Amino acids control cell growth via activation of the highly conserved kinase TORC1. Glutamine is a particularly important amino acid in cell growth control and metabolism. However, the role of glutamine in TORC1 activation remains poorly defined. Glutamine is metabolized through glutaminolysis to produce α-ketoglutarate. We demonstrate that glutamine in combination with leucine activates mammalian TORC1 (mTORC1) by enhancing glutaminolysis and α-ketoglutarate production. Inhibition of glutaminolysis prevented GTP loading of RagB and lysosomal translocation and subsequent activation of mTORC1. Constitutively active Rag heterodimer activated mTORC1 in the absence of glutaminolysis. Conversely, enhanced glutaminolysis or a cell-permeable α-ketoglutarate analog stimulated lysosomal translocation and activation of mTORC1. Finally, cell growth and autophagy, two processes controlled by mTORC1, were regulated by glutaminolysis. Thus, mTORC1 senses and is activated by glutamine and leucine via glutaminolysis and α-ketoglutarate production upstream of Rag. This may provide an explanation for glutamine addiction in cancer cells.

Hypoxia-inducible factor (HIF) prolyl hydroxylases (PHDs) are α-ketoglutarate (αKG)-dependent dioxygenases that function as cellular oxygen sensors. However, PHD activity also depends on factors other than oxygen, especially αKG, a key metabolic compound closely linked to amino-acid metabolism. We examined the connection between amino-acid availability and PHD activity. We found that amino-acid starvation leads to αKG depletion and to PHD inactivation but not to HIF stabilization. Furthermore, pharmacologic or genetic inhibition of PHDs induced autophagy and prevented mammalian target of rapamycin complex 1 (mTORC1) activation by amino acids in a HIF-independent manner. Therefore, PHDs sense not only oxygen but also respond to amino acids, constituting a broad intracellular nutrient-sensing network.

Analysis of phosphorylated histone protein H2AX (cH2AX) foci is currently the most sensitive method to detect DNA double-strand breaks (DSB). This protein modification has the potential to become an individual biomarker of cellular stress, especially in the diagnosis and monitoring of neoplastic diseases. To make cH2AX foci analysis available as a routine screening method, different software approaches for automated immunofluorescence pattern evaluation have recently been developed. In this study, we used novel pattern recognition algorithms on the AKLIDES V R platform to automatically analyze immunofluorescence images of cH2AX foci and compared the results with visual assessments. Dose-and time-dependent cH2AX foci formation was investigated in human peripheral blood mononuclear cells (PBMCs) treated with the chemotherapeutic drug etoposide (ETP). Moreover, the AKLIDES system was used to analyze the impact of different immunomodulatory reagents on cH2AX foci formation in PBMCs. Apart from cH2AX foci counting the use of novel pattern recognition algorithms allowed the measurement of their fluorescence intensity and size, as well as the analysis of overlapping cH2AX foci. The comparison of automated and manual foci quantification showed overall a good correlation. After ETP exposure, a clear dose-dependent increase of cH2AX foci formation was evident using the AKLIDES as well as Western blot analysis. Kinetic experiments on PBMCs incubated with 5 lM ETP demonstrated a peak in cH2AX foci formation after 4 to 8 h, while a removal of ETP resulted in a strong reduction of cH2AX foci after 1 to 4 h. In summary, this study demonstrated that the AKLIDES system can be used as an efficient automatic screening tool for cH2AX foci analysis by providing new evaluation features and facilitating the identification of drugs which induce or modulate DNA damage. V C 2013 International Society for Advancement of CytometryKey terms cH2AX foci; automated microscopy; image analysis; DNA double-strand breaks; etoposide; human PBMCs THE damage of DNA is a critical event able to affect cellular functions and development. Thus, it is essential for cells to maintain DNA integrity and repair such lesions effectively. Among different kinds of DNA lesion, double strand breaks (DSB) are considered to be the most critical type of DNA damage and misrepair can lead to tumorgenesis or cell death (1,2). To ensure detection and repair of DNA damage sites a variety of proteins are involved in different DNA damage response (DDR) pathways (3). After induction of DSB, the histone protein H2AX is rapidly phosphorylated at serine 139, termed cH2AX. Large amounts of cH2AX molecules form a focus in the

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Whilst heterozygous germline mutations in the ABRAXAS1 gene have been associated with a hereditary predisposition to breast cancer, their effect on promoting tumourigenesis at the cellular level has not been explored. Here, we demonstrate in patient-derived cells that the Finnish ABRAXAS1 founder mutation (c.1082G>A, Arg361Gln), even in the heterozygous state leads to decreased BRCA1 protein levels as well as reduced nuclear localization and foci formation of BRCA1 and CtIP. This causes disturbances in basal BRCA1-A complex localization, which is reflected by a restraint in error-prone DNA double-strand break (DSB) repair pathway usage, attenuated DNA damage response and deregulated G2-M checkpoint control. The current study clearly demonstrates how the Finnish ABRAXAS1 founder mutation acts in a dominant-negative manner on BRCA1 to promote genome destabilisation in heterozygous carrier cells.

BackgroundIn response to DNA double-strand breaks, the histone protein H2AX becomes phosphorylated at its C-terminal serine 139 residue, referred to as γ-H2AX. Formation of γ-H2AX foci is associated with recruitment of p53-binding protein 1 (53BP1), a regulator of the cellular response to DNA double-strand breaks. γ-H2AX expression in peripheral blood mononuclear cells (PBMCs) was recently proposed as a diagnostic and disease activity marker for multiple sclerosis (MS).ObjectiveTo evaluate the significance of γ-H2AX and 53BP1 foci in PBMCs as diagnostic and disease activity markers in patients with clinically isolated syndrome (CIS) and early relapsing-remitting MS (RRMS) using automated γ-H2AX and 53BP1 foci detection.MethodsImmunocytochemistry was performed on freshly isolated PBMCs of patients with CIS/early RRMS (n = 25) and healthy controls (n = 27) with γ-H2AX and 53BP1 specific antibodies. Nuclear γ-H2AX and 53BP1 foci were determined using a fully automated reading system, assessing the numbers of γ-H2AX and 53BP1 foci per total number of cells and the percentage of cells with foci. Patients underwent contrast enhanced 3 Tesla magnetic resonance imaging (MRI) and clinical examination including expanded disability status scale (EDSS) score. γ-H2AX and 53BP1 were also compared in previously frozen PBMCs of each 10 CIS/early RRMS patients with and without contrast enhancing lesions (CEL) and 10 healthy controls.ResultsThe median (range) number of γ-H2AX (0.04 [0–0.5]) and 53BP1 (0.005 [0–0.2]) foci per cell in freshly isolated PBMCs across all study participants was low and similar to previously reported values of healthy individuals. For both, γ-H2AX and 53BP1, the cellular focus number as well as the percentage of positive cells did not differ between patients with CIS/RRMS and healthy controls. γ-H2AX and 53BP1 levels neither correlated with number nor volume of T2-weighted lesions on MRI, nor with the EDSS. Although γ-H2AX, but not 53BP1, levels were higher in previously frozen PBMCs of patients with than without CEL, γ-H2AX values of both groups overlapped and γ-H2AX did not correlate with the number or volume of CEL.Conclusionγ-H2AX and 53BP1 foci do not seem to be promising diagnostic or disease activity biomarkers in patients with early MS. Lymphocytic DNA double-strand breaks are unlikely to play a major role in the pathophysiology of MS.

The efficacy of many chemotherapeutic agents relies on the preferential destruction of rapidly dividing cancer cells by inducing various kinds of DNA damage. The most dele-terious type of DNA lesions are DNA double-strand breaks (DSB), which can be detected by immunofluorescence staining of phosphorylated histone protein H2AX (cH2AX). Furthermore, cH2AX has been suggested as clinical pharmacodynamic bio-marker in chemotherapeutic cancer treatment. A great challenge in treating neoplastic diseases is the varying response behavior among cancer patients. Thus, intrinsic or drug-induced overexpression of efflux pumps often leads to multiple drug resistance (MDR) and treatment failure. In particular, inter-individual differences in expression levels of efflux pumps, such as the permeability glycoprotein (P-gp), were shown to correlate with cancer progression. Several efficient cytostatic drugs, including the DSB-inducing agent etoposide (ETP) are known P-gp substrates. In this respect, modulation of MDR by P-gp inhibitors, like the immunosuppressives cyclosporine A (CsA) and rapamycin (Rapa) have been described. Here, we investigated the application of cH2AX focus assay to monitor the impact of CsA and Rapa on ETP-induced cytotoxic-ity in human peripheral blood mononuclear cells. Evaluation of cH2AX foci was performed by the automated fluorescence microscopy and interpretation system AKLIDES. Compared to ETP treatment alone, our results revealed a significant rise in cH2AX focus number and percentage of DSB-positive cells after cells have been treated with ETP in the presence of either CsA or Rapa. In contrast, DSB levels of cells incu-bated with CsA or Rapa alone were comparable to focus number of untreated cells. Our results successfully demonstrated how automated cH2AX analysis can be used as fast and reliable approach to monitor drug resistance and the impact of MDR modula-tors during treatment with DSB-inducing cytostatics. V C 2015 International Society for Advancement of Cytometry Key terms DNA double-strand breaks; automated cH2AX foci analysis; drug efflux; MDR modulation ; etoposide; cytostatic drug resistance; P-gp inhibition ONE central molecule in recognition and repair mechanisms of DNA double-strand breaks (DSB) is the core histone protein H2AX. Upon DSB formation, thousands of adjacent H2AX molecules become rapidly phosphorylated at serin-139 and form a so-called cH2AX repair focus. Thereby, DSB can be visualized microscopically as discrete nuclear spot after indirect anti-cH2AX immunofluorescence staining (IIF) (1,2). Previously, we validated fully automated cH2AX foci analysis in human peripheral mononuclear cells (PBMCs) by the fluorescent interpretation system AKLIDES and demonstrated a good agreement between manual and automated cH2AX foci quantification (3-5). Furthermore, automated analysis of cH2AX foci by the AKLIDES platform was successfully used in different studies (4,6).

Detection of 8-OHdG-base damage has been a big challenge for decades, though different analytical methods are developed. The recent approaches that are used for quantitating either the total amount of base damage or the amount of base damage per cell from different sources of samples are not automated. We have developed a method for automated damage detection from a single cell and applied it to 8-OHdG quantitation.