Effect of adenosine 3′:5′-cyclic monophosphate and inhibition of protein kinase a on heat sensitivity in H35 hepatoma cells

Abstract

Purpose : To investigate the role of the cyclic adenosie 3′:5′-monophosphate (AMP) signal transduction pathway in heat-induced cell death and the development of thermotolerance. Methods and Materials : Reuber H35 rat hepatoma cells were heated after preincubation with various compounds known to modulate the cyclic AMP signal transduction pathway. Cell survival was determining by colony-forming ability. Results : Preincubation of H35 cells with forskolin, a stimulator of adenylate cyclase, in combination with IBMX (3-isobutyl-1-methylxanthine), an inhibitor of cyclic AMP phosphodiesterase, results in thermosensitization. Similar results are obtained with various cyclic AMP analogs. Maximum thermosensitization occurs with 0.5 mM dibutyryl cyclic AMP (DBcAMP) after a preincubation period of 5 h and heating in the presence of the drug. The same relative degree of thermosensitization is found with 8-Cl-cAMP, but a 10-fold lower concentration Thermosensitization by DBcAMP is prevented by H89, a specific inhibitor of cyclic AMP-dependent protein kinase (PKA). Without additional cyclic AMP-inducing factors, H89 induces thermoprotection. Node of the drug treatments are cytotoxic at 37°C. DBcAMP does not affect the development of heat-induced thermotolerance but it reduces its experession to an extent similarly found in the observed thermosensitization in nonthermotolerant cells. Conclusion: The results strongly indicate that the cyclic AMP signal transduction pathway is involved in the process of heat-induced cell death. DBcAMP reduces the expression of thermotolerance, but does not affect its induction.