RTS AND ITS ROLE IN CHEMOTHERAPY

描述：(申请者摘要)本申请的长期目标 目的是确定RTS基因的生理功能及其相关性 癌症治疗学。Rts是一种与人类胸腺苷酸重叠的基因。 合成酶(TS)基因，由DNA的相反链编码。RTS为 转录以产生一个RNA(RTS-α)，该RNA与 TS和另一种，RTS-beta，它不是。RTS基因编码至少 两种蛋白质，RTS-α和RTS-β。RTSB已被发现 耐5-氟尿嘧啶(H630-1， 结肠癌)、甲氨蝶呤(K562 B1a、慢性粒细胞白血病和 氟代脱氧尿苷加ZD 1694(HCT-8DF2，回盲部癌)。高程 甲氨蝶呤耐药组和氟代脱氧尿嘧啶核苷组rTSB蛋白水平 PLUS ZD 1694细胞系分别伴随有完整的和 部分丧失了TS活性的生长调节。RTSB蛋白水平也 随着细胞从对数生长期进入稳定期生长而增加。 免疫沉淀数据表明，rTSB和一种新发现的蛋白质， RTS与TS和二氢叶酸还原酶(DHFR)形成复合体。已被占用 申请人总共提交了大量的初步数据， 提示RTS蛋白似乎在TS的生长调节中发挥作用 并在细胞耐药性的发展过程中发挥作用。四个具体目标是 建议研究RTS如何影响TS基因的表达，细胞 生理和对TS抑制剂的敏感性。它们如下：1) RTS蛋白表达的扩展特性及鉴定 RTS蛋白与TS形成复合体的亚细胞定位 和DHFR分别作为生长的函数；2)改变的影响 RTS-α和RTS-β在转染人癌细胞中的表达 细胞生长、TS酶活性和TS蛋白表达的特性， RTS：TS复合体的形成和癌细胞随后对TS的敏感性 3)RTS-β和RTS-α在大肠杆菌中的表达和纯化 巴斯德毕赤酵母及纯化的RTS蛋白对TS的影响 和DHFR酶活性及体外对TS抑制剂的敏感性； 4)RTS基因的定位和克隆，为进一步研究RTS基因奠定基础 结构和组织。

DESCRIPTION: (Applicant's Abstract) The long-term goal of this application is to determine the physiologic function of the rTS gene and its relevance to cancer therapeutics. rTS is a gene which overlaps the human thymidylate synthase (TS) gene and is encoded by the opposite strand of DNA. rTS is transcribed so as to produce one RNA (rTS-alpha) which is complementary to TS and to another, rTS-beta, which is not. The rTS gene codes for at least two proteins, rTS-alpha and rTS-beta. rTSB has been found to be overexpressed in three cell lines resistant to: 5-fluorouracil (H630-1, colon cancer), methotrexate (K562 B1A, chronic myelogenous leukemia and fluorodeoxyuridine plus ZD 1694 (HCT-8DF2, ileocecal carcinoma). Elevation of rTSB protein levels in the methotrexate-resistant, and fluorodeoxyuridine plus ZD 1694 cell lines are accompanied, respectively, by a complete and partial loss of growth regulation of TS activity. rTSB protein levels also increase as cells progress from log to stationary phase growth. Immunoprecipitation data indicate that rTSB, and a newly discovered protein, rTS, are complexed with TS and dihydrofolate reductase (DHFR). Taken together, the applicant has presented substantial preliminary data that suggest that rTS proteins appear to play a role in growth regulation of TS and in the development of cellular drug resistance. Four specific aims are proposed to investigate how rTS may impact on TS gene expression, cellular physiology, and sensitivity to TS inhibitors. They are as follows: 1) Extended characterization of rTS protein expression and identification of and subcellular localization of complex formation of rTS proteins with TS and DHFR, respectively, as a function of growth; 2) Effect of altered expression of rTS-alpha and rTS-beta in transfected human cancer cells on properties of cell growth, TS enzyme activity and TS protein expression, formation of rTS:TS complex and subsequent sensitivity of cancer cells to TS inhibitors; 3) Expression and purification of rTS-beta and rTS-alpha from Pichia pastoris and to determine the effects of purified rTS protein on TS and DHFR enzyme activity and on sensitivity to TS inhibitors in vitro; and 4) Mapping and cloning of the rTS gene in order to characterize the rTS gene structure and organization.