RESUMO
Protein phosphatase magnesium/manganese-dependent 1D (PPM1D) is a p53-induced phosphatase that functions as a negative regulator of stress response pathways and has oncogenic properties. However, the functional role of PPM1D in bladder cancer (BC) remains largely unknown. In the present study, lentivirus vectors carrying small hairpin RNA (shRNA) targeting PPM1D were used to explore the effects of PPM1D knockdown on BC cell proliferation and tumorigenesis. shRNA-mediated knockdown of PPM1D significantly inhibited cell growth and colony forming ability in the BC cell lines 5637 and T24. Flow cytometric analysis showed that PPM1D silencing increased the proportion of cells in the G0/G1 phase. Downregulation of PPM1D also inhibited 5637 cell tumorigenicity in nude mice. The results of the present study suggest that PPM1D plays a potentially important role in BC tumorigenicity, and lentivirus-mediated delivery of shRNA against PPM1D might be a promising therapeutic strategy for the treatment of BC.
Assuntos
Animais , Humanos , Masculino , Fosfoproteínas Fosfatases/fisiologia , Interferência de RNA/fisiologia , RNA Interferente Pequeno/farmacologia , Neoplasias da Bexiga Urinária/patologia , Linhagem Celular Tumoral , Carcinogênese/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Vetores Genéticos , Lentivirus/genética , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosfoproteínas Fosfatases/genética , Reação em Cadeia da Polimerase em Tempo Real , Estresse Fisiológico/genética , Ensaio Tumoral de Célula-Tronco , Neoplasias da Bexiga Urinária/terapiaRESUMO
Protein phosphatase magnesium/manganese-dependent 1D (PPM1D) is a p53-induced phosphatase that functions as a negative regulator of stress response pathways and has oncogenic properties. However, the functional role of PPM1D in bladder cancer (BC) remains largely unknown. In the present study, lentivirus vectors carrying small hairpin RNA (shRNA) targeting PPM1D were used to explore the effects of PPM1D knockdown on BC cell proliferation and tumorigenesis. shRNA-mediated knockdown of PPM1D significantly inhibited cell growth and colony forming ability in the BC cell lines 5637 and T24. Flow cytometric analysis showed that PPM1D silencing increased the proportion of cells in the G0/G1 phase. Downregulation of PPM1D also inhibited 5637 cell tumorigenicity in nude mice. The results of the present study suggest that PPM1D plays a potentially important role in BC tumorigenicity, and lentivirus-mediated delivery of shRNA against PPM1D might be a promising therapeutic strategy for the treatment of BC.
Assuntos
Fosfoproteínas Fosfatases/fisiologia , Interferência de RNA/fisiologia , RNA Interferente Pequeno/farmacologia , Neoplasias da Bexiga Urinária/patologia , Animais , Carcinogênese/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Vetores Genéticos , Humanos , Lentivirus/genética , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosfoproteínas Fosfatases/genética , Proteína Fosfatase 2C , Reação em Cadeia da Polimerase em Tempo Real , Estresse Fisiológico/genética , Ensaio Tumoral de Célula-Tronco , Neoplasias da Bexiga Urinária/terapiaRESUMO
The neuronal cytoskeleton regulates numerous processes that occur in normal homeostasis. Under pathological conditions such as those of Alzheimer's disease (AD), major alterations in cytoskeleton organization have been observed and changes in both microtubules and actin filaments have been reported. Many neurodegenerative consequences of AD are linked to the production and accumulation of amyloid peptides (Aß) and their oligomers, produced from the internal cleavage of the amyloid-ß protein precursor. We previously reported that fibrillar Aß1-42 (fAß) treatment of hippocampal neurons induced an increase in Rac1 and Cdc42 activities linking fAß effects with changes in actin dynamics. Here we show fAß-induces increased activity of PAK1 and cyclin-dependent kinase 5, and that p21-activated kinase (PAK1) activation targets the LIMK1-cofilin signaling pathway. Increased cofilin dephosphorylation under conditions of enhanced LIM-Kinase 1 (LIMK1) activity suggests that fAß co-stimulates bifurcating pathways impacting cofilin phosphorylation. Overexpression of slingshot (SSH) prevents the augment of F-actin induced by fAß after 24 h, suggesting that fAß-induced changes in actin assembly involve both LIMK1 and SSH. These results suggest that fAb may alter the PAK1/LIMK1/cofilin axis and therefore actin organization in AD.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Actinas/fisiologia , Peptídeos beta-Amiloides/fisiologia , Amiloide/fisiologia , Neuropeptídeos/fisiologia , Fragmentos de Peptídeos/fisiologia , Fosfoproteínas Fosfatases/fisiologia , Proteína cdc42 de Ligação ao GTP/fisiologia , Proteínas rac de Ligação ao GTP/fisiologia , Animais , Células Cultivadas , Camundongos , Camundongos Transgênicos , Fosforilação , Ratos , Ratos Sprague-Dawley , Proteínas rac1 de Ligação ao GTPRESUMO
During Trypanosoma cruzi cell invasion, signal transduction pathways are triggered in parasite and host cells, leading to a rise in intracellular Ca2+ concentration. We posed the question whether calcineurin (CaN), in particular the functional regulatory subunit CaNB, a Ca2+-binding EF-hand protein, was expressed in T. cruzi and whether it played a role in cell invasion. Here we report the cloning and characterization of CL strain CaNB gene, as well as the participation of CaNB in cell invasion. Treatment of metacyclic trypomastigotes (MT) or tissue-culture trypomastigotes (TCT) with the CaN inhibitors cyclosporin or cypermethrin strongly inhibited (62-64%) their entry into HeLa cells. In assays using anti-phospho-serine/threonine antibodies, a few proteins of MT were found to be dephosphorylated in a manner inhibitable by cyclosporin upon exposure to HeLa cell extract. The phosphatase activity of CaN was detected by a biochemical approach in both MT and TCT. Treatment of parasites with antisense phosphorothioate oligonucleotides directed to TcCaNB-CL, which reduced the expression of TcCaNB and affected TcCaN activity, resulted in approximately 50% inhibition of HeLa cell entry by MT or TCT. Given that TcCaNB-CL may play a key role in cell invasion and differs considerably in its primary structure from the human CaNB, it might be considered as a potential chemotherapeutic target.
Assuntos
Calcineurina/fisiologia , Proteínas de Protozoários/fisiologia , Trypanosoma cruzi/patogenicidade , Fatores de Virulência/fisiologia , Sequência de Aminoácidos , Animais , Calcineurina/biossíntese , Calcineurina/genética , Inibidores de Calcineurina , Clonagem Molecular , Ciclosporina/farmacologia , Inibidores Enzimáticos/farmacologia , Inativação Gênica , Células HeLa , Humanos , Dados de Sequência Molecular , Oligonucleotídeos Antissenso , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/biossíntese , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/fisiologia , Filogenia , Proteínas de Protozoários/biossíntese , Piretrinas , Alinhamento de Sequência , Fatores de Virulência/biossínteseRESUMO
Tumor cell proliferation is frequently associated to genetic or epigenetic alterations in key cell cycle regulators. Most human tumors deregulate this pathway to sustain proliferation with independence of external mitogenic factors. In addition, the alteration of cell cycle proteins may confer genomic instability that results in additional mutations in these tumor cells. The frequent alteration of the cell cycle in tumor cells has launched the identification for critical cell cycle regulators as anticancer targets. The inhibition of some cell cycle kinases such as cyclin-dependent kinases (CDKs) or the Aurora and Polo mitotic kinases is currently under study in several preclinical and clinical trials. Similarly, the clinical success of microtubule poisons such as taxol has promoted new applied research in mitosis regulation. Recent investigations have suggested new targets of interest including additional kinases, phosphatases and other mitotic regulators such as microtubule motor proteins (kinesins). Current research in this area will undoubtedly result in new and improved targeted therapies for cancer treatment.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Ciclo Celular/efeitos dos fármacos , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Fuso Acromático/efeitos dos fármacos , Animais , Aurora Quinases , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/fisiologia , Segregação de Cromossomos/efeitos dos fármacos , Segregação de Cromossomos/fisiologia , Proteínas Inibidoras de Quinase Dependente de Ciclina/fisiologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/fisiologia , Dano ao DNA , Replicação do DNA/efeitos dos fármacos , Desenho de Fármacos , Previsões , Fase G1/efeitos dos fármacos , Genes cdc , Humanos , Camundongos , Modelos Biológicos , Proteínas Motores Moleculares , Proteínas de Neoplasias/fisiologia , Neoplasias/genética , Neoplasias/fisiopatologia , Fosfoproteínas Fosfatases/fisiologia , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/fisiologia , Transdução de Sinais/efeitos dos fármacos , Quinase 1 Polo-LikeRESUMO
BACKGROUND: The biochemical bases for hormone dependence in breast cancer have been recognized as an important element in tumor resistance, proliferation and metastasis. On this respect, dexamethasone (Dex) dependent protection against TNF-alpha-mediated cell death in the MCF-7 cell line has been demonstrated to be a useful model for the study of this type of cancer. Recently, cytoplasmic signaling induced by steroid receptors has been described, such as the activation of the PI3K/Akt and NF-kappaB pathways. We evaluated their possible participation in the Dex-dependent protection against TNF-alpha-mediated cell death. RESULTS: Cellular cultures of the MCF-7 cell line were exposed to either, TNF-alpha or TNF-alpha and Dex, and cell viability was evaluated. Next, negative dominants of PI3K and IkappaB-alpha, designed to block the PI3K/Akt and NF-kappaB pathways, respectively, were transfected and selection and evaluation of several clones overexpressing the mutants were examined. Also, correlation with inhibitor of apoptosis proteins (IAPs) expression was examined. Independent inhibition of these two pathways allowed us to test their participation in Dex-dependent protection against TNF-alpha-cytotoxicity in MCF-7 cells. Expression of the PI3K dominant negative mutant did not alter the protection conferred by Dex against TNF-alpha mediated cell death. Contrariwise, clones expressing the IkappaB-alpha dominant negative mutant lost the Dex-conferred protection against TNF-alpha. In these clones degradation of c-IAP was accelerated, while that of XIAP was remained unaffected. CONCLUSION: NF-kappaB, but not PI3K/Akt activation, is required for the Dex protective effect against TNF-alpha-mediated cell death, and correlates with lack of degradation of the anti-apoptotic protein c-IAP1.
Assuntos
Apoptose/efeitos dos fármacos , Dexametasona/farmacologia , NF-kappa B/fisiologia , Proteína Oncogênica v-akt/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/fisiopatologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Fosfatase 1 de Especificidade Dupla , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/fisiologia , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/fisiologia , NF-kappa B/genética , Proteína Oncogênica v-akt/genética , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/fisiologia , Proteína Fosfatase 1 , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/fisiologia , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/fisiologia , Transfecção , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/fisiologiaRESUMO
The specific signaling connections between the mitogen-activated protein kinases (MAPK) such as c-Jun N-terminal kinase (JNK-1) and phosphatases PP4 and M3/6, affecting the family of early nuclear factors, is complex and remains poorly understood. JNK-1 regulates cellular differentiation, apoptosis and stress responsiveness by up-regulating early nuclear factors such as c-Jun, a member of the activating protein (AP-1) family, and the Early Growth Factor (EGR-1). C-Jun, when phosphorylated by c-Jun N-terminal kinase (JNK-1) associates with c-Fos to form the AP-1 transcription factor that activates gene expression. We have investigated the regulation of the JNK-1 kinase by co-transfecting phosphatases PP4 and M3/6 in prostate cancer cell lines PC-3 and LNCaP, which have been previously stimulated with human EGF or cisplatin. Co-transfections of plasmids expressing the JNK-1 and the serine/threonine phosphatases PP4 resulted in a significant increase in JNK-1 activity in both PC3 and LNCaP cells. In contrast, co-transfection of JNK-1 with the dual specific phosphatase serine/threonine M3/6 showed only a marginal effect in JNK-1 activity. The phosphatase M3/6 also failed in blocking the induction of JNK-1 activity observed in presence of PP4. The higher activity of JNK-1 was associated with increased activities of the factors c-Jun/AP-1 and EGR-1. This suggests that JNK-1 activity in PC-3 and LNCaP cells requires not only active PP4 for stable maintenance but also suggests that the relative degree of phosphorylation of multiple cellular components is the determinant of JNK-1 stability.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Fosfoproteínas Fosfatases/fisiologia , Neoplasias da Próstata/enzimologia , Fator de Transcrição AP-1/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/enzimologia , Cisplatino/farmacologia , Proteína 1 de Resposta de Crescimento Precoce/efeitos dos fármacos , Ativação Enzimática , Fator de Crescimento Epidérmico/farmacologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Proteína Quinase 8 Ativada por Mitógeno/genética , Fator de Transcrição AP-1/efeitos dos fármacosRESUMO
Even though there is a large body of information concerning the harmful effects of alcohol on different organisms, the mechanism(s) that affects developmental programs, at a single-cell level, has not been clearly identified. In this respect, the spore-forming bacterium Bacillus subtilis constitutes an excellent model to study universal questions of cell fate, cell differentiation, and morphogenesis. Here, we demonstrate that treatment with subinhibitory concentrations of alcohol that did not affect vegetative growth inhibited the initiation of spore development through a selective blockage of key developmental genes under the control of the master transcription factor Spo0A approximately P. Isopropyl-beta-D-thiogalactopyranoside-directed expression of a phosphorylation-independent form of Spo0A (Sad67) and the use of an in vivo mini-Tn10 insertional library permitted the identification of the developmental SinR repressor and RapA phosphatase as the effectors that mediated the inhibitory effect of alcohol on spore morphogenesis. A double rapA sinR mutant strain was completely resistant to the inhibitory effects of different-C-length alcohols on sporulation, indicating that the two cell fate determinants were the main or unique regulators responsible for the spo0 phenotype of wild-type cells in the presence of alcohol. Furthermore, treatment with alcohol produced a significant induction of rapA and sinR, while the stationary-phase induction of sinI, which codes for a SinR inhibitor, was completely turned off by alcohol. As a result, a dramatic repression of spo0A and the genes under its control occurred soon after alcohol addition, inhibiting the onset of sporulation and permitting the evaluation of alternative pathways required for cellular survival.
Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/fisiologia , Etanol/farmacologia , Regulação Bacteriana da Expressão Gênica , Fosfoproteínas Fosfatases/fisiologia , Esporos Bacterianos/fisiologia , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/crescimento & desenvolvimento , Fatores de TranscriçãoRESUMO
Here we provide evidence for a critical role of PP2As (protein phosphatase 2As) in the transformation of Trypanosoma cruzi. In axenic medium at pH 5.0, trypomastigotes rapidly transform into amastigotes, a process blocked by okadaic acid, a potent PP2A inhibitor, at concentrations as low as 0.1 microM. 1-Norokadaone, an inactive okadaic acid analogue, did not affect the transformation. Electron microscopy studies indicated that okadaic acid-treated trypomastigotes had not undergone ultrastructural modifications, reinforcing the idea that PP2A inhibits transformation. Using a microcystin-Sepharose affinity column we purified the native T. cruzi PP2A. The enzyme displayed activity against 32P-labelled phosphorylase a that was inhibited in a dose-dependent manner by okadaic acid. The protein was also submitted to MS and, from the peptides obtained, degenerate primers were used to clone a novel T. cruzi PP2A enzyme by PCR. The isolated gene encodes a protein of 303 amino acids, termed TcPP2A, which displayed a high degree of homology (86%) with the catalytic subunit of Trypanosoma brucei PP2A. Northern-blot analysis revealed the presence of a major 2.1-kb mRNA hybridizing in all T. cruzi developmental stages. Southern-blot analysis suggested that the TcPP2A gene is present in low copy number in the T. cruzi genome. These results are consistent with the mapping of PP2A genes in two chromosomal bands by pulsed-field gel electrophoresis and chromoblot hybridization. Our studies suggest that in T. cruzi PP2A is important for the complete transformation of trypomastigotes into amastigotes during the life cycle of this protozoan parasite.
Assuntos
Fosfoproteínas Fosfatases/fisiologia , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/crescimento & desenvolvimento , Sequência de Aminoácidos , Animais , Fracionamento Químico , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Inibidores Enzimáticos/química , Flagelos/química , Genes de Protozoários , Genoma , Inibidores do Crescimento/química , Humanos , Dados de Sequência Molecular , Ácido Okadáico/química , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/genética , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2 , Transcrição Gênica , Trypanosoma cruzi/genética , Trypanosoma cruzi/ultraestruturaRESUMO
A gene, SIT4, was identified as corresponding to a serine/threonine protein phosphatase and when overexpressed confers lithium tolerance in galactose medium to the budding yeast Saccharomyces cerevisiae. This gene has been previously identified as a regulator of the cell cycle and involved in nitrogen sensing. It is shown that the transcription levels of SIT4 are induced by low concentrations of Li(+) in a time-dependent manner. Na(+) and K(+) at high concentrations, but not sorbitol, also induce transcription. As a response to Na(+) or Li(+) stress, yeast cells lower the intracellular K(+) content. This effect is enhanced in cells overexpressing SIT4, which also increase (86)Rb efflux after the addition of Na(+) or Li(+) to the extracellular medium. Another feature of SIT4-overexpressing cells is that they maintain a more alkaline pH of 6.64 compared with 6.17 in the wild type cells. It has been proposed that the main pathway of salt tolerance in yeast is mediated by a P-type ATPase, encoded by PMR2A/ENA1. However, our results show that in a sit4 strain, expression of ENA1 is still induced by monovalent cations, and overexpression of SIT4 does not alter the amount of ENA1 transcript. These results show that SIT4 acts in a parallel pathway not involving induction of transcription of ENA1 and suggest a novel function for SIT4 in response to salt stress.
Assuntos
Proteínas de Transporte de Cátions , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas Fosfatases/fisiologia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Adenosina Trifosfatases/metabolismo , Cátions , Ciclo Celular/fisiologia , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Resistência a Medicamentos , Galactose/metabolismo , Concentração de Íons de Hidrogênio , Íons , Lítio/farmacologia , Cloreto de Lítio/farmacologia , Potássio/farmacologia , Proteína Fosfatase 2 , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Radioisótopos de Rubídio/metabolismo , Sódio/farmacologia , Cloreto de Sódio/farmacologia , ATPase Trocadora de Sódio-Potássio , Sorbitol/farmacologia , Fatores de Tempo , Transcrição GênicaRESUMO
The binding of aldosterone (ALDO) to the mineralocorticoid receptor (MR) induces a conformational change of the protein referred to as 'transformation'. This feature can be evidenced in vivo by the capacity of the MR to interact with chromatin, and in vitro by the ability of the MR to bind to DNA strands or to shift the sedimentation coefficient (S) to lower values. The transformation process allows MR to work as a transcription factor after interacting with specific sequences of DNA. The signal transduction pathway for the MR transformation remains unknown. As a first step towards elucidating the mechanism of steroid-dependent MR transformation, we asked if the MR-signaling pathway is affected by the phosphorylation status of the MR-heterocomplex, and how that pathway may be regulated. Incubation of preformed [3H]ALDO-MR complex with bovine intestinal alkaline phosphatase led to an increase in the rate of MR-transformation (measured as 9.4-5.4S shift). This alkaline phosphatase-dependent MR transformation was inhibited by the specific alkaline phosphatase-type inhibitor levamisole, and was not evident in incubations performed with acid phosphatases. A direct correlation between the DNA-cellulose binding capacity of the [3H]ALDO-MR complex and the percentage of transformed 5.4S MR form was also observed. When rat kidney cytosol was incubated in the absence of both exogenous phosphatase and stabilizing agents (such as molybdate or vanadate), MR transformation also took place, in a time- and temperature-dependent process. In contrast with the inhibitory effect observed upon alkaline phosphatase-promoted transformation, levamisole was unable to inhibit the endogenous transforming activity of MR, suggesting that an endogenous phosphatase other than those which belong to the alkaline-type may be responsible for that transformation. Tautomycin, a polyketide produced by the soil bacteria Streptomyces which inhibits serine/threonine phosphatases of the PP1/PP2A subgroup, was able to inhibit the endogenous phosphatase activity in a concentration-dependent form (Ki(app)=7.35 nM). These results support the idea that the endogenous renal activity involved in the regulation of rat kidney MR transformation may be a protein phosphatase which belongs to the PP1/PP2A subgroup.