RETROVIRAL VECTOR-MEDIATED OVEREXPRESSION OF THE RII-BETA 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 RII beta re gulatory subunit of cAMP-dependent protein kinase is essential for cAM P-induced growth inhibition and differentiation of HL-60 human leukemi a cells. We constructed a retroviral vector for RII beta (MT-RII beta) by inserting human RII beta complementary DNA into the OT1521 retrovi ral vector plasmid that contains an internal mouse metallothionein-1 p romoter and a neomycin resistance gene. The PA317 packaging cell line was then transfected with MT-RII beta plasmid to produce the amphotrop hic stock of MT-RII beta retroviral vector. The infection with MT-RII beta and treatment with CdCl2, brought about growth arrest in HL-60 hu man leukemia and Ki-ras-transformed NIH 3T3 clone DT tells in monolaye r culture with no sign of toxicity. The growth inhibition correlated w ith the expression of RII beta and accompanied changes in cell morphol ogy; cells became flat, exhibiting enlarged cytoplasm. The growth of t hese cells in semisolid medium (anchorage-independent growth) was almo st completely suppressed. In contrast, overexpression of the Rl alpha subunit of protein kinase enhanced the cell proliferation in DT cells. The MT-RII beta-infected cells exhibited an increased sensitivity tow ard treatment with cAMP analogues, such as 8-Cl-cAMP and N-6-benzyl-cA MP, as compared with the parental noninfected cells. In MT-RII beta HL -60 cells, N-6-benzyl-cAMP treatment greatly enhanced the expression o f monocytic surface markers. These results suggest that the RII beta c AMP receptor, by binding to its ligand, cAMP, acts as a tumor suppress or protein exerting growth inhibition, differentiation, and reverse tr ansformation.