Maria Tsokos

We demonstrate a role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy. In particular, transient increased expression of Sirt1 is sufficient to stimulate basal rates of autophagy. In addition, we show that Sirt1(-/-) mouse embryonic fibroblasts do not fully activate autophagy under starved conditions. Reconstitution with wild-type but not a deacetylase-inactive mutant of Sirt1 restores autophagy in these cells. We further demonstrate that Sirt1 can form a molecular complex with several essential components of the autophagy machinery, including autophagy genes (Atg)5, Atg7, and Atg8. In vitro, Sirt1 can, in an NAD-dependent fashion, directly deacetylate these components. The absence of Sirt1 leads to markedly elevated acetylation of proteins known to be required for autophagy in both cultured cells and in embryonic and neonatal tissues. Finally, we show that Sirt1(-/-) mice partially resemble Atg5(-/-) mice, including the accumulation of damaged organelles, disruption of energy homeostasis, and early perinatal mortality. Furthermore, the in utero delivery of the metabolic substrate pyruvate extends the survival of Sirt1(-/-) pups. These results suggest that the Sirt1 deacetylase is an important in vivo regulator of autophagy and provide a link between sirtuin function and the overall cellular response to limited nutrients.

Based on our review of 35 cases and the literature, we found the spectrum of pulmonary neuroendocrine (NE) tumors to be too broad to fit into the traditional three-category classification scheme of typical carcinoid (TC), atypical carcinoid (AC), and small-cell lung carcinoma (SCLC). We found that a spectrum of high- and low-grade tumors exist between TC and SCLC and that in the past many of these tumors have been called AC. We chose to adhere to Arrigoni's definition of AC, as his original criteria characterized a low-grade tumor. For the higher grade non-small-cell tumors (NSCLC), we propose a fourth category of large-cell neuroendocrine carcinoma (LCNEC), which is characterized by: (a) light microscopic NE appearance; (b) cells of large size, polygonal shape, low nuclear-cytoplasmic ratio (N:C), coarse nuclear chromatin, and frequent nucleoli; (c) high mitotic rate [greater than 10/10 high-power fields (HPF)] and frequent necrosis; and (d) NE features by immunohistochemistry (IHC) or electron microscopy (EM). Thus, after deciding that a pulmonary NE tumor is high grade, the major diagnostic issue is separation of LCNEC from SCLC. This distinction is based not only on cell size, but on a variety of morphologic features. We studied 20 TC, six AC, five LCNEC, and four SCLC and characterized the clinical, light microscopic, EM, IHC, and flow cytometric features of each type of tumor. We did not find any advantage to IHC, EM, or flow cytometry over light microscopy in the subclassification or prediction of prognosis; however, these methods were useful in characterizing these four types of pulmonary NE tumors and in demonstrating their NE properties. LCNEC must be distinguished from a fifth category pulmonary NE tumor: NSCLC with NE features in which NE differentiation is not evident by light microscopy and must be demonstrated by EM or IHC. Although the prognosis of LCNEC appears to be intermediate between AC and SCLC, larger numbers of patients will be needed to demonstrate significant differences in survival.

BACKGROUND: We describe severe and unexpected multisystem toxicity that occurred during a study of the antiviral nucleoside analogue fialuridine (1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil, or FIAU) as therapy for chronic hepatitis B virus infection. METHODS: Fifteen patients with chronic hepatitis B were randomly assigned to receive fialuridine at a dose of either 0.10 or 0.25 mg per kilogram of body weight per day for 24 weeks and were monitored every 1 to 2 weeks by means of a physical examination, blood tests, and testing for hepatitis B virus markers. RESULTS: During the 13th week lactic acidosis and liver failure suddenly developed in one patient. The study was terminated on an emergency basis, and all treatment with fialuridine was discontinued. Seven patients were found to have severe hepatotoxicity, with progressive lactic acidosis, worsening jaundice, and deteriorating hepatic synthetic function despite the discontinuation of fialuridine. Three other patients had mild hepatotoxicity. Several patients also had pancreatitis, neuropathy, or myopathy. Of the seven patients with severe hepatotoxicity, five died and two survived after liver transplantation. Histologic analysis of liver tissue revealed marked accumulation of microvesicular and macrovesicular fat, with minimal necrosis of hepatocytes or architectural changes. Electron microscopy showed abnormal mitochondria and the accumulation of fat in hepatocytes. CONCLUSIONS: In patients with chronic hepatitis B, treatment with fialuridine induced a severe toxic reaction characterized by hepatic failure, lactic acidosis, pancreatitis, neuropathy, and myopathy. This toxic reaction was probably caused by widespread mitochondrial damage and may occur infrequently with other nucleoside analogues.

Recent evidence suggests that one mechanism whereby cytotoxic drugs, such as doxorubicin, kill tumors is the induction or up-regulation of Fas ligand (FasL) expression on the tumor cell surface. The ensuing engagement of Fas by FasL on adjacent cells leads to apoptosis. However, despite cytotoxic drug-induced FasL expression, Fas-sensitive tumors frequently resist chemotherapy, suggesting that they may possess a mechanism that prevents or inactivates Fas-FasL interactions. In the present work, we addressed the involvement of the FasL/Fas signaling pathway in doxorubicin-induced apoptosis and the ability of matrix metalloproteinases (MMPs) to proteolytically cleave FasL in tumor cells. Doxorubicin-induced apoptosis was inhibited by expression of soluble Fas or incubation of the tumor cells with MMP-7 but not with MMP-2 or MMP-9. Resistance to doxorubicin was also induced by expression in the tumor cells of constitutively active MMP-7 but not of a catalytically inactive mutant. Conversely, inhibition of MMP-7 expression in tumor cells by transfection of MMP-7 cDNA in antisense orientation resulted in sensitization to doxorubicin. MMP-7 efficiently cleaved recombinant FasL in vitro and reduced cell surface FasL expression. Our observations provide evidence that one mechanism whereby MMP-7 may promote tumor survival and resistance to doxorubicin is by cleaving FasL and reducing its effectiveness in triggering Fas-mediated apoptosis.