Involvement of ATM in homologous recombination after end resection and RAD51 nucleofilament formation.

Ataxia-telangiectasia mutated (ATM) is needed for the initiation of the double-strand break (DSB) repair by homologous recombination (HR). ATM triggers DSB end resection by stimulating the nucleolytic activity of CtIP and MRE11 to generate 3'-ssDNA overhangs, followed by RPA loading and RAD51 nucleofilament formation. Here we show for the first time that ATM is also needed for later steps in HR after RAD51 nucleofilament formation. Inhibition of ATM after completion of end resection did not affect RAD51 nucleofilament formation, but resulted in HR deficiency as evidenced by (i) an increase in the number of residual RAD51/γH2AX foci in both S and G2 cells, (ii) the decrease in HR efficiency as detected by HR repair substrate (pGC), (iii) a reduced SCE rate and (iv) the radiosensitization of cells by PARP inhibition. This newly described role for ATM was found to be dispensable in heterochromatin-associated DSB repair, as KAP1-depletion did not alleviate the HR-deficiency when ATM was inhibited after end resection. Moreover, we demonstrated that ATR can partly compensate for the deficiency in early, but not in later, steps of HR upon ATM inhibition. Taken together, we describe here for the first time that ATM is needed not only for the initiation but also for the completion of HR.

Expression of insulin-like growth factor II mRNA-binding protein 3 in squamous cell carcinomas of the head and neck.

BACKGROUND: Insulin-like growth factor II mRNA-binding protein 3 (IMP3) was found overexpressed in various cancer types suggesting its possible role in carcinogenesis. Analysis of IMP3 expression in head and neck squamous cell carcinomas (HNSCC) is rare so that we evaluated it using tissue microarray method. METHOD: Immunohistochemical analysis of IMP3 was performed on samples from over 400 patients. The expression was measured semiquantitative, subsequently divided into four categories (negative, weak, medium, or strong) and correlated with several available clinicopathologic parameters. RESULTS: For HNSCC, positive IMP3 expression was observed in patients with all tumor stages (pT1-4) and nodal stages (pN0-3), showing also significant statistical correlation (P=0.023 and P=0.0013, respectively). No further correlations were found. Separate analysis according to tumor localization (oral cavity, oropharyngeal, and laryngeal) showed a significant correlation of positive IMP3 expression and overall survival (P=0.038) only in patients with tumors of the oral cavity. Multivariate analysis showed IMP3 as an independent predictive marker for oral squamous cell carcinomas (OSCC). CONCLUSION: Insulin-like growth factor II mRNA-binding protein 3 (IMP3) expression might be used as an independent prognostic factor in the subgroup of OSCC.

Loss of PTEN-assisted G2/M checkpoint impedes homologous recombination repair and enhances radio-curability and PARP inhibitor treatment response in prostate cancer.

Here we report that PTEN contributes to DNA double-strand break (DSB) repair via homologous recombination (HR), as evidenced by (i) inhibition of HR in a reporter plasmid assay, (ii) enhanced sensitivity to mitomycin-C or olaparib and (iii) reduced RAD51 loading at IR-induced DSBs upon PTEN knockdown. No association was observed between PTEN-status and RAD51 expression either in-vitro or in-vivo in a tissue microarray of 1500 PTEN-deficient prostate cancer (PC) samples. PTEN depletion and sustained activation of AKT sequestered CHK1 in the cytoplasm, thus impairing the G2/M-checkpoint after irradiation. Consistently, AKT inhibition recovered the G2/M-checkpoint and restored HR efficiency in PTEN-depleted cells. We show that, although PTEN loss correlates with a worse prognosis, it may predict for improved response of PC patients to radiotherapy. Further, we provide evidence for the use of PTEN as a biomarker for predicting the response to PARP inhibitors as radiosensitizing agents in prostate cancer. Collectively, these data implicate PTEN in maintaining genomic stability by delaying G2/M-phase progression of damaged cells, thus allowing time for DSB repair by HR. Furthermore, we identify PTEN-status in PC as a putative predictor of (i) radiotherapy response and (ii) response to treatment with PARP inhibitor alone or combined with radiotherapy.

Targeting of ß1 integrins impairs DNA repair for radiosensitization of head and neck cancer cells.

ß1 Integrin-mediated cell-extracellular matrix interactions allow cancer cell survival and confer therapy resistance. It was shown that inhibition of ß1 integrins sensitizes cells to radiotherapy. Here, we examined the impact of ß1 integrin targeting on the repair of radiation-induced DNA double-strand breaks (DSBs). ß1 Integrin inhibition was accomplished using the monoclonal antibody AIIB2 and experiments were performed in three-dimensional cell cultures and tumor xenografts of human head and neck squamous cell carcinoma (HNSCC) cell lines. AIIB2, X-ray irradiation, small interfering RNA-mediated knockdown and Olaparib treatment were performed and residual DSB number, protein and gene expression, non-homologous end joining (NHEJ) activity as well as clonogenic survival were determined. ß1 Integrin targeting impaired repair of radiogenic DSB (γH2AX/53BP1, pDNA-PKcs T2609 foci) in vitro and in vivo and reduced the protein expression of Ku70, Rad50 and Nbs1. Further, we identified Ku70, Ku80 and DNA-PKcs but not poly(ADP-ribose) polymerase (PARP)-1 to reside in the ß1 integrin pathway. Intriguingly, combined inhibition of ß1 integrin and PARP using Olaparib was significantly more effective than either treatment alone in non-irradiated and irradiated HNSCC cells. Here, we support ß1 integrins as potential cancer targets and highlight a regulatory role for ß1 integrins in the repair of radiogenic DNA damage via classical NHEJ. Further, the data suggest combined targeting of ß1 integrin and PARP as promising approach for radiosensitization of HNSCC.

Role of activated astrocytes in neuronal damage: potential links to HIV-1-associated dementia.

HIV-1-associated dementia (HAD) is an important complication of HIV-1 infection. Reactive astrogliosis is a key pathological feature in HAD brains and in other central nervous system (CNS) diseases. Activated astroglia may play a critical role in CNS inflammatory diseases such as HAD. In order to test the hypothesis that activated astrocytes cause neuronal injury, we stimulated primary human fetal astrocytes with HAD-relevant pro-inflammatory cytokine IL-1beta. IL-1beta-activated astrocytes induced apoptosis and significant changes in metabolic activity in primary human neurons. An FITC-conjugated pan-caspase inhibitor peptide FITC-VAD-FMK was used for confirming caspase activation in neurons. IL-1beta activation enhanced the expression of death protein FasL in astrocytes, suggesting that FasL is one of the potential factors responsible for neurotoxicity observed in HAD and other CNS diseases involving glial inflammation. Our data presented here add to the developing picture of role of activated glia in HAD pathogenesis.

HIV-1 and IL-1 beta regulate Fas ligand expression in human astrocytes through the NF-kappa B pathway.

Reactive astrogliosis is a prominent pathological feature of HIV-1-associated dementia (HAD). We hypothesized that in HAD, astrocytes activated with proinflammatory stimuli such as IL-1beta express Fas ligand (FasL), a death protein. IL-1beta and HIV-1-activated astrocytes expressed FasL mRNA and protein. Luciferase reporter constructs showed that IL-1beta and HIV-1 upregulated FasL promoter activity (p<0.001). The NF-kappaB pathway was involved as shown by inhibition with SN50 and dominant negative IkappaBalpha mutants. Brain extracts from HAD patients had significantly elevated FasL levels compared to HIV-seropositive (p<0.001) and seronegative individuals (p<0.01). We propose that astrocyte expression of FasL may participate in neuronal injury in HAD.

Overexpression of human Ku70/Ku80 in rat cells resulting in reduced DSB repair capacity with appropriate increase in cell radiosensitivity but with no effect on cell recovery.

The effect of an overexpression of human Ku70/80 was studied using cells of the rat cell lines Rat-1 and R7080, the latter being transfected with the human cDNAs for Ku70 and Ku80. The overexpression was found to result in a 20% reduction of the DNA-PK activity. The kinetics of DSB repair, which was studied after exposure of the cells to 30 Gy of X rays, was biphasic and had identical half-times for Rat-1 and R7080 cells (tfast = 7 min and tslow = 135 min). However, there was a significant difference between the cell lines in the fractions of DSBs repaired with slow and fast kinetics. In R7080 cells, about twice as many DSBs were repaired with slow kinetics compared to Rat-1 cells (34% compared to 16%). A similar difference was found in the number of residual DSBs (3.6% compared to 2.0%). R7080 cells also showed a reduced capacity to repair chromosome damage as detected by the PCC technique. Concerning cell killing, R7080 cells were clearly more radiosensitive than Rat-1 cells (D0.1 = 6.4 compared to 10.5 Gy), and this increase in sensitivity correlated well with the increase in residual DSBs. The two cell lines, however, did not vary in cell recovery. For sublethal as well as potentially lethal damage, Rat-1 and R7080 cells showed identical recovery ratios. These data demonstrate that the overexpression of human Ku70/Ku80 led to a reduced capacity for DSB repair with an associated increase in cell sensitivity but with no effect on cell recovery.

Relationship between PCC fragments and cell killing studied in X-irradiated CHO, CHO-K1 cells and two radiosensitive mutants xrs1 and xrs5.

PURPOSE: To investigate the correlation between PCC fragments and cell killing. MATERIALS AND METHODS: Induction and repair of DNA fragments were measured in CHO, CHO-K1, xrs1 and xrs5 cells using the premature chromosome condensation (PCC) technique and cell survival was determined by a colony assay. RESULTS: The number of PCC fragments measured in cells immediately fused after X-irradiation was the same for CHO (3.4 +/- 0.16/cell/Gy) and CHO-K1 (3.6 +/- 0.12/cell/Gy) cells but significantly higher for xrs1 (4.9 +/- 0.07/cell/Gy) and xrs5 cells (7.0 +/- 0.4/cell/Gy). The repair curve of PCC fragments studied for CHO, CHO-K1 and xrs5 cells was best described by a monophasic exponential decline with a final plateau; the half-time of this decline was always about 30 min. The number of unrejoined PCC fragments, which was measured 14h after irradiation, increased linearly with dose. The steepest increase was found for xrs5 cells (5.5 +/- 0.3 fragments per cell and per Gy), the lowest for CHO/CHO-K1 (0.9 +/- 0.1; 1.0 +/- 0.1) and for xrs1 in between (3.3 +/- 0.1). For all four cell lines the relationship between cell killing and unrejoined fragments could be described by a single curve with a D0 of 2.5 +/- 0.4 unrejoined PCC fragments per lethal event. CONCLUSIONS: The data showed that the number of unrejoined PCC fragments can be used as an indicator of cellular radiosensitivity.

The absence of Ku but not defects in classical non-homologous end-joining is required to trigger PARP1-dependent end-joining.

Classical-non-homologous end-joining (C-NHEJ) is considered the main pathway for repairing DNA double strand breaks (DSB) in mammalian cells. When C-NHEJ is defective, cells may switch DSB repair to an alternative-end-joining, which depends on PARP1 and is more erroneous. This PARP1-EJ is suggested to be active especially in tumor cells contributing to their genomic instability. Here, we define conditions under which cells would switch the repair to PARP1-EJ. Using the end jining repair substrate pEJ, we revealed that PARP1-EJ is solely used when Ku is deficient but not when either DNA-PKcs or Xrcc4 is lacking. In the latter case, DSB repair, however, could be shuttled to PARP1-EJ after additional Ku80 down-regulation, which partly rescued the DSB repair in these mutants. We demonstrate here that PARP-EJ may work on DSB ends at high fidelity manner, as evident from the unchanged efficiency upon blocking end resection by either roscovitin or mirin. Furthermore, we demonstrate for that PARP-EJ is likewise involved in the repair of multiple DSBs (I-PpoI- and IR-induced). Importantly, we identified a chromatin signature associated with the switch to PARP1-EJ which is characterized by a strong enrichment of both PARP1 and LigIII at damaged chromatin. Together, these data indicate that Ku is the main regulator for the hierarchal organization between C-NHEJ and PARP1-EJ.

Tissue inhibitor of metalloproteinases-1 protects human neurons from staurosporine and HIV-1-induced apoptosis: mechanisms and relevance to HIV-1-associated dementia.

HIV-1-associated dementia (HAD)-relevant proinflammatory cytokines robustly induce astrocyte tissue inhibitor of metalloproteinases-1 (TIMP-1). As TIMP-1 displays pleotropic functions, we hypothesized that TIMP-1 expression may serve as a neuroprotective response of astrocytes. Previously, we reported that chronically activated astrocytes fail to maintain elevated TIMP-1 expression, and TIMP-1 levels are lower in the brain of HAD patients; a phenomenon that may contribute to central nervous system pathogenesis. Further, the role of TIMP-1 as a neurotrophic factor is incompletely understood. In this study, we report that staurosporine (STS) and HIV-1(ADA) virus, both led to induction of apoptosis in cultured primary human neurons. Interestingly, cotreatment with TIMP-1 protects neurons from apoptosis and reverses neuronal morphological changes induced by these toxins. Further, the anti-apoptotic effect was not observed with TIMP-2 or -3, but was retained in a mutant of the N-terminal TIMP-1 protein with threonine-2 mutated to glycine (T2G) that is deficient in matrix metalloproteinase (MMP)-1, -2 and -3 inhibitory activity. Therefore, the mechanism is specific to TIMP-1 and partially independent of MMP-inhibition. Additionally, TIMP-1 modulates the Bcl-2 family of proteins and inhibits opening of mitochondrial permeability transition pores induced by HIV-1 or STS. Together, these findings describe a novel function, mechanism and direct role of TIMP-1 in neuroprotection, suggesting its therapeutic potential in HAD and possibly in other neurodegenerative diseases.

Influence of age and sex in exhaled breath samples investigated by means of infrared laser absorption spectroscopy.

Breath gas analysis provides insight into human metabolism of healthy and ill individuals. As an innovative and non-invasive method, it opens up options to improve diagnostics, monitoring and treatment decisions. Mid-infrared laser absorption spectroscopy is utilized to detect CH(4), H(2)O, CO(2), NH(3) and CH(3)OH in exhaled human breath. An off-line approach using breath sampling by means of Tedlar bags is applied. The breath gas samples are measured within the population-based epidemiological Study of Health in Pomerania (SHIP-TREND) performed at the University of Greifswald. The study covers about 5000 adult subjects aged 20-79 years within 3 years. Besides breath gas analysis many other examinations are conducted. It is expected to find associations between distinct concentration levels of species in the exhaled breath and diseases assessed in this study. The study will establish reference values for exhaled breath components and serve as background population for case-control studies. In the long run, morbidity and mortality follow-ups will be conducted, which will answer the question whether end-expiratory breath gas components predict future diseases and death. As first results, we present data from 45 dialysis patients (23 males, 22 females) which were recruited in a preliminary study in preparation for SHIP-TREND.