Chaetocin induced chromatin condensation: effect on DNA repair signaling and survival.

PURPOSE: The aim of the present study was to evaluate the effect of the histone lysine-methyltransferase (HKMT) inhibitor chaetocin on chromatin structure and its effect on ionizing radiation (IR) induced DNA damage response. METHODS: Concentration and time-dependent effects of chaetocin on chromatin clustering and its reversibility were analyzed by immunofluorescent assays in the non-small cell lung carcinoma (NSCLC) cell lines H460 and H1299Q4 and in human skin fibroblasts. In addition, IR induced damage response (γH2AX, 53BP1, and pATM foci formation) was studied by immunofluorescent assays. The effect on survival was determined by performing single-cell clonogenic assays. RESULTS: Chaetocin significantly increased the radiation sensitivity of H460 (F test on nonlinear regression, p < .0011) and of H1299 (p = .0201). In addition, treatment with 15 nM chaetocin also decreased the total radiation doses that control 50% of the plaque monolayers (TCD50) from 17.2 ± 0.3 Gy to 7.3 ± 0.4 Gy (p < .0001) in H1299 cells and from 11.6 ± 0.1 Gy to 6.5 ± 0.3 Gy (p < .0001). Phenotypically, chaetocin led to a time and concentration-dependent clustering of the chromatin in H1299 as well as in fibroblasts, but not in H460 cells. This phenotype of chaetocin induced chromatin clustering (CICC) was reversible and depended on the expression of the HKMTs SUV39H1 and G9a. Treatment with siRNA for SUV39h1 and G9a significantly reduced the CICC phenotype. Immunofluorescent assay results showed that the CICC phenotype was enriched for the heterochromatic marker proteins H3K9me3 and HP1α. γH2AX foci formation was not affected, neither in cells with normal nor with CICC phenotype. In contrast, repair signaling with 53BP1 and pATM foci formation was significantly reduced in the CICC phenotype. CONCLUSIONS: Treatment with chaetocin increased the radiation sensitivity of cells in vitro and DNA damage response, especially of 53BP1 and ATM-dependent repair by affecting chromatin structure. The obtained results support the potential use of natural HKMT inhibitors such as chaetocin or other bioactive compounds in improving radiosensitivity of cancer cells.

Targeting ARID1A-mutant colorectal cancer: depletion of ARID1B increases radiosensitivity and modulates DNA damage response.

The SWI/SNF chromatin remodeling complex has been found mutated in a wide range of human cancers, causing alterations in gene expression patterns, proliferation and DNA damage response that have been linked to poor clinical prognosis. Here, we investigated weather knockdown of ARID1B, one of two mutually exclusive subunits within the SWI/SNF complex, can sensitize colorectal cancer cell lines mutated in the other subunit, ARID1A, to ionizing radiation (IR). ARID1A-mutated colorectal cancer (CRC) cell lines are selectively sensitized to IR after siRNA mediated ARID1B depletion, as measured by clonogenic survival. This is characterized by a decrease in the surviving cell fraction to 87.3% ± 2.1%, 86.0% ± 1.1% and 77.2% ± 1.5% per 1 Gy compared with control siRNA exposed cells in the dose range of 0-6 Gy for the LS180, RKO and SW48 lines, respectively (p < 0.0001, F-test). The magnitude of this dose modifying effect was significantly larger in ARID1A mutated than in non-mutated cell lines (Spearman rank correlation rs = 0.88, p = 0.02). Furthermore, initial formation of RAD51 foci at 4 h after IR, as a measure for homologous recombination repair, was significantly reduced in ARID1A-mutant CRC cell lines but not in the majority of wildtype lines nor in fibroblasts. These findings open up perspectives for targeting ARID1B in combination with radiotherapy to improve outcomes of patients with ARID1A-mutant CRC.

Effect of separase depletion on ionizing radiation-induced cell cycle checkpoints and survival in human lung cancer cell lines.

OBJECTIVES: This study is to evaluate the effect of separase depletion on cell cycle progression of irradiated and non-irradiated cells through the G(2)/M phases and consecutive cell survival. MATERIALS AND METHODS: Separase was depleted with siRNA in two human non-small cell lung carcinoma (NSCLC) cell lines. Cell cycle progression, mitotic fraction, DNA repair, apoptotic and clonogenic cell death were determined. RESULTS: By depletion of endogenous separase with siRNA in NSCLCs, we showed that separase affects progression through the G(2) phase. In non-irradiated exponentially growing cells, separase depletion led to an increased G(2) accumulation from 17.2% to 29.1% in H460 and from 15.7% to 30.9% in A549 cells and a decrease in mitotic cells. Depletion of separase significantly (P < 0.01) increased the fraction of radiation-induced G(2) arrested cells 30-56 h after irradiation and led to decrease in the mitotic fraction. This was associated with increased double-strand break repair as measured by gamma-H2AX foci kinetics in H460 cells and to a lesser extent in A549 cells. In addition, a decrease in the expression of mitotic linked cell death after irradiation was found. CONCLUSIONS: These results indicate that separase has additional targets involved in regulation of G(2) to M progression after DNA damage. Prolonged G(2) phase arrest in the absence of separase has consequences on repair of damaged DNA and cell death.

[Proapoptotic antibodies as new therapeutic agents for tumor treatment]. / Proapoptotische Antikörper als neue Tumortherapeutika.

To convert the concept already successful in mice into clinical practice and commercialize it, a human anti-CD95-antibody must be produced. In a second step experiments must be performed on various normal healthy cells and tissues to determine whether these human anti-CD95-antibodies administered in very low doses have any effect on human cells (particularly hepatocytes) or at least cause only minimal side effects. If these studies yield positive results, then clinical trials can be conducted in which increasing doses are given to exclude an acute hepatotoxic effect and then the effect exerted by the antibody in combination with irradiation on tumor growth can be investigated.The advantage of this concept lies in the fact that systemic stimulus (low doses of anti-CD95-antibodies) is highly intensified by local radiotherapy and only then initiates cell death. Since the anti-CD95-antibodies trigger apoptosis primarily in tumor endothelia, this approach could be employed not only for prostate cancer and melanomas, which have already been tested, but also for many other tumors.

Targeting of Rad51-dependent homologous recombination: implications for the radiation sensitivity of human lung cancer cell lines.

The aim of the present work was to study the role of Rad51-dependent homologous recombination in the radiation response of non-small-cell lung cancer (NSCLC) cell lines. A dose- and time-dependent increase in the formation of Rad51 and gamma-H2AX foci with a maximum at about 4 and 1 h after irradiation, followed by a decrease, has been found. The relative fraction of cells with persisting Rad51 foci was 20-30% in radioresistant and 60-80% in radiosensitive cell lines. In comparison, a higher fraction of residual Dsb was evident in cell lines with nonfunctional p53. Transfection with As-Rad51 significantly downregulates radiation-induced formation of Rad51 foci and increases apoptosis, but did not influence the rejoining of DNA double-strand breaks. Interestingly, wortmannin, a well-known inhibitor of nonhomologous end-joining, also inhibits Rad51 foci formation. In general, there was no correlation between the clonogenic survival at 2 Gy and the percentage of initial Rad51 or gamma-H2AX foci after ionising radiation (IR). The most reliable predictive factor for radiosensitivity of NSCLC cell lines was the relative fraction of Rad51 foci remaining at 24 h after IR. Although most of the Rad51 foci are co-localised with gamma-H2AX foci, no correlation of the relative fraction of persisting gamma-H2AX foci and SF2 is evident.