Open Collections

Regulation of the c-myc gene by cytokines and growth factors

University of British Columbia

The proto-oncogene transcription factor c-myc has long been associated with cell growth as this gene is frequently found in many tumors to have undergone amplification or rearrangements with other genes. These alterations result in unregulated expression of an otherwise normal gene product. While c-myc is required for growth and differentiation the mechanism as to how c-myc contributes to these processes is not understood. Treatment of a variety of cells with a growth factor results in the accumulation of c-myc mRNA and Myc protein although the proteins involved in regulating this process are unknown. Growth factor stimulation also results in increased Phosphatidylinositol 3-Kinase (PI 3-Kinase) activity, which is also required for growth, however the function of PI 3-Kinase in mitogenesis is not well understood although it has been shown to prevent apoptosis. During the course of this thesis several lines of evidence suggested that PI 3-Kinase might be involved in the regulation of c-myc gene expression. I show here that increased levels of c-myc mRNA induced by growth factor treatment is dependent on PI 3-Kinase activity. Two structurally and mechanistically distinct inhibitors of PI 3-Kinase, the synthetic molecule LY294002 or the fungal metabolite wortmannin, blocked the increases in c-myc mRNA induced by platelet derived growth factor (PDGF) treatment of NIH-3T3 fibroblasts, or mast cells stimulated with interleukin-3 (IL-3), I L - 4 or Steel locus factor (SLF). Another means of blocking PI 3-Kinase activity is the expression of a dominant interfering mutant of the regulatory subunit of PI 3-Kinase called Δp85. This mutant possesses the domains required for recruitment to activated receptors, and other intermediate proteins, but lacks the domain required for binding to the catalytic subunit of PI 3- Kinase. Transiently over-expressing this mutant protein prevents activation of the endogenous PI 3-Kinase by competing with the endogenous regulatory subunit for binding to these activated receptors and intermediate proteins. In these transient systems c-myc gene expression can not be monitored so a reporter plasmid containing the c-myc promoter fused to the firefly luciferase gene was also transiently introduced. Luciferase activity obtained by treatment of cells with IL-3 or IL-4 was blocked by the co-expression of the Δp85 mutant. These results show that PI 3- Kinase activity is required for expression of the c-myc gene and also suggest that the signaling pathway to the c-myc gene lies downstream of PI 3-Kinase. This suggests a novel function for PI 3-Kinase, in addition to its anti-apoptotic role, and possibly explains the requirement of PI 3- Kinase activity for mitogenesis. The role of molecules activated by PI 3-Kinase, in the regulation of the c-myc gene, was also investigated. One of these molecules is the protein serine/threonine kinase called mammalian Target of Rapamycin (mTOR) which is inhibited by the drug rapamycin but also by wortmannin or LY294002. Rapamycin failed to block the increased levels of c-myc mRNA induced by treatment with IL-3, IL-4 or SLF in hemopoietic cells or PDGF in NIH-3T3 fibroblasts. Thus c-myc induction correlated with PI 3-Kinase activity but not mTOR activity suggesting the signal transduction pathway, regulating the c-myc gene, lies downstream of PI 3- Kinase but does not involve mTOR.

Thesis/Dissertation

application/pdf

2009-07-03

For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.

http://hdl.handle.net/2429/10081

Experimental Medicine

University of British Columbia

UBCV

DSpace