myelin-basic-protein and Colonic-Neoplasms

Analysis of the calcium-dependent cell adhesion molecule E-cadherin has led to the identification of catenins, which are necessary for cadherin function. Growing evidence that cadherins and catenins are subjected to genetic alterations in carcinogenesis makes it especially important to understand protein-protein interactions within the cadherin-catenin complex. Here we report the identification and analysis of the alpha-catenin binding site in plakoglobin (gamma-catenin). Using N- and C-terminal truncations of plakoglobin, we identified a domain of 29 amino acids necessary and sufficient for binding alpha-catenin. The alpha-catenin binding site is fully encoded within exon 3 of plakoglobin but only partially represented in Armadillo repeat 1. This suggests that exons rather than individual Arm repeats encode functional domains of plakoglobin. Site-directed mutagenesis identified residues in the alpha-catenin binding site indispensable for binding in vitro. Analogous mutations in beta-catenin and Armadillo had identical effects. Our results indicate that single amino acid mutations in the alpha-catenin binding site of homologs of Armadillo could prevent a stable association with alpha-catenin, thus affecting cadherin-mediated adhesion.

Topics: alpha Catenin; Amino Acid Sequence; Animals; Armadillo Domain Proteins; Base Sequence; Binding Sites; Cadherins; Cell Adhesion Molecules; Cell Line; Colonic Neoplasms; Cytoskeletal Proteins; Desmoplakins; DNA Primers; Drosophila; Drosophila Proteins; gamma Catenin; Glutathione Transferase; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Myelin Basic Protein; Point Mutation; Polymerase Chain Reaction; Protein Binding; Protein Biosynthesis; Proteins; Recombinant Fusion Proteins; Restriction Mapping; Sequence Deletion; Trans-Activators; Transcription Factors; Tumor Cells, Cultured

In a prior study, we have shown that stable transfection of expression plasmids for protein kinases C beta 1 (PKC beta 1) or PKC beta 2 into differentiated colon cancer cells led to elevated levels of PKC beta 1 or PKC beta 2 protein and PKC beta kinase activities in the transfectants, without altering PKC alpha levels. PKC gamma is not found in these cells, so the major modulation was in PKC beta. PKC beta transfectant cells exhibited blocked differentiation, increased growth rate in athymic mice, and restoration of the basic fibroblast growth factor response pathway. In this study, we have extended the analysis of these PKC beta transfectants to the mitogen-activated protein kinase ERK3. Analysis of cell lysates on the mitogen-activated protein kinase substrate myelin basic protein by in gel kinase assay showed increased activity at 63 kDa, the size of ERK3, in each of two PKC beta 1 and each of two PKC beta 2 transfectants compared with the vector control transfectant. ERK3 was expressed at equal abundance in PKC beta 1, PKC beta 2, and control transfectant cells as demonstrated by Western blotting and by immunoprecipitation with anti-ERK3 monoclonal antibody. However, a > 10-fold increase in ERK3 activity in each PKC beta transfectant was shown by immunoprecipitation with anti-ERK3 monoclonal antibody followed by either immune complex kinase assay or by in gel kinase assay. Thus, while overexpression of transfected PKC beta does not lead to overexpression of ERK3, it does lead to constitutive activation of ERK3. A causal link between PKC beta overexpression and ERK3 activation was established because 12-O-tetradecanoylphorbol-13-acetate treatment down-regulated both PKC and ERK3 activities in both PKC beta 1 transfectants. ERK3 activity was found in nuclear and membrane fractions in each PKC beta transfectant, in contrast to controls, perhaps accounting for constitutive activation of ERK3 in cells with elevated levels of PKC beta 1 or PKC beta 2.

Topics: Animals; Blotting, Western; Calcium-Calmodulin-Dependent Protein Kinases; Colonic Neoplasms; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Gene Expression; Genetic Vectors; Humans; Isoenzymes; Mice; Mice, Nude; Mitogen-Activated Protein Kinase 6; Mitogen-Activated Protein Kinases; Myelin Basic Protein; Protein Kinase C; Recombinant Proteins; Transfection; Tumor Cells, Cultured

Phytohaemagglutinin (PHA), a well-known mitogen, and encephalitogenic factor (EF) are recognized by lymphocytes of patients with different malignant diseases as non-specific antigens. Utilizing these two antigens, the SCM (structuredness of the cytoplasmatic matrix) test offers a means of discrimination between malignant and non-malignant diseases. The SCM test can also be used as a specificity test since lymphocytes from donors with a given malignant disease react exclusively with the tumour-associated antigen (TAA) of that disease. Results from 73 donors (15 healthy patients, 38 patients with different types of malignant disorders and 20 patients with autoimmune diseases) indicate the predictive value of the test. First, the non-specific test was applied in order to establish whether the patients suffered from an active malignant disease. The lymphocytes of those patients which were found to suffer from an active malignant disorder were then exposed to different types of tumour tissues. Twenty-five out of the 38 patients with malignant disorders were found by the SCM test to have an active disease. The lymphocytes of 24 out of these 25 patients showed a positive reaction when exposed to tumour tissue of the same type of cancer of which they were found to suffer by other clinical tests, and displayed no reaction with any other tumour tissues for which they were tested. One patient, with an inconclusive value of the SCM test, showed no reaction with any type of tumour to which he was exposed. The remaining 13 patients, who were diagnosed by the test as non-cancerous, did not show any clinical evidence of malignancy at the time of the test, after their tumours had been excised. Eighteen out of 20 patients with autoimmune diseases showed negative results when tested by the general test and by the various specificity tests.

Topics: Antigens, Neoplasm; Breast Neoplasms; Colonic Neoplasms; Epitopes; Fluorescence Polarization; Humans; Lymphocyte Activation; Lymphocytes; Myelin Basic Protein; Neoplasms; Phytohemagglutinins

Leukocyte migration tests under agarose (Clausen technique) were performed in 28 patients tentatively diagnosed as having any malignancy with the use of a 3 M KCl-extract panel prepared from bronchogenic, gastric, colonic, renal, and mammary carcinoma, corresponding normal tissues, carcinoembryonic antigen (CEA), and human encephalitogenic protein (HEP). 17 out of 22 proven carcinoma patients showed sensitization by reaction with optimal concentrated KCl-extract of cancer from the same organ type as their own tumor. In some cases positive reactions could be observed also with normal tissue antigen (NTA) of tumor organ type (7/22) or with an additional carcinoma extract of organ type differing from patients own primary tumor (8/22). Gastrointestinal carcinomas, especially, showed sensitization to CEA (7/12) contrary to nongastrointestinal carcinomas (1/10). With HEP no positive reactivity could be found (0/10). With the use of tumor antigen panel (5 antigens) only few positive reactions (MI less than 0.80 or greater than 1.20) could be observed in 6 patients with nonmalignant diseases (1/30 tests) and 8 healthy blood donors (1/40 tests). A widespread individual screening program using tissue antigens for patients suspected of malignancies could give a pattern of reactivities and improve the recognition of cell-mediated sensitization against tumor tissues.

Topics: Aged; Antigens, Neoplasm; Breast Neoplasms; Carcinoembryonic Antigen; Cell Migration Inhibition; Colonic Neoplasms; Epitopes; Female; Humans; Immunity, Cellular; Kidney Neoplasms; Leukocytes; Lung Neoplasms; Male; Middle Aged; Myelin Basic Protein; Neoplasms; Sepharose; Stomach Neoplasms

The macrophage electrophoretic mobility (MEM) test was performed on guinea-pig macrophages treated with the interaction products of encephalitogenic protein and peripheral lymphocytes from 44 patients with colorectal cancer and 33 "healthy" controls. In 54/60 tests involving patients, statistically significant reductions in electrophoretic mobilities were observed, compared with 12/33 in controls. Our overall results on the reductions in macrophage mobilities by lymphocyte products are in accord with the work of some other workers, but not all. In contrast to many other studies, the standard procedures used here to express the results should permit an exchange of data on an international basis and allow a more rapid, general appraisal of the MEM test.

Topics: Adult; Aged; Animals; Cell Movement; Colonic Neoplasms; Electrophoresis; Female; Guinea Pigs; Humans; Lymphocytes; Macrophages; Male; Middle Aged; Myelin Basic Protein; Neoplasm Metastasis; Rectal Neoplasms