Kaposi's sarcoma-associated herpesvirus-induced upregulation of the c-kit proto-oncogene,as identified by gene expression profiling,is essential for the transformation of endothelial cells

Kaposi's sarcoma (KS), the most frequent malignancy afflicting AIDS patients, is characterized by spindle cell formation and vascularization. Infection with KS-associated herpesvirus (KSHV) is consistently observed in all forms of KS. Spindle cell formation can be replicated in vitro by infection of dermal microvascular endothelial cells (DMVEC) with KSHV. To study the molecular mechanism of this transformation, we compared RNA expression profiles of KSHV-infected and mock-infected DMVEC. Induction of several proto-oncogenes was observed, particularly the receptor tyrosine kinase c-kit. Consistent with increased c-Kit expression, KHSV-infected DMVEC displayed enhanced proliferation in response to the c-Kit ligand, stem cell factor (SCF). Inhibition of c-Kit activity with either a pharmacological inhibitor of c-Kit (STI 571) or a dominant-negative c-Kit protein reversed SCF-dependent proliferation. Importantly, inhibition of c-Kit signal transduction reversed the KSHV-induced morphological transformation of DMVEC. Furthermore, overexpression studies showed that c-Kit was sufficient to induce spindle cell formation. Together, these data demonstrate an essential role for c-Kit in KS tumorigenesis and reveal a target for pharmacological intervention.     

Response to oxidative stress caused by H(2)O(2) in Saccharomyces cerevisiae mutants deficient in trehalase genes

The role of trehalose as cell protector against oxidative stress induced by H(2)O(2) has been studied in Saccharomyces cerevisiae mutants in which the two trehalase genes ATH1 and NTH1 are deleted. The addition of low H(2)O(2) concentrations to proliferating cultures of either strain did not harm cell viability and induced a marked activity to Nth1p, but with no significant level of trehalose accumulation. This pattern was reversed after a more severe H(2)O(2) treatment that caused drastic cell killing. The most severe phenotype corresponded to the Delta nth1 mutant. Under these conditions, the increase in Nth1p was abolished and a three-fold rise in trehalose content was recorded concomitant with activation of the trehalose synthase complex. The behavior of the double-disruptant Delta ath1Delta nth1 mutant was identical to that of wild-type cells, although in exponential cultures Ath1p activity was virtually undetectable upon exposure to H(2)O(2). Furthermore, these strains displayed an adaptive response to oxidative stress that was independent of intracellular trehalose synthesis. Our data strongly suggest that trehalose storage in budding yeasts is not an essential protectant in cell defense against oxidative challenge.     

Purification and characterization of a prolyl aminopeptidase from Debaryomyces hansenii

A prolyl aminopeptidase (PAP) (EC 3.4.11.5) was isolated from the cell extract of Debaryomyces hansenii CECT12487. The enzyme was purified by selective fractionation with protamine and ammonium sulfate, followed by two chromatography steps, which included gel filtration and anion-exchange chromatography. The PAP was purified 248-fold, with a recovery yield of 1.4%. The enzyme was active in a broad pH range (from 5 to 9.5), with pH and temperature optima at 7.5 and 45 degrees C. The molecular mass was estimated to be around 370 kDa. The presence of inhibitors of serine and aspartic proteases, bestatin, puromycin, reducing agents, chelating agents, and different cations did not have any effect on the enzyme activity. Only iodoacetate, p-chloromercuribenzoic acid, and Hg(2+), which are inhibitors of cysteine proteases, markedly reduced the enzyme activity. The K(m) for proline-7-amido-4-methylcoumarin was 40 micro M. The enzyme exclusively hydrolyzed N-terminal-proline-containing substrates. This is the first report on the identification and purification of this type of aminopeptidase in yeast, which may contribute to the scarce knowledge about D. hansenii proteases and their possible roles in meat fermentation.     

Flavopiridol induces mitochondrial-mediated apoptosis in murine glioma GL261 cells via release of cytochrome c and apoptosis inducing factor

Glioblastoma (GBM) remains one of the most challenging solid cancers to treat due to its highly proliferative, angiogenic and invasive nature. The small molecule CDK inhibitor, flavopiridol, has demonstrated antitumor activity in human xenograft models and is currently in clinical trials showing efficacy in patients with advanced disease. We have developed an experimental animal model using the murine glioma GL261 cells as a novel in vivo system to screen potential therapeutic agents for GBM. Results of in vitro testing demonstrate that flavopiridol has several relevant clinical characteristics such as its ability to: 1. inhibit cell growth; 2. inhibit cell migration; 3. decrease expression of cyclin D1, CDK4 and p21; 4. induce apoptosis in cells with high levels of p27 expression; and 5. decrease the expression of the anti-apoptotic protein Bcl-2. The mechanism by which flavopiridol induces apoptosis is mitochondrial-mediated. We demonstrate by electron microscopy and immunohistochemistry that drug treatment induces mitochondrial damage that was accompanied by the release of cytochrome c into the cytosol together with the translocation of apoptosis inducing factor (AIF) into the nucleus. This finding in murine glioma cells differs from the mechanism of flavopiridolinduced cell death reported by us for human glioma cells (Alonso et al., Mol Cancer Ther 2003; 2:139) where drug treatment induced a caspase- and cytochrome c-independent pathway in the absence of detectable damage to mitochondria. In apoptotic human glioma cells only translocation of AIF into the nucleus occurred. Thus, the same drug kills different types of glioma cells by different mitochondrial-dependent pathways.     

Hepatic inflammatory mediators contribute to intestinal damage in necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a common and devastating gastrointestinal disease of premature infants. Along with pathological effects in the ileum, severe NEC is often accompanied by multisystem organ failure, including liver failure. The aim of this study was to determine the changes in hepatic cytokines and inflammatory mediators in experimental NEC. The well-established neonatal rat model of NEC was used in this study, and changes in liver morphology, numbers of Kupffer cells (KC), gene expression, and histological localization of IL-18, TNF-alpha, and inducible nitric oxide synthase were evaluated. Intestinal luminal TNF-alpha levels were also measured. Production of hepatic IL-18 and TNF-alpha and numbers of KC were increased in rats with NEC and correlated with the progression of intestinal damage during NEC development. Furthermore, increased levels of TNF-alpha in the intestinal lumen of rats with NEC was significantly decreased when KC were inhibited with gadolinium chloride. These results suggest an important role of the liver and the gut-liver axis in NEC pathogenesis.