IntroductionHuman T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia-lymphoma (ATLL) and tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). HTLV-1 uses several strategies for controlling the expression of its genome, including the production of 9 alternatively spliced transcripts ( Figure 1A). 1-6 Production of plus-strand transcripts is controlled by Tax at the level of transcription and by Rex at the level of nucleo-cytoplasmic export of unspliced and partially spliced mRNAs. 7,8 Regulation of the minus-strand HBZ transcripts, which lack elements responsive to Rex, remains to be determined.Current models suggest that plus-strand HTLV-1 mRNAs are expressed with a distinct timing during the course of the viral life cycle, with a switch from early (Rex-independent) to late (Rexdependent) transcripts. Although early studies showed a qualitative switch among classes of HTLV-1 mRNAs (multiply spliced vs unspliced), 9-12 detection of this phenomenon with quantitative transcript-specific methods has proven difficult. 13 To answer this question we used quantitative RT-PCR to quantify proviral expression during the spontaneous proviral reactivation observed in cells from infected patients. The results demonstrated a "two-phase" expression pattern. Using transfection of HTLV-1 molecular clones and subcellular RNA fractionation we demonstrated the Rex-dependency of the two-phase kinetics and determined the compartmentalization of the individual mRNAs, showing that more than 90% of the HBZ mRNAs were retained in the nucleus. Mathematical modeling 14 revealed the importance of a delay of Rex function compared with Tax, which was supported by experimental evidence of delayed accumulation and longer halflife of Rex. Methods Samples from HTLV-1-infected patientsPeripheral blood mononuclear cells (PBMCs) from ATLL and TSP/HAM patients were purified as in. 15 Patients are described in supplemental Table 1 (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). All samples were obtained from patients after informed consent in accordance with the Declaration of Helsinki, with approval from the Imperial College and King's College hospitals (London) Institutional Rreview Boards. Plasmids, cells, and transfectionsPlasmid pBS1-2-3 consists of the tax/rex cDNA (exons 1, 2, and 3 flanked by the 5Ј and 3ЈLTRs, from infectious molecular clone CS-HTLV-1 16 ) inserted in pBluescript (Stratagene). Plasmid ACH-Rex knockout (KO) was For personal use only. on May 10, 2018. by guest www.bloodjournal.org From derived from the HTLV-1 molecular clone ACH 17 by digestion with SphI followed by removal of 3Ј overhangs (including the Rex initiation codon) with T4 DNA polymerase and religation. Transfections were performed in the HeLa-derived cell line HLtat, 18 chosen for its high transfection efficiency. Quantitative RT-PCRRNA of PBMCs from infected patients and transfected cells was extracted and viral transcripts were quantitated as detailed in supp...

Human T-cell leukemia virus type 1 encodes a number of "accessory" proteins of unclear function; one of these proteins, p13 II , is targeted to mitochondria and disrupts mitochondrial morphology. The present study was undertaken to unravel the function of p13 II through (i) determination of its submitochondrial localization and sequences required to alter mitochondrial morphology and (ii) an assessment of the biophysical and biological properties of synthetic peptides spanning residues 9 -41 (p13 9 -41 ), which include the amphipathic mitochondrialtargeting sequence of the protein. p13 9 -41 folded into an ␣ helix in micellar environments. Fractionation and immunogold labeling indicated that full-length p13 II accumulates in the inner mitochondrial membrane. p13 9 -41 induced energy-dependent swelling of isolated mitochondria by increasing inner membrane permeability to small cations (Na ؉ , K ؉ ) and released Ca 2؉ from Ca 2؉ -preloaded mitochondria. These effects as well as the ability of full-length p13 II to alter mitochondrial morphology in cells required the presence of four arginines, forming the charged face of the targeting signal. The mitochondrial effects of p13 9 -41 were insensitive to cyclosporin A, suggesting that full-length p13 II might alter mitochondrial permeability through a permeability transition pore-independent mechanism, thus distinguishing it from the mitochondrial proteins Vpr and X of human immunodeficiency virus type 1 and hepatitis B virus, respectively.HTLV-1 1 is a complex retrovirus that is associated with two distinct pathologies, a leukemia/lymphoma of mature CD4 ϩ

Human T cell leukemia virus type 1 encodes an ''accessory'' protein named p13 II that is targeted to mitochondria and triggers a rapid flux of K ؉ and Ca 2؉ across the inner membrane. In this study, we investigated the effects of p13 II on tumorigenicity in vivo and on cell growth in vitro. Results showed that p13 II significantly reduced the incidence and growth rate of tumors arising from c-myc and Ha-ras-cotransfected rat embryo fibroblasts. Consistent with these findings, HeLa-derived cell lines stably expressing p13 II exhibited markedly reduced tumorigenicity, as well as reduced proliferation at high density in vitro. Mixed culture assays revealed that the phenotype of the p13 II cell lines was dominant over that of control lines and was mediated by a heat-labile soluble factor. The p13 II cell lines exhibited an enhanced response to Ca 2؉ -mediated stimuli, as measured by increased sensitivity to C2-ceramide-induced apoptosis and by cAMP-responsive element-binding protein (CREB) phosphorylation in response to histamine. p13 II -expressing Jurkat T cells also exhibited reduced proliferation, suggesting that the protein might exert similar effects in T cells, the primary target of HTLV-1 infection. These findings provide clues into the function of p13 II as a negative regulator of cell growth and underscore a link between mitochondria, Ca 2؉ signaling, and tumorigenicity.apoptosis ͉ calcium signaling ͉ tumorigenicity ͉ retrovirus H uman T cell leukemia virus type 1 (HTLV-1) possesses a complex genome that codes for Gag, Pol, Env, Tax, Rex, and a number of ''accessory'' proteins (1-4). Although the function of the accessory proteins has not yet been elucidated completely, they elicit an immune response in HTLV-1 infected individuals (5) and are required for efficient viral propagation in an animal model (6).One of the HTLV-1 accessory proteins, p13 II , accumulates in mitochondria by means of an amphipathic mitochondrial targeting signal and disrupts mitochondrial morphology (7). Biochemical analyses showed that p13 II is inserted in the inner mitochondrial membrane and alters mitochondrial conductance to Ca 2ϩ and K ϩ , leading to swelling and collapse of inner mitochondrial membrane potential (8).These effects suggest that p13 II might alter key mitochondrial functions such as energy production, redox status, and apoptosis, which could in turn disrupt the balance between cell death and proliferation. In this study, we investigated the impact of p13 II on cell growth in vitro and tumor growth in vivo by using the rat embryo fibroblast (REF) transformation model and cell lines expressing p13 II . Results showed that p13 II -expressing cells exhibit reduced tumorigenicity in vivo and slower proliferation at high density in vitro. p13 II -expressing cells also display perturbations in signal transduction pathways depending on Ca 2ϩ homeostasis. Materials and MethodsGeneration of HeLa Tet-On Cell Lines Expressing p13 II . The doxycyclininducible p13 II expression plasmid pTRE-p13 II -AU1 was constructed by inse...

The miR-200 family of microRNAs (miRNAs) includes miR-200a, miR-200b, miR-200c, miR-141 and miR-429, five evolutionarily conserved miRNAs that are encoded in two clusters of hairpin precursors located on human chromosome 1 (miR-200b, miR-200a and miR-429) and chromosome 12 (miR-200c and miR-141). The mature -3p products of the precursors are abundantly expressed in epithelial cells, where they contribute to maintaining the epithelial phenotype by repressing expression of factors that favor the process of epithelial-to-mesenchymal transition (EMT), a key hallmark of oncogenic transformation. Extensive studies of the expression and interactions of these miRNAs with cell signaling pathways indicate that they can exert both tumor suppressor- and pro-metastatic functions, and may serve as biomarkers of epithelial cancers. This review provides a summary of the role of miR-200 family members in EMT, factors that regulate their expression, and important targets for miR-200-mediated repression that are involved in EMT. The second part of the review discusses the potential utility of circulating miR-200 family members as diagnostic/prognostic biomarkers for breast, colorectal, lung, ovarian, prostate and bladder cancers.

The present study investigated the function of p13, a mitochondrial protein of human T-cell leukemia virus type 1 (HTLV-1). Although necessary for viral propagation in vivo, the mechanism of function of p13 is incompletely understood. Drawing from studies in isolated mitochondria, we analyzed the effects of p13 on mitochondrial reactive oxygen species (ROS) in transformed and primary T cells. In transformed cells (Jurkat, HeLa), p13 did not affect ROS unless the cells were subjected to glucose deprivation, which led to a p13-dependent increase in ROS and cell death. Using RNA interference we confirmed that expression of p13 also influences glucose starvation-induced cell death in the context of HTLV-1-infected cells. ROS measurements showed an increasing gradient from resting to mitogenactivated primary T cells to transformed T cells (Jurkat). Expression of p13 in primary T cells resulted in their activation, an effect that was abrogated by ROS scavengers. These findings suggest that p13 may have a distinct impact on cell turnover depending on the inherent ROS levels; in the context of the HTLV-1 propagation strategy, p13 could increase the pool of "normal" infected cells while culling cells acquiring a transformed phenotype, thus favoring lifelong persistence of the virus in the host. (Blood. 2010;116(1): 54-62) IntroductionHuman T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that infects an estimated 20 million people worldwide. HTLV-1 is the causative agent of adult T-cell leukemia/lymphoma (ATLL), an aggressive neoplasm of mature CD4ϩ T cells that is refractory to current therapies. ATLL arises in approximately 3% of HTLV-1-infected persons and is preceded by a decades-long clinical latency.Many aspects of HTLV-1 replication, persistence, and pathogenesis remain incompletely understood (reviewed in Verdonck et al 1 ). Studies so far have been focused primarily on the transcriptional activator Tax, which is essential for expression from the viral promoter, and the posttranscriptional factor Rex, which is required for expression of incompletely spliced viral transcripts. Tax also plays a critical role in T-cell transformation through its ability to deregulate the expression of a vast array of cellular genes and interfere with cell-cycle checkpoints, producing major alterations in cell proliferation and survival and promoting genetic instability (reviewed in Lairmore et al 2 ). Indeed, expression of Tax in mouse thymocytes is sufficient for induction of T-cell leukemia/ lymphoma. 3 However, the contrast between the powerful oncogenic properties of Tax and the low prevalence and long latency of ATLL suggests the existence of mechanisms that limit the transforming potential of the virus and favor its lifelong persistence in the host in the absence of disease.Recent studies indicate that the viral accessory proteins p12, p21, p30, HBZ, and p13 may also contribute to HTLV-1 replication and pathogenesis (reviewed in Nicot et al 4 ). The present study is focused on p13, an 87-amino acid accessory prote...

Human T-cell leukemia virus type-1 (HTLV-1) expresses an 87-amino acid protein named p13 that is targeted to the inner mitochondrial membrane. Previous studies showed that a synthetic peptide spanning an alpha helical domain of p13 alters mitochondrial membrane permeability to cations, resulting in swelling. The present study examined the effects of full-length p13 on isolated, energized mitochondria. Results demonstrated that p13 triggers an inward K(+) current that leads to mitochondrial swelling and confers a crescent-like morphology distinct from that caused by opening of the permeability transition pore. p13 also induces depolarization, with a matching increase in respiratory chain activity, and augments production of reactive oxygen species (ROS). These effects require an intact alpha helical domain and strictly depend on the presence of K(+) in the assay medium. The effects of p13 on ROS are mimicked by the K(+) ionophore valinomycin, while the protonophore FCCP decreases ROS, indicating that depolarization induced by K(+) vs. H(+) currents has different effects on mitochondrial ROS production, possibly because of their opposite effects on matrix pH (alkalinization and acidification, respectively). The downstream consequences of p13-induced mitochondrial K(+) permeability are likely to have an important influence on the redox state and turnover of HTLV-1-infected cells.

Malignant pleural mesothelioma (MPM) is a rare malignancy of mesothelial cells with increasing incidence, and in many cases, dismal prognosis due to its aggressiveness and lack of effective therapies. Environmental and occupational exposure to asbestos is considered the main aetiological factor for MPM. Inhaled asbestos fibres accumulate in the lungs and induce the generation of reactive oxygen species (ROS) due to the presence of iron associated with the fibrous silicates and to the activation of macrophages and inflammation. Chronic inflammation and a ROS-enriched microenvironment can foster the malignant transformation of mesothelial cells. In addition, MPM cells have a highly glycolytic metabolic profile and are positive in 18 F-FDG PET analysis. Loss-offunction mutations of BRCA-associated protein 1 (BAP1) are a major contributor to the metabolic rewiring of MPM cells. A subset of MPM tumours show loss of the methyladenosine phosphorylase (MTAP) locus, resulting in profound alterations in polyamine metabolism, ATP and methionine salvage pathways, as well as changes in epigenetic control of gene expression. This review provides an overview of the perturbations in metabolism and ROS homoeostasis of MPM cells and the role of these alterations in malignant transformation and tumour progression.

Approximately 20% of pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients are currently incurable due to primary or secondary resistance to glucocorticoid-based therapies. Here we employed an integrated approach to selectively kill T-ALL cells by increasing mitochondrial reactive oxygen species (ROS) using NS1619, a benzimidazolone that activates the K+ (BK) channel, and dehydroepiandrosterone (DHEA), which blunts ROS scavenging through inhibition of the pentose phosphate pathway. These compounds selectively killed T-ALL cell lines, patient-derived xenografts and primary cells from patients with refractory T-ALL, but did not kill normal human thymocytes. T-ALL cells treated with NS1619 and DHEA showed activation of the ROS-responsive transcription factor NRF2, indicating engagement of antioxidant pathways, as well as increased cleavage of OPA1, a mitochondrial protein that promotes mitochondrial fusion and regulates apoptosis. Consistent with these observations, transmission electron microscopy analysis indicated that NS1619 and DHEA increased mitochondrial fission. OPA1 cleavage and cell death were inhibited by ROS scavengers and by siRNA-mediated knockdown of the mitochondrial protease OMA1, indicating the engagement of a ROS-OMA1-OPA1 axis in T-ALL cells. Furthermore, NS1619 and DHEA sensitized T-ALL cells to TRAIL-induced apoptosis. In vivo, the combination of dexamethasone and NS1619 significantly reduced the growth of a glucocorticoid-resistant patient-derived T-ALL xenograft. Taken together, our findings provide proof-of-principle for an integrated ROS-based pharmacological approach to target refractory T-ALL.