aphidicolin and Ovarian-Neoplasms

Fragile sites are nonrandomly located gaps and/or breaks and their expres-sion can be induced by specific culture conditions. There are many reports in the literature that indicate that these sites can act as factors that predispose to specific chromosome aberrations and other complex rearrangement in the chromosome and their association with cancers. In the present study, the expression of the fragile sites induced by aphidicolin was evaluated on prometaphase chromosomes from peripheral blood lymphocytes of 55 patients with breast cancer patients belonging to different stages of the cancer, 25 patients with epithelial ovarian cancer, and 13 with non-small-cell lung cancer, 100 of their first-degree clinically healthy female relatives, and 100 normal age-matched healthy persons without a familial history of cancer. The frequency of expression of the fragile sites in cancer patients and their first-degree relatives was found to be statistically significant (P<0.05) than those of the controls. In different stages of breast cancer patients, 6q26 is the best-defined fragile site whereas 13q13 is confined to stage II and stage III patients only. The chromosomal aberration rate/cell in breast cancer patients was found to be 0.29+/-0.13, in epithelial ovarian cancer patients 0.38+/-0.14, and in non-small-cell lung cancer 0.29+/-0.11 as compared to 0.07+/-0.03 in controls, and was found to be statistically significant. Therefore, our results indicate that these fragile sites may be the unstable sites in the genome and, hence, can be used as suitable and reliable markers for genetic predisposition to breast cancer, epithelial ovarian cancer, and in non-small-cell lung cancer.

Topics: Aphidicolin; Breast Neoplasms; Carcinoma, Non-Small-Cell Lung; Chromosome Fragile Sites; Chromosome Fragility; Chromosome Mapping; Female; Genetic Predisposition to Disease; Humans; Lung Neoplasms; Lymphocytes; Neoplasm Staging; Ovarian Neoplasms

Loss of expression of mismatch repair (MMR) proteins leads to resistance of tumor cells to a variety of DNA-damaging agents, including bifunctional alkylating and monofunctional methylating agents such as cis-diaminedichloroplatinum II (CDDP) and N'-methyl-N-nitrosourea (MNU). It has been suggested that coupling to cell death does not occur in the absence of MMR, but instead, DNA lesions are bypassed during replication, giving a drug-tolerant phenotype. In the present study, we have used aphidicolin (Ap), an inhibitor of DNA polymerases, to study the role of replicative bypass in drug resistance mediated by loss of MMR. We have examined the survival of matched ovarian carcinoma cell lines with known MMR status after sequential treatment with CDDP or MNU and Ap. We show that Ap increases the sensitivity of MMR-deficient cell lines to CDDP and MNU to a greater extent than their MMR-proficient counterparts. Furthermore, loss of MMR correlates with loss of CDDP-induced G2 arrest, but this is partially restored after Ap treatment. These data support Ap sensitizing drug-resistant cancer cells that have lost MMR to CDDP and MNU and suggest that the potential use of Ap as a modulator of drug resistance should be targeted to MMR-defective tumors.

Topics: Adaptor Proteins, Signal Transducing; Antineoplastic Agents; Aphidicolin; Apoptosis; Base Pair Mismatch; Carrier Proteins; Cisplatin; DNA Damage; DNA Repair; DNA Replication; DNA, Neoplasm; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Inhibitors; Female; G2 Phase; Humans; Methylnitrosourea; MutL Protein Homolog 1; Neoplasm Proteins; Nuclear Proteins; Nucleic Acid Synthesis Inhibitors; Ovarian Neoplasms; Tumor Cells, Cultured; Tumor Stem Cell Assay

Repair of cisplatin-damaged DNA was investigated in a human ovarian carcinoma cell line (2008) and its cisplatin-resistant variant (C13*) using a host-cell reactivation (HCR) assay. The HCR of cisplatin-damaged adenovirus (Ad) was not significantly different in C13* cells compared to 2008 cells. The cisplatin concentrations required to reduce the amount of viral DNA replicated to 50% were 0.12 +/- 0.02 microM and 0.10 +/- 0.01 microM after 48 h of repair in 2008 and C13* cells respectively. Similarly, the cisplatin concentration required to reduce the expression of a reporter gene inserted in the viral DNA was not significantly altered in C13* cells compared to the parental line (IC50 values were 0.28 +/- 0.04 microM in 2008 cells and 0.17 +/- 0.06 microM in C13* cells after 48 h of repair). Pretreatment of the cells with cisplatin, immediately prior to Ad infection, did not significantly alter the HCR of cisplatin-damaged Ad in either cell type. In addition, a cisplatin-sensitive variant derived from the C13* cells, namely the RH4 cells, did not differ significantly from either the 2008 or C13* cells in their ability to reactivate cisplatin-damaged Ad. Furthermore, a component of the nucleotide excision repair (NER) pathway, DNA polymerase alpha, was investigated using the competitive inhibitor aphidicolin. The combination of cisplatin and aphidicolin resulted in similar synergistic growth inhibition in both the 2008 and C13* cells providing additional support to the HCR results which suggest that enhanced NER is not responsible for the cisplatin resistance in C13* cells.

Topics: Adenoviridae; Antineoplastic Agents; Aphidicolin; Carcinoma; Cell Survival; Cisplatin; DNA Adducts; DNA Damage; DNA Polymerase II; DNA Repair; DNA, Viral; Drug Synergism; Enzyme Inhibitors; Female; Humans; Ovarian Neoplasms; Tumor Cells, Cultured

Enhanced DNA repair has been observed in cisplatin-resistant ovarian cancer cell lines. This resistance can be modulated, on co-incubation with aphidicolin in established cell lines and animal tumour models, by inhibiting DNA polymerases. We describe a study of the in vitro modulation effect of aphidicolin on cisplatin and carboplatin using fresh cells harvested from biopsy samples or ascitic fluids from 25 patients with ovarian adenocarcinoma. The MTT assay was used to measure cell survival after drug exposure. Aphidicolin (up to 30 microM) showed no cytotoxicity when tested alone. Forty-seven comparisons were made between drug with and without aphidicolin, and 37 (79%) cases demonstrated a significant increase in sensitivity to the platinum agents on co-incubation. Overall, there was a median 10-fold (range 1.64- to 58.5-fold) increase in sensitivity. When patients were grouped according to in vitro sensitivity to platinum, aphidicolin had a significantly greater effect in the "resistant' group, causing a median 13.5-fold increase in sensitivity compared with 2.4-fold in the "sensitive' group. Furthermore, a positive correlation between the LC50 for platinum and the corresponding fold increase in sensitivity suggests that aphidicolin overcomes platinum resistance in fresh cells from primary tumours. These results encourage the further development of this interesting compound.

Topics: Adenocarcinoma; Antineoplastic Agents; Aphidicolin; Carboplatin; Cisplatin; DNA Repair; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Female; Humans; Ovarian Neoplasms; Tumor Cells, Cultured

Aphidicolin, an inhibitor of DNA polymerases alpha and delta, is cytotoxic in vitro against tumor cells. The poor solubility of aphidicolin has led to the development of aphidicolin glycinate (AG; NSC 303812), a water soluble ester currently in early clinical trials. The antitumor activity of AG was investigated in a series of transplantable murine tumors in vivo. The drug demonstrated activity against the i.p. implanted B16 melanoma, producing maximum increased life spans of 75% following i.p. administration every 3 h for three doses on days 1-9. Treatment schedules involving both single injections per day on days 1-9 and multiple injections per day on days 1, 5, and 9 were less effective, indicating that this antitumor activity is schedule dependent. Similarly, greater activity was observed against the i.p. M5076 sarcoma when three daily injections were given on days 1-9 (57% increased life span) than with a single injection either on days 1-9 (36% increased life span) or on days 1, 5, 9, and 13 (inactive). Further scheduling studies in the s.c. M5076 sarcoma model showed that a 7-day infusion was superior to both a 24-h infusion and a 7-day course of three bolus treatments per day. On the assumption that DNA polymerase inhibition is the basis for this antitumor activity, inhibition of DNA synthesis in BALB/c x DBA/2 F1 mice was investigated by measuring incorporation of [3H]thymidine (20 microCi, i.v.) into DNA of spleen and jejunum. At 2 h after administration of AG, inhibition of DNA synthesis was dose dependent (median inhibitory dose, 60 mg/kg in both tissues) and was > 99% at 300 mg/kg. The inhibition was rapid in onset; AG (100 mg/kg i.p.) produced maximal (> 98%) inhibition in both tissues at 30 min. Recovery occurred in the intestine within 16 h; in spleen recovery was delayed to 24 h, and was followed by a rebound incorporation at 48 h (203%). A comparison of the inhibition of thymidine incorporation in tumor cells (B16 melanoma and P388 leukemia) and normal jejunum revealed no significant differences in the extent of inhibition or the rapidity of recovery in these tissues. The rapid recovery of DNA synthesis inhibition supports the use of prolonged infusion schedules in clinical trials, but the lack of evidence of selectivity for tumor cells suggests that AG may be of limited therapeutic value as a single agent. Thus, we evaluated AG in combination with cisplatin in an in vivo model of cisplatin refractory human ovarian cancer.(ABSTRACT TRUNC

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Aphidicolin; Cisplatin; Disease Models, Animal; DNA Repair; DNA-Directed DNA Polymerase; Drug Administration Schedule; Drug Resistance; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Neoplasms, Experimental; Nucleic Acid Synthesis Inhibitors; Ovarian Neoplasms

Exposure of human ovarian cancer SW626 cell line to 0.08 mumol/l methotrexate or 25 mumol/l aphidicolin for 24 h caused no cytotoxicity but enhanced etoposide cytotoxicity. Methotrexate or aphidicolin treatment induced a reversible blockade at the beginning of S phase which was reversed upon drug removal with a consequent wave of synchronisation. The enhancement of etoposide cytotoxicity was not due to higher etoposide intracellular uptake in the methotrexate or aphidicolin-pretreated cells. The topoisomerase II content in methotrexate or aphidicolin pretreated SW626 cells was higher than in control cells assessed by western blotting or flow cytometry. The higher etoposide cytotoxicity observed after synchronization with methotrexate or aphidicolin was apparently unrelated to the number of drug-induced DNA-topoisomerase II complexes evaluated as DNA double strand breaks or DNA-protein crosslinks. These data support the view that etoposide-induced DNA-topoisomerase II complexes are more cytotoxic in cells which are in S-phase.

Topics: Aphidicolin; Cell Survival; DNA Topoisomerases, Type I; DNA, Neoplasm; Drug Synergism; Etoposide; Female; Humans; Methotrexate; Mitosis; Ovarian Neoplasms; Tumor Cells, Cultured; Tumor Stem Cell Assay

Aphidicolin, a reversible inhibitor of DNA polymerase alpha and delta, has recently been reported to reverse the resistance to cisplatin (DDP) of an ovarian cancer cell line. We investigated the pharmacokinetics of aphidicolin in mice and examined its activity either alone or in combination with DDP in the DDP-sensitive M5076 (M5) murine reticular cell sarcoma as well as in a DDP-resistant subline (M5/DDP). The drug was cleared from plasma very rapidly (clearance, 41.6 ml min-1 kg-1), showing a half-life of 15 min. Aphidicolin concentrations in the tumor were approximately 50% of those found in plasma at steady state. Using several dose schedules and continuous infusions we failed to detect significant antitumor activity for aphidicolin glycinate. Potentiation of the activity of DDP by aphidicolin glycinate was moderate in mice bearing M5 tumor as well as in those bearing M5/DDP tumor. These data do not support the possible clinical use of aphidicolin in combination with DDP. However, further studies should be carried out in different tumor models before this possibility is conclusively ruled out.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Aphidicolin; Cell Line; Cisplatin; Drug Administration Schedule; Drug Resistance; Drug Synergism; Female; Half-Life; Infusions, Intravenous; Injections, Intraperitoneal; Mice; Mice, Inbred C57BL; Ovarian Neoplasms

We have shown that cis diamminedichloroplatinum-(II) (DDP) resistant mutants can be isolated from the human ovarian carcinoma cell line A2780 using a single-step selection protocol with DDP. DDP resistant colonies were calculated to be present at a frequency of 1.7 x 10(-6)/viable cell using a fluctuation analysis. The mutational origin of these surviving colonies is inferred by the fact that their frequency is increased by treatment of the A2780 cells with the chemical mutagen ethyl methane-sulfonate, with a maximum frequency observed after a 3-day expression time. Independently isolated clones maintain, in the absence of selection, a DDP resistant phenotype up to 7-fold more resistant than the parental A2780 cells. The resistance modifiers aphidicolin and buthionine sulfoximine have no effect on the frequency of DDP resistant mutants. Therefore neither of these drugs appears to have an effect on increasing the sensitivity of DDP resistant mutants existing in a cell population prior to DDP exposure.

Topics: Aphidicolin; Buthionine Sulfoximine; Cell Survival; Cisplatin; Diterpenes; Drug Resistance; Female; Humans; Methionine Sulfoximine; Mutation; Ovarian Neoplasms; Phenotype; Tumor Cells, Cultured; Tumor Stem Cell Assay

Two recently established human ovarian carcinoma cell lines (JA-T and TR175) have been used to study the effects of aphidicolin glycinate (APG), a specific competitive inhibitor of DNA polymerase alpha (Ikegani et al. (1978) Nature, 275, 458-460), on the formation and removal of four platinum-DNA adducts. Logarithmically-growing cells were exposed to cis-diamminedichloroplatinum (II) (cisplatin) (10 micrograms, 33.4 microM) in the presence or absence of APG (5 or 50 micrograms/ml, 11.6 or 116 microM). Platinum-DNA adducts were quantitated using a competitive ELISA technique. No differences were observed between the initial levels of total DNA platination and of specific DNA adducts formed in the presence or absence of APG in either cell line. Following 18 h posttreatment incubation both lines showed some ability to remove each of the three main platinum-DNA lesions (Pt-GMP, Pt-AG and Pt-GG). However, the levels of these specific DNA adducts decreased over this time period, by similar rates with or without APG addition. It was also shown that the APG concentrations used had minimal inhibitory effects alone on growth or DNA synthesis during this 18 h posttreatment incubation period. Furthermore its addition did not significantly modify cisplatin-induced cytotoxicity, as judged by inhibition of growth or DNA synthesis over this time period. We therefore conclude that under these experimental conditions APG does not modulate 'repair' of cisplatin-induced DNA damage in logarithmically-growing cultures of these two apparently 'repair-proficient' human ovarian tumour cell lines.

Topics: Antibiotics, Antineoplastic; Aphidicolin; Cisplatin; Diterpenes; DNA; DNA Damage; DNA Polymerase II; DNA Repair; Female; Guanine; Humans; Ovarian Neoplasms; Platinum; Tumor Cells, Cultured

DNA damage was evaluated by flow cytometric (FCM) analysis of cells treated with L-phenylalanine mustard (L-PAM) and stained with anti-DNA monoclonal antibody (MAb) F7-26. DNA damage was rapidly repaired, as indicated by the loss of DNA immunoreactivity after removal of L-PAM. Two types of drug combinations were found to inhibit DNA repair. Combinations containing inhibitors of DNA polymerase (ara-C, aphidicolin) or these inhibitors and hydroxyurea inhibited DNA repair in A2780/PAM and A549 cells. The inhibition of DNA repair by combinations of DNA-damaging agents thioTEPA or cisplatin and DNA polymerase inhibitors is a novel observation based on the specificity of DNA damage assay with MAb F7-26. Combinations containing thioTEPA or cisplatin inhibited DNA repair in A549 but not in A2780/PAM cells. Drug combinations which inhibited DNA repair also significantly enhanced cell killing by L-PAM. Cell survival in cultures treated with L-PAM and efficient inhibitors was 2 to 3 orders of magnitude lower than was expected for additive survival. ThioTEPA and cisplatin play a dual role in combination chemotherapy by inducing DNA damage and inhibiting repair of DNA damage. FCM analysis of DNA repair may be a useful component of drug evaluation and could be applied to determine cell-type specific sensitivity to inhibitors of DNA repair.

Topics: Alkylating Agents; Antineoplastic Agents; Aphidicolin; Cell Line; Cell Survival; Cisplatin; Cytarabine; Diterpenes; DNA Damage; DNA Repair; Female; Humans; Lung Neoplasms; Melphalan; Nucleic Acid Synthesis Inhibitors; Ovarian Neoplasms; Thiotepa

We examined the effect of specific inhibitors of DNA polymerases alpha and delta, and beta, on cisplatin (DDP) cytotoxicity in DDP-sensitive and -resistant human 2008 ovarian carcinoma cells. Under conditions of continuous exposure to drug combinations, neither aphidicolin glycinate (AG) nor dideoxythymidine enhanced the cytotoxicity of DDP in either cell line as determined by clonogenic survival assays. However, when clonogenic survival was determined following short-term drug exposure, AG exhibited strong synergism with DDP in the DDP-resistant, but not the DDP-sensitive cells, as indicated by median effect analysis of the data. DNA polymerase alpha mRNA levels were the same in both cell lines under basal conditions. DDP-sensitive cells, but not DDP-resistant cells, were able to increase their expression of DNA polymerase alpha in response to DDP exposure. Levels of mRNA for DNA polymerase beta and for the human DNA repair gene ERCC-1 were not elevated in resistant cells, either under basal conditions or 18 hr after a 1 hr exposure to IC20 concentrations of DDP. In another human ovarian carcinoma cell line, A2780, AG and DDP were synergistic in both DDP-sensitive and -resistant variants in short-term exposure. We conclude that DNA polymerases alpha and/or delta play a role in the DDP sensitivity of human ovarian carcinoma cells.

Topics: Antibiotics, Antineoplastic; Aphidicolin; Blotting, Northern; Cell Line; Cell Survival; Cisplatin; Dideoxynucleosides; Diterpenes; DNA Polymerase I; DNA Polymerase II; Female; Humans; Ovarian Neoplasms; Plasmids; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

We have studied the effect of glutathione reduction by buthionine sulfoximine (BSO), a specific inhibitor of gamma -glutamyl cysteine synthetase, on DNA repair after cisplatin damage in an ovarian cancer cell line with in vitro induced resistance to cisplatin. In addition, we have examined the effect of aphidicolin, a specific inhibitor of DNA polymerase alpha, in combination with BSO on cisplatin-associated DNA repair. BSO treatment was found to partially inhibit DNA repair, and the addition of aphidicolin caused nearly a 100% inhibition in DNA repair activity. Treatment of cells with glutathione ester after BSO resulted in complete recovery of DNA repair activity or partial recovery if aphidicolin was present. The significance of these results to the chemosensitizing effects of BSO medicated glutathione reduction is discussed.

Topics: Aphidicolin; Buthionine Sulfoximine; Centrifugation, Density Gradient; Cisplatin; Diterpenes; DNA Polymerase II; DNA Repair; Drug Resistance; Female; Glutathione; Humans; Methionine Sulfoximine; Ovarian Neoplasms; Tumor Cells, Cultured

A human ovarian cancer cell line, A2780, derived from an untreated ovarian cancer patient and relatively sensitive to cisplatin was treated by stepwise incubation with cisplatin to produce a cisplatin-resistant variant, 2780CP. The relative abilities of these cell lines to repair cisplatin-induced damage to cellular DNA then was examined by measure of [3H]thymidine incorporation into normal density DNA separated from bromodeoxyuridine-substituted DNA on alkaline cesium chloride gradients. These studies revealed that primary cisplatin resistance present in 2780CP was associated with a near twofold-increased ability to repair damage induced by the drug under conditions where 2780CP was approximately 5-fold resistant to cisplatin. Aphidicolin, a specific inhibitor of DNA polymerase alpha, showed a dose-dependent capacity to inhibit DNA repair in this system with maximum inhibition of 63% at 4 micrograms/ml. It was also found that inhibition of DNA repair during and shortly after cisplatin exposure resulted in an approximately threefold increase in the cytotoxicity of cisplatin as monitored by clonogenic cell survival in the resistant but not the sensitive parental cell line.

Topics: Aphidicolin; Bromodeoxyuridine; Cell Line; Cisplatin; Diterpenes; DNA Repair; Drug Resistance; Female; Humans; Ovarian Neoplasms; Thymidine

Topics: Aphidicolin; Cisplatin; Diterpenes; DNA Repair; Dose-Response Relationship, Drug; Drug Resistance; Female; Humans; Ovarian Neoplasms; Tumor Cells, Cultured

Chromosomal fragile sites that are inducible by methotrexate and aphidicolin are frequent in the human population. To assess the frequency and distribution of these common fragile sites, we performed a cytogenetic survey on lymphocytes from subjects known to be particularly prone to breakage because of constitutional chromosomal instability, the possession of a rare fragile site, or Fanconi anemia. Furthermore, a group of cancer patients was included in this study in view of possible acquired chromosomal instability. Lymphocyte chromosomes from several healthy donors were analyzed under identical conditions. We found that methotrexate- and aphidicolin-induced fragile sites are widespread in the general population, showing a similar breakpoint distribution. Ten fragile sites (3p14, 16q23, 2q32, 6q25, 4p16, 4q31, 14q24, 1p31, 20p12, 7q21) were observed in at least 40% of the individuals among the different groups. Our data point out a significantly increased breakage induced by aphidicolin in lymphocytes from cancer patients and, to a lesser extent, from rare fragile sites carriers. These results suggest that common fragile sites are enhanced in some constitutional and acquired conditions.

Topics: Adenocarcinoma; Adolescent; Adult; Aged; Aphidicolin; Cells, Cultured; Chromosome Aberrations; Chromosome Disorders; Chromosome Fragile Sites; Chromosome Fragility; Diterpenes; Fanconi Anemia; Female; Heterozygote; Humans; Italy; Lymphocytes; Male; Methotrexate; Middle Aged; Ovarian Neoplasms