Establishment and Characterization of a Novel Murine Hepatoma Cell Line Allowing Concomitant Conditional Inactivation of Caspase-8 and IKKγ/NEMO

The aim of this study was to generate a hepatocyte-derived cell line in which the Casp8 and Nemo genes can be conditionally inactivated simultaneously to investigate the significance of the corresponding signaling pathways. To this end, we induced hepatocellular carcinoma in Casp8f/fNemof/f mice using diethylnitrosamine (DEN) and established an immortalized hepatoma cell line from explanted liver tumors, which is subsequently referred to as ΔCN60. ΔCN60 cells still retain floxed Casp8 and Nemo alleles, allowing for efficient Cre-mediated deletion to generate Casp8ΔNemoΔ derivatives.

Loss of both Caspase-8 and NEMO inhibits cell proliferation, increases expression of tumor and progenitor markers (AFP, CD133), reduces albumin expression, and blocks TNF-α-induced NF-κB p65 nuclear translocation. Casp8ΔNemoΔ ΔCN60 cells show increased RIP1 stability, altered RIP3 dynamics, and heightened sensitivity to prolonged TNF-α exposure, suggesting a shift towards necroptotic signaling. ΔCN60 serves as a versatile hepatoma model for investigating Caspase-8/NEMO/RIP1/RIP3-dependent pathways and TNF-α-induced liver damage, reducing the need for animal testing.