The research program of the Molecular Cytogenetics Section of the Laboratory of Experimental Carcinogenesis is aimed at the identification and characterization of genomic modifications associated with initiation and progression of the neoplastic phenotype. Using a combined approach based on integrated use of molecular cytogenetics and molecular biology, our group identified and examined a number of recurrent chromosome alterations that led to the discovery of several new cancer-relevant genes, and to the detection of alterations in a number of known ones. Analysis of these alterations may provide markers useful for the prognosis and diagnosis of cancer and potential targets for therapy.In the past year a recurrent region of deletion on the long arm of chromosome 16q23 identified in human hepatocellular carcinoma (HCC) by comparative genomic hybridization was characterized molecularly. The 16q32 region span the second most active fragile site (FS) FRA16D and contains WWOX (WW-domain containing oxidoreductase), a candidate tumor suppressor gene. WWOX mRNA and protein expression were absent or reduced in over 60% of HCC cell lines, showing that WWOX gene is frequently altered in liver cancer and raising the possibility that this gene is implicated in hepatocarcinogenesis. The organization of DNA and its replication at FSs renders them prone to breakage and recombination as well as preferential targets for mutagenic carcinogens and integration of oncogenic viruses. For many years, attempts to link FSs and cancer generated mostly circumstantial evidence, but recently ample evidence has accumulated that DNA breakage at FSs is associated with chromosomal translocations, amplification of proto-oncogenes, deletion of tumor suppressor genes, and integration of oncogenic viruses. Alterations of WWOX gene at FRA16D provide additional support for relating damage at FSs to alterations in cancer-related genes. Several years ago, from a critical region of deletion on the short arm of chromosome 8 suspected to harbor one or more tumor suppressor genes, we isolated the DLC-1 gene, which encodes a regulator of the Rho family of small GTPases. The Rho proteins control actin cytoskeleton organization, membrane trafficking, gene expression, and are involved in cell proliferation, malignant transformation, and metastasis. In the intervening years we showed that restoration of DLC-1 expression in cells derived from metastatic breast adenocarcinomas lacking endogenous gene expression caused significant growth inhibition and prevented the development of tumors in athymic nude mice. In the past year, evidence that DLC-1 also acts as a tumor suppressor gene in liver and lung cancer was provided. Transfection experiments demonstrated that the restoration of DLC-1 expression in HCC cells resulted in caspase 3 mediated apoptosis, inhibition of cell growth and invasiveness in vitro as well as a reduction of the ability of the cells to form tumors in athymic nude mice. In HCC exonic missense mutations and intronic insertions/deletions of DLC-1 were found only in subset of the tumors. Several single nucleotide polymorphisms (SNP) were identified that could be used for determining whether DLC-1 is linked to liver cancer susceptibility loci. Other investigators, using high-throughput SNP genotyping and gene expression profiling, identified DLC-1 as a strong candidate for a breast cancer susceptibility gene. In non-small cell lung carcinoma (NSCLC), we showed that DLC-1 is frequently inactivated by aberrant DNA methylation and restoration of its expression results in suppression of in vitro and in vivo tumor cell growth. Stable transfer of DLC-1 abolished the tumorigenicity of cultured cells derived from NSCLC in nude mice. Hypermethylation of the DLC-1 promoter region has been detected in a number of cases in various types of cancer, and the DLC-1 methylation profile may serve as a useful marker for early detection, prediction of cancer risk and prognosis of the disease. Also, it may represent a potential target for pharmacological re-expression as a novel method for lung cancer treatment. The mouse DLC-1 gene was also isolated, and the exon/intron organization was characterized. An intragenic polymorphic microsatellite marker was identified that was useful for linkage mapping and loss of heterozygosity analysis. To provide an animal model system for investigating the biological functions of DLC-1 in vivo, we successfully used homologous recombination in embryonic stem cells to generate mice with a disrupted DLC-1 gene. The generation of the DLC-1-knock out mice, and initial characterization of this model revealed that the DLC-1 protein is also essential for normal embryonic development. The mice heterozygous for the mutated DLC-1 allele will be useful for analysis of the role of DLC-1 in tumorigenesis.In addition to DLC-1, alterations of other cancer-related genes were detected by examining the gene expression profile in human NSCLC cells exposed to the demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC). Down-regulation of tyrosine protein kinase ABL2 (ABL2) gene and up-regulation of hint/protein kinase C inhibitor 1 (Hint/PKCI-1), DVL1, TIMP-1 and TRP-1 genes were recurrent in NSCLC cell lines. Transfer of Hint/PKCI-1 gene in cell lines exhibiting 5-aza-dC-induced up-regulation of the gene resulted in a significant in vitro growth inhibition and significantly reduced in vivo tumorigenicity of NSCLC cells. Cancer is considered a disorder of aging organisms. Various types of damage and mutations may accumulate during the lifetime rendering the cells more susceptible to developing cancer. Localization of double-strand breaks strand (DBS) by immunostaining with g-H2AX antibody and fluorescence in situ hybridization (FISH) with a telomeric probe on human and mouse cells undergoing senescence generated evidence on the genomic distribution of the DBS repair factors, the role of accumulation and repair of DNA damage during the process of aging and provided a useful marker for discerning human and animal aging cells. We previously localized a human mitochondrial topoisomerase1 gene and recent FISH localization of the murine homologue showed that genes for mitochondrial DNA topoisomerases (type IB) exist only in vertebrates. GC-binding factor 2 (GCF), a transcriptional repressor overexpressed in cancer cells that plays a role in regulating the expression of EGFR, was mapped at a region a frequent genomic alterations in several disorders and might be important in pathogenesis of malignant lymphomas.
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