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.