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 program combines molecular cytogenetics and molecular biology to study chromosome and gene alterations in solid tumors and hematological malignancies as well as in transformed cells. In the past year progress has been made in molecular genetics and cytogenetics of liver cancer, breast, and cervical carcinoma, in Burkitt's lymphoma (BL), as well as in liver tumors in transgenic mice. This resulted in 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. Deletion of the short arm of chromosome 8 is one of the most common alterations in liver tumors as well as in several other cancers. DLC-1 is a gene located at a critical region of deletions on chromosome 8p. Several lines of evidence indicate that DLC-1 acts as a tumor suppressor gene in more than one form of cancer. The mouse DLC-1 gene was isolated, and the exon/intron organization was characterized. An intragenic polymorphic microsatellite marker was identified that was useful for linkage mapping and LOH analysis. Fragments of the gene were cloned into a targeting vector that was successfully used to disrupt the gene by homologous recombination in embryonic stem (ES) cells to generate DLC-1 deficient mice. From the 11q13 region commonly amplified in several cancers, a fragment having homology with EMS1 oncogene was isolated and subsequently found to be amplified in some primary human hepatocellular carcinoma (HCC) and overexpressed in HCC cell lines in the absence of gene amplification. The EMS1 gene encodes cortactin, a cortical actin-associated protein that is a substrate for Src kinase and is involved in cytoskeleton organization. Alterations of the EMS1 gene that lead to overexpression of cortactin may be associated with tumor development of HCC. EMS1 amplification and overexpression is indicative of an unfavorable prognosis in several cancers and may have similar prognostic implications in liver cancer. Among the newly isolated genes is mouse GTF2IRD1 that was identified near the breakpoint of recurrent balanced chromosome translocation t (5; 6) in HCC that developed in MYC transgenic mice. GTF2IRD1 encodes a transcription factor that interacts with the retinoblastoma protein; and the 5 end of the gene is deleted and its expression greatly reduced in mice homozygous for the translocation. The balanced translocation t (5; 6) occurs at the earliest stage in tumor development and may play a critical role in the initiation of neoplastic transformation. In addition, the mouse GTF2IRD1 gene is one of the genes hemizygously deleted in Williams-Beuren syndrome. Since the transgenic mice line represent the first """"""""knock out"""""""" of one of the genes in the Williams-Beuren syndrome deletion, they may aid in understanding the pathogenesis of this disorder. Recurrent gain of chromosome 19 and double minute chromosomes (DM) derived from chromosome 19 has been identified in HCC developed in MYC transgenic mice. DM are cytological manifestations of high level DNA amplification. At the site of DNA amplification the MYCS and MXI1 genes are located, both known to interfere with MYC gene activity, and also the mouse RelA gene, which promote cell survival by inhibiting apoptosis, allowing MYC to drive proliferation of transformed cells. Further characterization of the PCR probes from DM showed that one of these genes, MYCS, a member of the intronless MYC was incorrectly assigned on chromosome 19. We provided the correct assignment MYCS on chromosome X. In the past, we localized the sites of breakage within the most active fragile site in human genome, FRA3B encompassing FHIT, a tumor suppressor gene implicated in a large number of cancers. A significantly higher frequency of loss of heterozygosity at the FHIT locus was observed for breast cancers with deleterious mutations of BRCA2. We examined a series of breast tumors with BRCA1 mutations and found that loss of FHIT expression was significantly more frequent in the BRCA1 cancers compared to sporadic breast tumors, suggesting that the BRCA1 pathway is also important in protecting the FRA3B/FHIT locus from damage. To further shed light on the association between repair and the FRA3B/FHIT locus, the incidence of apidicholin (apc)-induced gaps and breaks was examined in tumor cell lines and lymphoblastoid cell lines completely lacking mismatch repair as well as mouse repair deficient cell lines. The incidence of chromosomal lesions induced by apc was significantly greater in all repair-deficient cells than in control cells implicating proteins involved in mismatch repair and double strand break repair in maintenance of the integrity of fragile sites. Characteristic for BL are reciprocal translocations involving the MYC locus at fragile site 8C to any of the three immunoglobulin genes, resulting in MYC gene deregulation. In a comprehensive combined molecular cytogenetic study, we demonstrated two more complex t(8;14;18) and (7;8;14) translocations in addition to the reciprocal translocation t(8;14).These novel rearrangements in BL resulted in transposition of MYC sequences in a new genomic configuration. We also also observed partial duplication of the long arm of chromosome 1q23, the second most common alteration in BL and known to be associated with aggressive tumors and poor prognosis. While translocations involving the MYC gene are important in the initial stages of BL, duplication of 1q may be critical during progression and the acquisition of an invasive tumor phenotype. This region might harbor gene(s) associated with tumor cell invasiveness and serve as a prognostic marker in BL. The specificity of viral integration is fundamental in determining the biological significance of this phenomenon to various pathological conditions including cancer. Human papilloma viruses (HPV) are closely associated with cervical cancer. HPV16 integration in the genome of a rapidly progressive, lethal cervical cancer in a 39-year-old woman was localized on chromosome 14. HPV integration resulted in the disruption of the TNFAIP2 cytokine/retinoic acid-inducible gene, thus contributing to the rapid progression of cervical cancer. Acquisition of drug resistance is a major problem in cancer therapy. In a human colon carcinoma cell line rendered resistant to ecteinascidine a critical cytogenetic abnormality detected by SKY that led to the demonstration of novel mechanism of action for the drug. Thus, for the first time a specific cytogenetic abnormality was directly linked to the acquisition of drug resistance. The elucidation of the requirements for conversion of normal cells into neoplastic cells is critical in cancer research. Our cytogenetic analysis of neoplastically transformed human by the introduction of mutant alleles of a small number of genes, demonstrated that malignant transformation of normal human mammary cells required nonrandom chromosome changes involving the MYC gene, which is commonly altered in spontaneous breast cancer. In contrast the acquisition of tumorigenicity of kidney epithelial cells can occur in the absence of widespread genetic instability and aneuploidy. Introduction of a limited set of genes to induce a specific phenotype could be applied to other disorders and the resulting phenotype may be manifested by specific chromosome changes. The advances could lead to an improved early detection. A number of new cancer-related human, mouse, and rat genes have been mapped. These localizations will permit linkage analysis and facilitate both the identification of loci associated with genetic diseases of unknown etiology and examination of their involvement in cancer cell chromosome rearrangements. Among the mouse genes are: Sca-1, an antigen commonly used for purification of murine pluripotent hematopoietic cells, a member of the Ly-6 family and pancreatic phospholipase A2 which plays an important role in pancreatic and extrapancreatic tissue and UGRP, uteroglobin related protein which is down-regulated in lung and might be involved in inflammation. We also mapped the rat RMT-1 gene whose expression is correlated with mammary carcinogenesis. Among the human genes localized are: mitochondrial topoisomerase 1 gene; the claudin 18 gene which plays an important role in the structure and function of tight junctions in lung and stomach; and UGRP1, encoding uteroglobin protein 1, and is associated with an increased risk of asthma.
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