Mechanistic/mammalian target of rapamycin (mTOR) activity drives a number of key metabolic processes including growth and protein synthesis. characteristic of AML. Background Tumour cell growth is driven Oaz1 by active biosynthetic and glycolytic pathways [1] fuelling interest in finding anti-cancer uses for drugs which interfere with these processes [2C5]. Mechanistic/mammalian target of rapamycin (mTOR) is an element of the mTORC1 signalling complex which drives energy generation, macromolecule synthesis and cell growth [6C8]. Constitutive activation of mTOR is commonly found in tumour cells, but in quiescent normal cells mTOR activity and biosynthetic pathways are suppressed [1, 5]. This may happen in an energy-rich and nutrient-replete environment, such as in the case of circulating lymphocytes [9, 10], or may be a homeostatic response to nutrient or energy depletion in which AMPK is activated and mTOR subsequently inactivated to promote conservation of essential cell functions [1, 4, 11]. What remains unclear in these scenarios is the behaviour of the dormant cancer cell. Reversible exit from the cell cycle into the quiescent, G0 state is well described in somatic cells, and is characterised by small size and low RNA and protein synthesis [12, 13]. The mitogenic factors driving malignant transformation might be thought not to permit a state of true (G0) quiescence in cancer cells [13]. Nevertheless, in acute myeloid leukaemia, dormant (apparently quiescent) cells which retain proliferative potential have been described [14, 15]. A high proportion of circulating and bone marrow blasts in AML also have phenotypic features of dormancy, as measured by lack of Ki-67 [16]. Ki -67 is expressed in all active phases of the cell cycle including G1[17]. Standard chemotherapy for AML buy (R,R)-Formoterol tends to spare dormant leukaemia cells [16, 18], so it would be useful to characterise this subset in order to establish how best to target it. Do dormant leukaemia cells better resemble normal dormant cells or proliferating cancer cells? To further our understanding of Ki-67 leukaemia cells, particularly with regard to their metabolic activity and hence potential susceptibility to therapeutic inhibition of this activity, we have measured biomarkers of mTOR activation status in presentation samples, using flow cytometry. This technique has enabled us to examine mTOR activation concurrently with proliferation status at the single cell level. We have measured activation-related epitopes of mTOR, 4E-BP1 and ribosomal protein S6, in conjunction with Ki-67 or the transferrin receptor CD71 and maturation markers, in primary cells of pre-treatment samples from patients with AML. MTOR phosphorylation was measured at serine 2448. This phospho-epitope is lost when raptor is depleted, indicating its specificity for mTORC1 [19]. MTOR is phosphorylated at serine 2448 by p70S6 kinase: whereas the phosphorylation is not thought to be intrinsically activating, it can be used as an indicator of buy (R,R)-Formoterol the level of mTOR signalling because p70S6 kinase activity is, in turn, mTOR-dependent [20, 21]. S6 kinase also phosphorylates ribosomal protein S6 (rpS6) [22]. Antibodies to ribosomal protein S6 (rpS6) phosphorylated at serine 235/236 have been optimised for flow cytometry, where they are well-established as biomarkers for mTORC1 activity [23, 24]. A second major target of mTOR is 4E-BP1, which is directly phosphorylated by mTOR at T36/T45 [25]. 4E-BPs control protein synthesis [5, 26] and mediate mTORC1-dependent cell proliferation [27]. Conditional deletion of raptor from AML cells has revealed that mTORC1 deficiency increases the proportion of undifferentiated and self-renewing haematopoietic cells and decreases the proportion of differentiated cells in vivo [28], suggesting that mTOR might be most activated in the context of maturation rather than self-renewal. This would have implications for mTOR as a therapeutic target, and we therefore also measured mTOR activity in undifferentiated (CD34+CD38-) AML cells and those with monocytoid maturation compared to blast cells. Materials and Methods Cells AML patient samples were obtained buy (R,R)-Formoterol with written, informed consent according to the protocol approved by National Research Ethics Service Committee East MidlandsNottingham 1 and Nottingham University Hospitals NHS Trust. Cells from G-CSF-mobilised donor stem cell harvests (SCH) were obtained with written, informed consent according the protocol approved by National Research Ethics Service Committee East MidlandsNottingham 2 and Nottingham University Hospitals NHS Trust. Cells were processed by standard methods to mononuclear cell preparations. The KG1a myeloid leukaemia cell line was obtained from the buy (R,R)-Formoterol European Collection of Animal Cell Cultures (Salisbury, UK) and was maintained in.