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 G1 phase in proliferating primary rat hepatocytes, but to inhibit their entry into S phase and RB phosphorylation. In the present study, DBcAMP was also found to enhance the down-regulation of RB 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 RB 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 RB 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.
Okamoto
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Y
Department of Central Clinical Laboratory, Nara Medical University, Japan. yasuyuki@naramed-u.ac.jp
D017209:Apoptosis / Q000187:drug effects; D003994:Bucladesine / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects; D006801:Humans; D006868:Hydrolysis; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D016160:Retinoblastoma Protein / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D016160:Retinoblastoma Protein; D003994:Bucladesine; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases