Study of the novel interaction between MNT and c-REL and its impact on cell proliferation and viability

Abstract

MNT is a basic-helix-loop-helix-leucine zipper (bHLH-LZ) protein from the MXD family. MNT and other MXD proteins play an important role as MYC antagonists, which is one of the most frequently altered oncogenes in human cancer. Both MYC and MXD proteins heterodimerize with MAX, binding to E-boxes within regulatory regions of target genes, and generally activate (MYC) or repress (MXD) their transcription. However, some MAX-deficient cell lines and tumours with MAX mutations have been found, pointing out the existence of MYC and MXD functions which are MAX-independent. Our preliminary results in UR61 cells (derived from rat pheochromocytoma and deficient in MAX) suggest a possible interaction between MNT and c-REL that is MAX-independent. c-REL belongs to the REL/NF-kB family which takes part in several relevant biological processes. Here, performing co-immunoprecipitation assays, we confirm this interaction in other rodent cell lines (rat C6, mouse Neuro-2a) but not in the human cell lines tested so far (HeLa, 293T, Raji, K562, SH-SY5Y, T98G). In order to identify the MNT interaction domain, a MNT deletion mutant (ΔBr) was transfected into UR61 cells and the c-REL binding was analysed by immunoprecipitation with anti-MNT antibodies. The results showed that the basic region (including the DNA binding domain) is required for the c-REL-MNT interaction. Furthermore, we showed that MNT and c-REL silencing by siRNAs (generated with short-hairpin vectors) reduced cell proliferation and viability of UR61, C6 and H1417 cell line (human lung cancer cells deficient in MAX), as assessed by clonogenic assays and/or cell counting.