This research is an investigation of some of the biochemical processes involved in malignant cell differentiation. The case under consideration is the differentiation of mouse neuroblastoma cells in tissue culture. The differentiation inducer to be studied is cyclic adenosine 3':5'-monophosphate (cAMP). The immediate goals of this research are to: (1) determine the role of cAMP-dependent protein kinase as an effector of cAMP action in neuroblastoma differentiation; (2) study the mechanism of the increased expression of R?I?, a free cAMP-binding protein, and its functonal significance in neuroblastoma cell differentiation. Our approaches to define the action of cAMP-dependent protein kinase in neuroblastoma cell differentiation include: (a) to screen/select, isolate, and characterize protein kinase-minus mutants of the neuroblastoma cells and to use these mutants to relate the occurence of cAMP-dependent protein kinase with the cells' sensitivity towards cAMP; (b) the use of small unilamellar liposomes to deliver cyclic nucleotides intracellularly to examine the occupancy of cAMP-receptor protein, the activation of cAMP-dependent protein kinase, and the evocation of cAMP-regulated biological processes in intact neuroblastoma cells; (c) the use of microinjection techniques to introduce purified cAMP-dependent protein kinase or an inhibitor of the kinase into intact neuroblastoma cells, to demonstrate that the expression of various differentiated functions in these cells is causally related to the activity of cAMP-dependent protein kinase in these cells. In order to gain a better understanding of the mechanism that underlies the increased experssion of the R?I? cAMP-binding protein during neuroblastoma cell differentiation, we will study the rate of synthesis and degradation of this protein and quantitate the level of mRNA coding for this protein during the course of differentiation. (M)
