Expression of alternatively spliced human T-cell leukemia virus type 1 mRNAs is influenced by mitosis and by a novel cis-acting regulatory sequence

Human T-cell leukemia virus type 1 (HTLV-1) expression depends on the concerted action of Tax, which drives transcription of the viral genome, and Rex, which favors expression of incompletely spliced mRNAs and determines a 2-phase temporal pattern of viral expression. In the present study, we investigated the Rex dependence of the complete set of alternatively spliced HTLV-1 mRNAs. Analyses of cells transfected with Rex-wild-type and Rex-knockout HTLV-1 molecular clones using splice site-specific quantitative reverse transcription (qRT)-PCR revealed that mRNAs encoding the p30Tof, p13, and p12/8 proteins were Rex dependent, while the p21rex mRNA was Rex independent. These findings provide a rational explanation for the intermediate-late temporal pattern of expression of the p30tof, p13, and p12/8 mRNAs described in previous studies. All the Rex-dependent mRNAs contained a 75-nucleotide intronic region that increased the nuclear retention and degradation of a reporter mRNA in the absence of other viral sequences. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis revealed that this sequence formed a stable hairpin structure. Cell cycle synchronization experiments indicated that mitosis partially bypasses the requirement for Rex to export Rex-dependent HTLV-1 transcripts. These findings indicate a link between the cycling properties of the host cell and the temporal pattern of viral expression/latency that might influence the ability of the virus to spread and evade the immune system

Expression Of Mir-34a In T-cells Infected By Human T-lymphotropic Virus 1

Human T-lymphotropic virus 1 (HTLV-1) immortalizes T-cells and is the causative agent of adult T-cell leukemia/lymphoma (ATLL). HTLV-1 replication and transformation are governed by multiple interactions between viral regulatory proteins and host cell factors that remain to be fully elucidated. The present study investigated the impact of HTLV-1 infection on the expression of miR-34a, a microRNA whose expression is downregulated in many types of cancer. Results of RT-PCR assays showed that five out of six HTLV-1-positive cell lines expressed higher levels of miR-34a compared to normal PBMC or purified CD4+ T-cells. ATLL cell line ED, which did not express miR-34a, showed methylation of the miR-34a promoter. Newly infected PBMC and samples from 10 ATLL patients also showed a prominent increase in miR-34a expression compared to PBMC controls. The primary miR-34a transcript expressed in infected cell line C91PL contained binding motifs for NF-kappa B and p53. Pharmacological inhibition of NF-kappa B with Bay 11-7082 indicated that this pathway contributes to sustain miR-34a levels in infected cells. Treatment of infected cell lines with the p53 activator nutlin-3a resulted in a further increase in miR-34a levels, thus confirming it as a transcriptional target of p53. Nutlin-3a-treated cells showed downregulation of known miR-34a targets including the deacetylase SIRT1, which was accompanied by increased acetylation of p53, a substrate of SIRT1. Transfection of C91PL cells with a miR-34a mimic also led to downregulation of mRNA targets including SIRT1 as well as the pro-apoptotic factor BAX. Unlike nutlin-3a, the miR-34a mimic did not cause cell cycle arrest or reduce cell viability. On the other hand, sequestration of miR-34a with a sponge construct resulted in an increase in death of C91PL cells. These findings provide evidence for a functional role for miR-34a in fine-tuning the expression of target genes that influence the turnover of HTLV-1-infected cells

Detection of circulating immunosuppressive cytokines in malignant pleural mesothelioma patients for prognostic stratification.

Abstract Background No data on circulating biomarkers for the prognostic stratification of Malignant Pleural Mesothelioma (MPM) patients are available. We prospectively explored the prognostic role of circulating monocyte and cytokine levels and their dynamic change during chemotherapy. Patients and Methods MPM patients receiving a first line treatment based on a platinum compound plus pemetrexed were eligible. Blood samples were collected at the baseline and at the end of induction chemotherapy. CCL-2, IL-10 and TGF-β levels in plasma were quantified by Enzyme-Linked Immunosorbent Assay (ELISA); white blood cells, monocytes and platelets were evaluated by blood count test. Results Thirty-one patients were included in the study. Median overall survival (OS) was 12.13 months versus 9.6 months in patients with lower and higher monocytes count, respectively (p value = 0.02). We further stratified patients according to a combined score based on the association of IL-10, TGF-β levels and monocytes count. High combined score was associated with shorter OS and PFS in univariate and multivariate analysis. Chemotherapy induced an increase in monocytes, IL-10, but not TGF-β levels. Conclusion The prognostic value of circulating levels of multiple immunosuppressive cytokines and inflammatory cells should be confirmed in a wider validation set of MPM patients

Mitochondrial Alterations Induced by the p13II Protein of Human T-cell Leukemia Virus Type 1 CRITICAL ROLE OF ARGININE RESIDUES

Abstract Human T-cell leukemia virus type 1 encodes a number of "accessory" proteins of unclear function; one of these proteins, p13II, is targeted to mitochondria and disrupts mitochondrial morphology. The present study was undertaken to unravel the function of p13II 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 (p139–41), which include the amphipathic mitochondrial-targeting sequence of the protein. p139–41 folded into an α helix in micellar environments. Fractionation and immunogold labeling indicated that full-length p13II accumulates in the inner mitochondrial membrane. p139–41 induced energy-dependent swelling of isolated mitochondria by increasing inner membrane permeability to small cations (Na+, K+) and released Ca2+ from Ca2+-preloaded mitochondria. These effects as well as the ability of full-length p13II to alter mitochondrial morphology in cells required the presence of four arginines, forming the charged face of the targeting signal. The mitochondrial effects of p139–41 were insensitive to cyclosporin A, suggesting that full-length p13II 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

Repurposing Verapamil to Enhance Killing of T-ALL Cells by the mTOR Inhibitor Everolimus

New therapies are needed for patients with T-cell lymphoblastic leukemia (T-ALL) who do not respond to standard chemotherapy. Our previous studies showed that the mTORC1 inhibitor everolimus increases reactive oxygen species (ROS) levels, decreases the levels of NADPH and glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway (PPP), and induces apoptosis in T-ALL cells. Studies in T-ALL-xenografted NOD/SCID mice demonstrated that everolimus improved their response to the glucocorticoid (GC) dexamethasone. Here we show that verapamil, a calcium antagonist used in the treatment of supraventricular tachyarrhythmias, enhanced the effects of everolimus on ROS and cell death in T-ALL cell lines. The death-enhancing effect was synergistic and was confirmed in assays on a panel of therapy-resistant patient-derived xenografts (PDX) and primary samples from T-ALL patients. The verapamil-everolimus combination produced a dramatic reduction in the levels of G6PD and induction of p38 MAPK phosphorylation. Studies of NOD/SCID mice inoculated with refractory T-ALL PDX cells demonstrated that the addition of verapamil to everolimus plus dexamethasone significantly reduced tumor growth in vivo. Taken together, our results provide a rationale for repurposing verapamil in association with mTORC inhibitors and GC to treat refractory T-ALL