): Ras is a family of GTP-binding proteins which is responsible for mediating cellular proliferation, differentiation and apoptosis. ras is a widely mutated proto-oncogene in human tumors. Oncogenic Ras chronically binds GTP resulting in the constitutive activation of downstream MAPK, MAPK-like and MAPK- independent signaling cascades. The ability of oncogenic Ras to transform cells is due, in part, to the ability of MAPK signaling pathways to activate transcription factors (such as Ets, c-Myc, and c-Jun) which are required for proliferation. These Ras-responsive transcription factors are required for transformation; however, the mechanisms by which these proteins facilitate o n cogenesis have not been fully established. Although the NFkappa B transcription factor is known to play a critical role in regulating immune responses, recent evidence has suggested that NF kappa B may be important for oncogene-mediated transformation. Preliminary data presented in this proposal indicate that NF kappa B is required by oncogenic Ras to suppress programmed cell death. Data presented in this proposal indicate that oncogenic Ras requires the Rac to activate NF kappa B, but that Rac induces apoptosis in cells which lack the NF kappa B transcription factor. Although Rac is known to activate JNK and actin polymerization, Rac-induced cell killing (following the loss of NF kappa B activity) was associated with the ability of the p21 GTPase to activate the NADPH oxidase complex. Ras-transformed-cells, which lack NF kappa B transcriptional activity, displayed elevated intracellular reactive oxygen species (ROS, including increased superoxide levels), while the inhibition of NADPH oxidase rescued oncogenic Ras-mediated apoptosis. In addition, the superoxide generating cytokine, TNF alpha, required NF kappa B to upregulate inhibitor apoptosis proteins (IAPs) which block programmed cell death by inhibiting caspase-dependent cascades. Data presented also indicate that the Akt anti-apoptotic pathway, which has been implicated in the inactivation of the BAD pro-apoptotic protein, stimulated the transcriptional activity of NF kappa B. This finding suggests that the cell survival functions of Akt may be mediated through the upregulation of NF kappa B. The main objective of this grant is to understand the mechanisms by which Ras utilizes N F k appa B to overcome apoptotic pathways and facilitate cellular transformation. This will be accomplished by addressing three specific aims.
Aim 1 will elucidate whether NF kappa B is required to maintain the redox status of the cell following oncogenic Ras expression, and will identify NF kappa B-regulated gene products which are required to block Ras-induced apoptosis by re-establishing the antioxidant balance in the cell.
Aim 2 will address whether oncogenic Ras mediates apoptosis through a mechanism involving caspase-induced cytochrome c release, and will elucidate whether NF kappa B is required to upregulate IAP proteins in order to block H-Ras(V12)-induced apoptosis. Additionally, this aim will identify NF kappa B-regulated genes r e sponsible for potentiating transformation by inhibiting Ras-mediated apoptosis.
Aim 3 will identify the Akt signaling pathways utilized to provide NF kappa B-dependent transcription.
This aim will also determine whether the anti-apoptotic function of Akt requires NF kappa B. These studies will elucidate the mechanisms by which NF kappa B provides protection against apoptosis in response to oncogenic Ras and will potentially provide new insight into the development of anticancer therapies.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Scientist Development Award - Research & Training (K01)
Project #
5K01CA078595-05
Application #
6513298
Study Section
Subcommittee G - Education (NCI)
Program Officer
Eckstein, David J
Project Start
1999-08-19
Project End
2004-06-30
Budget Start
2002-07-01
Budget End
2003-06-30
Support Year
5
Fiscal Year
2002
Total Cost
$146,722
Indirect Cost
Name
University of Virginia
Department
Biochemistry
Type
Schools of Medicine
DUNS #
001910777
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
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