Cyclin-D1/CDK4 Complex in Hepatocyte Profliferation

Statement of Work for "The cyclin D1/cdk4 complex in hepatocyte proliferation." 1. Sample processing and data collection (Years 1,2) Table Isotope Resolved Metabolomics (SIRM) analysis of cells and media. Hepatocytes and hepatocellular carcinoma cell lines grown for 24 h in the presence of either [U-13C]-glucose or [U-13C,15N]-glutamine shipped from U. Minnesota to The Center for Environmental Systems Biochemistry (CESB) at UKY, Lexington, will be extracted according to a standard protocol ([1, 2]) for separating protein, polar and non-polar metabolites. The separate extracts will be processed for NMR, GC-MS and FT-ICR-MS according to our standard protocols [2-8]. In parallel, aliquots of culture media at different times to harvest (24 h) will be processed for NMR and GC-MS. NMR spectra will be recorded at 14.1 T under standard protocols [5, 8-11], which include 1D proton and 1D 1H{13C}-HSQC for polar cell extracts, and 1D proton for media extracts. Where necessary 2D TOCSY and HSQC spectra will be recorded on specific extracts as needed. SIRM analysis of mouse tissue and blood Liver and blood from groups of mice subjected to stable isotope tracer protocols developed at CREAM [12-14] will be obtained from Dr. Albrecht at U. Minnesota will be processed as per standardized protocols into polar and non-polar fractions at UofL. The samples will be prepared for GC-MS, FT-MS and NMR, and data acquired according to established standard protocols [1, 2] [5, 8-11]. 2. Data analysis (years 1,2,3) Media, blood metabolites For the different cell lines and mice with two tracers, there will be 156 media and blood analytical samples NMR: 1D 1H NMR are acquired and processed. NMR spectra require transformation, referencing, peak picking and integration, with normalization to DSS for absolute quantification, using either VNMRJ or MNova 8 (Mestrelab Research, Spain). Each molecule comprises several isotopomers, though for media we concentrate on glucose, glutamine, lactate, alanine (using the essential amino acids threonine and valine as a control). GS-MS requires derivatization of the samples using MTBSTFA [21]. The metabolites are identified and quantified by peak area with respect to authentic standards. The uptake rate of nutrients per unit tissue weight, production of labeled products (e.g. lactate, alanine) the fraction of glucose consumed that is converted to lactate, the enrichment factor of lactate are determined [5, 15-17]. Cells and tissue metabolites The extracted metabolites are processed as described for blood above. For tissue extracts, also nucleotide ribose, UDP-hexoses, PCho are determined. Where necessary, additional 2D NMR experiments may be carried out to quantify positional isotopomers in either lipids or polar entities. 2D experiments typically take ca. 8-16 h spectrometer time, and expert level data analysis. 2D analyses provide positional enrichments in nucleotide ribose (pentose phosphate pathways), lactate and alanine (glycolysis) and reporters of Krebs cycle activity and the malate-aspartate shuttle (viz. aspartate, Glu, Gln, glutathione), as well as pyrimidine nucleobases [5, 11, 16, 18, 19]. Metabolites are identified using in-house databases [5, 9, 11, 20]. FT- MS is carried out by direct infusion using the Advion autosampler system. The raw data are acquired at a resolution of >400,000, and are reduced to peaks that are assigned using accurate mass using in-house software, PREMISE [3, 7, 8, 22]. Isotopologues are quantified according to peak area, and normalized as mol fractions of each isotopologue. Corrections for natural abundance are made using our stripping software [3, 22, 23]. The number of polar metabolites typically analyzed is >200 compounds, which may be as many as 1000 quantifications including the isoptopomers and isotopologue distributions. The non-polar extracts analyzed by FT- MS typically yield hundreds of lipid species form many lipid classes. With the isotopologue distributions, this amounts to >2000 quantified and identified species per sample. These data are all acquired simultaneously. For initial data interpretations, the focus will be on the central glucose and glutamine metabolism, particularly carbon flow from glucose and glutamine through proline to probe the connection between Cyclin D1 and POX expression and activity [24]. As Cyclin D1 also affects nucleotide and lipid metabolism, the nucleotide synthesis pathways will be analyzed. These are the primary pentose phosphate pathways (ribose biosynthesis) and the nucleobase syntheses via aspartate (for pyrimidines) and the serine/glycine/folate pathways for purines[7, 11, 22, 25]. Lipid analysis will be analyzed by the FT-ICR-MS derived isotopologue distributions, and bile acids by NMR according to [26-28].