cytochalasin-b and Skin-Neoplasms

Although cisplatin is a frequently used cancer chemotherapeutic drug, its effectiveness is hindered by the development of resistance in cancer cells. In order to understand the reason(s) for this resistance, the mechanism of uptake of cisplatin into cells must be characterized. While several previous studies showed structural differences between cisplatin-sensitive and resistant cells, the influence of microfilaments, known to affect transport of molecules into cells, and the influence of certain biophysical characteristics of the plasma membrane needed clarification.. We show that resistant human epidermal carcinoma (KB-CP20) and liver carcinoma (BEL-7404-CP20) cells become relatively more resistant if their already weak microfilaments are degraded by cytochalasin B treatment (.5-2 microM). The sensitive counterparts of these cells with intact microfilaments are not significantly affected by this treatment. We also show that the "fluidity" of the plasma membrane and the membrane potential of the sensitive and resistant cells studied do not appear to influence the uptake of cisplatin into the cells.. Our results suggest that the status of the microfilament system influences the mechanism of uptake of cisplatin into cells.

Topics: Actin Cytoskeleton; Biological Transport; Carcinoma; Carcinoma, Hepatocellular; Cell Division; Cell Line, Tumor; Cell Membrane Permeability; Cisplatin; Cytochalasin B; Drug Resistance, Neoplasm; Humans; KB Cells; Liver Neoplasms; Membrane Fluidity; Membrane Potentials; Skin Neoplasms

It was previously found that L-tyrosine oxidation product(s) are cytotoxic, genotoxic and increase the sister chromatid exchange (SCE) levels in human melanoma cells. In this work, the micronucleus assay has been performed on human melanotic and amelanotic melanoma cell lines (Carl-1 MEL and AMEL) in the presence of 1.0, 0.5 and 0.1 mM L-tyrosine concentrations to investigate if melanin synthesis intermediate(s) increase micronuclei production. L-Tyrosine oxidation product(s) increased the frequency of micronuclei in melanoma cells; 0.1 mM phenylthiourea (PTU), an inhibitor of L-tyrosine oxidation by tyrosinase, lowered the micronucleus production to the control levels. The culture of melanoma cells with high L-tyrosine in the culture medium resulted in a positive response to an ELISA-based apoptotic test. For comparison the effect of L-tyrosine on micronuclei production in human amelanotic melanoma cells was also investigated; the micronucleus production in the presence of 1 mM L-tyrosine in the culture medium was lower than that found with melanotic melanoma cells of the same cell line. The data suggest that melanin synthesis intermediates arising from L-tyrosine oxidation may cause micronuclei production in Carl-1 human melanoma cells; the addition of PTU in the presence of L-tyrosine decreased the frequency of micronuclei to about the control values thus the inhibition of melanogenesis may have some clinical implication in melanotic melanoma.

Topics: Apoptosis; Cell Nucleus; Cytochalasin B; Dose-Response Relationship, Drug; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Humans; Melanoma; Melanoma, Amelanotic; Micronucleus Tests; Monophenol Monooxygenase; Phenylthiourea; Skin Neoplasms; Tumor Cells, Cultured; Tyrosine

Cytokinesis-block micronucleus assay (CB-MNA) was applied for comparison of radiation sensitivity of 25 human malignant melanomas in primary culture. Cells obtained from tumor specimens were irradiated (0-4.Gy) on dishes, incubated with cytochalasin B (2 micrograms/ml) to block cytokinesis, stained in situ and micronuclei (MN) scored in binucleate cells (BNC). Proportions of BNC in nonirradiated controls after fixed time of incubation (96 h) ranged from 2.3 to 38% indicating great differences (C.V. = 74%) in proliferative activity among tumors evaluated. No correlation was observed between proliferative activity and susceptibility of cells to induction of MN by radiation. The great inter-tumor heterogeneity was observed in respect of radiation sensitivity expressed either as normalized (Net) frequency (Fq) of BNC with MN or as number of MN per BNC. Both endpoints differed widely at 2 Gy and 4 Gy as well (Net FqBNC with MN = 0.28-25.4% or 1.5-45% and MN/BNC = 0.004-0.309 or 0.013-0.593 respectively at 2 Gy and 4 Gy) with coefficients of variation ranging from 44 to 57%. Extreme difference in MN frequency was also observed between one primary tumor and its metastasis indicating intra-tumor heterogeneity. Our results suggest that CB-MNA may contribute some clinically useful information for discriminating tumors that will eventually respond to radiotherapy and those that will probably not. However, studies aimed at comparison of MN induction in vitro with clinical radioresponsiveness of malignant melanomas are urgently required.

Topics: Antibodies, Monoclonal; Cell Division; Cell Nucleus; Cytochalasin B; Female; Flow Cytometry; Humans; Immunohistochemistry; Male; Melanoma; Micronucleus Tests; Ploidies; Radiation Tolerance; Skin Neoplasms; 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

Keratinocytes from mouse skin were cultured for a short period in vitro following single or multiple treatments at low dose levels in vivo with the known chromosome-damaging agent triethylenemelamine (TEM). The chemical was applied to the skin of HRA/Skh hairless mice at concentrations corresponding to those reported to initiate cancer in initiation-promotion assays. A significant dose-related depression in keratinocyte cell recovery occurred over the dose range 0.3-1 mg TEM/mouse (single or multiple treatments). Under the same conditions, a dose-related induction of micronuclei was observed using the cytokinesis-block method with cytochalasin B. A similar frequency of micronuclei was detected in binucleate cells from mice treated with single or multiple applications of TEM. Mice held for 12-48 h post-treatment, before removal of skin for in vitro culture, yielded highest micronuclei frequencies. These results indicate that the same target cell population, skin keratinocytes, can be used to investigate both genotoxicity and carcinogenesis, and that micronucleus induction in these cells may be a sensitive signal of skin cancer initiation.

Topics: Animals; Cells, Cultured; Cytochalasin B; Male; Mice; Mice, Hairless; Micronucleus Tests; Skin; Skin Neoplasms; Triethylenemelamine

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

Topics: Acetates; Animals; Anthralin; Cantharidin; Carcinogens; Cell Membrane; Chick Embryo; Cytochalasin B; Deoxyglucose; Fibroblasts; Glycoproteins; Lauric Acids; Neoplasms, Experimental; Oleic Acids; Palmitic Acids; Phenols; Phorbols; Skin Neoplasms; Stearic Acids; Tetradecanoylphorbol Acetate

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