Red Blood Cells Derived from Whole Blood Treated with Riboflavin and UV Light Maintain Adequate Cell Quality through 21 Days of Storage.

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.

A Genome-Wide Loss-of-Function Screen Identifies SLC26A2 as a Novel Mediator of TRAIL Resistance.

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.

Non-cell-autonomous Effects of Autophagy Inhibition in Tumor Cells Promote Growth of Drug-resistant Cells.

Autophagy, the mechanism by which cells deliver material to the lysosome, has been associated with resistance to anticancer drugs, leading autophagy inhibition to be widely studied as a potential chemosensitization strategy for cancer cells. This strategy is based on the idea that inhibition of autophagy will increase drug sensitivity and kill more cancer cells. Here we report an unintended negative effect of this strategy. When modeling the effect of drug resistance in a heterogeneous cancer cell population, we found that autophagy inhibition in drug-sensitive tumor cells causes increased growth of drug-resistant cells in the population through a mechanism involving caspase activation and prostaglandin E2 signaling. These results emphasize the importance of understanding how autophagy manipulation in a tumor cell can have both cell-autonomous and nonautonomous effects and suggest that attempts to chemosensitize by inhibiting autophagy could be enhanced by adopting methods aimed at reducing tumor repopulation.

Stat1 activation attenuates IL-6 induced Stat3 activity but does not alter apoptosis sensitivity in multiple myeloma.

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.

IGF-1 receptor tyrosine kinase inhibition by the cyclolignan PPP induces G2/M-phase accumulation and apoptosis in multiple myeloma cells.

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.

Ectopic and IFN-induced expression of Fas overcomes resistance to Fas-mediated apoptosis in multiple myeloma cells.

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.

Hepatitis C virus core protein induces an anergic state characterized by decreased interleukin-2 production and perturbation of mitogen-activated protein kinase responses.

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.

The hepatitis C virus core protein modulates T cell responses by inducing spontaneous and altering T-cell receptor-triggered Ca2+ oscillations.

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.

Retinoic acid inhibits in vitro development of mast cells but has no marked effect on mature human skin tryptase- and chymase-positive mast cells.

Stem cell factor plays a key role in the development of human mast cells via interaction with Kit receptor. We and other groups have previously shown that a number of cytokines can regulate the stem-cell-factor-dependent development of mast cells in vitro. In this study we investigated the effect of retinoic acid on human mast cells in vitro and in vivo. Retinoids are known to have strong modulatory effects on hematopoietic differentiation. We found that all-trans-retinoic acid, at concentrations as low as 1 nM, inhibits the stem-cell-factor-dependent differentiation of mast cells in vitro. This effect of retinoic acid was found to be on progenitor cells, whereas more mature mast cells were less affected. The use of specific agonists binding either to the RAR or the RXR nuclear receptors indicated involvement of both the RAR/RXR and RXR/RXR pathways in inhibiting mast cell differentiation. In contrast to the effects on mast cell progenitors, retinoic acid had no effect on the number of mature mast cells in skin organ cultures. Furthermore, topical treatment of normal skin with a retinoic-acid-containing cream caused an increase in the number of tryptase-positive mast cells, whereas the numbers of the major cutaneous mast cell type, tryptase- and chymase-positive mast cells, remained unaffected. Our results suggest that retinoic acid suppresses commitment of progenitor cells into the mast cell lineage and/or acts on early mast cell progenitors, whereas mature cutaneous mast cells are less susceptible to retinoic acid.