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

1.免疫荧光染色、免疫印迹、电镜或免疫电镜观察发现，5-氟脱氧尿苷单磷酸（FdUMP）处理具有类似唾液腺闰管细胞表型的人涎腺癌细胞系HSG，可诱导细胞分化为肌上皮细胞，表达肌球蛋白、S-100蛋白β链、胞饮小泡和肌丝。用~ 3 H标记的FdUMP进行Scatchard分析，发现HSG细胞膜上FdUMP受体的数量为10^6个/细胞（Kd= 3.7 × 10 ~ 6 μ <-10>M）。发现FdUMP对HSG细胞的体外生长抑制作用可被5 B/10 MAb所解除。这个我 ...更多信息 表明5 B/10单抗识别FdUMP受体的表位。因此，我们已经通过使用5 B/10 MAb作为配体的亲和层析或通过FdUMP交换反应分离了与FdUMP结合的受体蛋白（28 KDa和84 KDa）。另外，通过5 B/10单抗免疫荧光染色技术发现，FdUMP受体在涎腺、胃和结肠腺癌中表达频率较高。4.对FdUMP结合蛋白进行了氨基酸序列分析。结果表明，该序列为NLDLDSIIAEVKA，与细胞角蛋白5、6或8.5的氨基酸序列相对应。将该表达cDNA文库转染到HSG细胞中，并分离在FdUMP存在下增殖的细胞克隆。我们已经对从细胞克隆中回收的基因进行了DNA测序。因此，已经发现蛋白体基因和编码与中等大小的细丝相关的衔接蛋白的plectin基因与通过FdUMP受体的信号转导有关。少

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