MECHANISM OF CAMP ACTION IN GROWTH CONTROL, DIFFERENTIATION, AND GENE REGULATION

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The striking growth inhibitory effect of 8-Cl-cAMP has been related to its selective binding and activation of protein kinase isozymes: It binds to RII with a high affinity for Site B but with a low affinity for Site A, keeping type Il protein kinase in the holoenzyme form, while binding with moderately high affinity for both Site A and Site B to RI, facilitating dissociation of the RI subunit and down-regulation of type I protein kinase. The growth inhibition induced by 8-Cl-cAMP brought about various effects among the cell lines tested, including the suppression of oncogenes and transforming growth factor a (TGFalpha), and morphological changes, differentiation, and reverse transformation. Despite the appearance of markers of mature phenotype and definitive growth arrest, the 8-Cl-cAMP-treated leukemic cells exhibited no change in the cell cycle phase. 8-Cl-cAMP therefore produces growth inhibition while allowing the cells to progress through their normal cell cycle, albeit at a slower rate, and this may lead to eventual restoration of a balance between cell proliferation and differentiation in cancer cells. Thus, unlike cytotoxic drugs, 8-Cl-cAMP does not act to prevent mitosis but acts to alter the growth ratio, the ratio of cell births to cell deaths, via restoration of the RI/RII balance in cancer cells. The cellular events underlying growth inhibition and differentiation of cancer cells induced by 8-Cl-cAMP include a rapid nuclear translocation of RIIbeta, and such translocation of RIIbeta into the nucleus correlates with an increase n transcription factors in cancer cells that bind specifically to cAMP response element (CRE). Thus, the mechanism of action of 8-Cl-cAMP in the suppression of malignancy may involve the restoration of normal gene transcription in cancer cells where the RIIbeta cAMP receptor plays an important role. By the use of site-directed mutagenesis technique, the structure-function analysis of RI and Rll is currently underway. The RI and Rll are distinguished by their autophorylation and nuclear translocation properties. RII has an autophosphorylation site at a proteolytically sensitive hinge region around the R and C interaction site while RI has a pseudo-phosphorylation site. The Rll but not the RI contains a nuclear location signal, K K R K. The autophosphorylation and nuclear location sequences are either point-mutated in RIIbeta of introduced into RIalpha to specifically assess the role of these sequences in the growth regulatory function. These studies contribute to understanding the mechanism of cAMP control cell growth and differentiation and provide new approaches to the treatment of cancer.