aphidicolin and Colorectal-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

Chromosomal fragile sites are distributed all over the human genome. Aphidicolin mediated expression frequency of common fragile sites and other chromosomal changes were evaluated in prometaphase/metaphase chromosomes obtained from peripheral blood lymphocytes of colorectal cancer patients. The present study reveals first time high incidence i.e. 6 % of aphidicolin induced chromosome breaks / gaps designated as "common fragile sites" in cell population of clinically diagnosed patients of colorectal cancer patients in Nepalese population. These chromosomal changes including structural and numerical were compare to clinically healthy normal individual of same sex / age groups, act as controls for statistical analysis. The frequency of chromosomal aberration in cancer patients were significantly higher (p<0.001) when compare to normal individuals. The increased genetics instability probably either due to nutritional factor i.e. lack of folic acid component in diet--an essential component required for DNA synthesis or unknown environmental factor for such genetic disorder. The present study indicates aphidicolin high frequency of induced chromosome aberrations and "common fragile sites" because of late replication of DNA in mitosis in colorectal cancer patients suggesting these sites could be used as suitable marker for determining genetic predisposition in cancer patients.

Topics: Aphidicolin; Chromosome Aberrations; Chromosome Fragile Sites; Colorectal Neoplasms; Humans; Lymphocytes; Middle Aged

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

Topoisomerase I inhibitors have been shown to have clinical activity against human colorectal cancer. Previous studies showed that the cytotoxicity of camptothecin, a topoisomerase I inhibitor, occurs mainly in the S -phase of the cell cycle and is protectable by aphidicolin, an inhibitor of replicative DNA polymerase in some camptothecin-sensitive colorectal cells. Transcription factor E2F-1 regulates the G1/S transition, and recent studies have shown that E2F-1 potentiated the cytotoxicity of some cell-cycle-related drugs. Therefore, the present study was designed to investigate the effect of adenovirus-mediated E2F-1 gene transfer on chemosensitivity of colorectal cancer to camptothecin, in vitro and in vivo. Two human colorectal cancer cells, SW620 (mutant p53) and RKO (wild-type p53), were treated with camptothecin, alone or in combination with adenoviral vectors expressing beta-galactosidase (Ad-LacZ), or E2F-1 (Ad-E2F-1). E2F-1 overexpression was confirmed by Western blot analysis. Ad-E2F-1 gene transfer at low doses (less than the LD(20) dose) markedly increased the sensitivity of human colorectal cancer cells to camptothecin in vitro, which is because of induction of apoptosis. Aphidicolin did not have any protective effect on the Ad-E2F-1/camptothecin-mediated cytotoxicity. The level of topoisomerase I expression was not affected by combination treatment as well, suggesting that DNA replication and topoisomerase I activity may not account for the molecular mechanism of cell killing in response to Ad-E2F-1/camptothecin treatment. Fas and Fas ligand expression were not altered by treatment with camptothecin and/or Ad-E2F-1. Moreover, combination of camptothecin and Ad-E2F-1 has an additive antitumor effect in an in vivo nude mouse xenograft model. When combined with camptothecin, E2F-1 adenovirus therapy resulted in a 95.7% decrease in tumor size compared to control groups (P<.05). These results suggest a chemosensitization strategy that may have clinical utility in human colorectal cancer.

Topics: Adenoviridae; Animals; Antineoplastic Agents, Phytogenic; Aphidicolin; Apoptosis; Camptothecin; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Line, Tumor; Colorectal Neoplasms; Combined Modality Therapy; DNA Topoisomerases, Type I; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Genes, p53; Genetic Therapy; Genetic Vectors; Humans; Male; Mice; Mice, Inbred BALB C; Topoisomerase I Inhibitors; Transcription Factors

The frequency and distribution of aphidicolin induced common fragile sites was evaluated on chromosomes of peripheral blood lymphocytes in 10 breast and 10 colorectal cancer patients, and 10 healthy controls to determine correlation between specific fragile sites and cancer breakpoints. Fifty complete metaphases were screened from each culture and the results were evaluated by Student's t-test. The total number of fragile sites was found as 933 in breast cancer patients, 950 in colorectal cancer patients and 501 in control group. Both the number of aberrations per cell and number of aberrations per damaged cell were significantly higher in the patient groups. These findings indicate that genetic instability in the breast and colorectal cancer patients increased and fragile sites may play a critical role in the pathogenesis of breast and colorectal cancer.

Topics: Adenocarcinoma; Adult; Aged; Aphidicolin; Breast Neoplasms; Chromosome Aberrations; Chromosome Fragile Sites; Chromosome Fragility; Chromosomes, Human; Colorectal Neoplasms; Female; Humans; Lymphocytes; Male; Middle Aged

Mimosine (MIM) and aphidicolin (APH) are two agents frequently used in tissue culture-based experiments to achieve cell synchronization at late G1 and S phases. Following MIM or APH treatment of human cancer cell lines, a reversible growth arrest in late G1 and S phases of the cell cycle was correlated with moderate increases in p53 and p21 protein levels. Both p53-dependent and -independent increases in p21 were observed following treatment with either agent. However, a striking increase in p21 protein levels and a continuous elevation in both p53 and p21 protein levels were observed over 48 h after cells re-entered the cell cycle following the chemically-induced synchronization. In addition, the increase in p21 protein levels typically seen following treatment of cells with DNA damaging agents, was enhanced when cells were treated with genotoxic agents following MIM or APH synchronization. These findings suggest that caution should be exercised when interpreting results from experiments using cell synchronization agents, in particular, studies designed to investigate p53- and p21-regulatory pathways.

Topics: Aphidicolin; Carcinoma, Large Cell; Cell Cycle; Colorectal Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Doxorubicin; Enzyme Inhibitors; Flow Cytometry; Humans; Malondialdehyde; Mimosine; Tumor Cells, Cultured; Tumor Suppressor Protein p53