Transformation nonresponsive cells owe their resistance to lack of p65/nuclear factor-kappa B activation

Clonal variants of mouse epidermal JB6 cells that are genetically susceptib le (P+) or resistant (P-) to tumor promoter-induced neoplastic transformati on exhibit differential activator protein-1 (AP-1) response. Transactivatio n of AP-1 appears to be necessary but not sufficient to promote transformat ion in JB6 cells. Inhibition of AP-1 is invariably accompanied by inhibitio n of nuclear factor-kappaB (NF-kappaB) when transformation is suppressed, s uggesting that NF-kappaB may also play a role in neoplastic transformation. We report here that transactivation of NF-kappaB is inducible by tumor pro moters in P+ but not in P- JB6 cells. Inhibition of NF-kappaB using a nonde gradable mutant of I kappaB alpha suppressed inducible anchorage-independen t transformation of P+ JB6 cells, suggesting that NF-kappaB activation is r equired for tumor promotion, Induced degradation of I kappaB alpha occurred in both P+ and P- JB6 cells, indicating that failure to activate NF-kappaB in P- JB6 cells cannot be attributed to failure to degrade I kappaB alpha. Slightly higher levels of nuclear p65 were seen in P+ than in P-JB6 cells, The p65-specific DNA binding activity was also higher in P+ cells upon ind uction by tumor necrosis factor-alpha, suggesting that differential NF-kapp aB activation may be attributable to changes in p65 activity. Transactivati on of p65 protein was substantially higher in P+ than in P-JB6 cells, as de termined by assay of Ga14-p65 fusion constructs, Thus activated, p65 may be a limiting factor for NF-kappaB activation and transformation responses. S table expression of p65 in P- JB6 cells conferred not only inducible NF-kap paB and AP-1 activation but also transformation response to tumor promoters . Therefore, p65/NF-kappaB appears to be not only necessary for but also su fficient to confer tumor promotion response, Although stable expression of p65 in P- cells produced p65 increases in whole cell extracts, only the tra nsfectants exhibiting increased nuclear p65 showed transformation response. Thus, elevation of nuclear p65 appears to be a necessary step for a transf ormation response, The P-/p65 transfectants showing acquired transformation response also shelved elevated p65-specific transactivation response, thus recapitulating the NF-kappaB phenotypes seen in P+ cells. Expression of a transactivation-deficient mutant of Jun or dominant-negative extracellular signal-regulated kinase suppressed both AP-1 activation and p65-specific tr ansactivation in JB6 cells, suggesting that AP-1 activity is needed for p65 transactivation and consequently for NF-kappaB activation. Thus, the trans formation nonresponsive P- JB6 cells owe their resistance to lack of NF-kap paB activation and p65 transactivation that appears in turn to be attributa ble to insufficient AP-1 activation.