ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2008) 16 P334

The cAMP analog 8CL-cAMP enhances PKA RIIb, triggers apoptosis and blocks cells at S and G2 cell cycle phases after 4 days of treatment in the human adrenocortical cells H295R.

Zhor Bouizar2, Jerôme Berterat3 & Marthe Rizk-Rabin1

1Institut Cochin, Université Descartes, CNRS UMR8104, Paris 75014, France; 2INSERM U567, Paris 75014, France; 3Pôle Endocrinologie-Diabétologie Adultes-Enfants, Groupe Hospitalier Cochin St Vincent de Paul, 27 Rue du FG St Jacques Paris 75014, France.

Various alterations of the cyclic AMP (cAMP) signalling cascade has been observed in adrenocortical tumors. Changes of the PKA regulatory subunits expression in tumors may positively or negatively regulate proliferation. 8CL-cAMP, a site selective cAMP analogue, induces growth inhibition in a variety of human cancer cell lines. The aim of the study was to determine if 8CL-cAMP acts on adrenocortical carcinoma H295R cell growth.

We have determined the involvement of PKA, PKA R subunits in this activity and studied the mechanisms of action on cell cycle molecule targets and cell cycle distribution. Cells were incubated for 96 h or 7days with 8CL-cAMP (10−4 M). Cell viability and proliferation was evaluated by MTT assay and BrdU incorporation. Apoptosis was investigated by annexin FITC assay. Western blotting was performed, for PKA subunits, cyclin and cyclin dependent kinase protein expression. Cell cycle distribution was analysed by flow cytometry.

8CL-cAMP induces PKA activation, modulates the expression of PKA regulatory subunits by increasing the type II isozymes. The cAMP analogue reduces cell proliferation and enhances apoptosis in the H295R cells. These effects were associated with the accumulation of cells at S and G2 phases. This is consistent with the accumulation of cyclin A in the nucleus and cyclin B both in the cytosol and the nucleus. Cells blocked from progression through M phase by nocodazole (1 μM, 18 h) or G1/S phase by aphidicollin (2.5 μM, 24 h), revealed that addition of 8CL-cAMP resulted in a delay in S/G2 transition and a blockage in G2/M chekpoint.

These results imply that cAMP/PKA RIIb pathway regulates S and G2/M phase progression in H295R. Activation of PKA RIIb holoenzyme may be responsible for the growth arrest, by altering the cell cycle regulator pattern and the induction of apoptosis.

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