8-Br-cAMP up-regulates antioncogene expression in human retinoblastoma HXO-Rb44 cells / 中华实验眼科杂志

ABSTRACT

It is kno wn that 8-Br-cAMP is one of selective bi nding site analogues for cAMP RIIα to af fect cell growth through regulation of g ene expression.The p16,p21wafl,p53 a nd Rb are antioncogenes which affect cel l growth through control of cell cycle.T he aim of this study is to investigate t he 8-Br-cAMP effect on the expression of antioncogenes in human HXO-Rb44 cells. Methods Cultured HXO-Rb44 cells in RPMI -1 640 medium were divided into two aliquot s.8-Br-cAMP (2×10-5mol/L) was added i nto one aliquot for 24h as the experime ntal group(EG),the another aliquot witho ut 8-Br-cAMP as the control group(CG).Af ter 24h,the cell suspension was dropped onto the nitrocellulose membrane.The mR NA of p16,p21wafl,wild type(w)p53,mut ant type(m) p53 and Rb were used respec tively with biotin-labeled cDNA probes b y intact cell RNA dot blot.The immunorea ctivity(IR) of P16,P21wafl,PRb,PCN A,cdk2 and cdk4 were detected respecti vely with specific monoclonal antibodies on dot blot.ResultsThe mRNA dot blot s ignals of mp53 and protein dot blot of cdk2-IR,cdk4-IR and PCNA-IR in EG were weaker than those in CG(P＜0.05～0.01). W hile,the mRNA signals of p16,p21wafl,wp53 and Rb in EG were stronger than tho se in CG(P＜0.05～0.01).The intensity of ea ch protein dot blot was consistent with that of their RNA dot blot (except for w P53-IR and mP53-IR not to be done).Conc lusions(1)8-Br-cAMP could up-regul ate expression of antioncogenes includin g p16,p21wafl,wp53,Rb,and protein exp ression of P16,P21wafl and PRb.(2) 8-Br-cAMP could down-regulate mp53 gene expression and protein expression of cd k2,cdk4 and PCNA.The results suggest t hat 8-Br-cAMP could inhibit human HXO-Rb 44 cell growth through interfering rela ted gene expression of cell cycle.