PSME3 Knockout HCT 116 Cell Line

The PSME3 Knockout HCT 116 Cell Line is a CRISPR/Cas9-edited human colorectal carcinoma cell line with targeted disruption of the PSME3 gene, which encodes the proteasome activator PA28??. This model provides a stable loss-of-function system for dissecting the roles of the 20S proteasome activator in protein degradation and cell cycle progression. By eliminating PSME3 expression, it enables precise investigation of its function within the ubiquitin-proteasome system and associated signaling pathways.

HCT 116 is an epithelial colon cancer cell line harboring a KRAS G13D mutation, making it a standard model for oncogenic signaling and drug resistance research. These cells exhibit adherent growth, rapid proliferation, and a functional p53 pathway, offering a relevant context for studying colorectal carcinoma. The KRAS mutation drives constitutive MAPK and PI3K signaling, mimicking aggressive tumor phenotypes.

PSME3 (PA28??) forms a heptameric ring that binds to the 20S core proteasome, enhancing its catalytic activity independently of ubiquitin and ATP. PSME3 expression is upregulated by PI3K/AKT signaling and transcription factors E2F1 and MYC in proliferating cells. Once activated, PSME3?C20S complexes degrade key substrates including p53, cyclin B1, p21, and SRC-3. PSME3 also interacts with MDM2 to facilitate p53 polyubiquitination and degradation. Thus, PSME3 promotes cell cycle progression by removing G1 and G2/M checkpoint inhibitors and suppresses apoptosis through p53 clearance. Its knockout stabilizes these substrates, leading to cell cycle arrest and apoptosis, while also perturbing NF-??B signaling via altered proteasomal processing.

In KRAS-mutant colorectal cancer, PSME3 overexpression contributes to enhanced degradation of tumor suppressors and resistance to apoptosis. The PSME3 knockout in HCT 116 cells therefore disrupts this oncogenic mechanism, restoring p53 and cyclin B1/p21 levels and re-establishing checkpoint control. This sensitizes cells to apoptosis and may counteract KRAS-driven proliferation. Consequently, the model is valuable for elucidating proteasome-dependent drug resistance and for testing therapeutic strategies that target the proteasome system or reactivate tumor suppressive pathways.

Typical applications include Western blotting and RT-qPCR to detect accumulation of p53, cyclin B1, and p21; cell cycle analysis via flow cytometry to assess G1 or G2/M arrest; Annexin V assays for apoptosis; proteasome activity measurement using fluorogenic peptides; colony formation assays; and drug sensitivity testing with proteasome inhibitors like bortezomib. This knockout cell line is an essential tool for research on proteasome-mediated degradation, cell cycle regulation, colorectal cancer biology, and apoptosis. For further details, please contact Ascent Research.