title: 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.
abstract: We have recently shown, using antisense strategy, that the RII beta regulatory subunit of cAMP-dependent protein kinase is essential for cAMP-induced growth inhibition and differentiation of HL-60 human leukemia cells. We constructed a retroviral vector for RII beta (MT-RII beta) by inserting human RII beta complementary DNA into the OT1521 retroviral vector plasmid that contains an internal mouse metallothionein-1 promoter and a neomycin resistance gene. The PA317 packaging cell line was then transfected with MT-RII beta plasmid to produce the amphotrophic stock of MT-RII beta retroviral vector. The infection with MT-RII beta 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 RII beta 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 RI alpha subunit of protein kinase enhanced the cell proliferation in DT cells. The MT-RII beta-infected cells exhibited an increased sensitivity toward treatment with cAMP analogues, such as 8-Cl-cAMP and N6-benzyl-cAMP, as compared with the parental noninfected cells. In MT-RII beta HL-60 cells, N6-benzyl-cAMP treatment greatly enhanced the expression of monocytic surface markers. These results suggest that the RII beta cAMP receptor, by binding to its ligand, cAMP, acts as a tumor suppressor protein exerting growth inhibition, differentiation, and reverse transformation.
authors.affiliation: Cellular Biochemistry Section, National Cancer Institute, NIH, Bethesda, Maryland 20892.
authors.forename: G
authors.initials: G
authors.lastname: Tortora
authors.forename: A
authors.initials: A
authors.lastname: Budillon
authors.forename: H
authors.initials: H
authors.lastname: Yokozaki
authors.forename: T
authors.initials: T
authors.lastname: Clair
authors.forename: S
authors.initials: S
authors.lastname: Pepe
authors.forename: G
authors.initials: G
authors.lastname: Merlo
authors.forename: C
authors.initials: C
authors.lastname: Rohlff
authors.forename: Y S
authors.initials: YS
authors.lastname: Cho-Chung
mesh_terms: D016475:3T3 Cells; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000495:Allosteric Site; D000818:Animals; D002104:Cadmium / Q000494:pharmacology; D002454:Cell Differentiation / Q000502:physiology*; D002455:Cell Division; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology*; D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D015473:Leukemia, Promyelocytic, Acute / Q000473:pathology; D008668:Metallothionein / Q000235:genetics; D051379:Mice; D010641:Phenotype; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology*; D014407:Tumor Cells, Cultured
chemical_list: D054757:Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit; D007527:Isoenzymes; C517837:PRKAR2B protein, human; C517838:Prkar2b protein, mouse; D011993:Recombinant Fusion Proteins; D002104:Cadmium; D015124:8-Bromo Cyclic Adenosine Monophosphate; C071595:N(6)-benzyl-cyclic adenosine 5'-monophosphate; D008668:Metallothionein; C044258:8-chloro-cyclic adenosine monophosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases