Molecular Cytogenetics of Solid Tumors

实体瘤的分子细胞遗传学

• 批准号: 6559115
• 负责人: NICOLAE POPESCU
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6559115
• 关键词: animal genetic material tag; cancer risk; chemical structure function; chromosome aberrations; cytogenetics; family genetics; fluorescent in situ hybridization; gene targeting; genetic mapping; genetic markers; genetic screening; genetic susceptibility; genetically modified animals; human genetic material tag; human tissue; karyotype; laboratory mouse; molecular oncology; natural gene amplification; neoplasm /cancer; neoplasm /cancer genetics; neoplastic process; nucleic acid structure; oncoproteins; patient oriented research

The major objective of our research is to identify recurrent chromosomal alterations and to isolate new genes that are relevant to neoplastic development and may serve as targets for therapy. Our successful utilization of the advanced molecular cytogenetic procedures for multicolor spectral karyotyping (SKY), comparative genomic hybridization (CGH) and high resolution fluorescence in situ hybridization (FISH) led to the identification of novel genetic changes and isolation of genes in solid tumors and hematological malignancies. Continuous progress has been made in molecular genetics and cytogenetics of liver cancer through the identification of new recurrent genomic alterations and isolation of new genes relevant to the initiation and progression of neoplasia and possibly useful markers for prognosis and diagnosis of the disease. In both human and mouse hepatocellular carcinoma (HCC), at several sites of recurrent genomic changes, we have identified new genes and detected alterations of known cancer-related genes. Several regions of DNA copy number gain were identified by comparative genomic hybridization (CGH) and two regions of high level DNA amplifications on chromosomes 1p32 and 11q13 were detected by representational difference analysis.Ten amplification fragments, showing a 5- to 50-fold increase in DNA copy-number were cloned and used to screen a human lambda genomic DNA library to obtain probes for FISH mapping of the amplified fragments. Several clones were localized to chromosome 1p32 and 11q13 at regions that are frequently amplified in several types of cancer including HCC. The sequence of one amplification product matched that of the EMS1/cortactin gene, which is located in the 11q13 amplicon, and encodes a protein that is a substrate of SRC kinase. Two amplifed fragments were isolated and mapped to chromosome 1p32, a region showing copy-number gains in HCC cell lines. Both fragments were present in partially and completely sequenced chromosome 1 BAC clones. The sequence of one fragment is located about 195 kb from r1f, a zinc finger protein gene that is co-amplified and fused with L-MYC in some lung cancer cell lines and it lies in the middle of a 23-kb interval between two novel genes. One gene encodes a new member of the zinc finger protein family, and the other gene encodes a protein of unknown function that contains a zinc binding domain homologous to those identified in ARF GAP (ADP-ribosylation factor GTPase-activating protein) family proteins. The ARF GAP-like gene appeared to be ubiquitously expressed, while the zinc finger protein gene transcript was undetectable in normal human tissues but was over-expressed in HCC cell lines and is a strong candidate proto-oncogene. The cytogenetic changes in HCC from transgenic mice closely evoked those that occur during the progression of spontaneously human carcinomas. Gain of chromosome 19 is a recurrent alteration in both mouse HCC primary tumors and derived cell lines, as well as in preneoplastic liver lesions induced with diethylnitrosamine. Chromosome 19 carries susceptibility genes for liver tumors and possibly a proto-oncogene involved in hepatocarcinogenesis. In addition to the gain of chromosome 19 in the majority of tumor lines, in one tumor line we identified double minute chromosomes (DM) derived from chromosome 19. DM, small pairs of acentric chromatin bodies and homogeneously stained regions (HSR) are cytological manifestations of DNA amplification. A probe generated by PCR from the microdissected DM was localized by FISH on normal chromosome 19 at two sites separated by seven or eight medium-size G-bands. At one site of DNA amplification (19131.1-131.3) the ms-MYC gene and mouse RelA gene recently characterized in the Laboratory of Experimental Carcinogenesis were localized, while the second site, 19 D1-D2 harbors the MXI1 gene. Both ms-MYC and MXI1 are known to interfere with the myc gene, while the Re1A gene may promote cell survival by inhibiting apoptosis, and allow c-MYC to drive proliferation of transformed cells. We are using gene targeting to generate DLC-1 knockout mice to establish an animal model for studying the biological functions of DLC-1, a candidate tumor suppressor gene that we have isolated. The mouse DLC-1 gene was isolated and used to construct a targeting vector that was successfully employed to disrupt the gene by homologous recombination in embryonic stem cells. Chimeric mice derived from ES cells carrying the disrupted gene have shown germline transmission of the mutant DLC-1 allele. The DLC-1 (+/-) mice will be bred to produce mice homozygous for the targeted gene. The effect of DLC-1 deficiency on viability, development, and reproduction will be analyzed. The susceptibility of the DLC-1 (+/-) and (-/-) mice to hepatocarcinogenesis can be studied using the liver cancer models developed in LEC. A new constitutive translocation involving a common fragile site was characterized by FISH and molecular markers. In a family in which multifocal clear cell renal carcinoma (RCC) segregates with a balanced constitutional chromosome translocation, t(2;3) (q33;q21), possibly with translocation positions similar to those of the renal cell cancer-associated t(2;3) (q35;q21) reported in another family. YAC and BAC contigs encompassing the 2q and 3q breakpoints were constructed and BACs crossing the breakpoints were partially sequenced. All known regional markers, genes and ESTs were mapped relative to the contigs, as well as to the breakpoint sequences. Two single ESTs map within the 2q breakpoint BAC, while the repeat rich 3q breakpoint region is gene poor. By FISH, we determined that the 3q break is in 3q13, possibly near the border with 3q21 and 2q breakpoint is closely telomeric to the 2q31 FRA2G site. Characterization of full length cDNAs for the ESTs near the 2q break will determine if a gene (s) is altered by this familial RCC-associated chromosome translocation. Deletion of the short arm of chromosome 5 was the sole structural alteration identified in a case of testicular lymphoma and it may harbor a tumor suppressor gene implicated in this malignancy. In the past year two studies with profound implications on the mechanisms of malignant transformation and drug action were completed. In a human colon carcinoma cell line rendered resistant to ecteinascidine, a potent anticancer agent, we identified a cytogenetic abnormality that affects the locus of the gene implicated in the hereditary disease xeroderma pigmentosum. As a result, it has been discovered that the killing mechanism of Et743 is mediated by nucleotide excision repair. We also demonstrated that the malignant transformation of normal human mammary cells required nonrandom chromosome changes involving the MYC gene, which is commonly altered in spontaneous breast cancer. Thus, it was demonstrated for the first time how a limited, definable number of genes and nonrandom chromosome changes results in the generation of a tumorigenic phenotype. Several genes isolated in our laboratory and by others were mapped by FISH and include the murine mitochondrial aldehyde dehydrogenase gene involved in ethanol metabolism, sca-1, an antigen commonly used for purification of murine pluripotent hematopoietic cells; and a member of the Ly-6 family and pancreatic phospholipase A2 that plays an important role in pancreatic and extrapancreatic tissue. In human 13 UDP glucunosylransferase genes encoded at the UGT1 complex were mapped on chromosome 2.

我们研究的主要目的是识别复发性染色体改变，分离与肿瘤发展相关的新基因，并可能作为治疗靶点。我们成功地利用先进的分子细胞遗传学方法进行多色光谱核型（SKY），比较基因组杂交（CGH）和高分辨率荧光原位杂交（FISH），从而鉴定出新的遗传变化并分离出实体肿瘤和血液系统恶性肿瘤的基因。