The helicase HAGE prevents interferon-a-induced PML expression in ABCB5+ malignant melanoma-initiating cells by promoting the expression of SOCS1

The tumour suppressor PML (promyelocytic leukaemia protein) regulates several cellular pathways involving cell growth, apoptosis, differentiation and senescence. PML also has an important role in the regulation of stem cell proliferation and differentiation. Here, we show the involvement of the helicase HAGE in the transcriptional repression of PML expression in ABCB5 + malignant melanoma-initiating cells (ABCB5 + MMICs), a population of cancer stem cells which are responsible for melanoma growth, progression and resistance to drug-based therapy. HAGE prevents PML gene expression by inhibiting the activation of the JAK-STAT (janus kinase-signal transducers and activators of transcription) pathway in a mechanism which implicates the suppressor of cytokine signalling 1 (SOCS1). Knockdown of HAGE led to a significant decrease in SOCS1 protein expression, activation of the JAK-STAT signalling cascade and a consequent increase of PML expression. To confirm that the reduction in SOCS1 expression was dependent on the HAGE helicase activity, we showed that SOCS1, effectively silenced by small interfering RNA, could be rescued by re-introduction of HAGE into cells lacking HAGE. Furthermore, we provide a mechanism by which HAGE promotes SOCS1 mRNA unwinding and protein expression in vitro

Sulindac Sulfide Reverses Aberrant Self-Renewal of Progenitor Cells Induced by the AML-Associated Fusion Proteins PML/RARα and PLZF/RARα

Chromosomal translocations can lead to the formation of chimeric genes encoding fusion proteins such as PML/RARα, PLZF/RARα, and AML-1/ETO, which are able to induce and maintain acute myeloid leukemia (AML). One key mechanism in leukemogenesis is increased self renewal of leukemic stem cells via aberrant activation of the Wnt signaling pathway. Either X-RAR, PML/RARα and PLZF/RARα or AML-1/ETO activate Wnt signaling by upregulating γ-catenin and β-catenin. In a prospective study, a lower risk of leukemia was observed with aspirin use, which is consistent with numerous studies reporting an inverse association of aspirin with other cancers. Furthermore, a reduction in leukemia risk was associated with use of non-steroidal anti-inflammatory drug (NSAID), where the effects on AML risk was FAB subtype-specific. To better investigate whether NSAID treatment is effective, we used Sulindac Sulfide in X-RARα-positive progenitor cell models. Sulindac Sulfide (SSi) is a derivative of Sulindac, a NSAID known to inactivate Wnt signaling. We found that SSi downregulated both β-catenin and γ-catenin in X-RARα-expressing cells and reversed the leukemic phenotype by reducing stem cell capacity and increasing differentiation potential in X-RARα-positive HSCs. The data presented herein show that SSi inhibits the leukemic cell growth as well as hematopoietic progenitors cells (HPCs) expressing PML/RARα, and it indicates that Sulindac is a valid molecular therapeutic approach that should be further validated using in vivo leukemia models and in clinical settings

Disruption of PML Nuclear Bodies Is Mediated by ORF61 SUMO-Interacting Motifs and Required for Varicella-Zoster Virus Pathogenesis in Skin

Promyelocytic leukemia protein (PML) has antiviral functions and many viruses encode gene products that disrupt PML nuclear bodies (PML NBs). However, evidence of the relevance of PML NB modification for viral pathogenesis is limited and little is known about viral gene functions required for PML NB disruption in infected cells in vivo. Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes cutaneous lesions during primary and recurrent infection. Here we show that VZV disrupts PML NBs in infected cells in human skin xenografts in SCID mice and that the disruption is achieved by open reading frame 61 (ORF61) protein via its SUMO-interacting motifs (SIMs). Three conserved SIMs mediated ORF61 binding to SUMO1 and were required for ORF61 association with and disruption of PML NBs. Mutation of the ORF61 SIMs in the VZV genome showed that these motifs were necessary for PML NB dispersal in VZV-infected cells in vitro. In vivo, PML NBs were highly abundant, especially in basal layer cells of uninfected skin, whereas their frequency was significantly decreased in VZV-infected cells. In contrast, mutation of the ORF61 SIMs reduced ORF61 association with PML NBs, most PML NBs remained intact and importantly, viral replication in skin was severely impaired. The ORF61 SIM mutant virus failed to cause the typical VZV lesions that penetrate across the basement membrane into the dermis and viral spread in the epidermis was limited. These experiments indicate that VZV pathogenesis in skin depends upon the ORF61-mediated disruption of PML NBs and that the ORF61 SUMO-binding function is necessary for this effect. More broadly, our study elucidates the importance of PML NBs for the innate control of a viral pathogen during infection of differentiated cells within their tissue microenvironment in vivo and the requirement for a viral protein with SUMO-binding capacity to counteract this intrinsic barrier

Misguided Transcriptional Elongation Causes Mixed Lineage Leukemia

Investigation of the activity of a family of fusion proteins that cause aggressive leukemia suggests transcriptional elongation as a new mechanism for oncogenic transformation

EBV Tegument Protein BNRF1 Disrupts DAXX-ATRX to Activate Viral Early Gene Transcription

Productive infection by herpesviruses involve the disabling of host-cell intrinsic defenses by viral encoded tegument proteins. Epstein-Barr Virus (EBV) typically establishes a non-productive, latent infection and it remains unclear how it confronts the host-cell intrinsic defenses that restrict viral gene expression. Here, we show that the EBV major tegument protein BNRF1 targets host-cell intrinsic defense proteins and promotes viral early gene activation. Specifically, we demonstrate that BNRF1 interacts with the host nuclear protein Daxx at PML nuclear bodies (PML-NBs) and disrupts the formation of the Daxx-ATRX chromatin remodeling complex. We mapped the Daxx interaction domain on BNRF1, and show that this domain is important for supporting EBV primary infection. Through reverse transcription PCR and infection assays, we show that BNRF1 supports viral gene expression upon early infection, and that this function is dependent on the Daxx-interaction domain. Lastly, we show that knockdown of Daxx and ATRX induces reactivation of EBV from latently infected lymphoblastoid cell lines (LCLs), suggesting that Daxx and ATRX play a role in the regulation of viral chromatin. Taken together, our data demonstrate an important role of BNRF1 in supporting EBV early infection by interacting with Daxx and ATRX; and suggest that tegument disruption of PML-NB-associated antiviral resistances is a universal requirement for herpesvirus infection in the nucleus

In vivo starch digestion in small intestine

Background In mammals, starch is digested mostly in the small intestine. However, most in vivo starch digestion studies have been carried out using digesta from terminal ileum, colon, and feces, and thus cannot give unambiguous information on starch digestion in the small intestine. Furthermore, many in vitro studies have tried to stimulate the digestion in the small intestine without reference to in vivo studies of starch digestion in the small intestine. Therefore, current in vitro methods might not reflect in vivo digestion. Objective This study explores the in vivo starch digestion in the pig small intestine and compares it with a standard in vitro method. Design Digesta were collected from different sections of small intestine of seven pigs fed with a diet containing 47% (dry weight basis) raw normal maize starch. Starch contents of the digesta were analysed using Megazyme total starch kits. The pig diet was also digested using a modified in vitro method (Sopade and Gidley, Starch, 2009, 61, pp. 245). Starch was extracted from the in vivo and in vitro digesta following the method of Syahariza et al. (Carbohydr. Polymers, 2010, 82, pp. 14) and analyzed using size exclusion chromatography (SEC, fully branched and debranched using isoamylase) following the methods of Witt et al. (J. Agric. Food Chem., 2010, 58, pp 8444). Outcomes The starch digestion in mouth and stomach was minor compared with that in the small intestine, and raw normal maize starch was almost completely digested in the first half of the small intestine. The time evolution of the size distributions (fully branched and debranched) of starch molecules during in vivo and in vitro digestion analyzed using SEC showed a qualitative difference between the two digestion methods. The former showed a degradation of starch molecules to a more uniform size, whereas the latter show a complex mechanism, which preserved the size distribution of native starch at the beginning of the digestion before producing a multimodal distribution. Conclusion The heterogeneous nature of the in vivo digestion cannot be reproduced by current in vitro methods, which are more homogenous. Nutritional claims of starch products based on in vitro methods need to take account of this phenomenon. Source of funding This study is financially supported by the Australian Research Council (DP0985694). The small intestine digesta were provided by the High Fibre Grains Cluster (supported by a grant from the CSIRO Flagship Collaboration Fund via the Food Futures Flagship)

In vivo and in vitro starch digestion: Are current in vitro techniques adequate?

The time evolution of the size distributions of (fully branched and debranched) starch molecules during in vivo and in vitro digestion was analyzed using size exclusion chromatography (SEC) and compared. In vivo digesta were collected from the small intestine of pigs fed with raw normal maize starch; in vitro digestion was carried out on the same diet fed to the pigs using a method simulating digestion in the mouth, stomach, and small intestine. A qualitative difference was observed between the in vitro and the in vivo digestion. The former showed a degradation of starch molecules to a more uniform size, whereas the in vivo digestion preserved the size distribution of native starch before producing a multimodal distribution, the heterogeneous nature of which current in vitro methods do not reproduce. The use of in vitro digestion to infer in vivo digestion patterns and, hence, potential nutrition benefits need to take account of this phenomenon