RESUMEN
The molecular mechanism of telomerase activation induced by ionizing radiation (IR) remains poorly understood. We demonstrate that DNA damage induced by IR at doses of 2-5 Gy triggers activation of Akt, predominant to that of protein phosphatase 2A (PP2A), resulting in human telomerase reverse transcriptase (hTERT) phosphorylation and increased telomerase activity in Y79 cells. DNA damage induced by IR at doses greater than 10 Gy might trigger PP2A activation, predominant to that of Akt, resulting in hTERT dephosphorylation and decreased telomerase activity. Our results suggest that differential activation of Akt and PP2A may be responsible for telomerase regulation.
Asunto(s)
Daño del ADN , Reparación del ADN , Telomerasa/metabolismo , Androstadienos/farmacología , Apoptosis/efectos de la radiación , Línea Celular Tumoral , Activación Enzimática , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de la radiación , Expresión Génica , Histonas/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , Ácido Ocadaico/farmacología , Fosforilación , Procesamiento Proteico-Postraduccional , Retinoblastoma , Telomerasa/genética , Proteína p53 Supresora de Tumor/metabolismo , WortmaninaRESUMEN
Adult T-cell leukemia (ATL) is a heterogeneous tumor that is resistant to chemotherapy. Telomerase activity plays a critical role in tumorigenesis and is associated with the prognosis of ATL patients. Interleukin (IL)-2 commonly promotes tumor growth in chronic ATL cells. The signaling pathways involved in IL-2-regulated telomerase activation were studied in ATL cells derived from chronic ATL patients. IL-2 challenge enhanced tyrosine phosphorylation of Janus-activated kinase (JAK)1-3 and STAT5, and induced JAK1 and JAK2 to associate with STAT5 in IL-2-dependent ATL cells. Chromatin immunoprecipitation assays revealed that STAT5 directly bound to the human telomerase reverse transcriptase (hTERT) promoter. STAT5 short interfering RNA inhibited hTERT transcription in IL-2-stimulated ATL cells. Inhibitors of PI3K, HSP90, and mTOR reduced IL-2-induced hTERT mRNA, protein expression, and telomerase activity. AKT, HSP90, mTOR, S6 kinase, and hTERT immunoprecipitate from IL-2-stimulated cells contained telomerase activity, suggesting that hTERT directly interacts with, and is regulated by, these proteins. Binding of the p85 regulatory subunit of PI3K to JAK2 was enhanced in an IL-2-dependent manner, indicating that JAK2 propagates activation signals from the IL-2 receptor and links hTERT activation to both the STAT5 and PI3K pathways. Finally, IL-2-induced activation of telomerase and STAT5 was observed in primary leukemic cells. These results indicate that IL-2 stimulation induces hTERT activation through the JAK/STAT pathway and the JAK/PI3K/AKT/HSP90/mTORC1 pathway in IL-2-responsive ATL cells. These signaling proteins represent novel and promising molecular therapeutic targets for IL-2-dependent ATL.
Asunto(s)
Leucemia-Linfoma de Células T del Adulto/genética , Leucemia-Linfoma de Células T del Adulto/metabolismo , Transducción de Señal , Telomerasa/genética , Telomerasa/metabolismo , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Expresión Génica , Humanos , Interleucina-2/farmacología , Quinasas Janus/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos , Fosfatidilinositol 3-Quinasas/metabolismo , Unión Proteica , Proteínas/metabolismo , Factores de Transcripción STAT/metabolismo , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TORRESUMEN
We used two imatinib resistant cell lines, K562-ADM cells, which over-express P-glycoprotein (a product of the ABCB1 gene, more commonly known as MDR1), and K562-hTERT cells, which over-express the telomerase reverse transcriptase (TERT), as models to show that the acquisition of multidrug resistance in CML is associated with the enhanced phosphorylation of signal transducer and activator of transcription 5 (STAT5). The induction of P-glycoprotein expression that occurred in response to adriamycin treatment was accompanied by increased phosphorylation of BCR-ABL and STAT5, as well as increased telomerase protein expression. Intriguingly, a ChIP assay using an anti-STAT5 antibody revealed direct binding of STAT5 to the promoter regions of both the human TERT gene and the MDR1 gene in K562-ADM cells. Conversely, silencing of endogenous STAT5 expression by siRNA significantly reduced both the expression of P-glycoprotein and telomerase activity and resulted in the recovery of the imatinib sensitivity of K562-ADM cells. These findings indicate a critical role for STAT5 in the induction of P-glycoprotein and in the modulation of telomerase activity in drug-resistant CML cells. Furthermore, primary leukemic cells obtained from patients in blast crisis showed increased levels of phospho-STAT5, P-glycoprotein and telomerase. In contrast, none of these proteins were detectable in the cells obtained from patients in the chronic phase. Together, these findings indicate a novel mechanism that contributes toward multidrug resistance involving STAT5 as a sensor for cytotoxic drugs in CML patients.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Antineoplásicos/farmacología , Crisis Blástica/patología , Resistencia a Antineoplásicos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Piperazinas/farmacología , Pirimidinas/farmacología , Factor de Transcripción STAT5/metabolismo , Telomerasa/genética , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Benzamidas , Crisis Blástica/genética , Crisis Blástica/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/farmacología , Regulación Leucémica de la Expresión Génica , Humanos , Mesilato de Imatinib , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Telomerasa/metabolismo , Transcripción Genética , Activación TranscripcionalRESUMEN
We have shown previously that PI3K/Akt pathway is active after cell differentiation in HL60 cells. In the present study, we have investigated whether additional molecules, such as protein kinase C (PKC), are involved in the regulation, not only of telomerase, but also of leukemia cell differentiation. We show that PKC activates telomerase and is, itself, activated following VD3- or ATRA-induced differentiation of HL60 cells, as was observed for PI3K/Akt. To clarify the significance of PI3K/Akt and PKC pathway activation in leukemia cell differentiation, we examined the active proteins in either the downstream or upstream regulation of these pathways. In conjunction with the activation of Akt or PKC, mTOR and S6K were phosphorylated and the protein expression levels of Rictor were increased, compared with Raptor, following cell differentiation. Silencing by Rictor siRNA resulted in the attenuation of Akt phosphorylation on Ser473 and PKCα/ßII phosphorylation, as well as the inhibition of Rictor itself, suggesting that Rictor is an upstream regulator of both Akt and PKC. In addition, in cells induced to differentiate by ATRA or VD3, Nitroblue-tetrazolium (NBT) reduction and esterase activity, were blocked either by LY294002, a PI3K inhibitor, or by BIM, a PKC inhibitor, without affecting cell surface markers such as CD11b or CD14. Intriguingly, the silencing of Rictor by its siRNA also suppressed the reducing ability of NBT following VD3-induced cell differentiation. Taken together, our results show that Rictor associated with mTOR (mTORC2) regulates the activity of both Akt and PKC that are involved in cell functions such as NBT reduction and esterase activity induced by leukemia cell differentiation.
Asunto(s)
Diferenciación Celular/genética , Proteína Quinasa C/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Telomerasa/metabolismo , Factores de Transcripción/fisiología , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Cromonas/farmacología , Células HL-60 , Humanos , Morfolinas/farmacología , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , ARN Interferente Pequeño/farmacología , Proteína Asociada al mTOR Insensible a la Rapamicina , Proteína Reguladora Asociada a mTOR , Regulación hacia ArribaRESUMEN
BACKGROUND: The clinical course of human papillomavirus (HPV) associated with Bowenoid papulosis and condyloma acuminatum of anogenital tumors are still unknown. Here we evaluated molecules that are relevant to cellular proliferation and regulation of apoptosis in HPV associated anogenital tumors. METHODS: We investigated the levels of telomerase activity, and inhibitor of apoptosis proteins (IAPs) family (c-IAP1, c-IAP2, XIAP) and c-Myc mRNA expression levels in 20 specimens of Bowenoid papulosis and 36 specimens of condyloma acuminatum in anogenital areas. Overall, phosphorylated (p-) AKT, p-ribosomal protein S6 (S6) and p-4E-binding protein 1 (4EBP1) expression levels were examined by immunohistochemistry in anogenital tumors both with and without positive telomerase activity. RESULTS: Positive telomerase activity was detected in 41.7% of Bowenoid papulosis and 27.3% of condyloma acuminatum compared to normal skin (p < 0.001). In contrast, the expression levels of Bowenoid papulosis indicated that c-IAP1, c-IAP2 and XIAP mRNA were significantly upregulated compared to those in both condyloma acuminatum samples (p < 0.001, p < 0.001, p = 0.022, respectively) and normal skin (p < 0.001, p = 0.002, p = 0.034, respectively). Overall, 30% of Bowenoid papulosis with high risk HPV strongly promoted IAPs family and c-Myc but condyloma acuminatum did not significantly activate those genes. Immunohistochemically, p-Akt and p-S6 expressions were associated with positive telomerase activity but not with p-4EBP1 expression. CONCLUSION: Combined analysis of the IAPs family, c-Myc mRNA expression, telomerase activity levels and p-Akt/p-S6 expressions may provide clinically relevant molecular markers in HPV associated anogenital tumors.
Asunto(s)
Neoplasias del Ano/química , Enfermedad de Bowen/química , Proteínas de Ciclo Celular/análisis , Condiloma Acuminado/metabolismo , Neoplasias de los Genitales Femeninos/química , Neoplasias de los Genitales Masculinos/química , Proteínas Inhibidoras de la Apoptosis/análisis , Infecciones por Papillomavirus/complicaciones , Neoplasias Cutáneas/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias del Ano/genética , Neoplasias del Ano/patología , Neoplasias del Ano/virología , Apoptosis , Proteína 3 que Contiene Repeticiones IAP de Baculovirus , Western Blotting , Enfermedad de Bowen/genética , Enfermedad de Bowen/patología , Enfermedad de Bowen/virología , Proteínas de Ciclo Celular/genética , Proliferación Celular , Condiloma Acuminado/genética , Condiloma Acuminado/patología , Condiloma Acuminado/virología , Femenino , Neoplasias de los Genitales Femeninos/genética , Neoplasias de los Genitales Femeninos/patología , Neoplasias de los Genitales Femeninos/virología , Neoplasias de los Genitales Masculinos/genética , Neoplasias de los Genitales Masculinos/patología , Neoplasias de los Genitales Masculinos/virología , Humanos , Inmunohistoquímica , Proteínas Inhibidoras de la Apoptosis/genética , Japón , Masculino , Infecciones por Papillomavirus/genética , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/patología , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/análisis , Proteínas Proto-Oncogénicas c-myc/análisis , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas S6 Ribosómicas/análisis , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/virología , Telomerasa/análisis , Ubiquitina-Proteína Ligasas , Proteína Inhibidora de la Apoptosis Ligada a X/análisisRESUMEN
Imatinib mesylate (imatinib), previously known as STI571 (Gleevec), is currently utilized in the treatment of chronic myeloid leukemia (CML). However, its effect on telomerase activity and the correlation of this to its observed antitumor effect has yet to be defined. We investigated the effects of this agent on human telomerase reverse transcriptase (hTERT) expression and telomerase activity and found that it significantly down-regulated telomerase activity in both K562 cells and primary leukemic cells. The telomerase activity of primary leukemic cells from CML patients in blastic crisis showed less suppression than that of cells from patients in chronic phase. Additionally, data also demonstrate that inhibition of telomerase was due to the direct action of imatinib on hTERT transcription, rather than an increase in cell death. These results suggest a novel mechanism in the antitumor activity of imatinib and may provide a basis for future development of anti-telomerase therapies, as well as leading to better understanding of the regulation of telomerase in leukemic cells.
Asunto(s)
Antineoplásicos/uso terapéutico , Resistencia a Antineoplásicos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Piperazinas/uso terapéutico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Pirimidinas/uso terapéutico , Telomerasa/metabolismo , Apoptosis/efectos de los fármacos , Benzamidas , Crisis Blástica , Femenino , Regulación Leucémica de la Expresión Génica , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Masculino , Persona de Mediana Edad , Leucemia-Linfoma Linfoblástico de Células Precursoras B/enzimología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Regiones Promotoras Genéticas , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Telomerasa/antagonistas & inhibidores , Telomerasa/genética , Telómero/genética , Transcripción Genética/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Using three different differentiation agents (1alpha, 25 dihydroxyvitamin D3, all-trans-retinoic acid, and Am80), down-regulation of telomerase activity was found to be a common response during the monocytic or granulocytic differentiation of human acute myeloblastic leukemia cell line 60 (HL60) cells. Rapid down-regulation of telomerase transcription occurred during early differentiation of HL60 cells prior to G(1) arrest. Akt kinase activity was suppressed after 6 h of differentiation along with inhibition of telomerase activity, and the extent of the suppression that occurred while maintaining telomerase protein expression suggested the post-translational regulation of telomerase activity. Recombinant Akt dose-dependently increased telomerase activity, and telomerase was inhibited at the transcriptional and post-translational levels by LY294002, suggesting that PI-3K/Akt is one of the key signaling proteins involved in telomerase regulation. Each of the three differentiation agents caused a significant increase of signaling proteins (including Akt) at 3 days after the initiation of differentiation. Changes of acetyl-histone H4, which regulates transcription of the telomerase gene, were observed before the activation of Akt. This finding suggests that epigenetic control of telomerase transcription occurs before activation of Akt during the late stage of differentiation. These results indicate that telomerase activity is regulated by at least two mechanisms during granulocytic and monocytic differentiation, with one mechanism being transcriptional and the other being post-translational.
Asunto(s)
Diferenciación Celular/fisiología , Células HL-60/enzimología , Telomerasa/genética , Calcitriol/farmacología , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Cromatina/genética , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HL-60/efectos de los fármacos , Células HL-60/patología , Humanos , Telomerasa/metabolismo , Tretinoina/farmacologíaRESUMEN
Endonuclease G (Endo G), a sugar-nonspecific nuclease preferring single-stranded DNA (ssDNA), is responsible for major nuclease activity in mitochondria. If the enzyme provides an important nicking function for mitochondrial DNA (mtDNA) in vivo, then mitochondrial factors likely exist which modulate the enzyme's activity and prevent cleavage at single-stranded moieties of mtDNA. In the present paper, we report that specific membrane phospholipids, polyamines and single-stranded DNA-binding protein (SSB) appear to exert such effects in vitro. Phosphatidylcholine and phosphatidylethanolamine, the major constituents of the mitochondrial inner membrane, stimulated purified Endo G activity 5- to 10-fold. Spermine at 5-100 microM also stimulated activity about 4-fold. However, at more than 500 microM, the spermine largely inhibited the degradation of ssDNA and duplex DNA. Escherichia coli SSB, which has physicochemical properties analogous to those of mitochondrial SSB (mtSSB), markedly inhibited the degradation of phiX174 ssDNA by Endo G, indicating the possible involvement of mtSSB in vivo in protection of single-stranded regions of mtDNA from nucleolytic attacks.