CIP2A oncoprotein controls cell growth and autophagy through mTORC1 activation.

mTORC1 (mammalian target of rapamycin complex 1) integrates information regarding availability of nutrients and energy to coordinate protein synthesis and autophagy. Using ribonucleic acid interference screens for autophagy-regulating phosphatases in human breast cancer cells, we identify CIP2A (cancerous inhibitor of PP2A [protein phosphatase 2A]) as a key modulator of mTORC1 and autophagy. CIP2A associates with mTORC1 and acts as an allosteric inhibitor of mTORC1-associated PP2A, thereby enhancing mTORC1-dependent growth signaling and inhibiting autophagy. This regulatory circuit is reversed by ubiquitination and p62/SQSTM1-dependent autophagic degradation of CIP2A and subsequent inhibition of mTORC1 activity. Consistent with CIP2A's reported ability to protect c-Myc against proteasome-mediated degradation, autophagic degradation of CIP2A upon mTORC1 inhibition leads to destabilization of c-Myc. These data characterize CIP2A as a distinct regulator of mTORC1 and reveals mTORC1-dependent control of CIP2A degradation as a mechanism that links mTORC1 activity with c-Myc stability to coordinate cellular metabolism, growth, and proliferation.

BAMLET activates a lysosomal cell death program in cancer cells.

A complex of human alpha-lactalbumin and oleic acid (HAMLET) was originally isolated from human milk as a potent anticancer agent. It kills a wide range of transformed cells of various origins while leaving nontransformed healthy cells largely unaffected both in vitro and in vivo. Importantly, purified alpha-lactalbumins from other mammals form complexes with oleic acid that show biological activities similar to that of HAMLET. The mechanism by which these protein-lipid complexes kill tumor cells is, however, largely unknown. Here, we show that complex of bovine alpha-lactalbumin and oleic acid (BAMLET), the bovine counterpart of HAMLET, kills tumor cells via a mechanism involving lysosomal membrane permeabilization. BAMLET shows potent cytotoxic activity against eight cancer cell lines tested, whereas nontransformed NIH-3T3 murine embryonic fibroblasts are relatively resistant. BAMLET accumulates rapidly and specifically in the endolysosomal compartment of tumor cells and induces an early leakage of lysosomal cathepsins into the cytosol followed by the activation of the proapoptotic protein Bax. Ectopic expression of three proteins known to stabilize the lysosomal compartment, i.e. heat shock protein 70 (Hsp70), Hsp70-2, and lens epithelium-derived growth factor, confer significant protection against BAMLET-induced cell death, whereas the antiapoptotic protein Bcl-2, caspase inhibition, and autophagy inhibition fail to do so. These data indicate that BAMLET triggers lysosomal cell death pathway in cancer cells, thereby clarifying the ability of alpha-lactalbumin:oleate complexes to kill highly apoptosis-resistant tumor cells.

The temperature dependence and involvement of mitochondria permeability transition and caspase activation in damage to organotypic hippocampal slices following in vitro ischemia.

The aggravating effect of hyperglycemia on ischemic brain injury can be mimicked in a model of in vitro ischemia (IVI) using murine hippocampal slice cultures. Using this model, we found that the damage in the CA1 region following IVI in the absence or presence of 40 mm glucose (hyperglycemia) is highly temperature dependent. Decreasing the temperature from 35 to 31 degrees C during IVI prevented cell death, whereas increasing the temperature by 2 degrees C markedly aggravated damage. As blockade of the mitochondrial permeability transition (MPT) is equally effective as hypothermia in preventing ischemic cell death in vivo, we investigated whether inhibition of MPT or of caspases was protective following IVI. In the absence of glucose, the MPT blockers cyclosporin A and MeIle4-CsA but not the immunosuppressive compound FK506 diminished cell death. In contrast, following hyperglycemic IVI, MPT blockade was ineffective. Also, the pan-caspase inhibitor Boc-Asp(OMe)fluoromethyl ketone did not decrease cell death in the CA1 region following IVI or hyperglycemic IVI. We conclude that cell death in the CA1 region of organotypic murine hippocampal slices following IVI is highly temperature dependent and involves MPT. In contrast, cell death following hyperglycemic IVI, although completely prevented by hypothermia, is not mediated by mechanisms that involve MPT or caspase activation.

Selective sparing of hippocampal CA3 cells following in vitro ischemia is due to selective inhibition by acidosis.

A brief global ischemic insult to the brain leads to a selective degeneration of the pyramidal neurons in the hippocampal CA1 region while the neurons in the neighbouring CA3 region are spared. The reason for this difference is not known. The selective vulnerability of CA1 neurons to ischemia can be reproduced in vitro in murine organotypic slice cultures, if the ion concentrations in the medium during the anoxic/aglycemic insult are similar to that in the brain extracellular fluid during ischemia in vivo. As acidosis develops during ischemia, we studied the importance of extracellular pH for selective vulnerability. We found that cell death in the CA1 and CA3 regions was equally prevented by removal of calcium from the medium or following blockade of the N-methyl-D-aspartate (NMDA) receptor by D-2 amino-5-phosphonopentanoic-acid (D-APV). On the other hand, damage to the CA3 neurons markedly decreased with decreasing pH following in vitro ischemia, while the degeneration of CA1 neurons was less pH dependent. Patch-clamp recordings from pyramidal neurons in the CA1 and CA3 regions, respectively, revealed a pronounced inhibition of NMDA-receptor mediated excitatory postsynaptic currents (EPSCs) at pH 6.5 that was equally pronounced in the two regions. However, when changing pH from 6.5 to 7.4 the recovery of the EPSCs was significantly slower in the CA3 region. We conclude that acidosis selectively protects CA3 pyramidal neurons during in vitro ischemia, and differentially affects the kinetics of NMDA receptor activation, which may explain the difference in vulnerability between CA1 and CA3 pyramidal neurons to an ischemic insult.

Chelation of intracellular calcium reduces cell death after hyperglycemic in vitro ischemia in murine hippocampal slice cultures.

The aggravating effect of high glucose levels during cerebral ischemia has been extensively documented in clinical studies and in vivo models of global and focal ischemia. Detailed mechanistic studies of hyperglycemic ischemia have so far been hampered by the lack of in vitro models since glucose during anoxia in vitro is highly protective. We have previously reported glucose toxicity in murine hippocampal organotypic slice cultures exposed to anoxia in an acidotic medium containing high potassium and low calcium. In the present study, we compared the importance of calcium, nitric oxide and free radicals during in vitro ischemia (IVI) and hyperglycemic (40 mM) IVI. Extracellular calcium was a ubiquitous factor for cell death after IVI, but its removal from the medium had no effect on cell death after hyperglycemic IVI. When intracellular calcium was chelated by the 1,2-Bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA-AM) cell death appeared earlier but was mitigated in hyperglycemic IVI, while it was increased in glucose-free IVI. Addition of the nitric oxide synthase (NOS) inhibitor N(omega)-Nitro-L-arginine methyl ester hydrochloride (L-NAME) or the free radical scavengers N-tert-butyl-alpha-phenylnitrone (PBN), deferoxamine and N-acetyl-L-cysteine (NAC) did not affect cell damage in either paradigm. We conclude that the aggravating effect of hyperglycemia during in vitro ischemia is partially mediated by calcium ions released from intracellular stores.

Deletion of the adenosine A1 receptor gene does not alter neuronal damage following ischaemia in vivo or in vitro.

Extracellular adenosine is dramatically increased during cerebral ischaemia and is considered to be neuroprotective due to its inhibitory effect on synaptic transmission mediated by the adenosine A1 receptor (A1R). We investigated the importance of the A1R in a mouse model of global ischaemia and in a murine hippocampal slice culture model of in vitro ischaemia, using mice with the A1R gene deleted. In brains from mice lacking the A1R, damage induced by global ischaemia was similar to that in wild-type animals. In contrast, treatment with a selective A1R antagonist [8-cyclo-pentyl theophylline (8-CPT)], administered before the ischaemic insult in naive wild-type mice, exacerbated the neuronal damage following global ischaemia. Although the inhibitory action of adenosine on excitatory neurotransmission in hippocampal slices was lost in A1R knockout mice, there was no difference in damage between slices from wild-type and knockout mice after in vitro ischaemia. The results suggest that some effects of the A1R are compensated for in knockout animals.

Glucose but not lactate in combination with acidosis aggravates ischemic neuronal death in vitro.

BACKGROUND AND PURPOSE: Hyperglycemia aggravates brain damage in clinical stroke and in experimental in vivo models of cerebral ischemia. Elevated preischemic glucose levels, lactate production, and intracerebral acidosis correlate with increased brain damage. We have developed a murine hippocampal slice culture model of in vitro ischemia (IVI), suitable for studies of the mechanisms of neuronal death. In this model we investigated the individual contribution of glucose, pH, lactate, and combinations thereof as well as ionotropic glutamate receptor activation to the development of hyperglycemic ischemic cell death. METHODS: Murine organotypic hippocampal slice cultures were exposed to IVI in a medium with an ionic composition similar to that of the extracellular fluid in the brain during ischemia in vivo. Cell death was assessed by propidium iodide uptake. Ionotropic glutamate receptor blockade was accomplished by D-2-amino-5-phosphonopentanoic acid (D-APV) or 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX). RESULTS: The combination of high glucose levels and acidosis (pH 6.8), but not acidosis per se or the combination of lactate and acidosis during IVI, exacerbated damage. Cell death after hyperglycemic IVI was not diminished by blockade of ionotropic glutamate receptors. CONCLUSIONS: Aggravation of brain damage by hyperglycemia in vivo can be reproduced in hippocampal slice cultures in vitro. Our results demonstrate that glucose per se, but not lactate, in combination with acidosis mediates the detrimental hyperglycemic effect through a mechanism independent of ionotropic glutamate receptors.

Mouse hippocampal organotypic tissue cultures exposed to in vitro "ischemia" show selective and delayed CA1 damage that is aggravated by glucose.

Oxygen and glucose deprivation (OGD) in cell cultures is generally studied in a medium, such as artificial cerebrospinal fluid (CSF), with an ion composition similar to that of the extracellular fluid of the normal brain (2 to 4 mmol/L K+, 2 to 3 mmol/L Ca2+; pH 7.4). Because the distribution of ions across cell membranes dramatically shifts during ischemia, the authors exposed mouse organotypic hippocampal tissue cultures to OGD in a medium, an ischemic cerebrospinal fluid, with an ion composition similar to the extracellular fluid of the brain during ischemia (70 mmol/L K+, 0.3 mmol/L Ca2+; pH 6.8). In ischemic CSF, OGD induced a selective and delayed cell death in the CA1 region, as assessed by propidium iodide uptake. Cell death was glutamate receptor dependent since blockade of the N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors mitigated cell damage. Hyperglycemia aggravates ischemic brain damage whereas glucose in artificial CSF prevents oxygen deprivation-induced damage. The authors demonstrate that glucose in ischemic CSF significantly exacerbates cell damage after oxygen deprivation. This new model of "ischemia" can be useful in future studies of the mechanisms and treatment of ischemic cell death, including studies using genetically modified mice.