aphidicolin and Melanoma

The induction of micronuclei (MN) by incubation with different mutagenic agents was tested in diploid fibroblast cultures obtained from 15 probands with constitutively high MN rates (15.75-77.25 MN/500 cells; average 36.27 +/- 17.60 MN) and 15 probands (controls) with low MN rates (4-13.75 MN/500 cells; average 8.97 +/- 2.73 MN). In order to find out whether fibroblast cultures of individuals with increased spontaneous MN levels exhibit an increased sensitivity to various agents with different genotoxic mechanisms, we studied the induction of MN in these cell cultures by ultraviolet (UV) irradiation, mitomycin C (MMC), N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and benzo-(a)pyrene-diol-expoxid (BPDE). In addition, we tested aphidicolin (APC), a polymerase alpha inhibitor, which is a potent inducer of common fragile sites. Probands with spontaneously high MN showed an significantly increased sensitivity to UV (p less than or equal to 0.005), MMC (p less than or equal to 0.005), and BPDE (p less than or equal to 0.005). No significant differences were found for MNNG and APC as compared to controls.

Topics: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; Aphidicolin; Cells, Cultured; Fibroblasts; Humans; Melanoma; Methylnitronitrosoguanidine; Mitomycin; Mutagens; Reference Values; Skin; Skin Neoplasms; Ultraviolet Rays

After the joining of human large DNA replication intermediates and before the appearance of mature chromatin DNA, there exists a distinct stage--'the post-elongation stage'. This stage reappears during recovery of DNA synthesis simultaneously with the reappearance of a large DNA replication intermediate, 10 kb DNA.

Topics: Aphidicolin; Diterpenes; DNA; DNA Replication; Endonucleases; Humans; Melanoma; Single-Strand Specific DNA and RNA Endonucleases; Tumor Cells, Cultured

We have looked for the presence of single-stranded DNA in human melanoma cells. Single-stranded DNA was observed by lysis of cells in dilute alkali (to partly denature the DNA) followed by CsCl gradient centrifugations. In normally growing cells we did not observe single-stranded DNA whereas large amounts were present in cells treated with aphidicolin (an inhibitor of DNA polymerase alpha). The single-stranded DNA is much larger (greater than 20 kb) than Okazaki fragments. When the cells were washed free of aphidicolin, the single-stranded DNA was converted to high molecular weight DNA. Furthermore, when DNA synthesis is recovering after drug treatment, the single-stranded DNA disappears. The single-stranded DNA represents a transient step during the maturation of newly synthesized DNA.

Topics: Aphidicolin; Diterpenes; DNA Replication; DNA, Neoplasm; DNA, Single-Stranded; Humans; Melanoma; Molecular Weight; Tumor Cells, Cultured

Dacarbazine induces DNA lesions in cells synthesizing new DNA. When poly(ADP-ribosylation) is inhibited with 3-aminobenzamide, repair of the dacarbazine-induced DNA lesions is reduced. This is parallelled by increased cytotoxicity. Cells pre-treated with aphidicolin (to stop DNA synthesis) are resistant to dacarbazine.

Topics: Antineoplastic Agents; Aphidicolin; Benzamides; Cell Line; Dacarbazine; Diterpenes; DNA; DNA Repair; Drug Synergism; Humans; Melanoma; Poly(ADP-ribose) Polymerase Inhibitors

During DNA synthesis there is a distinct stage immediately after the joining of large DNA replication intermediates (post-elongation stage). The conversion of this DNA to mature DNA was analysed in cells treated with aphidicolin to stop the movement of the replication fork. In such cells mature DNA is formed. In contrast, UV-A, which induces a wide spectrum of DNA lesions, inhibits the conversion to mature DNA. The data indicate that the maturation of the post-elongation stage can be uncoupled from the movement of the replication fork.

Topics: Aphidicolin; Cell Line; Diterpenes; DNA Replication; DNA, Neoplasm; Humans; Melanoma; Ultraviolet Rays

During eukaryotic DNA synthesis there is formation of, in addition to Okazaki fragments, discrete 10-kilobase (kb) DNA replication intermediates. We have investigated the ligation of 10-kb DNA replication intermediates to high molecular weight DNA, using the drug 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthetase. In human melanoma cells treated with this inhibitor, there is an accumulation of 10-kb DNA. In contrast, in cells treated with aphidicolin, which inhibits DNA polymerase alpha, there is continued ligation of 10-kb DNA to high molecular weight DNA. Furthermore, using sequential treatment with aphidicolin and 3-aminobenzamide, one can observe the conversion of radiolabeled Okazaki fragments into 10-kb intermediates. The 10-kb DNA pieces are, however, not ligated to high molecular weight DNA in the presence of 3-aminobenzamide. Our results imply that functioning poly(ADP-ribose) synthetase is necessary for the ligation process.

Topics: Aphidicolin; Benzamides; Cell Line; Diterpenes; DNA Replication; DNA, Single-Stranded; Humans; Interphase; Melanoma; Molecular Weight; NAD+ Nucleosidase; Nucleic Acid Precursors; Poly(ADP-ribose) Polymerase Inhibitors

X irradiation of cells induces damage in the DNA, which can be detected as fragmentation of the DNA in alkali. To examine whether DNA polymerase alpha plays a role in the X-ray-induced fragmentation of the DNA, cells with and without functioning DNA polymerase alpha have been compared. We have used the drug aphidicolin, which is a specific inhibitor of polymerase alpha. The results show that DNA of aphidicolin-treated cells is more easily fragmented in alkali than DNA of untreated cells. This is paralleled by a lower repair replication in cells without functioning DNA polymerase alpha. Hence polymerase alpha is involved in the repair process of lesions induced by X irradiation.

Topics: Aphidicolin; Cell Line; Diterpenes; DNA; DNA Polymerase II; DNA Repair; DNA, Neoplasm; Humans; In Vitro Techniques; Melanoma; Radiation Genetics

Ascorbate-Cu2+ shows considerable cytotoxicity for human melanoma cells at a dose which has very little effect on human fibroblasts. Ascorbate itself inhibits DNA synthesis in melanoma cells but does not fragment the parental DNA. However, the combined action of ascorbate-Cu2+ generates fragmentation of the parental DNA due to the induction of alkali-labile bonds in the DNA. In contrast, if DNA polymerase alpha is inhibited by aphidicolin prior to treatment with ascorbate-Cu2+ one cannot detect the fragmentation of the DNA. The generated fragments show a discrete appearance in agarose gel electrophoresis with a single-stranded size of approximately 5 kb. When fibroblasts were analyzed using the same experimental protocol it was not possible to detect the fragmentation of the DNA.

Topics: Aphidicolin; Ascorbic Acid; Cells, Cultured; Copper; Diterpenes; DNA Polymerase II; DNA Replication; DNA, Neoplasm; Electrophoresis, Agar Gel; Fibroblasts; Humans; Melanoma

DNA replication intermediates in human melanoma cells have been investigated by using the drug aphidicolin, which inhibits DNA polymerase alpha. In untreated cells, Okazaki fragments and 10-kilobase (kb) DNA intermediates are formed. In aphidicolin-treated cells, the replication fork is stopped and there is no formation of DNA replication intermediates. However, 10-kb DNA intermediates formed before the drug blockade are ligated to high molecular weight DNA whereas already formed Okazaki fragments accumulate in the cell. Moreover, in cells released from aphidicolin inhibition there is preferential labeling of 10-kb DNA compared to Okazaki fragments. The 10-kb DNA and the Okazaki fragments, therefore, respond differently to aphidicolin.

Topics: Aphidicolin; Base Composition; Cell Line; Diterpenes; DNA Polymerase II; DNA Replication; DNA, Neoplasm; Humans; Kinetics; Melanoma; Molecular Weight; Nucleic Acid Synthesis Inhibitors

Aphidicolin inhibits DNA replication and growth of all tested human and murine neoplastic cells including leukemic T- and B-lymphocytes and melanocarcinoma cells. The concentration of aphidicolin causing 50% inhibition of DNA synthesis in all of the tested neoplastic cell lines is similar to that necessary to inhibit DNA synthesis in HeLa cells by 50%. The mechanism of inhibition of DNA synthesis in neoplastic cells is again due to the inhibition of DNA polymerase alpha by aphidicolin. Aphidicolin at a concentration 100 times higher than that causing 50% inhibition of DNA synthesis and cell growth had no effect on total protein synthesis, on the secretion of immunoglobulins, or on the expression of HLA antigens which are involved in relevant phenomena of the immune response.

Topics: Animals; Aphidicolin; Cell Division; Cell Line; Diterpenes; DNA Polymerase II; DNA Replication; HLA Antigens; Humans; Immunoglobulins; Leukemia; Lymphocytes; Melanoma; Mice; Multiple Myeloma; Protein Biosynthesis