RESUMO
Multiple myeloma (MM) is a clinically and genetically heterogenous cancer where tumour cells have dysregulated expression of a D-type cyclin, often in association with a recurrent IgH translocation. Patients whose tumour cells express cyclin D2, with the translocation t(4;14) or t(14;16), generally have more proliferative disease and inferior outcomes. The phosphatidylinositol-3-kinase (PI3K) pathway is a major regulator of D-type cyclin expression and cell cycle entry. We evaluated the effect of PI3K pathway blockade on cell cycle behaviour in MM cells, investigating differences between cyclin D2- and cyclin D1-expressing tumours. MM cell lines and primary bone marrow CD138(+) MM cells were exposed to the pan-PI3K/mTOR inhibitor, PI-103, and assessed for cell cycle profiles, [(3)H]-thymidine uptake and cell cycle proteins. We report, in both cell lines and primary MM cells, that PI-103 induced cell cycle arrest with downregulation of cyclin D2 and CDK4/6 in MM cells expressing cyclin D2 via t(4;14) or t(14;16) translocations. Cells expressing cyclin D1 via t(11;14) were insensitive to PI-103, despite exhibiting inhibition of downstream signalling targets. In primary MM cells, PI-103 enhanced the anti-proliferative effects of anti-MM agents. Treatment paradigms including blockade of the PI3K/mTOR pathway should be targeted at patients with IgH translocations associated with cyclin D2 overexpression.
RESUMO
We describe a new model of myeloma bone disease in which beta2m 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.
Assuntos
Modelos Animais de Doenças , Lentivirus/genética , Células-Tronco Mesenquimais/citologia , Mieloma Múltiplo/patologia , Mieloma Múltiplo/terapia , Osteoprotegerina/biossíntese , Animais , Osso e Ossos/metabolismo , Linhagem Celular , Terapia Genética/métodos , Humanos , Cinética , Lentivirus/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Tíbia/patologiaRESUMO
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 p27(Kip1), which are associated with pocket protein hypophosphorylation and E2F inactivation. Ectopic expression of p27(Kip1) 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 p27(Kip1) have opposing roles in these pathways and our data also suggest that cyclin D2 functions upstream of p27(Kip1) 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 p27(Kip1). Inhibition of PI3-K signalling by LY294002 down-regulated cyclin D2 and up-regulated p27(Kip1) 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 p27(Kip1) expression induced by BCR-engagement. These results indicate that BCR activation targets p27(Kip1) 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.
Assuntos
Apoptose/fisiologia , Linfócitos B/patologia , Proteínas de Ciclo Celular/fisiologia , Ciclinas/fisiologia , Proteínas de Ligação a DNA , Fase G1/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas , Receptores de Antígenos de Linfócitos B/fisiologia , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Anticorpos Anti-Idiotípicos/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Cromonas/farmacologia , Ciclina D2 , Inibidor de Quinase Dependente de Ciclina p27 , Ciclinas/biossíntese , Ciclinas/genética , Fatores de Transcrição E2F , Inibidores Enzimáticos/farmacologia , Fase G1/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Linfoma de Células B/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Morfolinas/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosfoproteínas/fisiologia , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Proteínas Recombinantes de Fusão/fisiologia , Proteína do Retinoblastoma/fisiologia , Proteína p107 Retinoblastoma-Like , Proteína p130 Retinoblastoma-Like , Fatores de Transcrição/antagonistas & inibidores , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologiaRESUMO
B lymphocytes from mice null for the Rho-family guanine-nucleotide exchange factor, Vav, are defective in their ability to proliferate in response to BCR cross-linking, but are able to proliferate normally in response to LPS. In addition, they have a depletion of CD5(+) (B1) lymphocytes and defective IgG class switching. This phenotype is reminiscent of that observed in mice null for the cell cycle regulatory protein, cyclin D2. We demonstrate here that the inability of vav(-/-) B cells to proliferate in response to BCR ligation is due to an inability to induce cyclin D2. In addition, we show that the proliferative defect of these cells occurs after the cells have entered early G1 phase. Analyses of potential down-stream signaling intermediates revealed differential activation of the stress-activated MAP kinases in the absence of Vav, normal activation of the ERK, MAPK, and phosphatidylinositol 3-kinase pathways, and defective intracellular calcium mobilization. We further demonstrate that intracellular calcium homeostasis is required for cyclin D2 induction, implicating a possible link with the defective calcium response of vav(-/-) B cells and their inability to induce cyclin D2.
Assuntos
Linfócitos B/imunologia , Divisão Celular/fisiologia , Ciclinas/biossíntese , Proteínas Oncogênicas/fisiologia , Receptores de Antígenos de Linfócitos B/fisiologia , Transdução de Sinais , Animais , Cálcio/metabolismo , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Ciclina D2 , Imunoglobulina M/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-vav , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
This study investigated the influence of expression of proteins of the INK4 family, particularly p16, on the growth and self-renewal kinetics of hematopoietic cells. First, retrovirus-mediated gene transfer (RMGT) was used to restore p16(INK4a) expression in the p16(INK4a)-deficient lymphoid and myeloid cell lines BV173 and K562, and it was confirmed that this inhibited their growth. Second, to sequester p16(INK4a) and related INK4 proteins, cyclin-dependent kinase 4 (CDK4) was retrovirally transduced into normal human CD34(+) bone marrow cells and then cultured in myeloid colony-forming cell (CFC) assays. The growth of CDK4-transduced colonies was more rapid; the cell-doubling time was reduced; and, upon replating, the colonies produced greater yields of secondary colonies than mock-untransduced controls. Third, colony formation was compared by marrow cells from p16(INK4a-/-) mice and wild-type mice. The results from p16(INK4a-/-) marrow were similar to those from CDK4-transduced human CFCs, in terms of growth rate and replating ability, and were partially reversed by RMGT of p16(INK4a). Lines of immature granulocytic cells were raised from 15 individual colonies grown from the marrow of p16(INK4a-/-) mice. These had a high colony-forming ability (15%) and replating efficiency (96.7%). The p16(INK4a-/-) cell lines readily became growth factor-independent upon cytokine deprivation. Taken together, these results demonstrate that loss of INK4 proteins, in particular p16(INK4a), increases the growth rate of myeloid colonies in vitro and, more importantly, confers an increased ability for clonal expansion on hematopoietic progenitor cells.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Animais , Divisão Celular/fisiologia , Linhagem Celular , Regulação da Expressão Gênica/fisiologia , Genes Supressores de Tumor , Humanos , CamundongosRESUMO
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 p27(Kip1) 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 p27(Kip1) expression in response to IL-3 and BCR-ABL expression, we studied the effects of ectopic expression of cyclin D2 and p27(Kip1) on cell proliferation and survival. Our results demonstrate that both cyclin D2 and p27(Kip1) 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 p27(Kip1) can cause cell cycle arrest and apoptosis in the BaF3 cells. Furthermore, our data also suggest that cyclin D2 functions upstream of p27(Kip1), 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.
Assuntos
Apoptose , Proteínas de Ciclo Celular/biossíntese , Ciclinas/biossíntese , Proteínas de Fusão bcr-abl/fisiologia , Células-Tronco Hematopoéticas/citologia , Interleucina-3/fisiologia , Proteínas Supressoras de Tumor , Animais , Benzamidas , Proteínas de Ciclo Celular/genética , Divisão Celular , Linhagem Celular , Sobrevivência Celular , Ciclina D2 , Inibidor de Quinase Dependente de Ciclina p27 , Ciclinas/genética , Ciclinas/fisiologia , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Fase G1 , Células-Tronco Hematopoéticas/efeitos dos fármacos , Mesilato de Imatinib , Interleucina-3/farmacologia , Piperazinas/farmacologia , Pirimidinas/farmacologia , RNA Mensageiro/biossíntese , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
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 p27(Kip1) but that down-regulation of other cyclin-dependent kinase (CDK) inhibitors, including p15(INK4b), p16(INK4a), p19( INK4d), and p21(Cip1) as well as other negative cell cycle regulators such as p53 and p19(ARF), implies that this senescence-related growth arrest is independent of the activity of p53, p19(ARF), p16(INK4a), and p21(Cip1), which are associated with replicative senescence. The p27(Kip1) binds to the cyclin/CDK2 complexes and causes a decrease in CDK2 kinase activity. We demonstrated that ectopic expression of p27(Kip1) 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 p27(Kip1)expression. In summary, these observations taken together suggest that p27(Kip1) is an important mediator of the permanent cell cycle arrest induced by PI3K inhibitors. Our data suggest that repression of CDK2 activity by p27(Kip1) is required for the PI3K-induced senescence, yet mouse embryo fibroblasts derived from p27(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 p27(Kip1). This is the first description that p130 may have a role in inhibiting CDK activity during senescence.
Assuntos
Proteínas de Ciclo Celular , Senescência Celular/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor , Animais , Divisão Celular/fisiologia , Células Cultivadas , Cromonas/farmacologia , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Fibroblastos , Camundongos , Morfolinas/farmacologiaRESUMO
Progression into G(1) in B lymphocytes is regulated by cyclins D2 and D3, components of the cell cycle machinery currently believed to have overlapping and potentially redundant roles in cell cycle control. To study the specific role of cyclin D2 in B lymphocyte proliferation, we examined B cells from cyclin D2(-/-) mice and demonstrate a specific requirement for cyclin D2 in BCR- but not CD40- or lipopolysaccharide-induced proliferation. Furthermore, conventional B cell development proceeds normally in the mutant mice; however, the CD5 B cell compartment is dramatically reduced, suggesting that cyclin D2 is important in CD5 B cell development as well as antigen-dependent B cell clonal expansion.
Assuntos
Linfócitos B/citologia , Ciclinas/imunologia , Proteínas Oncogênicas/farmacologia , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas , Animais , Anticorpos/farmacologia , Linfócitos B/efeitos dos fármacos , Western Blotting , Antígenos CD40/farmacologia , Antígenos CD5/análise , Antígenos CD5/metabolismo , Diferenciação Celular , Divisão Celular , Ciclina D2 , Ciclina D3 , Ciclinas/análise , Ciclinas/deficiência , Citometria de Fluxo , Imunoglobulinas/imunologia , Interleucina-4/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Proto-Oncogênicas c-bcrRESUMO
Cyclin D2 is the only D-type cyclin expressed in mature mouse B-lymphocytes, and its expression is associated with retinoblastoma protein (pRB) and pRB-related protein phosphorylation and induction of E2F activity, as B-cells enter the cell cycle following stimulation via surface IgM and/or CD40. Cyclin D-dependent kinase activity is required for cell proliferation, yet cyclin D2(-/-) mice have normal levels of mature B-lymphocytes. Here we show that B-lymphocytes from cyclin D2(-/-) mice can proliferate in response to anti-IgM and anti-CD40, but the time taken to enter S-phase is longer than for the corresponding cyclin D2(+/+) cells. This is due to the compensatory induction of cyclin D3, but not cyclin D1, which causes pRb phosphorylation on CDK4-specific sites. This is the first demonstration that loss of a D-type cyclin causes specific expression and functional compensation by another member of the family in vivo and provides a rationale for the presence of mature B-lymphocytes in cyclin D2(-/-) mice.
Assuntos
Linfócitos B/imunologia , Antígenos CD40/imunologia , Ciclinas/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Linfócitos B/metabolismo , Antígenos CD40/metabolismo , Células Cultivadas , Ciclina D2 , Ciclina D3 , Ciclinas/metabolismo , Ativação Linfocitária , Camundongos , Receptores de Antígenos de Linfócitos B/metabolismoRESUMO
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.
Assuntos
Linfócitos B/imunologia , Antígenos CD40/metabolismo , Proteínas de Transporte , Imunoglobulina M/metabolismo , Ativação Linfocitária , Receptores de Antígenos de Linfócitos B/fisiologia , Receptores Fc/fisiologia , Fatores de Transcrição/metabolismo , Animais , Linfócitos B/metabolismo , Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fator de Transcrição E2F3 , Fator de Transcrição E2F4 , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Fosforilação , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Fatores de Transcrição/análise , Fatores de Transcrição/biossínteseRESUMO
The aims of this investigation were (1) to examine IGF-binding protein-3 (IGFBP-3) mRNA levels in candidate tissues which might be important sources for blood IGFBP-3 (liver and skin) and in a target tissue for IGF-I action (skeletal muscle), and (2) to examine the effects of a single dose (500 micrograms) of GH or IGF-I on IGFBP-3 message levels in these tissues since temporal responses (4, 8 and 24 h after the single subcutaneous dose of peptide to GH-deficient dwarf rats) would indicate which peptide is the primary modulator of IGFBP-3 synthesis. Circulating IGF-I and IGFBP-3 concentrations were significantly increased (P < 0.05) by IGF-I and GH. GH treatment increased liver IGFBP-3 mRNA levels by 4 h (P < 0.001 over the 24 h) whereas IGF-I had no effect. Similarly, GH, but not IGF-I, increased muscle IGFBP-3 mRNA levels (P < 0.001 for the 24 h study period). However, both IGF-I and GH induced increases in skin IGFBP-3 mRNA abundance throughout the 24 h period (P < 0.001 and P < 0.01 respectively) and skin IGFBP-3 message abundance was greater that in the liver. Liver IGF-I mRNA levels were, as expected, increased after GH and tended to decrease after IGF-I treatment; muscle IGF-I mRNA was increased by GH (P < 0.001) and, interestingly, progressively increased by IGF-I (P < 0.05 for the 24 h period); skin IGF-I mRNA levels were unchanged by both peptides. The IGF-I induced increase in serum IGFBP-3 concentrations in the absence of an increase in hepatic IGFBP-3 mRNA levels and a paucity of liver IGF-I type 1 receptor mRNA imply that other sources of IGFBP-3 protein or synthesis must exist. The response of skin IGFBP-3 mRNA levels to both GH and IGF-I suggests that other cell types, such as fibroblast-derived cells, could be more important than the liver in the regulation of circulating reservoir IGFBP-3 in certain circumstances. In contrast to some current suggestions, the rapid and consistent GH-induced increase in IGFBP-3 message levels in all tissues studied implies that GH might have a direct function in the regulation of IGFBP-3 synthesis.
Assuntos
Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Fígado/metabolismo , Músculo Esquelético/metabolismo , Neuropeptídeos/farmacologia , RNA Mensageiro/metabolismo , Pele/metabolismo , Animais , Autorradiografia , Western Blotting , Hormônio do Crescimento/deficiência , Hormônio do Crescimento/farmacologia , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/biossíntese , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Masculino , Modelos Biológicos , Sondas RNA , RNA Antissenso , RNA Mensageiro/análise , Ratos , Ratos Mutantes , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismoRESUMO
Pregnancy is rarely associated with Cushing's syndrome. This case report describes the successful management of a Caesarean section under epidural anaesthesia in a patient with Cushing's syndrome. Maternal and fetal complications are reviewed from an anaesthetic perspective and alternative anaesthetic techniques discussed.