sepharose and Breast-Neoplasms

To explore the action of pingxiao capsules (PXC) and its significance in the treatment of late stage mammary cancer (LSMC).. One hundred and forty-two LSMC patients were randomized into four groups: the two single treated groups treated by endocrinotherapy (ET) alone (n = 27) and by chemotherapy alone (n=44) respectively, and the two PXC combined treated groups treated with PXC plus endocrinotherapy (n=27) or chemotherapy (n=44). The remission rate and progression time (TTP) of disease, the survival time and quality of life (QOL) of patients, and the adverse reaction were compared between the single treated groups and the combined treated groups.. The median progression time was obviously prolonged, and QOL improved in the combined treated groups than those in the single treated groups (P < 0.05), but no significant difference was found in the remission rate or adverse reaction between them.. PXC can improve QOL, prolong the progression time in patients of LSMC, and with less adverse reaction. It is worth spreading combination of PXC with chemo- or endocrino-therapy in clinical application for treatment of LSMC patients.

Topics: Adult; Aged; Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Drug Therapy, Combination; Drugs, Chinese Herbal; Female; Humans; Middle Aged; Paclitaxel; Phytotherapy; Sepharose; Tamoxifen

Recently, many types of 3D culture systems have been developed to preserve the physicochemical environment and biological characteristics of the original tumors better than the conventional 2D monolayer culture system. There are various types of models belonging to this culture, such as the culture based on non-adherent and/or scaffold-free matrices to form the tumors. Agarose mold has been widely used to facilitate tissue spheroid assembly, as it is essentially non-biodegradable, bio-inert, biocompatible, low-cost, and low-attachment material that can promote cell spheroidization. As no studies have been carried out on the development of a fluorescent bicellular tumoroid mimicking ductal carcinoma

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Female; Humans; Sepharose; Tumor Microenvironment

The progression of breast cancer involves cancer-cell invasions of extracellular matrices. To investigate the progression, 3D cell cultures are widely used along with different types of matrices. Currently, the matrices are often characterized using parallel-plate rheometry for matrix viscoelasticity, or liquid-like viscous and stiffness-related elastic characteristics. The characterization reveals averaged information and sample-to-sample variation, yet, it neglects internal heterogeneity within matrices, experienced by cancer cells in 3D culture. Techniques using optical tweezers and magnetic microrheometry have measured heterogeneity in viscoelasticity in 3D culture. However, there is a lack of probabilistic heterogeneity quantification and cell-size-relevant, microscale-viscoelasticity measurements at breast-tumor tissue stiffness up to ≃10 kPa in Young's modulus. Here, we have advanced methods, for the purpose, which use a magnetic microrheometer that applies forces on magnetic spheres within matrices, and detects the spheres displacements. We present probabilistic heterogeneity quantification using microscale-viscoelasticity measurements in 3D culture matrices at breast-tumor-relevant stiffness levels. Bayesian multilevel modeling was employed to distinguish heterogeneity in viscoelasticity from the effects of experimental design and measurement errors. We report about the heterogeneity of breast-tumor-relevant agarose, GrowDex, GrowDex-collagen and fibrin matrices. The degree of heterogeneity differs for stiffness, and phase angle (i.e. ratio between viscous and elastic characteristics). Concerning stiffness, agarose and GrowDex show the lowest and highest heterogeneity, respectively. Concerning phase angle, fibrin and GrowDex-collagen present the lowest and the highest heterogeneity, respectively. While this heterogeneity information involves softer matrices, probed by ≃30 μm magnetic spheres, we employ larger ≃100 μm spheres to increase magnetic forces and acquire a sufficient displacement signal-to-noise ratio in stiffer matrices. Thus, we show pointwise microscale viscoelasticity measurements within agarose matrices up to Young's moduli of 10 kPa. These results establish methods that combine magnetic microrheometry and Bayesian multilevel modeling for enhanced heterogeneity analysis within 3D culture matrices.

Topics: Bayes Theorem; Breast Neoplasms; Cell Culture Techniques, Three Dimensional; Collagen; Elastic Modulus; Extracellular Matrix; Female; Fibrin; Humans; Magnetic Phenomena; Sepharose

The response of cancer cells to hypoxic conditions found within the interior of a tumor mass is mediated through the hypoxia inducible factor (HIF) cascade and is thought to promote metastasis. However, given their distant proximity from blood vessels as compared to normoxic cells at the vascularised tumor periphery, it is uncertain if these cells can migrate through the tumor mass to gain access. Hypoxia was simulated by exposure to cobalt chloride or deferoxamine in normal (MCF10A) and cancerous [estrogen receptor (ER)-ve (pII), and ER +ve (YS1.2/ EII)] cells. In this report, HIF1α expression and localization was measured using western blotting, ELISA, and immunofluorescence, cell proliferation by MTT assay, motility and invasion by wound healing, live cell imaging, matrigel and co-culture in chambered slides. We found that the expression and nuclear translocation of HIF1α was significantly elevated by hypoxia, which inhibited cell proliferation, but significantly increased motility of pII cells and their penetration into and through a dense layer of adjacent EII cells, as well as their selective emergence out of a co-culture. These data suggest that endocrine resistant pII cancer cells, having undergone epithelial to mesenchymal transition are able to penetrate through other cell layers, with possible enhancement in response to hypoxia.

Topics: Basement Membrane; Breast Neoplasms; Cell Hypoxia; Cell Line, Tumor; Cell Movement; Cobalt; Coculture Techniques; Deferoxamine; Epithelial-Mesenchymal Transition; Estrogens; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; MCF-7 Cells; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Receptors, Estrogen; RNA, Small Interfering; Sepharose

Topics: Breast Neoplasms; Cell Death; Cell Survival; Culture Media; Female; Ferric Compounds; HeLa Cells; Hot Temperature; Humans; Hyperthermia, Induced; In Vitro Techniques; Magnetic Fields; Magnetic Iron Oxide Nanoparticles; Manganese Compounds; MCF-7 Cells; Phantoms, Imaging; Sepharose; Uterine Cervical Neoplasms; Zinc Compounds

Chemotaxis, the motion of cells directed by a gradient of chemoattractant molecules, guides cells in immune response, development, wound healing, and cancer. Unfortunately, this process is difficult to distinguish from chemokinesis, i.e., stimulated random cell motion. Chemotaxis is frequently inferred by determining how many cells cross a boundary in a chemotaxis assay, for example how many cells crawl into a chemoattractant-infused filter, or how many cells enter a defined region in an under-agarose assay or agarose spot assay. To mitigate possible ambiguity in whether motion observed in these assays is directed by the chemoattractant gradient or by chemokinesis, we developed a mathematical model to determine when such methods indeed indicate directed motion of cells. In contrast to previous analyses of chemotaxis assays, we report not just the gradients that arise in the assays but also resulting cell motion. We applied the model to data obtained from rigorous measurements and show, as examples, that MDA-MB-231 breast-cancer cells are at least 20 times less sensitive to gradients of EGF or CXCL12 than neutrophils are to formyl peptides; we then used this information to determine the extent to which gradient sensing increases the rate of boundary crossing relative to a random-motility control. Results show, for example, that in the filter assay, 2-4 times as many neutrophils pass through the filter when exposed to a gradient as when the gradient is absent. However, in the other combinations of cells and assays we considered, only 10-20% more cells are counted as having migrated in a directed, rather than random, motility condition. We also discuss the design of appropriate controls for these assays, which is difficult for the under-agarose and agarose spot assays. Moreover, although straightforward to perform with the filter assay, reliable controls are often not done. Consequently, we infer that chemotaxis is frequently over-reported, especially for cells like MDA-MB-231 cells, which move slowly and are relatively insensitive to gradients. Such results provide insights into the use of chemotaxis assays, particularly if one wants to acquire and analyze quantitative data.

Topics: Breast Neoplasms; Cell Movement; Chemokine CXCL12; Chemotactic Factors; Chemotaxis; Chemotaxis, Leukocyte; Epidermal Growth Factor; Eukaryotic Cells; Female; Humans; Models, Biological; Neutrophils; Sepharose

Properties of soft biological tissues are increasingly used in medical diagnosis to detect various abnormalities, for example, in liver fibrosis or breast tumors. It is well known that mechanical stiffness of human organs can be obtained from organ responses to shear stress waves through Magnetic Resonance Elastography. The Local Interaction Simulation Approach is proposed for effective modelling of shear wave propagation in soft tissues. The results are validated using experimental data from Magnetic Resonance Elastography. These results show the potential of the method for shear wave propagation modelling in soft tissues. The major advantage of the proposed approach is a significant reduction of computational effort.

Topics: Algorithms; Breast Neoplasms; Computer Simulation; Elasticity Imaging Techniques; Finite Element Analysis; Gelatin; Humans; Liver; Liver Cirrhosis; Models, Statistical; Phantoms, Imaging; Sepharose; Shear Strength; Software; Stress, Mechanical

The development of three-dimensional (3D) cultures is increasing, as they are able to provide the utility of in vitro models and the strength of testing in physiologically relevant systems. When cultured in a scaffold-free agarose hydrogel system, MCF-7 human breast carcinoma cells organize and develop into microtissues that contain a luminal space, in stark contrast to the flat morphology of MCF-7 two-dimensional (2D) monolayer cultures. Following exposure to 1nM E2, expression of typical estrogen-responsive genes, including progesterone receptor (PGR), PDZ containing domain 1 (PDZK1) and amphiregulin (AREG) is increased in both 2D and 3D cultures. When examining expression of other genes, particularly those involved in cell adhesion, there were large changes in 3D MCF-7 microtissues, with little to no change observed in the MCF-7 monolayer cultures. Together, these results indicate that while the initial estrogen-regulated transcriptional targets respond similarly in 2D and 3D cultures, there are large differences in activation of other pathways related to cell-cell interactions.

Topics: Breast Neoplasms; Cell Adhesion; Cell Communication; Cell Culture Techniques; Cluster Analysis; Estradiol; Estrogens; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Hydrogel, Polyethylene Glycol Dimethacrylate; MCF-7 Cells; Reverse Transcriptase Polymerase Chain Reaction; Sepharose; Signal Transduction

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Topics: Agar; Antineoplastic Agents; Breast Neoplasms; Carcinogenesis; Carcinoma in Situ; Carcinoma, Ductal, Breast; Cell Line, Tumor; Cell Proliferation; Disease Progression; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Female; Humans; Hydrolases; Neoplastic Stem Cells; Ornithine; Protein-Arginine Deiminase Type 2; Protein-Arginine Deiminases; Sepharose

In current clinical practice, there is no integrated 3D ultrasound (3DUS) guidance system clinically available for breast brachytherapy. In this study, the authors present a novel robot-assisted 3DUS system for real-time planning and guidance of breast interstitial high dose rate (HDR) brachytherapy treatment.. For this work, a new computer controlled robotic 3DUS system was built to perform a hybrid motion scan, which is a combination of a 6 cm linear translation with a 30° rotation at both ends. The new 3DUS scanner was designed to fit on a modified Kuske assembly, keeping the current template grid configuration but modifying the frame to allow the mounting of the 3DUS system at several positions. A finer grid was also tested. A user interface was developed to perform image reconstruction, semiautomatic segmentation of the surgical bed as well as catheter reconstruction and tracking. A 3D string phantom was used to validate the geometric accuracy of the reconstruction. The volumetric accuracy of the system was validated with phantoms using magnetic resonance imaging (MRI) and computed tomography (CT) images. In order to accurately determine whether 3DUS can effectively replace CT for treatment planning, the authors have compared the 3DUS catheter reconstruction to the one obtained from CT images. In addition, in agarose-based phantoms, an end-to-end procedure was performed by executing six independent complete procedures with both 14 and 16 catheters, and for both standard and finer Kuske grids. Finally, in phantoms, five end-to-end procedures were performed with the final CT planning for the validation of 3DUS preplanning.. The 3DUS acquisition time is approximately 10 s. A paired Student t-test showed that there was no statistical significant difference between known and measured values of string separations in each direction. Both MRI and CT volume measurements were not statistically different from 3DUS volume (Student t-test: p > 0.05) and they were significantly correlated to 3DUS measurement (Pearson test: MRI p < 0.05 and CT p < 0.001). The mean angular separation distance between catheter trajectories segmented from 3DUS and CT images was 0.42° ± 0.24°, while the maximum and mean trajectory separations were 0.51 ± 0.19 and 0.37 ± 0.17 mm, respectively. Overall, the new finer grid has performed significantly better in terms of dosimetric indices. The planning target volume dosimetric indices were not found statistically different between 3DUS and CT planning (Student t-test, p > 0.05). Both the skin and the pectoral muscle dosimetric indices were within ABS guidelines.. A novel robot-assisted 3DUS system was designed and validated. To their knowledge, this is the first system capable of performing real-time guidance and planning of breast multicatheter HDR brachytherapy treatments. Future investigation will test the feasibility of using the system in the clinic and for permanent breast brachytherapy.

Topics: Algorithms; Brachytherapy; Breast; Breast Neoplasms; Feasibility Studies; Humans; Imaging, Three-Dimensional; Magnetic Resonance Imaging; Mammography; Muscle, Skeletal; Phantoms, Imaging; Radiotherapy Dosage; Radiotherapy Planning, Computer-Assisted; Robotics; Sepharose; Skin; Tomography, X-Ray Computed; Ultrasonography, Mammary

Heat shock protein 90 (Hsp90) has been identified in the extracellular space and has been shown to chaperone a finite number of extracellular proteins involved in cell migration and invasion. We used chemical cross-linking and immunoprecipitation followed by tandem mass spectrometry (MS/MS) to isolate a complex containing Hsp90 and the matrix protein fibronectin (FN) from breast cancer cells. Further analysis showed direct binding of Hsp90 to FN using an in vitro co-immunoprecipitation assay, a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. Confocal microscopy showed regions of co-localisation of Hsp90 and FN in breast cancer cell lines. Exogenous Hsp90β was shown to increase the formation of extracellular FN matrix in the Hs578T cell line, whilst knockdown or inhibition of Hsp90 led to a reduction in the levels of both soluble and insoluble FN and could be partially rescued by addition of exogenous Hsp90β. Treatment of cells with novobiocin led to internalization of FN into vesicles that were positive for the presence of the lysosomal marker, LAMP-1. Taken together, the direct interaction between FN and Hsp90, as well as the decreased levels of both soluble and insoluble FN upon Hsp90 inhibition or knockdown, suggested that FN may be a new client protein for Hsp90 and that Hsp90 was involved in FN matrix assembly and/or stability. The identification of FN as a putative client protein of Hsp90 suggests a role for Hsp90 in FN matrix stability, which is important for a number of fundamental cellular processes including embryogenesis, wound healing, cell migration and metastasis.

Topics: Bacterial Proteins; Breast Neoplasms; Cell Line, Tumor; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix; Female; Fibronectins; Fluorescence; HSP90 Heat-Shock Proteins; Humans; MCF-7 Cells; Microscopy, Confocal; RNA Interference; Sepharose; Surface Plasmon Resonance; Tandem Mass Spectrometry

A dimeric 64-kDa hemagglutinin was isolated with a high yield from dried Phaseolus vulgaris cultivar "French bean number 35" seeds using a chromatographic protocol that involved Blue-Sepharose, Q-Sepharose, and Superdex 75. The yield was exceptionally high (1.1g hemagglutinin per 100g seed), which is around 10-85 times higher than other Phaseolus cultivars. Its N-terminal sequence resembled those of other Phaseolus hemagglutinins. The hemagglutinating activity of the hemagglutinin was stable in the pH range 6-8, and in the temperature range 0 degrees C-50 degrees C. It inhibited HIV-1 reverse transcriptase with an IC50 of 2microM. It suppressed mycelial growth in Valsa mali with an IC50 of 10microM. It inhibited proliferation of hepatoma HepG2 cells and breast cancer MCF-7 cells with an IC50 of 100 and 2microM, respectively. It had no antiproliferative effect on normal embryonic liver WRL68 cells.

Topics: Antifungal Agents; Antineoplastic Agents, Phytogenic; Ascomycota; Breast Neoplasms; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Female; Hemagglutinins; Hep G2 Cells; Hepatocytes; HIV Reverse Transcriptase; HIV-1; Humans; Inhibitory Concentration 50; Liver Neoplasms; Mycelium; Phaseolus; Plant Extracts; Plant Lectins; Reverse Transcriptase Inhibitors; Seeds; Sepharose

A dimeric 70-kDa chymotrypsin inhibitor with substantial N-terminal sequence homology to serine protease inhibitors was isolated from Acacia confusa seeds. The chymotrypsin inhibitor was purified using a protocol that entailed ion exchange chromatography on Q-Sepharose, SP-Sepharose and fast protein liquid chromatography-gel filtration on Superdex 75. The chymotrypsin inhibitor was unadsorbed on both Q-Sepharose and SP-Sepharose. Its chymotrypsin inhibitory activity was stable from pH 3 to 10 and from 0 to 50 degrees C. It exerted antiproliferative activity toward breast cancer MCF-7 cells with an IC(50) of 10.7+/-4.2 microM. It inhibited HIV-1 reverse transcriptase with an IC(50) of 8+/-1.5 microM. It was devoid of antifungal activity toward a variety of fungal species. The distinctive features of the chymotrypsin inhibitor included dimeric nature, a high molecular mass, lack of trypsin inhibitory activity, highly potent HIV-1 reverse transcriptase inhibitory activity, specific antitumor activity and relatively high pH-stability.

Topics: Acacia; Adsorption; Amino Acid Sequence; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Chromatography; Chymotrypsin; Dimerization; Dose-Response Relationship, Drug; Female; Fungi; Hep G2 Cells; HIV Reverse Transcriptase; Humans; Hydrogen-Ion Concentration; Inhibitory Concentration 50; Molecular Structure; Phytotherapy; Plant Proteins; Reverse Transcriptase Inhibitors; Seeds; Sepharose; Sequence Homology; Temperature

Mild hyperthermia is increasingly important for the activation of temperature-sensitive drug delivery vehicles. Noninvasive ultrasound thermometry based on a 2-D speckle tracking algorithm was examined in this study. Here, a commercial ultrasound scanner, a customized co-linear array transducer, and a controlling PC system were used to generate mild hyperthermia. Because the co-linear array transducer is capable of both therapy and imaging at widely separated frequencies, RF image frames were acquired during therapeutic insonation and then exported for off-line analysis. For in vivo studies in a mouse model, before temperature estimation, motion correction was applied between a reference RF frame and subsequent RF frames. Both in vitro and in vivo experiments were examined; in the in vitro and in vivo studies, the average temperature error had a standard deviation of 0.7°C and 0.8°C, respectively. The application of motion correction improved the accuracy of temperature estimation, where the error range was 1.9 to 4.5°C without correction compared with 1.1 to 1.0°C following correction. This study demonstrates the feasibility of combining therapy and monitoring using a commercial system. In the future, real-time temperature estimation will be incorporated into this system.

Topics: Algorithms; Animals; Breast Neoplasms; Female; Hyperthermia, Induced; Image Processing, Computer-Assisted; Mice; Neoplasm Transplantation; Phantoms, Imaging; Sepharose; Signal Processing, Computer-Assisted; Temperature; Thermography; Transducers; Ultrasonography

We report the results of a study carried out to investigate the effect of He-Ne laser (632.8 nm) pre-irradiation on DNA damage induced by continuous wave 1064 nm trapping beam exposure in MCF-7 cells. A significant decrease in % tail DNA (p < 0.05) was observed in MCF-7 cells pre-exposed to He-Ne laser beam. The dependence of the induced protection against 1064 nm trapping beam irradiation induced DNA damage on the time interval between the two irradiations as well as the He-Ne laser pre-irradiation parameters is presented.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; DNA Damage; DNA, Neoplasm; Female; Gels; Humans; Lasers, Gas; Optical Tweezers; Sepharose; Spectroscopy, Near-Infrared

The accurate mass and time (AMT) tag strategy has been recognized as a powerful tool for high-throughput analysis in liquid chromatography-mass spectrometry (LC-MS)-based proteomics. Due to the complexity of the human proteome, this strategy requires highly accurate mass measurements for confident identifications. We have developed a method of building a reference map that allows relaxed criteria for mass errors yet delivers high confidence for peptide identifications. The samples used for generating the peptide database were produced by collecting cysteine-containing peptides from T47D cells and then fractionating the peptides using strong cationic exchange chromatography (SCX). LC-tandem mass spectrometry (MS/MS) data from the SCX fractions were combined to create a comprehensive reference map. After the reference map was built, it was possible to skip the SCX step in further proteomic analyses. We found that the reference-driven identification increases the overall throughput and proteomic coverage by identifying peptides with low intensity or complex interference. The use of the reference map also facilitates the quantitation process by allowing extraction of peptide intensities of interest and incorporating models of theoretical isotope distribution.

Topics: Alkanesulfonates; Breast Neoplasms; Cell Line, Tumor; Chromatography, Affinity; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Data Display; Databases, Protein; Female; Humans; Peptides; Proteome; Proteomics; Reference Standards; Reproducibility of Results; Sepharose; Tandem Mass Spectrometry

To evaluate the advantages of using an active marker (active micro coil) for MR-guided breast biopsy procedures.. An add-on breast biopsy guidance device used with a standard breast coil was equipped with an active marker. The marker's position was determined with a dedicated MRI sequence. In combination with custom software, the biopsy planning process was reduced basically to defining the target in the diagnostic MR images. Automatic control scans verified the settings of the biopsy guidance device. To measure the targeting accuracy, x-ray control of the needle placement was performed in phantoms containing 36 small titanium cylinders. The reliability of the procedure was evaluated in 24 core needle biopsies on phantoms. Workflow enhancements were analyzed.. The root mean square deviation of the needle position from the target perpendicular to the needle axis was 1.25 mm, in three-dimensions it was 1.35 mm. All targets were sampled successfully. The duration of a phantom biopsy was nine minutes.. The use of an active marker can offer advantages for MR-guided breast biopsies in terms of handling and procedure time as well as accuracy.

Topics: Biomarkers; Biopsy; Biopsy, Needle; Breast; Breast Diseases; Breast Neoplasms; Female; Humans; Image Processing, Computer-Assisted; Magnetic Resonance Imaging; Phantoms, Imaging; Sepharose; X-Rays

Herein we report a method for studying slow acting pharmaceutical COX-2 inhibitors in the MCF-7 breast cancer cell line where cells are grown in a three-dimensional format within an agarose matrix. The cancer cells are suspended in agarose and plated in a cell culture dish, where they will form small multicellular 'tumors' in the agarose. The time frame for conducting experiments is up to 2.5 weeks, and gives ample time for COX-2 inhibitors to induce cell death. Etodolac was used for these experiments, and was found to induce cell death in a time dependent manner over a 2.5-week period. This is in contrast to the cell line grown in monolayer and treated with the same concentrations of etodolac. This method is appropriate for determining mechanisms of cell death caused by COX-2 inhibition.

Topics: Breast Neoplasms; Cell Culture Techniques; Cell Death; Cell Division; Cell Line, Tumor; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Etodolac; Humans; Isoenzymes; Membrane Proteins; Prostaglandin-Endoperoxide Synthases; Sepharose; Time Factors

ADAM 12 is a member of a family of disintegrin-containing metalloproteases that have been implicated in a variety of diseases including Alzheimer's disease, arthritis, and cancer. We purified ADAM 12 from the urine of breast cancer patients via Q-Sepharose anion exchange and gelatin-Sepharose affinity chromatography followed by protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Four peptides were identified that spanned the amino acid sequence of ADAM 12. Immunoblot analysis using ADAM 12-specific antibodies detected an approximately 68-kDa band identified as the mature form of ADAM 12. To characterize catalytic properties of ADAM 12, full-length ADAM 12-S was expressed in COS-7 cells and purified. Substrate specificity studies demonstrated that ADAM 12-S degrades gelatin, type IV collagen, and fibronectin but not type I collagen or casein. Gelatinase activity of ADAM 12 was completely abrogated by zinc chelators 1,10-phenanthroline and EDTA and was partially inhibited by the hydroxamate inhibitor Marimastat. Endogenous matrix metalloprotease inhibitor TIMP-3 inhibited activity. To validate our initial identification of this enzyme in human urine, 117 urine samples from breast cancer patients and controls were analyzed by immunoblot. The majority of samples from cancer patients were positive for ADAM 12 (67 of 71, sensitivity 0.94) compared with urine from controls in which ADAM 12 was detected with significantly lower frequency. Densitometric analyses of immunoblots demonstrated that ADAM 12 protein levels were higher in urine from breast cancer patients than in control urine. In addition, median levels of ADAM 12 in urine significantly increased with disease progression. These data demonstrate for the first time that ADAM 12 is a gelatinase, that it can be detected in breast cancer patient urine, and that increased urinary levels of this protein correlate with breast cancer progression. They further support the possibility that detection of urinary ADAM 12 may prove useful in the development of noninvasive diagnostic and prognostic tests for breast and perhaps other cancers.

Topics: ADAM Proteins; ADAM12 Protein; Adult; Aged; Amino Acid Sequence; Animals; Blotting, Western; Breast Neoplasms; Caseins; Catalysis; Chelating Agents; Chromatography, Affinity; Chromatography, Ion Exchange; Collagen Type I; Collagen Type IV; COS Cells; Databases as Topic; Densitometry; Disease Progression; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Extracellular Matrix; Female; Fibronectins; Gelatin; Humans; Hydroxamic Acids; Immunoblotting; Membrane Proteins; Metalloendopeptidases; Middle Aged; Molecular Sequence Data; Neoplasm Metastasis; Peptides; Phenanthrolines; Plasmids; Recombinant Proteins; Sensitivity and Specificity; Sepharose; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity; Ultracentrifugation; Zinc

Bone metastasis is the major reason for death caused by breast cancer. We used human breast cancer (MCF-7) cells that are poorly metastatic but show highly inducible migration to determine bone-derived factors that induce migration of initially non-disseminating breast cancer cells. We have found that a lipid fraction from human osteoblast-like MG63 cell-conditioned medium (MG63CM) contains a migration-inducing factor for MCF-7 cells. In this fraction, we have identified oxysterol (OS) as a lipid mediator for tumor cell migration. In MCF-7 cells, insulin-like growth factor 1 elevates the expression of OS-binding protein-related protein 7. Binding of OS to OS-binding protein or OS-binding protein-related protein is known to trigger elevation of sphingomyelin, a sphingolipid that organizes lipid microdomains in the cell membrane. In MCF-7 cells, OS increases the intracellular concentration of sphingomyelin and other phospholipids and induces the translocation of the small GTPase p21Ras to GM1- and cholesterol-rich membrane areas. The induction of migration by MG63CM is prevented by incubation of MG63 cells with mevinolin, a statin-type cholesterol biosynthesis inhibitor that depletes the conditioned medium of OS. Osteoblast-derived OS may, thus, be a yet unrecognized lipid mediator for bone metastasis of breast cancer and a new target for anti-metastasis chemotherapy with statins.

Topics: Arachidonic Acid; Aspirin; Breast Neoplasms; Cell Membrane; Cell Movement; Cholesterol; Chromatography, High Pressure Liquid; Coculture Techniques; Culture Media, Conditioned; Dinoprostone; Electrophoresis, Polyacrylamide Gel; HSP70 Heat-Shock Proteins; Humans; Insulin-Like Growth Factor I; Lipid Metabolism; Lovastatin; MAP Kinase Signaling System; Microscopy, Fluorescence; Neoplasm Metastasis; Osteoblasts; Phospholipids; Phosphorylation; Prostaglandins B; Protein Transport; Proto-Oncogene Proteins p21(ras); Reverse Transcriptase Polymerase Chain Reaction; Sepharose; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sphingolipids; Sterols; Trypsin; Tumor Cells, Cultured

We previously reported that the tamoxifen treatment caused a decrease in lipoprotein lipase (LPL) activity and an increase in LPL mass. We hypothesized that tamoxifen may increase the quantity of inactive LPL.. Lipoprotein lipase in post-heparin plasma usually exists in both monomeric and dimeric forms, which may be separated on a heparin-Sepharose column with different salt concentrations. Lipoprotein lipase in post-heparin plasma from postmenopausal patients with hypertriglyceridemia treated with or without tamoxifen was incubated with or without 4-hydroxy-tamoxifen (4-OHT), the monomers and dimers were separated on a heparin-Sepharose column and their masses were measured.. The masses of total LPL and dimeric LPL of tamoxifen-treated patients were significantly higher than those of control subjects. Monomeric LPL of tamoxifen-treated patients passed more slowly through the heparin-Sepharose column compared with that of control subjects. The ratio of monomeric LPL to dimeric LPL of tamoxifen-treated patients was 0.61, significantly lower than that of control subjects, which was 1.45 (p<0.01). In addition, monomeric LPL incubated with 4-OHT passed more slowly through the heparin-Sepharose column compared with that incubated without 4-OHT.. Tamoxifen influences the affinity of LPL for heparin.

Topics: Breast Neoplasms; Cholesterol; Chromatography, Agarose; Estrogen Antagonists; Female; Heparin; Humans; Hyperlipoproteinemia Type IV; Lipoprotein Lipase; Postmenopause; Sepharose; Tamoxifen; Triglycerides

Histone deacetylase inhibitors induce hyperacetylation of the amino-terminal lysine residues of the core nucleosomal histones, which results in chromatin remodeling and altered gene expression. Present studies demonstrate that exposure to a novel hydroxamic acid analogue histone deacetylase inhibitor, LAQ824, induced p21WAF1 and p27KIP1 and caused growth arrest and apoptosis of human breast cancer SKBR-3 and BT-474 cells that possess amplification and overexpression of Her-2/neu. Treatment with LAQ824 depleted the mRNA and protein levels of Her-2/neu-encoded Her-2, which was associated with attenuation of pAKT, c-Raf-1, and phosphorylated mitogen-activated protein kinase levels. LAQ824 also induced the acetylation of heat shock protein (hsp) 90, resulting in inhibition of its binding to ATP, which has been shown to impair the chaperone association of hsp 90 with its client proteins, Her-2, AKT, and c-Raf-1. Consistent with this, treatment with LAQ824 shifted the binding of Her-2 from hsp 90 to hsp 70, promoting proteasomal degradation of Her-2. Thus, LAQ824 depletes Her-2 through two mechanisms: attenuation of its mRNA levels and promotion of its degradation by the proteasome. Following LAQ824 treatment, the cell membrane association, autotyrosine phosphorylation, and colocalization of Her-2 with HER-3 also declined. Cotreatment with LAQ824 significantly increased trastuzumab-induced apoptosis of BT-474 and SKBR-3 cells. This was associated with greater attenuation of Her-2, c-Raf-1, and pAKT levels. LAQ824 also enhanced taxotere-induced, epothilone B-induced, and gemcitabine-induced apoptosis of BT-474 and SKBR-3 cells. These findings suggest that LAQ824 is active against human breast cancer cells and has the potential to improve the efficacy of trastuzumab, taxotere, gemcitabine, and epothilone B against breast cancer with Her-2/neuamplification.

Topics: Annexin A5; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antimetabolites, Antineoplastic; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Northern; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Chromatin; Coloring Agents; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Endopeptidases; Deoxycytidine; Detergents; Docetaxel; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors; Epothilones; Flow Cytometry; Gemcitabine; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Microscopy, Fluorescence; Multienzyme Complexes; Phosphorylation; Precipitin Tests; Proteasome Endopeptidase Complex; Receptor, ErbB-2; Reverse Transcriptase Polymerase Chain Reaction; Sepharose; Taxoids; Tetrazolium Salts; Thiazoles; Time Factors; Trastuzumab

During mitotic arrest induced by paclitaxel, most of the mitochondrial Bcl-2 is phosphorylated. This mitotic arrest is transient; exit from mitosis, due to mitotic slippage, occurs and Bcl-2 is rapidly dephosphorylated. In the present study, we characterized PP1 as the cytosolic phosphatase involved in Bcl-2 dephosphorylation. When mitochondria and cytosol prepared from mitotic arrested cells were incubated in vitro, the proportion of phosphorylated forms of Bcl-2 in mitochondria remained unchanged. In contrast, cytosol prepared from cells during mitotic slippage led to a dose-dependent loss of phosphorylated forms of Bcl-2. Depletion of these cytosol extracts by microcystin-Sepharose maintained Bcl-2 phosphorylated forms, indicating that this cytosol possessed phosphatase activity. Furthermore, the dephosphorylation of Bcl-2 by cytosol prepared from cells exiting mitotic block was inhibited by okadaic acid, at a dose known to inhibit PP1, and by inhibitor 2, a specific inhibitor of PP1 and by immunodepletion of PP1. Finally, we showed that PP1 is associated with mitochondrial Bcl-2 in vivo. Taken together, these results demonstrate that PP1 is directly involved in Bcl-2 dephosphorylation during mitotic slippage.

Topics: Antineoplastic Agents; Breast Neoplasms; Cytosol; Enzyme Inhibitors; Female; Humans; Microcystins; Mitochondria; Mitosis; Paclitaxel; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphorylation; Precipitin Tests; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Sepharose; Tumor Cells, Cultured

Two in vitro models are compared to investigate whether cellular configuration or composition of the matrix in which the cells are cultured influences growth and/or prognostic parameters. T47D, MCF-7 and Hs578T breast cancer cell lines were cultured on two different matrices (agarose and collagen). Growth curves, biological markers (Ki-67, p53 and bcl-2) and the expression of hemostatic parameters were studied. The tested hemostatic parameters were urokinase-type plasminogen activator, tissue-type plasminogen activator and plasminogen activator inhibitor as fibrinolytic parameters and von Willbrand factor, tissue factor, antithrombin III, factor X and factor Xa as coagulation parameters. We found that T47D and MCF-7 formed spheroids in both matrices. Hs578T did not form spheroids; instead, the cells remained single cells in the agarose matrix and grew invasively through the collagen matrix. Expression of the biological markers was similar for spheroids and monolayers. In contrast, a clear difference in expression of hemostatic factors by spheroids and monolayers was found.

Topics: Biomarkers; Blood Coagulation Factors; Breast Neoplasms; Cell Division; Cells, Cultured; Collagen; Female; Hemostasis; Humans; Ki-67 Antigen; Kinetics; Plasminogen Inactivators; Prognosis; Proto-Oncogene Proteins c-bcl-2; Sepharose; Tissue Plasminogen Activator; Tumor Suppressor Protein p53; Urokinase-Type Plasminogen Activator

We studied the influence of 10 synthetic glycoamine analogs on colony formation in 0.3 and 0.9% agarose by metastatic human breast carcinoma (MDA-MB-435) and melanoma (TXM-13) cells. Nine synthetic analogs significantly inhibited the colony formation in 0.9% agarose of MDA-MB-435 human breast carcinoma cells; five compounds caused a 73-83% reduction of colony formation. Seven synthetic glycoamines caused a significant inhibition of colony formation in 0.9% agarose by TXM-13 melanoma cells with the inhibitory effect ranging from 71 to 87%. The 50% inhibition (I50) doses and relative activity rank of the compounds were similar for both breast carcinoma and melanoma cell lines. The murine B16 melanoma cell aggregation assay was employed to elucidate the potential mechanism(s) of the inhibitory activity of synthetic glycoamines. The relative activity ranks of the compounds based on the independently determined I50 doses for both cell aggregation and clonogenic growth assays were very similar for the four most active synthetic analogs and clearly indicated the importance of hydrophobic amino acid in mediating the bioactivity of synthetic glycoamines. In both experimental systems (clonogenic growth in agarose and cell aggregation assay) the leading compound was N-(1-deoxy-D-fructos-1-yl)-D-leucine (Fru-D-Leu) and the least active analog was N-(l-deoxy-D-fructos-1-yl)-glycine (Fru-Gly). These results show that synthetic glycoamines may act by competing for specific carbohydrate-lectin interactions, particularly those involving beta-galactoside-specific lectins expressed on metastatic cells.

Topics: Amino Sugars; Animals; Binding, Competitive; Breast Neoplasms; Carbohydrate Sequence; Carcinoma; Colony-Forming Units Assay; Female; Galactosides; Glucosamine; Humans; Lectins; Magnetic Resonance Spectroscopy; Mass Spectrometry; Melanoma; Mice; Molecular Sequence Data; Molecular Structure; Neoplasm Metastasis; Sepharose; Structure-Activity Relationship; Tumor Cells, Cultured; Tumor Stem Cell Assay

The purification of DNA polymerases (RNA-directed DNA polymerases and DNA-directed DNA polymerases) on poly(U)-Sepharose 4B from a breast tumour cell line (T-47D) is reported. The elution of these enzymes was followed in each fraction by activity measurements with the four primer-templates poly(rA)-oligo(dT)12-18, poly(dA) oligo(dT)12-18, poly(rC)-oligo(dG)12-18 and poly(rCm)-oligo (dG)12-18. The control of the polymerase purification by chromatography was performed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the pooled active enzymatic fractions.

Topics: Breast Neoplasms; Cell Line; Chromatography, Ion Exchange; DNA-Directed DNA Polymerase; Electrophoresis, Polyacrylamide Gel; Humans; Poly U; Sepharose

1. In previous studies we have isolated and characterized mucin-type glycopeptides from mouse and human melanoma cells. 2. These glycopeptides have clusters of oligosaccharides of the type (NeuNAc)0-2----[Gal----GalNAc] linked to serine and or threonine suggesting an apparent similarity to glycophorin. 3. We now report the interaction of polyclonal anti-glycophorin antibodies with various cultured cells. Antisera to highly purified glycophorin A were raised in rabbits. 4. Human melanoma cells (HM7), human breast cells (HBL-100) and two lines of human breast cancer cells (MCF-7 and MDA-MB-231) showed medium to very strong cell surface fluorescence pattern after staining with rabbit anti-glycophorin F(ab')2 and FITC-conjugated goat anti-rabbit F(ab')2. 5. Immunodiffusion, immunoelectrophoresis and affinity chromatography on anti-glycophorin IgG-Sepharose 4B of detergent extracts of metabolically labeled cultured cells gave further evidence for the presence of glycophorin-like components in these cells. 6. Glycoproteins of MCF-7 cells interacting with anti-glycophorin antibodies were affinity purified and partially characterized.

Topics: Breast Neoplasms; Cell Line; Cells, Cultured; Chromatography, Affinity; Erythrocyte Membrane; Fluorescent Antibody Technique; Glycophorins; Humans; Immune Sera; Immunodiffusion; Membrane Glycoproteins; Sepharose; Sialoglycoproteins

The purpose of this study was to determine whether the degree of anchorage-independent growth of human tumor cells in increasing concentrations of agarose correlated with the capacity of the cells to produce experimental metastases in nude mice. Human melanoma, breast carcinoma, and colon carcinoma cells from parental lines and variants selected in vivo for metastasis and in vitro cloned lines were plated into medium containing 0.3%, 0.6%, 0.9%, or 1.2% of agarose. These cells were also injected into nude mice: intravenously for melanoma, into the mammary fat pad for breast carcinoma, and into the spleen for colon carcinoma. Production of tumor cell colonies in dense agarose (greater than 0.6%) correlated with production of experimental metastases in the lung (melanoma, breast carcinoma) or liver (colon carcinoma). We conclude that the degree of anchorage-independent growth of tumor cells can predict their biological behavior and metastatic potential in vivo. Thus, this technique may be useful for the isolation of metastatic cells from heterogeneous human neoplasms.

Topics: Animals; Breast Neoplasms; Clone Cells; Colonic Neoplasms; Culture Media; Humans; Lung Neoplasms; Melanoma; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Sepharose; Tumor Cells, Cultured

A high resolution and quantitative method for isoelectric focusing has been developed to separate the isoforms of estrogen and progesterone receptors in human mammary tumor cytosols stabilized by sodium molybdate. Agarose gels (0.5%) were used. Six samples can be analyzed on one gel in about 2 h, and 35-microliters samples are sufficient to determine the estrogen receptor isoform pattern. The constant yields and the reproducibility of data allow a quantitative analysis of these receptors. Four estrogen receptor isoforms have been observed (pI 4.7, 5.5, 6, and 6.5), isoforms with pI 4.7 and 6.5 being present in all tumors. After incubation at 28 degrees C in high ionic strength, the comparison of isoelectric focusing and high-performance size exclusion chromatography patterns of estrogen receptor confirms the oligomeric structure of the pI 4.7 isoform and suggests a monomeric structure for the pI 6.5 isoform. Under the same conditions of analysis, only one progesterone receptor isoform has been detected with pI 4.7.

Topics: Aged; Aged, 80 and over; Breast Neoplasms; Chromatography, High Pressure Liquid; Female; Humans; Hydrogen-Ion Concentration; Isoelectric Focusing; Receptors, Estrogen; Receptors, Progesterone; Sepharose

The growth of the human metastatic cell line (T-47D) in a chemically defined medium (DM) is shown to be dependent on the presence of three traditional growth factors: epidermal growth factor, insulin, and transferrin. The addition of thrombin further stimulates its growth. The mitogenic action on a human mammary tumor cell line from epithelial origin is a novel action of thrombin. Cells in the DM show striking morphological changes which are dramatically enhanced by the addition of thrombin. These observations are part of a pleiotropic response to the growth factors: the protein content of the cells increases in the defined medium; the 2DG gels of the 35S- and 32P-labeled proteins show important changes in spots, several of which are probably of cytoskeletal origin. It is also shown that cells in a semisolid growth factor-supplemented medium have growth advantages over their counterparts grown with serum. All the phenotypic changes mentioned above reveal the important role of growth factors in the growth and behavior of this mammary cell line. The results obtained with thrombin indicate a new site of action of this enzyme which may be important in the metastatic spread of human mammary tumor cells.

Topics: Breast Neoplasms; Cell Division; Culture Media; DNA Replication; Drug Synergism; Epidermal Growth Factor; Gene Expression Regulation; Humans; Insulin; Neoplasm Proteins; Phosphorylation; Sepharose; Thrombin; Transferrin; Tumor Cells, Cultured

Several affinity chromatography reagents have been proposed for purification of progesterone receptor (PgR), and significant results have been achieved with some of these. None, however, have approached the results achieved in affinity chromatography of estrogen receptor. We have therefore synthesized a number of new 19-nortestosterone derivatives capable of chemically stable linkage with Sepharose beads, and have identified one with very high PgR affinity for further study. We first synthesized the epoxides of 17 alpha-allyl nortestosterone, by analogy with the estradiol derivatization of Greene and Jensen. The relative affinity of these epoxides for PgR from T47D human breast cancer cells, however, was only around 5% that of R5020, and affinity beads prepared from them bound very little PgR. We then reacted appropriately protected 17 alpha-ethynyl-nortestosterone with a series of diiodo alkanes, and found that 17 alpha-(6'-iodohex-1'-ynyl)nortestosterone had an affinity of 22% relative to R5020, equal to the affinity of progesterone itself. Reaction with Thiopropyl-Sepharose 6B yielded hexynyl-nortestosterone-Sepharose beads with a ligand density of about 7 micromoles/ml beads. One-hundred microliter of these beads adsorbed 71% of the PgR present in 1 ml of cytosol from T47D cells. This adsorption was inhibited by 10 microM progesterone but not cortisol, indicating the specificity of the binding. Comparisons with NADAC and Sterogel, other affinity beads used for PgR purification, show that the former takes up much less receptor, while the latter takes up and releases similar amounts of receptor but more extraneous protein, and is less stable. We therefore believe that hexynyl-nortestosterone-Sepharose, having a high density of a high affinity ligand, and having chemically and biochemically stable covalent bonds, should be a good reagent for affinity purification of PgR.

Topics: Animals; Breast Neoplasms; Cell Line; Chemical Phenomena; Chemistry; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Female; Humans; Nandrolone; Promegestone; Rats; Rats, Inbred Strains; Receptors, Progesterone; Sepharose; Uterus

Plasma from 182 patients with different malignant diseases was tested for riboflavin binding by immunoglobulins, which have been recently identified as major carriers of this micronutrient. A wide range of binding (5.9 to 130 pmole/ml plasma) was observed, and significant elevations were found for patients having breast cancer (21.2 +/- 1.9, P less than 0.05) and melanoma (25.7 +/- 1.9, P less than 0.001) compared to controls (15.5 +/- 1.9). The proteins responsible for a majority of the higher binding were identified as immunoglobulins, based on their elution from gel filtration columns and the removal of 57-88% of the non-albumin binding by treating of plasma with Protein A-agarose. The binding was only weakly related to the total concentration of immunoglobulins (r = 0.11 by linear regression analysis), however, and is apparently due to a subclass that is elevated in some types of cancer. Elevated levels of these immunoglobulins may contribute to the lower urinary levels and clearance of riboflavin in cancer.

Topics: Breast Neoplasms; Chromatography, Gel; Female; Humans; Immunoglobulin G; In Vitro Techniques; Male; Melanoma; Neoplasms; Nephelometry and Turbidimetry; Protein Binding; Riboflavin; Sepharose

Leukocyte migration inhibition (LMI) in patients with breast cancer was examined by using 3 M-KCl tissue extracts of breast tumors. Tumor extracts were obtained from 12 breast carcinomas of various histological types. In 20 of 65 (30.8%) leukocyte preparations from 25 patients with breast cancer migration was inhibited by breast cancer tissue extracts. In 26 leukocyte preparations from eight healthy persons and 22 preparations from eight patients with benign diseases of the breast migration was not inhibited by the breast cancer extracts. In only two of 47 (4.3%) leukocyte preparations from 12 patients with other cancers migration was inhibited by the extracts. The occurrence of LMI was the highest in leukocytes from the patients with breast cancer showing marked nuclear pleomorphism (10/19, 52.6%) or showing marked mononuclear cell infiltration (6/12, 50.0%). These results suggest that 3 M-KCl tissue extracts of breast cancer may contain tumor-associated antigens in solubilized form and that there may be a correlation between LMI and nuclear pleomorphism of cancer cells of the leukocyte donor as well as mononuclear cell infiltration in the tumor of the leukocyte donor.

Topics: Adenofibroma; Agar; Antigens, Neoplasm; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Cell Migration Inhibition; Cell Nucleus; Cell Separation; Female; Humans; Leukocytes; Lymphocytes; Mitotic Index; Neoplasm Staging; Sepharose; Staining and Labeling; Tissue Extracts

We tested the ability of a serum-free medium containing insulin, transferrin, 17 beta-estradiol, dexamethasone, triiodothyronine, prostaglandin F2 alpha, and fibronectin (HBCA medium) to support the continuous growth and passage of five human breast carcinoma cell lines on a collagen matrix. Doubling times of the cell lines (20 to 44 hr) were similar in HBCA and serum-supplemented media. The gross morphology of the cell lines was not altered in the serum-free medium. Insulin, transferrin, and the collagen matrix were the most essential factors required for optimal growth of the cell lines. Estradiol appeared to stimulate the growth of cell lines, both with and without estrogen receptors. HBCA medium supplemented with low concentrations of bovine serum albumin, Fraction V (0.5%, v/v), supported the clonal growth of three cell lines in soft agarose with colony-forming efficiencies superior to that observed with standard serum-supplemented medium. Deleting estradiol from HBCA medium reduced the colony-forming efficiency of the three cell lines. HBCA medium may be useful in studying hormonal regulation and improving the in vitro growth of human breast cancer.

Topics: Breast Neoplasms; Cell Division; Cell Line; Clone Cells; Collagen; Culture Media; Culture Techniques; Female; Hormones; Humans; Sepharose

Leukocyte migration tests under agarose (Clausen technique) were performed in 28 patients tentatively diagnosed as having any malignancy with the use of a 3 M KCl-extract panel prepared from bronchogenic, gastric, colonic, renal, and mammary carcinoma, corresponding normal tissues, carcinoembryonic antigen (CEA), and human encephalitogenic protein (HEP). 17 out of 22 proven carcinoma patients showed sensitization by reaction with optimal concentrated KCl-extract of cancer from the same organ type as their own tumor. In some cases positive reactions could be observed also with normal tissue antigen (NTA) of tumor organ type (7/22) or with an additional carcinoma extract of organ type differing from patients own primary tumor (8/22). Gastrointestinal carcinomas, especially, showed sensitization to CEA (7/12) contrary to nongastrointestinal carcinomas (1/10). With HEP no positive reactivity could be found (0/10). With the use of tumor antigen panel (5 antigens) only few positive reactions (MI less than 0.80 or greater than 1.20) could be observed in 6 patients with nonmalignant diseases (1/30 tests) and 8 healthy blood donors (1/40 tests). A widespread individual screening program using tissue antigens for patients suspected of malignancies could give a pattern of reactivities and improve the recognition of cell-mediated sensitization against tumor tissues.

Topics: Aged; Antigens, Neoplasm; Breast Neoplasms; Carcinoembryonic Antigen; Cell Migration Inhibition; Colonic Neoplasms; Epitopes; Female; Humans; Immunity, Cellular; Kidney Neoplasms; Leukocytes; Lung Neoplasms; Male; Middle Aged; Myelin Basic Protein; Neoplasms; Sepharose; Stomach Neoplasms