Su Z Z, Yemul S, Stein C A, Fisher P B
Department of Urology, Institute of Cancer Research, Columbia University, College of Physicians and Surgeons, New York, New York 10032, USA.
Oncogene. 1995 May 18;10(10):2037-49.
The early gene expression changes mediating carcinogen enhancement of viral transformation (CET) remain to be elucidated. A model cell culture system has been developed that is now permitting a molecular analysis of CET. Pretreatment of cloned rat embryo fibroblast (CREF) cells with methyl methanesulfonate (MMS) prior to infection with the cold-sensitive host-range type 5 adenovirus mutant, H5hr1, results in a dose-dependent increase in viral transformation. The present study investigates the role of immediate-early response genes, specifically c-fos, in the CET process. MMS pretreatment, alone or in combination with infection with H5hr1 temporally and differentially increases c-fos, c-jun, jun-B, jun-D and c-myc steady-state mRNA levels. Maximum induction occurs with c-fos and c-jun 8 to 12 h posttreatment and the magnitude of response is generally greatest in CREF cells pretreated with MMS and then infected with H5hr1. Enhancement in RNA levels is observed in the presence of cycloheximide indicating that ongoing protein synthesis is not required for induction of c-fos, c-jun, jun-B or c-myc expression. Nuclear run-on analysis indicates an enhancement in transcriptional rates for c-fos, c-jun, jun-B and c-myc in CREF cells treated with MMS or MMS plus infection with H5hr1. A requirement for elevated c-fos in the early stages of CET is indicated by the ability of c-fos antisense oligonucleotides to prevent the CET process. Direct evidence implicating early increases in c-fos as a mediator of the CET process is demonstrated by stably expressing mouse mammary tumor virus promoter-regulated human sense and antisense c-fos genes in CREF cells. Induction of c-fos sense expression by dexamethasone (DEX) in the absence of MMS treatment results in enhanced c-fos mRNA, Fos protein, AP-1 DNA-binding activity and H5hr1-induced transformation and CET. Induction of c-fos expression by DEX in stable c-fos-sense CREF constructs also results in elevated levels of c-jun, jun-B and c-myc mRNA and protein. Conversely, induction of c-fos antisense expression prevents the increase in c-fos mRNA, Fos protein and AP-1 DNA-binding activity and eliminates CET. In the antisense-c-fos constructs, increases in c-jun, jun-B and c-myc mRNA and protein normally induced by MMS also are not apparent. Thus, induction or inhibition in c-fos expression affects the level of expression of additional immediate-early response genes, including c-jun, jun-B and c-myc.(ABSTRACT TRUNCATED AT 400 WORDS)
介导致癌物增强病毒转化（CET）的早期基因表达变化仍有待阐明。现已开发出一种模型细胞培养系统，它使得对CET进行分子分析成为可能。在用冷敏感宿主范围5型腺病毒突变体H5hr1感染之前，用甲磺酸甲酯（MMS）预处理克隆大鼠胚胎成纤维细胞（CREF），会导致病毒转化呈剂量依赖性增加。本研究调查即刻早期反应基因，特别是c-fos，在CET过程中的作用。单独的MMS预处理或与H5hr1感染联合使用，会在时间上和差异上增加c-fos、c-jun、jun-B、jun-D和c-myc的稳态mRNA水平。c-fos和c-jun在处理后8至12小时出现最大诱导，并且在用MMS预处理然后感染H5hr1的CREF细胞中反应幅度通常最大。在放线菌酮存在的情况下观察到RNA水平升高，这表明c-fos、c-jun、jun-B或c-myc表达的诱导不需要正在进行的蛋白质合成。细胞核连续分析表明，在用MMS或MMS加H5hr1感染处理的CREF细胞中，c-fos、c-jun、jun-B和c-myc的转录速率增强。c-fos反义寡核苷酸能够阻止CET过程，这表明在CET早期阶段需要升高的c-fos。通过在CREF细胞中稳定表达小鼠乳腺肿瘤病毒启动子调控的人有义及反义c-fos基因，证明了直接证据表明c-fos的早期增加作为CET过程的介导物。在没有MMS处理的情况下，地塞米松（DEX）诱导c-fos有义表达会导致c-fos mRNA、Fos蛋白、AP-1 DNA结合活性增强，以及H5hr1诱导的转化和CET增强。在稳定的c-fos有义CREF构建体中，DEX诱导c-fos表达也会导致c-jun、jun-B和c-myc mRNA及蛋白水平升高。相反，诱导c-fos反义表达会阻止c-fos mRNA、Fos蛋白和AP-1 DNA结合活性的增加，并消除CET。在反义c-fos构建体中，通常由MMS诱导的c-jun、jun-B和c-myc mRNA及蛋白增加也不明显。因此，c-fos表达的诱导或抑制会影响包括c-jun、jun-B和c-myc在内的其他即刻早期反应基因的表达水平。（摘要截断于400字）
