Characterisation Of Antigens Detected By A New Mouse Monoclonal Antibody Ibmr3

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Date
2002-04
Authors
Hara Yuka
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Abstract
Monoclonal antibody IBMR3 (MAb IBMR3) was generated against synthetic peptides of the published amino acid sequences of the human interleukin-4 receptor (hlL-4R) with an initial intention to further study the role of interlukin-4 (IL-4) and its receptor in tumourigenesis. In the current study, MAb IBMR3 was used to analyse the expression of IBMR3 antigen (Ag) in normal and transformed mononuclear cells, established cell lines and tumour tissues to obtain an overview of its expression pattern and evaluate its possible application in medicine: This thesis describes various investigations including; flow cytometric analyses of norma! and transformed haematopoietic cells and established cell lines, biochemical and inununohistochemical analyses of cell lines and frozen tissues, as well as transient transfection analysis of epithelial cell line. \ For flow cytometric studies, IBMR3 Ag was found to be predominantly present in the cytoplasm of normal peripheral blood mononulcear cells (PBMC) and leukaemic cells. Leukaemic cells generally expressed higher levels of cytoplasmic IBMR3 Ag [AML: 65%, ALL-B: 85%, ALL-T: 69% (mean %)] compared to PBMC [lymphocytes: 42%, monocytes: 49.7%]. Higher cytoplasmic expression was more frequent in certain types of leukaemia (CLL-B) and particular subclasses of lymphocytes (CD19+cells). Surface IBMR3 Ag expression was essentially absent on lymphocytes (6.5%, MFI=69.5) but was observed on monocytes (20%, MFI=442). Among nineteen cases of leukaemia patients (eleven AML, six ALL-B, two ALL-T), significant upregulation of surface expression was seen in two cases of AML patients. Furthermore, this Ag was found to be JDodulatable since mitogen activation of PBMC resulted in a significant upregulation of the surface Ag expression [PHA: non-activated (1.18%), activated (47.5%), ConA: nonactivated (0.59%), activated (42.8%)]. Flow cytometric and biochemical analyses of various established cell lines showed that higher expression of cytoplasmic IBMR3 Ag was observed in poorly differentiated cells such as Hep2 cells (97%, MFI=58) compared to those with more differentiated morphology (HT29 cells: 59.9%, MFI=23). In these cell lines, MAb IBMR3 detected a heterogeneous population of molecules with molecular weight ranging from 25kD to 230kD. Poorly differentiated Hep7 cells expressed these molecules with higher intensity than cells with well-differentiated morphology, HepG2 cells. For immunohistochemical analysis of breast tissues, the results revealed strong IBMR3 Ag expression in malignant tissues but not in normal counterpart from the same patients, suggesting that IBMR3 Ag may be differentially expressed in normal and malignant breast tissues. In addition, we analysed whether the modulation of IL-4R expression had any effect on IBMR3 Ag expression in epithelial cell line. For this, plasmid encoding the IL-4R gene was transfected into HT29 colorectal cell line, leading to a transient overexpression of its gene product. Surface IBMR3 Ag expression was upregulated following transfection (sham transfected: 3.9%, MFI=128, transfected: 16.7%, MFI=273) and the pattern was similar to that of gp200-MR6 Ag. The latter molecule has been found to be functionally associated with hIL-4R and suspected to have a role in tumourigenesis. These results may postulate the possible association of upregulated IBMR3 Ag expression in malignant cells and cells with poorly differentiated morphology. Taken together, one can speculate that IBMR3 Ag might have a role in tumour development and that its expression pattern may have a significant value in cancer prognosis.
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Flow cytometric analyses of normal , and transformed haematopoietic cells
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