Retroviral Vector-mediated Overexpression of the R11j3 Subunit of the cAMP-dependent Protein Kinase Induces Differentiation in Human Leukemia Cells and Reverts the Transformed Phenotype of Mouse Fibroblasts

We have recently shown, using antisense strategy, that the R11j3 regulatory subunit of cAMP-dependent protein kinase is essential for cAMP-induced growth inhibition and differentiation of HL-6O human leukemia cells. We constructed a retroviral vector for RII 3 (MT-RIIf ) by inserting human RII 3 complementary DNA into the OTi 521 retroviral vector plasmid that contains an internal mouse metallothionein-i promoter and a neomycin resistance gene. The PA31 7 packaging cell line was then transfected with MT-Rll plasmid to produce the amphotrophic stock of MT-RIIf3 retroviral vector. The infection with MT-RlIfJ and treatment with CdCl2 brought about growth arrest in HL-60 human leukemia and Ki-ras-transformed NIH 3T3 clone DT cells in monolayer culture with no sign of toxicity. The growth inhibition correlated with the expression of RIIIJ and accompanied changes in cell morphology; cells became flat, exhibiting enlarged cytoplasm. The growth of these cells in semisolid medium (anchorage-independent growth) was almost completely suppressed. In contrast, overexpression of the RIa subunit of protein kinase enhanced the cell proliferation in DT cells. The MTRII -infected cells exhibited an increased sensitivity toward treatment with cAMP analogues, such as 8-ClcAMP and M-benzyl-cAMP, as compared with the parental noninfected cells. In MT-RII Ht-60 cells, Mbenzyl-cAMP treatment greatly enhanced the expression of monocytic surface markers. These results suggest that the RIIJ3 cAMP receptor, by binding to its ligand, cAMP, acts as a tumor suppressor protein exerting growth inhibition, differentiation, and reverse transformation.