My long-term goal is to identify the genes and gen products responsible for the proliferation and malignant transformation of human melanocytes. My approach is based on knowledge concerning the growth factor requirements of normal and malignant human melanocytes in culture. Normal human melanocytes, unlike pigment cells from metastatic melanomas, do not survive in culture, neither in serum-supplemented nor in defined medium. Until recently, normal human melanocytes could proliferate well in culture only in the presence of 12-0-tetradecanoyl-phorbol-13-acetate (TPA) in synergy with substances that increase intracellular levels of cyclic-adenosine-monophosphate (cAMP). We demonstrated that mitogenic activity in normal tissues and melanoma cell extracts was immunologically related to basic fibroblast growth factor (bFGF) and that bFGF was the only mitogen for melanocytes that could substitute for TPA. It is thus possible that abnormal production of bFGF has a role in the uncontrolled growth of transformed melanocytes. Other gene products might be those conferring independence from cAMP stimulators.
My specific aims are therefore: I. To determine at which stage of malignant transformation melanomas becomes independent of and/or produce bFGF, and what is the mechanism of bFGF induction. II. To find out whether the genes that confer independence form TOA, bFGF, and stimulators of cAMP to melanomas are the genes for bFGF and cAMP-dependent protein kinases. III. To identify the receptor for bFGF in melanocytes and to compare its expression in normal and malignant melanocytes. IV. To compare the expression of cAMP-dependent protein kinases in normal and malignant melanocytes. The techniques to be employed are cultured normal and malignant melanocytes, antibodies to bFGF and subunits of protein kinase A, Northern blot analysis with cDNA probes to mRNAs of bFGF and subunits of protein kinase A, cellular DNA transfection, and transfection with specific constructs encoding bFGF and the catalytic subunit of protein kinase A.
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