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 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.
Tortora
G
G
Cellular Biochemistry Section, National Cancer Institute, NIH, Bethesda, Maryland 20892.
Budillon
A
A
Yokozaki
H
H
Clair
T
T
Pepe
S
S
Merlo
G
G
Rohlff
C
C
Cho-Chung
Y S
YS
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
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