Jun N-terminal kinase (JNK) is a stress-activated protein kinase that can be induced by inflammatory cytokines, bacterial endotoxin, osmotic shock, UV radiation, and hypoxia. We report the identification of an anthrapyrazolone series with significant inhibition of JNK1, -2, and -3 (K i ‫؍‬ 0.19 M). SP600125 is a reversible ATPcompetitive inhibitor with >20-fold selectivity vs. a range of kinases and enzymes tested. In cells, SP600125 dose dependently inhibited the phosphorylation of c-Jun, the expression of inflammatory genes COX-2, IL-2, IFN-␥, TNF-␣, and prevented the activation and differentiation of primary human CD4 cell cultures. In animal studies, SP600125 blocked (bacterial) lipopolysaccharideinduced expression of tumor necrosis factor-␣ and inhibited anti-CD3-induced apoptosis of CD4 ؉ CD8 ؉ thymocytes. Our study supports targeting JNK as an important strategy in inflammatory disease, apoptotic cell death, and cancer.

Nitric oxide (NO) is a pleiotropic regulator, critical to numerous biological processes, including vasodilatation, neurotransmission and macrophage-mediated immunity. The family of nitric oxide synthases (NOS) comprises inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS). Interestingly, various studies have shown that all three isoforms can be involved in promoting or inhibiting the etiology of cancer. NOS activity has been detected in tumour cells of various histogenetic origins and has been associated with tumour grade, proliferation rate and expression of important signaling components associated with cancer development such as the oestrogen receptor. It appears that high levels of NOS expression (for example, generated by activated macrophages) may be cytostatic or cytotoxic for tumor cells, whereas low level activity can have the opposite effect and promote tumour growth. Paradoxically therefore, NO (and related reactive nitrogen species) may have both genotoxic and angiogenic properties. Increased NO-generation in a cell may select mutant p53 cells and contribute to tumour angiogenesis by upregulating VEGF. In addition, NO may modulate tumour DNA repair mechanisms by upregulating p53, poly(ADP-ribose) polymerase (PARP) and the DNA-dependent protein kinase (DNA-PK). An understanding at the molecular level of the role of NO in cancer will have profound therapeutic implications for the diagnosis and treatment of disease.

well as SCF Skp2 -and p27-dependent cell killing. This programmed cell death was caspase-independent, and instead occurred through activation of autophagy. In models of multiple myeloma, CpdA overcame resistance to dexamethasone, doxorubicin, and melphalan, as well as to bortezomib, and also acted synergistically with this proteasome inhibitor. Importantly, CpdA was active against patient-derived plasma cells and both myeloid and lymphoblastoid leukemia blasts, and showed preferential activity against neoplastic cells while relatively sparing other marrow components. These findings provide a rational framework for further development of SCF Skp2 IntroductionThe proteasome inhibitor bortezomib (VELCADE, Millennium Pharmaceuticals, Cambridge, MA) has demonstrated significant activity both as a single agent 1,2 and in combination with other drugs 3 against multiple myeloma (MM), validating the ubiquitinproteasome pathway as a target for cancer therapy. Its broad impact on cellular proteolysis may also have deleterious effects, however, including induction of antiapoptotic heat-shock proteins (HSPs) at the molecular level, and peripheral neuropathy at the clinical level. 4,5 One approach that may overcome these drawbacks would be to target E3 ligases, which are responsible for transfer of ubiquitin moieties to target proteins prior to proteasomal degradation. Since each E3 ligase serves a small subset of protein clients, this would selectively stabilize only those specific proteins, thus minimizing unwanted effects on other targets. 6 SCF complex ligases are the largest family of E3 ligases, and consist of 4 components, including S-phase kinase-associated protein-1 (Skp1), Cullin-1 (Cul1), regulator of cullins-1 (Roc1), and a variable F-box protein. 7 A recent crystal structure of the SCF containing the F-box protein Skp2 (SCF Skp2 ) revealed that Cul1 served as a scaffold binding a Skp1/Skp2 complex at its N-terminus to recruit substrates, while Roc1 bound at the C-terminus to recruit E2 ubiquitinconjugating enzymes. 8,9 Since the F-box protein determines the specificity of SCF ligases, it represents a target that could provide the greatest potential selectivity.Incorporation of Skp2 into an SCF complex confers the ability to induce ubiquitination of several targets, including p27 Kip1 , p21 Cip1 , and p57 Kip2 . As a critical cell-cycle regulator, p27 arrests cell division and inhibits G 1 /S transition, and cellular p27 Kip1 levels are largely modulated through the ubiquitin-proteasome pathway. After phosphorylation at Thr187, p27 is recruited to SCF Skp2 in the nucleus to be polyubiquitinated for degradation through the 26S proteasome. [10][11][12][13][14] Mutation or silencing of p27 in human cancers is extremely rare, 15 but loss of p27 Kip1 protein is a common event due to enhanced proteolysis, and has been associated with an aggressive phenotype and a poor prognosis in a variety of malignancies. [16][17][18][19][20][21] In multiple myeloma, clinical studies have shown that patients with low p27 expressio...

We have created a transgenic mouse with a hypomorphic allele of the vesicular monoamine transporter 2 (Vmat2) gene by gene targeting. These mice (KA1) have profound changes in monoamine metabolism and function and survive into adulthood. Specifically, these animals express very low levels of VMAT2, an endogenous protein which sequesters monoamines intracellularly into vesicles, a process that, in addition to being important in normal transmission, may also act to keep intracellular levels of the monoamine neurotransmitters below potentially toxic thresholds. Homozygous mice show large reductions in brain tissue monoamines, motor impairments, enhanced sensitivity to dopamine agonism, and changes in the chemical neuroanatomy of the striatum that are consistent with alterations in the balance of the striatonigral (direct) and striatopallidal (indirect) pathways. The VMAT2-deficient KA1 mice are also more vulnerable to the neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in terms of nigral dopamine cell death. We suggest that the mice may be of value in examining, long term, the insidious damaging consequences of abnormal intracellular handling of monoamines. On the basis of our current findings, the mice are likely to prove of immediate interest to aspects of the symptomatology of parkinsonism. They may also, however, be of use in probing other aspects of monoaminergic function and dysfunction in the brain, the latter making important contributions to the pathogenesis of schizophrenia and addiction.

Nitric oxide (NO) is a pleiotropic signalling molecule that binds to cytochrome c oxidase (complex IV) reversibly and in competition with oxygen. This action of NO has both physiological and pathophysiological consequences. Here we report that endogenously generated NO, which disrupts the respiratory chain, may cause changes in mitochondrial calcium flux. This induces cleavage of the endoplasmic reticulum (ER) stress-regulated transcription factor p90 ATF6 into an active p50 form. Cleavage depends on a calcium-dependent serine protease through a regulated intramembrane proteolysis (RIP) process. p50 ATF6 then translocates to the nucleus to upregulate expression of the ER-resident molecular chaperone, glucose-regulated protein 78 (Grp78). The increase in Grp78 provides significant cytoprotection against toxic agents, including thapsigargin, a selective ER calcium-ATPase inhibitor. Cytoprotection is abolished after treatment with cyclosporin A (CsA), which disrupts mitochondrial calcium signalling, or with the calcium chelator BAPTA-AM. The NO-mediated ER stress response is diminished in rho(0) cells devoid of mitochondrial DNA, consistent with our evidence that NO-dependent mitochondrial disruption is coupled to the ER stress response.