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.

Targeted next-generation sequencing of locally advanced squamous cell carcinomas of the head and neck reveals druggable targets for improving adjuvant chemoradiation.

BACKGROUND: Despite clear differences in clinical presentation and outcome, squamous cell carcinomas of the head and neck (SCCHN) arising from human papilloma virus (HPV) infection or heavy tobacco/alcohol consumption are treated equally. Next-generation sequencing is expected to reveal novel targets for more individualised treatment. PATIENTS AND METHODS: Tumour specimens from 208 patients with locally advanced squamous cell carcinoma of the hypopharynx, oropharynx or oral cavity, all uniformly treated with adjuvant cisplatin-based chemoradiation, were included. A customised panel covering 211 exons from 45 genes frequently altered in SCCHN was used for detection of non-synonymous point and frameshift mutations. Mutations were correlated with HPV status and treatment outcome. RESULTS: Mutational profiles and HPV status were successfully established for 179 cases. HPV- tumours showed an increased frequency of alterations in tumour suppressor genes compared to HPV+ cases (TP53 67% versus 4%, CDKN2A 18% versus 0%). Conversely, HPV+ carcinomas were enriched for activating mutations in driver genes compared to HPV- cases (PIK3CA 30% versus 12%, KRAS 6% versus 1%, and NRAS 4% versus 0%). Hotspot TP53 missense mutations in HPV- carcinomas correlated with an increased risk of locoregional recurrence (hazard ratio [HR] 4.3, 95% confidence interval [CI] 1.5-12.1, P=0.006) and death (HR 2.2, 95% CI 1.1-4.4, P=0.021). In HPV+ SCCHN, driver gene mutations were associated per trend with a higher risk of death (HR 3.9, 95% CI 0.7-21.1, P=0.11). CONCLUSIONS: Distinct mutation profiles in HPV- and HPV+ SCCHN identify subgroups with poor outcome after adjuvant chemoradiation. Mutant p53 and the phosphoinositide 3-kinase pathway were identified as potential druggable targets for subgroup-specific treatment optimisation.

Recombinant activation domains of virion protein 16 and human estrogen receptor generate transcriptional interference in vitro by distinct mechanisms.

Overexpression of transcriptional activators in transfection assays may inhibit their own activity or interfere with trans-activation by different sequence-specific transcription factors. In this study we show that this phenomenon of transcriptional interference (squelching) can be mimicked in vitro by adding recombinant activation domains to nuclear extracts. We demonstrate that the acidic activation domain of virion protein 16 interferes both with basal transcription from TATA-box promoters and promoters activated by various trans-activators in two different mammalian cell-free transcription systems. This suggests that virion protein 16 interacts with and thereby sequesters a basal transcription factor. In contrast the recombinant activation function 2 (AF-2) of human estrogen receptor does not affect basal promoter activity but inhibits TATA promoters activated by human progesterone receptor (hPR) or Sp 1 as well as the beta-globin and adenovirus major late promoter. By analyzing the effects of AF-2 on DNA binding of hPR and Sp1 we found that AF-2 inhibits the DNA binding activity of hPR, but not Sp1. Our data suggest that the recombinant AF-2 squelches Sp1 trans-activation by sequestering a common coactivator(s), whereas hPR function might be inhibited due to competition for a common cofactor stabilizing hPR dimers or through the formation of inactive heterodimers between AF-2 and hPR.

Endothelial Caveolin-1 regulates the radiation response of epithelial prostate tumors.

The membrane protein caveolin-1 (Cav1) recently emerged as a novel oncogene involved in prostate cancer progression with opposed regulation in epithelial tumor cells and the tumor stroma. Here we examined the role of stromal Cav1 for growth and radiation response of MPR31-4 prostate cancer xenograft tumors using Cav1-deficient C57Bl/6 mice. Syngeneic MPR31-4 tumors grew faster when implanted into Cav1-deficient mice. Increased tumor growth on Cav1-deficient mice was linked to decreased integration of smooth muscle cells into the wall of newly formed blood vessels and thus with a less stabilized vessel phenotype compared with tumors from Cav1 wild-type animals. However, tumor growth delay of MPR31-4 tumors grown on Cav1 knockout mice to a single high-dose irradiation with 20 Gray was more pronounced compared with tumors grown on wild-type mice. Increased radiation-induced tumor growth delay in Cav1-deficient mice was associated with an increased endothelial cell apoptosis. In vitro studies using cultured endothelial cells (ECs) confirmed that the loss of Cav1 expression increases sensitivity of ECs to radiation-induced apoptosis and reduces their clonogenic survival after irradiation. Immunohistochemical analysis of human tissue specimen further revealed that although Cav1 expression is mostly reduced in the tumor stroma of advanced and metastatic prostate cancer, the vascular compartment still expresses high levels of Cav1. In conclusion, the radiation response of MPR31-4 prostate tumors is critically regulated by Cav1 expression in the tumor vasculature. Thus, Cav1 might be a promising therapeutic target for combinatorial therapies to counteract radiation resistance of prostate cancer at the level of the tumor vasculature.

Repair of ionizing radiation induced DNA double-strand breaks (dsb) at the c-myc locus in comparison to the overall genome.

PURPOSE: The aim of this study was to determine the repair of radiation-induced DNA double-strand breaks (dsb) in actively transcribed regions and in the overall genome. MATERIALS AND METHODS: Pulsed-field gel electrophoresis was performed on Sfi I restriction enzyme digested DNA, labelled with a c-myc probe and on non-specifically 14C-labelled DNA of the adenocarcinoma cell line COLO320HSR after 400 Gy irradiation with 7 MeV electrons and repair incubation. RESULTS: At the 130 kbp c-myc locus 68 +/- 5% of all dsb induced at a dose of 400 Gy were repaired by a fast mechanism with a repair half time of 9.4 +/- 3.2 min in comparison to the overall genome where all dsb induced at 400 Gy were repaired with a half time of 86 +/- 23 min. The fraction of residual dsb was about 30% higher in the c-myc locus than in the overall genome. CONCLUSIONS: This study demonstrates intragenomic heterogeneity in half times of dsb repair with faster repair at the c-myc locus. In addition, differences in the residual dsb were found to represent region specific heterogeneity in residual damage or to possibly be attributed to the different assays used. The approach with gene probing can distinguish between correct and incorrect rejoining of dsb within the resolution of the experiments (< or = 20 kbp), in contrast to the assay at the overall genome.

Induction of DNA double-strand breaks by ionizing radiation at the c-myc locus compared with the whole genome: a study using pulsed-field gel electrophoresis and gene probing.

Ionizing radiation-induced double-strand breaks (dsb) in a human colon carcinoma-derived cell line COLO320HSR were determined from the fragment size distribution of non-specifically labelled DNA and Sfi I restriction enzyme-digested DNA uniformly labelled with a c-myc probe. The dose-effect relation for the induction of DNA dsb was linear with no significant difference between slopes for the curves in the whole genome (7.2 +/- 0.3 x 10(-9) dsb/bp/Gy) and in the 130 kbp restriction fragments containing c-myc (6.5 +/- 0.5 x 10(-9) dsb/bp/Gy). The size distribution of the c-myc fragments showed deviations from the random-breakage model, indicating heterogeneity of dsb induction at this locus.

Protection of DNA from radiation-induced double-strand breaks: influence of replication and nuclear proteins.

PURPOSE: To study the protective effect of histone and non-histone proteins on double-strand break (dsb) induction in replicating S-phase DNA as well as bulk DNA of plateau phase human tumour cells. MATERIALS AND METHODS: Induction of dsb was studied in two human adenocarcinoma cell lines: Colo320HSR and MCF-7. To assess the influence of chromatin structure on radiation-induced DNA dsb, different nuclear preparations of cells, either continuously labelled with 14C or pulse labelled with 3H, were assessed by pulsed-field gel electrophoresis (PFGE). RESULTS AND CONCLUSIONS: Stepwise removal of DNA-bound proteins from the chromatin increased the amount of radiation-induced dsb in both cell lines. However, the protective effect of DNA-associated proteins on dsb induction was significantly reduced in DNA of replicating S-phase cells compared with bulk DNA of plateau phase cells. These data show that proteins associated with the DNA have a different protective effect on radiation-induced dsb, rendering replicating DNA with open chromatin structure more sensitive to dsb induction by ionizing radiation.

Radiation-induced apoptosis in human non-small-cell lung cancer cell lines is secondary to cell-cycle progression beyond the G2-phase checkpoint.

PURPOSE: To characterize the relationship between cell-cycle progression and radiation-induced apoptosis in NSCLC cell lines with different p53 status. MATERIALS AND METHODS: Cell lines with functional (H460, A549) and non-functional p53 (H661 and H520) were irradiated with 20 Gy. Multiparameter flow-cytometry was used to follow the progression of synchronized cells through the cell cycle after irradiation. RESULTS: Delayed apoptosis was observed after cell-cycle progression beyond the G2 block, either in the late G2/M-phase of the same cell cycle being irradiated (H661, H520) or in the G1-phase of the subsequent cell cycle (H460, A549). The apoptotic fraction in H661 and H520 was 60-80% at 144h after irradiation, higher than in A549 and H460 (5 and 35%, respectively). As an alternative to apoptosis in cells cycling beyond the G2 restriction point, hyperploid cells were generated by all cell lines. Inhibition of cell-cycle progression through the G2/M-phase efficiently reduced the induction of late apoptosis. After irradiation in S-phase, 50-60% of cells with functional p53 remained arrested at the G2 restriction point until 144 h post-irradiation, while only 20% of the H661 or H520 did so. CONCLUSIONS: These data characterize radiation-induced apoptosis in NSCLC cell lines as a removal pathway of clonogenically inactivated cells secondary to cell-cycle progression beyond G2/M, and is unlikely to be a critical factor for cellular radiation sensitivity.

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.

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.

Radiation-induced apoptosis in human sarcoma and glioma cell lines.

Six human soft-tissue sarcoma and 14 glioma cell lines, exhibiting considerable differences in radioresponsiveness and histological grade of differentiation of the parental tumour, were examined with respect to apoptosis development after irradiation with 60Co gamma-rays. After test doses of 6 and 25 Gy, significant changes characteristic of apoptosis occurring within 6 to 30 hr were exhibited by only 2 differentiated sarcoma cell lines, EL7 and ESS2. The characteristic internucleosomal fragmentation of DNA was detected as early as 6 hr after exposure of subconfluent monolayer cultures to 6 Gy. It was limited to cells that had detached from the culture plate, whereas adherent cells showed random degradation of DNA, namely after higher doses (25Gy) or longer incubation times (30 hr). As assessed by fluorescence microscopy of unfixed cultures stained with Hoechst 33342 and propidium iodide, the proportion of cells showing apoptotic bodies in non-irradiated controls was < 0.1% and 0.3% for EL7 and ESS2, respectively. The dose-response relationship for apoptosis was determined at 9 hr post-irradiation. After 2 Gy, the percentage of apoptotic cells was elevated to 3.4% in EL7 and 4.5% in ESS2 cultures. Saturation was obtained above 6 Gy, with 8.4% apoptosis in EL7 and 15% in ESS2 after 25 Gy. Taken together, rapid ionizing-radiation-induced apoptosis seems to be limited to a subgroup of sarcomas and is unlikely to occur in gliomas.

[Mechanisms of post-isometric relaxation of skeletal muscles as a method of rehabilitation]. / O mekhanizmakh postizometricheskoi relaksatsii skeletnykh myshts kak metode reabilitatsii.

The authors demonstrate that a merited place among the methods of manual therapy belongs to PIR--postisometric relaxation of the muscles. It is shown that the relaxation of the muscle converted to isometric regime of work is achieved on the basis of the anatomic and biomechanical principles of its action. The use of PIR is advisable, which is evident in the light of the theory of portal pain control.

[Prerequisites for the manual therapy of diseases of the cervical spine]. / K preposylkam manual'noi terapii pri zabolevaniiakh sheinogo otdela pozvonochnika.

The authors examine the anatomical and biomechanical peculiarities of the upper and lower cervical vertebromotor segments. A differential administration of the manual therapy methods to influence various levels of the cervical spine is substantiated. It has been demonstrated that the mobilization of the atlanto-occipital joints should be carried out by means of flexion and extension movements and lateral inclinations. It is supposed that the atlanto-occipital complex of joints may be affected by means of combination of axial traction and rotation of the cervical spine. The manipulations on the lower cervical vertebromotor segments require a combined application of lateral inclinations and rotation.