cytochalasin-b and Carcinoma--Squamous-Cell

C57BL mice bearing EL4 tumors and C3H / He mice bearing SCC VII tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. Three hours after oral administration of l-p-boronophenylalanine-(10)B (BPA), or 30 min after intraperitoneal injection of sodium borocaptate-(10)B (BSH) or l-p-boronophenylalaninol (BPA-ol), a newly developed (10)B-containing alpha-amino alcohol, the tumors were irradiated with thermal neutron beams. For the combination with mild temperature hyperthermia (MTH) and / or tirapazamine (TPZ), the tumors were heated at 40 degrees C for 30 min immediately before neutron exposure, and TPZ was intraperitoneally injected 30 min before irradiation. The tumors were then excised, minced and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling ( = quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. Meanwhile, 6 h after irradiation, tumor cell suspensions obtained in the same manner were used for determining the apoptosis frequency in Q cells. The MN and apoptosis frequency in total (P + Q) tumor cells were determined from tumors that were not pretreated with BrdU. Without TPZ or MTH, BPA-ol increased both frequencies most markedly, especially for total cells. However, as with BPA, the sensitivity difference between total and Q cells was much larger than with BSH. On combined treatment with both MTH and TPZ, this sensitivity difference was markedly reduced, similarly to when BPA was used. MTH increased the (10)B uptake of all (10)B-compounds into both tumor cells. BPA-ol has good potential as a (10)B-carrier in neutron capture therapy, especially when combined with both MTH and TPZ.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Apoptosis; Boranes; Boron Neutron Capture Therapy; Bromodeoxyuridine; Carcinoma, Squamous Cell; Combined Modality Therapy; Cytochalasin B; Drug Screening Assays, Antitumor; Fluorescent Antibody Technique, Indirect; Hindlimb; Hyperthermia, Induced; Injections, Intraperitoneal; Interphase; Lymphoma; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Micronucleus Tests; Molecular Structure; Neutrons; Phenylalanine; Radiation-Sensitizing Agents; Radiometry; Tirapazamine; Triazines

Response of quiescent (Q) and total tumor cells in solid tumors to reactor neutron beam irradiation with two different cadmium (Cd) ratios was examined in terms of micronucleus (MN) frequency and apoptosis frequency, using four different tumor cell lines.. C57BL mice bearing EL4 tumors, C3H/He mice bearing SCC VII or FM3A tumors, and Balb/c mice bearing EMT6/KU tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. Thirty min after i.p. injection of sodium borocaptate-10B (BSH), or 3 h after oral administration of p-boronophenylalanine-10B (BPA), the tumors were irradiated with neutron beams. The tumors without 10B-compound administration were irradiated with neutron beams or gamma-rays. This neutron beam irradiation was performed using neutrons with two different Cd ratios. The tumors were then excised, minced, and trypsinized. The tumor cell suspensions thus obtained were incubated with cytochalasin-B (a cytokinesis blocker), and the MN frequency in cells without BrdU labeling (=Q cells) was determined using immunofluorescence staining for BrdU. Meanwhile, for apoptosis assay, 6 h after irradiation, tumor cell suspensions obtained in the same manner were fixed, and the apoptosis frequency in Q cells was also determined with immunofluorescence staining for BrdU. The MN and apoptosis frequencies in total (P + Q) tumor cells were determined from the tumors that were not pretreated with BrdU.. Without 10B-compounds, the sensitivity difference between total and Q cells was reduced by neutron beam irradiation. Under our particular neutron beam irradiation condition, relative biological effectiveness (RBE) of neutrons was larger in Q cells than in total cells, and the RBE values were larger for low Cd-ratio than high Cd-ratio neutrons. With 10B-compounds, both frequencies were increased for each cell population, especially for total cells. BPA increased both frequencies for total cells more than BSH did. Nevertheless, the sensitivity of Q cells treated with BPA was lower than that of Q cells treated with BSH. Whether based on the MN frequency or the apoptosis frequency, similar results concerning the sensitivity difference between total and Q cells, the values of RBE, and the enhancement effect by the use of 10B-compound were obtained.. Apoptosis frequency, as well as the MN frequency, can be applied to our method for measuring the Q cell response to reactor neutron beam irradiation within solid tumor in which the ratio of apoptosis to total cell death is relatively high, as in EL4 tumor. The absolute radiation dose required to achieve the same endpoint for Q cells is much higher than that for total cells when combined with 10B-compound, especially with BPA.

Topics: Animals; Apoptosis; Borohydrides; Boron Compounds; Boron Neutron Capture Therapy; Bromodeoxyuridine; Cadmium; Carcinoma, Squamous Cell; Cytochalasin B; Fluorescent Antibody Technique; Lymphoma; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Micronucleus Tests; Neoplasms; Neutrons; Phenylalanine; Radiation Tolerance; Radiation-Sensitizing Agents; Radiobiology; Radioisotopes; Relative Biological Effectiveness; Sarcoma, Experimental; Sulfhydryl Compounds; Tumor Cells, Cultured

C3H/He mice bearing SCC VII tumours received 5-bromo-2'-deoxyuridine (BrdU) continuously for 5 days via implanted mini-osmotic pumps to label all proliferating (P) cells. The mice then received gamma-ray irradiation, or administration of tirapazamine (TPZ), cisplatin or bleomycin. At various time points after each treatment, tumour-bearing mice were irradiated with a series of test doses of gamma-rays, while alive or after being killed, to obtain hypoxic fractions (HFs) in the tumours. Immediately after gamma-ray test irradiation, the tumours were excised, minced and trypsinized. Tumour cell suspensions obtained were incubated with cytochalasin-B, a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labelling (i.e. quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. MN frequency in the total (P + Q) tumour cells was determined from the tumours that were not pre-treated with BrdU. MN frequency of BrdU-unlabelled cells was then used to calculate the surviving fraction of the unlabelled cells from the regression line for the relationship between the MN frequency and the surviving fraction of total tumour cells. TPZ and cisplatin reduced the HF after treatment, especially in Q cells, and this tendency was particularly marked with TPZ. In contrast, bleomycin increased the HF after treatment. Both reoxygenation following gamma-ray irradiation or bleomycin treatment and a subsequent return to pre-treatment levels of HF following TPZ or cisplatin treatment (rehypoxiation) occurred more rapidly in total (P + Q) cells than in Q cells. Based on our previous report that total (P + Q) and Q cells within this tumour have large acutely and chronically HFs, respectively, we conclude that acute hypoxic cells play a major role in reoxygenation and rehypoxiation in SCC VII tumours.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Bleomycin; Bromodeoxyuridine; Carcinoma, Squamous Cell; Cell Hypoxia; Cisplatin; Cytochalasin B; Injections; Mice; Micronucleus Tests; Tirapazamine; Triazines; Tumor Cells, Cultured

We analyzed the time-course of changes in the sensitivity of total (proliferating + quiescent and quiescent (Q) cell populations within solid tumors in situ following a neutron capture reaction and compared it with that after gamma-ray irradiation.. After continuous labeling of proliferating cells with BrdU for 5 days, mice bearing SCC VII tumors received thermal neutron irradiation with or without a (10)B-labeled compound (sodium [(10)B]borocaptate, BSH, or DL-p-[(10)B]boronophenylalanine, BPA), or gamma-ray irradiation. From 5 min to 72 h after treatment, tumors were excised, minced, and trypsinized. Cell suspensions were incubated for 48 h with the cytokinesis blocker cytochalasin-B. The micronucleus frequency for BrdU-unlabeled cells, Q cells at treatment, was then determined by immunofluorescence staining for BrdU. The micronucleus frequency for total cells was obtained from tumors that had not been pretreated with BrdU labeling. The sensitivity was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (micronucleus frequency).. Overall, Q cells showed greater repair capacities than total cells. gamma-Ray irradiation and neutron irradiation with BPA induced larger repair capacities in each cell population. In contrast, thermal neutron irradiation without a (10)B-labeled compound induced the smallest repair capacity in both cell populations. The use of a (10)B-labeled compound, especially BPA, widened the difference in sensitivity between total and Q cells, resulted in an increase in repair capacity in both cell populations, and made the repair patterns of the two cell populations look like those induced by gamma-ray irradiation.. Differences in sensitivity and repair patterns following the neutron capture reaction were thought to depend on differences in the distribution of the (10)B-labeled compound between the proliferating and Q cell populations.

Topics: Animals; Borohydrides; Boron Compounds; Boron Neutron Capture Therapy; Bromodeoxyuridine; Carcinoma, Squamous Cell; Cell Division; Cytochalasin B; DNA Damage; Dose-Response Relationship, Radiation; Female; Gamma Rays; Mice; Mice, Inbred C3H; Micronucleus Tests; Neoplasm Transplantation; Phenylalanine; Sensitivity and Specificity; Sulfhydryl Compounds; Tumor Cells, Cultured

In neutron capture therapy, whose effectiveness depends on the tumor distribution of neutron capture compound and the neutron energy distribution, controlling quiescent tumor cells with clonogenic potential is critical for therapeutic gain, as is the case in conventional radio- and chemotherapy. Tumor-bearing mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating cells. After administration of sodium borocaptate-10B (BSH), dl-p-boronophenylalanine-10B (BPA) or gadodiamide hydrate (Omniscan), the tumors were irradiated with neutrons of different cadmium (Cd) ratio, and then isolated and incubated with cytochalasin-B (a cytokinesis blocker). The micronucleus (MN) frequency in cells without BrdU labeling (quiescent cells) was determined using immunofluorescence staining for BrdU, and that for total cells was obtained from tumors not pretreated with BrdU. Without drugs, quiescent cells showed lower MN frequencies than total cells, but neutron irradiation reduced gamma-ray sensitivity difference between the two. Relative biological effectiveness (RBE) of neutrons compared with gamma-rays was greater in quiescent cells than in total cells, and low Cd ratio neutrons tended to exhibit large RBE values. With neutron capture compounds, MN frequency for each cell population was increased, especially when high Cd ratio neutrons were used. BPA increased the MN frequency for total cells to a greater extent than BSH. However, the sensitivity of quiescent cells treated with BPA was lower than that in BSH-treated quiescent cells. This tendency was clearly observed in high Cd ratio neutrons. Omniscan only slightly increased the MN frequency in both cell populations, compared with irradiation alone, without drugs. From the viewpoint of increasing the quiescent cell sensitivity, tumors should be irradiated with high Cd ratio neutrons after BSH administration.

Topics: Animals; Borohydrides; Boron; Boron Compounds; Boron Neutron Capture Therapy; Bromodeoxyuridine; Cadmium Radioisotopes; Carcinoma, Squamous Cell; Contrast Media; Cytochalasin B; Dose-Response Relationship, Radiation; Female; Fluorescent Antibody Technique, Indirect; Gadolinium; Gadolinium DTPA; Injections, Intraperitoneal; Isotopes; Mice; Mice, Inbred C3H; Micronucleus Tests; Neoplasm Transplantation; Phenylalanine; Radiation-Sensitizing Agents; Sulfhydryl Compounds; Tumor Cells, Cultured

Parathyroid hormone-related protein (PTHrP) has been identified as a causative factor in the pathogenesis of humoral hypercalcemia of malignancy (HHM). The regulation and mechanisms of PTHrP secretion in most normal and malignant cells are unknown. PTHrP secretion, mRNA expression, and transcription were measured in neoplastic human squamous carcinoma cells (A253) and normal human foreskin keratinocytes (NHFK) by radioimmunoassay, RNase protection assay, and transient transfections of the 5'-flanking region of human PTHrP in a luciferase expression vector. Mechanisms of PTHrP secretion were investigated using chemicals (monensin, colchicine, cytochalasin B, guanosine 5'-[gamma-thio]triphosphate (GTPgammaS)) that interfere with or facilitate intracellular transport. Monensin inhibited PTHrP secretion in both NHFK and A253 cells. Ultrastructurally, monensin caused dilatation of rough endoplasmic reticulum and the formation of numerous cytoplasmic secretory vacuoles in both cell lines. Colchicine decreased PTHrP production in NHFK cells and stimulated PTHrP production and mRNA levels in A253 cells. Colchicine also stimulated transcription of the PTHrP-luciferase reporter gene. Cytochalasin B stimulated PTHrP secretion and mRNA expression in A253 cells, but had no effect in NHFK cells. GTPgammaS had no effect on PTHrP secretion in either cell line. It was concluded that PTHrP secretion is dependent on the constitutive movement of secretory vesicles to the cytoplasmic membrane and regulation of PTHrP secretion and mRNA expression are altered in squamous carcinoma cells compared to normal human keratinocytes in vitro.

Topics: Actin Cytoskeleton; Biological Transport; Carcinoma, Squamous Cell; Cells, Cultured; Colchicine; Cytochalasin B; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Keratinocytes; Microtubules; Monensin; Neoplasm Proteins; Parathyroid Hormone-Related Protein; Proteins; RNA, Messenger; Skin; Transcription, Genetic

Mice bearing transplantable solid tumors received 10 intraperitoneal administrations of 5-bromo-2'-deoxyuridine (BrdU) to label the proliferating (P) tumor cells, and were then irradiated with 60Co gamma-rays or injected with cis-diamminedichloroplatinum (II) (cisplatin). The tumor cells were isolated and incubated with cytochalasin-B (a cytokinesis blocker). The micronucleus (MN) frequency in the cells without BrdU labeling, which were regarded as quiescent (Q) cells in the tumor, was determined using immunofluorescence staining for BrdU. The MN frequency in the total (P + Q) tumor cell population was determined from tumors that were not pretreated with BrdU. Pretreatment with tirapazamine, a bioreductive agent, could enhance the sensitivity of tumor cells, including Q cells, to radiation more markedly than mitomycin C pretreatment as judged from an in vivo assay immediately after irradiation. Post-irradiation administration of tirapazamine produced a large post-irradiation radiosensitizing effect on both the total and Q tumor cell populations in vivo. Cisplatin treatment combined with tirapazamine demonstrated that tirapazamine also has a chemosensitizing potential for both the total and Q tumor cell populations. We confirmed that the sensitivity of Q cell populations to radiation and chemotherapy using cisplatin can be enhanced by combined treatment with tirapazamine.

Topics: Animals; Antineoplastic Agents; Bromodeoxyuridine; Carcinoma, Squamous Cell; Cell Division; Cisplatin; Cobalt Radioisotopes; Cytochalasin B; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Gamma Rays; Mice; Mice, Inbred C3H; Micronuclei, Chromosome-Defective; Micronucleus Tests; Mitomycin; Mitotic Index; Tirapazamine; Triazines

Adherence of Aeromonas caviae to HEp-2 and Caco-2 cell monolayers was investigated with 24 clinical isolates. Growth phase, temperature, multiplicity of infection and length of incubation affected adherence. Treatment of the bacteria with trypsin, sodium metaperiodate, mechanical shearing and the addition of cytochalasin B and cycloheximide to the monolayer significantly reduced the adherence capabilities of the strains investigated. The use of chloramphenicol to inhibit protein synthesis reduced the adhesive capabilities of bacteria grown in liquid medium and those subjected to mechanical shearing. Light microscopy, scanning and transmission electron microscopy were employed in the investigation of bacteria-bacteria and bacteria-monolayer interactions and indicated similarities with the aggregative adherence patterns of the Enterobacteriaceae. The presence of extracellular bacterial appendages and their correlation with increased adhesive capacity may indicate a role in the process of adherence.

Topics: Aeromonas; Anti-Bacterial Agents; Bacterial Adhesion; Caco-2 Cells; Carcinoma, Squamous Cell; Chloramphenicol; Cycloheximide; Cytochalasin B; Humans; Laryngeal Neoplasms; Microscopy, Electron, Scanning; Mitogens; Periodic Acid; Protein Synthesis Inhibitors; Stress, Mechanical; Temperature; Trypsin; Tumor Cells, Cultured

Expression of the cell surface protein intercellular adhesion molecule-1 (ICAM-1) is a prerequisite for the interaction of a large variety of cells with leukocytes. Constitutive expression of ICAM-1 in human epidermoid carcinoma cells is low, but is inducible through inflammatory cytokines including interferon gamma (INF gamma). Disruption of the actin cytoskeleton with dehydrocytochalasin B (CB) increased constitutive and potentiated INF gamma-induced ICAM-1 cell surface expression, but did not alter formation of soluble ICAM-1. Actinomycin D inhibited CB-induced ICAM-1 surface expression and CB increased the steady-state levels of ICAM-1 transcripts. However, CB did not alter the glyceralaldehyde-3-phosphate dehydrogenase mRNA levels and the ICAM-1 mRNA half-life. These studies indicate that in human epidermoid carcinoma cells the actin cytoskeleton regulates ICAM-1 transcription and cell surface expression.

Topics: Actins; Carcinoma, Squamous Cell; Cell Line; Cytochalasin B; Cytoskeleton; Dactinomycin; Gene Expression; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Intercellular Adhesion Molecule-1; Interferon-gamma; Recombinant Proteins; RNA, Messenger; Tumor Cells, Cultured

The state of polymerization of actin and the organization of actin filaments is widely believed to be related to cellular transformation. Since the intracellular monomer (G) and filamentous (F) actin content reflects the state of microfilament polymerization, we measured the G/total actin ratio in primary cultures of normal and malignant human keratinocytes. In normal keratinocytes the mean value of this ratio was 0.30 +/- 0.03 (mean +/- SE, n = 15), while in basal cell carcinoma (BCC) keratinocytes it was 0.49 +/- 0.03 (n = 8) and in squamous cell carcinoma keratinocytes (SCC) 0.5 +/- 0.07 (n = 4), indicating a 1.7-fold increase of the G/total actin ratio in malignant cells. These results imply that the proportion of polymerized actin is decreased markedly in malignant keratinocytes, suggesting alterations of microfilament structures which probably occur during the transformation process. This was supported by the morphological changes of microfilament structures as assessed by fluorescence microscopy. A different distribution of actin filaments in normal and malignant cells became evident; stress-fibres were converging in patches at several points in SCC cells, when compared to normal keratinocytes. Furthermore, incubation of normal and malignant keratinocytes with cytochalasin B indicated differences in the resistance of their microfilament networks. After 1 h exposure to 10(-6) and 10(-5) M cytochalasin B, microfilaments in normal cells appeared to be less affected than their counterparts in neoplastic cells. Even in a high excess of cytochalasin B (10(-4) M), normal keratinocytes preserved their shape, while both basal cell and SCC were totally disrupted.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Actin Cytoskeleton; Actins; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Cells, Cultured; Cytochalasin B; Humans; Keratinocytes; Microscopy, Fluorescence; Skin; Skin Neoplasms; Tumor Cells, Cultured

We established an in vitro cytokinesis-block micronucleus assay of human tumours for estimation of the proportion of cells undergoing mitosis (the dividing fraction, DF), the time for the number of nuclei to double and the radiosensitivity in terms of the micronucleus frequency, based on a concept described previously. Under certain conditions, the nuclear number doubling time (NNDT) was considered to represent the potential doubling time. Tumour specimens obtained at surgery were disaggregated into single-cell suspensions and were directly cultured in the presence of cytochalasin B with or without irradiation. At various intervals, the percentage of multinucleate cells (the plateau value represented the DF), the average number of nuclei per cell and the number of micronuclei in binucleate cells were determined. DF and NNDT values were obtained in 58 of the 73 tumours investigated, and the micronucleus frequency was obtained in 54 of these 58 tumours. The DF ranged from 4.1% to 71% and the NNDT ranged from 3.1 to 83 days. A DF > or = 20% was associated with a higher recurrence rate in patients undergoing curative operation. A correlation was found between the NNDT and the time to relapse in patients with recurrent disease. The average number of micronuclei per binucleate cell at 2 Gy of irradiation (after subtraction of the value at 0 Gy) ranged from 0.052 to 0.35. Tumours which produced more micronuclei after irradiation showed a better response to radiotherapy. This assay can be readily performed on human tumours and appears to have promise as a predictive assay for radiation therapy.

Topics: Adenocarcinoma; Brain Neoplasms; Carcinoma; Carcinoma, Squamous Cell; Carcinoma, Transitional Cell; Cell Division; Cell Nucleus; Cell Survival; Cytochalasin B; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Humans; Micronucleus Tests; Neoplasms; Predictive Value of Tests; Prognosis; Radiation Tolerance; Remission Induction; Sarcoma; Treatment Outcome; Tumor Cells, Cultured

Avidin-Biotin Peroxidase complex technique, ABV staining, was employed by using monoclonal anti-keratin antibody HK2 in this study. The organization and dynamics of keratins in both interphase and mitotic T56 and HeLa cells were analysed. We also observed the effects of microtubule (MT) and microfilament (MF) inhibitors, colchicine and cytochalasin B, on the organization of keratin filaments in T56 and HeLa cells. The results showed that a significant alteration in the structural organization and distribution of keratin filaments occurred during mitosis, and an extensive rearrangement of keratin networks of the two cell lines was induced in interphase after the MT and MF were disrupted by combined treatment with the two drugs, colchicine and cytochalasin B; the keratin networks turned into a star-like lattice rapidly within 1-2h. Neither colchicine nor cytochalasin B alone elicited significant organizational change in the keratin networks of the two cell lines.

Topics: Carcinoma, Squamous Cell; Colchicine; Cytochalasin B; HeLa Cells; Humans; Immunoenzyme Techniques; Immunohistochemistry; Keratins; Tongue Neoplasms; Tumor Cells, Cultured

By the use of rhodamine-phalloidin, the distribution of actin in A-431 cells during the action of epidermal growth factor (EGF) has been studied. Changes in the pattern of staining are observed in 30-60 s after addition of the EGF. Microvilli and wrinkles are created on the cell surface. Following a 5-10 min action of EGF, rhodamine-phalloidin stained intensely ruffles and cell borders. After 60 min, the ruffling of cell surface disappeared, and actin was seen concentrating on the cell borders only. Electron microscopy of the EGF-treated A-431 cells lysed by Triton X-100 also revealed some vigorous fibrillar bunches on the cell edges.

Topics: Actins; Carcinoma, Squamous Cell; Cell Line; Cytochalasin B; Cytoskeleton; Epidermal Growth Factor; Humans; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Staining and Labeling; Time Factors; Tumor Cells, Cultured; Vinblastine

Capping of the EGF receptor (EGF-R) on the surface of suspended and adherent epidermoid carcinoma cells, A431, is studied. It was induced at 20 degrees C after treating cells with monoclonal antibody to the EGF receptor followed by the second antibody conjugated with FITC. Accumulation of cortical actin under the caps was detected by rhodamine-phalloidin. Destruction of the actin stress-fiber-like bundles was observed during incubation of cells with the ligands at 0 degrees C. Two processes appear to take place at 20 degrees C: redistribution of the EGF-R with cortical actin into the caps within 15-30 min and reconstruction of cytoplasmic actin bundles over 45-60 min. Dihydrocytochalasin B prevented cap formation in adherent cells, but small patches of EGF-R colocalized with actin aggregates under plasma membrane were observed. The function of different actin-containing cytoskeleton structures in the process of capping is discussed.

Topics: Actin Cytoskeleton; Actins; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Membrane; Cytochalasin B; ErbB Receptors; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Antibody Technique; Humans; Receptor Aggregation; Thiocyanates; Tumor Cells, Cultured

The correlation between cell survival curve and dose response curve of MN frequency following irradiations was studied using cytokinesis-block method. Both dose response curves were analyzed by linear quadratic model, i.e. SF = exp (- alpha D - beta D2) and MN frequency = aD + bD2 + c. A good correlation between alpha/beta and a/b ratios was observed in repeated paired experiments (gamma = 0.97). When the cells were treated with BUdR, alpha-type radiosensitizer, a value in dose response curve of MN frequency increased but b value did not. In 10 renal cell carcinomas, the linear correlations between cell surviving fractions and MN frequencies were observed. When radioresponses of 5 esophageal cancer cell lines were evaluated with MN assay, wide range of a/b ratios was found. These data present that MN frequency assay using CB method is available as a tool of rapid assay of radiosensitivity of cells.

Topics: Animals; Carcinoma, Renal Cell; Carcinoma, Squamous Cell; Cell Survival; Cytochalasin B; Dose-Response Relationship, Radiation; Esophageal Neoplasms; Humans; Kidney Neoplasms; Mice; Micronucleus Tests; Predictive Value of Tests; Radiation Tolerance; Tumor Cells, Cultured

Epidermal growth factor (EGF) receptor capping results from the interaction between the receptors and polyvalent ligands in A-431 cells examined in suspension at 22 degrees C. Colocalization of actin and spectrin with the ligand-receptor complexes during the redistribution was shown using double immunofluorescence. The obtained data show that the cortical microfilaments are involved in capping. EGF receptors become associated with the Triton-insoluble cytoskeleton as a consequence of ligand binding. EGF-receptor capping is not sensitive to the action of cytochalasin B. Capping in A-431 cells is discussed as a new model for studying the redistribution of the ligand-receptor complex.

Topics: Actins; Antibodies; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Line; Cytochalasin B; Cytoskeleton; Epidermal Growth Factor; ErbB Receptors; Humans; Immune Sera; Ligands; Receptor Aggregation; Spectrin; Tumor Cells, Cultured

Human epidermoid carcinoma A431 cells attach more rapidly to collagen type-I and -IV substrates than to surfaces coated with laminin or fibronectin. The diminished intercellular interaction and rounding up manifested when these cells are exposed to epidermal growth factor (EGF) in tissue culture plastic or collagen films is not shown when the assay is performed on 3-dimensional collagen. In the latter substrate, cells exposed to EGF reveal greater cell cohesion with interdigitations and desmosomal junctions, compared to the limited intercellular interaction detected in similarly treated cells assayed in tissue culture substrate. Although the protein synthesis inhibitor, cycloheximide, did not prevent these EGF-mediated changes, metabolic labelling indicated a substrate-dependent effect of EGF on the synthesis of proteins associated with the Triton-insoluble cytoskeletal matrix. The involvement of cytoskeleton components in the EGF effects on intercellular adhesion was shown by its susceptibility to cytochalasin B, known to disorganize actin-containing microfilaments. Some of the mechanisms of epithelial morphogenesis and the influence of extracellular matrix components on cell-receptor/growth-factor interactions may now be suitably analyzed by examining the EGF effects on A431 cells grown on different substrata.

Topics: Carcinoma, Squamous Cell; Cell Adhesion; Cell Communication; Cells, Cultured; Collagen; Cytochalasin B; Epidermal Growth Factor; Extracellular Matrix; Gels; Humans; Neoplasm Proteins; Polyethylene Glycols; Tritium; Trypsin

Topics: Carcinoma, Squamous Cell; Cell Cycle; Cell Line; Cytochalasin B; Diploidy; DNA Replication; Fetus; Humans; Lung; Lung Neoplasms; Nasopharyngeal Neoplasms

Epidermal growth factor (EGF) receptors can be spontaneously and selectively transferred from donor plasma membranes to recipient receptorless fibroblasts in the absence of any added fusogenic agent. Studies on the time and temperature dependence of this transfer indicate that it is due to preferential insertion of the EGF receptor over the other plasma membrane proteins. The inserted receptor is exceptionally stable to dissociation or damage. The number of receptors inserted increased with increasing amounts of donor membranes and then reached a plateau, which also suggests the existence of saturable receptor 'docking' sites in recipient cells. It is interesting that both human and murine receptors are selectively inserted into the mutant mouse cell membrane. This suggests that the parts of the receptor molecule responsible for insertion are similar in murine and human receptors, and that a 'docking' factor present in the mouse recipient cells may accept both human and murine receptors.

Topics: Animals; Biological Transport; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Colchicine; Cycloheximide; Cytochalasin B; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Liver; Membrane Proteins; Mice; Receptors, Cell Surface; Temperature; Tunicamycin

The cytoplasmic fine structures of both normal human cultured keratinocytes (NHK) and squamous cell carcinoma cells (HSC) were examined by electron microscopy using the whole-cell preparation method and stereo-viewing techniques. The presence of cytoplasmic fine network (CFN) was confirmed in both NHK and HSC, but the structures of the two were found to be radically different. In particular, the mitochondria showed a number of distinct morphological differences. The introduction of cytochalasin B and colchicine into HSC partially destroyed the CFN, and, as a result, the morphology of the HSC mitochondria changed to become similar to those of NHK. It seems that the CFN may have an important role in determining the shape of the cell organelles, such as mitochondria, and that the shape of the mitochondria may perhaps be used as an indication of cell malignancy.

Topics: Carcinoma, Squamous Cell; Cells, Cultured; Colchicine; Cytochalasin B; Cytoskeleton; Epidermis; Humans; Keratins; Mitochondria; Skin Neoplasms

Topics: Carcinoma, Squamous Cell; Cell Line; Cytochalasin B; Humans; In Vitro Techniques; Melanoma; Skin Neoplasms

Tissue culture monolayers of seven human intracranial tumours comprising 2 astrocytomas, 3 meningiomas, 1 secondary squamous cell carcinoma and 1 secondary adenocarcinoma were examined by a double immunofluorescent staining technique to demonstrate Concanavalin A (Con A) surface receptors and cytoplasmic actin in the same cell. Tumour cells, treated with fluoresceinisothiocyanate-labelled Con A (FITC-Con A) showed staining in cell margins or in a random distribution over the cell surface. Incubating the cells with FITC-Con A at 37 degrees for increasing periods of time resulted first in staining of clusters and later of perinuclear globules. Cells, pretreated with 4% paraformaldehyde at 4 degrees for 10 min or with cytochalasin B at 37 degrees for 30 min showed staining restricted to cell margins. In the cytochalasin B-treated cells, the peripheral staining was in the form of coarse clusters. Double fluorochrome studies showed that the anti-actin antibody (AAA) staining occurred in sites closely related to those stained by FITC-Con A both in untreated as well as in cytochalasin B-treated cells. The findings suggest that Con A receptors, as an example of a stable cell membrane determinant in human tumour cells, are associated with actin and that their mobility on the cell surface is dependent on an intact cytoplasmic actin system.

Topics: Actins; Adenocarcinoma; Astrocytoma; Brain Neoplasms; Carcinoma, Squamous Cell; Colchicine; Cytochalasin B; Cytoplasm; Fluorescent Antibody Technique; Humans; In Vitro Techniques; Meningioma; Receptors, Concanavalin A; Receptors, Drug

The occurrence and structure of microfilaments in epidermal cancers induced in mice by treatment with 3,4-benzpyrene were investigated with the electron microscope. With malignant change, pleomorphic, undifferentiated cells with a cortical zone of microfilaments became increasingly abundant. The microfilaments were 40 A in diameter and occupied the cortex of the cells beneath the plasma membrane, extended into cell processes, and were situated in the cores of microvilli. At high magnification, the filamentous areas were formed by an interconnected meshwork of filaments which in favorable planes had a polygonal arrangement. When exposed to high concentrations of cytochalasin B, the microfilaments became clumped and moderately disrupted. At the same time, the processes and microvilli of the cells were blunted. The structure of these filaments and their sensitivity to cytochalasin B place them in a class of microfilaments believed to be related to cell motility. Their presence in malignant cells may be correlated with the motile, invasive properties of these cells.

Topics: Acetone; Animals; Benz(a)Anthracenes; Benzopyrenes; Carcinoma; Carcinoma, Squamous Cell; Cell Movement; Cytochalasin B; Female; Mice; Mice, Inbred BALB C; Microscopy, Electron; Skin; Skin Neoplasms