Transformation nonresponsive cells owe their resistance to lack of p65/nuclear factor-kappaB activation.
Clonal variants of mouse epidermal JB6 cells that are genetically susceptible (P+) or resistant (P-) to tumor promoter-induced neoplastic transformation exhibit differential activator protein-1 (AP-1) response. Transactivation of AP-1 appears to be necessary but not sufficient to promote transformation in JB6 cells. Inhibition of AP-1 is invariably accompanied by inhibition of nuclear factor-kappaB (NF-kappaB) when transformation is suppressed, suggesting that NF-kappaB may also play a role in neoplastic transformation. We report here that transactivation of NF-kappaB is inducible by tumor promoters in P+ but not in P- JB6 cells. Inhibition of NF-kappaB using a nondegradable mutant of IkappaBalpha suppressed inducible anchorage-independent transformation of P+ JB6 cells, suggesting that NF-kappaB activation is required for tumor promotion. Induced degradation of IkappaBalpha 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 IkappaBalpha. 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 induction by tumor necrosis factor-alpha, suggesting that differential NF-kappaB activation may be attributable to changes in p65 activity. Transactivation of p65 protein was substantially higher in P+ than in P- JB6 cells, as determined by assay of Gal4-p65 fusion constructs. Thus activated, p65 may be a limiting factor for NF-kappaB activation and transformation responses. Stable expression of p65 in P- JB6 cells conferred not only inducible NF-kappaB and AP-1 activation but also transformation response to tumor promoters. Therefore, p65/NF-kappaB appears to be not only necessary for but also sufficient to confer tumor promotion response. Although stable expression of p65 in P- cells produced p65 increases in whole cell extracts, only the transfectants exhibiting increased nuclear p65 showed transformation response. Thus, elevation of nuclear p65 appears to be a necessary step for a transformation response. The P-/p65 transfectants showing acquired transformation response also showed 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 transactivation in JB6 cells, suggesting that AP-1 activity is needed for p65 transactivation and consequently for NF-kappaB activation. Thus, the transformation nonresponsive P- JB6 cells owe their resistance to lack of NF-kappaB activation and p65 transactivation that appears in turn to be attributable to insufficient AP-1 activation.
Hsu
T C
TC
Frederick Cancer Research and Development Center, National Cancer Institute, Frederick, MD 21702, USA.
Nair
R
R
Tulsian
P
P
Camalier
C E
CE
Hegamyer
G A
GA
Young
M R
MR
Colburn
N H
NH
D000818:Animals; D002273:Carcinogens / Q000494:pharmacology; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002471:Cell Transformation, Neoplastic / Q000187:drug effects / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins; D051379:Mice; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D052002:NF-kappa B p50 Subunit; D012867:Skin / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000502:physiology; D051996:Transcription Factor RelA; D015533:Transcriptional Activation; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D002273:Carcinogens; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D018808:Transcription Factor AP-1; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D004247:DNA; D013755:Tetradecanoylphorbol Acetate