aphidicolin and Lung-Neoplasms

Checkpoint kinase 1 (CHK1) is a key mediator of the DNA damage response that regulates cell-cycle progression, DNA damage repair, and DNA replication. Small-molecule CHK1 inhibitors sensitize cancer cells to genotoxic agents and have shown single-agent preclinical activity in cancers with high levels of replication stress. However, the underlying genetic determinants of CHK1 inhibitor sensitivity remain unclear. We used the developmental clinical drug SRA737 in an unbiased large-scale siRNA screen to identify novel mediators of CHK1 inhibitor sensitivity and uncover potential combination therapies and biomarkers for patient selection. We identified subunits of the B-family of DNA polymerases (

Topics: Aphidicolin; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Checkpoint Kinase 1; Colorectal Neoplasms; DNA Damage; DNA Polymerase beta; DNA Polymerase I; DNA Polymerase II; Drugs, Investigational; Enzyme Inhibitors; Gene Knockdown Techniques; Heterocyclic Compounds, 4 or More Rings; Humans; Lung Neoplasms; Neoplasm Proteins; Poly-ADP-Ribose Binding Proteins; Retinoids; RNA, Small Interfering

p21(Cip1/WAF1) is a known inhibitor of the short-gap filling activity of proliferating cell nuclear antigen (PCNA) during DNA repair. In agreement, p21 degradation after UV irradiation promotes PCNA-dependent repair. Recent reports have identified ubiquitination of PCNA as a relevant feature for PCNA-dependent DNA repair. Here, we show that PCNA ubiquitination in human cells is notably augmented after UV irradiation and other genotoxic treatments such as hydroxyurea, aphidicolin and methylmethane sulfonate. Intriguingly, those DNA damaging agents also promoted downregulation of p21. While ubiquitination of PCNA was not affected by deficient nucleotide excision repair (NER) and was observed in both proliferating and arrested cells, stable p21 expression caused a significant reduction in UV-induced ubiquitinated PCNA. Surprisingly, the negative regulation of PCNA ubiquitination by p21 does not depend on the direct interaction with PCNA but requires the cyclin dependent kinase binding domain of p21. Taken together, our data suggest that p21 downregulation plays a role in efficient PCNA ubiquitination after UV irradiation.

Topics: Antineoplastic Agents; Aphidicolin; Cell Cycle; Cell Proliferation; Cells, Cultured; Colorectal Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; DNA Damage; DNA Repair; Down-Regulation; Enzyme Inhibitors; Fibroblasts; Humans; Hydroxyurea; Lung Neoplasms; Methyl Methanesulfonate; Mutagens; Proliferating Cell Nuclear Antigen; Ubiquitin; Ultraviolet Rays

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

Accumulating evidence indicates that small cell lung cancer (SCLC) is defective in many of the regulatory mechanisms that control cell cycle progression. The purpose of this study was to determine the effects of flavopiridol, a pan-cyclin-dependent kinase inhibitor, on growth and apoptosis of SCLC cell lines.. Cell growth was monitored using 3-(4,5dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (MTT) and clonogenic assays. Induction of apoptosis was assessed using multiple assays, including flow cytometric determination of DNA content and mitochondrial membrane potential, terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL), and Western blot analysis of procaspase 3 and poly(ADP-ribose) polymerase cleavage.. Flavopiridol induced growth inhibition and cytotoxicity in multiple SCLC cell lines, with an IC(50) of 50-100 nM and an LD(50) of 150-200 nM in 72-h MTT assays. The cytotoxicity seen in the MTT assay proved to be apoptosis by several criteria. Interestingly, inhibition of caspase activation with the caspase inhibitor Boc-Asp(OMe)-CH(2)F reduced TUNEL labeling by 40% but did not have any effect on the loss of mitochondrial membrane potential (detected as early as 4 h after drug exposure) or cytotoxicity in MTT assays. These results suggest that the primary event in flavopiridol-induced apoptosis involves induction of mitochondrial dysfunction. Cells synchronized with aphidicolin at the G(1)-S border and treated with flavopiridol during S phase showed a marked increase in apoptosis compared with an asynchronous population or a population treated during G(2)-M. Despite the increased apoptosis, a significant proportion of synchronized cells proceeded through S, G(2)-M, and into G(1) phase in the presence of flavopiridol, demonstrating that a high-grade cell cycle arrest is not required for apoptosis. Cells synchronized at the G(1)-S border treated with a short exposure to flavopiridol also showed more than a 10-fold decrease in clonogenicity compared with asynchronous cells treated identically.. Taken together, these data demonstrate that flavopiridol potently and selectively induces SCLC apoptosis preferentially during S phase, in a manner that involves early mitochondrial dysfunction without a requirement for a high-grade block to cell cycle progression. Furthermore, clonogenicity data suggests that prior S phase synchronization could be a highly effective way of enhancing the efficacy of bolus or short infusions of flavopiridol in the clinical setting.

Topics: Aphidicolin; Apoptosis; Blotting, Western; Carcinoma, Small Cell; Caspase 3; Caspases; Cell Division; Enzyme Inhibitors; Flavonoids; Flow Cytometry; Humans; In Situ Nick-End Labeling; Lung Neoplasms; Membrane Potentials; Mitochondria; Piperidines; Poly(ADP-ribose) Polymerases; S Phase; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured; Tumor Stem Cell Assay

Mutations in oncogenes and tumor suppressor genes are critical in the development of cancer. A major pathway for the formation of mutations is the replication of unrepaired DNA lesions. To better understand the mechanism of translesion replication (TLR) in mammals, a quantitative assay for TLR in cultured cells was developed. The assay is based on the transient transfection of cultured cells with a gapped plasmid, carrying a site-specific lesion in the gap region. Filling in of the gap by TLR is assayed in a subsequent bioassay, by the ability of the plasmid extracted from the cells, to transform an Escherichia coli indicator strain. Using this method it was found that TLR through a synthetic abasic site in the adenocarcinoma H1299, the osteogenic sarcoma Saos-2, the prostate carcinoma PC3, and the hepatoma Hep3B cell lines occurred with efficiencies of 92 +/- 6%, 32 +/- 2%, 72 +/- 4%, and 26 +/- 3%, respectively. DNA sequence analysis showed that 85% of the bypass events in H1299 cells involved insertion of dAMP opposite the synthetic abasic site. Addition of aphidicolin, an inhibitor of DNA polymerases alpha, delta, and epsilon, caused a 4.4-fold inhibition of bypass. Analysis of two XP-V cell lines, defective in DNA polymerase eta, showed bypass of 89%, indicating that polymerase eta is not essential for bypass of abasic sites. These results suggest that in human cells bypass of abasic sites does not require the bypass-specific DNA polymerase eta, but it does require at least one of the replicative DNA polymerases, alpha, delta, or epsilon. The quantitative TLR assay is expected to be useful in the molecular analysis of lesion bypass in a large variety of cultured mammalian cells.

Topics: Adenocarcinoma; Aphidicolin; Base Sequence; Deoxyadenine Nucleotides; DNA Damage; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; Humans; Lung Neoplasms; Nucleic Acid Synthesis Inhibitors; Plasmids; Transformation, Genetic; Tumor Cells, Cultured

Fragile sites are non-staining gaps and breaks in specific points of chromosomes that are inducible by various culture conditions. Previous studies have shown that various clastogenic agents increase expression of fragile sites. In this study, the expression of common fragile sites induced by aphidicolin was evaluated on prometaphase chromosomes obtained from peripheral blood lymphocytes. Chromosomal aberrations and fragile site expression of 60 individuals, including 20 patients with non-small cell lung cancer (NSCLC), 20 of their clinically healthy family members, and 20 age-matched normal healthy controls without history of any cancer type were studied. Both the proportion of damaged cells (P < 0.001) and the mean number of gaps and breaks per cell (P < 0.001) were significantly higher in both the patients and relatives' groups when compared with the control group. However, they were insignificant when the patients were compared to their relatives (P > 0.05). We determined four aphidicolin type common fragile sites in our study. These sites in patients with NSCLC and relatives were the following: 1p21, 2q33, 3p14, and 16q23. In these fragile sites, 2q33, 3p14, and 16q23 sites were statistically significant when compared with control group (P < 0.001, P < 0.0005, and P < 0.05, respectively). Consequently, we believe that fragile site studies may be helpful to detection of cancer risk.

Topics: Adolescent; Adult; Aged; Aphidicolin; Carcinoma, Non-Small-Cell Lung; Child; Chromosome Aberrations; Chromosome Fragile Sites; Chromosome Fragility; Chromosome Mapping; Family Health; Female; Genetic Predisposition to Disease; Humans; Lung Neoplasms; Male; Middle Aged; Models, Genetic; Models, Statistical; Mutation; Smoking

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

Alveolar epithelial cell (AEC) injury and repair are important in the pathogenesis of oxidant-induced lung damage. Keratinocyte growth factor (KGF) prevents lung damage and mortality in animals exposed to various forms of oxidant stress, but the protective mechanisms are not yet established. Because DNA strand break (DNA-SB) formation is one of the earliest cellular changes that occurs after cells are exposed to an oxidant stress, we determined whether KGF reduces H2O2-induced pulmonary toxicity by attenuating AEC DNA damage. KGF (10-100 ng/ml) decreased H2O2 (0.05-0.5 mM)-induced DNA-SB formation in cultured A549 and rat alveolar type II cells measured by an alkaline unwinding, ethidium bromide fluorometric technique. The protective effects of KGF were independent of alterations in catalase, glutathione (GSH), or the expression of bcl-2 and bax, two protooncogenes known to regulate oxidant-induced apoptosis. Actinomycin D and cycloheximide abrogated protective effects of KGF. Furthermore, protection by KGF was completely blocked by 1) genistein, a tyrosine kinase inhibitor; 2) staurosporine and calphostin C, protein kinase C (PKC) inhibitors; and 3) aphidicolin, butylphenyl dGTP, and 2',3'-dideoxythymidine 5'-triphosphate, inhibitors of DNA polymerase. We conclude that KGF attenuates H2O2-induced DNA-SB formation in cultured AECs by mechanisms that involve tyrosine kinase, PKC, and DNA polymerases. These data suggest that the ability of KGF to protect against oxidant-induced lung injury is partly due to enhanced AEC DNA repair.

Topics: Animals; Aphidicolin; Cell Death; Cycloheximide; Dactinomycin; DNA Damage; DNA Repair; Enzyme Inhibitors; Fibroblast Growth Factor 10; Fibroblast Growth Factor 7; Fibroblast Growth Factors; Genistein; Growth Substances; Humans; Hydrogen Peroxide; Kinetics; Lung Neoplasms; Naphthalenes; Protein Kinase C; Protein-Tyrosine Kinases; Pulmonary Alveoli; Rats; Staurosporine; Tumor Cells, Cultured

Flavopiridol (NSC 649890, L86-8275), a potent inhibitor of cyclin-dependent kinase 1/p34cdc2 phosphorylation and kinase activity, is currently undergoing Phase I clinical testing as a potential antineoplastic agent. Previous studies have suggested that flavopiridol is cytostatic but not cytotoxic when applied to exponentially growing cells. In the present study, various human tumor cell lines were assayed for trypan blue exclusion and ability to form colonies after exposure to flavopiridol under a variety of growth conditions. When log phase A549 non-small cell lung cancer cells were examined 72 h after the start of a 24-h flavopiridol exposure, as many as 90% of the cells accumulated trypan blue. A 24-h exposure to 250-300 nM resulted in trypan blue uptake in 50% of A549 cells at 72 h and a 50% reduction in colony formation. Similar results were observed in HCT8 ileocecal adenocarcinoma, T98G glioblastoma, MCF-7 breast adenocarcinoma, and HL-60 leukemia cells. With A549 cells, identical results were obtained in actively growing logarithmic phase cells and growth-arrested confluent cells. Treatment with the DNA synthesis inhibitor aphidicolin only minimally affected the cytotoxicity of flavopiridol. In contrast, the RNA synthesis inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole or the protein synthesis inhibitor cycloheximide reduced the cytotoxicity of flavopiridol. These results suggest that: (a) flavopiridol is not only cytostatic, but also cytotoxic to a variety of human tumor cell lines; (b) flavopiridol is equally active against cycling and noncycling A549 cells; and (c) RNA and protein synthesis appear to play a role in flavopiridol-induced cytotoxicity.

Topics: Antineoplastic Agents; Aphidicolin; Apoptosis; Cell Cycle; Cell Survival; Cycloheximide; Flavonoids; Humans; Lung Neoplasms; Piperidines; 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

Among common fragile sites, fra(3)(p14.2) is the most expressed either spontaneously or after treatment with aphidicolin (APC) in lymphocyte cultures. Because recurrent chromosomal abnormalities involving the short arm of chromosome 3 in tumor tissue are present in various malignancies, including lung cancer, the induction of fra(3)(p14.2) elicited by APC was investigated with the aim of detecting possible interindividual polymorphism in its expression that might be relevant to predisposition toward cancer-related events. Thirty-four patients affected with various lung cancers (14 squamous cell carcinomas, 13 adenocarcinomas, and seven small cell carcinomas) and 14 controls (patients undergoing routine routine follow-up after coronary by-pass) were included in this study. The frequency of fra(3)(p14.2) expression was not significantly different among the patients grouped either by disease or by sex and age. It was estimated that fra(3)(p14.2) accounts for about 20% of total breakage in APC-treated lymphocyte cultures from the general population.

Topics: Adenocarcinoma; Aphidicolin; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Cells, Cultured; Chromosome Fragile Sites; Chromosome Fragility; Chromosomes, Human, Pair 3; Diterpenes; Humans; Karyotyping; Lung Neoplasms; Lymphocytes