Abasic sites (apurinic/apyrimidinic, AP sites) are the most common DNA lesions generated by both spontaneous and induced base loss. In a previous study we have shown that circular plasmid molecules containing multiple AP sites are efficiently repaired by Chinese hamster extracts in an in vitro repair assay. An average patch size of 6.6 nucleotides for a single AP site was calculated. To define the exact repair patch, a circular DNA duplex with a single AP site was constructed. The repair synthesis carried out by hamster and human cell extracts was characterized by restriction endonuclease analysis of the area containing the lesion. The results indicate that, besides the repair events involving the incorporation of a single nucleotide at the lesion site, repair synthesis occurred also 3' to the AP site and involved a repair patch of approximately 7 nucleotides. This alternative repair pathway was completely inhibited by the presence in the repair reaction of a polyclonal antibody raised against human proliferating cell nuclear antigen. These data give the first evidence that mammalian cell extracts repair natural AP sites by two distinct pathways: a single nucleotide gap filling reaction targeted at the AP site and a proliferating cell nuclear antigen-dependent pathway that removes a short oligonucleotide containing the abasic site and 3'-flanking nucleotides.

We reported previously that Bcl-2 is paradoxically downregulated in paclitaxel-resistant cancer cells. We reveal here that paclitaxel directly targets Bcl-2 in the loop domain, thereby facilitating the initiation of apoptosis. Molecular modeling revealed an extraordinary similarity between the paclitaxel binding sites in Bcl-2 and B-tubulin, leading us to speculate that paclitaxel could be mimetic of an endogenous peptide ligand, which binds both proteins. We tested the hypothesis that paclitaxel mimics Nur77, which, like paclitaxel, changes the function of Bcl-2. This premise was confirmed by Nur77 interacting with both paclitaxel targets (Bcl-2 and B-tubulin) and a peptide sequence mimicking the Nur77 structural region, thus reproducing the paclitaxel-like effects of tubulin polymerization and opening the permeability transition pore channel in mitochondria. This discovery could help in the development of novel anticancer agents with nontaxane skeleton as well as in identifying the clinical subsets responsive to paclitaxel-based therapy.

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Taxanes act by inhibiting microtubule dynamics; in this study, we have investigated mitochondria as an additional target of taxanes. We incubated isolated mitochondria in the presence of taxanes with or without stimulation of the mitochondrial respiratory state. Results showed that they rapidly induced the loss of ⌬ m after stimulation of the respiratory state. CPM values after Bcl-2 immunoprecipitation was 62.8-fold higher than those of the control antibody, thereby indicating the involvement of Bcl-2 in paclitaxel binding. Then, we established a panel of A2780 cell lines resistant to increasing doses of paclitaxel alone or to high doses of paclitaxel/cyclosporin A (A2780 TC cells). In both cases, Bcl-2 expression was consistently down-regulated, whereas levels of other members of the Bcl-2 family, such as Bax and Bcl-x, did not change in paclitaxel-resistant cell lines. When A2780TC cells were stably transfected with a Bcl-2 construct, paclitaxel sensitivity was partially restored, thereby supporting a direct role of Bcl-2 down-regulation in the maintenance of drug-resistance. Finally, we examined Bcl-2 by immunohistochemistry in a small subset of ovarian cancer paclitaxel-resistant patients and we noticed that the protein is down-regulated in this clinical setting with respect to the expression levels found in drug-sensitive tumors. These findings demonstrate that Bcl-2 is an additional intracellular target of taxanes and that its down-regulation is involved in taxane resistance.Taxanes are natural products derived from trees of the genus Taxoidaceae. The first taxane introduced in cancer therapy was paclitaxel, firstly isolated from Taxus brevifolia (Schiff et al., 1979). The clinical success of taxanes is dependent on the excellent response rate in second-line treatment of relapsing/resistant cancers and on the efficacy of taxanes in the multichemotherapeutic approach of ovarian and breast cancer (Verweij et al., 1994).In earlier studies, the microtubule network appeared as the main target of paclitaxel (Schiff et al., 1979;Manfredi et al., 1982). In fact, taxanes bind to ␤-tubulin subunits, thereby disrupting normal turnover of the microtubules. The final consequence is the arrest of the cell cycle in M phase with formation of aberrant mitosis and the activation of cell death pathways (Jordan et al., 1993). Along with arrest in M phase of the cell cycle, taxanes have also been reported to induce post-translational serine phosphorylation of the Bcl-2 protein (Haldar et al., 1995). The BCL2 gene is the homologous of the nematode CED-9 gene product (Hengartner and Horvitz, 1994) and is capable of prolonging cell survival by inhibiting apoptotic cell death. Overexpression of Bcl-2 has been observed in follicular lymphoma, where this protein is deregulated by chromosomal translocation, and in a large number of human tumors, including breast, lung, and prostate cancer.Disagreement exists on the levels of Bcl-2 and resistance to taxanes. A strong suggestion for a direct role of Bcl-2 in mediating paclitax...

BackgroundMicroRNAs in solid malignancies can behave as predictors of either good or poor outcome. This is the case with members of the miR-200 family, which are the primary regulators of the epithelial to mesenchymal transition and have been reported to act as both oncogenes and tumor suppressors. This study assessed the role of miR-200c as regulator of class III β-tubulin (TUBB3), a factor associated with drug-resistance and poor prognosis in ovarian cancer.MethodsExpression of miR-200c was assessed in a panel of ovarian cancer cell lines with inherent or acquired drug-resistance. Stable overexpression of miR-200c was obtained in A2780 and Hey cell lines. Crosslinking-coupled affinity purification method and ribonucleic-immunoprecipitation assay were used to characterise the complexes between miR-200c, HuR and 3′UTR region of TUBB3 mRNA. Nanofluidic technology and immunohistochemistry were used to analyze the expression of HuR, TUBB3 and miR-200c in 220 ovarian cancer patients.ResultsIn a panel of ovarian adenocarcinoma cell lines, we observed a direct correlation between miR-200c expression and chemoresistance. In A2780 cells miR-200c targeted TUBB3 3′UTR, while a positive correlation was observed between miR-200c and TUBB3 expression in most of the other cell lines. Through the analysis of 3′UTR-associated complexes, we found that the miR-200c can increase the association of the RNA binding protein HuR with TUBB3 mRNA, whereas HuR binding enhanced TUBB3 mRNA translation. Most importantly, in our analysis on 220 ovarian cancer patients we observed that overexpression of miR-200c correlated with poor or good outcome depending on the cellular localization of HuR.ConclusionThis study suggests a model for the combined regulatory activity of miR-200c and HuR on TUBB3 expression in ovarian cancer. When HuR is nuclear, high expression of miR-200c inhibits TUBB3 expression and results in a good prognosis, whereas when HuR occurs in cytoplasm, the same miRNA enhances TUBB3 expression and produces a poor outcome. These findings reveal the usefulness of multidimensional analysis in the investigation of the prognostic role of miRNA expression.