Scanlon K J, Jiao L, Funato T, Wang W, Tone T, Rossi J J, Kashani-Sabet M
Department of Medical Oncology, City of Hope National Medical Center, Duarte, CA 91010.
Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10591-5. doi: 10.1073/pnas.88.23.10591.
The c-fos gene product Fos has been implicated in many cellular processes, including signal transduction, DNA synthesis, and resistance to antineoplastic agents. A fos ribozyme (catalytic RNA) was designed to evaluate the effects of suppressing Fos protein synthesis on expression of enzymes involved in DNA synthesis, DNA repair, and drug resistance. DNA encoding the fos ribozyme (fosRb) was cloned into the pMAMneo expression plasmid, and the resultant vector was transfected into A2780DDP cells resistant to the chemotherapeutic agent cisplatin. The parental drug-sensitive A2780S cells were transfected with the pMMV vector containing the c-fos gene. Morphological alterations were accompanied by significant changes in pharmacological sensitivity in both c-fos- and fosRb-transfected cells. pMAMneo fosRb transfectants revealed decreased c-fos gene expression, concomitant with reduced thymidylate (dTMP) synthase, DNA polymerase beta, topoisomerase I, and metallothionein IIA mRNAs. In contrast, c-myc expression was elevated after fos ribozyme action. Insertion of a mutant ribozyme, mainly capable of antisense activity, into A2780DDP cells resulted in smaller reductions in c-fos gene expression and in cisplatin resistance than the active ribozyme. These studies establish a role for c-fos in drug resistance and in mediating DNA synthesis and repair processes by modulating expression of genes such as dTMP synthase, DNA polymerase beta, and topoisomerase I. These studies also suggest the utility of ribozymes in the analysis of cellular gene expression.
原癌基因c-fos的产物Fos参与了许多细胞过程，包括信号转导、DNA合成以及对抗肿瘤药物的耐药性。设计了一种fos核酶（催化RNA），以评估抑制Fos蛋白合成对参与DNA合成、DNA修复和耐药性的酶表达的影响。将编码fos核酶（fosRb）的DNA克隆到pMAMneo表达质粒中，并将所得载体转染到对化疗药物顺铂耐药的A2780DDP细胞中。将含有c-fos基因的pMMV载体转染亲代药物敏感的A2780S细胞。形态学改变伴随着c-fos和fosRb转染细胞药理学敏感性的显著变化。pMAMneo fosRb转染子显示c-fos基因表达降低，同时胸苷酸合酶（dTMP）、DNA聚合酶β、拓扑异构酶I和金属硫蛋白IIA的mRNA水平降低。相反，fos核酶作用后c-myc表达升高。将主要具有反义活性的突变核酶插入A2780DDP细胞中，与活性核酶相比，c-fos基因表达和对顺铂的耐药性降低幅度较小。这些研究确定了c-fos在耐药性以及通过调节dTMP合酶、DNA聚合酶β和拓扑异构酶I等基因的表达来介导DNA合成和修复过程中的作用。这些研究还表明核酶在细胞基因表达分析中的实用性。
