C-FOS IS A POSITIVE REGULATOR OF CARCINOGEN ENHANCEMENT OF ADENOVIRUSTRANSFORMATION

The early gene expression changes mediating carcinogen enhancement of viral transformation (GET) remain to be elucidated. A model cell cultu re system has been developed that is now permitting a molecular analys is of GET. Pretreatment of cloned rat embryo fibroblast (CREF) cells w ith methyl methanesulfonate (MMS) prior to infection with the cold-sen sitive host-range type 5 adenovirus mutant, H5hr1, results in a dose-d ependent increase in viral transformation, The present study investiga tes the role of immediate-early response genes, specifically c-fos, in the CET process, MMS pretreatment, alone or in combination with infec tion with H5hr1 temporally and differentially increases c-fos, c-jun, jun-B, jun-D and c-myc steady-state mRNA levels, Maximum induction occ urs 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 t hen 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 antisens e oligonucleotides to prevent the CET process, Direct evidence implica ting early increases in c-fos as a mediator of the CET process is demo nstrated by stably expressing mouse mammary tumor virus promoter-regul ated human sense and antisense c-fos genes in CREF cells, Induction of c-fos sense expression by dexamethasone (DEX) in the absence of MMS t reatment results in enhanced c-fos mRNA, Fos protein, AP-1 DNA-binding activity and H5hr1-induced transformation and GET. 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. Convers ely, induction of c-fos antisense expression prevents the increase in c-fos mRNA, Fos protein and AP-I DNA-binding activity and eliminates G ET. 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, T hus, induction or inhibition in c-fos expression affects the level of expression of additional immediate-early response genes, including c-j un, jun-B and c-myc. These results demonstrate a pivotal role for the early induction of c-fos and possibly c-fos responsive genes, in the C ET process.