The use of a novel MUC1 antibody to identify cancer stem cells and circulating MUC1 in mice and patients with pancreatic cancer.

BACKGROUND AND OBJECTIVES: MUC1 is over-expressed and aberrantly glycosylated in >60% of human pancreatic cancer (PC). Development of novel approaches for detection and/or targeting of MUC1 are critically needed and should be able to detect MUC1 on PC cells (including cancer stem cells) and in serum. METHODS: The sensitivity and specificity of the anti-MUC1 antibody, TAB 004, was determined. CSCs were assessed for MUC1 expression using TAB 004-FITC on in vitro PC cell lines, and on lineage(-) cells from in vivo tumors and human samples. Serum was assessed for shed MUC1 via the TAB 004 EIA. RESULTS: In vitro and in vivo, TAB 004 detected MUC1 on >95% of CSCs. Approximately, 80% of CSCs in patients displayed MUC1 expression as detected by TAB 004. Shed MUC1 was detected serum in mice with HPAF-II (MUC1(high) ) but not BxPC3 tumors (MUC1(low)). The TAB 004 EIA was able to accurately detect stage progression in PC patients. CONCLUSIONS: The TAB 004 antibody may be explored as a therapeutic targeting agent for CSCs in PC. The TAB 004 EIA detected circulating MUC1 in a stage-dependent manner in patients with PC and thus may be explored as a PC stage diagnostic biomarker.

Cancer-associated MUC1 mucin inhibits human T-cell proliferation, which is reversible by IL-2.

A number of adenocarcinomas abundantly express and secrete underglycosylated MUC1 mucin. Underglycosylation exposes tandem repeat peptide sequences on cancer-associated MUC1 mucin that are normally cryptic. High levels of MUC1 mucin are correlated with a poor prognosis and immunosuppression in adenocarcinoma patients. In this report we show that cancer-associated MUC1 mucin, affinity-purified from ascites fluids of cancer patients, and synthetic tandem repeats of MUC1 mucin core peptide can suppress human T-cell proliferative responses. This MUC1 mucin-induced suppression of T-cell responses can be reversed by the addition of exogenous IL-2 or anti-CD28 monoclonal antibody. These results are consistent with other studies showing that lymphocytes present in the vicinity of tumor cells are anergic and can be reactivated with exogenous interleukin-2. Overcoming MUC1 mucin-induced immunosuppression with IL-2 combined with active specific immunotherapy might be an effective immunotherapeutic strategy against human adenocarcinomas.

Purification of clinical-grade recombinant HSP65-MUCI fusion protein.

HSP65-MUCI is a fusion protein between BCG (Bacille Calmette-Guerin)-derived HSP65 (heat-shock protein 65) and human MUCI (mucin I) VNTR (variable number of tandem repeats)-domain peptides that has shown antitumour efficacy. China's Food and Drug Administration has recently approved a Phase I clinical trial using HSP65-MUCI for the treatment of MUCI-positive breast cancer. In order to produce sufficient quantities of clinical-grade HSP65-MUCI, we established a pilot-scale purification scheme comprising two steps of column chromatography: HIC (hydrophobic-interaction chromatography) and IEX (ion-exchange chromatography). The pH values of the buffers used in homogenization and HIC were adjusted to pH 9.0 to maintain protein stability and prevent protein degradation. Using this manufacturing process, we obtained clinical-grade HSP65-MUCI with a yield of 400 mg per 70 g of wet cell pellet and >96% purity.

An ultrasensitive aptasensor based on self-enhanced Au nanoclusters as highly efficient electrochemiluminescence indicator and multi-site landing DNA walker as signal amplification.

Gold nanoclusters (Au NCs) have been shown to be prospective nanoscale electrochemiluminescence (ECL) materials that are being extensively explored in bioanalysis. However, the low ECL efficiency of Au NCs has been a bottleneck barrier for their better bioapplications. To overcome this disadvantage, a low oxidation potential co-reactant N,N-diisopropylethylenediamine (DPEA) was first used to prepare self-enhanced Au NCs (Au-DPEA NCs) for drastically enhancing the ECL efficiency of Au NCs in this study. In addition, an efficient multi-site landing DNA walker with multidirectional motion track and rapid payloads release compared to directional DNA walker was constructed for converting target mucin 1 (MUC1) to intermediate DNA and achieving significant signal amplification. On the basis of the Au-DPEA NCs as efficient ECL signal labels and multi-site landing DNA walker as signal amplification strategy, an ECL aptasensor was established for the ultrasensitive detection of MUC1 in the range from 1 fg mL-1 to 1 ng mL-1 with a limit of detection down to 0.54 fg mL-1. The results demonstrated that the present study opened a new research direction for the development of high-efficiency Au NCs indicator as well as ultrasensitive ECL sensing platform for applications in clinical and bioanalysis.

Photovoltaics, plasmonics, plastic antibodies and electrochromism combined for a novel generation of self-powered and self-signalled electrochemical biomimetic sensors.

This work describes further developments into the self-powered and self-signalled biosensing system that merges photovoltaic cells, plastic antibodies and electrochromic cells into a single target. Herein, the plasmonic effect is introduced to improve the photoanode features of the photovoltaic cell, a dye sensitized solar cell (DSSC), and better electrocatalytic features are introduced in the electrode containing the sensing element. In brief, the DSSC had a counter-electrode of poly(3,4-ethylenedioxythiophene) on an FTO glass modified by a plastic antibody of 3,4-ethylenedioxythiophene and pyrrol. The photoanode had dye sensitized TiO2 modified with gold nanoparticles (AuNPs) to increase the cell efficiency, aiming to improve the sensitivity of the response of hybrid device for the target biomarker. The target biomarker was carcinoembryonic antigen (CEA). The response of the hybrid device evidenced a linear trend from 0.1 ng/mL to 10 µg/mL, with an anionic slope of 0.1431 per decade concentration. The response of the plastic antibody for CEA revealed great selectivity against other tumour markers (CA 15-3 or CA 125). The colour response of the electrochromic cell was also CEA concentration dependent and more sensitive when the hybrid device was set-up with a photoanode with AuNPs. A more intense blue colour was obtained when higher concentrations of CEA were present. Overall, this improved version of the self-powered and self-signalled set-up has zero-requirements and is particularly suitable for point-of-care analysis (POC). It is capable of screening CEA in real samples and differentiating clinical levels of interest. This concept opens new horizons into the current cancer screening approaches.

Aptasensors as a new sensing technology developed for the detection of MUC1 mucin: A review.

Mucin 1 protein (MUC1) is a membrane-associated glycoprotein overexpressed in the majority of human malignancies and considered as a predominant protein biomarker in cancers. Owing to the crucial role of MUC1 in cancer dissemination and metastasis, detection and quantification of this biomarker is of great importance in clinical diagnostics. Today, there exist a wide variety of strategies for the determination of various types of disease biomarkers, especially MUC1. In this regard, aptamers, as artificial single-stranded DNA or RNA oligonucleotides with catalytic and receptor properties, have drawn lots of attention for the development of biosensing platforms. So far, various sensitivity-enhancement techniques in combination with a broad range of smart nanomaterials have integrated into the design of novel aptamer-based biosensors (aptasensors) to improve detection limit and sensitivity of analyte determination. This review article provides a brief classification and description of the research progresses of aptamer-based biosensors and nanobiosensors for the detection and quantitative determination of MUC1 based on optical and electrochemical platforms.

Simplified approach for in-vitro production and purification of cell derived Cancer Antigen 15-3.

Cancer antigen 15-3 (CA15-3) is a key biomarker, currently used for understanding the onset and prognosis of breast cancer. In present investigation, CA15-3 has been purified from the culture supernatant of breast cancer T47-D cell line with 76% yield and 3350 fold purification. Isolated CA15-3 was analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting (western blotting), chemiluminescence immunoassay (CLIA) and Fourier-transform infrared spectroscopy (FTIR). CA15-3 is a monomeric protein with an apparent molecular mass in between ∼250-350kDa. The FTIR spectroscopy revealed similar profiles of T47-D derived CA15-3 and commercially available CA15-3 protein. With the easy availability of T47-D cell line and a simple purification approach described here will support for the large scale production of CA15-3 to be used for various clinical and diagnostic applications.

Multiplexed aptasensor for simultaneous detection of carcinoembryonic antigen and mucin-1 based on metal ion electrochemical labels and Ru(NH3)63+ electronic wires.

In this paper, a dual-target electrochemical aptasensor has been developed for simultaneous detection of carcinoembryonic antigen and mucin-1 based on metal ion electrochemical labels and Ru(NH3)63+ electronic wires. When targets are present, the interaction between targets and their respective aptamers leads to the dissociation of double-strand DNA because the targets have higher affinity to its aptamer than the complementary strand. And the qualitative and quantitative analyses of the two targets are realized by the differential pulse voltammetry (DPV) peaks generated by metal ion electrochemical labels. For the effective loading of a large number of metal ions, Au/bovine serum albumin (Au/BSA) nanospheres are employed as carriers to develop Au/BSA-metal ions. After Ru(NH3)63+ complexes are embedded into double-strand DNA to form the electronic wires, the electrical conductivity and the electron transfer of the detection system are greatly improved. The detection limit of the proposed assay was calculated as 3.33fM ranging from 0.01pM to 100nM. Therefore, this novel sensing assay provides a new and sensitive platform for detecting several targets simultaneously in biochemical research and clinical diagnosis.

Disposable electrochemical detection of breast cancer tumour marker CA 15-3 using poly(Toluidine Blue) as imprinted polymer receptor.

In this work, electrically-conducting poly(Toludine Blue) was employed for the first time as synthetic receptor film, prepared by Molecular Imprinting strategies and using electrochemical methods, for the specific screening of breast cancer biomarker Carbohydrate Antigen 15-3 (CA 15-3). The protein imprinted poly(Toluidine Blue) film was grown in a pre-formed Toluidine Blue (TB) tailed SAM at the AuSPE surface, which greatly enhanced the stability against degradation of the Molecular Imprinted Polymer (MIP) film at the electrode surface. The MIP receptor film recognition ability towards the protein was investigated by fitting data to Freundlich isotherm. The binding affinity (KF) obtained for the MIP system was significantly higher (~ 12-fold) to that obtained for the NIP system, demonstrating the success of the approach in creating imprinted materials that specifically respond to CA 15-3 protein. The incubation of the MIP modified electrode with increasing concentration of protein (from 0.10 U mL-1 to 1000 U mL-1) resulted in a decrease of the ferro/ferricyanide redox current. The device displayed linear response from 0.10 U mL-1 to 100 U mL-1 and LODs below 0.10 U mL-1 were obtained from calibration curves built in neutral buffer and diluted artificial serum, using DPV technique, enabling the detection of the protein biomarker at clinically relevant levels. The developed MIP biosensor was applied to the determination of CA 15-3 in spiked serum samples with satisfactory results. The developed device provides a new strategy for sensitive, rapid, simple and cost-effective screening of CA 15-3 biomarker. Importantly, the overall approach seems suitable for point-of-care (PoC) use in clinical context.

Ultrasensitive electrochemiluminescence immunoassay for simultaneous determination of CA125 and CA15-3 tumor markers based on PAMAM-sulfanilic acid-Ru(bpy)32+ and PAMAM-CdTe@CdS nanocomposite.

A multiplex ultrasensitive electrochemiluminescence (ECL) immunoassay was developed for the simultaneous determination of two different tumor markers, cancer antigen 153 (CA 15-3) and cancer antigen 125 (CA 125) using polyamidoamine dendrimer-quantum dots (PAMAM-QDs) and PAMAM-sulfanilic acid-Ru(bpy)32+ as the signal probes and Fe3O4-SiO2 as a magnetic bead. The CdTe@CdS- QDs and Ru(bpy)32+ at the presence of tripropyl amine (TPA) as coreactant generate ECL at an applied voltage of + 1.2V (vs Ag/AgCl) in two different wavelengths 500 and 620nm, respectively. Based on this strategy, the simultaneous detection of two tumor markers in single run carried out. This dual signal amplification technique was achieved by employing Fe3O4@SiO2-dendrimer as immunosensing platform and PAMAM as the carrier for immobilizing CdTe@CdS and Ru(bpy)32+ probes. Experimental results illustrated that the designed immunosensor can be used to sequentially detection of CA 125 and CA 15-3 markers with the wide linear ranges of 1µU/mL to 1U/mL and 0.1mU/mL to 100U/mL with very low detection limits of 0.1µU/mL and 10µU/mL, respectively. The application of the immunosensor for simultaneous detection of CA125 and CA15-3 in human serum samples was evaluated and the obtained results were found to be in acceptable agreement with the those obtained with an ELISA assay as reference method. The proposed ECL immunosensor can provide a simple, sensitive and reliable approach for the simultaneous detection of tumor markers in clinical samples.

Diagnostic accuracy of epithelial membrane antigen for malignant effusions: a meta-analysis.

BACKGROUND AND OBJECTIVES: Body cavity fluid examination sometimes presents a diagnostic challenge in cytology practice. This meta-analysis was undertaken to comprehensively assess the diagnostic potential of epithelial membrane antigen (EMA) in malignant effusions. MATERIALS AND METHODS: All relevant original articles about EMA in the diagnosis of malignant effusions published up to July 1, 2014 were retrieved. The overall sensitivity, specificity, positive and negative likelihood ratio, diagnostic odds ratio, and summary receiver operating characteristic (SROC) curve were pooled to evaluate the diagnostic value of EMA for malignant effusions using the Meta-Disc 1.4 and STATA 12.0 statistical software. RESULTS: Eleven studies met the inclusion criteria for the meta-analysis and the summary estimates for EMA in the diagnosis of malignant effusions were as follows: sensitivity 0.9 (95% CI 0.83-0.87), specificity 0.87 (95% CI 0.96-0.99), positive likelihood ratio 5.8 (95% CI 15.59-36.37), negative likelihood ratio 0.15 (95% CI 0.07-0.20) and diagnostic odds ratio 52.63 (95% CI 20.91-132.49). The SROC curve indicated that the maximum joint sensitivity and specificity (Q-value) was 0.88; the area under the curve was 0.94. CONCLUSION: The present meta-analysis indicated that EMA may be a useful diagnostic tool with good sensitivity and specificity for differentiating malignant effusions from benign effusions.

Ultrasensitive Nanoimmunosensor by coupling non-covalent functionalized graphene oxide platform and numerous ferritin labels on carbon nanotubes.

An ultrasensitive electrochemical nanostructured immunosensor for a breast cancer biomarker carbohydrate antigen 15-3 (CA 15-3) was fabricated using non-covalent functionalized graphene oxides (GO/Py-COOH) as sensor probe and multiwalled carbon nanotube (MWCNTs)-supported numerous ferritin as labels. The immunosensor was constructed by immobilizing a monoclonal anti-CA 15-3 antibody on the GO modified cysteamine (Cys) self-assembled monolayer (SAM) on an Au electrode (Au/Cys) through the amide bond formation between the carboxylic acid groups of GO/Py-COOH and amine groups of anti-CA 15-3. Secondary antibody conjugated MWCNT-supported ferritin labels (Ab2-MWCNT-Ferritin) were prepared through the amide bond formation between amine groups of Ab2 and ferritin and carboxylic acid groups of MWCNTs. The detection of CA 15-3 was based on the enhanced bioelectrocatalytic reduction of hydrogen peroxide mediated by hydroquinone (HQ) at the GO/Py-COOH-based sensor probe. The GO/Py-COOH-based sensor probe and Ab2-MWCNT-Ferritin labels were characterized using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscope (SEM), transmission electron microscope (TEM), and x-ray photoelectron spectroscopy (XPS) techniques. Using differential pulse voltammetry (DPV) technique, CA 15-3 can be selectively detected as low as 0.01 ± 0.07 U/mL in human serum samples. Additionally, the proposed CA 15-3 immunosensor showed excellent selectivity and better stability in human serum samples, which demonstrated that the proposed immunosensor has potentials in proteomic researches and diagnostics.

Sensitive electrochemiluminescence detection for CA15-3 based on immobilizing luminol on dendrimer functionalized ZnO nanorods.

In this study, we constructed a novel electrochemiluminescence (ECL) immunosensor for sensitive and selective detection of carbohydrate antigen 15-3 (CA15-3) by using polyamidoamine (PAMAM)-functionalized ZnO nanorods (ZNs-PAMAM) as carriers. PAMAM dendrimers with hyper-branched and three-dimensional structure were used as linked reagents for co-immobilization of luminol and CA15-3 detection antibody on the ZNs to prepare the signal probe. In addition, ZNs could hasten the decomposition of H2O2 to generate various reactive oxygen species (ROSs) which accelerated the ECL reaction of luminol with amplified ECL intensity. Compared with luminol in the detection solution, the ECL efficiencies of luminol could be improved by immobilizing luminol on the electrode due to the smaller distance between luminescence reagent and the electrode surface. Moreover, the electrodepositing gold nanoparticles (AuNPs) on the bare glass carbon electrode (GCE) with enhanced surface area could capture a large amount of primary anti-CA15-3 to improve the sensitivity of the immunosensor. Under the optimized experimental conditions, a wide linear range of 0.1-120 U mL(-1) was acquired with a relatively low detection limit of 0.033 U mL(-1) (S/N=3) for CA15-3.

Establishment of a heterotypic 3D culture system to evaluate the interaction of TREG lymphocytes and NK cells with breast cancer.

Three-dimensional (3D) culture approaches to investigate breast tumour progression are yielding information more reminiscent of the in vivo microenvironment. We have established a 3D Matrigel system to determine the interactions of luminal phenotype MCF-7 cells and basal phenotype MDA-MB-231 cells with regulatory T lymphocytes and Natural Killer cells. Immune cells were isolated from peripheral blood using magnetic cell sorting and their phenotype validated using flow cytometry both before and after activation with IL-2 and phytohaemagglutinin. Following the establishment of the heterotypic culture system, tumour cells displayed morphologies and cell-cell associations distinct to that observed in 2D monolayer cultures, and associated with tissue remodelling and invasion processes. We found that the level of CCL4 secretion was influenced by breast cancer phenotype and immune stimulation. We further established that for RNA extraction, the use of proteinase K in conjunction with the Qiagen RNeasy Mini Kit and only off-column DNA digestion gave the best RNA yield, purity and integrity. We also investigated the efficacy of the culture system for immunolocalisation of the biomarkers oestrogen receptor-α and the glycoprotein mucin 1 in luminal phenotype breast cancer cells; and epidermal growth factor receptor in basal phenotype breast cancer cells, in formalin-fixed, paraffin-wax embedded cultures. The expression of these markers was shown to vary under immune mediation. We thus demonstrate the feasibility of using this co-culture system for downstream applications including cytokine analysis, immunolocalisation of tumour biomarkers on serial sections and RNA extraction in accordance with MIQE guidelines.

Membrane-associated mucins in normal human conjunctiva.

PURPOSE: To examine the presence of specific membrane-associated mucins in normal human conjunctiva. METHODS: Glycoconjugates were extracted from membranes with two detergents: octylglucoside and Triton X114. Mucins were separated by cesium chloride density gradient centrifugation. Size was assessed by gel filtration on Sepharose CL2B and charge by ion-exchange chromatography on MonoQ. Cross reaction with antibodies against mucin gene products was assessed in blots of electrophoresis gels. RESULTS: Extraction of total tissue membranes yielded material with a buoyant density typical of mucins. Gel filtration showed material reacting with antimucin antibodies in a range of molecular sizes. Agarose electrophoresis confirmed the presence of MUC1 and MUC4 and the absence of MUC2 or MUC5AC. Isolation of membrane mucins by sequential, exhaustive extraction with octylglucoside followed by Triton X114 suggested the existence of mucins in different membrane environments. Reagents to carbohydrate epitopes revealed high mobility material, comigrating with MUC1 and MUC4. Low mobility membrane-bound mucins did not cross-react with any antibodies to mucin genes known to be expressed in human conjunctiva. CONCLUSIONS: Membrane-associated mucins are distinct from secreted mucins in normal human conjunctiva. MUC1 and MUC4 mature products decorate the membranes of conjunctival epithelial cells. Their segregation between octyl glucoside and the detergent and aqueous phases of Triton X114 suggests a variety of membrane anchoring modes.

Purification and characterization of a human pancreatic adenocarcinoma mucin.

Pancreatic mucins consist of core proteins that are decorated with carbohydrate structures. Previous studies have identified at least two physically distinct populations of mucins produced by a pancreatic adenocarcinoma cell line (HPAF); one is the MUC1 core protein, which includes an oligosaccharide structure identified by a monoclonal antibody (MAb) recognizing the DU-PAN-2 epitope. In this study, we purified and characterized a second mucin fraction, which also shows reactivity with the DU-PAN-2 antibody, but which has an amino acid composition that is not consistent with the MUC1 core protein. This new mucin was purified by ammonium sulfate precipitation, molecular sieve chromatography, and density gradient centrifugation. It eluted in the void volume of a Sepharose 4B column together with an associated low molecular weight protein, which could be further resolved. The mucin is highly polyanionic due to numerous sulfated and sialylated saccharide chains. Carbohydrate analyses of the purified mucin showed the presence of galactose, glucosamine, galactosamine, and sialic acid, but no mannose, glucose, or uronic acid. The purified and deglycosylated mucin shows no reactivity with anti-MUC1 apomucin antibody, but reacts with antiserum against deglycosylated tracheal mucins and antiserum against the MUC4 tandem repeat peptide. Analysis of mucin expression in HPAF cells revealed high levels of MUC1 and MUC4 mRNA, and moderate levels of MUC5AC and MUC5B mRNA. The amino acid composition of the purified mucin shows a high degree of similarity to the MUC4 core protein.

Detection and isolation of MUC1 mucin from larynx squamous cell carcinoma.

UNLABELLED: The progression from uncontrolled cell proliferation to invasion and metastasis of epithelial tumors is partially understood. Alteration of epithelial mucin expression have been described in different malignant localizations but only few attempts have been made to identify mucin expression in malignant laryngeal tumors. In the present report, results are shown of studies on the expression of mucins and carbohydrate related antigens in laryngeal cancer and on the isolation of MUC1 mucin from this tumor tissue. Malignant laryngeal specimens were processed for immunohistochemical analysis and for extranuclear membrane fractions (ENM) which were obtained by ultracentrifugation. Subsequently, ENM samples were centrifuged in density-gradient; the analysis of fractions was performed by means of SDS-PAGE and Western-blotting. The panel of monoclonal antibodies (MAbs) included anti MUC1 mucin, anti Lewis x, anti sialyl Lewis x, anti Lewis y, anti MUC-5B, anti oral mucin (gp230), anti Tn hapten, anti p53 and anti cytokeratins. By immunohistochemistry, it was possible to detect MUC1 mucin, Lewis x and Lewis y showing strong reactions while sialyl-Lewis x and Tn antigen only reacted weakly in a few cells; cytokeratins were detected in all samples. In ENM derived fractions obtained by CsCl centrifugation, MUC1 was demonstrated by Western blotting. CONCLUSIONS: (1) laryngeal cancer antigenic expression comprises mostly MUC1 mucin, Lewis x, Lewis y as well as Tn antigen and (2) the methodology here employed is useful to isolate MUC1 from tumor samples.

Analysis of peptide and lipopeptide content in liposomes.

PURPOSE: To evaluate several methods for extraction of peptides from liposomal formulations as a first step in their quantification, and to determine the encapsulation efficiency for a panel of 8 peptides. METHODS: Eight peptides were chosen due to their importance in the field of vaccine development. Three different extraction media were examined: 25% ethanol, 98% ethanol, and 100% methanol. After extraction from liposomes, peptide content was measured using reverse phase HPLC. RESULTS: The effectiveness of the extraction media for peptide recovery varied considerably for the different peptides studied. In general, more hydrophilic peptides were recovered to a greater extent using 25% ethanol while more hydrophobic peptides were more thoroughly recovered using 98% ethanol. Encapsulation efficiencies (EE) ranged from 1% to 99% for the different peptides. No strong correlation was found between the average hydrophobicity values for the peptides and their EE. CONCLUSIONS: The most effective solvent for the extraction of a peptide from liposomes depends on the physicochemical properties of the peptide. Although the peptide sequence characteristics may provide guidance on the choice of the extraction media, only peptide recovery experiments will be able identify the optimal medium for extraction.

Osteoclastic giant cell tumor of the pancreas: an immunohistochemical study.

A case of an osteoclastic giant cell tumor of the pancreas is presented. Immunohistochemical studies were performed, which showed keratin (CAM, AE1) and epithelial membrane antigen positivity in the tumor cells. The findings support an epithelial origin for this tumor.

MUC1 expression in human breast cancer cells is altered by the factors affecting cell proliferation.

Increased expression of the epithelial mucin MUC1 has been linked to tumor aggressiveness in human breast carcinoma. In the present study, we have investigated if the factors affecting cells proliferation could influence MUC1 mucin biosynthesis and shedding from cell surface into the culture medium in two human breast cancer cell lines: MCF-7 (ER+) and MDA-MB-231 (ER-). Using MCF-7 line we found that estradiol at a concentration of 10(-7) M increased [3H]glucosamine incorporation into mucin in cell lysate approximately twofold in comparison with control cultures, and a similar increase was observed in the culture medium. The selective estrogen receptor modulator, tamoxifen (at concentrations of 10(-6) M and 10(-5) M) had a little inhibitory effect. MDA-MB-231 cells in culture were stimulated with phorbol ester PMA, the protein kinase C activator. We noted that PMA greatly stimulated MUC1 synthesis and its shedding to culture medium and that this effect was abolished by protein kinase C specific inhibitor--bisindolylmaleimide.