ANALYSIS OF THE HUMAN α6 INTEGRIN PROMOTER

细胞与基质的相互作用在肿瘤转移中起重要作用。整联蛋白α6亚基与β1或β4亚基结合形成层粘连蛋白受体，层粘连蛋白是基底膜的主要成分。整合素α6亚单位的表达被认为是肿瘤细胞通过基底膜侵袭的重要因素。为了研究整合素α6基因的调控机制，我们克隆了人α6整合素5'端侧翼序列。启动子区含有TATA样序列、Sp1和NF-κB的共有结合位点。在转录起始位点上游350-360 bp处存在一个GRE/PRE结合位点，以及Ap 1和c-myc结合位点。我们分析了Ap 1和c-myc结合位点周围CpG位点的甲基化。从培养的乳腺癌细胞系提取的DNA显示出较高的甲基化比前列腺癌细胞系的限制性内切酶消化后，以及通过亚硫酸氢钠修饰的测序分析的Southern印迹。因此，人α6整合素基因启动子Ap 1和c-myc结合位点的DNA甲基化可能在一定程度上调节了乳腺癌细胞中基因的表达。我们进一步分析了人α6整合素启动子DNA甲基化对乳腺癌病例存档病理标本中该基因表达的影响。为了检测癌组织中细胞粘附分子和细胞外基质基因的表达，我们建立了一种用PCR扩增的单链DNA探针进行原位杂交的方法。我们在各种国际和国内会议上展示了我们的数据，并在学术期刊上发表了大量科学论文。

Cell to matrix interactions play important roles in tumor metastasis. integrin α6 subunit associates with β1 or β4 subunit to form receptors for laminin, a major component of the basement membrane. The expression of integrin α6 subunit is thought to be important for tumor cell invasion through the basement membrane. To investigate the regulatory mechanism of integrin α6 gene we previously cloned 5' flanking region of the human α6 integrin. The promoter region contained a TATA-like sequence, consensus binding sites for Sp1 and NF-κB. A putative GRE/PRE, together with Ap1 and c-myc binding sites were located around 350-360 bp upstream of the transcription start site. We analyzed the methylation of CpG loci around Ap1 and c-myc binding sites. DNA extracted from cultured breast cancer cell lines showed higher methylation than that from prostate cancer cell lines by Southern blotting after the restriction enzyme digestion as well as by the sodium bisulfite-modified sequencing analysis. Thus DNA methylation around Ap1 and c-myc binding sites of the human α6 integrin gene promoter might regulate, in part, the gene expression in breast cancer cells.We established the methods to detect methylated-cytosine residue of gene promoter from formalin-fixed paraffin-embedded histological specimen. We furthermore continue to analyze the effects of DNA methylation of human α6 integrin promoter on expression of the gene using archived pathological samples of the cases with breast cancer. To assess the expression of genes of cell adhesion molecules and extracellular matrices on cancer tissues, we set up the system of in situ hybridization using PCR-derived single stranded DNA probes. We have presented our data at various international as well as domestic meetings, and publish a lot of scientific papers for academic journals.