not express MT but have approximately 4-fold greater Zn levels compared with non-malignant breast (MCF-10A) cells. Zinc accumulation coincided with ZnT2 over-expression and increased vesicular Zn pools. In this study, we hypothesized that ZnT2 suppression would eliminate protection from Zn accumulation and result in cytotoxicity in malignant breast tumor cells. Suppression of ZnT2 significantly increased cytoplasmic Zn pools (1.6-fold) as assessed with a Zn-responsive reporter assay containing four metal response elements (4X-MRE) fused to luciferase. Increased cytoplasmic Zn pools activated apoptosis in a caspase-independent manner. We observed significant generation of reactive oxygen species (ROS) (2.3-fold), lysosomal swelling and cathepsin D leakage in ZnT2-attenuated compared with ZnT2-expressing cells. Most importantly, tumor cell viability and tumor formation were significantly decreased (approximately 25%) in ZnT2-attenuated cells compared with ZnT2-expressing cells. Our data indicate that ZnT2 over-expression protects malignant MT-null breast tumor cells from Zn hyper-accumulation by sequestering Zn into intracellular vesicles. Moreover, our results implicate Zn compartmentalizing mechanisms as novel targets for breast cancer therapy.

Topics: Apoptosis; Blotting, Western; Breast; Breast Neoplasms; Caspases; Cation Transport Proteins; Female; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Lysosomes; Metallothionein; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Tumor Cells, Cultured; Zinc

The purpose of the study was to evaluate the clinical significance of the intensity of metallothionein (MT-I/II) expression and its relationship to two different proliferation markers, Ki-67 antigen and minichromosome maintaince 2 protein (MCM-2) in 117 patients with invasive ductal breast carcinoma (IDC). A significantly higher MT-I/II expression was noted in the grade 3 (G3) carcinomas as compared to those of G1 and G2. A positive correlation was observed between the MT-I/II expression and both proliferation markers, Ki-67 (r=0.20, p=0.0343) and MCM-2 (r=0.25, p=0.0065). Also a strong positive correlation was noted between Ki-67 and MCM-2 expression (r=0.52, p<0.0001). No significant correlations were found between the analyzed markers and tumour size, lymph node metastasis, oestrogen expression (ER), progesterone receptor (PR) or human epidermal growth-factor receptor (HER-2) expression. Out of the three studied markers only the high expression of Ki-67 exhibited a negative impact on patient overall and event free survival and was an independent prognostic factor. MT-I/II and MCM-2 protein expression was not correlated with poor patient outcome, although MCM-2 expression correlated (Fisher's exact test) positively with grade of malignancy (p=0.0018) and negatively with ER (p=0.0002) and PR (p=0.0056) expression. To conclude, MT-I/II may play a key role in IDC proliferation, but is not a useful prognostic factor of this disease.

Topics: Breast Neoplasms; Carcinoma, Ductal; Cell Cycle Proteins; Female; Humans; Ki-67 Antigen; Metallothionein; Middle Aged; Minichromosome Maintenance Complex Component 2; Neoplasm Invasiveness; Nuclear Proteins

The presence of a live cell cohabiting within another cell has fascinated scientists for many decades. Far from being a spurious event, many have attempted to uncover the molecular mechanism underlying this phenomenon. In this study, we observed anchorage-dependent MCF-7 cells internalizing neighboring epithelial cells (entosis) after siRNA-mediated silencing of the Metallothionein-2A (MT-2A) gene. MTs belong to a family of low-molecular weight proteins, which bind metal ions endogenously and its over-expression has been reported in a variety of cancers that include breast, prostate, and colon. We provide microscopic evidence at light and ultrastructural levels of the occurrence of entosis after altering MT expression in a subpopulation of MCF-7 breast cancer cells by silencing the MT-2A gene. Our results demonstrate that adheren junctions may play important roles in the formation of cell-in-cell cytostructure after MT-2A gene downregulation and the entotic process does not appear to involve genes associated with autophagy. Interiorized cells often underwent lysosomal degradation within the cytoplasmic body of the engulfing cell. It would appear that a subset of breast cancer cells could die via entosis after MT-2A gene silencing.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Down-Regulation; Entosis; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Metallothionein; Microscopy, Electron, Transmission; RNA, Messenger; RNA, Small Interfering; Transfection; Tumor Stem Cell Assay

Our study aimed at examining significance of metallothionein (MT) expression in ductal breast cancers by determination of a relationship between expression of MT protein (MT-1/2) and selected prognostic factors, including grade of histological differentiation (G), expression of Ki-67 proliferative antigen, expression of estrogen receptors (ER) and progesterone receptors (PgR) and expression of HER-2 receptor. Material for the studies involved 54 samples of invasive ductal breast cancer, manifesting malignancy grades of G1-G3. In paraffin sections of examined tumours immunohistochemical reactions were performed using specific antibodies directed to MT, Ki-67, ER, PgR or HER-2. Intensity of MT-specific immunohistochemical reactions was measured using the semiquantitative IRS scale of Remmele. Intensity of colour reactions targeted at Ki-67, ER, PgR was evaluated scoring proportions of positive cells, while HER-2-specific reactions were evaluated in the scale of 0-3 points. The lowest level of MT expression was detected in breast cancer cases of G1 malignancy grade (G1 vs G3 p=0.020). A positive correlation between MT and Ki-67 antigen expression (r=0.32, p=0.019) was disclosed. Moreover, MT expression exhibited negative correlations with expression of ER (r=-0.35, p=0.008) and PgR (r=-0.27, p=0.046). No relationships could be detected between expression of MT and expression of HER-2 (r=0.12, p=0.37). The obtained results suggest that MT expression might be helpful in prognostic evaluation of ductal breast cancers.

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Female; Humans; Metallothionein; Prognosis

Metallothioneins (MTs) are a group of metal-binding proteins involved in cell proliferation, differentiation and apoptosis. The MT-2A isoform is generally the most abundant isoform among the 10 known functional MT genes. In the present study, we observed that down-regulation of the MT-2A gene in MCF-7 cells via siRNA-mediated silencing inhibited cell growth by inducing cell cycle arrest in G1-phase (G1-arrest) and a marginal increase in cells in sub-G1-phase. Scanning electron microscopic examination of the cells with silenced expression of MT-2A (siMT-2A cells) revealed essentially normal cell morphology with presence of scattered apoptotic cells. To elucidate the underlying molecular mechanism, we examined the expression of cell cycle related genes in MT-2A-silenced cells and found a higher expression of the ataxia telangiectasia mutated (ATM) gene concomitant with a lower expression of the cdc25A gene. These data suggest that MT-2A could plausibly modulate cell cycle progression from G1- to S-phase via the ATM/Chk2/cdc25A pathway.

Topics: Ataxia Telangiectasia Mutated Proteins; Blotting, Western; Breast Neoplasms; cdc25 Phosphatases; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Checkpoint Kinase 2; DNA-Binding Proteins; Down-Regulation; Female; Flow Cytometry; G1 Phase; Gene Expression; Gene Silencing; Humans; Metallothionein; Microscopy, Electron, Scanning; Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; S Phase; Signal Transduction; Tumor Suppressor Proteins

Metallothionein (MT) plays a role in fundamental cellular processes such as proliferation, apoptosis and differentiation. We examined MT expression in women with invasive breast ductal carcinoma who underwent mastectomy/lumpectomy without neo-adjuvant treatment. We showed that MT was over-expressed in 87.9% of breast cancer tissues examined, with the mean percentage of positive cells at 30%. There were two patterns of MT expression: predominantly cytoplasmic in 75.9% and nuclear in 24.1% of MT-positive cases. Higher MT scores were associated with poorer histological grade (p = 0.009) but were independent of age, tumour size and oestrogen receptor status. For patients who were treated with adjuvant chemotherapy (cyclophosphamide/methotrexate/5 fluorouracil- or doxorubicin-based regimes), those with high MT expression had a significantly lower recurrence-free survival (p = 0.048), suggesting a role of MT in predicting disease recurrence. Down-regulation of MT in MCF-7 cells by silencing the MT-2A gene (the most abundantly expressed of the 10 known functional MT isoforms) increased chemosensitivity of the cells to doxorubicin. To examine the mechanisms underlying these clinical data, we used siRNAs to decrease MT-2A mRNA expression and protein expression. In MT down-regulated cells challenged with the IC(50) concentration of doxorubicin, we observed a significant reduction in cell viability. Cell cycle analysis also revealed a corresponding increase in apoptosis in the MT down-regulated cells following doxorubicin exposure, showing that down-regulation of MT increased susceptibility to doxorubicin cytotoxicity. The data suggest that MT could be a potential marker of chemoresistance and a molecular therapeutic target.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents, Hormonal; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Disease-Free Survival; Doxorubicin; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Metallothionein; Middle Aged; Neoplasm Staging; Prognosis; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering

To evaluate the efficacy of the application of various chemotherapy schemes based on the immunohistochemical study of expression patterns of proteins associated with the drug resistance P-glycoprotein (P-gp), glutathione-S-transferase (GST), metallothioneins (MT) of breast cancer (BC) patients with the triple-receptor-negative (RE(-), RP(-), HER-2/neu(-)) cancer.. P-gp, GST and MT expression in BC-biopsy samples from 60 BC patients was evaluated by immunohistochemical analysis. The results of the clinical observations showed that 3-years relapse-free survival rate of the patients of with P-gp, GST and MT-positive tumors treated with taxoter + adriablastin / taxoter + cyclophosphamide (TA/TC), gemcitabine + carboplatin, or TC + bevacizumab was 61.5%, 78.6% and 81.2% respectively, vs 41.2% in the control group with P-gp, GST and MT-negative tumors treated with adriablastin + cyclophosphamide (p<0.05), while overrall suvival rates were 84.4%, 92.6% and 93.8% respectively vs 70.6% in the control group (p<0.05).. The study points on the possibility to elevate the efficiency of polychemotherapy by its individualization based on the expression patterns of P-gp, GST and MT on tumor cells of the patients with the triple-receptor-negative BC.

Topics: Antineoplastic Combined Chemotherapy Protocols; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biomarkers, Tumor; Breast Neoplasms; Drug Resistance, Neoplasm; Female; Gene Expression; Glutathione Transferase; Humans; Immunohistochemistry; Metallothionein; Receptor, ErbB-2; Receptors, Estrogen; Receptors, Progesterone; Survival Analysis

5-Azactydine inhibits cell growth by direct cytotoxic action as well as by inhibition of DNA methyl transferase enzyme. Inhibitors of DNMT have been reported to potentiate the therapeutic activity of cisplatin in vitro. Dose dependent bone marrow toxicity, neurotoxicity and nephrotoxicity are the major side effects of cisplatin, limiting its use as an effective chemotherapeutic agent. The present study was aimed to reduce the nephrotoxic potential of cisplatin without compensating its potency. To best of our knowledge, this is the first report which shows that the combination of 5-azacytidine with cisplatin leads to remarkable reduction in nephrotoxicity, by involving inhibition of cisplatin induced metallothionein expression. 5-Azacytidine treatment with cisplatin leads to maximum reduction in tumor size in DMH induced colon cancer and tumor volume in DMBA induced breast cancer bearing SD rats. This combination regimen prevents phosphorylation and acetylation of histone H3 which may be involved in inhibition of aberrant gene expression in colon tumors. Further, 5-azacytidine potentiated cisplatin induced antitumor activity by involving decreased expression of pAKT, DNMT1 and an increased expression of p38 in colon tumors. Thus, combination of 5-azactydine with cisplatin attenuates the cisplatin induced nephrotoxicity and potentiates the anti-cancer activity which can have profound clinical implications.

Topics: Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents; Azacitidine; Blood Urea Nitrogen; Blotting, Western; Breast Neoplasms; Cell Nucleus; Cisplatin; Colonic Neoplasms; Creatinine; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; Drug Synergism; Drug Therapy, Combination; Female; Histones; Kidney Diseases; Male; Mammary Neoplasms, Experimental; Metallothionein; Neoplasms, Experimental; p38 Mitogen-Activated Protein Kinases; Proliferating Cell Nuclear Antigen; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley

To compare the molecular profile and cell cycle of sensitive and resistant to doxorubicin MCF-7 breast cancer cells upon exposition to conventional or liposome-encapsulated forms of doxorubicin.. capital EM, Cyrilliccapital TE, Cyrilliccapital TE, Cyrillic-test, immunocytochemistry, flow cytometry.. In sensitive MCF-7 cells the exposure to conventional but not liposomal form of doxorubicin decreased metallothionein (MT) expression demonstrating activation of MT-detoxification system. In doxorubicin-resistant cells (MCF-7/Dsmall o, Cyrillicsmall ha, Cyrillic) MT expression drastically decreased. Conventional but not liposomal form of doxorubicin reduced the levels of expression of steroid hormones receptors on MCF-7 sensitive cells. The exposure of MCF-7 cells to conventional form of doxorubicin resulted in the decrease of p53 expression and the increase of Bcl-2 expression. In MCF-7/Dsmall o, Cyrillicsmall ha, Cyrillic cells neither conventional nor liposomal form of doxorubicin altered Bcl-2 expression. The exposure of MCF-7 but not MCF-7/Dsmall o, Cyrillicsmall ha, Cyrillic to doxorubicin in conventional form caused cell cycle arrest in G0/G1. Upon exposure to doxorubicin in liposomal form, cell cycle blockage in G0/G1 phase was observed in both sensitive and resistant sublines.. The differential effects of the conventional and liposomal forms of doxorubicin in sensitive and resistant cells have been demonstrated. Exposure of MCF-7 and MCF-7/Dsmall o, Cyrillicsmall ha, Cyrillic cells to doxorubicin in liposomal form alters the molecular profile and cell distribution over cell cycle phases.

Topics: Antibiotics, Antineoplastic; Biomarkers, Tumor; Breast Neoplasms; Cell Cycle; Doxorubicin; Drug Resistance, Neoplasm; Female; Flow Cytometry; Humans; Immunoenzyme Techniques; Metallothionein; Proto-Oncogene Proteins c-bcl-2; Receptors, Steroid; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Breast carcinoma is the most common malignancy in women. Estrogen is an important growth factor for breast tumor that plays an important role in regulating the proliferation and differentiation of normal and malignant mammary epithelial cells. Nuclear morphometry and metallothioneins (MTs) are indicators of proliferation that have been used as predictors of prognosis in many tumors. The present study aimed to study mean nuclear area (MNA) and MT; estrogen receptor (ER) expression in fibroadenoma (FA), ductal carcinoma in situ (DCIS), and infiltrating ductal carcinoma (IDC) of the breast. Also MNA and MT expression will be correlated with histologic grade and ER status in breast carcinoma. Breast tissues from 18 patients with FA, 10 patients with DCIS, and 40 patients with IDC were used in this study. MNA and MT expression; as proliferation markers; were investigated and correlated with ER status. All cases of FA, 7 out of 10 cases (70%) of DCIS and 23 out of 40 cases (57.5%) of IDC were positive for ER. MNA of cancer cells was significantly larger than that of normal and benign breast tissue. A significant direct correlation was found between MNA and histologic grades. MNA of ER-negative carcinomas was significantly larger than that of ER-positive tumors. In normal and benign breast tissue, myoepithelial cells consistently expressed the MT protein. Four out of 10 DCIS cases (40%) and 24 out of 40 cases of IDC cases (60%) were positively stained for MT. MT positivity was directly correlated with histologic grade of IDC. There was a highly significant inverse correlation between MT and ER overexpression. From this study, it is concluded that in invasive ductal carcinoma of the breast, the large MNA and MT overexpression are correlated with histologic grades and ER negativity. Therefore, large MNA and MT overexpression may be possible important indicators for more aggressive and less differentiated breast carcinoma.

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Cell Nucleus; Female; Fibroadenoma; Humans; Immunohistochemistry; Metallothionein; Receptors, Estrogen

Recent studies have shown that only breast cancer epithelial cells with intact p53 can induce metallothionein (MT) synthesis after exposure to metals. In this study, the potential role of p53 on regulation of MT was investigated. Results demonstrate that zinc and copper increased metal response elements (MREs) activity and MTF-1 expression in p53 positive MN1 and parental MCF7 cells. However, inactivation of p53 by treatment with pifithrin-alpha or the presence of inactive p53 inhibited MRE-dependent reporter gene expression in response to metals. MTF-1 levels remained unchanged after treatment with zinc in cells with nonfunctional p53. The introduction of wild-type p53 in MDD2 cells, containing nonfunctional p53, enhanced the ability of zinc to increase MRE-dependent reporter gene expression. The cellular level of p21Cip1/WAF1 was increased in MDD2 cells after p53 transfection, confirming the presence of active p53. The treatment of MN1 and parental MCF7 with trichostatin A led to a sixfold increase in the MRE activity in response to zinc. On the contrary, MRE activity remained unaltered in MDD2 cells with inactive p53. The above results demonstrate that activation of p53 is an important factor in metal regulation of MT.

Topics: Benzimidazoles; Breast Neoplasms; Carbocyanines; Cations, Divalent; Cell Line, Tumor; Copper; DNA-Binding Proteins; Female; Fluorescent Dyes; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Luciferases; Membrane Potentials; Metallothionein; Metals, Heavy; Mitochondria; Plasmids; Response Elements; Time Factors; Transcription Factor MTF-1; Transcription Factors; Transcription, Genetic; Transfection; Tumor Suppressor Protein p53; Zinc

Previous reports have shown that metallothionein (MT) may modulate p53 activity through zinc exchange. However, little is known on a direct interaction between MT and p53 in cells. The results demonstrate an interaction between MT and p53 can occur in vitro. The complex between MT and p53 was observed in breast cancer epithelial cells with both wild and inactive type of p53. Furthermore, it was shown that wt-p53 was preferentially associated with Apo-MT. Our data suggest that co-expression of MT and p53 and their complex formation in tumor cells may be involved in regulation of apoptosis in these cells.

Topics: Apoptosis; Breast Neoplasms; Epithelial Cells; Female; Humans; Metallothionein; Protein Binding; Tumor Suppressor Protein p53

This laboratory has proposed that the third isoform of the metallothionein gene family (MT-3) might be a biomarker for the development of human bladder cancer. Immunohistochemical staining of MT-3 on archival diagnostic specimens showed that only 2 of 63 (3.17%) benign bladder specimens had even weak reactivity for the MT-3 protein. In contrast, 103 of 107 (96.26%) high-grade urothelial cancers and 17 of 17 (100%) specimens of carcinoma in situ stained positive for the MT-3 protein. For low-grade bladder cancer it was shown that 30 of 48 specimens (62.5%) expressed the MT-3 protein. Using a cell culture model (UROtsa), it was demonstrated that expression of the MT-3 protein was not required for malignant transformation of urothelial cells by either Cd(+2) or As(+3). In contrast, it was shown that the cells transformed by Cd(+2) and As(+3) that did not express the MT-3 gene in cell culture, gained expression of MT-3 when grown as heterotransplants in nude mice. The gain in MT-3 expression when cells were grown as heterotransplants was also shown to occur for the MCF-7, T-47D, Hs 578t, MDA-MB-231 breast cancer, and the PC-3 prostate cancer cell lines. An analysis of MT-3 mRNA and protein expression suggested that a posttranscriptional mechanism was responsible for accumulation of the MT-3 protein. The results provide strong evidence that MT-3 could be a biomarker for the development of high-grade bladder cancer and that the expression of the MT-3 gene is not involved in the in vitro malignant transformation of UROtsa cells by Cd(+2) and As(+3).

Topics: Animals; Arsenic; Biomarkers, Tumor; Breast Neoplasms; Cadmium; Cell Transformation, Neoplastic; Cells, Cultured; Female; Humans; Immunohistochemistry; Male; Metallothionein; Metallothionein 3; Mice; Neoplasm Transplantation; Prostatic Neoplasms; Protein Isoforms; RNA, Messenger; Transplantation, Heterologous; Urinary Bladder Neoplasms

Determination of oestrogen receptor alpha (ER) represents at present the most important predictive factor in breast cancers. Data of ours and of other authors suggest that promising predictive/prognostic factors may also include pS2, metallothionein (MT) and CD24. Present study aimed at determining prognostic and predictive value of immunohistochemical determination of ER, pS2, MT, and CD24 expression in sections originating from 104 patients with breast cancer. An univariate and multivariate analysis was performed. Both univariate and multivariate analyses demonstrated that cytoplasmic-membranous expression of CD24 (CD24c-m) represents a strong unfavourable prognostic factor in the entire group and in most of the subgroups of patients. In several subgroups of the patients also a prognostic value was demonstrated of elevated expression of pS2 and of membranous expression of CD24. Our studies demonstrated that all patients with good prognostic factors (higher ER and pS2 expressions, lower MT expression, CD24c-m negativity) survived total period of observation (103 months). The study documented that cytoplasmic-membranous expression of CD24 represented an extremely strong unfavourable prognostic factor in breast cancer. Examination of the entire panel of the studied proteins permitted to select a group of patients of an exceptionally good prognosis.

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; CD24 Antigen; Cytoplasm; Disease Progression; Disease-Free Survival; Estrogen Receptor alpha; Female; Humans; Immunohistochemistry; Metallothionein; Middle Aged; Multivariate Analysis; Neoplasm Invasiveness; Predictive Value of Tests; Prognosis; Retrospective Studies; Survival Rate; Trefoil Factor-1; Tumor Suppressor Proteins

Breast cancer is the most common cancer in women, with a general upward trend in incidence. Basic and clinical breast cancer research has continued at a rapid pace, in the endeavor to understand the biology of the disease so as to improve management of patients. Besides traditional pathological indicators, expression of molecular markers in breast cancer has also been comprehensively investigated. This paper will focus on the prognostic utility of metallothioneins (MTs), a family of low molecular weight metal binding proteins encoded by at least 10 functional MT genes that are associated with cell proliferation in breast cancer. Evidence that MT is a potential prognostic biomarker for breast cancer is supported by many reports in the literature. Expression of the MT protein has been detected by immunohistochemistry in a significant portion of invasive ductal breast cancers. MT expression has also been well studied in association with traditional clinico-pathological parameters of breast cancers. Generally, higher MT expression in breast cancers is predictive of worse patient outcomes. The relationship of MT isoforms to histological grade, estrogen receptor (ER) status, and prognosis will also be discussed.

Topics: Biomarkers, Tumor; Breast Neoplasms; Immunohistochemistry; Metallothionein; Models, Molecular; Prognosis; RNA, Messenger

The generation of proper immune response in the tumor environment seems to be essential in antitumor defense. RCAS1 expression has been shown to participate in the regulation of immune cytotoxic activity, metallothionein participates in the protection of cells against immune mediated apoptosis. Since MT and RCAS1 expression is observed within healthy tumor environment we aimed to focus on the proteins expression in tumor and its healthy adjacent tissue in invasive ductal breast cancer regarding the immune cells presence and activity.. RCAS1, metallothionein, CD3, CD56, CD4, CD25, CD69, CD68 and CD16 antigens expression was assessed by immunohistochemistry in invasive ductal breast cancer. Tissue samples were obtained from 45 patients and were grouped according to the presence of lymph nodes metastases. Two groups were obtained: with and without lymph nodes metastases.. Significant differences were observed in RCAS1 and metallothionein expression in tumor and significant differences in metallothionein expression in healthy stroma regarding the presence of lymph nodes metastases. The significantly higher RCAS1 expression was noticed in tumor in comparison to stroma in patients with the presence of lymph nodes metastases. No such difference was observed in patients without the metastases. Significantly higher metallothionein expression was identified in tumor than in stroma in both groups of patients, with and without lymph nodes metastases. These changes in RCAS1 and metallothionein expression were significantly related with the changes in the number and activity of immune cells.. RCAS1 and metallothionein expression in breast cancer healthy stroma seems to be essential for the coexistence of cytotoxic immune cells and normal epithelial cells. The loss of the ability to compensate the growing cytotoxic immune response in the environment might participate in the development of tumor spread.

Topics: Analysis of Variance; Antigens, CD; Antigens, Neoplasm; Apoptosis; Breast Neoplasms; Carcinoma, Ductal, Breast; Female; Gene Expression Regulation, Neoplastic; Humans; Immune Tolerance; Immunity, Cellular; Lymphatic Metastasis; Mammary Glands, Human; Metallothionein; Statistics, Nonparametric

Studies have shown, using immunohistochemical staining, that the MT-1 and MT-2 proteins (MT-1/2) are overexpressed in a substantial subset of ductal breast cancers, that overexpression occurs early in the disease process, and that this overexpression is indicative of a poor prognosis. Normal ductal breast epithelium fails to immunostain for the MT-1/2 protein, whereas the myoepithelial cells of the ducts stain intensely. There is no information regarding the expression of the mRNAs for the eight active MT-1 and MT-2 genes in normal breast duct epithelium. Microdissection of normal breast samples was used to obtain total RNA from enriched populations of ductal epithelium and myoepithelium. Analysis by reverse-transcription polymerase chain reaction (RT-PCR) demonstrated that the identity of the MT isoform-specific genes expressed (MT-2A and MT-1X) and their relative levels of expression were similar between the myoepithelial and ductal components. These findings indicate that the ductal and myoepithelial components express similar amounts of MT-2A and MT-1X mRNAs, but that they have distinctly different expression of the MT-1/2 protein. Confluent cultures of MCF-10A breast epithelial cells were exposed to Cd(+2) to test for evidence of post-transcriptional regulation of MT-1/2 protein accumulation in ductal epithelium. It was demonstrated that Cd(+2) elicited only a marginal induction of MT-1E, MT-1X, or MT-2A mRNAs, whereas, there was a marked increase in MT-1/2 protein, reaching levels of 6% of total cell protein under conditions of extended exposure. This study suggests that the mechanism underlying the finding of increased MT-1/2 protein expression in ductal breast cancer may involve, to some degree, the post-transcriptional regulation of MT-1/2 protein expression.

Topics: Breast; Breast Neoplasms; Cadmium; Cell Line, Tumor; Cell Survival; Epithelium; Female; Gene Expression Regulation; Humans; Immunohistochemistry; Metallothionein; Neoplasm Proteins; Paraffin Embedding; Protein Processing, Post-Translational; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

The primary aims of this study were to examine the expression of metallothionein (MT) in 123 primary invasive breast carcinomas and the in situ components of these carcinomas and to assess the association between MT expression and certain socio-demographic and clinico-pathologic characteristics. MT expression was assessed using immunohistochemical procedures and semi-quantified using an immunoreactivity score.. Results showed that 57.7% of the invasive tumors and 43.3% of the in situ carcinomas in the study were MT-positive. Chi-squared analyses showed that MT expression was significantly higher in the tumors of women categorized as being of 'other' race and of women with tumors of high histological grade.. The results of this study suggest that MT is a biomarker of tumor differentiation and aggressiveness and that MT expression may differ by race.

Topics: Adult; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Female; Humans; Immunohistochemistry; Maryland; Metallothionein; Middle Aged; Socioeconomic Factors

Elevated expression of the low molecular weight metallothionein (MT) proteins can be found typically in breast cancer cases with less favourable prognosis. The MT gene has been described to be potentially down-regulated by estrogen receptor alpha. The present study is aimed at examining the predictive value of MT expression for results of tamoxifen treatment in breast cancer in relation to steroid receptor status. Sixty patients with primary invasive ductal breast cancers with post-operative tamoxifen treatment were enrolled in the study. In paraffin sections of the studied tumours immmunohistochemical reactions were performed using antibodies directed against MT, estrogen receptors (ER) and progesterone receptors (PgR). Results of the immunohistochemical reactions and of clinical observations were analysed using multivariate progression analysis based on the Cox proportional hazard model. Elevated MT expression was demonstrated to be typical for cases with documented relapse of the disease (P<0.001) or terminated by death (P=0.03). Decreased ER expression was found to be typical for cases of a higher grade (P=0.02) and cases terminated by death (P=0.006). The multivariate analysis showed that elevated MT expression was characteristic for cases with shorter overall survival time (P=0.04). The data showed that MT carried an independent, and also independent from ER status, unfavourable predictive value as far as results of tamoxifen treatment were concerned.

Topics: Age Factors; Analysis of Variance; Antineoplastic Agents, Hormonal; Biomarkers; Breast Neoplasms; Carcinoma, Ductal, Breast; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Metallothionein; Middle Aged; Tamoxifen

An accumulation of genetic alterations forming the field of cancerization is an important event for the transformation from normal to cancer cell in multistep carcinogenesis. Histopathologically healthy tumor adjacent tissue might be considered as a cancerization field which is typified by genetic changes required for the development of cancer. Metallothionein (MT) is considered to be a protective and anti-apoptotic protein. The aim of our study was to evaluate the MT expression in head and neck squamous cells carcinoma and breast adenocarcinoma and their histologically healthy adjacent tissue.. We have sampled 29 tissue samples in total derived from head and neck cancers and 29 samples of their clear surgical margins, 33 breast adenocarcinomas and 33 clear surgical margins. Antibody recognizing MT-1 was used for immunohistochemical analysis.. MT expression was revealed in 85,7% of head and neck cancers and 94% of breast adenocarcinomas. It was found in all tumor adjacent tissue. MT expression was statistically significantly higher in tumor adjacent tissue than in cancer tissue in cases with the presence of lymph node metastases in both, breast adenocarcinoma and head and neck squamous cell carcinoma. Generally stroma seems to respond to the presence of cancer by the expression of MT, even in tissues which normally do not express MT.. MT might be a normal or protective reaction of healthy adjacent tissue to the presence of tumor.

Topics: Adenocarcinoma; Adult; Aged; Breast Neoplasms; Carcinoma, Squamous Cell; Female; Head and Neck Neoplasms; Humans; Immunohistochemistry; Lymphatic Metastasis; Male; Metallothionein; Middle Aged; Stromal Cells

The metallothioneins (MTs) are a group of proteins which, due to their unique structure, fulfil numerous functions in the cell. They participate in growth, differentiation, and reparative processes, protect cells against free radicals, and are responsible for heavy metal homeostasis. Their involvement has been reported in the multidrug resistance to cytostatic drugs. Numerous reports document MT presence in cells of various tumors, including breast cancer. Augmented expression of MTs has been reported in less differentiated tumors. MT expression used to be linked to higher proliferative activity of tumor cells, shorter survival of the patients, and tamoxifen-resistance. The present study aimed at examining the relation between MT expression and the manifestation of proliferation exponents (Ki67, nucleolar organizers--AgNORs) in cells of ductal breast cancer of G2 grade of malignancy.. Reactions were performed to detect MTs (clone E9), Ki67 (clone MIB-1) (immunocytochemistry), and AgNORs (silver impregnation) in paraffin sections of breast cancers in G2 grade originating from 60 females. Results of the reactions were subjected to statistical analysis using Statistica 98 PL software.. Statistical analysis (Spearman's rank correlation) demonstrated no relationships between the studied markers (p>0.05).. There is no correlation between metallothionein expression and proliferation and between Ki67 and AgNORs in ductal breast cancers of G2 grade of differentiation.

Topics: Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Division; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Metallothionein; Neoplasm Staging

Studies indicate that smoking is significantly related to breast cancer mortality. One hypothesis is that this association is due to the elevated expression of proteins associated with resistance to cancer therapies. The metallothioneins (MTs) are a family of proteins that appear to play a role in conferring resistance to certain cancer therapies. This study was carried out to assess whether smoking was associated with MT expression in breast carcinomas. Archival breast tissues with accompanying clinical and epidemiological information were collected from three different cancer centers for 123 women diagnosed with invasive breast cancer. MT expression was assessed using immunohistochemical procedures and semi-quantified using an immunoreactivity score that was obtained by multiplying the percentage of MT-positive tumor cells by MT staining intensity. The results showed that 27.6% of the women were current smokers. Among women whose tissues were collected at the cancer center where enrolled women had primarily early stage, low grade breast cancer, smokers had increased odds, although not significantly, of having a MT-positive tumor compared to non-smokers, independent of cancer stage. This association was not observed among the women whose tissues were collected from the other cancer centers. These findings suggest that among specific groups of women, smoking at the time of breast cancer diagnosis may be associated with an increase in breast tissue MT.

Topics: Adult; Aged; Breast Neoplasms; Carcinoma; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Metallothionein; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Retrospective Studies; Smoking

The most important immunocytochemical prognostic and predictive factors in cases of breast cancer include estrogen receptor alpha (ER) and progesterone receptor (PgR). The present study aimed at examining the relationship between the manifestation intensity of proliferation markers (Ki-67 and nucleolar organizer regions--AgNORs) on one hand, and expression of ER and PgR on the other in a uniform group of invasive ductal breast cancers of G2 grade. Moreover, the study aimed at examining the relationship between the above mentioned markers and expression of metallothionein (MT). The studies were performed on samples of invasive ductal breast cancers of G2 grade, originating from 60 females. In paraffin sections originating from the studied cases immunocytochemical reactions were performed using monoclonal antibodies to ER, PgR, Ki-67 and MT, and silver staining was conducted to localize AgNORs. The obtained results were subjected to statistical analysis using Statistica software. Results indicate that manifestation of AgNORs does not correlate with any of the studied antigens (ER, PgR, Ki-67, MT) (p>0.05). Moreover, no relationship could be demonstrated between the intensity of MT expression and proliferation markers or steroid receptor status (p>0.05). A negative correlation was shown between the expression of ER and Ki-67 (p=0.0009). The most intense proliferative activity was demonstrated in cases of breast cancer showing PgR expression but no ER expression (p=0.015), while the lowest proliferative activity was detected in breast cancers with expression of both ER and PgR (p<0.05).

Topics: Biomarkers, Tumor; Breast Neoplasms; Cell Proliferation; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Metallothionein; Nucleolus Organizer Region; Receptors, Estrogen; Receptors, Progesterone; Retrospective Studies

Isoflavone, genistein, was shown to have antioxidant and antitumor activities. We have reported the stimulatory effect of genistein on the expression of antioxidant and metal-binding protein, metallothionein IIA (MTIIA), in human intestinal Caco-2 cells. Genistein has been shown to up-regulate the expression of other genes through estrogen response element (ERE) but the ERE sequence is not in the MTIIA promoter. In this paper, we investigated the ERE-independent mechanism that mediates the stimulatory effect of genistein. Genistein enhanced the expression of human MTIIA promoter (up to -426)-containing reporter genes, thus supporting a promoter-specific transcriptional regulation. A shorter MTIIA promoter (-83 to +27) was found to be able to mediate the full reporter gene response to genistein in a dose- and time-dependent fashion. Further deletion and mutation analysis revealed that the GC-rich Sp1 binding sequence was the target of the stimulation. Genistein was known to bind to estrogen receptors (ER). When cells were cotransfected with ER beta, the stimulatory effect of genistein on the reporter gene containing the GC-rich promoter sequence increased further and a similar result was observed for breast cancer MCF-7 cells. Inhibitors of protein kinase A could block the response to genistein but the phosphorylation of Sp1 protein per se was not affected by the genistein treatment. Our observation could help to evaluate the biological significance of genistein, which is used widely as a supplement.

Topics: Antineoplastic Agents; Base Sequence; Binding Sites; Breast Neoplasms; Caco-2 Cells; Copper; Dose-Response Relationship, Drug; Enzyme Inhibitors; Estrogen Receptor alpha; Estrogen Receptor beta; GC Rich Sequence; Gene Expression Regulation; Genistein; Humans; Intestinal Mucosa; Intestines; Metallothionein; Molecular Sequence Data; Mutation; Phosphorylation; Promoter Regions, Genetic; Receptors, Estrogen; Response Elements; Sp1 Transcription Factor; Tumor Cells, Cultured

The third isoform of metallothionein (MT-3) is overexpressed in some breast cancers and its expression is associated with a poor disease outcome. In the PC-3 prostate cancer cell line, MT-3 expression has been shown to inhibit cell growth and increase drug resistance. The goal of the present study was to determine if MT-3 overexpression would influence the growth of human breast cancer cell lines. To determine this, the coding sequence of the MT-3 gene was stably transfected into two estrogen receptor positive (MCF-7 and T-47D) and two estrogen receptor negative cell lines (Hs578T and MDA-MB-231) having no basal expression of MT-3. Cell growth was determined by counting DAPI-stained nuclei, cadmium resistance by the colony formation assay, MT mRNA expression by RT-PCR, and MT protein by immuno-blot. It was demonstrated that MCF-7 and Hs578T cells that overexpress the MT-3 gene were growth inhibited compared to untransfected cells. In contrast, T-47D and MDA-MB-231 cells that overexpress MT-3 were not growth inhibited. Stable transfection of the MT-1E gene had no effect on the growth of any of the four cell lines. It was also demonstrated that the overexpression of both MT-3 and MT-1E only increased the resistance of MCF-7 cells to Cd(+2). In all instances, stable transfection of the MT-3 or MT-1E gene had no effect on the expression of the other MT isoforms. The study shows that MT-3 can influence the growth of some breast cancer cell lines.

Topics: Breast Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Isomerism; Metallothionein; Receptors, Estrogen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transfection; Tumor Cells, Cultured

Metallothionein (MT), a low-molecular weight protein with pleiotropic functions, is believed to play an important role in tumorigenesis. The aim of this study was to compare the expression of functional MT-1 and MT-2 mRNA isoforms in five breast cancer cell lines ranging from noninvasive MCF7 breast cancer cells to highly aggressive MDA-MB-231 breast cancer cells together with breast myoepithelial cells in vitro by conventional semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. The MT-2A isoform was observed to be differentially upregulated in the invasive phenotype. The MT-1E isoform was found to be present in estrogen receptor-negative breast cancer cell lines (MDA-MB-231 and Hs578T) but not detectable in the estrogen receptor-positive cell lines (T47D, MCF7, and ZR75-1 cells). Only the myoepithelial cells exhibited the presence of the MT-1G transcript. Direct sequencing of the RT-PCR products revealed the occurrence of a variant MT-1H isoform with changes in amino acid residues in the protein sequence and notable differences in the predicted secondary protein structure. The observations in this study are relevant to the development of novel approaches to metastatic breast cancer disease, and may herald the search for novel MT mutants and the elucidation of their biological roles.

Topics: Breast Neoplasms; Cell Line; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Metallothionein; Mutation; Neoplasm Metastasis; Protein Isoforms; Protein Structure, Secondary; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Expression of the hormone-related proteins hsp27, pS2, and also of cathepsin D (CD) and metallothionein (MT) was studied by immunohistochemistry and analyzed against clinical data in breast cancer. Archived material of paraffin-embedded breast carcinoma tissues from a cohort of 134 patients with primary invasive breast cancer was used. Hsp27 and pS2 (>10% of tumor cells stained) were found to be expressed in 63.6% and 37.6% of cases, respectively, and were correlated negatively with grading (P=0.006 and 0.01) and positively with estrogen receptors (ER) (P=0.04 and 0.04). pS2 expression was correlated with lymph node status (P=0.02), tumor size (P=0.01), progesterone receptor (PR) content (P=0.02), hsp27 (P=0.015) and bcl-2 protein (P=0.001). An inverse relationship between pS2 expression and the expression of p53 protein (P=0.005) and proliferation-associated index MIB1 (P<0.0001) was noted. Stromal cathepsin D was positively correlated with tumor grade (P=0.01), PCNA (P=0.007), MIB1 (P=0.001) and p53 (P=0.01), and negatively with ER (P=0.04) and bcl-2 (P<0.0001). MT was correlated positively with stromal CD (P=0.007) and inversely with PgR (P=0.04). Univariate analysis showed CD expression to be a positive prognostic factor for survival (P=0.035), with borderline significance, while MT was more strongly positive (P=0.01). However, none of the proteins studied was found to be related to disease outcome in univariate analysis. Our data show that hsp27, pS2 and stromal CD expression may reflect tumor differentiation and the functional status of ER in breast cancer, but stromal CD and tumor MT expression were the only factors found that may be of limited prognostic value.

Topics: Adult; Aged; Aged, 80 and over; Analysis of Variance; Biomarkers, Tumor; Biopsy, Needle; Breast Neoplasms; Carcinoma, Ductal, Breast; Cathepsin D; Chi-Square Distribution; Cohort Studies; Female; Heat-Shock Proteins; Humans; Immunohistochemistry; Metallothionein; Middle Aged; Neoplasm Invasiveness; Neoplasm Proteins; Probability; Prognosis; Proteins; Sensitivity and Specificity; Survival Analysis; Trefoil Factor-1; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

The expression and induction of metallothionein has been associated with protection against oxidative stress and apoptosis. This study examines the effect of tumour suppressor protein p53 on metallothionein expression following CdCl2 treatment in eight human epithelial breast cancer cell lines differing in p53 and oestrogen-receptor status. Cells were treated with 10 microM CdCl2 for 24 h and metallothionein protein levels were measured by cadmium binding assay. MCF7 cells which are p53-positive (p53+) and oestrogen-receptor-positive showed a large induction in metallothionein synthesis by 10.79+/-1.36-fold. Other breast cancer cell lines which are p53-negative (p53-) and oestrogen-receptor-negative or weakly oestrogen-receptor-positive showed a small induction ranging from 1.40+/-0.10 to 3.65+/-0.30-fold. RT-PCR analysis showed an induction of metallothionein mRNA in MCF7 cells by about 1.61+/-0.08-fold, while in HCC1806 cells (p53-, oestrogen-receptor-negative) by 1.11+/-0.13-fold, and in MDA-MB-231 (p53-, oestrogen-receptor-negative) by 1.25+/-0.06-fold. Metallothionein localisation was determined by immunohistochemical staining. Prior to metal treatment, metallothionein was localised mainly in the cytoplasm of MCF7 and MDA-MB-231 cells. After treatment with 10 microM CdCl2 for 24 h, MCF7 cells showed intense nuclear and cytoplasmic staining for metallothionein, while MDA-MB-231 cells showed staining in the cytoplasm with weak nuclear staining. Apoptosis induced by 10-40 microM CdCl2 at time points between 4 and 48 h was examined with TUNEL assay. In MCF7 cells, apoptosis increased with higher concentrations of CdCl2, it peaked at 6-8 h and appeared again at 48 h for all concentrations of CdCl2 tested. In MDA-MB-231 cells, apoptosis remained at low levels for 10-40 microM CdCl2 at all time points. Studies on cadmium uptake showed similar uptake and accumulation of cadmium at 8 and 24 h in all the cell lines. The data demonstrate that treatment of epithelial breast cancer cells with 10 microM CdCl2 for 24 h caused a greater induction of metallothionein protein and mRNA expression in p53+ and oestrogen-receptor-positive cells as compared to p53- and oestrogen-receptor-negative or weakly oestrogen-receptor-positive cells. This effect may be associated with the occurrence of apoptosis and suggests a role for p53 and oestrogen-receptor on the expression and induction of metallothionein in epithelial cells.

Topics: Apoptosis; Breast Neoplasms; Cadmium Chloride; Cell Survival; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; In Situ Nick-End Labeling; Metallothionein; Receptors, Estrogen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Transfection; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Metallothioneins (MTs) belong to a family of cysteine-rich, metal-binding intracellular proteins, which have been linked with cell proliferation. In this study, expression levels of the 8 known MT-1 and MT-2 functional isoforms in human invasive ductal breast cancer specimens were determined by RT-PCR. The expression profiles of the MT protein and MT-2A mRNA were further evaluated in 79 cases of human invasive ductal breast carcinoma by immunohistochemistry and in situ hybridization, and correlated with cancer cell proliferation (determined by Ki-67 nuclear antigen immunolabeling). MT-1A, MT-1E, MT-1F, MT-1G, MT-1H, MT-1X and MT-2A but not MT-1B, were detected in breast cancer tissue samples. The MT-2A mRNA transcript was the highest among all the isoforms detected. A positive correlation was observed between MT-2A mRNA and MT protein expression with Ki-67 labeling (P = 0.0003 and P < 0.0001, respectively) but not with apoptosis (P = 0.1244 and P = 0.8189, respectively). Co-localization of the MT protein and Ki-67 nuclear antigen in breast cancer cells was demonstrated by double immunofluorescence staining. There was also significantly higher MT protein and MT-2A mRNA expression in histological grade 3 tumors than in histological grade 1 and 2 tumors. The finding that MT 2A appears to be the main isoform associated with cell proliferation in invasive ductal breast cancer tissues, may have therapeutic implications.

Topics: Adult; Aged; Breast Neoplasms; Cell Division; Female; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization; In Situ Nick-End Labeling; Metallothionein; Microscopy, Confocal; Middle Aged; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Metallothioneins (MTs), a group of cysteine-rich proteins with a small molecular mass, are known to have metalloregulatory functions. MT gene expression has been demonstrated to be cell type-specific and differentially regulated (possibly related to their germ layer origin and different functional states). In vitro studies suggest that MT-2A, MT-IE, and MT-1F isoforms may be related to breast cancer. In this study, data on MT-2A, MT-1E, MT-1F mRNA analysis via reverse transcription-polymerase chain reaction in invasive ductal breast cancer tissues and their adjacent benign breast tissues from 27 mastectomies are presented. Expression of mRNA in all the three MT isoforms was detected in both cancerous and adjacent benign breast tissues (with MT-2A mRNA expression being the highest). MT-1F expression was significantly higher in benign breast tissues compared with the breast cancers (P=0.017). In situ hybridization confirmed the expression of MT-2A mRNA in the myoepithelial cells of the breast tissues. Immunohistochemical localization of the MT protein revealed that myoepithelial cells consistently expressed the MT protein, while the cancer cells expressed MT with great variation. Based on our immunohistochemical and mRNA analysis, it is likely that the three MT isoforms are specifically expressed in myoepithelial cells of benign breast tissues and cancer cells of the invasive ductal breast cancer tissues. As MT expression occurs in myoepithelial cells and ductal breast cancer cells, our finding supports the proposition that loss of myoepithelial cells in invasive mammary cancers may be compensated in part by changes in the tumor cells, which may subsequently be the basis for studying the role of MT in breast physiology and carcinogenesis. Differential MT-1F expression in breast myoepithelial cells warrants further study.

Topics: Breast; Breast Neoplasms; Carcinoma, Ductal, Breast; Epithelial Cells; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization; Metallothionein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Immunohistochemical expression of metallothioneins (MTs), a group of intracellular metal-binding proteins, is well documented in breast cancer. However, there is a paucity of information on the expression of the different MT isoforms in breast cancer tissues. The dichotomous association of MT overexpression with tumour types and progression led us to examine the role of the MT-1F mRNA isoform in breast cancer. We evaluated MT expression in 48 primary invasive ductal breast cancer tissues by immunohistochemistry, and the corresponding MT-1F mRNA expression via a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. The specificity of the RT-PCR products was confirmed by direct cycle sequencing and restriction enzyme digestion. Immunohistochemical analysis of MT revealed a significantly higher MT expression in histological grade 3 tumours as compared to grade 1 and 2 tumours (p = 0.021). Similarly, MT-1F mRNA expression was found to be significantly higher in grade 3 tumours (p < 0.001). The results suggest that the MT-1F isoform influences histological differentiation in invasive ductal breast cancer. The converse is also true in that the histological grade may determine the level of MT-1F expression in breast cancer.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Carcinoma, Ductal, Breast; DNA, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Isomerism; Metallothionein; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Restriction Mapping; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sequence Analysis, DNA

Metallothioneins (MTs) protect the cell against reactive forms of oxygen, ionizing radiation, pharmacological agents and mutagens. Metallothioneins are also responsible for neoplastic cell resistance to cytostatic drugs. The aim of this study was to determine the MT level in cell fractions as well as to determine whether there is any change in the concentration of these proteins in a neoplastic cell, and in which cell fractions this change takes place. The neoplastic tissue examined was histopathologically ductal carcinoma invasive, and the control tissue was mastopathic tissue with proliferated connective and glandular tissue. The level of MTs was determined using cadmium isotope (109Cd). It was determined that there was an increase in MT level in the neoplastic tissue, the highest level being found in the mitochondrial fraction obtained from the control and mastopathic tissues. The greatest changes in MT concentrations in breast carcinoma were observed in the nuclear and cytosol fractions. In the nuclear fraction in the breast carcinoma tissue, the MT level was almost three times that of the control group.

Topics: Adult; Breast Neoplasms; Cadmium Radioisotopes; Carcinoma, Ductal, Breast; Cell Nucleus; Cytosol; Female; Humans; Metallothionein

The aim of this study was to assess the presence and clinical significance of metallothionein (MT) in primary invasive ductal carcinoma of the breast. We investigated 96 cases of routinely fixed and paraffin-embedded primary breast carcinomas with the immunohistochemical method. Positive staining for MT was observed in 52.1% of specimen. In most cases both nuclear and cytoplasmic staining was seen. A statistically significant association was found between MT-positive staining and nuclear grade (p < 0.01). MT-positive cases had a significantly poorer prognosis when compared with the MT-negative cases (p < 0.01). We concluded that the MT expression may be of a prognostic value for primary invasive ductal carcinoma of the breast and is possibly an indicator of more aggressive and less differentiated carcinoma cells.

Topics: Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Female; Humans; Immunohistochemistry; Metallothionein; Middle Aged; Neoplasm Invasiveness; Prognosis

Metallothioneins (MTs), a group of ubiquitous metalloproteins, comprise isoforms encoded by ten functional genes in humans. Different MT isoforms possibly play different functional roles during development or under various physiological conditions. The MT-1E isoform mRNA has been recently shown to be differentially expressed in oestrogen receptor (OR)-positive and OR-negative breast cancer cell lines. In this study, we evaluated MT-1E mRNA expression via semi-quantitative RT-PCR in 51 primary invasive ductal breast cancer tissues, concurrently with OR-positive and progesterone receptor (PR)-positive MCF7 cells, OR-negative and PR-negative MDA-MB-231 cells and PR-transfected MDA-MB-231 breast cancer cells (ABC28). We demonstrated significantly higher MT-1E mRNA expression in OR-negative compared with OR-positive breast cancer tissues (P = 0.026). MCF7 cells lacked MT-1E mRNA expression, while both OR- and PR-negative MDA-MD-231 cells exhibited a high level of MT-1E mRNA expression. The level of MT-1E mRNA expression in progesterone-treated and -untreated ABC28 cells remained similar as the parental cell line MDA-MB-231-C2 cells. The results suggest that MT-1E may have specific and functional roles in OR-negative invasive ductal breast cancers, possibly mediated via effector genes downstream of the oestrogen receptor, but not through the PR pathway.

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; DNA Restriction Enzymes; Electrophoresis, Agar Gel; Female; Gene Expression Regulation, Neoplastic; Genetic Markers; Humans; Immunohistochemistry; Isomerism; Metallothionein; Receptors, Estrogen; Receptors, Progesterone; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity; Transfection; Tumor Cells, Cultured

Maspin is a recently described member of the serpin family of protease inhibitors that is consistently expressed at high levels in mammary myoepithelial cells. This feature was used in the immunohistochemical evaluation of tubular carcinoma (TC) and radial sclerosing lesion (RSL) of the breast, and compared with other markers of myoepithelial cells. Ten cases of TC and 11 cases of RSL were studied for the expression of maspin, alpha-smooth muscle actin (alpha-SMA), metallothionein (MT), and S-100 protein by immunohistochemistry. Myoepithelial cells stained strongly and diffusely for maspin creating a pattern of an outer continuous ring surrounding the epithelium of tubules of all RSLs. This pattern was absent in all TCs; however, the single-layered epithelium comprising the tubules of two TCs was positive for maspin with a moderate to strong intensity. Myoepithelial cells were not positive for MT in a consistent manner. Benign nonproliferative epithelium stained focally and weakly for maspin in four of 11 cases of RSL and was negative for MT in all 11 cases. Foci of mild to moderate epithelial hyperplasia noted in five of 11 cases of RSL stained diffusely with a weak to moderate intensity for maspin and focally with a strong intensity for MT. alpha-SMA was consistently expressed in myoepithelial cells but also in stromal myofibroblasts and blood vessels, creating a pattern that was less satisfactory than maspin in distinguishing RSL from TC. Immunohistochemical staining for S-100 protein was of no differential diagnostic value. In conclusion, immunohistochemical staining for maspin is diagnostically useful and superior to MT, S-100, and alpha-SMA, in distinguishing RSL from TC. The epithelial immunoreactivity for maspin in two of 10 TCs merits further investigation from a prognostic viewpoint.

Topics: Adenocarcinoma; Biomarkers, Tumor; Breast; Breast Neoplasms; Diagnosis, Differential; Epithelium; Female; Genes, Tumor Suppressor; Humans; Immunohistochemistry; Metallothionein; Proteins; Sclerosis; Serpins

Expression of metallothionein (MT) isoforms by a human breast cancer cell line, PMC42, which retains many characteristics of normal breast epithelial cells and expresses functional estrogen receptors, was examined because it has been proposed that human breast cancer cells which are estrogen receptor positive can be differentiated from those which are estrogen receptor negative, by failure to express MT-1E [J.A. Friedline, S.H. Garrett, S. Somji, J.H. Todd, D. A. Sens, Differential expression of the MT-1E gene in estrogen-receptor positive and -negative breast cancer cell lines, Am. J. Pathol. 152 (1998) 23-27]. Using RT-PCR, PMC42 cells were found to transcribe genes for the MT isoforms IE, IX and 2A but not 1A or 1H. In order to examine which of the expressed isoforms might protect against metal toxicity, the cells were challenged with high concentrations of zinc and copper. Using competitive RT-PCR, cells resistant to 500 microM zinc showed 7+/-2 fold (SD, n=3) increases in expression of MT-1X and 6+/-3 fold increases in expression of MT-2A compared to control cells in normal media. For cells resistant to 250 microM copper the corresponding increases were 37+/-13 and 60+/-20 fold, whilst for control cells treated with 250 microM copper for only 6 h, increases were 10+/-3 and 6+/-3 fold. There was only a low level of expression of MT-1E in untreated cells and but a >120 fold increase in copper- resistant cells. Thus estrogen receptor positive cells cannot, in general, be differentiated from estrogen receptor negative cells by failure to express MT-1E, as suggested by Friedline et al. (1998). Increased expression of MT-1E, as well as MT-1X and MT-2A, protects against metal toxicity in PMC42 breast cancer cells.

Topics: Breast Neoplasms; Copper; DNA Primers; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Electrophoresis, Agar Gel; Gene Deletion; Gene Expression Regulation, Neoplastic; Humans; Metallothionein; Metals; Protein Isoforms; Receptors, Estrogen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Zinc

Cellular drug resistance and increased metastatic potential are the major obstacles in the successful treatment of cancer with chemotherapy. The aim of this study was to investigate whether the immunohistochemical expression of two proteins implicated in drug resistance (P-glycoprotein and metallothionein) and the product of the suppressor gene nm23 could be related to prognosis in breast cancer. Seventy-two patients with palpable or occult breast carcinoma, not treated with chemotherapy or endocrine therapy, were examined. Immunohistochemical methods were used to determine the expression of P-glycoprotein (PG), metallothionein (MT), nm23, as well as the estrogen receptor (ER), the p53 status, and the Ki67 index. The results were correlated with clinical and morphological features. Cytoplasmic and membrane-specific immunostainings of PG were seen exclusively in tumor cells and identified in 14 of 72 cases (19.4%). Only a statistically significant association with metastases, (p = 0.06) and recurrences (p = 0.1) was observed. MT-positive reaction was identified in the cytoplasm of the tumor cells in 47 (65.3%) cases. Statistical significance was associated with metastases (p = 0.07), but not with death or recurrences. Specific immunostaining of nm23 protein was seen only in the cytoplasm of tumor cells. A positive reaction was observed in 55 of 72 (89.3%) cases. Although a significant association between nm23 protein expression and other morphologic and immunohistochemical variables did not exist, we observed a higher morbidity in patients with the MT-positive/nm23-negative tumor phenotype. Univariate analysis for survival selected the following variables: histologic grade (p = 0.001), ER (p = 0.002), mitotic index (p = 0.005), Ki 67 index (p = 0.068), MT (p = 0.046) and PG (p = 0.085). The Cox model provided the following independent variables: histologic grade (p = 0.021) and metallothionein (p = 0.03). These data confirm the prognosis observed in patients with PG or metallothionein expression as well as the independence of these two variables. It also suggests that nm23 is not necessarily involved in the development of an invasive phenotype.

Topics: Adult; Aged; Aged, 80 and over; ATP Binding Cassette Transporter, Subfamily B, Member 1; Breast Neoplasms; Carcinoma; Female; Humans; Immunohistochemistry; Metallothionein; Middle Aged; Monomeric GTP-Binding Proteins; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Prognosis; Survival Analysis; Transcription Factors

The p53 tumor suppressor protein is a transcription factor that binds DNA in a sequence-specific manner through a protein domain stabilized by the coordination of zinc within a tetrahedral cluster of three cysteine residues and one histidine residue. We show that cadmium, a metal that binds thiols with high affinity and substitutes for zinc in the cysteinyl clusters of many proteins, inhibits the binding of recombinant, purified murine p53 to DNA. In human breast cancer MCF7 cells (expressing wild-type p53), exposure to cadmium (5-40 microM) disrupts native (wild-type) p53 conformation, inhibits DNA binding, and down-regulates transcriptional activation of a reporter gene. Cadmium at 10-30 microM impairs the p53 induction in response to DNA-damaging agents such as actinomycin D, methylmethane sulfonate, and hydrogen peroxide. Exposure to cadmium at 20 microM also suppresses the p53-dependent cell cycle arrest in G(1) and G(2)/M phases induced by gamma-irradiation. These observations indicate that cadmium at subtoxic levels impairs p53 function by inducing conformational changes in the wild-type protein. There is evidence that cadmium is carcinogenic to humans, in particular for lung and prostate, and cadmium is known to accumulate in several organs. This inhibition of p53 function could play a role in cadmium carcinogenicity.

Topics: Animals; Biological Transport; Breast Neoplasms; Cadmium Chloride; Cell Compartmentation; Cell Nucleus; DNA Damage; Down-Regulation; Female; G1 Phase; Gamma Rays; Humans; Metallothionein; Mice; Protein Binding; Protein Conformation; Recombinant Proteins; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The goal of this study was to determine which of the 10 functional metallothionein (MT) genes are expressed in four human breast cancer cell lines and whether expression varies among the cell lines. Using reverse transcription polymerase chain reaction (RT-PCR) technology, it was shown that there was no expression of mRNA for the MT-1A, MT-1B, MT-1F, MT-1G, MT-1H, MT-3, and MT-4 genes in any of the four cell lines. All four cell lines were shown to express mRNA for the MT-2A and MT-1X genes. The expression level of mRNA for the MT-2A gene demonstrated modest differences among the cell lines, whereas expression of the MT-1X gene was consistent. In contrast, mRNA for the MT-1E gene was expressed in only two of the four cell lines and expression correlated to the estrogen receptor status of the cell lines. The two estrogen-receptor-positive cell lines showed no mRNA expression for the MT-1E gene. In the two estrogen-receptor-negative cell lines, mRNA expression for the MT-1E gene was elevated with expression levels similar to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. The cellular content of MT protein was also shown to be elevated in the estrogen-receptor-negative cell lines that express MT-1E mRNA. These results suggest a possible relationship between estrogen receptor status and MT-1E gene expression in human breast cancer.

Topics: Breast Neoplasms; Female; Gene Expression; Humans; Metallothionein; Polymerase Chain Reaction; Receptors, Estrogen; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured

The antiapoptotic response and enhanced cellular proliferation observed in neoplastic cells on overexpression of metallothionein (MT) have been well documented. We have investigated the mechanisms associated with this phenomenon by using MT inducers that increased MT transcripts and stimulated growth in MCF-7 cells. A MT antisense phosphorothioate oligonucleotide inhibited growth induction by >50%, suggesting a potential role of MT in mediating the mitogenic effects of these agents. Mobility shift assays using oligonucleotides encompassing the consensus nuclear factor kappaB (NFkappaB) binding site and anti-MT antibody revealed activation and a specific interaction of NFkappaB with MT. Cotransfection experiments using expression and reporter constructs demonstrated that MT caused transactivation of NFkappaB. Gel shift assays using purified proteins showed a specific interaction between MT and the p50 subunit of NFkappaB. These data indicate that MT may be involved in the interaction of NFkappaB with the DNA-binding domain and further suggest a potential role for NFkappaB in mediating the antiapoptotic effects of MT.

Topics: Apoptosis; Binding Sites; Breast Neoplasms; DNA; Estradiol; Female; Gene Expression Regulation, Neoplastic; Humans; Metallothionein; Mitosis; NF-kappa B; Oligonucleotides, Antisense; Transcriptional Activation; Tumor Cells, Cultured; Zinc Sulfate

Topics: Animals; Antibodies; Antibody Specificity; Breast; Breast Neoplasms; Female; Humans; Immunohistochemistry; Male; Metallothionein; Rabbits; Rats

Although the relationship among different biologic markers of breast cancer has been shown to be important in predicting cancer behavior, expression of these markers can be an attribute of the population under study. Breast cancer is the most common malignancy among Egyptian women. We have studied a number of prognostic tumor markers in infiltrating ductal carcinoma in a group of Egyptian women and have correlated our results with traditional histologic parameters of behavior such as tumor nuclear grade and lymph node status. Seventy-five cases of infiltrating ductal breast cancer were evaluated from pathology archives. Formalin-fixed paraffin-embedded sections were immunohistochemically stained for PCNA, p53, c-erB-2, metallothionein, cathepsin-D, and GST-pi using specific antibodies and a standard avidin-biotin method. Most high-grade tumors were associated with higher PCNA expression and p53 abnormality. There was a significant difference between node-negative and node-positive tumors with regard to their metallothionein content; other markers, however, did not differ significantly between node-negative and node-positive tumors. PCNA expression, metallothionein expression, and p53 mutation appear to be markers of aggressive tumor behavior in Egyptian women with breast cancer.

Topics: Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Cathepsin D; Egypt; Female; Histocytochemistry; Humans; Metallothionein; Middle Aged; Prognosis; Proliferating Cell Nuclear Antigen; Receptor, ErbB-2; Tumor Suppressor Protein p53

Immunocytochemically detectable MT and p53 have been found more commonly in comedo DCIS of the breast with high-grade cytology. The aim of this study is to confirm these findings and to investigate the relationship between MT and p53 in a single large series of cases of DCIS of the breast. To this end, 127 cases of DCIS were classified histologically according to architecture, cytonuclear differentiation (grade), presence and extent of intraduct necrosis, and using the Van Nuys system. Sections were immunostained for p53 and MT (E9) using established techniques, and the extent and intensity of staining were assessed semi-quantitively. The results confirmed that there was generally more MT and p53 positivity in poorly differentiated (grade 3) DCIS with extensive necrosis and that MT expression was greater in grade 2 lesions than p53 expression. However, overall there was no statistically significant correlation between p53 and MT staining. The results indicate that MT and p53 overexpression may arise from independent mechanisms in early breast neoplasia.

Topics: Breast Neoplasms; Carcinoma in Situ; Carcinoma, Ductal, Breast; Cell Transformation, Neoplastic; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Metallothionein; Necrosis; Tumor Suppressor Protein p53

The treatment of breast cancer by retinoic acid (RA) may be mediated by lipid peroxidation. Expression of metallothionein (MT) in cancer cells, however, can protect against lipid peroxidation by scavenging hydroxyl radicals. In this study, a two-by-six factorial design was used to investigate the interactive effects of all-trans-RA and zinc (Zn)-induced MT on the growth of two human breast cancer cell lines differing in basal expression of MT and estrogen receptors; MCF7 cells express estrogen receptor, BT-20 cells do not. Cells were treated with Zn to induce MT and then treated with six RA concentrations. Cell proliferation, lipid peroxidation, MT protein, MT mRNA and glutathione concentrations were measured. BT-20 cells expressed higher constitutive MT concentrations than MCF7 cells. MT was significantly increased by Zn treatment in BT-20 cells but not in MCF7 cells. Low RA concentrations stimulated growth proliferation but higher concentrations inhibited cell proliferation. Elevated RA concentrations increased lipid peroxidation as measured by thiobarbituric acid reactive substances. There was a significant negative correlation between lipid peroxidation and cell proliferation. Growth inhibition and lipid peroxidation were reduced by Zn pretreatment in BT-20 cells but not in MCF7 cells. RA increased MT levels in both cell lines, which suggests that RA may generate free radicals which will induce MT mRNA expression. Glutathione did not appear to be a significant factor. Therefore, induction of MT by Zn may modulate the growth inhibitory effects of RA in human breast cancer cells. One mechanism of growth inhibition may be through increased lipid peroxidation. Induction of MT by RA may be one explanation for acquired RA resistance in cancer.

Topics: Breast Neoplasms; Cell Division; Female; Glutathione; Humans; Lipid Peroxidation; Metallothionein; RNA, Messenger; Tretinoin; Tumor Cells, Cultured; Zinc

Sodium butyrate-induced differentiation of breast cancer cell lines was used to identify protein tyrosine phosphatases (PTPs) involved in differentiation and growth inhibition of breast cancer cells. Of 42 PTPs analyzed, 31 were expressed in the ZR75-1 breast cancer cell line. Expression of four PTPs (DEP-1, SAP, PTP gamma, and PAC) was regulated in ZR75-1 cells undergoing differentiation. Expression of two of these PTPs (DEP-1 and SAP) was also regulated in the SKBr-3 cell line undergoing differentiation. In view of its marked induction with differentiation in an estrogen receptor (ER)-positive and an ER-negative breast cancer cell line, DEP-1 was investigated for a role in growth inhibition or induction of differentiation in breast cancer cells. A DEP-1 cDNA construct under control of a constitutively active cytomegalovirus promoter was transfected into the ZR75-1, SKBR-3, and MCF-7 breast cancer cell lines, and resistant colonies were selected with G418. DEP-1 expression inhibited the development of resistant colonies by 3-5-fold in all three lines compared to transfection with vector alone. Three stable MCF-7 cell lines expressing DEP-1 under control of an inducible metallothionein promoter were then established. In these lines, induction of DEP-1 expression inhibited breast cancer cell growth by 5-10-fold. These data describe PTPs expressed and regulated in breast cancer cell lines during differentiation and identify one PTP, DEP-1, that inhibits the growth of breast cancer cells in vitro.

Topics: Blotting, Northern; Breast Neoplasms; Butyrates; Butyric Acid; Cell Differentiation; Cell Division; Cell Line; Cell Membrane; Cytomegalovirus; Enzyme Induction; Female; Gene Expression Regulation, Enzymologic; Humans; Kinetics; Metallothionein; Polymerase Chain Reaction; Promoter Regions, Genetic; Protein Tyrosine Phosphatases; Receptor-Like Protein Tyrosine Phosphatases, Class 3; Recombinant Proteins; Transcription, Genetic; Tumor Cells, Cultured

Metallothioneins are ubiquitous low molecular weight proteins characterised by high cysteine content and affinity for binding heavy metals. Abnormal metallothionein function and expression have been implicated in various disease states, including neoplasia. The aim of this study was to investigate metallothionein expression in human breast carcinoma. Sections of routinely fixed and processed blocks of tumour from 100 consecutive cases of primary operable breast carcinoma were stained for metallothionein using a recently developed monoclonal antibody and a standard immunohistochemical technique. Expression was scored on the basis of microscopical assessment of percentage of tumour cells staining. One patient was lost to follow-up and excluded from the study. A significant association (P < 0.0001) was observed between metallothionein expression and tumour type, with low levels being observed in tumours of good prognostic type. There was also a significant association with local recurrence (P < 0.02) and a significant difference (P < 0.02) in both survival and disease-free interval between tumours showing low and high levels of expression, the latter indicating a poor prognosis. No relationship was observed with patient age, tumour size, lymph node stage, histological grade, vascular invasion, menopausal status or oestrogen receptor status. The assessment of metallothionein expression in human breast cancer appears to provide prognostic information and may have important implications for understanding its development.

Topics: Breast Neoplasms; Female; Humans; Immunohistochemistry; Metallothionein; Receptors, Estrogen

Metallothionein (MT) is a low-molecular-weight protein with a high affinity for group II metal ions, especially zinc and copper. MT serves as an intracellular reservoir of these ions, but may also be involved in the detoxification of certain toxic metal ions such as cadmium. In addition, high MT contents might protect tumour cells from alkylating agents and irradiation. The aim of this study was to evaluate the prognostic significance of immunohistochemically detected MT overexpression in patients with primary breast carcinoma: 478 patients with primary breast carcinoma diagnosed during the period 1980-1985 were included. Formalin-fixed and paraffin-embedded tissue was used. Immunoreactivity for MT was found to be independent of the length of formalin fixation if the sections were microwave processed before incubation with the primary antibody. Patients were divided into two groups: those with MT overexpression (more than 10% positive tumour cells) and those with low expression (less than 10% positive tumour cells). MT overexpression was correlated with postmenopausal status, large tumour size, presence of lymph node metastases, high number of mitoses, severe nuclear pleomorphism, high histological grade (poor differentiation), and absence of PgR. In univariate analysis of survival data, MT overexpression was a predictor of poor overall survival in the entire group of patients. In multivariate analysis, MT overexpression failed to be of prognostic significance, whereas classical histopathological parameters such as tumour size, histological grade, and PgR were of independent prognostic significance.

Topics: Analysis of Variance; Breast Neoplasms; Carcinoma, Ductal, Breast; Female; Humans; Immunohistochemistry; Lymphatic Metastasis; Metallothionein; Mitosis; Postmenopause; Prognosis; Receptors, Progesterone; Survival Rate

In a previous study immunocytochemically detectable metallothionein (MT) expression in tumor cells of invasive duct carcinoma of the breast was shown to be associated with a more aggressive behavior and these findings have been subsequently confirmed by others. The aim of this study was to examine the prevalence and significance of MT positivity in preinvasive duct carcinoma in situ (DCIS). Fifty-five specimens of pure screen-detected DCIS were stained immunocytochemically for MT using the antibody E9. The intensity and distribution of MT staining were assessed using a semiquantitative method resulting in intensity distribution (ID) scores allowing duct by duct analysis in relation to architectural and cytological features of the DCIS. In general, myoepithelial cells around benign and malignant structures stained uniformly strongly for MT. Staining in DCIS was analyzed by architecture irrespective of cytology and by nuclear grade irrespective of architecture. The results showed that MT staining was significantly greater in comedo (ducts with necrosis) DCIS (ID = 97) compared with noncomedo (ducts without necrosis) DCIS (ID = 56) (P = .05 by Mann Whitney U statistic) and that low cytological grade (ID = 50) was associated with less MT staining than was high cytological grade (ID = 92) (P = .05 by Mann Whitney U statistic). These observations thus are consistent with the previously observed association between MT positivity and more aggressive behavior in invasive duct carcinoma of the breast.

Topics: Aged; Breast Neoplasms; Carcinoma in Situ; Carcinoma, Ductal, Breast; Humans; Metallothionein; Middle Aged; Neoplasm Proteins

Breast cancer cell lines have been shown to secrete transforming growth factor alpha (TGF alpha) and other polypeptide growth factors in response to estrogen (E2) stimulation. In this study, we investigated whether cellular-derived TGF alpha mediates the growth-stimulatory effects of E2 in ER-positive breast cancer cells. To test this hypothesis, we introduced an antisense TGF alpha mRNA expression vector under control of a human metallothionein promoter into E2-responsive T47D human breast cancer cells. In stably transfected cells, cadmium induced antisense mRNA and reduced expression of TGF alpha mRNA and protein in antisense clones (AS1). TGF alpha expression was increased in sense clones (S2), while wild-type T47D cells (W3) or pSV2 neomycin resistance-transfected cells showed no change in TGF alpha expression in response to cadmium. The basal proliferative capacity of antisense transfected cells was equivalent to that of the wild-type. E2 increased TGF alpha synthesis and cell proliferation in transfected and wild-type cells. In AS1 cells, the simultaneous addition of cadmium with E2 blocked most of the E2-induced increase in TGF alpha mRNA and protein and nearly abolished the stimulatory effects of E2 on DNA synthesis and cell number. In contrast, no reduction in cell proliferation was observed with cadmium in antisense-transfected cells with a low level of antisense expression or in the S2 or W3 cells. Our results are compatible with the hypothesis that autocrine production of TGF alpha may be an important contributor to E2-induced growth in T47D cells.

Topics: Breast Neoplasms; Cell Division; Cell Line; Culture Media, Conditioned; DNA, Neoplasm; Estradiol; Gene Expression; Genetic Vectors; Humans; Metallothionein; Promoter Regions, Genetic; Receptors, Estrogen; Receptors, Progesterone; RNA, Messenger; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

Metallothioneins (MTs) are low-molecular-weight proteins with specific binding for group II metal ions. MTs are involved in the detoxification of metals, but can also play a role in protection of the cell against certain anticancer agents and from damage of irradiation. High expression of MTs in primary breast carcinomas has been found to be associated with poorer prognosis. Expression of MT (MT) was examined immunohistochemically in 160 breast carcinomas and their concomitant lymph node metastases. The immunoreactivity appeared to be independent of the length of fixation when the section was microwaved before incubation with the primary antibody, a monoclonal antibody E-9. The findings were correlated with various histopathological factors, disease-free survival and over-all survival. Patients were divided into two groups, those with MT over-expression (above 10% of positive tumour cells), and those with low MT expression (below 10% positive). MT over-expression was found to be correlated with postmenopausal status and inversely with positive progesterone receptor status (PgR). MT over-expression showed statistically significant correlation with poor over-all survival. No differences in survival were seen between pre- and postmenopausal patients. PgR was in univariate analysis a poor prognostic parameter. In one fourth of the patients, the lymph node metastases showed increased MT expression compared with the primary tumour. These patients had a poorer, but not statistically significant different survival. MT expression was not correlated to chemo- or radiation therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Breast Neoplasms; Carcinoma; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Disease-Free Survival; Female; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Lymphatic Metastasis; Metallothionein; Neoplasm Proteins; Survival Rate

Metallothioneins (MTs) are a set of low molecular weight proteins with a high binding affinity to metal ions. MT over-expression has been recently demonstrated in invasive ductal carcinoma of the breast with poor clinical prognosis. In the present study, MTs have been immunohistochemically investigated in normal human breast tissue and a variety of benign, pre-invasive, and malignant breast lesions. In normal breast tissue, MTs were present in myoepithelial cells whereas the vast majority of luminal cells were MT negative. In lesions without increased cancer risk (adenosis and scleradenosis), MT was only immunolocalized in myoepithelial cells. In papillomas, MT was also found exclusively in myoepithelial cells. In most cases of epitheliosis, both the luminal and myoepithelial cells expressed MT. Atypical lobular hyperplasia, lobular carcinoma in situ, and 13/15 invasive lobular carcinomas showed no MT over-expression. The two invasive lobular carcinomas with MT over-expression were classified as pleomorphic lobular carcinomas with apocrine differentiation. In contrast to lobular cancerization, 12/24 ductal in situ carcinomas and 9/20 invasive ductal carcinomas showed MT over-expression. In situ components found within invasive ductal carcinomas usually reflected the MT status of their invasive counterpart. It is concluded from our immunohistochemical results that breast carcinoma cases with MT overexpression arise from lesions which also show MT overexpression. Thus MT expression in carcinomas may be regarded as a genuine feature of the tumour cells and seems not to be related to endogenous or exogenous factors known to induce MT synthesis.

Topics: Breast; Breast Neoplasms; Carcinoid Tumor; Carcinoma; Carcinoma, Ductal, Breast; Epithelial Cells; Epithelium; Female; Fibrocystic Breast Disease; Humans; Immunohistochemistry; Metallothionein; Papilloma, Intraductal; Reference Values; Risk Factors

Metallothionein (MT) is a cysteine-rich, low molecular weight protein that binds zinc, copper, and cadmium. It is present in a number of normal cells including hepatocytes particularly during fetal and early postnatal life. It has been suggested that developmental profile of MT is similar to other oncofetal gene products and hence, it could be used as a marker for aggressive tumour behaviour. In order to test that hypothesis, we used a monoclonal antibody to MT and immunohistochemically evaluated formalin-fixed, paraffin-embedded tissues from 79 breast carcinomas. In non-neoplastic breast tissue, a strong nuclear and cytoplasmic staining was observed in myoepithelial cells. Positive staining for MT was present in 35 (44%) of breast carcinomas. In most positive cases, nuclear, or both nuclear and cytoplasmic staining was seen. All positive tumours were invasive ductal carcinomas, including a medullary and a metaplastic carcinoma. None of the mucinous, lobular, or intraductal papillary carcinomas reacted for MT. A statistically significant association was found between MT immunostaining and histological grade (P < 0.01) as well as with nuclear grade (P < 0.01). We also observed an inverse relationship between MT staining and oestrogen receptor content of tumours (P < 0.01). Similarly, a statistically significant association was found between moderate and strong MT immunostaining and decreased overall survival and shorter disease-free survival (P < 0.01). MT immunostaining was also predictive of a worse prognosis in the subgroup of lymph node negative (P < 0.001) and oestrogen receptor negative patients (P < 0.01). No statistically significant association was found between MT staining and size of tumour or the presence of lymph node metastasis. We conclude that MT staining may be a useful marker of less differentiated and more aggressive carcinomas of the breast.

Topics: Breast Neoplasms; Carcinoma; Humans; Immunohistochemistry; Lymphatic Metastasis; Metallothionein; Receptors, Estrogen

The antibody to the metal-binding low molecular weight protein metallothionein (MT) stains preferentially the proliferative edge of epithelial tumors in paraffin sections. The present report demonstrates its usefulness as an epithelial cell marker in DNA flow cytometry of archival specimens. Nine control breast (mammoplasty) specimens, 10 control colonic specimens (resection edges), 12 adenocarcinomas of breast, and 13 adenocarcinomas of colon were analyzed by DNA flow cytometry after MT and DNA staining. The average percentage of cells stained by MT ranged from 12% to 27% in these groups of specimens, which contain epithelial as well as stromal and inflammatory cells. Comparing cell turnover, measured as S-phase fraction (SPF) in unstained and MT-stained preparations, it was 10% and 20%, respectively, in control tissues and 10% and 30%, respectively, in adenocarcinomas. The SPF is lower in unstained preparations because of dilution by noncycling inflammatory and stromal cells. Immunohistochemical staining of various tissues for MT showed specific staining of epithelial cells. Evaluation of aneuploid malignant epithelial cells detected in six breast and eight colonic adenocarcinomas showed that on average, 47% of cells were stained with MT and that their SPF increased by about 50% when MT staining was compared with the unstained preparations. The results suggest that MT stains epithelial cells adequately for ploidy and cell cycle evaluation and that it may stain preferentially the proliferating cell compartment, which is considered to be an index of malignancy.

Topics: Adenocarcinoma; Biomarkers, Tumor; Breast Neoplasms; Cell Compartmentation; Cell Division; Colonic Neoplasms; DNA, Neoplasm; Epithelium; Female; Flow Cytometry; Humans; Metallothionein

Metallothioneins (MTs) are ubiquitous low-molecular-weight proteins with a high affinity for heavy metal ions such as zinc, copper and cadmium. MT over-expression has been associated with resistance against anticancer drugs. In the present study we investigated 86 cases (45 cases of tumour category pT1 and 41 of category pT2) of routinely fixed and paraffin-embedded primary breast carcinomas immunohistochemically with a monoclonal antibody to an epitope of MT shared by its I and II isoforms. Immunohistochemically demonstrated MT over-expression was found in the invasive components of 7 of 32 pT1 and 17 of 28 pT2 invasive ductal carcinomas, whereas all 26 invasive lobular carcinomas gave weak or negative results. Fourteen of 17 pT2 and 2 of 7 pT1 invasive ductal carcinomas with MT over-expression developed metastases during follow-up with poor prognostic outcome. In contrast only 3 of 11 pT2 and none of the 25 pT1 cases without MT over-expression had a poor clinical course (P < 0.001). It is concluded that MT over-expression is associated with significantly poor prognosis particularly in pT2 invasive ductal breast carcinomas.

Topics: Adult; Aged; Antibodies, Monoclonal; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Enzyme-Linked Immunosorbent Assay; Humans; Immunohistochemistry; Metallothionein; Middle Aged; Prognosis; Receptors, Estrogen; Receptors, Progesterone

In addition to retinoblastoma and osteosarcoma, mutation of both alleles of the RB1 gene occurs frequently in several other types of tumors. In order to evaluate the role of RB1 in cancer, the wild type RB1 gene was introduced into the RB1-deleted breast cancer cell line MDA-468-S4 and retinoblastoma cell lines WERI-Rb1 and Y-79. The RB1 complementary DNA was under control of the inducible murine metallothionein promoter in MDA-468-S4 and the thymidine kinase promoter in the retinoblastoma lines. The protein, p110RB1, produced from the exogenously introduced gene appeared normal by immunoprecipitation, Western blot analysis, and nuclear localization and also showed normal cell cycle-dependent phosphorylation and an ability to bind to E1a protein. No changes in growth rate or morphology were observed in either of the reconstituted cell types. Expression of p110RB1 in MDA-468-S4 did not affect anchorage-independent growth when measured by colony formation in soft agar. Although the ability of WERI-Rb1 cells expressing p110RB1 to form colonies in methylcellulose was reduced, the reconstituted retinoblastoma cell lines formed intraocular tumors in immunodeficient mice with the same efficiency as the RB1-negative parent cell lines and the tumors produced by the RB1-reconstituted cells continued to express p110RB1. These experimental results suggest that the malignant phenotype is little affected by the replacement of p110RB1 and that RB1 is a relatively weak tumor suppressor gene.

Topics: Animals; Breast Neoplasms; Cell Cycle; Cell Division; Cell Line; Chromosome Deletion; Eye Neoplasms; Female; Genes, Retinoblastoma; HeLa Cells; Humans; Metallothionein; Phenotype; Plasmids; Promoter Regions, Genetic; Recombinant Fusion Proteins; Restriction Mapping; Retinoblastoma; Thymidine Kinase; Transfection

Topics: Adenocarcinoma; Biomarkers, Tumor; Breast; Breast Neoplasms; Female; Humans; Immunoenzyme Techniques; Metallothionein; Ovarian Neoplasms; Ovary; Receptors, Estrogen; Receptors, Progesterone; Uterine Neoplasms; Uterus

Topics: Breast Neoplasms; Cadmium; Cells, Cultured; Female; Humans; Mercury; Metalloproteins; Metallothionein; Neoplasm Proteins; Protein Binding; Time Factors; Zinc