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1.
Am J Pathol ; 175(4): 1653-61, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19717643

RESUMO

B-cell lymphomas, the most frequent human immune system malignancies, often contain dysregulated TCL1 oncogene expression. TCL1 transgenic (TCL1-tg) mice develop a spectrum of B-cell malignancies, supporting an oncogenic role for TCL1 in B cells. Our prior global survey of DNA methylation patterns in TCL1-tg B-cell lymphomas identified many lymphoma-specific candidate hypermethylated genes, including Stk39. The Stk39 encoded protein, sterile 20-like-related proline-alanine-rich kinase (SPAK), regulates cell stress responses, and microarray studies identified reduced SPAK expression in metastatic prostate and treatment-resistant breast cancers, suggesting that its loss may have a role in cancer progression. Here we identified DNA hypermethylation and SPAK silencing in TCL1-tg B-cell lymphomas and SPAK silencing without DNA methylation in multiple subtypes of human B-cell lymphomas. SPAK knockdown by shRNA protected B cells from caspase-dependent apoptosis induced by DNA double-strand breaks but not apoptosis in response to osmotic or oxidative cell stressors. Caspase 3 activation by cleavage was impaired with SPAK repression in DNA damaged B cells. Interestingly, c-Jun NH(2)-terminal kinase is potentially activated by SPAK and pharmacological inhibition of c-Jun NH(2)-terminal kinase in SPAK-expressing B cells recapitulated the cell-protective phenotype of SPAK knockdown. Taken together, these data indicate that SPAK loss in B-cell lymphomas promotes increased cell survival with DNA damage and provides a potential mechanism for increased resistance to genotoxic stress in cancer.


Assuntos
Apoptose , Dano ao DNA , Inativação Gênica , Linfoma de Células B/enzimologia , Linfoma de Células B/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Caspase 3/metabolismo , Quebras de DNA de Cadeia Dupla , Metilação de DNA , Ativação Enzimática , Humanos , Linfoma de Células B/genética , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas/metabolismo
2.
Blood ; 113(11): 2478-87, 2009 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-19147787

RESUMO

B-cell lymphoma is the most common immune system malignancy. TCL1 transgenic mice (TCL1-tg), in which TCL1 is ectopically expressed in mature lymphocytes, develop multiple B- and T-cell leukemia and lymphoma subtypes, supporting an oncogenic role for TCL1 that probably involves AKT and MAPK-ERK signaling pathway augmentation. Additional, largely unknown genetic and epigenetic alterations cooperate with TCL1 during lymphoma progression. We examined DNA methylation patterns in TCL1-tg B-cell tumors to discover tumor-associated epigenetic changes, and identified hypermethylation of sprouty2 (Spry2). Sprouty proteins are context-dependent negative or positive regulators of MAPK-ERK pathway signaling, but their role(s) in B-cell physiology or pathology are unknown. Here we show that repression of Spry2 expression in TCL1-tg mouse and human B-cell lymphomas and cell lines is associated with dense DNA hypermethylation and was reversed by inhibition of DNA methylation. Spry2 expression was induced in normal splenic B cells by CD40/B-cell receptor costimulation and regulated a negative feedback loop that repressed MAPK-ERK signaling and decreased B-cell viability. Conversely, loss of Spry2 function hyperactivated MAPK-ERK signaling and caused increased B-cell proliferation. Combined, these results implicate epigenetic silencing of Spry2 expression in B lymphoma progression and suggest it as a companion lesion to ectopic TCL1 expression in enhancing MAPK-ERK pathway signaling.


Assuntos
Linfócitos B/fisiologia , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Linfoma de Células B/genética , Proteínas de Membrana/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linfócitos B/metabolismo , Antígenos CD40/metabolismo , Antígenos CD40/fisiologia , Metilação de DNA/fisiologia , Feminino , Inativação Gênica/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases , Células Tumorais Cultivadas
3.
Transplantation ; 83(8): 1114-21, 2007 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-17452903

RESUMO

BACKGROUND: Posttransplant lymphoproliferative disease (PTLD) is a serious complication of solid organ and bone marrow transplantation and is closely associated with Epstein-Barr virus (EBV) infection. We have previously shown that rapamycin (RAPA) directly inhibits the in vitro and in vivo proliferation of EBV-infected B lymphoblastoid cell lines (SLCL), derived from patients with PTLD, by arresting cells in the G1 phase of the cell cycle. The aim of this study is to elucidate the mechanism by which RAPA causes cell cycle arrest in EBV+ B cells. METHODS: SLCL were cultured without or with RAPA (10 ng/ml) and G1-associated cell cycle proteins were analyzed by immunoblot and densitometric analysis. CDK complexes were immunoprecipitated and incubated with retinoblastoma protein (Rb) substrate. Kinase activity of the complex was determined by Western blot with anti-phospho-Rb antibodies. RESULTS: We show that RAPA decreased both Cyclin D2 and Cyclin D3 protein levels. Furthermore, RAPA decreased the protein levels of cyclin dependent kinase 4 (CDK4) and increased the expression of the CDK inhibitor p27. In contrast, expression of the CDK inhibitor p21 was markedly inhibited by RAPA in the SLCL. Finally, in vitro kinase assays revealed that downstream hyperphosphorylation of Rb by CDK complexes was also decreased by RAPA. CONCLUSION: The results presented here elucidate key targets of RAPA-induced cell cycle arrest, provide insight into the growth pathways of EBV+ B-cell lymphomas, and demonstrate the potential for RAPA as a therapeutic option in the treatment of PTLD and other EBV+ lymphomas.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Herpesvirus Humano 4/fisiologia , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Sirolimo/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Fosforilação , Proteína do Retinoblastoma/metabolismo
4.
Cancer Lett ; 248(2): 198-210, 2007 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-16934922

RESUMO

TCL1 is an AKT kinase coactivator that, when dysregulated, initiates mature lymphocyte malignancies in humans and transgenic mice. While TCL1 augments AKT pathway signaling, additional TCL1 interacting proteins that may contribute to cellular homeostasis or transformation are lacking. Here, an exoribonuclease, PNPase, was identified in a complex with TCL1. The AKT interaction domain on TCL1 bound either RNase PH repeat domain of PNPase without influencing its RNA degrading activity, which was compatible with predicted docking models for a TCL1-PNPase complex. Our data provide a novel protein interaction for mammalian PNPase that may impact TCL1 mediated transformation.


Assuntos
Exorribonucleases/química , Exorribonucleases/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Western Blotting , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoprecipitação , Plasmídeos , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade
5.
Mol Cell Biol ; 26(22): 8475-87, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16966381

RESUMO

We recently identified polynucleotide phosphorylase (PNPase) as a potential binding partner for the TCL1 oncoprotein. Mammalian PNPase exhibits exoribonuclease and poly(A) polymerase activities, and PNPase overexpression inhibits cell growth, induces apoptosis, and stimulates proinflammatory cytokine production. A physiologic connection for these anticancer effects and overexpression is difficult to reconcile with the presumed mitochondrial matrix localization for endogenous PNPase, prompting this study. Here we show that basal and interferon-beta-induced PNPase was efficiently imported into energized mitochondria with coupled processing of the N-terminal targeting sequence. Once imported, PNPase localized to the intermembrane space (IMS) as a peripheral membrane protein in a multimeric complex. Apoptotic stimuli caused PNPase mobilization following cytochrome c release, which supported an IMS localization and provided a potential route for interactions with cytosolic TCL1. Consistent with its IMS localization, PNPase knockdown with RNA interference did not affect mitochondrial RNA levels. However, PNPase reduction impaired mitochondrial electrochemical membrane potential, decreased respiratory chain activity, and was correlated with altered mitochondrial morphology. This resulted in FoF1-ATP synthase instability, impaired ATP generation, lactate accumulation, and AMP kinase phosphorylation with reduced cell proliferation. Combined, the data demonstrate an unexpected IMS localization and a key role for PNPase in maintaining mitochondrial homeostasis.


Assuntos
Mitocôndrias/enzimologia , Mitocôndrias/fisiologia , Membranas Mitocondriais/enzimologia , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , Trifosfato de Adenosina/metabolismo , Apoptose , Linhagem Celular , Citocromos c/metabolismo , Células HeLa , Homeostase , Humanos , Modelos Biológicos , Polirribonucleotídeo Nucleotidiltransferase/genética , Polirribonucleotídeo Nucleotidiltransferase/fisiologia , RNA/metabolismo , Interferência de RNA , RNA Mitocondrial , Ribonucleases/metabolismo , Ribonucleases/fisiologia
6.
Cancer Res ; 63(15): 4472-80, 2003 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-12907620

RESUMO

EBV-infected B-cell lymphomas are a potentially life-threatening complication in bone marrow and solid organ transplant recipients. Immunosuppressive drugs required to prevent allograft rejection also impair anti-EBV T-cell immunity, thereby increasing the risk of EBV-associated disease. Here we demonstrate that the immunosuppressant rapamycin (RAPA) has a strong antiproliferative effect in vitro on B-cell lines derived from organ transplant recipients with EBV-associated posttransplant lymphoproliferative disorder (PTLD). Furthermore, RAPA significantly inhibits or delays the growth of solid tumors established from EBV-infected B-cell lines in a xenogeneic mouse model of PTLD. RAPA acts via cell cycle arrest, induction of apoptosis, and, most importantly, inhibition of interleukin 10 secretion, a necessary autocrine growth factor. The reduced interleukin 10 production is accompanied by corresponding decreases in the constitutive activation of the growth-promoting transcription factors signal transducer and activator of transcription 1 and 3. Thus, RAPA can limit B-cell lymphoma growth while simultaneously providing immunosuppression to prevent graft rejection in patients who are otherwise at risk for EBV-associated PTLD. Moreover, these findings may have application to other EBV-associated malignancies.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Interleucina-10/antagonistas & inibidores , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/virologia , Sirolimo/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/efeitos dos fármacos , Infecções por Vírus Epstein-Barr/etiologia , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/prevenção & controle , Herpesvirus Humano 4 , Humanos , Imunossupressores/farmacologia , Interleucina-10/biossíntese , Interleucina-10/metabolismo , Interleucina-10/fisiologia , Janus Quinase 1 , Linfoma de Células B/metabolismo , Linfoma de Células B/prevenção & controle , Masculino , Camundongos , Proteínas Tirosina Quinases/metabolismo , Fator de Transcrição STAT3 , Transdução de Sinais/efeitos dos fármacos , Transativadores/metabolismo , Transplante/efeitos adversos , Ensaios Antitumorais Modelo de Xenoenxerto
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