sodium-dodecyl-sulfate and Breast-Neoplasms

GDC-0810, administered orally, was used in Phase I and II clinical studies to treat estrogen receptor positive breast cancers. It contains N-methyl-D-glucamine (NMG) salt of GDC-0810 with 10% sodium lauryl sulfate (SLS) as a surfactant and 15% sodium bicarbonate (NaHCO. RapidFACT®, a streamlined, data-driven drug product optimization platform was used to bridge Phase I/II and Phase III formulations of GDC-0810. Five prototype formulations, varying in either the form of active pharmaceutical ingredient and/or the levels of the excipients SLS and NaHCO. The study successfully identified a Phase III formulation with a reduced SLS content, which when administered following a low-fat meal, gave comparable pharmacokinetic exposure to the Phase I/II formulation administered under the same conditions.. Our novel 'Make and Test in Parallel' approach enabled optimization of GDC-0810 formulation in a time- and cost-efficient fashion.

Topics: Administration, Oral; Aged; Antineoplastic Agents; Biological Availability; Breast Neoplasms; Cinnamates; Cross-Over Studies; Drug Compounding; Excipients; Female; Food-Drug Interactions; Humans; Indazoles; Meglumine; Middle Aged; Receptors, Estrogen; Sodium Dodecyl Sulfate; Surface-Active Agents

The low distribution of hydrophobic anticancer drugs in patients is one of the biggest limitations during conventional chemotherapy. SDS-based polyelectrolyte multicore nanocarriers (NCs) prepared according to the layer by layer (LbL) procedure can release paclitaxel (PTX), and selectively kill cancer cells. Our main objective was to verify the antitumor properties of PTX-loaded NCs and to examine whether the drug encapsulated in these NCs retained its cytotoxic properties. The cytotoxicity of the prepared nanosystems was tested on MCF-7 and MDA-MB-231 tumour cells and the non-cancerous HMEC-1 cell line in vitro. Confocal microscopy, spectrophotometry, spectrofluorimetry, flow cytometry, and RT PCR techniques were used to define the typical hallmarks of apoptosis. It was demonstrated that PTX encapsulated in the tested NCs exhibited similar cytotoxicity to the free drug, especially in the triple negative breast cancer model. Moreover, SDS/PLL/PTX and SDS/PLL/PGA/PTX significantly reduced DNA synthesis. In addition, PTX-loaded NCs triggered apoptosis and upregulated the transcription of Bax, AIF, cytochrome-c, and caspase-3 mRNA. Our data demonstrate that these novel polyelectrolyte multicore NCs coated with PLL or PLL/PGA are good candidates for delivering PTX. Our discoveries have prominent implications for the possible choice of newly synthesized, SDS-based polyelectrolyte multicore NCs in different anticancer therapeutic applications.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Electrolytes; Humans; Mitochondria; Nanoparticles; Paclitaxel; Sodium Dodecyl Sulfate

In the course of screening new anticancer natural products, an edible forest mushroom Suillus luteus (L. Ex Franch). Gray was found to have potent cytotoxicity against several human cancer cells. However, the lipophilic sample made some countercurrent chromatography solvent systems emulsify, which caused difficulties in the separation of its cytotoxic components. Here, we found that the addition of an organic salt sodium dodecyl sulfate could efficiently shorten the settling time of the mushroom sample solutions by eliminating the emulsification of two-phase solvent systems. Moreover, we found that sodium dodecyl sulfate could play a new "salting-in" role and made the partition coefficients of the solutes decrease with the increased concentrations. Thus, a sodium dodecyl sulfate based salting-in countercurrent chromatography method has been successfully established for the first time for preparative isolation of a cytotoxic principle of the mushroom. The active component was identified as isosuillin. Whole results indicated that sodium dodecyl sulfate could be used as an efficient salting-in reagent for two-phase solvent system selection and targeted countercurrent chromatography isolation. It is very useful for current natural products isolation and drug discovery.

Topics: Agaricales; Antineoplastic Agents; Biological Products; Breast Neoplasms; Cell Line, Tumor; Chromatography, High Pressure Liquid; Countercurrent Distribution; DNA; Drug Discovery; Drug Screening Assays, Antitumor; Humans; Salts; Sodium Dodecyl Sulfate; Solvents; Water

Three surfactant-assisted shotgun methods using acid labile surfactants, sodium-3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)-methoxyl]-1-propanesulfonate (RapiGest) and 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate (PPS), and sodium dodecyl sulfate (SDS) were investigated for their applicability to membrane proteome analysis. It is shown that RapiGest is a preferred reagent for handling membrane proteomes of Escherichia coli and MCF7 cells for liquid chromatography tandem mass spectrometry (LC MS/MS) analysis of tryptic digests. The RapiGest method allowed identification of more peptides and proteins than the SDS and PPS methods and there was no apparent bias for the type of peptides and proteins identified by the RapiGest and SDS methods, while a slightly higher proportion of hydrophilic peptides and proteins were identified by the PPS method. The performance of the SDS and PPS methods is similar in terms of the numbers of peptides and proteins identified. Since the SDS method required the removal of SDS using a technique such as strong-cation exchange (SCX), we further investigated the effect of SCX on sample loss through analyzing the digest of an enriched E. coli membrane fraction as well as a standard protein, bovine serum albumin (BSA). The results showed that extensive sample loss (as much as 62%) was encountered during the SCX cleaning step. We then applied the RapiGest method in combination with two-dimensional LC MS/MS to characterize the E. coli membrane proteome. In total, 1626 unique proteins (5799 unique peptides) were identified with a peptide false discovery rate of 2.4%. About 60% of the identified proteins with known cellular locations were found to be membrane proteins. Among them, about 75% were integral membrane proteins. This work represents one of the most comprehensive profiles of E. coli membrane proteome generated by a proteomic technique.

Topics: Acids; Breast Neoplasms; Cation Exchange Resins; Cell Line, Tumor; Chromatography, Liquid; Escherichia coli; Female; Humans; Membrane Proteins; Proteomics; Serum Albumin, Bovine; Sodium Dodecyl Sulfate; Spectrometry, Mass, Electrospray Ionization; Surface-Active Agents

Chains of magnetosomes extracted from AMB-1 magnetotactic bacteria are shown to be highly efficient for cancer therapy when they are exposed to an alternative magnetic field. When a suspension containing MDA-MB-231 breast cancer cells was incubated in the presence of various amounts of extracted chains of magnetosomes, the viability of these cells remained high in the absence of an alternative magnetic field. By contrast, when this suspension was exposed to an alternative magnetic field of frequency 183 kHz and field strengths of 20, 40, or 60 mT, up to 100% of these cells were destroyed. The antitumoral activity of the extracted chains of magnetosomes is demonstrated further by showing that they can be used to fully eradicate a tumor xenografted under the skin of a mouse. For that, a suspension containing ∼1 mg of extracted chains of magnetosomes was administered within the tumor and the mouse was exposed to three heat cycles of 20 min, during which the tumor temperature was raised to ∼43 °C. We also demonstrate the higher efficiency of the extracted chains of magnetosomes compared with various other materials, i.e., whole inactive magnetotactic bacteria, individual magnetosomes not organized in chains, and two different types of chemically synthesized superparamagnetic iron oxide nanoparticles currently tested for alternative magnetic field cancer therapy. The higher efficiency of the extracted chains of magnetosomes compared with that of the other nanoparticles is attributed to three factors: (i) a specific absorption rate higher for the magnetosomes than for the chemically synthesized superparamagnetic iron oxide nanoparticles, (ii) a more uniform heating for the chains of magnetosomes than for the individual magnetosomes and (iii) the ability of the chains of magnetosomes to penetrate within the cancer cells or bind at the cell membrane following the application of the alternative magnetic field, which enables efficient cell destruction. Biodistribution studies revealed that extracted chains of magnetosomes administered directly within xenografted breast tumors progressively left the tumors during the 14 days following their administration and were then eliminated in large proportion in the feces.

Topics: Animals; Bacteria; Breast Neoplasms; Cell Line, Tumor; Citric Acid; Edetic Acid; Female; Hot Temperature; Humans; Magnetic Field Therapy; Magnetosomes; Mice; Nanoparticles; Polyethylene Glycols; Sodium Dodecyl Sulfate; Time Factors

We describe simultaneous analysis of naphthalene-2,3-dicarboxaldehyde (NDA)-amino acid and NDA-biogenic amine derivatives by CE in conjunction with light-emitting diode-induced fluorescence detection using poly(ethylene oxide) (PEO) solutions containing sodium dodecyl sulfate (SDS). After sample injection, via EOF 0.1% PEO prepared in 100mM TB solution (pH 9.0) containing 30 mM SDS entered a capillary filled with 0.5M TB solution (pH 10.2) containing 40 mM SDS. Under this condition, 14 NDA-amino acid and NDA-amine derivatives were separated within 16 min, with high efficiency ((1.0-3.2)x10(5) theoretical plates) and sensitivity (LODs at S/N=3 ranging from 2.06 to 19.17 nM). In the presence of SDS and PEO, analytes adsorption on the capillary wall was suppressed, leading to high efficiency and reproducibility. The intraday analysis RSD values (n=3) of the mobilities for the analytes are less than 0.52%. We have validated the practicality of this approach by quantitative determination of 9 amino acids in breast cancer cells (MCF-7) and 10 amino acids in normal epithelial cells (H184B5F5/M10). The concentrations of Tau and Gln in the MCF-7 cells were different than those in the H184B5F5/M10 cells, respectively. Our results show the potential of this approach for cancer study.

Topics: Amino Acids; Biogenic Amines; Breast Neoplasms; Cell Line, Tumor; Electrophoresis, Capillary; Female; Fluorescence; Humans; Limit of Detection; Naphthalenes; Polyethylene Glycols; Reproducibility of Results; Sodium Dodecyl Sulfate

The water-soluble peptide, melittin, was modified with an anionic agent, sodium dodecyl sulfate by hydrophobic ion-pairing. Investigations showed that the formed complex was very soluble in organic solvent, especially, in dimethylsulfoxide and dehydrated alcohol. Furthermore, the physiochemical properties of the complex in the solid state or in an aqueous medium were characterized using octanol/water partition measurement, Fourier transform infrared spectroscopy, and differential scanning calorimetry. The complex was formulated into poly(D,L-lactide-co-glycolide acid) nanoparticles by an emulsion solvent diffusion method. It was found that the nanoparticles of about 130 nm in size can be produced with a high encapsulation efficiency, and the entrapment of nanoparticles prepared with the formed complex increased from about 50% to nearly 100% compared with that for pure melittin. Moreover, the growth inhibitory effects of modified melittin and melittin-loaded nanoparticles in breast cancer MCF-7 cells were not changed comparing with free melittin as determined by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay.

Topics: Breast Neoplasms; Calorimetry, Differential Scanning; Cell Line, Tumor; Chemistry, Pharmaceutical; Dimethyl Sulfoxide; Drug Carriers; Female; Humans; Hydrophobic and Hydrophilic Interactions; Lactic Acid; Melitten; Nanoparticles; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Sodium Dodecyl Sulfate; Solubility; Solvents; Spectroscopy, Fourier Transform Infrared; Surface-Active Agents

One of the prominent cell cycle-related modifications of histone proteins whose function is correlated with chromosome condensation is the phosphorylation of histone H3. In this work we used immunofluorescence labeling on human MCF-7 cells with the antibody that was specific for phosphorylated histone H3 at Ser10 to examine the cellular distribution of this protein. The acid-soluble proteins from interphase and mitotic cells were separated by SDS-PAGE and the transferred proteins were probed with the antibody. A strong H3-specific band was only detected in the acid-soluble proteins from mitotic cells, demonstrating the correlation between H3 phosphorylation and mitosis. With confocal microscopy on whole cells, our results showed that mitotic phosphorylation of H3 initiated in discrete foci near the nuclear envelope in early prophase cells. Following initiation, H3 phosphorylation appeared to spread throughout the condensing chromatin and reached maximum in early metaphase cells. Dephosphorylation of H3 began in anaphase cells and was complete immediately prior to detectable chromosome decondensation in telophase cells. There was a precise spatial and temporal correlation between H3 phosphorylation and initial stages of chromatin condensation. The possible functions of the singular phosphorylation of the amino-terminus of H3 were discussed.

Topics: Breast Neoplasms; Electrophoresis, Polyacrylamide Gel; Female; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Histones; Humans; Phosphorylation; Sodium Dodecyl Sulfate; Tumor Cells, Cultured

Carboxy-terminally deleted forms of STAT5 have been described to be generated in vivo either by proteolytic processing or by differential splicing mechanisms. By comparing two different cell extraction procedures, we can show that in the mammary gland carboxy-terminally deleted forms are produced in vitro and are not detectable in extracts prepared by SDS lysis.

Topics: Alternative Splicing; Amino Acid Sequence; Animals; Artifacts; Blotting, Western; Breast Neoplasms; Cell Extracts; Cell Fractionation; DNA-Binding Proteins; Endopeptidases; Female; Hot Temperature; Mammary Glands, Animal; Mice; Milk Proteins; Molecular Sequence Data; Precipitin Tests; Pregnancy; Protein Isoforms; Response Elements; RNA; Sodium Dodecyl Sulfate; STAT5 Transcription Factor; Trans-Activators

Lumican mRNA has been identified as being differentially expressed between different regions of the same human breast tumor. In situ hybridization study of 26 independent breast tumors confirmed the presence of lumican mRNA in fibroblast-like cells within stroma and showed a significant increase of its expression in tumor compared to adjacent normal stroma (P < 0.001). Higher lumican expression was associated with higher tumor grade, lower estrogen receptor levels in the tumor, and younger age of the patients (P < 0.05). Reverse transcription-PCR analysis of total RNA extracted from 19 independent breast tissues exhibiting lesions that are thought to parallel tumor progression also suggests that this proteoglycan is differentially expressed during tumor progression.

Topics: Adult; Aged; Aged, 80 and over; Breast; Breast Neoplasms; Chondroitin Sulfate Proteoglycans; Electrophoresis, Polyacrylamide Gel; Female; Humans; In Situ Hybridization; Keratan Sulfate; Lumican; Middle Aged; Polymerase Chain Reaction; RNA, Messenger; Sodium Dodecyl Sulfate; Transcription, Genetic; Tumor Cells, Cultured

Matrix metalloproteinases (MMPs) have been implicated in mechanisms of metastasis in experimental cancer models and in human malignancies. In this study, we used substrate gel electrophoresis (zymography) to determine the frequency of detection of MMPs in urine of patients with a variety of cancers. Three molecular weight classes of urinary MMPs, Mr 72,000, Mr 92,000, and high molecular weight (Mr > or = 150,000) species, were detected reproducibly and correlated with disease status. The Mr 72,000 and Mr 92,000 species were identified as MMP-2 and MMP-9, respectively, by Western blot analysis. The presence of biologically active MMP-2 (P < 0.001) or MMP-9 (P = 0.002) was an independent predictor of organ-confined cancer, and the high molecular weight species (P < 0.001) was an independent predictor of metastatic cancer. This is the first study to demonstrate that analysis of urinary MMPs may be useful in determining disease status in a variety of human cancers, both within and outside of the urinary tract.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Collagenases; Electrophoresis, Polyacrylamide Gel; Female; Gelatinases; Humans; Kidney Neoplasms; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Metalloendopeptidases; Middle Aged; Neoplasm Proteins; Neoplasms; Prostatic Neoplasms; Sensitivity and Specificity; Sodium Dodecyl Sulfate; Substrate Specificity; Urinary Bladder Neoplasms

Actin and associated proteins at the cytoskeleton-plasma membrane interface stabilize the membrane bilayer, control cell shape, and delimit specialized membrane domains. To identify membrane proteins that bind directly to F-actin, we have developed a blot overlay assay with 125I-labeled F-actin. In the soil amoebae, Dictyostelium discoideum, the major proteins reactive in this assay are p30a, a 34-kD peripheral membrane protein that is concentrated in filopodia and at sites of cell-cell adhesion, and ponticulin, a 17-kD transmembrane glycoprotein required for efficient chemotaxis and for control of pseudopod dynamics. Proteins with apparent molecular masses of approximately 34- and approximately 17-kD also are observed on F-actin blot overlays of many mammalian cell lines. However, in mammalian cells, the most prominent F-actin binding proteins in this assay exhibit apparent molecular masses of 78-, 80-, 81-, approximately 120-, and 205-kD. Bovine neutrophils contain the 78-, 81-, and 205-kD proteins, all of which co-isolate with a plasma membrane-enriched fraction. We have previously identified the 78-, 80-, and 81-kD proteins as moesin, radixin, and ezrin, respectively. These proteins, which are members of the protein 4.1 superfamily, colocalize with actin in cell surface extensions and have been implicated in the protrusion of microvilli, filopodia, and membrane ruffles. The 205-kD protein (p205) appears to be absent from current databases, and its characteristics are still under investigation. We here report that the 120-kD protein is drebrin, a submembranous actin-binding protein originally identified as a developmentally regulated brain protein. Thus, it appears that F-actin blot overlays provide an efficient assay for simultaneous monitoring of a subset of F-actin binding proteins, including p30a, ponticulin, moesin, radixin, ezrin, p205, and drebrin.

Topics: 3T3 Cells; Actins; Amino Acid Sequence; Animals; Blotting, Western; Brain; Breast Neoplasms; Cattle; Chick Embryo; Dictyostelium; Electrophoresis, Polyacrylamide Gel; HeLa Cells; Humans; Iodine Radioisotopes; Mammals; Membrane Proteins; Mice; Microfilament Proteins; Neuroblastoma; Neuropeptides; Neutrophils; Sodium Dodecyl Sulfate; Tumor Cells, Cultured

Human breast carcinoma MCF-7/AdrVp cells display a novel multidrug resistance phenotype that is characterized by the overexpression of a 95-kDa membrane glycoprotein (p95) and by marked reduction in intracellular anthracycline accumulation, without overexpression of P-glycoprotein or the multidrug resistance protein MRP. p95 is also highly expressed in multidrug-resistant NCI-H1688 cells derived from a human small cell lung carcinoma. Deglycoslyated p95 from NCI-H1688 cells was isolated by two-dimensional gel electrophoresis and then digested with trypsin. The tryptic peptides were analyzed by mass spectrometry and microsequencing. These analyses identified p95 to be identical to NCA-90, the nonspecific cross-reacting antigen related to the carcinoembryonic antigen (CEA). Further confirmation that p95 is indeed NCA-90 was obtained by Northern and Western blot studies using probes or antibodies specific for p95, NCA-90, or CEA family members. Western blot studies also revealed that CEA itself is overexpressed in MCF-7/AdrVp cells compared to parental MCF-7/W cells. The enforced expression of NCA-90 protein in HeLa cells stably transfected with NCA-90 cDNA did not result in increased resistance of the transfected cells to daunorubicin or a decrease in daunorubicin accumulation in the transfected cells compared to cells transfected only with the expression vector. However, a recent report by H. Kawaharata et al. (Int. J. Cancer, 72: 377-382, 1997) of diminished accumulation, retention, and cytotoxicity of doxorubicin in EJNIH3T3 cells in which enforced expression of CEA was accomplished leaves open the possibility that the overexpression of CEA, possibly in combination with that of NCA-90, could account at least in part for the drug resistant phenotype displayed by MCF-7/AdrVp cells.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Antibiotics, Antineoplastic; Antigens, Neoplasm; Blotting, Western; Breast Neoplasms; Carcinoembryonic Antigen; Cell Adhesion Molecules; Cross Reactions; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Electrophoresis, Gel, Two-Dimensional; Epitopes; HeLa Cells; Humans; Isoelectric Focusing; Membrane Glycoproteins; Mice; Molecular Sequence Data; Neoplasm Proteins; Phenotype; Sodium Dodecyl Sulfate; Tumor Cells, Cultured

HMB-45 is an anti-melanoma monoclonal antibody widely used in diagnostic pathology owing to its great specificity in identifying poorly differentiated melanomas. In this study, by a series of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblots with the enhanced chemiluminescent (ECL) detection method on the HU-214 melanoma cell line, we identified the antigen of HMB-45 in a protein or proteins of 30-35 kDa. Although this result is in discrepancy with the previous literature which identified the antigen as a protein of 7 or 10 kDa, a family of proteins of 25-70 kDa of as a protein of 100 kDa (gp100), the present data indicate that the antigen signal we found might be specific. Furthermore, immunoblots on neuraminidase-treated cell lysates show, in agreement with already published data, that the antigen might be a sialated glycoprotein with the sialic acid involved in the epitope. Immunoblots on partially purified melanosomes confirmed the presence of the antigen in these organelles.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Blotting, Western; Breast Neoplasms; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Humans; Immunohistochemistry; Luminescent Measurements; Melanocytes; Melanoma; Melanoma-Specific Antigens; Neoplasm Proteins; Sialic Acids; Sodium Dodecyl Sulfate; Tumor Cells, Cultured

The expression of both 92- and 72-kDa gelatinases has been studied in 20 samples of human breast carcinoma by the technique of gelatin zymography. This technique allowed the relative amount of each gelatinase to be determined in small samples of tissue (< 10 mg). More importantly, active and latent forms of the two gelatinases were resolved. Two samples (10-20 mg) were cut from each piece of tumour in order to monitor the variability of gelatinase distribution within that section of tumour. The 72-kDa latent progelatinase was present in 15 of the 20 tumours, with trace amounts in two others. The 62-kDa activated form of this gelatinase was detected in all 15 of the tumours in which the latent form was present. The 92-kDa latent progelatinase was present in 11 of the 20 tumours, with trace amounts in four others. However, the 82-kDa activated form of this gelatinase was only clearly detected in two tumours, although three others showed the presence of trace amounts. The ratio of active to latent forms of the 72-kDa gelatinase ranged from 0.9 to 3.6. There were no marked correlations between gelatinase expression and established staging and prognostic markers. Analysis of three samples of fibroadenoma revealed only very low levels of gelatinase expression. On the basis of these results, activation of the 72-kDa progelatinase appears to be a more common event in invasive breast carcinoma than activation of the 92-kDa progelatinase. However, neither proteinase showed a correlation with metastatic progression, as measured by lymph node involvement.

Topics: Biomarkers, Tumor; Breast Neoplasms; Collagenases; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Female; Humans; Isoenzymes; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Metalloendopeptidases; Molecular Weight; Neoplasm Invasiveness; Neoplasm Proteins; Protein Denaturation; Sensitivity and Specificity; Sodium Dodecyl Sulfate

A gradient elution reversed-phase high-performance liquid chromatographic method was developed for the direct serum injection analysis of porphyrins based on the use of eluent containing an anionic surfactant (sodium dodecyl sulfate) at a concentration below the critical micelle concentration to elute the serum proteins at the column void volume. Separation and detection performances were tested with a mixture of porphyrin standards containing uro-, heptacarboxylic-, hexacarboxylic-, pentacarboxylic-, copro-, zinc proto- and mesoporphyrin in a model serum consisting of 50 mg/ml bovine serum albumin. Average limit of detection is 0.06 pmol with a 10-microliter injection volume using fluorimetric excitation at the Soret band of porphyrins. The utility of this method for the direct serum injection analysis of porphyrins in human serum was evaluated by investigating serum samples from individuals suffering from iron-deficiency anemia and breast cancer.

Topics: Anemia, Hypochromic; Breast Neoplasms; Chromatography, High Pressure Liquid; Detergents; Humans; Porphyrins; Sodium Dodecyl Sulfate

We have developed a method for the efficient transfer of histones from acetic acid-urea-Triton X-100 (AUT)-polyacrylamide minislab gels to nitrocellulose. The AUT gel was equilibrated with 50 mM acetic acid and 0.5% sodium dodecyl sulfate and then with 62.5 mM Tris-HCl, pH 6.8, and 2.3% sodium dodecyl sulfate. An alkaline transfer buffer [25 mM 3-(cyclohexylamino)-1-propanesulfonic acid, pH 10, with 20% methanol] was used to electrophoretically transfer the strongly basic proteins from AUT or sodium dodecyl sulfate gels to nitrocellulose. The applicability of this approach in the immunochemical detection of ubiquitinated histone species is demonstrated.

Topics: Animals; Blotting, Western; Breast Neoplasms; Cell Line; Chickens; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Female; Histones; Humans; Indicators and Reagents; Molecular Weight; Polyethylene Glycols; Sodium Dodecyl Sulfate; Urea

We have reported the production of human monoclonal antibodies (mAb), by the fusion of lymph node lymphocytes from a primary carcinoma patient with murine myeloma cells. Seven heterohybridomas showed reactivity with class III antigens, and five hybridomas (1G12, 2D4, 4H5, 5D10 and 3B10) were reactive with class II antigens. One of these human mAbs (1G12) was intensively studied and results are presented here. 1G12 reacted strongly and specifically with five mammary carcinoma cell lines and showed no cross-reactivity with seven normal fibroblast cells. It continuously produced human mAbs (IgM) at a rate of 4.5-12.5 micrograms/ml over a period of 2 years. Human mAb 1G12 (IgM) was purified by either a combination of anion-exchange chromatography (ABx) and gel filtration (Superose 6) or affinity chromatography (agarose). Immunohistological analysis of frozen tissue sections was performed with biotinylated 1G12. All mammary carcinomas analysed (n = 26) were positive, while the connective tissue of 36 different patients was completely negative with 1G12. In normal breast, endometrium and intestine only a weak or moderate staining of the epithelial cells was observed. Normal oesophagus, small bowel, cervix, uterus, lung and skin were completely negative. Partly purified tumour antigen recognized by 1G12 had a molecular mass of 1-2 MDa and showed strong binding with Ulex europaeus lectin I and Bauhina purpurea agglutinin, indicative for the glucoprotein nature of antigens. These results show that human mAb 1G12 may be useful for the analysis of tumour-associated antigen of mammary carcinoma patients. In further studies the therapeutic and diagnostic application of 1G12 should be analysed in more detail.

Topics: Animals; Anions; Antibodies, Monoclonal; Antigens, Neoplasm; Biotin; Breast Neoplasms; Chromatography, Gel; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Female; Humans; Immunohistochemistry; Mammary Neoplasms, Experimental; Mice; Neoplasms, Experimental; Sodium Dodecyl Sulfate

In order to analyse the molecular weight polymorphism of the estrogen receptor (ER) in MCF-7 cells, we have developed a procedure which allowed in situ linkage of ER by (3H) tamoxifen aziridine and provided labelled proteins in conditions which minimized protease activities. After labelling, cell lysis was performed in SDS buffer containing various concentrations of [symbol: see text]-mercaptoethanol. Proteins extracted with phenolic solution and precipitated by cold acetone were analysed by SDS PAGE. It appears that beside the form of 67 kDa already described, binding entities of tamoxifen aziridine were also present at a molecular mass of 110 kDa and 45 kDa. On the other hand, investigations on the effect of 12-0-Tetradecanoyl Phorbol 13-Acetate (TPA) showed that TPA induces a decrease of the 67 kDa entity.

Topics: Breast Neoplasms; Endopeptidases; Humans; Intracellular Fluid; Molecular Weight; Receptors, Estrogen; Sodium Dodecyl Sulfate; Tamoxifen; Tetradecanoylphorbol Acetate; Tritium; Tumor Cells, Cultured

An enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of human urokinase-type plasminogen activator (u-PA) in plasma and serum. Microtiter plates were coated with a monoclonal antibody and incubated with standard or sample. Bound u-PA was quantitated with polyclonal antibodies conjugated with biotin, followed by avidin-peroxidase. The assay was 10 times as sensitive as previously reported immunoassays, the detection limit being approximately 1 pg u-PA in a volume of 100 microliter, with a linear dose-response up to 15 pg u-PA. The assay detected active u-PA and its inactive proenzyme form equally well, and the recovery of both forms was higher than 90% in plasma. It also detected u-PA complexed with plasminogen activator inhibitor type 1. Various structurally related proteins, including t-PA, were tested, but no reaction was observed with proteins other than u-PA and its amino-terminal fragment. The intra-assay and interassay coefficients of variation for determination of u-PA in plasma were 7.6% and 8.4%, respectively. The ELISA was used to measure the concentration of u-PA in plasma from 34 healthy donors and 92 patients with breast cancer with a varying extent of disease. The mean value for the healthy donors was 1.1 +/- 0.3 ng/ml (SD) of u-PA in plasma. This value is substantially lower than those previously reported. The mean value for the patients with breast cancer was 1.3 +/- 0.4 ng/ml. This moderate increase was statistically significant at the 1% level. Approximately one quarter of the patients had plasma u-PA concentrations above the range observed for the healthy controls. There was a positive correlation between the mean u-PA plasma concentration and the extent of disease in different groups of patients.

Topics: Adolescent; Adult; Aged; Antibodies, Monoclonal; Blood Donors; Breast Neoplasms; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Female; Humans; Indicators and Reagents; Male; Middle Aged; Plasminogen Activators; Sodium Dodecyl Sulfate; Urokinase-Type Plasminogen Activator

Topics: Adolescent; Adult; Aged; Benzalkonium Compounds; Breast Neoplasms; Croton Oil; Dermatitis, Contact; Female; Humans; Lung Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Neoplasms; Skin Tests; Sodium Dodecyl Sulfate