Involvement of Proteases in the Action of IFN-γon WISH Cells

Abstract

This study investigates the possible involvement of serine proteases in interferon-gamma (IFN-gamma) activity on WISH cells. It was observed that inhibition of (3)H-thymidine incorporation induced by IFN-gamma was abrogated by the serine protease inhibitors Nalpha-tosyl-L-lysyl-chloromethane and soybean trypsin inhibitor, both of which act mainly on trypsin. Phenylmethyl sulfonyl fluoride also had a partial inhibitory effect. Other protease inhibitors specific to the cysteine, the aspartic, and the metalloprotease families were not effective. Kinetic analysis revealed that a trypsin-like protease is involved in IFN-gamma activity for up to 7 h. Trypsin-like activity induced by IFN-gamma was detected in the particulate fraction but not in the cytosolic fraction, whereas chymotrypsin activity was not enhanced in either the cytosolic or particulate fractions under similar conditions. Following separation on a gelatin substrate gel, two trypsin-like protease activities located in the particulate fraction were found to increase in response to IFN-gamma treatment. Hence, it seems that a specific membrane-associated trypsin-like protease activity induced by IFN-gamma may play a role in the action of the cytokine on thymidine incorporation in WISH cells.

Exposure to Interferon γ Decreases Levels and Activity of Key Cell Cycle Proteins Resulting in Severe Growth Arrest of the Human Non-Transformed Cell Line, WISH

“…It is believed that the IFN effects growth arrest through cellular functions of gene products, whose synthesis is induced or repressed by the IFN. The molecular changes include reduction in the activity of cyclin dependent kinase 2 (CDK2) in mouse embryonic fibroblasts (Bromberg et al, 1996), induction of expression of CDK2 inhibitor p21 in A431 cells (Chin et al, 1996), in glioblastoma cell lines T98G, SNB-19 and U-373 (Kominsky et al, 1998), in macrophages (Xaus et al, 1999) and in breast carcinoma MCF7 cells (Gooch et al, 2000), inhibition of cyclin A gene transcription in vascular smooth muscle cells (Sibinga et al, 1999), repression of cdk2 gene expression by the interference of interferon regulatory factor 1 (IRF1) with SP1dependent transcriptional activation of cdk2 promoter (Xie et al, 2003), hyperphosphorylation of retinoblastoma protein in mammary epithelial cells (Harvat and Jetten, 1996), and reduction in thymidine incorporation in WISH cells (Aharon et al, 2002). IFNg has also been found to induce or sensitise target cells to apoptosis through the induction of caspase 8 by IRF1 in Ewing tumor, neuroblastoma or medulloblastoma (Fulda and Debatin, 2002) or by downregulating the levels of p21 in human hepatocellular carcinoma cells (Detjen et al, 2003).…”