Overview: Melanoma cells, unlike normal melanocytes, are in a perpetual mode of cell division regardless of extracellular inhibiting signals. Substantial evidence implicates contributions from at least two major processes to this aberrant behavior: a) loss of p16INK4a functional inhibition of cyclin dependent kinases 4 and 6 (CDK4/6); and b) aberrant expression of bFGF (basic fibroblast growth factor). Hypothesis: The two processes are intricately linked and cooperatively induce constitutive CDK activity and the accumulation of E2F transcription factor, both being known rate-limiting reactions in cell cycle progression.
Specific Aims :
Aim number 1: To establish the role of CDK inhibitors (CKI) and E2F transcription factor in bFGF mediated growth dysregulation. We will examine the oncogenic effect of ectopic bFGF or E2F expression in melanocytes deficient for genes encoding specific CKI. To this end we will infect wild-type or knockout mouse melanocytes, deleted for CKI p16, p2l or p27, or the transcription factor E2F1 with a tetracycline(tet)-inducible retroviral vector encoding bFGF or E2F. The effect of transient versus sustained-expression of the ectopic genes on cell proliferation and tumorigenicity will be determined.
Aim number 2. To identify the effector molecules for bFGF-mediated autonomous growth. CDK and E2F activities will be monitored as the melanocytes are induced to produce bFGF. Included are determinations of retinoblastoma tumor suppressor protein (Rb) abundance and phosphorylation levels, histone 1 phosphorylating activity, cyclin-associated kinase activity, E2F transcriptional activity, E2F/DNA complex association and expression of E2F-responsive genes.
Aim number 3. To determine if inactivation of Rb and accumulation of E2F can be attributed to aberrant bFGF expression in autonomously proliferating human melanoma cells. We will examine the critical intermediates downregulated by disruption of the bFGF/FGF1 pathway in human melanoma cells in which the FGFR1 or bFGF have been neutralized by transfection with tet-inducible retroviral vector encoding a dominant-negative variant of the FGFR1 (dnflg) or anti-sense bFGF, respectively. Significance: Our unique cellular systems will enable us to systematically study the molecular changes induced by genetic alterations and aberrant expression of a growth factor identified in melanomas. Our functional studies may provide a basis for the design of new therapeutical agents for the treatment of patients with metastatic melanomas.
| Halaban, R; Cheng, E; Smicun, Y et al. (2000) Deregulated E2F transcriptional activity in autonomously growing melanoma cells. J Exp Med 191:1005-16 |
