cAMP inhibits the formation of and induces regression of some experimental tumors and reverses the phenotype of several transformed cell lines. All eukaryotic effects of cAMP are mediated by binding to regulatory subunits (R-subunits) of cAMP- dependent protein kinase (Pk-A). In the previous grant periods, we found changes in the R-subunit (RII) of the type II Pk-A isozyme during neoplastic mouse lung development. These changes include a loss of high affinity binding of the photoaffinity analog, 8-N3(32P)-cAMP, altered regulation of RII autophosphorylation by cAMP,and an inability of cAMP to cause dissociation of tumor Pk-A. We herein address the hypothesis that the altered RII in lung tumors represents an important mechanism by which these cells escape the constraints of normal growth regulation. The biological consequences of altered RII will be investigated by examining changes in those proteins which bind RII, using a Western blotting procedure, and by determining the intracellular location of tumor RII by immunocytochemistry. The amount and intracellular location of free catalytic subunits, which have been dissociated from the Pk-A holoenzyme by agents which raise intracellular cAMP levels in vitro, will be compared between normal lung cells and cells derived from lung tumors. Among those inbred strains of mice we have examined, lung RII from one strain had characteristics of tumor RII; this normal RII will be characterized extensively to better understand the range of possible RII microheterogeneity and its relationship to neoplastic growth. Genetic differences will also be exploited by isolating bronchiolar Clara cells from an inbred strain whose tumors are mainly derived from this cell type, and comparing the RII of those cells, at different times after urethan administration in vivo, with Clara cell RII from inbred strains whose Clara cells are not at risk for neoplasia. RII will be used as a monitor for comparing cell populations which are fixed at a benign stage with those which progress to malignancy, for comparing tumorigenic and non-tumorigenic type 2 cells (the other cell of origin of lung adenomas) and proliferating and non-proliferating type 2 cells.
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