APRIL promotes cell-cycle progression in primary multiple myeloma cells: influence of D-type cyclin group and translocation status.

A proliferation-inducing ligand (APRIL) promotes survival and drug resistance in multiple myeloma (MM) cell lines. We studied the effect of APRIL on cell-cycle behavior in primary MM cells and correlated our findings with D-type cyclin expression by immunohistochemistry and/or Western blotting. In MM cases, expressing cyclin D2 APRIL significantly increased the percentage of CD138(+) cells in S + G(2)/M phase (from 8.4% ± 1.9% to 14.3% ± 2.6%, n = 15, P < .01), whereas a lesser effect was seen in cases expressing cyclin D1 (n = 18). Cell-cycle response to APRIL was most marked for cyclin D2-expressing cases with IgH translocations (P < .01) and was accompanied by increased expression of cyclin D2, CDK4, CDK6, and phospho-retinoblastoma protein. Cell-cycle proteins in cyclin D1(+) cells were not modulated by APRIL. Surface expression of B-cell maturation antigen and transmembrane activator and calcium-modulating cyclophilin ligand interactor was not significantly different between cyclin D1(+) and D2(+) MM cells. We observed activation of nuclear factor-κB and PI3-kinase pathways in response to APRIL in both cyclin D1(+) and D2(+) MM cells. In conclusion, APRIL stimulates G(1)/S progression in cyclin D2(+) MM cells bearing IgH translocations but has minimal effect on cyclin D1(+) cells, suggesting MM cells from different cyclin D/translocation classes rely on different mechanisms for cell-cycle re-entry.

p27Kip1 and p130 cooperate to regulate hematopoietic cell proliferation in vivo.

To investigate the potential functional cooperation between p27Kip1 and p130 in vivo, we generated mice deficient for both p27Kip1 and p130. In p27Kip1-/-; p130-/- mice, the cellularity of the spleens but not the thymi is significantly increased compared with that of their p27Kip1-/- counterparts, affecting the lymphoid, erythroid, and myeloid compartments. In vivo cell proliferation is significantly augmented in the B and T cells, monocytes, macrophages, and erythroid progenitors in the spleens of p27Kip1-/-; p130-/- animals. Immunoprecipitation and immunodepletion studies indicate that p130 can compensate for the absence of p27Kip1 in binding to and repressing CDK2 and is the predominant CDK-inhibitor associated with the inactive CDK2 in the p27Kip1-/- splenocytes. The finding that the p27Kip1-/-; p130-/- splenic B cells are hypersensitive to mitogenic stimulations in vitro lends support to the concept that the hyperproliferation of splenocytes is not a result of the influence of their microenvironment. In summary, our findings provide genetic and molecular evidence to show that p130 is a bona fide cyclin-dependent kinase inhibitor and cooperates with p27Kip1 to regulate hematopoietic cell proliferation in vivo.

Control of cell cycle exit and entry by protein kinase B-regulated forkhead transcription factors.

AFX-like Forkhead transcription factors, which are controlled by phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) signaling, are involved in regulating cell cycle progression and cell death. Both cell cycle arrest and induction of apoptosis are mediated in part by transcriptional regulation of p27(kip1). Here we show that the Forkheads AFX (FOXO4) and FKHR-L1 (FOXO3a) also directly control transcription of the retinoblastoma-like p130 protein and cause upregulation of p130 protein expression. Detailed analysis of p130 regulation demonstrates that following Forkhead-induced cell cycle arrest, cells enter G(0) and become quiescent. This is shown by a change in phosphorylation of p130 to G(0)-specific forms and increased p130/E2F-4 complex formation. Most importantly, long-term Forkhead activation causes a sustained but reversible inhibition of proliferation without a marked increase in apoptosis. As for the activity of the Forkheads, we also show that protein levels of p130 are controlled by endogenous PI3K/PKB signaling upon cell cycle reentry. Surprisingly, not only nontransformed cells, but also cancer cells such as human colon carcinoma cells, are forced into quiescence by Forkhead activation. We therefore propose that Forkhead inactivation by PKB signaling in quiescent cells is a crucial step in cell cycle reentry and contributes to the processes of transformation and regeneration.

BCR targets cyclin D2 via Btk and the p85alpha subunit of PI3-K to induce cell cycle progression in primary mouse B cells.

The p85alpha subunit of PI3-K and Btk are two crucial components of the B-cell receptor (BCR) signalling pathway. In the present study, we showed that primary splenic B cells from p85alpha null and xid (Btk-deficient) mice fail to induce cyclin D2 expression and enter early G1, but not S phase of the cell cycle in response to BCR engagement. Furthermore, these Btk or p85alpha null B cells displayed increased cell death compared with wild type following BCR engagement. These findings are further confirmed by studies showing that specific pharmacological inhibitors of Btk (LFM-A13), PI3-K (LY294002 and Wortmannin) and PLCgamma (U73122) also block cyclin D2 expression and S phase entry following BCR stimulation, as well as triggering apoptosis. Collectively, these data provide evidence for the concept that the B-cell signalosome (p85alpha, Btk, BLNK and PLCgamma) is involved in regulating cyclin D2 expression in response to BCR engagement. PKC and intracellular calcium are two major downstream effectors of the B-cell signalosome and can be activated by PMA and ionomycin, respectively. In small resting (G0) B cells, costimulation with PMA and ionomycin, but not PMA or ionomycin alone, induces cyclin D2 expression and cell-cycle progression. Consistent with this, we also showed that the BCR-mediated cyclin D2 induction could be abolished by pretreatment of resting B cells with specific inhibitors of capacitative Ca(2+) entry (SK&F 96365) or PKC (Gö6850). Our present results lead us to propose a model in which the B-cell signalosome targets cyclin D2 via the Ca(2+) and PKC-dependent signalling cascades to mediate cell-cycle progression in response to BCR engagement.

Cyclin D2 controls B cell progenitor numbers.

Cyclin D2 affects B cell proliferation and differentiation in vivo. It is rate-limiting for B cell receptor (BCR)-dependent proliferation of B cells, and cyclin D2-/- mice lack CD5+(B1) B lymphocytes. We show here that the bone marrow (BM) of cyclin D2-/- mice contains half the numbers of Sca1+B220+ B cell progenitors but normal levels of Sca1+ progenitor cells of other lineages. In addition, clonal analysis of BM from the cyclin D2-/- and cyclin D2+/+ mice confirmed that there were fewer B cell progenitors (B220+) in the cyclin D2-/- mice. In addition, the colonies from cyclin D2-/- mice were less mature (CD19lo) than those from cyclin D2+/+ mice (CD19Hi). The number of mature B2 B cells in vivo is the same in cyclin D2-/- and cyclin D2+/+ animals. Lack of cyclin D2 protein may be compensated by cyclin D3, as cyclin-dependent kinase (cdk)6 coimmunoprecipitates with cyclin D3 but not cyclin D1 from BM mononuclear cells of cyclin D2-/- mice. It is active, as endogenous retinoblastoma protein is phosphorylated at the cdk6/4-cyclin D-specific sites, S807/811. We conclude that cyclin D2 is rate-limiting for the production of B lymphoid progenitor cells whose proliferation does not depend on BCR signaling.

Functional regulation of D-type cyclins by insulin-like growth factor-I and serum in multiple myeloma cells.

D-type cyclin genes are universally dysregulated in multiple myeloma (MM), but the functional consequences are unclear as D-type cyclin gene expression does not correlate with proliferation or disease progression. We examined the protein expression and regulation of D-type cyclins and other cell cycle regulators in human myeloma cell lines and primary CD138(+) plasma cells (PCs). Cyclin D1, cyclin D2, cyclin dependent kinase (CDK) 4, CDK6, p27(Kip1) p18(INK4C) and retinoblastoma protein (pRb) were absent in normal PCs, heterogeneously expressed in primary MM cells and positively correlated with disease activity/progression. Cyclins D1 and D2 complexed with both CDK4 and CDK6, suggesting that both phosphorylate pRb in MM. Furthermore, cyclin D2 expressed via either t(14;16) or t(4;14) IgH translocations was functionally upregulated by fetal calf serum or insulin-like growth factor-I, leading to pRb phosphorylation and cell cycle entry/progression, and in some cases inversely correlated with p27(Kip1). However, pRb phosphorylation and cell cycle progression mediated by cyclin D1 expressed via t(11;14) was less dependent on exogenous stimuli. These data suggest that the presence or absence of specific IgH translocations underlying aberrant D-type cyclin expression may influence their response to mitogens in the bone marrow microenvironment. We showed for the first time that D-type cyclins are functionally regulated in MM, differentially responsive to exogenous growth factors and upregulated with disease progression.

Phosphatidylinositol 3-kinase is required for the transcriptional activation of cyclin D2 in BCR activated primary mouse B lymphocytes.

Induction of cyclin D2 is essential for mediating cell cycle entry in B cells activated by BCR cross-linking. In the present study we show that, like B lymphocytes lacking cyclin D2, the p85alpha subunit of phosphatidylinositol 3-kinase (PI3K) or other components of the B cell signalosome, p110delta-null B cells fail to induce cyclin D2 and enter early G1 but not S phase of the cell cycle. The inhibitors of PI3K activity, LY294002 and Wortmannin, also abrogate cyclin D2 induction by BCR cross-linking, confirming that the class IA PI3K is necessary for cyclin D2 induction in response to BCR stimulation. Furthermore, using both p85alpha-null and p110delta-null B cells and inhibitors of PI3K, this study demonstrates for the first time, that BCR cross-linking induces cyclin D2 mRNA expression via transcriptional activation of the cyclin D2 promoter and that this transcriptional activation of cyclin D2 requires PI3K activity. Moreover, we identify a region between nucleotides -1624 and -1303 of the cyclin D2 promoter containing elements responsive to anti-IgM, which are PI3K dependent. Further characterisation of signalling intermediates downstream of the BCR revealed a perturbation of MAPK signalling pathways in p85alpha-null and p110delta-null B cells, and our data suggests that cross-talk exists between the PI3K and JNK pathways.

Vav-dependent and vav-independent phosphatidylinositol 3-kinase activation in murine B cells determined by the nature of the stimulus.

We show in this study that B cell activation following high avidity ligation of IgM or coligation of membrane Ig with CD19 elicits similar levels of Ca(2+) flux using different mechanisms. Each form of activation requires the function of Vav and PI3K. However, Vav regulates Ca(2+) flux independently of PI3K following anti-IgM cross-linking. By contrast, Vav function is essential for PI3K activation following membrane Ig (mIg)/CD19 coligation. Inhibition of PI3K revealed anti-IgM-stimulated Ca(2+) flux has a PI3K-independent component, while Ca(2+) flux following mIg/CD19 coligation is totally PI3K dependent. The p85alpha and p110delta subunits of PI3K both participate in anti-IgM and mIg/CD19 coligation-induced Ca(2+) flux, although the defects are not as severe as observed after pharmacological inhibition. This may reflect the recruitment of additional PI3K subunits, as we found that p110alpha becomes associated with CD19 upon B cell activation. These data show that the nature of the Ag encountered by B cells determines the contribution of Vav proteins to PI3K activation. Our results indicate that the strong signals delivered by multivalent cross-linking agents activate B cells in a qualitatively different manner from those triggered by coreceptor recruitment.

H2O2 induces a transient multi-phase cell cycle arrest in mouse fibroblasts through modulating cyclin D and p21Cip1 expression.

To defend against the potential damages induced by reactive oxygen species, proliferating cells enter a transient cell cycle arrest. We treated mouse fibroblasts with H(2)O(2) and found that sublethal doses of H(2)O(2) induced a transient multi-phase cell cycle arrest at the G(1), S, and G(2) phases but not the M phase. Western blot analysis demonstrated that this transient cell cycle arrest is associated with the down-regulation of cyclins D1 and D3 and up-regulation of the CKI p21(Cip1) expression. We also demonstrate that the induction in p21(Cip1) expression by H(2)O(2) is at least partially mediated at the transcriptional level and can occur in the absence of p53 function. Further immunoprecipitation kinase and immunodepletion assays indicated that in response to H(2)O(2) treatment, the down-regulation of cyclin Ds expression are associated with repression of cyclin D-CDK4, whereas the accumulation of p21(Cip1) is responsible for the inhibition of cyclin E and A-CDK2 activity and associated with the down-regulation of cyclin B-CDC2 activity. These data could account for the cell cycle arrest at the G(1), S, and G(2) phases following H(2)O(2) stimulation. Deletion of p21(Cip1), restoration of cyclin D expression, or overexpression of cyclin E alone is insufficient to effectively overcome the cell cycle arrest caused by sublethal doses of H(2)O(2). By contrast, overexpression of the human Herpesvirus 8 K cyclin, which can mimic the function of cyclin D and E, is enough to override this transient cell cycle arrest. On the basis of our findings, we propose a model in which moderate levels of H(2)O(2) induce a transient multi-phase cell cycle arrest at least partially through up-regulation of p21(Cip1) and down-regulation of cyclin D expression.