A requirement for cyclin D2 in G 1 -to-S phase progression has been definitively established in mature B cells stimulated via the B cell antigen receptor (BCR). However, the identity of constituents of the BCR signaling cascade that leads to cyclin D2 accumulation remains incomplete. We report that inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-1/2 blocked BCR-induced activation of extracellular signal-regulated kinase (ERK). Inhibition of the MEK1/2-ERK pathway was sufficient to abrogate BCR-induced cyclin D2 expression at the mRNA and protein levels. Disruption of endogenous heat shock protein 90 (hsp90) function with geldanamycin abrogated BCR-induced cyclin D2 expression and proliferation. Geldanamycin effects were attributed to a selective depletion of cellular Raf-1 that interrupted BCR-coupled activation of MEK1/2 and ERK. By contrast, signaling through the phosphatidylinositol 3-kinase and protein kinase C pathways was not affected, suggesting that disruption of hsp90 function did not cause a general impairment of BCR signaling. These results suggest that the MEK1/2-ERK pathway is essential for BCR signaling to cyclin D2 accumulation in ex vivo splenic B lymphocytes. Furthermore, these findings imply that hsp90 function is required for BCR signaling through the Raf-1-MEK1/2-ERK pathway but not through the phosphatidylinositol 3-kinase-or protein kinase C-dependent pathways.

Phosphatidylinositol 3-kinase (PI-3K) has been linked to promitogenic responses in splenic B cells following B cell Ag receptor (BCR) cross-linking; however identification of the signaling intermediates that link PI-3K activity to the cell cycle remains incomplete. We show that cyclin D2 induction is blocked by the PI-3K inhibitors wortmannin and LY294002, which coincides with impaired BCR-mediated mitogen-activated protein/extracellular signal-related kinase kinase (MEK)1/2 and p42/44ERK phosphorylation on activation residues. Cyclin D2 induction is virtually absent in B lymphocytes from mice deficient in the class IA PI-3K p85α regulatory subunit. In contrast to studies with PI-3K inhibitors, which inhibit all classes of PI-3Ks, the p85α regulatory subunit is not required for BCR-induced MEK1/2 and p42/44ERK phosphorylation, suggesting the contribution of another PI-3K family members in MEK1/2 and p42/44ERK activation. However, p85α−/− splenic B cells are defective in BCR-induced IκB kinase β and IκBα phosphorylation. We demonstrate that NF-κB signaling is required for cyclin D2 induction via the BCR in normal B cells, implicating a possible link with the defective IκB kinase β and IκBα phosphorylation in p85α−/− splenic B cells and their ability to induce cyclin D2. These results indicate that MEK1/2-p42/44ERK and NF-κB pathways link PI-3K activity to Ag receptor-mediated cyclin D2 induction in splenic B cells.

An effective humoral response requires that a given B lymphocyte population express a repertoire of receptors capable of recognizing a distinct array of antigens, while at the same time disregarding self-antigens. Mature B cells interacting with antigen via their B cell antigen receptors (BCRs) enter G(1) phase of the cell cycle and, depending on the strength of the signal, can commit to S phase entry. Input from co-receptors, which may function to either enhance or inhibit BCR signals, also influence the decision to proliferate. We review herein recent advances in the biochemistry of G(1)-cyclin holoenzymes that function to integrate BCR-coupled signaling pathways to the phosphorylation (and inactivation) of the retinoblastoma gene product (pRb) in splenic B lymphocytes (B-2 cells). We also highlight differences in the control of G(1)-to-S phase progression between B-2 cells and peritoneal CD5+ B cells (B-1 cells).

Cross-linking the B cell Ag receptor (BCR) to surface Fc receptors for IgG (FcγR) inhibits G1-to-S progression; the mechanism by which this occurs is not completely known. We investigated the regulation of three key cell cycle regulatory components by BCR-FcγR co-cross-linking: G1-cyclins, cyclin-dependent kinases (Cdks), and the retinoblastoma gene product (Rb). Rb functions to suppress G1-to-S progression in mammalian cells. Rb undergoes cell-cycle-dependent phosphorylation, leading to its inactivation and thereby promoting S phase entry. We demonstrate in this paper for the first time that BCR-induced Rb phosphorylation is abrogated by co-cross-linking with FcγR. The activation of Cdk4/6- and Cdk2-dependent Rb protein kinases is concomitantly blocked. FcγR-mediated inhibition of Cdk2 activity results in part from an apparent failure to express Cdk2 protein. By contrast, inhibition of Cdk4/6 activities is not due to suppression of Cdk4/6 or cyclins D2/D3 expression or inhibition of Cdk-activating kinase activity. Cdk4- and Cdk6-immune complexes recovered from B cells following BCR-FcγR co-cross-linking are devoid of coprecipitated D-type cyclins, indicating that inhibition of their Rb protein kinase activities is due in part to the absence of bound D-type cyclin. Thus, BCR-derived activation signals that up-regulate D-type cyclin and Cdk4/6 protein expression remain intact; however, FcγR-mediated signals block cyclin D-Cdk4/6 assembly or stabilization. These results suggest that assembly or stabilization of D-type cyclin holoenzyme complexes 1) is an important step in the activation of Cdk4/6 by BCR signals, and 2) suffice in providing a mechanism to account for inhibition of BCR-stimulated Rb protein phosphorylation by FcγR.

Students learn science by actually performing science activities. The 12 laboratories described in this article assist students in applying the fundamental techniques germane to the field of forensic science to "solve" contrived cases and present "evidence" in a mock trial. Moreover, students are also confronted with some of the legal and ethical issues concerning the validity, reliability, and application of some forensic techniques. The pedagogical design of the laboratory course provides a rich, challenging, and interdisciplinary academic experience intended to augment and compliment the didactic forensic lecture portion of the course. This laboratory course was designed to engender, embody, and articulate one of the University's directive goals to support interdisciplinary teaching, research, and programming. Because we developed the laboratories on minimal funds, we demonstrated that it could be cost-effective. And thus, we recommend a laboratory science course be included as part of the curriculum of all forensic nursing students and practitioners.