Janet Glassford

Control of Cell Cycle Exit and Entry by Protein Kinase B-Regulated Forkhead Transcription Factors

ABSTRACT 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 p27kip1. 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 G0 and become quiescent. This is shown by a change in phosphorylation of p130 to G0-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.

Inhibition of the phosphoinositide 3-kinase pathway induces a senescence-like arrest mediated by p27Kip1.

A senescence-like growth arrest is induced in mouse primary embryo fibroblasts by inhibitors of phosphoinositide 3-kinase (PI3K). We observed that senescence-like growth arrest is correlated with an increase in p27Kip1 but that down-regulation of other cyclin-dependent kinase (CDK) inhibitors, including p15INK4b, p16INK4a, p19 INK4d, and p21Cip1 as well as other negative cell cycle regulators such as p53 and p19ARF, implies that this senescence-related growth arrest is independent of the activity of p53, p19ARF, p16INK4a, and p21Cip1, which are associated with replicative senescence. The p27Kip1 binds to the cyclin/CDK2 complexes and causes a decrease in CDK2 kinase activity. We demonstrated that ectopic expression of p27Kip1 can induce permanent cell cycle arrest and a senescence-like phenotype in wild-type mouse embryo fibroblasts. We also obtained results suggesting that the kinase inhibitors LY294002 and Wortmannin arrest cell growth and induce a senescence-like phenotype, at least partially, through inhibition of PI3K and protein kinase B/Akt, activation of the forkhead protein AFX, and up-regulation of p27Kip1expression. In summary, these observations taken together suggest that p27Kip1 is an important mediator of the permanent cell cycle arrest induced by PI3K inhibitors. Our data suggest that repression of CDK2 activity by p27Kip1 is required for the PI3K-induced senescence, yet mouse embryo fibroblasts derived fromp27 Kip1−/− mice entered cell cycle arrest after treatment with LY294002. We show that this is due to a compensatory mechanism by which p130 functionally substitutes for the loss of p27Kip1. This is the first description that p130 may have a role in inhibiting CDK activity during senescence.

BCR-ABL and interleukin 3 promote haematopoietic cell proliferation and survival through modulation of cyclin D2 and p27Kip1 expression.

Although it is evident that BCR-ABL can rescue cytokine-deprived hematopoietic progenitor cells from cell cycle arrest and apoptosis, the exact mechanism of action of BCR/ABL and interleukin (IL)-3 to promote proliferation and survival has not been established. Using the pro-B cell line BaF3 and a BaF3 cell line stably overexpressing BCR-ABL (BaF3-p210), we investigated the proliferative signals derived from BCR-ABL and IL-3. The results indicate that both IL-3 and BCR-ABL target the expression of cyclin Ds and down-regulation of p27Kip1 to mediate pRB-related pocket protein phosphorylation, E2F activation, and thus S phase progression. These findings were further confirmed in a BaF3 cell line (TonB.210) where the BCR-ABL expression is inducible by doxycyclin and by using the drug STI571 to inactivate BCR-ABL activity in BaF3-p210. To establish the functional significance of cyclin D2 and p27Kip1 expression in response to IL-3 and BCR-ABL expression, we studied the effects of ectopic expression of cyclin D2 and p27Kip1 on cell proliferation and survival. Our results demonstrate that both cyclin D2 and p27Kip1 have a role in BaF3 cell proliferation and survival, as ectopic expression of cyclin D2 is sufficient to abolish the cell cycle arrest and apoptosis induced by IL-3 withdrawal or by BCR-ABL inactivation, while overexpression of p27Kip1 can cause cell cycle arrest and apoptosis in the BaF3 cells. Furthermore, our data also suggest that cyclin D2 functions upstream of p27Kip1, cyclin E, and cyclin D3, and therefore, plays an essential part in integrating the signals from IL-3 and BCR-ABL with the pRB/E2F pathway.

BCR signals target p27 Kip1 and cyclin D2 via the PI3-K signalling pathway to mediate cell cycle arrest and apoptosis of WEHI 231 B cells

Cross-linking of the B cell antigen receptor (BCR) on immature WEHI 231 B cells results in G1 cell cycle arrest and apoptosis. Here we investigated the molecular mechanisms that are necessary and sufficient for these changes to occur. We show that BCR stimulation of WEHI 231 cells results in down-regulation of cyclin D2 and up-regulation of p27Kip1, which are associated with pocket protein hypophosphorylation and E2F inactivation. Ectopic expression of p27Kip1 by TAT-fusion protein or retroviral transduction is sufficient to cause G1 cell cycle arrest, followed by apoptosis. In contrast, over-expression of cyclin D2 overcomes the cell cycle arrest and apoptosis induced by anti-IgM, indicating that down-regulation of cyclin D2 is necessary for the cell cycle arrest and apoptosis activated by BCR stimulation. Thus, cyclin D2 and p27Kip1 have opposing roles in these pathways and our data also suggest that cyclin D2 functions upstream of p27Kip1 and the pRB pathway and therefore plays an essential part in integrating the signals from BCR with the cell cycle machinery. We next investigated which signal transduction pathways triggered by the BCR regulate cell proliferation and apoptosis via cyclin D2 and p27Kip1. Inhibition of PI3-K signalling by LY294002 down-regulated cyclin D2 and up-regulated p27Kip1 expression at both protein and RNA levels, mimicking the effects of BCR-stimulation. Furthermore, ectopic expression of a constitutively active form of AKT blocked the cell cycle arrest and apoptosis triggered by anti-IgM and also abrogated down-regulation of cyclin D2 and up-regulation of p27Kip1 expression induced by BCR-engagement. These results indicate that BCR activation targets p27Kip1 and cyclin D2 to mediate cell cycle arrest and apoptosis and that down-regulation of PI3-K/AKT activity post BCR stimulation is necessary for these to occur.

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

The p85α 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 p85α 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 p85α 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 PLCγ (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 (p85α, Btk, BLNK and PLCγ) 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 Ca2+ 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 Ca2+ and PKC-dependent signalling cascades to mediate cell-cycle progression in response to BCR engagement.

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 Ca2+ flux using different mechanisms. Each form of activation requires the function of Vav and PI3K. However, Vav regulates Ca2+ 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 Ca2+ flux has a PI3K-independent component, while Ca2+ flux following mIg/CD19 coligation is totally PI3K dependent. The p85α and p110δ subunits of PI3K both participate in anti-IgM and mIg/CD19 coligation-induced Ca2+ 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 p110α 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.

A new xenograft model of myeloma bone disease demonstrating the efficacy of human mesenchymal stem cells expressing osteoprotegerin by lentiviral gene transfer

We describe a new model of myeloma bone disease in which β2m NOD/SCID mice injected with KMS-12-BM cells develop medullary disease after tail vein administration. Micro-computed tomography analysis demonstrated significant bone loss in the tibiae and vertebrae of diseased animals compared to controls, with loss of cortical bone (P<0.01), as well as trabecular bone volume, thickness and number (P<0.05 for all). Bone marrow of diseased animals demonstrated an increase in osteoclasts (P<0.01) and reduction in osteoblasts (P<0.01) compared to control animals. Both bone loss and osteoclast increase correlated with the degree of disease involvement. Mesenchymal stem cells (MSCs) were lentivirally transduced to express human osteoprotegerin (hOPG). Systemic administration of OPG expressing MSC reduced osteoclast activation (P<0.01) and trabecular bone loss in the vertebrae (P<0.05) and tibiae of diseased animals, to levels comparable to non-diseased controls. Because of its predominantly medullary involvement and quantifiable parameters of bone disease, the KMS-12-BM xenogeneic model provides unique opportunities to test therapies targeted at the bone marrow microenvironment.

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.

Modulation of E2F activity in primary mouse B cells following stimulation via surface IgM and CD40 receptors.

Since signals via CD40 and the B cell receptor are known to synergize to induce B cell activation, we have analyzed the pocket protein / E2F complexes in mouse B lymphocytes following stimulation by anti‐IgM, anti‐CD40, alone or together. We find that E2F4 and DP1 form the predominant E2F heterodimers in the G0 and G1 phases of the cell cycle, complexed with hypophosphorylated p130. During late G1 and S phase this complex is replaced by at least three different E2F complexes, one of which is an E2F complex containing p107 or pRB as well as two “free” E2F complexes consisting of E2F4 / DP1 and E2F1 – 3 / DP1. These effects were mirrored by the levels and phosphorylation status of the three pocket proteins. We also observed an increase in electrophoretic mobility of DP1 and E2F4 as B cells progressed from G0 into early G1, resulting from their dephosphorylation. This is known to correlate with a decrease in DNA binding capacity of these proteins and could also be important for derepression of genes negatively regulated through E2F sites in their promoters. These results therefore indicate that the pRB / E2F pathway integrates proliferative signals emanating from the sIgM and CD40 receptors.

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.