The mechanism of IFN-gamma induced HL-60 cell apoptosis and the downregulation of double-stranded RNA-dependent protein kinase (PKR) by phorbol ester

Interferons (IFNs) play key roles in mediating antiviral and antigrowth responses and in modulating the immune response. Type I and Type II IFNs signal through distinct but related pathways to exert their biological functions. Extensive studies have been done to investigate the biological roles of IFN. This dissertation study continued the investigation of the effects of IFN-alpha, beta and gamma in two directions to further explore their biological activities.; Direction I. We studied the mechanism of IFN-gamma induced HL-60 cell apoptosis. The ultimate goal of our study has been to find an alternative treatment for IFN-alpha resistant leukemia patients. Three malignant hematological cell lines, HL-60, K562 and U937 were chosen for the study. These cells were treated with 1000 units/ml IFN-alpha, beta, and gamma respectively. Trypan blue exclusion and MTT assays showed that only IFN-gamma can affect cell proliferation in HL-60 cells. All subtypes of IFN displayed similar effects on K562 and U937 cells. Further investigation revealed that IFN-gamma treatment led to the degradation of poly (ADP-ribose) polymerase (PARP) in HL-60 cells.; Direction II. We studied the regulation of double-stranded RNA-dependent protein kinase (PKR) induced by IFN-alpha. We found that PKR phosphorylation is down-regulated by phorbol 12-myristate-13-acetate (PMA). PKR is one of the important IFN-stimulated genes (ISGs), and it is also one of the key mediators of IFN action against viruses. PMA is one of the tumor promoters. Understanding the regulation of PKR activity is important for using PKR as a tool to discover and develop novel therapeutic tools for viral infection, cancer and immune dysfunction. Our lab previously reported that PMA can down-regulate another important ISG, RNase L [Chase et al, 2003]. Through our study, we found that PMA decreased the level of phosphorylated PKR in a dose- and time-dependent manner in IFN-treated mouse fibroblast cells. Polyinosinic- polycytidylic acid (poly I:C) treatment did not overcome PMA-induced reduction of PKR phosphorylation. A monoclonal antibody to mouse PKR was utilized for Western analysis, and the result revealed that the decrease of PKR phosphorylation was a result of PKR protein degradation. (Abstract shortened by UMI.)...