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12. Retroviral vector-mediated overexpression of the RIIbeta subunit of the cAMP-dependent protein kinase induces differentiation in human leukemia cells and reverts the transformed phenotype of mouse fibroblasts

Author

Giampaolo Tortora, Budillon A, Yokozaki H, Clair T, Pepe S, Merlo G, Rohlff C, Ys, Cho-Chung, Tortora, Giampaolo, Budillon, A., Clair, T., Pepe, Stefano, Yokozaki, H., Merlo, G., Rohlff, C., and Cho Chung, Y. S.

Subjects
RIIbeta subunit, Recombinant Fusion Proteins, Genetic Vectors, 8-Bromo Cyclic Adenosine Monophosphate, Mice, Leukemia, Promyelocytic, Acute, Cyclic AMP-Dependent Protein Kinase RIIbeta Subunit, Cyclic AMP, Tumor Cells, Cultured, Animals, Humans, PKA, transformation, Promoter Regions, Genetic, Cell Differentiation, 3T3 Cells, Cyclic AMP-Dependent Protein Kinases, Gene Expression Regulation, Neoplastic, Isoenzymes, Phenotype, Retroviridae, Metallothionein, Allosteric Site, Cell Division, Cadmium, Signal Transduction
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.

Published
1994

15. An antisense oligodeoxynucleotide that depletes RI alpha subunit of cyclic AMP-dependent protein kinase induces growth inhibition in human cancer cells

Author

Yokozaki H, Budillon A, Giampaolo Tortora, Meissner S, Sl, Beaucage, Miki K, and Ys, Cho-Chung

Subjects
oligodeoxynucleotide, Molecular Sequence Data, Down-Regulation, Breast Neoplasms, Oligonucleotides, Antisense, Gene Expression Regulation, Enzymologic, Receptors, Cyclic AMP, Neuroblastoma, oligodeoxynucleotide, AMP, Stomach Neoplasms, Colonic Neoplasms, Tumor Cells, Cultured, Humans, Amino Acid Sequence, RNA, Messenger, Protein Kinase Inhibitors, Protein Kinases, Cell Division, AMP
Abstract

Enhanced expression of the RI alpha subunit of cyclic AMP-dependent protein kinase type I has been correlated with cancer cell growth. We provide evidence that RI alpha is a growth-inducing protein that may be essential for neoplastic cell growth. Human colon, breast, and gastric carcinoma and neuroblastoma cell lines exposed to a 21-mer human RI alpha antisense phosphorothioate oligodeoxynucleotide (S-oligodeoxynucleotide) exhibited growth inhibition with no sign of cytotoxicity. Mismatched sequence (random) S-oligodeoxynucleotides of the same length exhibited no effect. The growth inhibitory effect of RI alpha antisense oligomer correlated with a decrease in the RI alpha mRNA and protein levels and with an increase in RII beta (the regulatory subunit of protein kinase type II) expression. The growth inhibition was abolished, however, when cells were exposed simultaneously to both RI alpha and RII beta antisense S-oligodeoxynucleotides. The RII beta antisense S-oligodeoxynucleotide alone, exhibiting suppression of RII beta along with enhancement of RI alpha expression, led to slight stimulation of cell growth. These results demonstrate that two isoforms of cyclic AMP receptor proteins, RI alpha and RII beta, are reciprocally related in the growth control of cancer cells and that the RI alpha antisense oligodeoxynucleotide, which efficiently depletes the growth stimulatory RI alpha, is a powerful biological tool toward suppression of malignancy.

Published
1993

18. Unhydrolyzable analogues of adenosine 3':5'-monophosphate demonstrating growth inhibition and differentiation in human cancer cells

Author

Yokozaki H, Giampaolo Tortora, Pepe S, Maronde E, Hg, Genieser, Jastorff B, and Ys, Cho-Chung

Subjects
Cell Differentiation, Receptors, Cyclic AMP, Enzyme Activation, Isomerism, Leukemia, Promyelocytic, Acute, Colonic Neoplasms, Cyclic AMP, Tumor Cells, Cultured, Humans, analogues, denosine 3':5'-monophosphate, Protein Kinases, analogues, Cell Division, denosine 3':5'-monophosphate
Abstract

A set of adenosine 3':5'-monophosphate (cAMP) analogues that combine exocyclic sulfur substitutions in the equatorial (Rp) or the axial (Sp) position of the cyclophosphate ring with modifications in the adenine base of cAMP were tested for their effect on the growth of HL-60 human promyelocytic leukemia cells and LS-174T human colon carcinoma cells. Both diasteromeres of the phosphorothioate derivatives were growth inhibitory, exhibiting a concentration inhibiting 50% of cell proliferation of 3-100 microM. Among the analogues tested, Rp-8-Cl-cAMPS and Sp-8-Br-cAMPS were the two most potent. Rp-8-Cl-cAMPS was 5- to 10-fold less potent than 8-Cl-cAMP while Sp-8-Br-cAMPS was approximately 6-fold more potent than 8-Br-cAMP. The growth inhibition was not due to a block in a specific phase of the cell cycle or due to cytotoxicity. Rp-8-Cl-cAMPS enhanced its growth-inhibitory effect when added together with 8-Cl-cAMP and increased differentiation in combination with N6-benzyl-cAMP. The binding kinetics data showed that these Sp and Rp modifications brought about a greater decrease in affinity for Site B than for Site A of RI (the regulatory subunit of type I cAMP-dependent protein kinase) and a substantial decrease of affinity for Site A of RII (the regulatory subunit of type II protein kinase) but only a small decrease in affinity for Site B of RII, indicating the importance of the Site B binding of RII in the growth-inhibitory effect. These results show that the phosphorothioate analogues of cAMP are useful tools to investigate the mechanism of action of cAMP in growth control and differentiation and may have practical implication in the suppression of malignancy.

Published
1992

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