Cloning of genes expressing in the early stage of rat bladder carcinogenesis induced by BBN

我们一直在研究人尿路上皮癌细胞中肿瘤抑制基因的失活。除了使用人类肿瘤组织进行分析外，实验模型应该提供癌症发生的早期阶段或癌前病变的数据。最近发展起来的差异RT-PCR技术使我们能够检测在这种原发病变中高表达的基因的表达。也有可能从通过差异RT-PCR方法分离的克隆中鉴定人类同源物。从用BBN治疗6个月的大鼠膀胱的增生性病变、乳头状瘤和原位癌中提取总的细胞质RNA。以正常膀胱粘膜为对照，用多对随机引物和寡核苷酸片段进行差异显示。选择了42个条带，因为它们被特定地发现或它们的强度增加。使用Northern印迹或定量RT-PCR方法确认每个皮损中的特异性表达。由于来自原发灶的RNA数量有限，定量RT-PCR对此类评估是有效的。对隔离克隆的进一步鉴定正在进行中。

We have been studying inactivation of tumor suppressor genes in human urothelial cancer cells. Along with analyzes using human tumor tissues, experimental models should provide data in the early stage of carcinogenesis or in precancerous lesions. A recently developed technique of differential RT-PCR allows us to detect expression of genes highly expressed in such primary lesions. It would also be possible to identify human homologues from clones isolated by the differential RT-PCR method. Total cytoplasmic RNA was isolated from hyperplastic lesions, papilloma, and carcinoma in situ of the rat bladder treated with BBN for six months. Differential display was performed with several sets of random primers and oligo dTs using a normal bladder mucosa as a control. Forty-two bands were chosen because they were specificly found or their intensity was increased. Confirmation of specific expression in each lesions was performed using Northern blot or quantitative RT-PCR assay. Due to a limited amount of RNA from the primary lesions, quantitative RT-PCR was effective for such evaluation. Further characterization of the isolated clones are ongoing.