1. Differential Response of C-RAF and B-RAF Melanoma Lines to Multiple Chemotherapy Treatments Shawna Dougherty Department of Biological Sciences, York College of Pennsylvania Introduction Melanoma is a disease in which malignant cells are found in the melanocytes on the surface of the skin. Melanoma can spread quickly to other parts of the body through the lymph system or through the blood and frequently ends in death. The American Cancer Society reported 62,190 new cases of melanoma and 7,910 deaths from melanoma in the year 2006 in the United States alone. Even with the urgent warning about melanoma, it is still on the rise. While a family history of melanoma and exposure to ultraviolet irradiation have been known for years as risk factors in melanoma development, the exact molecular pathway melanoma takes in cell growth has only recently been revealed. The RAS-RAF-MEK-ERK pathway is a signaling pathway which typically stimulates normal cell growth. Mutations in the pathway often lead to abnormal or excessive cell growth. Typically mutations occur in the B-RAF protein of the cell growth, however recent studies show that melanoma lines can also mutate in the RAS protein forcing the growth to take the C-RAF growth pathway. No current treatments substantially enhance patient survival once metastasis has occurred; it is now thought that different chemotherapy treatments could produce different results depending on the specific pathway taken to produce the tumor. Expected results Research design Literature Cited 1.Dumaz, N., Hayward, R., Martin, J., Ogilvie, L., Douglas, H., Curtin, J., Boris, B., Springer, C., Marais, R. 2006. In Melanoma, RAS Mutations Are Accompanied by Switching Signaling from BRAF to CRAF and Disrupted Cyclic AMP Signaling. Cancer Research. [serial online] 66: (19) Available from: Cancer Research database. 2.Kaufman, H. L. The Melanoma Book. New York: Penguin Group Inc., 2005. 3.P.T. Wan, S.M. Roe and D. Barford. 2006. Investigating the Molecular Mechanism of Oncogenic Mutations of B-RAF. Cell, (116) 855- 867. 4.Lackey K., Cory M., Davis R. 2000. The discovery of potent cRaf1 kinase inhibitors. Bioorg. Med. Chem. Lett. (10) 223–226. 5.Caspase-3 Fluorometric Assay Kit, Catalog No. 907-014, Simplify Your Science. 6.Chin, Paul C., Liu, Li, Morrison, Bradley E., Siddiq, Ambreena, Ratan, Rajiv R., Bottiglieri, Teodoro & D'Mello, Santosh R. 2004. The c- Raf inhibitor GW5074 provides neuroprotection in vitro and in an animal model of neurodegeneration through a MEK-ERK and Akt-independent mechanism. Journal of Neurochemistry 90 (3), 595-608. Acknowledgments Ron Kaltreider, PhD for his advise and guidance throughout the project. Objective The hypothesis of this experiment is that C-RAF and B-RAF mutated melanoma lines will react differently to different mechanistic chemotherapy drugs. To determine if cell apoptosis differs in C- RAF and B-RAF mutated melanoma lines in vitro when treated with different known forms of chemotherapy drugs. The goal is to improve treatment strategies for specific melanoma lines and identify specific MEK protein kinase activation. Figure 1: Melanoma development. Taken from: www.plwc.org Images of Melanoma. Taken from: www.healthgate.partners.org Figure 2: MEK pathway including Caspase 3 and apoptosis stages of the cell cycle. Proposed alteration of RAF pathway. Image adapted from: www.dundee.ac.uk/biomedres/clarke.htm RAS C-RAFB-RAF MEK1/2 Images of Melanoma Classifications. Taken from University Health Care Systems Cell Culture (1) 37° incubation C-RAF lines (Sbcl2, WM852, WM1366, WM1791c.) B-RAF lines (A375, WM266.4, Colo829, SkMel28. Plate 4,000 cells in 96 well cell plate 37 ° incubation Day 1 Day 2 C-RAF inhibitor GW5074 B-RAF inhibitor Nexavar® (BAY43-9006) Chemotherapy Drugs (2.0x10 -7 - 2.0x10 3 μm) DTIC, BCNU, Velban 4 Hours Detection Caspase-3 Fluorometric Assay Kit to determine effectiveness via apoptosis (5) Procedure followed according to manual Excitation at 360nm Emission at 440nm Review of Literature RAS is a small G-protein, guanine nucleotide binding protein, involved in second messenger cascades (1) RAS acts as a switch that activates the RAF, MEK, and ERK cascade of protein kinases Three known forms of RAF, A-RAF, B-RAF and C-RAF exist, of these C-RAF and B-RAF appear to be the most relevant for melanoma (1) B-RAF mutations occur in 50% to 70% RAS mutations occur in 15% to 20% of melanoma cases which cause the selective activation of C-RAF Three common chemotherapy drugs used to treat melanoma are Dacarbazine (DTIC), Carmustine (BCNU), and Vinblastine (Velban) (2) DTIC is a purine analog interfering DNA and RNA synthesis BCNU interferes with DNA, RNA, and protein synthesis by alkylation and cross-linking Velban prevents microtubule depolarization Specific inhibitors exist for B-RAF (BAY 43- 9006, 3) and C-RAF (GW5074, 4) Expected Conclusions C-RAF mutations are expressed in neuronal tissue (6) and linked to brain melanoma (2). We predict BCNU would have greater efficacy on C-RAF mutants due to the drug’s ability to cross into the brain (2). DTIC is the most effective chemotherapy drug known for melanoma. Apoptosis in B- RAF will be greater than C-RAF since 50- 70% of melanoma lines have B-RAF mutations.