ALTERED EXPRESSION OF TRANSCRIPTION FACTOR FOR CD19 GENE IN HUMAN MYELOMA CELLS

骨髓瘤细胞表面不表达CD19，而正常浆细胞表达CD19。我们认为CD19的这种表达缺失与人骨髓瘤的发生有关，并进行了这项研究。我们的研究表明，骨髓瘤细胞CD19表达的缺失是由于CD19基因表达缺失所致。此外，还发现CD19基因表达的缺失是由于Pax-5基因表达改变所致。凝胶漂移实验也证实Pax-5基因编码的BSAP在骨髓瘤细胞中没有检测到功能活性。由于Pax-5基因在人骨髓瘤细胞中的表达发生改变，我们试图克隆和分析人Pax-5基因的调控区，以阐明Pax-5基因表达的调控机制。我们用基因步行法克隆了人Pax-5基因5‘上游1050bpDNA片段，并对该片段中预测的结合基序进行了分析。该片段含有Ikaros-2(Ik-2)、Lyf-1(Ik-1)、bHLH、E-47、SOX-5等结合基序。在该DNA片段的缺失突变实验中，其基本启动子活性位于Pax-5基因上游-70~-820碱基之间。因此，我们的研究表明，在不表达CD19的人骨髓瘤细胞中，Pax-5基因的表达发生了变化，我们克隆了人Pax-5基因5‘端1050bp5’上游区域，并对其进行了功能分析。对克隆的人Pax-5基因启动子区域的进一步分析将有助于了解人骨髓瘤的致癌机制。

Myeloma cells do not express CD19 on their surface, while normal plasma cells do. We considered that this loss of CD19 expression is involved in oncogenesis of human myeloma, and performed this project. Our study clarified that loss of CD19 expression on myeloma cells is derived from loss of expression of CD19 gene. Furthermore, it was revealed that loss of CD19 gene expression result from altered expression of Pax-5 gene. It was also confirmed that the functional activity of BSAP encoded by Pax-5 gene was not detected from myeloma cells by gel shift assay. Since expression of Pax-5 gene is altered in human myeloma cells, we tried to clone and analyze regulatory region of human Pax-5 gene in order to clarify the regulatory mechanism of Pax-5 gene expression. We cloned 1,050 bp 5' upstream region of human Pax-5 gene by PCR-mediated gene walking method, and analyzed predicted binding motifs in this DNA fragment. This fragment contained binding motifs of Ikaros-2 (Ik-2), Lyf-1 (Ik-1), bHLH,E-47, Sox-5 et al. In the experiment of deletion mutants of this DNA fragment, the basal promoter activity was located in the region from-70 bp to -820 bp upstream of Pax-5 gene. Therefore, our study showed that the expression of Pax-5 gene is altered in human myeloma cells that do not express CD19, and we cloned and functionally analyzed 1,050 bp 5' upstream region of human Pax-5 gene. The further analysis of this cloned promoter region of human Pax-5 gene will contribute to the understanding of oncogenic mechanism of human myeloma.