ENHANCED DEGRADATION OF I-KAPPA-B-ALPHA CONTRIBUTES TO ENDOGENOUS ACTIVATION OF NF-KB IN HS294T MELANOMA-CELLS

The expression of the CXC chemokine MGSA is often deregulated during v iral infection, chronic inflammation, and melanoma tumor progression. In Hs294T melanoma cells, the increased constitutive expression of MGS A is due to increased gene transcription. Moreover, nuclear extracts f rom unstimulated Hs294T cells contain 19-fold more immunoreactive NF-k appa B p65 than that observed in normal retinal pigment epithelial (AR PE) cells, This increase in NF-kappa B p65 correlates with increased N F-kappa B DNA binding activity in Hs294T nuclear extracts. After stimu lation with interleukin 1, Western and electrophoretic mobility shift assay analysis indicate that in both cell types, additional activated NF-kappa B p65 is translocated to the nucleus. However, the rate of po stinduction repression of NF-kappa B DNA binding is delayed in Hs294T melanoma cells compared to ARPE cells. Western analysis of whole-cell lysates from both Hs294T and ARPE cells indicates that protein levels of the inhibitor of NF-kappa B, I-kappa B alpha, are 3-fold lower in H s294T cells. The decrease in I-kappa B alpha cannot be attributed to a lterations in the transcription or translation of I-kappa B alpha. Rat her, the posttranslational processing has been altered, In Hs294T cell s, the half-life of the I-kappa B alpha protein is 45 min, compared to 120 min in ARPE cells. These results indicate that in Hs294T melanoma cells the equilibrium between I-kappa B alpha degradation and resynth esis has been altered, leading to constitutive nuclear translocation a nd activation of NF-kappa B. Similar mechanisms could also operate in other tumorigenic processes, as well as in viral and chronic inflammat ory disorders, to produce high constitutive and unregulated chemokine expression.