Accumulation Dynamics of Various Proteasome Forms in Colorectal Adenocarcinoma Cells Treated with Interferon-Gamma

Abstract

Most intracellular proteins are degraded by proteasomes. In addition to constitutive proteasomes (cPs), cells form non-constitutive proteasomes (ncPs), which contain alternative catalytic subunits. These proteasomes play a key role in immunological reactions and cellular response to stress. The ncPs synthesis is activated by interferon-γ (IFN-γ), however, studies of the proteasome pool dynamics following IFN-γ treatment have not been conducted with high temporal resolution and visualization of cPs and ncPs in the same cells. To characterize accumulation of proteasome subunits, two-dimensional (2D) and three-dimensional (3D) cultures of a human colorectal adenocarcinoma reporter cell line expressing the fluorescently tagged cP subunit β5 and ncP subunit β5i were used. The expression of proteasome subunits in 2D cell culture was assessed using real-time PCR, Western blotting, flow cytometry, fluorescence microscopy, and automated fluorescence microscopy after treatment with IFN-γ for 2, 4, 8, 10, 24, 28, 32, 48, 52, 56, and 72 h. The incorporation of subunits into proteasomes was studied by Western blotting and an activity-based probe. In parallel, changes in tumor spheroid fluorescence were examined 24, 48, and 72 h after the start of IFN-γ treatment. In 2D culture, increased levels of mRNA of ncP subunits were detected after 4 h of incubation with the cytokine. Accumulation and incorporation of ncP subunits into proteasomes were observed following 8 h of incubation, reaching maximum at 56-72 h post IFN-γ administration. Notably, starting from 48 h, a decrease in the amount of cP subunit β5 was detected within cells. In 3D models, significant changes in spheroid fluorescence occurred only after 48 h of incubation with IFN-γ. However, at the periphery of treated spheroids, the β5i subunit expression began to increase after 24 h, indicating delayed cytokine penetration into the spheroid interior. These results clarify the dynamics of IFN-γ-induced proteasome pool reorganization. The obtained data can be used to analyze the effects of drugs and modify IFN-γ-based treatment protocols.