The weak per turbation of the transcription regulatory network, leading to a biased expression of those early response genes that were involved in the cell death pathways, was presumably a direct outcome of the sparse nature of the BCR signal ing network in these cells. We believe that successful extraction of the core BCR dependent moreover regulatory network that enforced cell cycle arrest in CH1 cells represents a key highlight Inhibitors,Modulators,Libraries of our study. Its significance lies in the fact that this network encompasses pathways emanating from the BCR to the key signaling intermediates, and then also those extend ing from these intermediates to the TFs that were criti cal for inducing expression of the pro apoptotic genes. This could be achieved by employing an in silico based network approach that combined the data on BCR acti vated signaling events, with that on modulation of TF activities.

Further, this approach also enabled us to inte grate the DOR motif that Inhibitors,Modulators,Libraries linked these TFs to the effec tor genes. Importantly here, the effector genes responsible for causing G1 arrest could first be identi fied through a comparison of the early gene expression profile between CH1 and mature B cells, and then func tionally verified in experiments involving their selective depletion by siRNA. Having delineated the core BCR dependent molecu lar network that specified the G1 arrest, we could then test the effects of specific perturbations so as to iden tify the key signaling intermediates involved in driving this response. By using a panel of pharmacological inhibitors against different kinases, we localized p38 and CAMKII as the likely targets.

Such an inference could be derived from our observations that, of the inhibitors tested, only those specific for either of these kinases were capable of at least partially reversing anti IgM induced G1 arrest of the cells. A subsequent Inhibitors,Modulators,Libraries examination of the expression profile of the effector gene subset revealed that p38 inhibition was more effective at inhibiting induction of these genes, thus identifying p38 as the central regulator of the anti IgM induced cell cycle arrest response. Conclusions Interestingly, Inhibitors,Modulators,Libraries the mechanism by which p38 exerted such a prominent effect Inhibitors,Modulators,Libraries involved a novel feedback loop that controlled signal amplification at a level that was imme diately proximal to the BCR.

As we showed, p38 directly regulated activity of the BCR associated phosphatase SHP 1, which in turn influenced the activity of Lyn, the earliest intermediate involved in BCR signaling. Thus, p38 mediated attenuation of SHP 1 activity led to increased basal levels of Lyn phosphorylation, thereby rendering it less sensitive to BCR activation. The selec tive and transient activation meantime of the signaling network then was direct consequence of the dampening of the initiating signal from the BCR.