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1.
Biochim Biophys Acta ; 1859(8): 983-93, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27295567

RESUMEN

Chemotherapy-induced emergence of drug resistant cells is frequently observed and is exemplified by the expression of family of drug resistance proteins including, multidrug resistance protein 1 (MDR1). However, a concise mechanism for chemotherapy-induced MDR1 expression is unclear. Mechanistically, mutational selection, epigenetic alteration, activation of the Wnt pathway or impaired p53 function have been implicated. The present study describes that the surviving fraction of cisplatin resistant cells co- upregulate MDR1, BMI1 and acetyl transferase activity of TIP60. Using complementary gain and loss of function approaches, we demonstrate that the expression of MDR1 is positively regulated by BMI1, a stem-cell factor classically known as a transcriptional repressor. Our study establishes a functional interaction between TIP60 and BMI-1 resulting in upregulation of MDR1 expression. Chromatin immunoprecipitation (ChIP) assays further establish that the proximal MDR1 promoter responds to cisplatin in a BMI1 dependent manner. BMI1 interacts with a cluster of E-box elements on the MDR1 promoter and recruits TIP60 resulting in acetylation of histone H2A and H3. Collectively, our data establish a hitherto unknown liaison among MDR1, BMI1 and TIP60 and provide mechanistic insights into cisplatin-induced MDR1 expression resulting in acquired cross-resistance against paclitaxel, doxorubicin and likely other drugs. In conclusion, our results advocate utilizing anti-BMI1 strategies to alleviate acquired resistance to chemotherapy.


Asunto(s)
Antineoplásicos/farmacología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Histona Acetiltransferasas/genética , Complejo Represivo Polycomb 1/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/agonistas , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Acetilación/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacología , Doxorrubicina/farmacología , Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/genética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Histona Acetiltransferasas/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Lisina Acetiltransferasa 5 , Paclitaxel/farmacología , Complejo Represivo Polycomb 1/agonistas , Complejo Represivo Polycomb 1/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Transducción de Señal
2.
Mol Cancer ; 16(1): 56, 2017 03 07.
Artículo en Inglés | MEDLINE | ID: mdl-28270146

RESUMEN

BACKGROUND: The polycomb group protein, BMI1 plays important roles in chromatin modification, stem cell function, DNA damage repair and mitochondrial bioenergetics. Such diverse cellular functions of BMI1 could be, in part, due to post-translational modifications, especially phosphorylation. To date, AKT has been reported as a kinase that by site specific phosphorylation of BMI1 modulates its oncogenic functions. METHODS: Immunoprecipitation in conjunction with kinase assay and mass spectrometry was used to determine association with and site specific phosphorylation of BMI1 by CK2α. Functional implications of the BMI1/CK2α axis was examined in cancer cells utilizing siRNA and exogenous gene expression followed by biochemical and phenotypic studies. Correlations between expression of CK2α and BMI1 were determined from cell lines and formalin fixed paraffin embedded tissues representing the normal fallopian tube epithelium and high grade serous ovarian cancer samples. RESULTS: Here we report that CK2α, a nuclear serine threonine kinase, phosphorylates BMI1 at Serine 110 as determined by in-vitro/ex-vivo kinase assay and mass spectrometry. In ovarian cancer cell lines, expression of CK2α correlated with the phospho-species, as well as basal BMI1 levels. Preventing phosphorylation of BMI1 at Serine 110 significantly decreased half-life and stability of the protein. Additionally, re-expression of the phosphorylatable but not non-phosphorylatable BMI1 rescued clonal growth in endogenous BMI1 silenced cancer cells leading us to speculate that CK2α-mediated phosphorylation stabilizes BMI1 and promotes its oncogenic function. Clinically, compared to normal fallopian tube epithelial tissues, the expression of both BMI1 and CK2α were significantly higher in tumor tissues obtained from high-grade serous ovarian cancer patients. Among tumor samples, the expression of BMI1 and CK2α positively correlated (Spearman coefficient = 0.62, P = 0.0021) with each other. CONCLUSION: Taken together, our findings establish an important regulatory role of CK2α on BMI1 phosphorylation and stability and implicate the CK2α/BMI1 axis in ovarian cancer.


Asunto(s)
Complejo Represivo Polycomb 1/metabolismo , Quinasa de la Caseína II/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Mutación , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Fosforilación , Complejo Represivo Polycomb 1/genética , Unión Proteica , Proteolisis , Transducción de Señal
3.
FASEB J ; 30(12): 4042-4055, 2016 12.
Artículo en Inglés | MEDLINE | ID: mdl-27613804

RESUMEN

The polycomb complex proto-oncogene BMI1 [B lymphoma Mo-MLV insertion region 1 homolog (mouse)] is essential for self-renewal of normal and cancer stem cells. BMI1-null mice show severe defects in growth, development, and survival. Although BMI1 is known to exert its effect in the nucleus via repression of 2 potent cell-cycle regulators that are encoded by the Ink4a/Arf locus, deletion of this locus only partially rescues BMI1-null phenotypes, which is indicative of alternate mechanisms of action of BMI1. Here, we show that an extranuclear pool of BMI1 localizes to inner mitochondrial membrane and directly regulates mitochondrial RNA (mtRNA) homeostasis and bioenergetics. These mitochondrial functions of BMI1 are independent of its previously described nuclear functions because a nuclear localization-defective mutant BMI1 rescued several bioenergetic defects that we observed in BMI1-depleted cells, for example, mitochondrial respiration, cytochrome c oxidase activity, and ATP production. Mechanistically, BMI1 coprecipitated with polynucleotide phosphorylase, a ribonuclease that is responsible for decay of mtRNA transcripts. Loss of BMI1 enhanced ribonuclease activity of polynucleotide phosphorylase and reduced mtRNA stability. These findings not only establish a novel extranuclear role of BMI1 in the regulation of mitochondrial bioenergetics, but also provide new mechanistic insights into the role of this proto-oncogene in stem cell differentiation, neuronal aging, and cancer.-Banerjee Mustafi, S., Aznar, N., Dwivedi, S. K. D., Chakraborty, P. K., Basak, R., Mukherjee, P., Ghosh, P., Bhattacharya, R. Mitochondrial BMI1 maintains bioenergetic homeostasis in cells.


Asunto(s)
Diferenciación Celular/fisiología , Homeostasis/fisiología , Mitocondrias/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Proliferación Celular/fisiología , Células Cultivadas , Humanos , Ratones , Ratones Noqueados , Células Madre Neoplásicas/metabolismo , Complejo Represivo Polycomb 1/genética , Proto-Oncogenes Mas
4.
Antioxid Redox Signal ; 20(18): 2891-906, 2014 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-24180415

RESUMEN

AIMS: The human mutation R120G in the αB-crystallin (CRYAB) causes a multisystemic disease that is characterized by hypertrophic cardiomyopathy and cytoplasmic protein aggregates. In transgenic mice, human R120GCRYAB (hR120GTg) expression in heart sequentially modifies the REDOX status, in part by the activation of the nuclear factor, erythroid derived 2, like 2 (Nrf2). Thioredoxin system (TS) components are NRF2 target genes, so it could be hypothesized that TS was affected in hR120GTg mice. RESULTS: Transgenic hearts overexpressed thioredoxin 1 (Trx1), which was identified by isotope coded affinity tag-mass spectrometry, among hundreds of peptides displaying an increased reduced/oxidized ratio. Coupled to this higher level of reduced cysteines, the activity of thioredoxin reductase 1 (TrxR1) was augmented by 2.5-fold. Combining mutiple experimental approaches, the enzymatic regulation of TrxR1 by a histone deacetylase 3 (HDAC3)-dependent level of acetylation was confirmed. In vitro and in vivo functional tests established that TrxR1 activity is required to mitigate aggregate development, and this could be mediated by Bcl-2-associated athanogene 3 (BAG3) as a potential TS substrate. INNOVATION AND CONCLUSIONS: This study uncovers the compartmentalized changes and the involvement of TS in the cardiac stress response elicited by misfolded proteins such as R120GCRYAB. Our work suggests that R120GCRYAB triggers a defensive pathway acting through the newly identified interacting partners HDAC3, TrxR1, and BAG3 to counter aggregate growth. Therefore, those interactors may function as modifier genes contributing to the variable onset and expressivity of such human diseases. Furthermore, our work underscores the potential organismal effects of pharmacological interventions targeting TS and HDAC.


Asunto(s)
Cardiomiopatía Hipertrófica/genética , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Oxidación-Reducción , Agregación Patológica de Proteínas/genética , Cadena B de alfa-Cristalina/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Histona Desacetilasas/metabolismo , Humanos , Marcaje Isotópico , Espectrometría de Masas , Ratones , Ratones Transgénicos , Factor 2 Relacionado con NF-E2/metabolismo , Tiorredoxina Reductasa 1/metabolismo , Tiorredoxinas/metabolismo
5.
PLoS One ; 5(1): e8719, 2010 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-20090934

RESUMEN

BACKGROUND: Resveratrol is known to downregulate the high endogenous level of Heat shock protein 70 (Hsp70) in Chronic Myelogenous Leukemia (CML) K562 cells and induce apoptosis. Since Heat Shock Factor 1 (HSF1) controls transcription of Hsp70, we wanted to probe the signaling pathways responsible for transcriptional activation of HSF1. METHODOLOGY/PRINCIPAL FINDINGS: Cells exposed to 40microM Resveratrol rapidly abolished serine473 phosphorylation of Akt and significantly reduced its kinase activity. Inactivation of Akt pathway by Resveratrol subsequently blocked serine9 phosphorylation of Gsk3beta. Active non-phosphorylated Gsk3beta rendered HSF1 transcriptionally inactive and reduced Hsp70 production. Blocking PI3K/Akt activity also demonstrated similar effects on Hsp70 comparable to Resveratrol. Inactivation of Gsk3beta activity by inhibitors SB261763 or LiCl upregulated Hsp70. Resveratrol significantly modulated ERK1/2 activity as evident from hyper phosphorylation at T302/Y304 residues and simultaneous upregulation in kinase activity. Blocking ERK1/2 activation resulted in induction of Hsp70. Therefore, increase in ERK1/2 activity by Resveratrol provided another negative influence on Hsp70 levels through negative regulation of HSF1 activity. 17-allylamino-17-demethoxygeldanamycin (17AAG), a drug that inhibits Hsp90 chaperone and degrades its client protein Akt concomitantly elevated Hsp70 levels by promoting nuclear translocation of HSF1 from the cytosol. This effect is predominantly due to inhibition of both Akt and ERK1/2 activation by 17AAG. Simultaneously treating K562 with Resveratrol and 17AAG maintained phosho-ERK1/2 levels close to untreated controls demonstrating their opposite effects on ERK1/2 pathway. Resveratrol was found not to interfere with Bcr-Abl activation in K562 cells. CONCLUSION/SIGNIFICANCE: Thus our study comprehensively illustrates that Resveratrol acts downstream of Bcr-Abl and inhibits Akt activity but stimulates ERK1/2 activity. This brings down the transcriptional activity of HSF1 and Hsp70 production in K562 cells. Additionally, Resveratrol can be used in combination with chemotherapeutic agents such as 17AAG, an Hsp90 inhibitor reported to induce Hsp70 and hence compromise its chemotherapeutic potential.


Asunto(s)
Regulación hacia Abajo/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Estilbenos/farmacología , Western Blotting , Línea Celular Tumoral , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Microscopía Fluorescente , Fosforilación , Resveratrol
6.
Cell Stress Chaperones ; 14(6): 579-89, 2009 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-19291423

RESUMEN

Chinese hamster lung fibroblasts V79 cells were treated with heat stress for 4 weeks with short duration (15 min) heat shock every alternate day in culture. It was observed that Hsp 70 and the antioxidant enzyme MnSOD became overexpressed during the chronic heat stress period. Both p38 MAPK and Akt became phosphorylated by chronic heat stress exposure. Simultaneous exposure to SB203580, a potent and specific p38MAPK inhibitor drastically inhibited the phosphorylation of p38MAPK and Akt. Furthermore, exposure to SB203580 also blocked the increase in Hsp70 and MnSOD levels and the elevated SOD activity brought about by chronic heat stress. Heat shock factor 1 (HSF1) transcriptional activity and nuclear translocation of HSF1 were prominently augmented by chronic heat stress, and this amplification is markedly reduced by concomitant exposure to SB203580. Also, activations of p38MAPK and Akt and upregulations of Hsp70 and MnSOD were observed on exposure to heat shock for a single exposure of longer duration (40 min). siRNA against p38MAPK notably reduced Akt phosphorylation by single exposure to heat stress and drastically diminished the rise in Hsp70 and MnSOD levels. Similarly, siRNA against Akt also eliminated the augmentation in Hsp70 and MnSOD levels but p38MAPK levels remained unaffected. Heat stress produced reactive oxygen species (ROS) in V79 fibroblasts. N-acetyl cysteine blocked the increase in phosphorylation of p38MAPK, amplification of Hsp70, and MnSOD levels by heat stress. Therefore, we conclude that heat stress-activated p38MAPK which in turn activated Akt. Akt acted downstream of p38MAPK to increase Hsp70 and MnSOD levels.Concise summary: Thermal injury of the skin over a long period of time has been associated with development of cancerous lesions. Also, in many cancers, the cytoprotective genes Hsp70 and MnSOD have been found to be overexpressed. Therefore, we considered it important to identify the signaling elements upstream of the upregulated survival genes in heat stress. We conclude that heat stress activated p38MAPK which in turn activated Akt. Akt mediated an augmentation in Hsp70 and MnSOD levels working downstream of p38MAPK.


Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Respuesta al Choque Térmico/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Superóxido Dismutasa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Acetilcisteína , Animales , Línea Celular , Cricetinae , Cricetulus , Proteínas de Unión al ADN/metabolismo , Fibroblastos/fisiología , Depuradores de Radicales Libres/metabolismo , Factores de Transcripción del Choque Térmico , Imidazoles , Fosforilación/fisiología , Proteínas Proto-Oncogénicas c-akt/genética , Piridinas , ARN Interferente Pequeño , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Regulación hacia Arriba/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/genética
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