This study was aimed to characterize the mitochondrial and extra-mitochondrial oxygen consuming reactions in human CD34؉ hematopoietic stem cells. Cell samples were collected by apheresis following pre-conditioning by granulocyte colony-stimulating factor and isolated by anti-CD34 positive immunoselection. Polarographic analysis of the CN-sensitive endogenous cell respiration revealed a low mitochondrial oxygen consumption rate. Differential absorbance spectrometry on whole cell lysate and two-dimensional blue native-PAGE analysis of mitoplast proteins confirmed a low amount of mitochondrial respiratory chain complexes thus qualifying the hematopoietic stem cell as a poor oxidative phosphorylating cell type. Confocal microscopy imaging showed, however, that the intracellular content of mitochondria was not homogeneously distributed in the CD34؉ hematopoietic stem cell sample displaying a clear inverse correlation of their density with the expression of the CD34 commitment marker. About half of the endogenous oxygen consumption was extra-mitochondrial and completely inhibitable by enzymatic scavengers of reactive oxygen species and by diphenylene iodinium. By spectral analysis, flow cytometry, reverse transcriptase-PCR, immunocytochemistry, and immunoprecipitation it was shown that the extra-mitochondrial oxygen consumption was contributed by the NOX2 and NOX4 isoforms of the O 2 . producer plasma membrane NAD(P)H oxidase with low constitutive activity. A model is proposed suggesting for the NAD(P)H oxidase a role of O 2 sensor and/or ROS source serving as redox messengers in the activation of intracellular signaling pathways leading (or contributing) to mitochondriogenesis, cell survival, and differentiation in hematopoietic stem cells.

Hepatitis C virus (HCV) infection induces a state of oxidative stress that is more pronounced than that in many other inflammatory diseases. In this study we used well-characterized cell lines inducibly expressing the entire HCV open-reading frame to investigate the impact of viral protein expression on cell bioenergetics. It was shown that HCV protein expression has a profound effect on cell oxidative metabolism, with specific inhibition of complex I activity, depression of mitochondrial membrane potential and oxidative phosphorylation coupling efficiency, increased production of reactive oxygen and nitrogen species, as well as loss of the Pasteur effect. Importantly, all these effects were causally related to mitochondrial calcium overload, as inhibition of mitochondrial calcium uptake completely reversed the observed bioenergetic alterations. H epatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, affecting more than 170 million people worldwide. 1 There is no protective vaccine, and current therapies show limited efficacy for many patients with chronic hepatitis C. 2 HCV is an enveloped, positive-strand RNA virus. Its genome encodes a polyprotein of more than 3,000 amino acids that is cleaved co-and posttranslationally at the endoplasmic reticulum (ER) by host and viral proteases, yielding 3 structural (core, E 1 , and E2) and 7 nonstructural (p7, NS2 to NS5B) proteins. 3 HCV nonstructural proteins, together with replicating viral RNA and altered cellular membranes, form a membrane-associated replication complex. 3 A minor fraction of the viral proteins has been found to localize to mitochondria, 4-7 where the NS3-4A complex inactivates a newly discovered mitochondrion-dependent innate immunity antiviral signaling pathway. 8,9 A mounting body of evidence points toward alterations of mitochondrial oxidative metabolism by HCV, albeit the mechanism has remained elusive. 10,11 In the present study we used tetracycline-regulated cell lines 12 to analyze the impact of HCV protein expression on mitochondrial bioenergetics. 13 These cell lines allow the regulated expression and correct processing of all viral proteins in a well-defined cellular context. The data reported here extend our previous preliminary observations 14 and provide new mechanistic insight, correlating mitochondrial oxidative stress to the deregulation of ER stress-dependent Ca 2ϩ homeostasis by HCV proteins. Materials and Methods Cell

Hepatitis C virus (HCV) infection induces

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An endogenous molecular clockwork drives various cellular pathways including metabolism and the cell cycle. Its dysregulation is able to prompt pathological phenotypes including cancer. Besides dramatic metabolic alterations, cancer cells display severe changes in the clock phenotype with likely consequences in tumor progression and treatment response. In this study, we use a comprehensive systems-driven approach to investigate the effect of clock disruption on metabolic pathways and its impact on drug response in a cellular model of colon cancer progression. We identified distinctive time-related transcriptomic and metabolic features of a primary tumor and its metastatic counterpart. A mapping of the expression data to a comprehensive genome-scale reconstruction of human metabolism allowed for the in-depth functional characterization of 24 h-oscillating transcripts and pointed to a clock-driven metabolic reprogramming in tumorigenesis. In particular, we identified a set of five clock–regulated glycolysis genes, ALDH3A2, ALDOC, HKDC1, PCK2, and PDHB with differential temporal expression patterns. These findings were validated in organoids and in primary fibroblasts isolated from normal colon and colon adenocarcinoma from the same patient. We further identified a reciprocal connection of HKDC1 to the clock in the primary tumor, which is lost in the metastatic cells. Interestingly, a disruption of the core-clock gene BMAL1 impacts on HKDC1 and leads to a time-dependent rewiring of metabolism, namely an increase in glycolytic activity, as well as changes in treatment response. This work provides novel evidence regarding the complex interplay between the circadian clock and metabolic alterations in carcinogenesis and identifies new connections between both systems with pivotal roles in cancer progression and response to therapy.