Identification of a tumor-associated mutant form of the NF-κB RelA gene with reduced DNA-binding and transactivating activities

Abstract

Alterations of NF-kB family members have been found to be associated with various forms of lymphoid malignancies. In order to determine whether alterations of the RelA gene are involved in lymphomagenesis, we analysed a large and representative panel (200 cases) of such tumors. Southern blot analysis did not reveal any rearrangements or locus ampli®cation, suggesting that structural alterations of the RelA gene may represent rare events in lymphoid neoplasia. By means of PCR-SSCP analysis, we were able to identify a single point mutation leading to amino acid substitution (codon 494, Glu-Asp) in the transactivating (TA) domain in one case of multiple myeloma. The mutated allele was expressed in the pathological bone marrow sample but not in the peripheral blood cells of the patient. We demonstrate that the RelA protein carrying this speci®c mutation (called RelA 494D ) has less transactivating ability than the normal RelA protein. Interestingly, the mutated protein has a lower a nity for kB binding sites both as a homodimer or in association with the NFKB1/p50 subunit. Transfection experiments using a Gal4-RelA 494D fusion protein indicated that the mutation does not alter the intrinsic transactivating ability of the TA domain of RelA. Furthermore, in vitro translated RelA 494D is able to dimerize e ciently with other NF-kB members, such as p50, cREL and IkBa. Our data therefore suggest that this mutation may alter the speci®c structural conformation needed for the DNA interaction of RelA, and provide insights into the amino acid sequences involved in mediating the biological activities of RelA.

KiSS-1 Represses 92-kDa Type IV Collagenase Expression by Down-regulating NF-κB Binding to the Promoter as a Consequence of IκBα-induced Block of p65/p50 Nuclear Translocation

“…Further, our data suggest that the diminished synthesis of MMP-9 in the KiSS-1 transfectants can be partly accounted for by decreased promoter binding of NF-B. trans-Activation of gene expression by NF-B has been ascribed to multiple mechanisms including the degradation of the IBs, increased DNA binding affinity, and increased transacting potential (55)(56)(57)(58). The findings, herein, point to a decreased binding of the p65 and p50 NF-B proteins to the MMP-9 promoter, although reduced trans-acting potential of the bound transcription factor, as previously reported for c-Rel or p65, cannot be ruled out at this stage (56).…”

Mutations in the NF-κB signaling pathway: implications for human disease

“…However, in one multiple myeloma patient, a somatic cell mutation in RELA resulted in a C-terminal amino-acid substitution (E494D) that alters the DNA-binding and transactivation properties of RelA (Trecca et al, 1997).…”

Targeting NF-κB in hematologic malignancies

“…9 In contrast to c-REL, amplifications and/or chromosomal rearrangements affecting the REL A locus, which encodes the NF-kB p65 subunit, are rare in lymphomas. 22 No genetic alterations of the REL B locus have been described. 5 However, chromosomal rearrangements that affect the p52 (NF-kB2) locus at the 10q24 chromosomal region have been detected in T-cell nonHodgkin's lymphomas, and also in chronic B-lymphocytic leukemia (B-CLL), and MM.…”