CD40-Mediated Apoptosis in Murine B-Lymphoma Lines Containing Mutated p53

Abstract

Crosslinking CD40 induces normal B-cells to proliferate and differentiate but causes many tumor cell lines to undergo apoptosis. As p53 is required for many apoptotic pathways, we analyzed the effects of CD40 ligation and their correlation with p53 function in four murine B-lymphoma lines. A20 and M12 cells showed complete growth inhibition and an increase in the proportion of apoptotic cells upon CD40 crosslinking, but WEHI 231 and WEHI 279 cells were unaffected. IκBα protein levels were reduced in all four lines in response to CD40 crosslinking, indicating that CD40 signaling was normal. NFκB activity was quantitated by reporter and gel shift assays and found to be equivalent in all four cell lines. P53 reporter constructs were activated in WEHI231 but not A20 or M12 cells, suggesting that p53 in the latter two cell lines may be mutated. These data are supported by the lack of detectable p21 mRNA in A20 and M12 cells. P21 mRNA was increased to detectable levels in M12 cells upon CD40 ligation; however, blocking this effect with the p53 inhibitor pifithrin had no effect on CD40-mediated apoptosis. Sequencing showed that p53 in A20 and M12 cells contained point mutations leading to amino acid substitutions in DNA binding regions, but was unmutated in WEHI231 and WEHI 279. These results suggest that CD40-mediated apoptosis can occur in the absence of functional p53.