MUTANT OF THE WEHI-231 B-LYMPHOCYTE LINE THAT IS RESISTANT TO PHORBOLESTERS IS STILL SENSITIVE TO ANTIGEN RECEPTOR-MEDIATED GROWTH-INHIBITION

In order to gain a better understanding of the pathways linking recept or immunoglobulin (sig) crosslinking to downstream B lymphocyte respon ses, mutants were generated that were defective in sig-associated sign aling pathways. The murine B lymphoma WEHI-231 has proven to be a usef ul model for studies of sig-mediated signal transduction. Signaling th rough sIgM using antireceptor antibodies (anti-mu) leads to growth arr est and apoptosis of this continuously proliferating cell line. Direct activation of protein kinase C (PKC) with phorbol esters also can med iate this response in WEHI-231. This negative growth response is a use ful characteristic that can be exploited to generate signaling mutants that are resistant to the growth-inhibiting effects of anti-mu or pho rbol ester. Using this approach, we selected a mutant, PR30-3, in whic h signaling was blocked downstream of the phorbol diester response ele ment, presumably PKC. Although no longer responsive to phorbol ester s timulation, PR30-3 is not defective in PKC expression or function. Wes tern 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 W EHI-231 express equivalent levels of sIgM expression and nearly indist inguishable second messenger responses in the form of increases in [Ca 2+](i) and inositol phospholipid hydrolysis. Both PR30-3 and WEHI-231 I demonstrate rapid induction of tyrosine kinase activation following sIgM signaling, although there is a reproducible difference in the abi lity to phosphorylate a 40-kDa substrate in PR30-3. Interestingly, tyr osine phosphorylation of this substrate is induced by phorbol ester st imulation in the wild-type but not the mutant PR30-3. We observed that phorbol ester stimulation of PR30-3 induces the expression of the ear ly response gene c-Sos, previously shown to be PKC dependent in this c ell line. These results indicate that the signaling component(s) defec tive in PR30 lie downstream of PKC but upstream of the commitment poin t for growth inhibition and cell death. Finally, although PR30-3 is re sistant to the inhibitory effects of phorbol ester, proliferation is n onetheless still inhibited in response to anti-mu stimulation. These r esults suggest that the growth inhibitory response of WEHI-231 to anti -mu and phorbol ester involves different pathways. (C) 1994 Academic P ress, Inc.