RESUMEN
BACKGROUND: The Mirasol system for whole blood (WB) is a non-toxic, non-mutagenic pathogen reduction technology (PRT) that treats WB units with riboflavin (vitamin B2) and ultraviolet (UV) light to alter nucleic acids, thereby reducing pathogen infectivity and inactivating white blood cells. This study evaluates the quality of red blood cells (RBCs) derived from WB treated with the Mirasol system. STUDY DESIGN AND METHODS: Paired units of WB were collected from 61 healthy donors. One unit per donor was treated with riboflavin and UV light and the other was used as an untreated control. RBCs were processed from the WB units and stored in AS-3 at 1-6°C for 21 days and sampled for in vitro analyses of RBC quality parameters. RESULTS: Several statistically significant differences were observed between test and control units, but values were overall within normal clinical ranges. After leukoreduction, the residual leukocyte count and RBC recovery met FDA requirements. The RBC units derived from treated WB maintained haemolysis below 1% through 21 days of storage. CONCLUSION: RBCs derived from WB treated with the Mirasol system meet accepted FDA guidelines for RBC quality through 21 days of storage at 1-6°C.
RESUMEN
TRAIL is a potent death-inducing ligand that mediates apoptosis through the extrinsic pathway and serves as an important endogenous tumor suppressor mechanism. Because tumor cells are often killed by TRAIL and normal cells are not, drugs that activate the TRAIL pathway have been thought to have potential clinical value. However, to date, most TRAIL-related clinical trials have largely failed due to the tumor cells having intrinsic or acquired resistance to TRAIL-induced apoptosis. Previous studies to identify resistance mechanisms have focused on targeted analysis of the canonical apoptosis pathway and other known regulators of TRAIL receptor signaling. To identify novel mechanisms of TRAIL resistance in an unbiased way, we performed a genome-wide shRNA screen for genes that regulate TRAIL sensitivity in sublines that had been selected for acquired TRAIL resistance. This screen identified previously unknown mediators of TRAIL resistance including angiotensin II receptor 2, Crk-like protein, T-Box Transcription Factor 2, and solute carrier family 26 member 2 (SLC26A2). SLC26A2 downregulates the TRAIL receptors, DR4 and DR5, and this downregulation is associated with resistance to TRAIL. Its expression is high in numerous tumor types compared with normal cells, and in breast cancer, SLC26A2 is associated with a significant decrease in relapse-free survival.Implication: Our results shed light on novel resistance mechanisms that could affect the efficacy of TRAIL agonist therapies and highlight the possibility of using these proteins as biomarkers to identify TRAIL-resistant tumors, or as potential therapeutic targets in combination with TRAIL. Mol Cancer Res; 15(4); 382-94. ©2017 AACR.
Asunto(s)
Proteínas de Transporte de Anión/genética , Resistencia a Antineoplásicos , Neoplasias/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Proteínas de Transporte de Anión/metabolismo , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , ARN Interferente Pequeño/genética , Transportadores de Sulfato , Regulación hacia ArribaRESUMEN
BACKGROUND: Multiple myeloma (MM) is at present an incurable malignancy, characterized by apoptosis-resistant tumor cells. Interferon (IFN) treatment sensitizes MM cells to Fas-induced apoptosis and is associated with an increased activation of Signal transducer and activator of transcription (Stat)1. The role of Stat1 in MM has not been elucidated, but Stat1 has in several studies been ascribed a pro-apoptotic role. Conversely, IL-6 induction of Stat3 is known to confer resistance to apoptosis in MM. METHODS: To delineate the role of Stat1 in IFN mediated sensitization to apoptosis, sub-lines of the U-266-1970 MM cell line with a stable expression of the active mutant Stat1C were utilized. The influence of Stat1C constitutive transcriptional activation on endogenous Stat3 expression and activation, and the expression of apoptosis-related genes were analyzed. To determine whether Stat1 alone would be an important determinant in sensitizing MM cells to apoptosis, the U-266-1970-Stat1C cell line and control cells were exposed to high throughput compound screening (HTS). RESULTS: To explore the role of Stat1 in IFN mediated apoptosis sensitization of MM, we established sublines of the MM cell line U-266-1970 constitutively expressing the active mutant Stat1C. We found that constitutive nuclear localization and transcriptional activity of Stat1 was associated with an attenuation of IL-6-induced Stat3 activation and up-regulation of mRNA for the pro-apoptotic Bcl-2 protein family genes Harakiri, the short form of Mcl-1 and Noxa. However, Stat1 activation alone was not sufficient to sensitize cells to Fas-induced apoptosis. In a screening of > 3000 compounds including bortezomib, dexamethasone, etoposide, suberoylanilide hydroxamic acid (SAHA), geldanamycin (17-AAG), doxorubicin and thalidomide, we found that the drug response and IC50 in cells constitutively expressing active Stat1 was mainly unaltered. CONCLUSION: We conclude that Stat1 alters IL-6 induced Stat3 activity and the expression of pro-apoptotic genes. However, this shift alone is not sufficient to alter apoptosis sensitivity in MM cells, suggesting that Stat1 independent pathways are operative in IFN mediated apoptosis sensitization.
Asunto(s)
Apoptosis , Interleucina-6/farmacología , Mieloma Múltiple/metabolismo , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismo , Transporte Activo de Núcleo Celular , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Núcleo Celular/metabolismo , Análisis por Conglomerados , Resistencia a Antineoplásicos/genética , Expresión Génica , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Mieloma Múltiple/genética , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT3/genética , Transcripción Genética , Receptor fas/metabolismoRESUMEN
Emerging evidence suggests the insulin-like growth factor-1 receptor (IGF-1R) to be an important mediator of tumor-cell survival and resistance to cytotoxic therapy in multiple myeloma (MM). Recently, members of the cyclolignan family have been shown to selectively inhibit the receptor tyrosine kinase (RTK) activity of the IGF-1R beta-chain. The effects of the cyclolignan picropodophyllin (PPP) were studied in vitro using a panel of 13 MM cell lines and freshly purified tumor cells from 10 patients with MM. PPP clearly inhibited growth in all MM cell lines and primary MM samples cultured in the presence or absence of bone marrow stromal cells. PPP induced a profound accumulation of cells in the G(2)/M-phase and an increased apoptosis. Importantly, IGF-1, IGF-2, insulin, or IL-6 did not reduce the inhibitory effects of PPP. As demonstrated by in vitro kinase assays, PPP down-regulated the IGF-1 RTK activity without inhibiting the insulin RTK activity. This conferred decreased phosphorylation of Erk1/2 and reduced cyclin dependent kinase (CDK1) activity. In addition, the expression of mcl-1 and survivin was reduced. Taken together, we suggest that interfering with the IGF-1 RTK by using the cyclolignan PPP offers a novel and selective therapeutic strategy for MM.
Asunto(s)
Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , Fase G2/efectos de los fármacos , Mieloma Múltiple/tratamiento farmacológico , Podofilotoxina/análogos & derivados , Receptor IGF Tipo 1/antagonistas & inhibidores , Proteína Quinasa CDC2/metabolismo , Proliferación Celular/efectos de los fármacos , Activación Enzimática , Humanos , Proteínas Inhibidoras de la Apoptosis , Insulina/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Interleucina-6/farmacología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Mieloma Múltiple/irrigación sanguínea , Mieloma Múltiple/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/metabolismo , Fosforilación , Podofilotoxina/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/antagonistas & inhibidores , Receptor de Insulina/metabolismo , Survivin , Células Tumorales CultivadasRESUMEN
Multiple myeloma (MM) is an as-yet incurable B-cell malignancy. Increased survival in vitro is a hallmark of MM cells, implying that a therapeutic potential may lie in circumventing antiapoptotic signals. We have previously reported that interferons (IFNs) sensitize MM cells to Fas/CD95-mediated apoptosis. In the present study, we explore the mechanism underlying this effect. In a wide screening of apoptosis-related genes, Apo2L/TRAIL (tumor necrosis factor [TNF]-related apoptosis inducing ligand) and Fas were identified as IFN targets. Sensitization to Fas-mediated apoptosis by IFNs was not affected by blocking Apo2L/TRAIL, suggesting that Apo2L/TRAIL is not a key mediator in this process. In contrast, we found that an elevated Fas expression was functionally linked to increased susceptibility to Fas-mediated apoptosis. This was further supported by the finding that IFN treatment enhanced Fas-mediated caspase-8 activation, one of the earliest signaling events downstream receptor activation. In addition, IFN treatment attenuated the interleukin 6 (IL-6)-dependent activation of signal transducer and activator of transcription 3 (Stat3), interfering with a known survival pathway in MM that has previously been linked with resistance to Fas-mediated apoptosis. Taken together, our results show that IFN-induced up-regulation of Fas sensitizes MM cells to Fas-mediated apoptosis and suggest that attenuation of Stat3 activation may be a potentially important event in this process.
Asunto(s)
Apoptosis/genética , Interferones/farmacología , Mieloma Múltiple/patología , Receptor fas/genética , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , Caspasa 8 , Caspasas/metabolismo , Proteínas de Unión al ADN/efectos de los fármacos , Humanos , Interleucina-6 , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Mieloma Múltiple/tratamiento farmacológico , Factor de Transcripción STAT3 , Ligando Inductor de Apoptosis Relacionado con TNF , Transactivadores/efectos de los fármacos , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/fisiología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Alterations of cytokine responses are thought to favor the establishment of persistent hepatitis C virus (HCV) infections, enhancing the risk of liver cirrhosis and hepatocellular carcinoma. Here we demonstrate that the expression of the HCV core (C) protein in stably transfected T cells correlates with a selective reduction of interleukin-2 (IL-2) promoter activity and IL-2 production in response to T-cell receptor triggering, whereas the activation of IL-4, IL-10, gamma interferon, and tumor necrosis factor alpha was moderately increased. This altered cytokine expression profile was associated with a perturbation of mitogen-activated protein (MAP) kinase responses. Extracellular regulated kinase and p38 were constitutively phosphorylated in C-expressing cells, while triggering of the costimulatory c-Jun N-terminal kinase (JNK) signaling cascade and activation of the CD28 response element within the IL-2 promoter appeared to be impaired. The perturbations of MAP kinase phosphorylation could be eliminated by cyclosporine A-mediated inhibition of nuclear factor of activated T cells, suggesting that the inactivation of JNK signaling and hyporesponsiveness to IL-2 induction were downstream consequences of C-induced Ca(2+) flux in a manner that mimics the induction of clonal anergy.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Interleucina-2/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Linfocitos T/efectos de los fármacos , Proteínas del Núcleo Viral/farmacología , Anergia Clonal , Activación Enzimática , Hepacivirus , Humanos , Células Jurkat , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Linfocitos T/patología , Linfocitos T/virología , Células Tumorales CultivadasRESUMEN
Alterations of cytokine responses are thought to favor the establishment of persistent hepatitis C virus (HCV) infection, enhancing the risk of liver cirrhosis and hepatocellular carcinoma. Expression of the HCV core (C) protein modulates transcription of the IL-2 promoter in T lymphocytes by activating the nuclear factor of activated T lymphocyte (NFAT) pathway. Here we report on the effect of HCV C on Ca2+ signaling, which is essential for activation of NFAT. Expression of HCV C correlated with increased levels of cytosolic Ca2+ and spontaneous Ca2+ oscillations in transfected Jurkat cells. Triggering of the T-cell receptor induced a prolonged Ca2+ response characterized by vigorous high frequent oscillations in a high proportion of the responding cells. This was associated with decreased sizes and accelerated emptying of the intracellular calcium stores. The effect of HCV C on calcium mobilization was not dependent on phospholipase C-gamma 1 (PLC-gamma) activity or increased inositol 1,4,5-trisphosphate (IP3) production and did not require functional IP3 receptors, suggesting that insertion of the viral protein in the endoplasmic reticulum membrane may be sufficient to promote Ca2+ leakage with dramatic downstream consequences on the magnitude and duration of the response. Our data suggest that expression of HCV C in infected T lymphocytes may contribute to the establishment of persistent infections by inducing Ca2+ oscillations that regulate both the efficacy and information content of Ca2+ signals and are ultimately responsible for induction of gene expression and functional differentiation.