Dominant-negative Rac1 suppresses Ras-induced apoptosis possibly through activation of NFkappaB in Ha-ras oncogene-transformed NIH/3T3 cells.
We investigated the involvement of Rac1 in Ha-ras-overexpression-induced apoptosis using a murine NIH/3T3-derived cell line (designated 7-4), which contains an inducible Ha-ras oncogene under the regulation of Escherichia coli lac operator-repressor system. Ha-ras overexpression was induced by isopropyl beta-D-thiogalactoside (IPTG). To reveal the role of endogenous Rac1, the dominant negative Rac1(Asn17) gene was transfected into the 7-4 cells. Using two cell lines 7-4 Racd2 and 7-4 Racd3 (7-4 derivates) stably expressing Rac1(Asn17), we demonstrate that suppression of Rac1 activity blocked Ha-ras-overexpression-induced apoptosis under a serum-depleted condition, indicating that Rac1 activity is required for a Ras-mediated apoptosis pathway. Cell-cycle analysis revealed that dominant-negative Rac1 partially shifted cell population from S-phase to G0/G1 phase in the cells overexpressing Ha-ras. In contrast to other reports, we showed activation of the transcription factor NFkappaB in the two cell lines expressing dominant-negative Rac1. All together, our results demonstrate that Ha-ras-overexpression- induced apoptosis can be blocked by dominant-negative Rac1, possibly through decreased S-phase accumulation and increased NFkappaB activity.
Chou
Chao-Kai
CK
Department of Microbiology, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan.
Liang
Kuo-Hsuan
KH
Tzeng
Ching-Chang
CC
Huang
Guan-Cheng
GC
Chuang
Jih-I
JI
Chang
Tsuey-Yu
TY
Liu
Hsiao-Sheng
HS
D016475:3T3 Cells; D000818:Animals; D017209:Apoptosis; D015152:Blotting, Northern; D002461:Cell Line, Transformed; D024202:Electrophoretic Mobility Shift Assay; D005799:Genes, Dominant / Q000502:physiology*; D011905:Genes, ras / Q000502:physiology*; D006801:Humans; D047468:Immunoprecipitation; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D051379:Mice; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016196:S Phase; D015398:Signal Transduction; D019076:Transgenes / Q000502:physiology; D020830:rac1 GTP-Binding Protein / Q000502:physiology*
D016328:NF-kappa B; C107953:RAC1 protein, human; D012333:RNA, Messenger; D007544:Isopropyl Thiogalactoside; D020830:rac1 GTP-Binding Protein