Here we report the identification and characterization of a novel protein, RelA-associated inhibitor (RAI), that binds to the NF-B subunit p65 (RelA) and inhibits its transcriptional activity. RAI gene was isolated in a yeast two-hybrid screen using the central region of p65 as bait. We confirmed the physical interaction in vitro using recombinant proteins as well as in vivo by immunoprecipitation/Western blot assay. RAI gene encodes a protein with homology to the C-terminal region of 53BP2 containing four consecutive ankyrin repeats and an Src homology 3 domain. RAI mRNA was preferentially expressed in human heart, placenta, and prostate. Despite its similarity to 53BP2, RAI did not interact with p53 in a yeast two-hybrid assay. RAI inhibited the action of NF-B p65 but not that of p53 in transient luciferase gene expression assays. Similarly, RAI inhibited the endogenous NF-B activity induced by tumor necrosis factor-␣. RAI specifically inhibited the DNA binding activity of p65 when co-transfected in 293 cells. RAI protein appeared to be located in the nucleus and colocalized with NF-B p65 that was activated by TNF-␣. These observations indicate that RAI is another inhibitor of NF-B in addition to IB proteins and may confer an alternative mechanism of regulation. Nuclear factor B (NF-B)1 is a sequence-specific DNAbinding protein complex which regulates the expression of viral genomes, including the human immunodeficiency virus, and a variety of cellular genes, particularly those involved in immune and inflammatory responses (1-4). The members of the NF-B family in mammalian cells include the proto-oncogene c-Rel, p50/p105 (NFB1), p65 (RelA), p52/p100 (NFB2), and RelB. All of these proteins share a conserved 300-amino acid region known as the Rel homology domain which is responsible for DNA binding, dimerization, and nuclear translocation of NF-B (5, 6). In most cells, Rel family members form heteroand homodimers with distinct specificities in various combinations. A common feature of the regulation of the NF-B family is their sequestration in the cytoplasm as inactive complexes with a class of inhibitory molecules known as IBs (2, 7). Treatment of cells with a variety of inducers such as phorbol esters, interleukin 1, and tumor necrosis factor-␣ (TNF-␣) results in dissociation of the cytoplasmic complexes and translocation of NF-B to the nucleus (8, 9). The dissociation of NF-B⅐IB complexes is known to be triggered by the phosphorylation and subsequent degradation of IB proteins (10 -12). This exposes the nuclear localization sequence in the remaining NF-B heterodimer, leading to nuclear translocation and subsequent binding of NF-B to DNA regulatory elements of the target genes. The p65 subunit is a major component of NF-B complexes and is responsible for trans-activation (13).Here we report the identification, cloning, and characterization of a novel RelA-binding protein, called RAI for "RelAassociated inhibitor." The RAI cDNA encodes a protein with high structural homology to the C-terminal 200 amino acid seg...
Nuclear factor kB (NF-kB) is a transcription factor that controls the expression of many cellular and viral genes. The p65 (RelA) subunit plays a critical role as a transcriptional activator and recent observations have highlighted its role in the control of apoptosis. Here we report that 53BP2, a protein previously identi®ed by interaction with wild type p53 and Bcl-2, also binds to p65 in a yeast two-hybrid system. This speci®c interaction was con®rmed by pull-down assay in vitro and by a mammalian two-hybrid assay in vivo. We observed that full-length 53BP2 fused to GFP had a punctate distribution in cytoplasm, predominantly in perinuclear region whereas the N-terminal 53BP2 localized in cytoplasm and C-terminal 53BP2 localized in the nucleus. Furthermore, we found that overexpression of GFP-53BP2 induced apoptosis in transiently transfected cells. Neither the N-terminal nor the Cterminal of 53BP2 fused to GFP induced cell death. Interestingly, co-transfection with a p65 expression plasmid signi®cantly inhibited 53BP2-induced cell death. The previous ®ndings that 53BP2 bound to p53 and Bcl-2 together with our present observations suggest that 53BP2 may play a central role in the regulation of apoptosis and cell growth.
Emodin is an active component of a traditional Chinese and Japanese medicine isolated from the root and rhizomes of Rheum palmatum L. Here, we show that emodin significantly induces cytotoxicity in the human myeloma cells through the elimination of myeloid cell leukemia 1 (Mcl-1). Emodin inhibited interleukin-6 -induced activation of Janus-activated kinase 2 (JAK2) and phosphorylation of signal transducer and activator of transcription 3 (STAT3), followed by the decreased expression of Mcl-1. Activation of caspase-3 and caspase-9 was triggered by emodin, but the expression of other antiapoptotic Bcl-2 family members, except Mcl-1, did not change in the presence of emodin. To clarify the importance of Mcl-1 in emodininduced apoptosis, the Mcl-1 expression vector was introduced into the human myeloma cells by electroporation. Induction of apoptosis by emodin was almost abrogated in Mcl-1 -overexpressing myeloma cells as the same level as in parental cells, which were not treated with emodin. In conclusion, emodin inhibits interleukin-6 -induced JAK2/ STAT3 pathway selectively and induces apoptosis in myeloma cells via down-regulation of Mcl-1, which is a good target for treating myeloma. Taken together, our results show emodin as a new potent anticancer agent for the treatment of multiple myeloma patients. [Mol Cancer Ther 2007;6(3):987 -94]
Aim: To clarify the mechanism of the anticancer effect of genistein, we examined the effect of genistein on telomerase activity in prostate cancer cells. We hypothesized that genistein may exert its anticancer effect by modifying telomerase activity in prostate cancer cells. Methods: Prostate cancer (LNCaP) cells were cultured with genistein and the number of viable cells was counted. Growth medium was also collected to measure prostate-specific antigen (PSA) concentration. Polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP) assay and reverse transcriptase (RT)-PCR analysis were performed to investigate telomerase activity and the expression of human telomerase reverse transcriptase (hTERT), c-myc and p21 mRNA. To examine the possibility that hTERT transcriptional activity is modulated by genistein, transient cell transfection studies were performed by using luciferase reporter assay. Telomere repeat amplification protocol (TRAP) assay and PCR analysis of hTERT were performed in androgen independent cells, DU-145. Results: Cell growth of LNCaP was inhibited by genistein and PSA secretion was similarly reduced. In TRAP assay, the telomerase activity of LNCaP cells was reduced by genistein. Reverse transcriptase-PCR analysis revealed that the expression of hTERT and c-myc mRNA was downregulated by genistein, whereas p21 mRNA increased in response to genistein. Luciferase reporter assay revealed that genistein reduced the transcriptional activity of hTERT. In DU-145 cells, telomerase activity and the expression of hTERT mRNA were also reduced by genistein. Conclusion:The current study elucidated the molecular mechanism of cell growth inhibition by genistein. The antiproliferative effects of genistein seem to be exerted on the hTERT transcriptional activity via different molecular pathways.
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