Molecular cytogenetic analysis of a human breast metastasis model: identification of phenotype-specific chromosomal rearrangements.

We have previously characterized an experimental system in which the role of candidate metastasis-related genes can be screened and tested. Monoclonal cell lines M4A4 and NM2C5 originated from the MDA-MB-435 breast tumor cell line but have opposite metastatic capabilities in vivo. To investigate gross genetic changes present in this model, we performed a detailed molecular cytogenetic evaluation of the parental cell line, the M4A4 and NM2C5 cell lines, and related clones of metastatic phenotype.

The interrelationship between DRIM gene expression and cytogenetic and phenotypic characteristics in human breast tumor cell lines.

Background: In order to facilitate the identification of genes involved in the metastatic phenotype we have previously developed a pair of cell lines from the human breast carcinoma cell line MDA-MB-435, which have diametrically opposite metastatic potential in athymic mice. Differential display analysis of this model previously identified a novel gene, DRIM (down regulated in metastasis), the decreased expression of which correlated with metastatic capability. DRIM encodes a protein comprising 2785 amino acids with significant homology to a protein in yeast and C.

Functional genomic comparison of lineage-related human bladder cancer cell lines with differing tumorigenic and metastatic potentials by spectral karyotyping, comparative genomic hybridization, and a novel method of positional expression profiling.

We have recently characterized T24T, an invasive and metastatic variant of the T24 human bladder cell line, resulting in a model for bladder cancer progression. To gain additional insight into the repertoire of genetic changes that may be responsible for the invasive and metastatic phenotype, we used spectral karyotyping (SKY) in combination with comparative genomic hybridization (CGH) in these cells. To assess the functional significance of the genetic differences found between the two cell lines, we have developed a positional expression profiling (PEP) method for comparing gene expression data obtained from oligonucleotide microarrays based upon chromosomal position.

Analysis of the regulation of the A33 antigen gene reveals intestine-specific mechanisms of gene expression.

The A33 antigen is a transmembrane protein expressed almost exclusively by intestinal epithelial cells. The level of its expression is robust and uniform throughout the rostrocaudal axis of the human and mouse intestines. In the colon, strong expression is found in the basolateral membranes of both the proliferating cells in the lower regions of the crypts and the differentiating cells in the upper regions of crypts.

Comprehensive karyotyping of the HT-29 colon adenocarcinoma cell line.

The tumor cell line HT-29 was derived from a primary adenocarcinoma of the rectosigmoid colon. HT-29 is hypertriploid (3n+) and has accumulated numerous chromosomal structural aberrations. To identify material involved in chromosome rearrangements, we performed a comprehensive cytogenetic analysis using G-banding, spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH).

p190-A, a human tumor suppressor gene, maps to the chromosomal region 19q13.3 that is reportedly deleted in some gliomas.

To date, two distinct genes coding for Ras GAP-binding phosphoproteins of 190kDa, p190-A and p190-B, have been cloned from mammalian cells. Rat p190-A of 1513 amino acids shares 50% sequence identity with human p190-B of 1499 amino acids. We have previously demonstrated, using rat p190-A cDNA, that full-length p190-A is a tumor suppressor, reversing v-Ha-Ras-induced malignancy of NIH 3T3 cells through both the N-terminal GTPase (residues 1-251) and the C-terminal Rho GAP (residues 1168-1441) domains.

CT10: a new cancer-testis (CT) antigen homologous to CT7 and the MAGE family, identified by representational-difference analysis.

Assays relying on humoral or T-cell-based recognition of tumor antigens to identify potential targets for immunotherapy have led to the discovery of a significant number of immunogenic gene products, including cancer-testis (CT) antigens predominantly expressed in cancer cells and male germ cells. The search for cancer-specific antigens has been extended via the technique of representational-difference analysis and SK-MEL-37, a melanoma cell line expressing a broad range of CT antigens. Using this approach, we have isolated CT antigen genes, genes over-expressed in cancer, e.

Isolation of DICE1: a gene frequently affected by LOH and downregulated in lung carcinomas.

In the development and progression of sporadic tumors multiple tumor suppressor genes are inactivated that may be distinct from predisposing cancer genes. Previously, a tumor suppressor locus on human chromosome 13q14 that is distinct from the retinoblastoma predisposing gene 1 (RB1) has been identified in lung, head and neck, breast, ovarian and prostate tumors. By an approach that combines genomic difference cloning and positional cloning we isolated the cDNA of a novel gene (DICE1) located at 13q14.

Characterization of the GAGE genes that are expressed in various human cancers and in normal testis.

The GAGE-1 gene was identified previously as a gene that codes for an antigenic peptide, YRPRPRRY, which was presented on a human melanoma by HLA-Cw6 molecules and recognized by a clone of CTLs derived from the patient bearing the tumor. By screening a cDNA library from this melanoma, we identified five additional, closely related genes named GAGE-2-6. We report here that further screening of this library led to the identification of two more genes, GAGE-7B and -8.

Isoforms of the human PDZ-73 protein exhibit differential tissue expression.

Patients with renal and colon cancer frequently develop IgG autoantibodies toward the NY-CO-38/PDZ-73 antigen, a protein of 652 amino acids (73 kDa) which contains three copies of the PDZ protein-protein interaction domain. The gene encoding PDZ-73 mapped to chromosome 11p15.4-p15.

Localization of serine kinases, SRPK1 (SFRSK1) and SRPK2 (SFRSK2), specific for the SR family of splicing factors in mouse and human chromosomes.

The serine- and arginine-rich (SR) splicing factors play an important role in both constitutive and alternative pre-mRNA splicing, and the functions of these splicing factors are regulated by phosphorylation. We have previously characterized SRPK1 (SFRSK1) and SRPK2 (SFRSK2), which are highly specific protein kinases for the SR family of splicing factors. Here we report the chromosomal localization of the mouse and human genes for both kinases.

Identification of a human gene encoding a homologue of Saccharomyces cerevisiae EXO1, an exonuclease implicated in mismatch repair and recombination.

The EXO1 gene was identified in Saccharomyces cerevisiae as a gene encoding an exonuclease that interacts with MSH2 and functions in mismatch repair and genetic recombination. To understand the role of EXO1 in higher eukaryotes, we identified the human EXO1 gene. The hEXO1 predicted amino acid sequence shares 26.

Identification of a new MAGE gene with tumor-specific expression by representational difference analysis.

Human genes expressed exclusively in tumors and male germ line cells, such as those of the MAGE, BAGE, and GAGE families, encode antigens recognized by T lymphocytes, which are potentially useful for antitumor immunotherapy. To identify new genes of this type, we generated cDNA populations enriched in sequences expressed only in testis and melanoma, using the representational difference analysis approach. A testis cDNA library enriched by subtraction with cDNA from four other normal tissues was hybridized with radiolabeled melanoma cDNA enriched by subtraction with normal skin cDNA.

Cloning and characterization of three human forkhead genes that comprise an FKHR-like gene subfamily.

Alveolar rhabdomyosarcomas are associated with unique chromosomal translocations t(2;13) and t(1;13), which arise from fusion of the genes for the paired box proteins PAX3 and PAX7, respectively, to the FKHR (forkhead in rhabdomyosarcoma) gene on chromosome 13q14. Here we report the identification and characterization of three novel human forkhead genes with similarity to FKHR. The three genes (HGMW-approved symbols FKHRP1, FKHRL1, and FKHRL1P1) map to chromosomal regions 5q35.

Genetic instability leads to loss of both p53 alleles in a human glioblastoma.

Little is known about the relationship between genetic recombination mechanisms and loss of tumour suppressor genes in solid tumours. Here, we demonstrate deletion and truncation of both p53 alleles in a primary human glioblastoma and a derived cell line as the combined result of a t(17;20) reciprocal translocation and a 1.1 Mbp genomic deletion on chromosome 17p, starting in intron 4 of the p53 gene and ending at the telomeric CA-repeat marker D17S960.

Chromosomal mapping and mutational analysis of the coding region of the glycogen synthase kinase-3alpha and beta isoforms in patients with NIDDM.

Activation of glycogen synthesis in skeletal muscle in response to insulin results from the combined inactivation of glycogen synthase kinase-3 (GSK-3) and activation of the protein phosphatase-1, changing the ratio between the inactive phosphorylated state of the glycogen synthase to the active dephosphorylated state. In a search for genetic defects responsible for the decreased insulin stimulated glycogen synthesis seen in patients with non-insulin-dependent diabetes mellitus (NIDDM) and their glucose-tolerant first-degree relatives we have performed mutational analysis of the coding region of the 2 isoforms of GSK-3alpha and GSK-3beta in 72 NIDDM patients and 12 control subjects. No structural changes were detected apart from a few silent mutations.

Genomic cloning and localization of CTAG, a gene encoding an autoimmunogenic cancer-testis antigen NY-ESO-1, to human chromosome Xq28.

CTAG was initially cloned from an esophageal squamous cell carcinoma cDNA expression library by immunoscreening with autologous patient's serum. CTAG mRNA is expressed in a proportion of human cancers in a lineage-nonspecific fashion, whereas its expression in normal tissues is restricted to testis and ovary only. This expression pattern suggests that the CTAG product (NY-ESO-1) is an aberrantly activated tumor antigen and can potentially be an antigenic target for tumor vaccination.

Allelic deletion mapping on chromosome 5 in human carcinomas.

We analysed allelic deletions on chromosome 5 in microdissected human non-small cell lung cancers. Thirty-four primary squamous cell carcinomas, 15 primary adenocarcinomas and five regional lymph node metastases were investigated for loss of heterozygosity (LOH) in chromosomal region 5p15-q21. The sites analysed included the APC tumor suppressor gene at 5q21, five polymorphic microsatellite markers and the putative tumor suppressor locus del-27, that was assigned to chromosomal region 5p13-12 by fluorescence in situ hybridization (FISH) analysis.

Chromosomal localization of mouse and human genes encoding the splicing factors ASF/SF2 (SFRS1) and SC-35 (SFRS2).

The mammalian SR-type splicing factors ASF/SF2 and SC-35 play crucial roles in pre-mRNA splicing and have been shown to shift splice site choice in vitro. We have mapped the ASF/SF2 gene in mice and humans and the SC-35 gene in mice. Somatic cell hybrid mapping of the human ASF/SF2 gene (SFRS1 locus) reveals that it resides on chromosome 17, and fluorescence in situ hybridization refines this localization to 17q21.

Localization of the human B-type natriuretic peptide precursor (NPPB) gene to chromosome 1p36.

Cardiac myocytes synthesize and secrete a family of peptide hormones with potent natriuretic, diuretic, and vasodilatory properties. These peptides are derived from precursor molecules that are encoded by two different genes, the atrial natriuretic peptide precursor A (NPPA) and the B-type natriuretic peptide or natriuretic peptide precursor B (NPPB). A human genomic clone for the NPPB gene was used to determine the chromosomal location of the NPPB gene.

Localization of short/branched chain acyl-CoA dehydrogenase (ACADSB) to human chromosome 10.

Short/branched chain acyl-CoA dehydrogenase, SBCAD (gene symbol ACADSB), is a member of the acyl-CoA dehydrogenase family of genes with activity toward the short/branched chain acyl-CoA derivatives as well as short/straight chain acyl-CoAs. Southern blot analysis of DNA from a panel of human/rodent somatic cell hybrids localized ACADSB to human chromosome 10, and fluorescence in situ hybridization experiments confirmed the chromosomal assignment and refined the subchromosomal localization to 10q25-q26.