Suppression of malignancy targeting the intracellular signal transducing proteins of cAMP: the use of site selective cAMP analogs, antisense strategy, and gene-transfer.

An hypothesis has been presented suggesting that two isoforms of cAMP receptor proteins are crucial effectors in tumorigenesis. The evidence in support of this hypothesis shows that: (1) cAMP transduces dual controls, both positive and negative, on cell growth and differentiation. (2) Such dual controls are respectively governed by two isoforms of cAMP receptor proteins, the type I and type II regulatory subunits of cAMP-dependent protein kinase. (3) In normal physiology, the functional balance of these cAMP receptor isoforms is strictly controlled to meet either stimulation or inhibition of cell growth as it is required, whereas such control is lost in cancer cells. (4) Cancer cells can also be made to differentiate and acquire growth control when the functional balance of these intracellular signal transducers of cAMP is restored by the use of site-selective cAMP analogs, antisense strategy, or gene transfer, suggesting new approaches to cancer therapy

Cooperative effect of 8-Cl-cAMP and rhGM-CSF on the differentiation of HL-60 human leukemia-cells.

In HL-60 leukemia cells the site-selective cAMP analog, 8-Cl-cAMP, at a dose of 5 microM produced growth inhibition with no signs of toxicity, whereas granulocyte-macrophage colony stimulating factor (GM-CSF) exerted an early transient increase of cell proliferation which was followed by differentiation toward monocytes. 8-Cl-cAMP in combination with GM-CSF blocked the growth stimulation due to GM-CSF and demonstrated a synergistic effect on the differentiation of HL-60 cells. The early proliferative effect of GM-CSF was correlated with an increased expression of type I regulatory subunit of cAMP-dependent protein kinase (RI alpha). Treatment with an RI alpha antisense oligodeoxynucleotide suppressed the GM-CSF-inducible cell proliferation and differentiation. Conversely, an RII beta antisense oligodeoxynucleotide, which suppresses the RII beta and causes a compensatory increase in RI alpha level, greatly enhanced the early proliferative input and the differentiation induced by GM-CSF. These results provide an insight into the mechanism of action of GM-CSF and the rationale for a combination differentiation therapy with 8-Cl-cAMP and GM-CSF

Unhydrolyzable analogs of Adenosine 3'/5'-monophosphate demonstrating growth-inhibition and differentiation in human cancer-cells.

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