Heat shock protein 90 (Hsp90) is a molecular chaperone that plays a key role in the conformational maturation of oncogenic signalling proteins, including HER-2/ErbB2, Akt, Raf-1, Bcr-Abl and mutated p53. Hsp90 inhibitors bind to Hsp90, and induce the proteasomal degradation of Hsp90 client proteins. Although Hsp90 is highly expressed in most cells, Hsp90 inhibitors selectively kill cancer cells compared to normal cells, and the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) is currently in phase I clinical trials. However, the molecular basis of the tumour selectivity of Hsp90 inhibitors is unknown. Here we report that Hsp90 derived from tumour cells has a 100-fold higher binding affinity for 17-AAG than does Hsp90 from normal cells. Tumour Hsp90 is present entirely in multi-chaperone complexes with high ATPase activity, whereas Hsp90 from normal tissues is in a latent, uncomplexed state. In vitro reconstitution of chaperone complexes with Hsp90 resulted in increased binding affinity to 17-AAG, and increased ATPase activity. These results suggest that tumour cells contain Hsp90 complexes in an activated, high-affinity conformation that facilitates malignant progression, and that may represent a unique target for cancer therapeutics.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory condition of the central nervous system with similarities to multiple sclerosis. In both diseases, circulating leukocytes penetrate the blood-brain barrier and damage myelin, resulting in impaired nerve conduction and paralysis. We sought to identify the adhesion receptors that mediate the attachment of circulating leukocytes to inflamed brain endothelium in EAE, because this interaction is the first step in leukocyte entry into the central nervous system. Using an in vitro adhesion assay on tissue sections, we found that lymphocytes and monocytes bound selectively to inflamed EAE brain vessels. Binding was inhibited by antibodies against the integrin molecule alpha 4 beta 1, but not by antibodies against numerous other adhesion receptors. When tested in vivo, anti-alpha 4 integrin effectively prevented the accumulation of leukocytes in the central nervous system and the development of EAE. Thus, therapies designed to interfere with alpha 4 beta 1 integrin may be useful in treating inflammatory diseases of the central nervous system, such as multiple sclerosis.

Emerging evidence suggests that an amplifiable protease cascade consisting of multiple aspartatespecific cysteine proteases (ASCPs) Apoptosis is a fundamental biochemical cell-death pathway essential for normal tissue homeostasis, cellular differentiation, and development within a multicellular organism (for review, see refs. 1-3). Members of the growing family of aspartate-specific cysteine proteases (ASCPs) that include mammalian interleukin 13 converting enzyme (ICE) (4,5), Nedd2 (ICH-1) (6,7), CPP32 (8), Mch2 (9), Mch3 (10), TX (ICH-2, ICErel-II) (11)(12)(13), and ICErel-Ill (13) have been implicated as mediators of all apoptotic cell death (for review, see ref. 14).Cytotoxic T lymphocytes (CTL) induce apoptosis in their target cells possibly by activating members of the ASCP family (15). This hypothesis was supported by the observation that granzyme B, the CTL granule aspartate-specific serine protease, can cleave CPP32 (16). Recent observations suggested that the Fas-interacting protein FADD/MORT1 (17,18)

Activation of caspase-3 requires proteolytic processing of the inactive zymogen into p18 and p12 subunits. We generated a rabbit polyclonal antiserum, CM1, which recognizes the p18 subunit of cleaved caspase-3 but not the zymogen. CM1 demonstrated an apparent specificity for activated caspase-3 by specifically immunolabeling only apoptotic but not necrotic cortical neurons in vitro. In the embryonic mouse nervous system, CM1 immunoreactivity was detected in neurons undergoing programmed cell death and was markedly increased in Bcl-x L -deficient embryos and decreased in Bax-deficient embryos. CM1 immunoreactivity was absent in the nervous system of caspase-3-deficient mouse embryos and in neurons cultured from caspase-3-deficient mice. Along with neuronal somata, extensive neuritic staining was seen in apoptotic neurons. These studies indicate that caspase-3 is activated during apoptosis in the developing nervous system in vivo and that CM1 is a useful reagent for its in situ detection.

Progressive cerebral deposition of extracellu- (15,16). A related issue concerns the mechanism whereby the normal, age-related deposition of (AP in selected brain regions and vessels is augmented in AD. The local proteolytic processing of the precursor and the role of amyloidassociated proteins, such as the serine protease inhibitor a1-antichymotrypsin (24), require elucidation.Although molecular studies of 8APP have advanced rapidly, the native precursor protein in human brain and other tissues has not been detected and characterized. To begin to address some of the questions posed above, we produced antibodies to synthetic peptides with sequences predicted from amyloidogenic and nonamyloidogenic regions of J3APP and have identified forms of the precursor molecule in brain, nonneural tissues, and cultured cells of several species. METHODSPreparation of Antibodies to Synthetic Peptides. Several peptides were synthesized according to the sequence deduced from a P3APP cDNA (14). Those used in this study were: amino acid residues 597-624 (peptide (31-28), comprising the first 28 residues of PAP; residues 676-695 (peptide C1), comprising the 20 C-terminal amino acids of PAPP; and residues 681-695, a second C-terminal peptide (C2), comprising the last 15 residues. The latter peptide and an antiserum to it were the gifts of Tsuyoshi Ishii (Psychiatric Research Institute, Tokyo). Peptide C1 was HPLC-purified and coupled to edestin. Peptide C2 had been coupled to keyhole limpet hemocyanin. Peptide p1-28 was injected uncoupled. Antisera were assayed by serial dilutions on dot blots of unconjugated peptide. Previously characterized antibodies used in this study included two antisera (A and C) to the -4-kDa P3AP purified from amyloid-rich fractions of AD cortex (12,25), two antisera (Fl and Ph) to the -4-kDa ,8AP purified from AD meningovascular amyloid (10), a paired helical filament-specific antiserum having no reaction with amyloid deposits (26), and an antiserum to heat-stable microtubule-associated proteins (principally X and MAP 2) purified from fetal human brain (25).Abbreviations: AD, Alzheimer disease; flAP, f8-amyloid protein; f3APP, 13-amyloid precursor polypeptide. 7341The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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The human proto-oncogene bcl-2 and its Caenorhabditis elegans homologue ced-9 inhibit programmed cell death. In contrast, members of the human interleukin-1beta converting enzyme (ICE) family of cysteine proteases and their C. elegans homologue CED-3 promote the death program. Genetic experiments in C. elegans have shown that ced-9 is formally a negative regulator of ced-3 function, but neither those studies nor others have determined whether CED-9 or Bcl-2 proteins act biochemically upstream or downstream of CED-3/ICE proteases. CPP32, like all known members of the CED-3/ICE family, is synthesized as a proenzyme that is subsequently processed into an active protease with specificity for cleavage at Asp-X peptide bonds. In this report, we demonstrate that the CPP32 proenzyme is proteolytically processed and activated in Jurkat cells induced to die by Fas ligation. CPP32 activation is blocked by cell-permeable inhibitors of aspartate-directed, cysteine proteases, suggesting that pro-CPP32 is cleaved by active CPP32 or by other ICE family members. Heterologous expression of Bcl-2 in Jurkat cells prevents Fas-induced cell death as well as proteolytic processing and activation of CPP32. Thus, Bcl-2 acts at or upstream of the CPP32 activation step to inhibit apoptosis induced by Fas stimulation.

Neuronal apoptosis occurs during nervous system development and after pathological insults to the adult nervous system. Inhibition of CED3/ICE-related proteases has been shown to inhibit neuronal apoptosis in vitro and in vivo, indicating a role for these cysteine proteases in neuronal apoptosis. We have studied the activation of the CED3/ICE-related protease CPP32 in two in vitro models of mouse cerebellar granule neuronal cell death: K+/serum deprivation-induced apoptosis and glutamate-induced necrosis. Pretreatment of granule neurons with a selective, irreversible inhibitor of CED3/ICE family proteases, ZVAD-fluoromethylketone, specifically inhibited granule neuron apoptosis but not necrosis, indicating a selective role for CED3/ICE proteases in granule neuron apoptosis. Extracts prepared from apoptotic, but not necrotic, granule neurons contained a protease activity that cleaved the CPP32 substrate Ac-DEVD-aminomethylcoumarin. Induction of the protease activity was prevented by inhibitors of RNA or protein synthesis or by the CED3/ICE protease inhibitor. Affinity labeling of the protease activity with an irreversible CED3/ICE protease inhibitor, ZVK(biotin)D-fluoromethylketone, identified two putative protease subunits, p20 and p18, that were present in apoptotic but not necrotic granule neuron extracts. Western blotting with antibodies to the C terminus of the large subunit of mouse CPP32 (anti-CPP32) identified p20 and p18 as processed subunits of the CPP32 proenzyme. Anti-CPP32 specifically inhibited the DEVD-amc cleaving activity, verifying the presence of active CPP32 protease in the apoptotic granule neuron extracts. Western blotting demonstrated that the CPP32 proenzyme was expressed in granule neurons before induction of apoptosis. These results demonstrate that the CED3/ICE homolog CPP32 is processed and activated during cerebellar granule neuron apoptosis. CPP32 activation requires macromolecular synthesis and CED3/ICE protease activity. The lack of CPP32 activation during granule neuron necrosis suggests that proteolytic processing and activation of CED3/ICE proteases are specific biochemical markers of apoptosis.