Expression of E-cadherin in bone and soft tissue sarcomas: a possible role in epithelial differentiation

The cadherin-mediated cell-cell adhesion system is now known to play a critical role in both the morphogenesis of cancer cells and suppression of their invasion. However, the pattern of expression of E-cadherin, the major cadherin of epithelial cells in bone and soft tissue sarcomas, remains unclear. This prompted us to study E-cadherin expression in a variety of bone and soft tissue sarcomas. Using the monoclonal antibody HECD-1, raised against the extracellular domain of E-cadherin, we observed immunoreactivity in 1 pleomorphic rhabdomyosarcoma, 2 of 5 diffuse mesotheliomas, 4 of 5 clear cell sarcomas, 1 of 5 epithelioid sarcomas, and 10 synovial sarcomas. Other types of bone and soft tissue sarcoma (4 osteosarcomas, 4 chondrosarcomas, 3 primitive neuroectodermal tumors, 1 fibrosarcoma, 4 malignant fibrous histiocytomas, 5 liposarcomas, 4 leiomyosarcomas, 6 alveolar and 5 embryonal rhabdomyosarcomas, 4 angiosarcomas, 4 malignant peripheral nerve sheath tumors, 2 extraskeletal myxoid chondrosarcomas, 2 extraskeletal osteosarcomas, and 3 alveolar soft part sarcomas) were completely negative for E-cadherin. Our findings indicate that E-cadherin is expressed in certain kinds of soft tissue sarcomas, especially those with epithelioid features, suggesting that E-cadherin plays a role in the constitution of their architecture.     

Optimedin: a novel olfactomedin-related protein that interacts with myocilin

Mutations in the MYOC gene may lead to juvenile open-angle glaucoma with high intraocular pressure, and are detected in about 4% of people with adult onset glaucoma. Most of these mutations are found in the third exon of the gene encoding the olfactomedin-like domain located at the C terminus of the protein. Another olfactomedin-related protein, known as noelin or pancortin, is involved in the generation of neural crest cells. Here we describe the identification of a novel olfactomedin-related gene, named optimedin, located on chromosome 1p21 in humans. Optimedin and noelin are both expressed in brain and retina. However, unlike noelin, rat optimedin is also highly expressed in the epithelial cells of the iris and the ciliary body in close proximity to the sites of Myoc expression. In the human eye, optimedin is expressed in the retina and the trabecular meshwork. Both optimedin and myocilin are localized in Golgi and are secreted proteins. The presence of mutant myocilin interferes with secretion of optimedin in transfected cells. Optimedin and myocilin interact with each other in vitro as judged by the GST pulldown, co-immunoprecipitation and far-western binding assays. The C-terminal olfactomedin domains are essential for interaction between optimedin and myocilin, while the N-terminal domains of both proteins are involved in the formation of protein homodimers. We suggest that optimedin may be a candidate gene for disorders involving the anterior segment of the eye and the retina.     

Analysis of E-cadherin in diffuse-type gastric cancer using a mutation-specific monoclonal antibody

In-frame deletions from the E-cadherin mRNA, coding for a homophilic cell adhesion molecule, are characteristic for diffuse-type gastric carcinomas. Using immunohistochemical analysis the mutant form cannot be distinguished from normal E-cadherin, making results difficult to interpret. In this study, a rat monoclonal antibody, designated E-cad delta 9-1, was generated against a peptide spanning the fusion junction region between exons 8 and 10. This new epitope is present in an E-cadherin variant that lacks exon 9 from the mRNA due to different splice-site gene mutations. Using Western blotting and immunohistochemistry of E-cadherin-transfected cells, we demonstrate that E-cad delta 9-1 specifically reacts with E-cadherin lacking exon 9 but not with the wild-type protein. No immunoreactivity was observed in 31 nontumorous and embryonal tissues analyzed. In gastric carcinoma specimens known to express mutant E-cadherin mRNA lacking exon 9, E-cad delta 9-1 targets exclusively tumor cells in routine formalin-fixed and paraffin-embedded material from biopsies, primary tumors, and lymph node metastases. In a retrospective series of 172 diffuse-type gastric carcinomas expressing E-cadherin, E-cad delta 9-1 reacted with 22 tumors (13%). This new tumor marker-monoclonal antibody system could open novel avenues for selective diagnosis and specific therapy of a subgroup of diffuse-type gastric cancer patients.     

Loss of cell-cell contact is induced by integrin-mediated cell-substratum adhesion in highly-motile and highly-metastatic hepatocellular carcinoma cells

The cadherin-mediated cell-cell adhesion system plays a critical role in normal development and morphogenesis. Inactivation of this system is thought to be responsible for cancer invasion and metastasis. A human hepatocellular carcinoma (HCC) cell line, KYN-2, was observed to have great potential for intrahepatic metastasis when orthotopically implanted into the liver of SCID mice. In vitro cultures of KYN-2 cells showed that they formed trabecular structures in suspension but lost tight cell-cell adhesion and became scattered when attached to a substratum such as collagen or fibronectin. In response to adhesion to the substratum, subcellular colocalization of E-cadherin and actin filaments were shown to be reduced, and a significant amount of alpha-catenin was dissociated from the E-cadherin-catenin complex in KYN-2 cells. These changes of cell-cell adhesion were blocked by inhibitory monoclonal antibodies against beta1 and beta5 integrins. We found that c-Src was coimmunoprecipitated with E-cadherin-catenin complex and was tyrosine-dephosphorylated and activated in the adherent cells. The tyrosine dephosphorylation of c-Src was induced by cell adhesion to the substratum and inhibited by addition of inhibitory monoclonal antibodies against beta1 and beta5 integrins. These findings indicate that integrin-mediated cell-substratum adhesion inhibits cadherin-mediated cell-cell adhesion, possibly through c-Src activation, and suggest that this cross-talk mediates transient inactivation of the cadherin system and plays an important role in intrahepatic metastasis of human HCC. Modulation of this interaction might provide a new approach to prevent metastasis and recurrence of HCC.     

Immunohistochemistry and angiography in adenomatous hyperplasia and small hepatocellular carcinomas

The sinusoidal structure and blood supply of 38 liver nodules less than 2 cm In diameter were Investigated. There were 18 cases of adenomatous hyperplasia (AH) and 20 cases of hepatocetlular carcinoma (HCC). Growth pattern, encapsulation and vascularity were examined, and Immunohistochemistry performed for factor VIII related antigen (factor VIII), type IV collagen (collagen IV), lamlnln and CD68. There were significant differences between AH and small HCC, except for the expression of CD68. There were differences In tumor size, vasculartty and the components of the basement membrane between AH and small, well differentiated HCC. The cases of AH were supplied by the portal system and maintained the sinusoidal structure, but small well-differentiated HCC were supplied by a mixture of portal and arterial vessels. In spite of their small size, moderately and poorly differentiated HCC had capillary and were supplied by branches of the hepatic artery.     

Detection of genes expressed in primary colon cancers by in situ hybridisation: overexpression of RACK 1.

AIMS: The isolation of various genes that are expressed in a region specific manner is considered useful for research in molecular pathology. In situ hybridisation (ISH) was used in a screening procedure to isolate these genes efficiently, using colon cancer as a model. METHODS: Suppression subtractive hybridisation (SSH) between colon cancer tissue samples and corresponding non-cancerous tissues was performed. Genes showing high expression in the cancers were selected using macro-DNA array analysis. As a final screening procedure, conventional ISH was performed to isolate genes expressed specifically in colon cancers. RESULTS: Sixty nine clones were selected by SSH and macro-DNA array analyses. These clones were then analysed by ISH to examine their expression patterns. ISH screening revealed that all the clones screened showed more intense signals in colon cancers than in non-cancerous tissues. Among them, RACK 1, which is a protein kinase C receptor and a homologue of the G protein beta subunit, was expressed intensely in colon cancer cells. RACK 1 expression was evaluated in multiple samples by ISH, and the results confirmed that RACK 1 was universally overexpressed in cells of all 11 colon cancers examined. CONCLUSIONS: Many genes, including RACK 1, expressed in colon cancer cells can be isolated efficiently by this method, and their precise expression pattern can be evaluated. These results indicate that ISH is an excellent technique for systemic screening of genes expressed in a region specific manner.     

Identification of human antitumor cytotoxic T lymphocytes epitopes of recoverin, a cancer-associated retinopathy antigen, possibly related with a better prognosis in a paraneoplastic syndrome

Cancer-associated retinopathy (CAR) is a rare paraneoplastic syndrome, and the recoverin-specific autoantibody is suggested to contribute to the pathogenesis of retinopathy, including apoptosis of retinal cells. Because it is known that CAR(+) cancer patients have a preferable prognosis, we hypothesized that aberrantly expressed recoverin in cancer cells can become a target of cytotoxic T lymphocytes (CTL). Here we tested nine recoverin-derived HLA-A24-binding peptides for their capacity to elicit antitumor CTL. We observed recoverin-specific CTL responses in two HLA-A24(+) CAR(+) cancer patients. In addition, the CTL responses were obtained from three of ten CAR(-) cancer patients and two of six healthy individuals. The CTL precursor frequency of CAR(+) cancer patients and that of CAR(-) cancer patients was higher than that of healthy individuals. Of nine recoverin peptides, R49 (QFQSIYAKF), R49.2 (QFQSIYAKFF), and R64 (AYAQHVFRSF) were discovered to induce the peptide-specific CTL. Taken together, our present data suggest that peripheral activation of recoverin-specific antitumor CTL is likely to contribute to the preferable prognosis of CAR(+) cancer patients. Moreover, in cases other than CAR(+) cancer patients, recoverin may offer the opportunity to design epitope-based immunotherapeutic approaches for treating HLA-A24(+) cancer patients with a recoverin-expressing tumor.     

Polymorphism of germ-line immunoglobulin VH genes correlates with allotype and idiotype markers

The polymorphic nature of the immunoglobulin V_H genes was investigated by Southern blot analysis of liver DNA of sixteen different mouse strains and hybridization with V_H probes. Differences in restriction enzyme pattern (REP) were observed and six different patterns of restriction fragments were found for the sixteen strains analyzed. No equivalent polymorphism was observed in another multigene family, the actins. The six patterns correlate with immunoglobin constant region allotypes (Igh-1). Experiments with Igh-1-congenic strains suggest that the V_H REP is linked to immunoglobulin constant region haplotype. Mouse strains which share inherited idiotypes also share identical V_H restriction pattern. This provides a structural basis for the genetic linkage between idiotypes and allotypes. It also indicates that different strains carry different VH gene repertoires, which may be the basis for the expression of different inherited idiotypes in various strains. We propose that a V_H group is a set of linked genes that are coinherited as a cluster with the constant region genes and that V_H and C_H can be regarded as an extended haplotype.     

A fluorescent amplified fragment length polymorphism study of Salmonella enterica serovar Sofia, the major Salmonella serovar isolated from chickens in Australia

Fluorescent amplified fragment length polymorphism (FAFLP) analysis was performed on 68 isolates of Salmonella enterica subsp. salamae serovar Sofia (S. Sofia). Fifty eight isolates were obtained over a period of approximately 15 years from a range of human, chicken industry and environmental sources throughout Australia. A further ten isolates were identified from human and poultry sources in Israel from 1972 to 1987. Analysis of FAFLP profiles for fragments between 50 to 500 base pairs in length indicated distinct clusters of isolates. All but seven isolates clustered into four groups of >90% similarity and all isolates displayed at least 70% similarity with each other. No cluster could be attributed to a particular geographical, temporal or source-of-isolation origin. It is concluded that S. Sofia is genetically variable with certain clones persisting over time but no group appears unique to Australia.     

Aberrant expression of beta-catenin and mutation of exon 3 of the beta-catenin gene in renal and urothelial carcinomas

The present study attempted to clarify the significance of aberrant expression of beta-catenin protein and mutation of exon 3 of the beta-catenin gene in renal and urothelial carcinogenesis. beta-Catenin expression was examined immunohistochemically and mutation of the beta-catenin gene was analyzed by polymerase chain reaction-single strand conformation polymorphism (SSCP) and direct sequencing. beta-Catenin immunoreactivity was observed at the cell membrane in all 30 renal cell carcinomas (RCC) examined, and no RCC showed a mobility-shifted SSCP band. Of 46 transitional cell carcinomas (TCC) examined, there was reduced expression of beta-catenin, as compared with its expression in non-cancerous transitional epithelium, in 22 cases (48%) and beta-catenin accumulation in the nucleus in five cases (11%). Of four renal pelvis TCC examined, point mutation of exon 3 of the beta-catenin gene at codon 45 resulting in amino acid substitution (Ser to Phe) was detected in one (25%). The incidence of reduced expression of beta-catenin correlated significantly with the growth pattern (superficial type vs invasive type) of TCC (P < 0.05). These data indicate that: (1) aberrant beta-catenin expression may be at least partly involved in urothelial carcinogenesis, but less significantly so in renal carcinogenesis, and (2) it may be associated with the progression of TCC showing invasive growth.