Detection of Novel Drug Receptor and Mechanism of Induction of Differentiation and Apoptosis in Human Salivary Cancer Cells

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1.It has been found by immunofluorescent staining technique, immunoblotting, electron microscopy or immunoelectron microscopy that the treatment of human salivary cancer cell line HSG having the phenotype similar to salivary intercalated duct cells with 5-fluorodeoxyuridine monophosphate (FdUMP) results in the cellular differentiation into myoepithelial cells with the expression of myosin, beta chain of S-100 protein, pinocytic vesicles and myofilaments. In addition, it has been detected by electron microscopy, agarose gel electrophoresis or 3'-OH nick-end labelling that apoptosis is induced in the treated cells.2.It has been found by Scatchard analysis using ^3H-labelled FdUMP that HSG cells have 10^6 receptors for FdUMP/cell on cell membrane (Kd=3.7x10^<-10>M).3.We have prepared the murine monoclonal antibody (MAb) directed to the FdUMP receptors on HSG cell membranes (5B/10). It has been found that the in vitro growth inhibition of HSG cells by FdUMP is released by 5B/10 MAb. This i … More ndicates that 5B/10 MAb recognizes the epitope of FdUMP receptors. Thus, we have isolated the receptor proteins binding FdUMP (28KDa and 84KDa), by affinity chromatography using 5B/10 MAb as a ligand or by FdUMP exchange reaction. In addition, it has been found by immunofluorescent staining technique using 5B/10 MAb that the FdUMP receptors are frequently expressed in the adenocarcinoma arising in salivary gland, stomach or colon.4.We have performed the amino acid sequencing of FdUMP-binding protein. Consequently, the sequence was decoded NLDLDSIIAEVKA,which corresponded to the amino acid sequence of cytokeratin 5,6 or 8.5.We have constructed anti-sense oriented cDNA library, using mRNA isolated from HSG cells. This expression cDNA library was transfected into HSG cells and the cell clones proliferating in the presence of FdUMP were isolated. We have performed DNA sequencing for the genes recovered from the cell clones. Consequently, it has been found that proteosome gene and plectin gene encoding the adaptor protein associated with intermediate-sized filaments are related with the signal tranduction via FdUMP receptor. Less