Charles E. Rogler - US grants

The long-term objective is to determine how persistent WHV infection of the liver leads to hepatocellular carcinoma (HCC) and study factors regulating persistent infections in woodchucks and in hepatocyte cultures. The specific aims will be: (1) To determine the function of nuclear WHV DNA by isolating nucleoprotein (NP) complexes containing MHV DNA from the nuclei of chronically infected hepatocytes and: (a) Determine the structure of the complexes and the molecular forms of viral DNA in them. (b) Determine if replicative intermediates can be identified in the DNA from NP complexes, paying special attention to complexes containing CCC forms of the virus. If such molecules are identified, determine their origin and mechanism of replication. (c) Determine the endogenous DNA and RNA polymerase activities of the NP complexes and identify the transcriptional templete. (2) Study the mechanism of integration of WHV DNA by cloning integrations and homologous cell sequences and sequencing the viral-cell junctions and search for "novel" forms of WHV using molecular cloning. (3) Attempt to establish persistent infections in woodchucks by perturbing the liver during acute infection in ways that will stimulate regeneration and/or suppress the immune response. (4) Utilize bio-matrix system to establish tissue cultures of chronically infected woodchuck hepatocytes. WHV is the only animal model which mimics the human disease with regard to the progression from chronic active hepatitis to HCC. The presence of viral integrations in human and woodchuck tumors has implicated their role in HCC. An understanding of how free WHV DNA is maintained in the nucleus, how it integrates in cellular DNA and how experimental treatments affect it may enable treatments for the prevention or cure of chronic infections to be devised. The methods used will include (1) isolation of NP complexes containing WHV DNA from hepatocyte nuclei by testing and modifying the selective nuclear extraction method for isolating SV40, polyoma and adenovirus NP complexes. Molecular cloning of viral integrations will be carried out by constructing libraries of recombinant DNA in lambda phage vectors and screening libraries with cloned WHV probe. Experimentally infected animals will be treated by partial hepatectomy and immunosuppresants in attempts to establish chronic infections.

The overall emphasis will be to investigate the role of HBV and WHV in hepatic oncogenesis. The significance and specificity of chromosome aberrations and Insulin-like growth factor II activation associated with HCC in hepadna virus carriers will be investigated. Cell culture assays and "in vivo" hepatocarcinogenesis studies in woodchucks will test mechanisms of HBV and WHV induced carcinogenesis. Specific studies to be conducted include: 1. Analysis of loss or amplification of alleles from specific chromosomes in primary human HCC. In this study we will determine (a) the extent and frequency of deletions in chromosome llp in HCCs using the loss of restriction fragment length polymorphisms (RFLPs) as an assay for loss of alleles, (b) the frequency of loss or amplification of alleles in chromosomes other than 11 and (c) the copy number, hemizygous, homozygous (reduplicated) or amplified, of the polymorphic alleles remaining after loss of their homologue and the relationship (if any) of HBV integration to loss of alleles on chromosomes containing these integrations. 2. Determine the frequency, specificity and mechanisms of transcriptional activation of IGF-II in primary woodchuck HCCs. The abundance and sizes aof IGFD-II RNAs intissues at stages of hepatic oncogenesis, inwoodchucks will be studied along with the specificity of IGF-II activation, and the mechanism of IGF-II transcription by sequencing cDNA clones of IGF-II mRNAs from woodchuck HCCs. 3. Analyze the activity of integrated HBV and WHY DNA using biological assays for tumorigenesis and transcription. Cell lines contain HBV and WHV constructs will be used to address the following questions (a) can HBV and WHV function in a "Hit and Run" tumorigenesis mechanism and do they affect the mutation rate of cellular genes and (b) how do HBV integrations containing enhancer elements affect transcription of cellular genes flanking HBV. 4. Experimental manipulation of HCC in woodchucks and isolation of woodchuck hepatocyte cell lines. Partial hepatectomy and CC14 and phenobarbitol treatments will be used to manipulate HCC in woodchucks. Preneoplastic nodules and HCCs will be analyzed for WHV integration and IGF-II activation to study events proceeding HCC. Woodchuck hepatocyte cell cultures will be initiated using newly developed methods of Kitagawa to study WHV replication and progression to HCC.

OVERVIEW: The overall goals of the research program have been to understand the molecular-genetic basis for hepatocarcinogenesis in hepadnavirus carrier. Hepadnaviral DNA integrations are agents of genetic change which can promote the process of hepatocarcinogenesis. Our recent data have established several features of the integration process. First, single and multiple integrations occur continuously through successive cell generations. Second, the integration frequency can vary dramatically in subclones of the same cell line. Third, integrations can be amplified and can also be lost from successive generations of cells. Fourth, the loss of an integration can be accompanied by the loss of cellular DNA associated with the integration. These results provide the basis for integrations to function as activators of protooncogenes, as well as agents of the loss of tumor suppressor genes during hepatocarcinogenesis. The specific aims of the current proposal will study the mechanisms responsible for the processes described above. SPECIFIC AIM 1: Studies on the molecular mechanisms associated with the acquisition and loss of DHBV integrations in LMH-D2 chicken hepatoma cells. SPECIFIC AIM 2: Studies on the effects of DHBV mutations which alter the structure of viral DNA's, or the amplification of nuclear CCC DHBV DNA's, on integrations in clonal populations of cells. SPECIFIC AIM 3: Studies on the effects of genotoxic agents, likely to be encountered during persistent infection, on the net accumulation or loss of DHBV integrations in LMH cells. SPECIFIC AIM 4: Studies on the acquisition, loss, or rearrangement of DHBV integrations during the growth of hepatomas in immunocompromized hosts. SPECIFIC AIM 5: Reconstitution of nu/nuupa mouse liver with woodchuck hepatocytes. Recent advances in the use of transgenic mice and hepatocyte transplantation now make it feasible for us to attempt to produce an animal model in which we will be able to clonally amplify and isolate woodchuck hepatocytes from all stages of carcinogenic progression.

The overall goal of the proposed studies is to directly access the role of IGF-II in hepatocarcinogenesis. IGF-II is primarily expressed in fetal tissues (1) and a gene knockout experiment has demonstrated that its expression is necessary for normal fetal growth (2). In the proposed studies, two transgenic mouse models, immortalized hepatocyte cell lines, and an IGF-II knockout mouse will be utilized. Transgenic mouse model utilizes the Major Urinary protein (MUP) promoter (3) to drive expression of a human IGF-II cDNA in the liver of adolescent and adult mice. The second transgenic mouse model utilizes SV40 Ag driven by the MUP promoter to induce malignant progression in the liver (4,5). The Specific Aims of this proposal are: 1. To utilize recently developed IGF-II transgenic mouse lines study the effect of IGF-II expression in adolescent and adult mice and determine: (A) The time course of transgene expression, the hormone inducibility of transgene expression and characterize IGF-II induced liver pathology; (B) Whether the oncogenicity of chemical hepatocarcinogens and/or tumor promoters is enhanced or repressed by the presence of IGF-II in adult mouse liver; (C) Whether homozygous knockout of the IGF-II gene can protect the liver from hepatocarcinogenesis; (D) Whether naturally occurring maternal imprinting of the IGF-II gene serves a cancer suppressor or protective function. 2. To study the role of IGF-II in malignant progression of immortalized hepatocytes by determining whether: (A) IGF-II transforms or otherwise alters the growth properties of immortalized hepatocytes and (B) whether the ability of IGF-II to alter hepatocellular phenotype varies with the stage of hepatocytic carcinogenesis in SV40 T Ag transgenic mice (i.e. original hepatocytes, small cell foci, neoplastic nodules). 3. To study the effect of IGF-II expression on the growth requirements and properties of primary transgenic hepatocytes. We will determine whether IGF-II expression in transgenic hepatocytes substitutes for the Insulin or EGF requirements of primary cultured hepatocytes, and whether the growth properties or phenotype of the cells is altered in vitro. 4. If a causative role for IGF-II in hepatocarcinogenesis is established by the above experiments, then we will study the structure and function of IGF-II polypeptides which accumulate in HCC. Studies will include: (A) Characterization of the structure of the 15 Kd IGF-II polypeptide in HCCs; (B) Search for and characterization of IGF-II-receptor complexes and (C) Initial studies on the signal transduction capabilities of IGF-II-receptor complexes.

molecular biology; liver disorder; molecular genetics; biomedical facility; gene targeting; genetic library; subtraction hybridization; polymerase chain reaction; nucleic acid sequence;

DESCRIPTION (provided by applicant): A multidisciplinary program is presented by The Albert Einstein College of Medicine (AECOM) that will make comprehensive genomics, bioinformatics and biostatistical resources available to an exceptionally strong research base of over 25 NIDDK-funded investigators, focused on three major health problems: diabetes mellitus and obesity, chronic liver injury and chronic progression of kidney diseases. AECOM has established a state-of-the-art microarray core facility since 1998. The Facility is responsible for acquisition and management of cDNA clone-sets (25k human and 33k mouse clones available in 2001) and for production of affordable, high-quality cDNA microarrays that include currently 17k human and 9k mouse arrays. The program for the new Biotechnology Center proposed in this application will build on the strength of this existing resource and provide NIDDK investigators with genomics, bioinformatics and biostatistics services and resources that are essential for a comprehensive functional genomics program and are currently not available. The Center proposes to provide: - comprehensive on-site consultational and educational services for microarray users - cost-efficient, quality-controlled microarray hybridization and microarray scanning services - quantitative real-time PCR services for quality control and cross validation of microarray services, and for cost-efficient second-step analysis of selected genes in large numbers of samples - support for implementation of laser capture microscopy in conjunction with gene expression profiling (microarray and/or real-time PCR) for micro-tissue and cell-type-specific gene expression analysis in situ - comprehensive microarray database development and management including web-based public access - integration of gene expression database with state-of-the-art genomic/bioinformatic viewing tools for filtering annotated genomic, proteomic and transcriptomic information - implementation of biostatistical analyses for data normalization, replicate and/or ordered microarray data sets, unsupervised data mining (clustering), and supervised data analysis for classification and class prediction problems. The Center will be directed by a Principal Investigator/Director (Dr. Erwin Bottinger), and governed and advised by an Internal Advisory Committee. A Scientific Advisory Panel of expert consultants will assist in review, development and implementation of new functional genomics and bioinformatics methodologies and services.

In the past year, the emphasis has advanced beyond the mechanics of doing eDNA microarrays toward quantitative real time PCR and a strong bioinformatics approach for analysis of microarray data. In addition, we were able to purchase an ABI Prism 7000 Sequence Detection System that can conduct real time PCR analysis. This instrument has found a broad base of use in the Liver Center and has greatly increased core usage. The quantitative real time PCR approach is used to confirm eDNA microarray data and as a transcription discovery tool. We have provided a full spectrum of support services for this instrument as outlined in the Progress Report. We have also expanded our bioinformatics services and have brought on-line second and third generation programs for processing microarray data. The laboratories of Drs. Charles and Leslie Rogler hired Dr. Raquel Norel to head up their bioinformatics efforts.