The cell-cycle inhibitor p27 is a potential tumor suppressor, but its gene has never been found inactivated in human tumors. Because cell-cycle regulation of p27 cellular abundance occurs at the post-transcriptional level, we analyzed p27 protein expression and degradation in human colorectal carcinomas. Proteasome-mediated degradation activity of p27 was compared with its protein levels in a subset of tumor samples. We found that carcinomas with low or absent p27 protein displayed enhanced proteolytic activity specific for p27, suggesting that low p27 expression can result from increased proteasome-mediated degradation rather than altered gene expression. Patients whose tumors expressed p27 had a median survival of 151 months, whereas patients who lacked p27 (10%) had a median survival of 69 months. By multivariate analysis, p27 was found to be an independent prognostic marker. Lack of p27 was associated with poor prognosis (2.9 risk ratio for death; P = 0.003). The absence of p27 protein expression is thus a powerful negative prognostic marker in colorectal carcinomas, particularly in stage II tumors, and thereby may help in the selection of patients who will benefit from adjuvant therapy. These data suggest that aggressive tumors may result from the selection of a clone or clones that lack p27 due to increased proteasome-mediated degradation.

Release of the excitatory neurotransmitter glutamate and the excessive stimulation of N-methyl-Daspartate (NMDA)-type glutamate receptors is thought to be responsible for much of the neuronal death that occurs following focal hypoxia-ischemia in the central nervous system. Our laboratory has identified endogenous sulfated steroids that potentiate or inhibit NMDA-induced currents. Here we report that 3␣-ol-5␤-pregnan-20-one hemisuccinate (3␣5␤HS), a synthetic homologue of naturally occurring pregnanolone sulfate, inhibits NMDA-induced currents and cell death in primary cultures of rat hippocampal neurons. 3␣5␤HS exhibits sedative, anticonvulsant, and analgesic properties consistent with an action at NMDA-type glutamate receptors. Intravenous administration of 3␣5␤HS to rats (at a nonsedating dose) following focal cerebral ischemia induced by middle cerebral artery occlusion significantly reduces cortical and subcortical infarct size. The in vitro and in vivo neuroprotective effects of 3␣5␤HS demonstrate that this steroid represents a new class of potentially useful therapeutic agents for the treatment of stroke and certain neurodegenerative diseases that involve over activation of NMDA receptors.Exposure of neurons to the excitatory neurotransmitter glutamate causes an increase in the concentration of intracellular free Ca 2ϩ (1) and initiates the process of excitotoxic cell death (2). There are at least three pharmacologically distinct ionotropic glutamate receptors that differ in their sensitivity to the selective agonists N-methyl-D-aspartate (NMDA), ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and kainate. Whereas excessive activation of NMDA, AMPA, or kainate receptors results in neurotoxicity, specific inhibition of NMDA receptors is sufficient to attenuate most of the neuronal death that develops after in vitro hypoxia, exposure to glutamate, in vivo ischemia, or hypoglycemia (3). As Ca 2ϩ passes through the NMDA receptor operated ion channel, it is believed that this receptor subserves a crucial role in mediating excitotoxicity. Therefore, it has been proposed that over activation of NMDA receptors may be an obligatory phase preceding neuronal death that occurs following focal cerebral ischemia induced stroke in humans.Neuroactive steroids have been shown to directly modulate excitatory and inhibitory amino acid receptor function (4-7). Therefore the possibility is raised that certain steroids might be useful neuroprotective agents. The endogenous neurosteroid pregnenolone sulfate probably acts as a positive allosteric modulator of the NMDA receptor by enhancing NMDAinduced inward currents and the subsequent rise in cytoplasmic free calcium (8, 9), whereas 3␣-ol-5␤-pregnan-20-one sulfate (3␣5␤S) is a negative modulator of NMDA-induced currents (6) and inhibits NMDA-stimulated increases in intracellular calcium (10). The present study examines the in vitro and in vivo neuroprotective activity of 3␣-ol-5␤-pregnan-20-one hemisuccinate (3␣5␤HS) ( Fig. 1; a synthetic analog of the endogenou...

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