transforming-growth-factor-alpha and Chromosome-Deletion

Topics: Adenocarcinoma; Chromosome Deletion; Cytokines; Epidermal Growth Factor; Genes, Tumor Suppressor; Humans; Neoplasm Staging; Oncogenes; Stomach Neoplasms; Transforming Growth Factor alpha; Transforming Growth Factor beta

Trp53(+/-) mice overexpressing Tgfalpha in a pancreas-specific manner represent a well-established animal model for pancreatic cancer. In this study we analyzed 38 pancreatic adenocarcinomas of these mice for secondary genomic changes by comparative genomic hybridization (CGH), loss of heterozygosity (LOH) analysis, real-time PCR, and methylation-specific analysis. CGH screening of the tumors revealed a recurrent pattern of genomic changes. In more than 50% of the tumors, chromosome 11 was affected. The gain of the proximal part spans about 16 cM, including the genes for Egfr, Rel, and Stk10. The distal part of chromosome 11, which contains the Trp53 locus, was deleted. LOH analysis proved that almost all tumors segregate the wild-type Trp53 allele. The Cdkn2a locus on chromosome 4 was inactivated by hypermethylation in 55% of all tumors. In addition, two other changes were detected in a mutually exclusive manner: overrepresentation of part of chromosome 15, or more rarely, loss of the distal part of chromosome 14. Together these data suggest the induction of a uniform pattern of secondary genomic changes in this transgenic tumor model for pancreatic cancer.

Topics: Adenocarcinoma; Animals; Chromosome Deletion; Crosses, Genetic; Disease Models, Animal; DNA Methylation; Gene Amplification; Gene Silencing; Genes, p16; Genes, p53; Humans; Loss of Heterozygosity; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Nucleic Acid Hybridization; Pancreatic Neoplasms; Polymerase Chain Reaction; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

Identification of specific and primary chromosomal alterations during the course of neoplastic development is an essential part of defining the genetic basis of cancer. We have developed a transgenic mouse model for liver neoplasia in which chromosomal lesions associated with both the initial stages of the neoplastic process and the acquisition of malignancy can be analyzed. Here we analyze chromosomal alterations in 11 hepatocellular carcinomas from the c-myc/TGF-alpha double-transgenic mice by fluorescent in situ hybridization with whole chromosome probes, single-copy genes, and 4'-6-diamidino-2-phenylindole (DAPI-) and G-banded chromosomes and report nonrandom cytogenetic alterations associated with the tumor development. All tumors were aneuploid and exhibited nonrandom structural and numerical alterations. A balanced translocation t(5:6)(G1;F2) was identified by two-color fluorescent in situ hybridization in all tumors, and, using a genomic probe, the c-myc transgene was localized near the breakpoint on derivative chromosome der 6. Partial or complete loss of chromosome 4 was observed in all tumors with nonrandom breakage in band C2. Deletions of chromosome 1 were observed in 80% of the tumors, with the most frequent deletion at the border of bands C4 and C5. An entire copy of chromosome 7 was lost in 80% of the tumors cells. Eighty-five percent of the tumor cells had lost one copy of chromosome 12, and the most common breakpoint on chromosome 12 occurred at band D3 (28%). A copy of chromosome 14 was lost in 72%, and band 14E1 was deleted in 32% of the tumor cells. The X chromosome was lost in the majority of the tumor cells. The most frequent deletion on the X chromosome involved band F1. We have previously shown that breakages of chromosomes 1, 6, 7, and 12 were observed before the appearance of morphologically distinct neoplastic liver lesions in this transgenic mouse model. Thus breakpoints on chromosome 4, 9, 14, and X appear to be later events in this model of liver neoplasia. This is the first study to demonstrate that specific sites of chromosomal breakage observed during a period of chromosomal instability in early stages of carcinogenesis are later involved in stable rearrangements in solid tumors. The identification of the 5;6 translocation in all of the tumors has a special significance, being the first balanced translocation reported in human and mouse hepatocellular carcinoma and having the breakpoint near a tumor susceptibility gene an

Topics: Age Factors; Animals; Carcinoma, Hepatocellular; Chromosome Breakage; Chromosome Deletion; Chromosome Mapping; Genes, myc; In Situ Hybridization, Fluorescence; Karyotyping; Liver Neoplasms; Mice; Mice, Transgenic; Transforming Growth Factor alpha; Translocation, Genetic

Loss of genetic material on chromosome 10 is regarded as a prominent feature in the genesis of glioblastomas. To use chromosome 10 deletions as diagnostic markers for glioblastomas we investigated, if the loss of chromosome 10 material could be restricted on the region 10q21-26. By PCR microsatellite analysis on frozen tissue and paraffin material from the ZULCH brain tumor collection we found (1) loss of heterozygosity in 10q21-26 in 75% of the investigated DNA from frozen tissue and (2) an interstitial loss in the region of the microsatellite marker D10S186. The combined immunohistochemical analysis of overexpression of EGFR, EGF and TGF alpha with LOH on chromosome 10 showed that chromosome 10 deletions are not exclusively bound to EGFR overexpression.

Topics: Brain Neoplasms; Chromosome Deletion; Chromosome Mapping; Chromosomes, Human, Pair 10; DNA, Neoplasm; DNA, Satellite; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Glioblastoma; Glioma; Humans; Immunohistochemistry; Paraffin; Polymerase Chain Reaction; Transforming Growth Factor alpha

Pseudomonas exotoxin A is composed of three structural domains that mediate cell recognition (I), membrane translocation (II), and ADP-ribosylation (III). Within the cell, the toxin is cleaved within domain II to produce a 37-kDa carboxyl-terminal fragment, containing amino acids 280-613, which is translocated to the cytosol and causes cell death. In this study, we constructed a mutant protein (PE37), composed of amino acids 280-613 of Pseudomonas exotoxin A, which does not require proteolysis to translocate. PE37 was targeted specifically to cells with epidermal growth factor receptors by inserting transforming growth factor-alpha (TGF-alpha) after amino acid 607 near the carboxyl terminus of Pseudomonas exotoxin A. PE37/TGF-alpha was very cytotoxic to cells with epidermal growth factor receptors. It was severalfold more cytotoxic than a derivative of full-length Pseudomonas exotoxin A containing TGF-alpha in the same position, probably because the latter requires intracellular proteolytic processing to exhibit its cytotoxicity, and proteolytic processing is not 100% efficient. Deletion of 2, 4, or 7 amino acids from the amino terminus of PE37/TGF-alpha greatly diminished cytotoxic activity, indicating the need for a proper amino-terminal sequence. In addition, a mutant containing an internal deletion of amino acids 314-380 was minimally active, indicating that other regions of domain II are also required for the cytotoxic activity of Pseudomonas exotoxin A.

Topics: ADP Ribose Transferases; Bacterial Toxins; Base Sequence; Binding, Competitive; Breast Neoplasms; Cell Line; Cell Survival; Chromosome Deletion; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Exotoxins; Female; Humans; Kinetics; Molecular Sequence Data; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Protein Biosynthesis; Protein Processing, Post-Translational; Pseudomonas aeruginosa; Pseudomonas aeruginosa Exotoxin A; Recombinant Fusion Proteins; Transforming Growth Factor alpha; Virulence Factors

Malignant human glioma D-298 MG amplifies a rearranged epidermal growth factor receptor (EGFR) gene (c-erbB proto-oncogene), resulting in an in-frame deletion of 83 amino acids in domain IV of the extracellular domain of the EGFR. EGF and transforming growth factor-a (TGF-a) bound to the mutant EGFR with high affinity and enhanced the intrinsic mutant EGFR kinase activity. The mutant EGFR was capable of transducing EGF-stimulated glioma cell proliferation and invasiveness in an in vitro three-dimensional spheroid model. The deletion-mutant EGFR in D-298 MG is capable of being activated by growth factor; this suggests that overexpression of this mutant EGFR protein rather than structural alteration may be the more significant biologic event.

Topics: Amino Acid Sequence; Base Sequence; Cell Line; Chromosome Deletion; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Gene Rearrangement; Glioma; Humans; Kinetics; Molecular Sequence Data; Phosphorylation; Protein-Tyrosine Kinases; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogenes; Receptor, ErbB-2; Transforming Growth Factor alpha

Pseudomonas exotoxin (PE) contains 613 amino acids that are arranged into 3 structural domains. PE exerts its cell-killing effects in a series of steps initiated by binding to the cell surface and internalization into endocytic vesicles. The toxin is then cleaved within domain II near arginine-279, generating a C-terminal 37-kDa fragment that is translocated into the cytosol where it ADP-ribosylates elongation factor 2 and arrests protein synthesis. In this study, we have focused on the functions of PE which are encoded by domain II. We have used the chimeric toxin TGF alpha-PE40 to deliver the toxin's ADP-ribosylating activity to the cell cytosol. Deletion analysis revealed that sequences from 253 to 345 were essential for toxicity but sequences from 346 to 364 were dispensable. Additional point mutants were constructed which identified amino acids 339 and 343 as important residues while amino acids 344 and 345 could be altered without loss of cytotoxic activity. Our data support the idea that domain II functions by first allowing PE to be processed to a 37-kDa fragment and then key sequences such as those identified in this study mediate the translocation of ADP-ribosylation activity to the cytosol.

Topics: Adenosine Diphosphate Ribose; ADP Ribose Transferases; Amino Acid Sequence; Bacterial Toxins; Biological Transport, Active; Chimera; Chromosome Deletion; Electrophoresis, Polyacrylamide Gel; Exotoxins; Models, Molecular; Molecular Sequence Data; Mutation; Plasmids; Pseudomonas aeruginosa; Pseudomonas aeruginosa Exotoxin A; Transforming Growth Factor alpha; Virulence Factors