Dibutyryl cyclic AMP-induced enhancement of RB protein degradation in human hepatoma cells

Dibutyryl cyclic AMP (DBcAMP) was previously reported to enhance the down-regulation of the retinoblastoma (RB) protein during GI phase in proliferating primary mt hepatocytes, brit to inhibit their entry into S phase and RS phosphorylation. In the present study, DBcAMP was also found to enhance the down-regulation of RE protein in the human hepatoma cells PLC/PRF/5 after hydroxyurea-induced synchronization at G1/S phase. One hour after synchronization, CPP32 activity was detected in the cells and was further enhanced in the presence of DBcAMP. CPP32-specific cleavage of the RE protein was also detected and enhanced by the addition of DBcAMP in a dose-dependent manner DNA analysis by flow cytometry after. serum starvation-induced synchronization at G0/G1 phase revealed that DBcAMP elicited an apoptotic peak after the S phase. Based on these findings, DBcAMP was suspected of inducing apoptosis by RE protein degradation during G1/S transition and thereby inhibit the growth of PLC/PRF/5 cells. Under serum-deficient culture conditions, addition of the CPP32 inhibitor DEVD or the ICE inhibitor YVAD enhanced cell growth but did not abolish the DBcAMP-induced growth inhibition. On the other hand antisense oligodeoxynucleotides against Bcl-2 mRNA showed a growth inhibitory effect on PLC/PRF/5 cells, but did not show an additive effect on the DBcAMP-induced growth inhibition. DBcAMP itself inhibited bcl-2 protein expression. DBcAMP-induced growth inhibition may be mediated by different mechanisms, including apoptosis.