Cloning Of CD40 Mutants And Their Selective Expression On B Cell Malignancy And Epithelial Tumor

CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily and is expressed on a wild range of cell types including B lymphocytes, dendritic cells, macrophages, haematopoietic progenitors, endothelial and epithelial cells, fibroblasts and carcinoma cells. CD40 plays a considerable role in B cell malignancies. However, multiple myeloma cells expressed CD40 antigen strongly, and the function of CD40 on these cells was the subject of debate. CD40 is well expressed by a variety of human carcinomas including bladder, breast and ovarian cancers cells. In contrast to leucocytes where the role of CD40/CD40L pair has been systematically investigated, their function remains surprisingly obscure even despite the fact that CD40 is initially described on carcinomas.In this research, we found that CD40 expressed a particular mutant in some cell lines and freshly isolated tumor cells, which is a C-to-A transition (H78Q) at nucleotide 234 (hmuCD40-1). This amino acid located in the 2^nd cysteine-rich extramembrane region of CD40. This region was very important to ligate with CD40L (CD154). CD40-H78 was highly conserved across diverse species. It indicated that this amino acid might play a critical role in keeping normal structure and function of CD40. The modeling of three-dimensional structure showed this mutant might change the partial epitope of CD40. Transfected results confirmed our presumptions, with hmuCD40-l displaying distinct antibody recognizability compared with hwtCD40. This mutant expressed not only on some B cell malignancies but also on some epithelial tumor cells. Interestingly, there was no detectable mutant expression on normal B cells, activated tonsillar cells, endothelial and epithelial cells. Furthermore, we found the expression of hmuCD40-1 was parallel to the grade of malignancy in some tumor. This suggested that the mutant should expresse on tumor cells selectively. We also found that hmuCD40-l was translocated to CD40 signalosome when CD40 was activated. Perhaps the mutant was correlated to tumorformation.It had been reported another single base substitution (TCA—?TTA) of the open reading frame of CD40 from RPMI 8226 cells, a MM cell line, resulting in the conversion of amino acid (Serl24Leu). These authors suggested that RPMI 8226 cells expressed a genetically altered CD40 receptor that may contribute to its unresponsiveness to CD40L binding. By comparison, other authors demonstrated CD40 activation of RPMI 8226 increased Gl/S transition and tumor cell proliferation at 37°C. So we cloned the hmuCD40-2 in RPMI 8226. To confirm the effect of different type of CD40-induced proliferation on RPMI 8226, we cultured the tumor cells with sCD40L, L929/CD40L, soluble 5C11, an agonistic anti-CD40 mAb, plate-bound 5C11 and their respective controls. Data showed that only plate-bound antibody could inhibit cell proliferation and induced cell apoptosis obviously.In summary, we suggested that hmuCD40 expressed on some tumor cells. Further research is needed to explain the possible applications of the CD40 point mutation as a potential molecular marker for tumor earlier diagnosis and treatment. Perhaps the tumor cells could not activate T cells efficiently due to the decrease of affinity of CD40L to hmuC40.