aphidicolin and Carcinoma

Cell migration requires precise control, which is altered or lost when tumor cells become invasive and metastatic. beta-catenin plays a dual role in this process: as a member of adherens junctions it is essential to link cadherins to the cytoskeleton thereby allowing tight intercellular adhesion, and as a member of the Wnt-signaling pathway, beta-catenin is translocated into the nucleus and serves together with the LEF1/TCF-transcription factors to drive gene expression necessary for the epithelial-to-mesenchymal transition (EMT). Activated beta-catenin signaling has been implicated in the genesis of a variety of tumors. Here we demonstrate a pivotal function for beta-catenin signaling in epithelial cell migration and tumorigenesis. Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) induce beta-catenin signaling under conditions where they stimulate cell motility. Ectopic expression of either stabilized beta-catenin or a regulatable form of activated beta-catenin induces cell migration in different cell types and cooperates with EGF and HGF in this process. Activation of beta-catenin signaling induces expression of the new target gene osteopontin during migration. Cells expressing stabilized beta-catenin also exhibit significantly increased capability to form tumors in a nude mouse xenograft model. The data suggest that a critical threshold of beta-catenin signaling, activated by cooperative mechanisms, may be important during the EMT and tumorigenesis.

Topics: Animals; Aphidicolin; beta Catenin; Carcinoma; Cell Line; Cell Movement; Cycloheximide; Cytoskeletal Proteins; Enzyme Inhibitors; Epidermal Growth Factor; Epithelial Cells; Gene Targeting; Mice; Mice, Nude; Neoplasms; Osteopontin; Protein Synthesis Inhibitors; Rats; Sialoglycoproteins; Signal Transduction; Trans-Activators; Transcriptional Activation; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Detailed investigations have addressed the infection pathway of recombinant adenovirus (Ad) gene transfer vectors, but little attention has been paid to the influence of cell physiology on the outcome of Ad infection. Based on observations that Ad infection of clonal cell populations show cell-to-cell variability in the extent of capsid binding, we hypothesized that the cell cycle may influence the outcome of Ad infection. To address this hypothesis, we evaluated Ad association with cells in both unsynchronized and pharmacologically synchronized cell populations. In unsynchronized cell populations, elevated Ad association with cells correlated with expression of cyclin B1, a marker of entry into the M phase of mitosis. The same analysis conducted on cell populations that were synchronized at M phase (using paclitaxel or nocodazole) or at S phase (using aphidicolin) confirmed that M phase cells bound three- to sixfold more capsid compared with unsynchronized cells, which are primarily in the G(1) and G(2) phases. The elevated association of vectors with cells translated into 2.5- to 4-fold greater transgene expression 24 hours after infection. Assessment of cell surface expression of Ad receptors demonstrated that both the high-affinity coxsackie-adenovirus receptor for Ad fiber protein and the low-affinity alpha(v) integrin receptor for Ad penton base protein showed increased cell surface expression at M phase (1.5-fold and 2- to 3-fold increases, respectively). These data demonstrate that Ad infection of a homogenous population of cells can vary depending on the cell cycle stage, with enhanced Ad binding and expression correlating with the enhanced expression of Ad receptors during M phase. These observations have relevance to understanding the mechanisms of gene transfer by Ad vectors and should help in the design of in vivo gene transfer strategies.

Topics: Adenoviridae; Antigens, CD; Aphidicolin; Capsid; Carcinoma; Cell Cycle; Coxsackie and Adenovirus Receptor-Like Membrane Protein; Cyclin B; Cyclin B1; Gene Expression; Gene Transfer Techniques; Genetic Vectors; Integrin alphaV; Lung Neoplasms; Nocodazole; Paclitaxel; Receptors, Virus; Transgenes; Tumor Cells, Cultured

Chlorambucil was previously found to be a specific inhibitor of total histone synthesis without affecting total cellular protein synthesis. In this study, we used S and G2 phase HEp-2 cancer cells to further analyze and specifically localize this effect. One hour (S phase), 4 hours (S phase) and 9 hours (G2 phase) after release from an aphidicolin double block synchronization procedure, cells were preincubated for 60 min with 30 microM chlorambucil and then radiolabeled for another 60 min in the continued presence of the agent. At the end of each of these time intervals, cells in almost mid-S phase, late S phase and toward the end of the G2 phase were obtained for analysis. It was found that chlorambucil partially inhibits total histone synthesis nonspecifically as to the variants being synthesized (S phase and basal variants) but only during the first half of the S phase. DNA synthesis is also inhibited partially, but during the second half of the S phase. The position during the S phase where chlorambucil exerts its effect on total histone synthesis, the degree of this effect and its uncoupling with DNA synthesis inhibition, indicate that it is temporally linked with the onset of S phase histone transcription and not with DNA synthesis initiation as occurs with agents, such as hydroxyurea.

Topics: Antineoplastic Agents, Alkylating; Aphidicolin; Carcinoma; Cell Division; Chlorambucil; DNA, Neoplasm; Electrophoresis; G2 Phase; Histones; Humans; S Phase; Thymidine; Tumor Cells, Cultured

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

Previous cell line comparisons indicated that neither S-phase fraction nor topoisomerase I (top1) levels are sufficient to predict camptothecin (CPT) cytotoxicity (F. Goldwasser el al., Cancer Res., 55: 2116-2121, 1995.). To identify new determinants for CPT activity, two mutant p53 human colon cancer cell lines, SW620 and KM12, that were previously reported to have similar top1 levels and differential sensitivity to CPT were studied. No difference in the kinetics of top1-mediated DNA single-strand breaks or DNA synthesis inhibition were observed after 1 h exposure to 1 microM CPT. Pulse-labeling alkaline elution showed deficiency of damaged replicon elongation in the more sensitive SW620 cells. Consistentiy, flow cytometry analyses showed that KM12 was arrested in G2, whereas SW620 cells were irreversibly blocked in S phase. Aphidicolin protection was minimal in KM12 and more pronounced in the more sensitive SW620 cells. Thus, CPT appears to have two cytotoxic mechanisms, one protectable by aphidicolin and present in SW620 and the other not protectable by aphidicolin and common to both cell lines. SW620 exhibited also a greater capacity to break through the G2 checkpoint after DNA damage. Consistently, SW620 cells failed to down-regulate cyclin B-cdc2 kinase activity, whereas KM12 cells down-regulated cyclin B/cdc2 kinase activity within 30 min to 20 % of control level after CPT treatment. Analysis of the 7 human colon carcinoma cell lines of the NCI Anticancer Drug Screen showed that defects in replicon elongation and G2 breakthrough capability correlate with sensitivity to CPT. Our results suggest that misrepair of damaged replicons and/or alterations in DNA damage checkpoints is critical to defining chemosensitivity to CPT-induced top1-cleavable complexes and that CPT appears to have two cytotoxic mechanisms, one protectable by aphidicolin, and the other not.

Topics: Antineoplastic Agents, Phytogenic; Aphidicolin; Camptothecin; Carcinoma; CDC2 Protein Kinase; Colonic Neoplasms; Cyclins; DNA Damage; DNA Repair; DNA Replication; Enzyme Inhibitors; G2 Phase; Humans; S Phase; Topoisomerase I Inhibitors; Tumor Cells, Cultured

Prior reports demonstrated more than additive cytotoxic effects of cis-diamminedichloroplatinum(II) (CDDP) and 1-beta-D-arabinofuranosylcytosine (ara-C) in LoVo colon carcinoma cells. We have extended these findings by analyzing mechanisms that may underlie the effect of ara-C on CDDP-induced cytotoxicity. In contrast to a previous study, ara-C neither enhances DNA interstrand cross-link formation by CDDP nor affects the excision of platinum from DNA. Features peculiar to ara-C, such as its misincorporation into DNA, probably contribute since more than additive cytotoxic effects do not occur by combinations of CDDP with inhibitors of DNA synthesis that are not incorporated into DNA. Also, while ara-C does not significantly enhance the degree of inhibition of DNA synthesis caused by CDDP, the recovery of DNA synthesis after drug removal is significantly slowed when cells are exposed to both drugs. These findings contrast with those obtained with CDDP and aphidicolin (the latter agent resembles ara-C in competing with dCTP for binding to DNA polymerase alpha but, unlike ara-C, is not incorporated into DNA). Lastly, ara-C is incorporated into LoVo cell DNA undergoing replicative synthesis as well as into DNA undergoing repair synthesis after CDDP-induced induced DNA damage.

Topics: Aphidicolin; Carcinoma; Cell Line; Cell Survival; Cisplatin; Colonic Neoplasms; Cytarabine; Diterpenes; DNA Damage; DNA Repair; Humans

Adenovirus types 2 and 5 DNA synthesized in vivo and in vitro in the presence of aphidicolin were studied. Inhibition of adenoviral DNA synthesis by aphidicolin was only 70% even at a concentration of 30 micrograms/ml of aphidicolin, at which the cellular DNA synthesis was completely inhibited. When initiation of the viral DNA synthesis was synchronized with hydroxyurea and labeled with [3H]thymidine for 60 min, the viral DNA synthesized in the presence of 30 micrograms/ml of aphidicolin was not of full length (35 kb) but small (approximately 12 kb) by analysis of alkaline sucrose density gradient centrifugation. When initiation of the viral DNA synthesis was not synchronized, the viral DNAs ranging from full size to 12 kb were synthesized in the presence of aphidicolin, indicating that the nascent DNAs longer than about 12 kb can continue to elongate in the presence of aphidicolin. This 12 kb DNA was not derived from the degradation products of newly synthesized full size adenoviral DNA. The viral DNA synthesis was restored and the full size of adenoviral DNA was attained within 15 min following removal of aphidicolin. About 20% of the entire viral genome length from the 5'-end was not inhibited by aphidicolin, while the synthesis of interior fragments of the adenoviral DNA was markedly inhibited by aphidicolin, judging from the electrophoretic pattern on neutral agarose gel after digestion of DNA with Hind III. These results indicate that aphidicolin inhibits adenoviral DNA replication at the internal region located approximately 20-30% from both terminals.

Topics: Adenoviruses, Human; Antiviral Agents; Aphidicolin; Carcinoma; Cell Line; Cell Transformation, Viral; Diterpenes; DNA Replication; DNA, Viral; Humans; Kinetics; Mouth Neoplasms

Aphidicolin-resistant mutants of mouse FM3A cells were isolated and characterized. Most of the mutants were of a type showing associated resistance to arabinosyladenine, arabinosylcytosine, deoxyadenosine, and excess thymidine. This phenotype could also be observed in a variant line selected by resistance to a low level of arabinosylcytosine. In cell-cell hybrids, aphidicolin resistance as well as this cross-resistance behaved a codominant traits. The mutants had an increased dATP pool and decreased ability to incorporate labeled deoxycytidine into macromolecules. Genetic and biochemical evidence suggested that the mutation conferring the pleiotropic phenotype resulted from a change in ribonucleotide reductase activity such that the enzyme was desensitized to the allosteric negative effector dATP. This alteration of the enzyme could account for the marked change in deoxynucleotide pools and for the aphidicolin resistance of the mutants.

Topics: Adenosine Triphosphate; Animals; Aphidicolin; Carcinoma; Cell Line; Diterpenes; Drug Resistance; Mammary Neoplasms, Experimental; Mice; Mice, Inbred C3H; Mutation; Phenotype; Ribonucleotide Reductases