demecolcine and Melanoma

This work presents the development of a protocol based on the dynamic laser speckle designed to monitor the reaction of cancer cells of line MEL-RC08 to the application of the drug Colcemid in two different concentrations: 0.2 and 0.4 μg/mL. The protocol was designed using the forward scattering approach with an He-Ne laser of 632.8 nm illuminating the samples, a control, and two variations of Colcemid, being monitored along 8 h. The data were analyzed numerically in the time and in the frequency domain, and the results presented the ability of the technique to monitor the action of the drug, particularly Colcemid (0.4 μg/mL).

Topics: Analysis of Variance; Antineoplastic Agents, Phytogenic; Cell Cycle; Cell Line, Tumor; Demecolcine; Diagnostic Imaging; Humans; Image Processing, Computer-Assisted; Lasers; Melanoma

Analysis of the expression of a number of known genes in cultured human cells has revealed UVB-induced changes that may be specific for melanocytic cells. The response of c-fos, p53 and HIV-LTR reporter constructs to UVB and UVC was reduced in MM96L melanoma cells compared to HeLa. Cell cycle arrest produced by UVA, gamma radiation, cisplatin or the antimetabolite deoxyinosine differed from that of UVB. Cell cycle analysis after multiple doses of UVB raised the possibility that UVB-induced pRb depletion could result in increased mutation and thus enhanced tumourigenesis of irradiated melanocytes in skin subjected to a defined pattern of UVB exposure. To extend the analysis of gene expression in cultured melanocytic cells to uncharacterised genes, promoter trap cell clones containing unknown genes 'tagged' by a beta-galactosidase reporter construct were generated from MM96L cells. Altered gene expression in clones treated with a panel of DNA-damaging agents was quantitated by measurement of beta-galactosidase activity. Of the clones containing 'tagged' endogenous promoters induced by UVB, 52% were induced only by UVB and not by other DNA-damaging agents (cisplatin, N-methyl-N-nitro-nitrsoguanidine, fotemustine). One third of the clones were also activated by TPA suggesting that general DNA damage responses involving PKC are activated less frequently than unique pathways of gene activation. Overall, 60% of the 50 clones that responded to the panel of agents were induced by only one of the agents, indicating that a high proportion of genes are induced by agent-specific mechanisms. In the long term, promoter trapping may allow the full repertoire of UVB-inducible genes to be characterised.

Topics: Antineoplastic Agents; beta-Galactosidase; Cell Cycle; Cisplatin; Demecolcine; Genes, fos; Genes, p53; HeLa Cells; HIV Long Terminal Repeat; Humans; Hydroxyurea; Inosine; Melanocytes; Melanoma; Methylnitronitrosoguanidine; Neoplasms, Radiation-Induced; Nitrosourea Compounds; Organophosphorus Compounds; Proto-Oncogene Proteins c-fos; Recombinant Proteins; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured; Ultraviolet Rays

Cytogenetic and molecular studies have implicated one or more tumor suppressor genes on the long arm of human chromosome 11 in the malignant progression of several human solid tumors, including malignant melanoma and carcinomas of the breast, cervix, ovary, and lung. Microcell-mediated chromosome transfer of an intact copy of chromosome 11 into tumor cell lines has provided additional evidence of tumor suppressor gene function in melanoma, breast cancer, and cervical cancer. However, sublocalization of the region(s) conferring the tumor suppressive effect has been difficult. To facilitate mapping of tumor suppressor gene(s) on chromosome 11, we have generated a panel of 25 mouse donor cell lines containing neo-tagged fragments of human chromosome 11q which can be transferred into cell lines to test for tumor suppressor activity. The chromosome fragments in these cell lines have been characterized by fluorescence in situ hybridization with probes to human DNA and to the centromere of chromosome 11, and also by analysis of microsatellite markers spanning chromosome 11. Finally, to demonstrate the usefulness of these cell lines as donors for microcell-mediated chromosome transfer, two fragments were transferred into the human melanoma cell line UACC 903. This panel of selectable subchromosomal fragments, derived from the long arm of human chromosome 11, will be useful for the regional localization of tumor suppressors and other genes by means of functional assays.

Topics: Animals; Antineoplastic Agents, Phytogenic; Chromosomes, Human, Pair 11; Demecolcine; DNA Fragmentation; Dose-Response Relationship, Radiation; Gene Transfer Techniques; Genes, Tumor Suppressor; Genetic Markers; Humans; Hybrid Cells; Melanoma; Mice; Tumor Cells, Cultured

The interaction of daunomycin (DAU), an anthracyclinic antibiotic employed as antitumoral agent, with microtubules, has been investigated by cytochemical and morphological methods on a human melanoma cell line (H14). Results obtained indicated that DAU was able to modulate the microtubule reassembly in cells treated with colcemid; such an effect proved to be dose-dependent. In particular, it has been observed that a low dose of DAU (0.05 microM) seemed to favor the microtubule reassembly whereas a higher dose (0.10 microM) impaired this process. In addition, when the anthracyclinic antibiotic was employed together with colcemid, both the cell detachment and the depolymerization of microtubules induced by the mitotic poison were hampered. These effects were dose-dependent and were better accomplished when DAU was used at an equimolar or at higher dose than that employed for the antimicrotubular agent. Moreover, the treatment of cells with DAU alone induced the stabilization of the microtubules, making them more resistant to the action of antimicrotubular agents. This effect could in part explain the antagonistic action exerted by DAU against colcemid. These observations seem to confirm that the microtubular network is an important target involved in the mechanism of action of the anthracyclinic antibiotics.

Topics: Cell Adhesion; Daunorubicin; Demecolcine; Dose-Response Relationship, Drug; Humans; Melanoma; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Microtubules; Tumor Cells, Cultured

Interleukin 1 (IL-1) has been shown to have antiproliferative or cytocidal effects on several tumor cell lines and this effect is closely related to the induction of terminal differentiation of the target tumor cells. In this study we analyzed the antiproliferative effect of recombinant human IL-1 alpha on a human melanoma cell line A375 in relation to cell cycle. Nutrient-starved cells, most of which were in G0 + G1, were stimulated by culturing in fresh medium, causing them to enter S. IL-1 treatment induced a slight decrease in the first cell cycle progression from G0 + G1 to S. In addition IL-1 retarded progression of the cells through G2M and inhibited progression of the second cell cycle from G0 + G1 to S. Therefore we concluded that IL-1 exerts its antiproliferative effect by arresting the cells in G0 + G1.

Topics: Cell Cycle; Cell Division; Cell Line; Clone Cells; Demecolcine; DNA Replication; Humans; Interleukin-1; Interphase; Kinetics; Melanoma; Recombinant Proteins; Thymidine

Mitotic cells selectively harvested after several h of colcemid treatment are routinely used to obtain synchronized cell cultures. DNA flow cytometry shows that when colcemid-treated B16 mitotic cells divide, they give rise to daughter cells in G1, some of which contain abnormal amounts of DNA. Two subpopulations appear to exist, one having a DNA content distribution expected of G1 cells, another having a mean DNA content about 0.8 of expected and an SD of DNA content more than 5 times expected. The effect was dependent on dose and duration of exposure to colcemid. Colcemid was more cytotoxic to cells in G2 + M than to G1 + S phase cells, and it slowed the progression of G1 cells to S. These effects of colcemid were much greater in aneuploid B16 melanoma cells than in pseudodiploid Chinese hamster ovary (CHO) cells.

Topics: Aneuploidy; Animals; Cell Division; Cell Line; Cricetinae; Demecolcine; Diploidy; DNA; Interphase; Melanoma; Mice; Mitosis; Vinblastine

Fifteen malignant melanomas were subjected to chromosomal analysis, in order to determine whether the number of chromosomes in a tumor cell could be correlated to a clinical prognosis. The protocol involved a direct technique which utilized a Colcemid blockade of spindle formation in mitosis to allow study of metaphase chromosomes. The direct method was chosen to reveal chromosome changes in the tumor cell in situ rather than changes in the cultured cell. Two tumors yielded chromosome spreads which could be counted and correlated with a clinical prognosis. Failure to obtain other adequate chromosome spreads were accounted for by the presence of tissue necrosis and the absence of viable tumor cells.

Topics: Adult; Aged; Chromosome Aberrations; Demecolcine; Female; Humans; Karyotyping; Male; Melanoma; Metaphase; Middle Aged; Prognosis; Skin Neoplasms

Topics: Animals; Colchicine; Cowpox; Demecolcine; Humans; Immunotherapy, Active; Melanoma; Skin Neoplasms; Smallpox; Vaccines; Variola virus