Cyclin A is a c-Jun target gene and is necessary for c-Jun-induced anchorage-independent growth in RAT1a cells.
Overexpression of c-Jun enables Rat1a cells to grow in an anchorage-independent manner. We used an inducible c-Jun system under the regulation of doxycycline in Rat1a cells to identify potential c-Jun target genes necessary for c-Jun-induced anchorage-independent growth. Induction of c-Jun results in sustained expression of cyclin A in the nonadherent state with only minimal expression in the absence of c-Jun. The promoter activity of cyclin A2 was 4-fold higher in Rat1a cells in which c-Jun expression was induced compared with the control cells. Chromatin immunoprecipitation demonstrated that c-Jun bound directly to the cyclin A2 promoter. Mutation analysis of the cyclin A2 promoter mapped the c-Jun regulatory site to an ATF site at position -80. c-Jun was able to bind to this site both in vitro and in vivo, and mutation of this site completely abolished promoter activity. Cyclin A1 was also elevated in c-Jun-overexpressing Rat1a cells; however, c-Jun did not regulate this gene directly, since it did not bind directly to the cyclin A1 promoter. Suppression of cyclin A expression via the introduction of a cyclin A antisense sequences significantly reduced the ability of c-Jun-overexpressing Rat1a cells to grow in an anchorage-independent fashion. Taken together, these results suggest that cyclin A is a target of c-Jun and is necessary but not sufficient for c-Jun-induced anchorage-independent growth. In addition, we demonstrated that the cytoplasmic oncogenes Ras and Src transcriptionally activated the cyclin A2 promoter via the ATF site at position -80. Using a dominant negative c-Jun mutant, TAM67, we showed that this transcriptional activation of cyclin A2 requires c-Jun. Thus, our results suggest that c-Jun is a mediator of the aberrant cyclin A2 expression associated with Ras/Src-induced transformation.
Katabami
Motoo
M
Department of Cell and Cancer Biology, NCI, National Institutes of Health, Rockville, Maryland 20850, USA.
Donninger
Howard
H
Hommura
Fumihiro
F
Leaner
Virna D
VD
Kinoshita
Ichiro
I
Chick
Jeffrey F B
JF
Birrer
Michael J
MJ
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002448:Cell Adhesion; D002453:Cell Cycle; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D047369:Chromatin Immunoprecipitation; D019925:Cyclin A / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D056751:Cyclin A2; D003593:Cytoplasm / Q000378:metabolism; D004252:DNA Mutational Analysis; D004318:Doxycycline / Q000494:pharmacology; D005799:Genes, Dominant; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D009154:Mutation; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D051381:Rats; D013997:Time Factors; D014162:Transfection
C503722:Ccna2 protein, rat; D019925:Cyclin A; D056751:Cyclin A2; D016376:Oligonucleotides, Antisense; D016755:Proto-Oncogene Proteins c-jun; D049452:Green Fluorescent Proteins; D008156:Luciferases; D004318:Doxycycline