A mutant of the WEHI-231 B lymphocyte line that is resistant to phorbol esters is still sensitive to antigen receptor-mediated growth inhibition

Abstract

In order to gain a better understanding of the pathways linking receptor immunoglobulin (sIg) crosslinking to downstream B lymphocyte responses, mutants were generated that were defective in sIg-associated signaling pathways. The murine B lymphoma WEHI-231 has proven to be a useful model for studies of sIg-mediated signal transduction. Signaling through sIgM using anti-receptor antibodies (anti-mu) leads to growth arrest and apoptosis of this continuously proliferating cell line. Direct activation of protein kinase C (PKC) with phorbol esters also can mediate this response in WEHI-231. This negative growth response is a useful characteristic that can be exploited to generate signaling mutants that are resistant to the growth-inhibiting effects of anti-mu or phorbol ester. Using this approach, we selected a mutant, PR30-3, in which signaling was blocked downstream of the phorbol diester response element, presumably PKC. Although no longer responsive to phorbol ester stimulation, PR30-3 is not defective in PKC expression or function. Western blot analyses of cellular lysates shows the mutant to express the PKC isoforms alpha, beta, and delta, at levels not markedly different from wild-type WEHI-231. PR30-3 expresses active PKC as shown by its ability to phosphorylate a PKC-specific peptide in vitro. PR30-3 and WEHI-231 express equivalent levels of sIgM expression and nearly indistinguishable second messenger responses in the form of increases in [Ca2+]i and inositol phospholipid hydrolysis. Both PR30-3 and WEHI-231 demonstrate rapid induction of tyrosine kinase activation following sIgM signaling, although there is a reproducible difference in the ability to phosphorylate a 40-kDa substrate in PR30-3. Interestingly, tyrosine phosphorylation of this substrate is induced by phorbol ester stimulation in the wild-type but not the mutant PR30-3. We observed that phorbol ester stimulation of PR30-3 induces the expression of the early response gene c-fos, previously shown to be PKC dependent in this cell line. These results indicate that the signaling component(s) defective in PR30 lie downstream of PKC but upstream of the commitment point for growth inhibition and cell death. Finally, although PR30-3 is resistant to the inhibitory effects of phorbol ester, proliferation is nonetheless still inhibited in response to anti-mu stimulation. These results suggest that the growth inhibitory response of WEHI-231 to anti-mu and phorbol ester involves different pathways.