Hiroshi Yokozaki

Role of site-selective cAMP analogs in the control and reversal of malignancy

Two isoforms of cAMP receptor protein, RI and RII, the regulatory subunits of cAMP-dependent protein kinase, transduce opposite signals, the RI being stimulatory and the RII being inhibitory of cell proliferation. In normal cells RI and RII exist at a specific physiological ratio whereas in cancer cells such physiological balance of these receptor proteins is disrupted. Reversal and suppression of malignancy can be achieved when the physiologic ratio of these intracellular signal transducers of cAMP is restored as shown by the use of site-selective cAMP analogs, antisense oligodeoxynucleotides or gene transfer, suggesting new approaches to cancer control.

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.

Site-selective 8-C1-cAMP which causes growth inhibition and differentiation increases DNA (CRE)-binding activity in cancer cells

Control mechanisms of normal differentiation are disrupted in cancer cells but can be restored by treatment with site‐selective cAMP analogs. The cellular events associated with such changes entail compartmental redistribution of the cAMP‐dependent protein kinase type II regulatory subunit, RIIβ. The results of this study indicate that the molecular mechanisms of action involve changes in specific DNA‐binding activity of putative transcription factors. Gel retardation analyses revealed that nuclear extracts from cells of various human cancer cell lines [colon cancer (LS‐174T), gastric cancer (TMK‐1), and leukemia (K‐562)] and rodent pheochromocytoma (PC12) show a concentration‐dependent increase in binding activity to a synthetic DNA that contained the cAMP‐responsive element 5′‐TGACGTCA‐3′ after treatment with 8‐C1‐cAMP. Such an increase in cAMP‐responsive element binding activity was not observed in the 8‐C1‐cAMP‐unresponsive MKN‐1 gastric cancer cells. These findings indicate that the antitumor activity of site‐selective cAMP analogs may reside in the induction of transcription factors that restore normal gene regulation in cancer cells.

Inhibitory Effect of 8‐Chloro‐cyclic Adenosine 3′,5′‐Monophosphate on Cell Growth of Gastric Carcinoma Cell Lines

A cAMP analogue, 8‐chloro‐cAMP (8‐Cl‐cAMP), selectively binds to site 1 receptor of type II regulatory subunit (RII) of cAMP‐dependent protein kinase. The effects of 8‐Cl‐cAMP on human gastric carcinoma cell lines were studied. Twenty μM 8‐CI‐cAMP clearly inhibited cell growth in six cell lines (TMK‐1, KATO‐III, MKN‐7, ‐28, ‐45, and ‐74) but not in MKN‐1. Cell population in the G1 phase was increased in KATO III cells, which were most responsive to 8‐Cl‐cAMP, while cell cycle progression in TMK‐1 and MKN‐1 cells was apparently not influenced by 8‐Cl‐cAMP. The various changes induced by 8‐Cl‐cAMP were further analyzed in TMK‐1 cells. Decrease of type I regulatory subunit (RI) of cAMP‐dependent protein kinase and translocation of RII from cytosol to nucleus were induced by 8‐Cl‐cAMP treatment. 8‐CI‐cAMP increased the level of cAMP‐response element (CRE) binding protein in addition to inducing FOS mRNA, whose promoter contains CRE. 8‐Cl‐cAMP decreased the expression of mRNA for transforming growth factor‐α (TGF‐α), while the expression of epidermal growth factor receptor was not changed. Expression of HRAS and MYC mRNAs was slightly increased, whereas the amounts of HRAS and MYC proteins remained unchanged. Our results overall suggest that 8‐Cl‐cAMP might be a useful tool for antitnmor therapy of gastric cancers and that cell growth inhibition by 8‐Cl‐cAMP might account for the decrease of TGF‐α expression by tumor cells.