cytochalasin-b and Breast-Neoplasms

The response of two well-characterized human breast cancer cell lines (MCF-7 and MDA-MB-231) to a series of nutrient deficiencies is investigated with a label-free cell assay platform. The motivation of the research is to analyze adaptive responses of tumor cell metabolism and to find limiting conditions for cell survival. The platform measures extracellular values of pH and dissolved oxygen saturation to provide data of extracellular acidification rates and oxygen uptake rates. Additional electric cell substrate impedance sensing and bright-field cell imaging supports the data interpretation by providing information about cell morphological parameters. A sequential administration of nutrient depletions does not cause metabolic reprogramming, since the ratios of oxygen uptake to acidification return to their basal values. While the extracellular acidification drops sharply upon reduction of glucose and glutamine, the oxygen uptake is not affected. In contrast to other published data, cell death is not observed when both glucose and glutamine are depleted and cell proliferation is not inhibited, at least in MCF-7 cultures. It is assumed that residual concentrations of nutrients from the serum component are able to maintain cell viability when delivered regularly by active flow like in the cell assay platform, and, in a similar way, under physiological conditions.

Topics: Actins; Antineoplastic Agents; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Survival; Cytochalasin B; Glucose; Glucose Transporter Type 1; Glutamine; Humans; Hydrogen-Ion Concentration; Lactic Acid; MCF-7 Cells; Metabolome; Oxazines; Oxygen; RNA, Messenger; Xanthenes

F18 2-Fluoro 2-deoxyglucose (FDG) has been the gold standard in positron emission tomography (PET) oncologic imaging since its introduction into the clinics several years ago. Seeking to complement FDG in the diagnosis of breast cancer using radio labeled fructose based analogs, we investigated the expression of the chief fructose transporter-GLUT 5 in breast cancer cells and human tissues. Our results indicate that GLUT 5 is not over-expressed in breast cancer tissues as assessed by an extensive immunohistochemistry study. RT-PCR studies showed that the GLUT 5 mRNA was present at minimal amounts in breast cancer cell lines. Further knocking down the expression of GLUT 5 in breast cancer cells using RNA interference did not affect the fructose uptake in these cell lines. Taken together these results are consistent with GLUT 5 not being essential for fructose uptake in breast cancer cells and tissues.

Topics: Base Sequence; Breast Neoplasms; Cell Line, Tumor; Cytochalasin B; DNA Primers; Female; Fructose; Gene Knockdown Techniques; Glucose Transporter Type 5; Humans; Immunohistochemistry; RNA, Small Interfering

Chromosomal instability could be one of primary causes for malignant cell transformation. The objective of the present study was to evaluate the spontaneous genetic damages in circulated lymphocytes of newly diagnosed cancer patients by using cytokinesis-block micronucleus (CBMN) assay, with respect to the factors that might affect micronucleus frequency (i.e. age, gender, smoking habits and cancer sites). Micronuclei (MN) are small nuclei that are originated from chromosome fragments or whole chromosomes. The analyzed samples included 44 untreated cancer patients (19 females and 25 males with mean age of 60.89 years) with different cancer sites (12 patients with breast cancer, 5 with uterine cancer and 27 with cancer of pharynx). Control group included 40 healthy donors (28 females and 12 males with mean age of 43.95 years). The mean baseline MN frequency was significantly higher (p < 0.001) in cancer patients (15.18 +/- 5.05 MN/1000 BN cells ranging from 4 to 27) than the baseline frequency in healthy controls (6.45 +/- 2.75 MN/1000 BN cells, ranging from 1 to 11). There was no gender difference in baseline MN frequency in cancer patients and healthy controls. Moreover, the MN frequency did not significantly differ among cancer sites, and between smokers and non-smokers in both patient and control samples. In conclusion, untreated cancer patients may be associated with an increase of chromosomal instability in peripheral blood lymphocytes, irrespective of gender, cigarette smoking and cancer sites.

Topics: Adult; Aged; Aged, 80 and over; Aging; Breast Neoplasms; Chromosomal Instability; Cytochalasin B; Cytokinesis; Female; Humans; Leukocytes, Mononuclear; Male; Micronuclei, Chromosome-Defective; Micronucleus Tests; Middle Aged; Neoplasms; Organ Specificity; Pharyngeal Neoplasms; Sex Characteristics; Smoking; Uterine Neoplasms

Recently we have described an HPMA copolymer conjugate carrying both the aromatase inhibitor aminoglutethimide (AGM) and doxorubicin (Dox) as combination therapy. This showed markedly enhanced in vitro cytotoxicity compared to the HPMA copolymer-Dox (FCE28068), a conjugate that demonstrated activity in chemotherapy refractory breast cancer patients during early clinical trials. To better understand the superior activity of HPMA copolymer-Dox-AGM, here experiments were undertaken using MCF-7 and MCF-7ca (aromatase-transfected) breast cancer cell lines to: further probe the synergistic cytotoxic effects of AGM and Dox in free and conjugated form; to compare the endocytic properties of HPMA copolymer-Dox-AGM and HPMA copolymer-Dox (binding, rate and mechanism of cellular uptake); the rate of drug liberation by lysosomal thiol-dependant proteases (i.e. conjugate activation), and also, using immunocytochemistry, to compare their molecular mechanism of action. It was clearly shown that attachment of both drugs to the same polymer backbone was a requirement for enhanced cytotoxicity. FACS studies indicated both conjugates have a similar pattern of cell binding and endocytic uptake (at least partially via a cholesterol-dependent pathway), however, the pattern of enzyme-mediated drug liberation was distinctly different. Dox release from PK1 was linear with time, whereas the release of both Dox and AGM from HPMA copolymer-Dox-AGM was not, and the initial rate of AGM release was much faster than that seen for the anthracycline. Immunocytochemistry showed that both conjugates decreased the expression of ki67. However, this effect was more marked for HPMA copolymer-Dox-AGM and, moreover, only this conjugate decreased the expression of the anti-apoptotic protein bcl-2. In conclusion, the superior in vitro activity of HPMA copolymer-Dox-AGM cannot be attributed to differences in endocytic uptake, and it seems likely that the synergistic effect of Dox and AGM is due to the kinetics of intracellular drug liberation which leads to enhanced activity.

Topics: Aminoglutethimide; Antineoplastic Agents; Apoptosis; beta-Cyclodextrins; Breast Neoplasms; Cell Survival; Chlorpromazine; Cytochalasin B; Doxorubicin; Endocytosis; Female; Flow Cytometry; Humans; Immunohistochemistry; Ki-67 Antigen; Kinetics; Liver; Lysosomes; Methacrylates; Microscopy, Confocal; Proto-Oncogene Proteins c-bcl-2; Tetrazolium Salts; Thiazoles

The micronucleus assay (MNT) in human lymphocytes is frequently used to assess chromosomal damage as a consequence of environmental mutagen exposure, to assess the effect of mutagens or to search for reduced DNA repair capacity after a mutagenic challenge. We have established an automated scoring procedure for the cytokinesis blocked MNT based on computerized image analysis (Metasystems Metafer 4 version 2.12). To evaluate the results we used the reproducibility of counts, established a dose-response curve for gamma-irradiation and used the ability of the system to differentiate between breast cancer patients and controls as a biological reference, a difference which we had observed before by visual counting. Blood cultures were irradiated with gamma-rays (2 Gy) at the beginning and treated with cytochalasin B during the last 24 h. The slides were stained with Giemsa for visual counting and with DAPI for automated analysis. Our test sample consisted of 73 persons (27 with breast cancer and 26 female and 20 male controls). A comparison between visual counting (controls, mean MN frequency 313) and automated counting (mean MN frequency 106) in slides from the same culture revealed a large drop for the automated counts. However, the automated counts were as reproducible as the visual counts [coefficient of variation (CV) on the sample approximately 20%; CV on repeated counts of the same slides approximately 5%] and both counts were highly correlated. Furthermore, the discrimination between cases and controls improved for automated counting of slides from the same cultures [visual odds rato (OR) < or = 4.0, P = 0.009; automated OR > 16, P < 0.0001], with a strong dependence on the set of parameters used. This improvement was confirmed in a validation sample of an additional 21 controls and 20 cases (OR = 11, P = 0.0018) performed as a prospective or diagnostic test.

Topics: Algorithms; Automation; Breast Neoplasms; Case-Control Studies; Cell Nucleus; Cells, Cultured; Chromosomes; Cytochalasin B; DNA Repair; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Female; Fluorescent Dyes; Gamma Rays; Humans; Image Processing, Computer-Assisted; Lymphocytes; Male; Micronucleus Tests; Mutagens; Odds Ratio; Regression Analysis; Reproducibility of Results; Time Factors

Tetraspanins (or proteins from the transmembrane 4 superfamily, TM4SF) form membrane complexes with integrin receptors and are implicated in integrin-mediated cell migration. Here we characterized cellular localization, structural composition, and signaling properties of alpha3beta1-TM4SF adhesion complexes. Double-immunofluorescence staining showed that various TM4SF proteins, including CD9, CD63, CD81, CD82, and CD151 are colocalized within dot-like structures that are particularly abundant at the cell periphery. Differential extraction in conjunction with chemical cross-linking indicated that the cell surface fraction of alpha3beta1-TM4SF protein complexes may not be directly linked to the cytoskeleton. However, in cells treated with cytochalasin B alpha3beta1-TM4SF protein complexes are relocated into intracellular vesicles suggesting that actin cytoskeleton plays an important role in the distribution of tetraspanins into adhesion structures. Talin and MARCKS are partially codistributed with TM4SF proteins, whereas vinculin is not detected within the tetraspanin-containing adhesion structures. Attachment of serum-starved cells to the immobilized anti-TM4SF mAbs induced dephosphorylation of focal adhesion kinase (FAK). On the other hand, clustering of tetraspanins in cells attached to collagen enhanced tyrosine phosphorylation of FAK. Furthermore, ectopic expression of CD9 in fibrosarcoma cells affected adhesion-induced tyrosine phosphorylation of FAK, that correlated with the reorganization of the cortical actin cytoskeleton. These results show that tetraspanins can modulate integrin signaling, and point to a mechanism by which TM4SF proteins regulate cell motility.

Topics: Actins; Antigens, CD; Breast Neoplasms; Cell Adhesion; Cell Adhesion Molecules; Culture Media, Serum-Free; Cytochalasin B; Cytoskeleton; Extracellular Matrix Proteins; Fibrosarcoma; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Integrin alpha3beta1; Integrins; Intercellular Junctions; Intracellular Signaling Peptides and Proteins; Membrane Glycoproteins; Membrane Proteins; Myristoylated Alanine-Rich C Kinase Substrate; Nocodazole; Organelles; Phosphorylation; Protein-Tyrosine Kinases; Proteins; Signal Transduction; Solubility; Talin; Tetraspanin 29; Tumor Cells, Cultured; Vinculin

To investigate whether radiation-induced micronucleus formation as expressed by the cytochalasin-blocked Mn-assay correlates with cellular radiosensitivity measured by a colony assay in primary fibroblast cultures from cancer patients.. Studies were made on skin fibroblasts from 36 breast cancer patients. The micronucleus assay was performed using treatment with cytochalasin-B to create binucleate cells. Response was scored in terms of the percentage of binucleate cells with micronuclei, also as the number of micronuclei per binucleate cell. The data were related to previously published results of cell survival measurements on these cell lines.. Neither endpoint for micronucleus formation showed a correlation with radiosensitivity by the colony assay. The fraction of fibroblasts that reach mitosis without micronuclei also failed to correlate with cell survival.. Among these primary fibroblast cell lines radiation-induced micronucleus formation was not associated with radiosensitivity as measured by a colony assay.

Topics: Adult; Aged; Biopsy; Breast Neoplasms; Cell Survival; Cells, Cultured; Combined Modality Therapy; Cytochalasin B; Female; Fibroblasts; Humans; Micronucleus Tests; Middle Aged; Mitosis; Regression Analysis; Skin

When human granulocytes were stimulated with the chemotactic peptide FNLPNTL (N-formyl-norleucyl-leucyl-phenylalanyl-norleucyl-tyrosinyl- lysin; in the presence of cytochalasin B), proteolytic enzymes were released which prevented activation of tumor-cell derived pro-uPA by plasmin. Elastase was identified by use of eglin C (elastase inhibitor) and an inhibitory monoclonal antibody to elastase as the functional proteolytic enzyme in these granulocyte supernatants. Purified human granulocyte elastase cleaves pro-uPA at amino acid position lle159-lle160 thus generating an enzymatically inactive two-chain form of uPA, as judged by N-terminal amino acid sequence analysis. An additional minor elastase-mediated cleavage site was detected at position Thr165-Thr166. This form of uPA was indistinguishable by SDS-PAGE from plasmin-generated enzymatically active HMW-uPA. Action of plasmin on the proenzyme form of uPA (pro-uPA) generates an enzymatically active uPA-molecule (high molecular weight form; HMW-uPA) which is cleaved at amino acid position Lys158-lle159 (Mr = 33,000 (B-chain) and 22,000 (A-chain). Thus elastase cannot substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active HMW-uPA. Enzymatically active HMW-uPA, however, was not affected by elastase. Elastase-containing granulocytes were identified by immunohistochemical staining of elastase in breast cancer tissue. Granulocytes were located close to the tumor cells and also in the tumor stroma surrounding the tumor nests. These tumor cells contain pro-uPA. Evidently, the conversion of tumor cell pro-uPA into enzymatically active HMW-uPA is controlled by elastase released from granulocytes into the tumor tissue.

Topics: Breast Neoplasms; Chemotactic Factors; Cytochalasin B; Endopeptidases; Enzyme Activation; Enzyme Precursors; Fibrinolysin; Granulocytes; Humans; Immunohistochemistry; Oligopeptides; Pancreatic Elastase; Thrombin; Urokinase-Type Plasminogen Activator

The effects of doxorubicin (adriamycin, ADR) and daunorubicin (daunomycin, DAU), two anthracyclinic antibiotics, on a human breast carcinoma cell line (CG5) were studied by cytochemical and morphological methods. Both ADR and DAU were capable of inducing the multinucleation and spreading phenomena, associated with a decrease of the cell growth rate. DAU appeared to be more effective than ADR at the tested concentrations (10(-5), 5 x 10(-5) mM), in affecting the cell growth as well as in inducing multinucleation. As revealed by scanning electron microscopy, spreading and multinucleation were accompanied by a remarkable redistribution of surface structures. Moreover, a dose- and time-dependent rearrangement of the underlying cytoskeletal components was clearly detected. In addition, both ADR and DAU at 5 x 10(-5) mM seemed to favor the rebuilding of microtubules after treatment with colcemid, while a higher dose (10(-4) mM) exerted the opposite effect. Furthermore, both anthracyclines prevented the action of the antimicrotubular agent. When recovered after treatment with cytochalasin B, in presence of ADR (or DAU) (5 x 10(-5), 10(-4) mM), cells showed a microfilament pattern rearranged differently as compared to that of cells recovered in anthracycline-free medium. The results reported here strongly suggest the involvement of actin and tubulin in CG5 cell response to ADR and DAU treatments. Thus, the cytoskeletal apparatus is confirmed as another target involved in the mechanism of action of anthracyclines.

Topics: Actins; Antibiotics, Antineoplastic; Breast Neoplasms; Cell Transformation, Neoplastic; Cytochalasin B; Cytoskeleton; Daunorubicin; Demecolcine; Doxorubicin; Humans; Microscopy, Electron; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Tubulin; Tumor Cells, Cultured

Micronucleation was induced by vincristine, colcemid, and colcemid in combination with cytochalasin B in cells of a human metastatic breast carcinoma cell line (MDA MB 231). Cells treated with the latter combination were enucleated subsequently by centrifugation in the presence of cytochalasin B. The resulting "microcell" fraction was fused with mitotic human primary fibroblasts or mitotic HeLa cells using polyethyleneglycol (PEG). The success of the microcell-mediated chromosome transfer thus could be demonstrated as premature condensation in the mitotic recipient of the transferred micronuclei. This technique of cytogenetic analysis allowed a fast and simple control of the influence of different conditions on micronucleation and fusion of micronuclei with recipient cells. It could be shown that microcell-mediated chromosome transfer from human tumor cells into human normal, as well as human tumor, recipient cells is practicable if the techniques figured out by the present study are employed.

Topics: Breast Neoplasms; Cell Fusion; Cell Line; Cell Nucleus; Chromosomes, Human; Cytochalasin B; Demecolcine; Female; Genetic Engineering; Genetic Vectors; HeLa Cells; Humans; Neoplasms; Polyethylene Glycols; Vincristine

The relation of in vitro properties to tumorigenicity was studied using eight sublines of the human breast cancer cell line MCF-7. Four of the eight were tumorigenic in estrogen-treated nude mice. The sublines differed for each of the in vitro properties measured, and no property correlated perfectly with tumorigenicity. Cytochalasin B-induced multinucleation was a property of all four tumorigenic sublines but of only one of the four nontumorigenic ones. Anchorage-independent growth and concanavalin A-mediated hemadsorption levels were higher in all sublines than reported levels for nontransformed fibroblasts and normal human or mouse mammary epithelial cells. The production of both plasminogen activator and a plasminogen-independent fibrinolytic activity showed no relationship to tumorigenicity but was higher in those sublines producing more invasive tumors. It appears that no one of these in vitro properties is sufficient to make a subline tumorigenic. Rather, the first three properties studied here and, perhaps, also production of plasminogen activator may each be necessary, but not sufficient, to make a subline tumorigenic. In addition, properties such as production of plasminogen activator and other proteases, while perhaps not essential to tumorigenicity, may confer characteristics, such as invasiveness, on the tumors produced by a given subline.

Topics: Animals; Breast Neoplasms; Cell Line; Concanavalin A; Cytochalasin B; Female; Hemadsorption; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Plasminogen Activators

An experimental in vitro or in vivo tumour model should be unchanged represent the biological properties (e.g. histology, proliferation). Changes of tumour cell populations were determined by means of DNA-distribution and multinucleation after cytochalasin B treatment. Flow cytometry measurements on cell cultures in 50 ml glass culture flasks reveal reduction of polyploid cells after collagenase treatment of human mammary carcinomas. Selection of cell populations are responsible for the failed induction of multinucleation by cytochalasin B. In organ cultures the composition of cell population prior to and after 48 hours could maintained. The improved penetration could be demonstrated by autoradiographic measurements of 3H-thymidine incorporation. Thickness, surface and improved penetration of metabolites of vital tissue slices seem to be also important for cell movement and cell division. In 10 out of 12 experiments an earlier cell migration and proliferation could be observed from vital slices than from tissue pieces. Organ cultures represent sufficiently carcinoma in vivo and are more suitable than other mentioned in vitro cell culture methods.

Topics: Autoradiography; Breast Neoplasms; Cells, Cultured; Cytochalasin B; DNA, Neoplasm; Flow Cytometry; Humans; Models, Biological; Organ Culture Techniques

In order to study the biological nature of various mammary tumors, differences in the formation of polynuclear cells after the administration of cytochalasin B were investigated in cultures of human mammary tumors and normal mammary gland. When cytochalasin B was applied to the cultures, polynuclear cells increased in all cancer cases (6.1% on average), but relatively little effect was seen in cases of benign tumors and normal mammary gland (less than 1.1% on average). From these results, it appears that the difference in polynucleation on treatment with cytochalasin B may be useful as a biological means to distinguish human malignant and benign mammary tumors.

Topics: Breast; Breast Neoplasms; Cell Nucleus; Cells, Cultured; Cytochalasin B; Female; Fibrocystic Breast Disease; Humans

When supernates from the established human breast cancer cell line MCF-7 were applied to fetal rat long bones that had been labeled with 45Ca and devitalized to remove endogenous bone cells, mineral was released from the bones. The release of bone mineral by MCF-7 supernates was associated with increased basal release of hydrolytic enzyme activity by the tumor cells. The basal release of lysosomal enzymes and collagenolytic activity by MCF-7 cells with approximately twice that of mouse 3T3 cells, which did not cause mineral release by the fetal rat bones. Release of hydrolytic enzymes and bone mineral-releasing activity was increased by colchicine and vinblastine, drugs that inhibit microtubule assembly, but not affected by lumicolchicine. Time-course experiments performed on MCF-7 cells with or without colchicine showed that release of cathepsin D and collagenolytic activity was associated more closely with release of bone mineral and degradation of bone matrix than was the release of N-acetylglucosaminidase. The release of previously incorporated [3H]proline from the bones exposed to MCF-7 cell cultures was more closely associated with release of collagenolytic activity by MCF-7 cells than with release of cathepsin D or N-acetylglucosaminidase. These data suggest that breast cancer-mediated bone resorption in vitro is positively correlated with release of hydrolytic enzymes by the tumor cells, and release of these enzymes is enhanced by disassembly of microtubules.

Topics: Acetylglucosaminidase; Animals; Bone and Bones; Breast Neoplasms; Calcium; Cell Line; Colchicine; Cytochalasin B; Humans; Hydrolases; Microbial Collagenase; Microtubules; Rats