The ITCH Knockout SK-HEP-1 Polyclonal Cells comprise a CRISPR/Cas9-edited polyclonal population of human hepatic adenocarcinoma cells in which the ITCH locus has been disrupted. This loss-of-function model maintains a heterogeneous genetic background while providing consistent ITCH ablation, enabling functional investigation of ITCH-dependent ubiquitination and its regulatory roles in signaling cascades without requiring clonal isolation.
SK-HEP-1 is a well-characterized human hepatic adenocarcinoma epithelial cell line originally isolated from a male patient. Exhibiting adherent morphology, it is widely employed as an in vitro model for hepatocellular carcinoma (HCC) and endothelial barrier studies. The malignant hepatic context of SK-HEP-1 makes it particularly suitable for interrogating tumor cell-intrinsic signaling, drug sensitivity, and ubiquitin-mediated regulatory mechanisms.
ITCH (Itchy E3 ubiquitin-protein ligase) is a HECT-type E3 ligase that transfers ubiquitin to substrate proteins, directing them toward proteasomal degradation or modulating non-proteolytic signaling. ITCH activity is regulated by upstream kinases including JNK1/2 (MAPK8/9) and Src, as well as by adaptor proteins Ndfip1 and Ndfip2, which facilitate enzyme-substrate interactions. Key substrates encompass transcription factors c-Jun and JunB, p53 family members p63 and p73, the adaptor TRAF6, kinase RIPK1, and SMAD7. Consequently, ITCH serves as a central node in JNK, NF-??B, Notch, and TGF-?? pathways. In hepatocellular carcinoma, ITCH has been implicated in controlling cell proliferation, apoptosis, and inflammatory responses through these substrates.
Disruption of ITCH in SK-HEP-1 liver cancer cells is predicted to stabilize pro-survival and pro-inflammatory targets such as c-Jun and RIPK1, leading to enhanced JNK/AP-1 transcriptional activity and NF-??B signaling. This may result in altered growth, resistance to apoptosis, and modified cytokine responses. The model is particularly relevant for investigating the dual roles of ITCH as a tumor suppressor or oncogene in HCC, and for exploring ITCH deficiency-related autoimmune phenotypes within a cancer context.
This polyclonal knockout population is suited for ubiquitination and co-immunoprecipitation assays to characterize ITCH-substrate complexes; phospho-JNK and c-Jun Western blotting to monitor JNK pathway activation; NF-??B luciferase reporter and Annexin V apoptosis assays; and cell viability and migration analyses. The model also supports drug screening for ubiquitin-proteasome modulators, transcriptomic profiling by RNA-seq, and protein stability studies using cycloheximide chase. For further technical information, please contact Ascent Research.
