We determined the levels and functions of p53, nuclear factor-kappa B (NF-kB; a key transcriptional regulator), and mot-2 (a p53 inhibitor) and their relationships in arsenite-induced transformed HELF cells by two-dimensional electrophoresis, reverse-transcriptase polymerase chain reaction, Western blot, immunofluorescence, and co-immunoprecipitation assays.
We found decreased expression of NF-kB repressing factor (NKRF; an inhibitor of NF-kB-mediated gene transcription), increased expression of mot-2, and increased activation of NF-kB.
KEY WORDS: arsenite, mot-2, NF-kB, p53, signal transduction, tumorigenesis.
The oligonucleotides for mot-2 siRNA were 5'-GGAUUGUCACUGAUCUAAU-3' and 5'-AUUAGAUCAGUGACAAUCC-3' (Sigma).
We used mot-2 primers (forward, 5' -CGAGTCAGATTGGAGCAT-3'; reverse, 5' -GACCATAGGCAAGAGCAG-3') for PCR amplification.
We used the following antibodies: NF-kB repressing factor (NKRF), CBP [cyclic AMP responsive element binding protein (CREB) binding protein], mot-2 (a p53 inhibitor), and [beta] actin (all from Sigma); and NF-kB inhibitor (IkB[alpha], phosphorylated IkB[alpha] [p-IkB[alpha] (serine 32)], RelA (a subunit of NF-kB), phosphorylated RelA (p-RelA; serine 536), wild-type p53, p-p53 (serine 15), and proliferating cell nuclear antigen (PCNA) (all from Cell Signaling Technology).
The immunoprecipitants were analyzed by Western blots with mot-2, RelA, or p53 antibodies.
Of these, we selected NKRF (an NF-kB inhibitor) and mot-2 (a p53 inhibitor) for further analysis.
NF-kB activation as a regulator of mot-2 in arsenite-exposed HELP cells.
Involvement of NF-kB and mot-2 in the activation and translocation of p53 in arsenite-treated HELF cells.
Arsenite increased the binding of mot-2 to p53, which was attenuated by blocking of NF-kB activity (Figure 5A,B).
mot-2, which is expressed in many cell types and tissues, possesses diverse activities, including oncogenic transformation.