Feras M. Ghazawi

Valproic acid and butyrate induce apoptosis in human cancer cells through inhibition of gene expression of Akt/protein kinase B

BackgroundIn eukaryotic cells, the genomic DNA is packed with histones to form the nucleosome and chromatin structure. Reversible acetylation of the histone tails plays an important role in the control of specific gene expression. Mounting evidence has established that histone deacetylase inhibitors selectively induce cellular differentiation, growth arrest and apoptosis in variety of cancer cells, making them a promising class of anticancer drugs. However, the molecular mechanisms of the anti-cancer effects of these inhibitors have yet to be understood.ResultsHere, we report that a key determinant for the susceptibility of cancer cells to histone deacetylase inhibitors is their ability to maintain cellular Akt activity in response to the treatment. Also known as protein kinase B, Akt is an essential pro-survival factor in cell proliferation and is often deregulated during tumorigenesis. We show that histone deacetylase inhibitors, such as valproic acid and butyrate, impede Akt1 and Akt2 expression, which leads to Akt deactivation and apoptotic cell death. In addition, valproic acid and butyrate induce apoptosis through the caspase-dependent pathway. The activity of caspase-9 is robustly activated upon valproic acid or butyrate treatment. Constitutively active Akt is able to block the caspase activation and rescues cells from butyrate-induced apoptotic cell death.ConclusionOur study demonstrates that although the primary target of histone deacetylase inhibitors is transcription, it is the capacity of cells to maintain cellular survival networks that determines their fate of survival.

Interplay of bromodomain and histone acetylation in the regulation of p300-dependent genes

The bromodomain is an evolutionarily conserved motif found in many transcriptional activators including p300 which contains an intrinsic histone acetyltransferase activity and is a general coactivator for many transcription factors. One mode of bromodomain action is to serve as a binding module to recognize specific acetyl-lysine residue of histones during chromatin remodeling and transcriptional activation. The function of p300 is required for diverse sets of gene expression. However, it is not known whether the p300 bromodomain is involved in the expression of all or only subset of p300-dependent genes. In this study, we examined the impact of either wild type or a bromo-deficient p300 on the expression of several p300-dependant genes. The effects of histone acetylation on the expression of these genes were also assessed by targeting histone deacetylase activities with an inhibitor approach. We show that the impact of these inhibitors on the transcriptional activation of p300-dependent genes are impaired in cells containing the bromo-deficient p300, indicating that the interplay of p300 and histone acetylation in p300-dependent gene transcription requires the bromodomain. We also observed an increase in the expression of bromo-deficient p300 at the level of transcription possibly to compensate for the loss of p300 function. However, the high level of bromo-deficient p300 is not able to maintain the basal level of histone acetylation. Thus, the bromodomain is important for p300 to maintain the basal level of histone acetylation and to induce the transcriptional activation of p300-dependent genes. Nevertheless, the requirement of bromodomain and histone acetylation in p300-dependent gene transcription is determined by a gene specific manner.

IL-7 downregulates IL-7Rα expression in human CD8 T cells by two independent mechanisms.

Interleukin (IL)‐7 is an essential nonredundant cytokine, and throughout the lifespan of a T‐cell signaling via the IL‐7 receptor influences cell survival, proliferation and differentiation. It is therefore no surprise that expression of the IL‐7 receptor alpha‐chain (CD127) is tightly regulated. We have previously shown that IL‐7 downregulates expression of CD127 at the cell surface and now elucidate the kinetics of that suppression and demonstrate that IL‐7 downregulates CD127 transcripts and surface protein in primary human CD8 T cells by two separate pathways. We show that IL‐7 induces the initial reduction in cell‐surface CD127 protein independent of transcriptional suppression, which is delayed by 40–60 min. Although IL‐7‐mediated downregulation of CD127 transcripts is dependent on Janus kinase (JAK)/STAT5, the early downregulation of surface CD127 protein is independent of JAK activity. The data further illustrate that low levels of IL‐7 induce smaller and transient decreases in CD127 transcripts and surface protein, whereas higher concentrations induce more profound and sustained suppression. Such flexibility in receptor expression likely allows for fine‐tuned immune responses in human CD8 T cells in different microenvironments and in response to different immunological challenges.

Immune tolerance properties of the testicular tissue as a viral sanctuary site in ART-treated HIV-infected adults.

Objective:HIV persistence in long-lived infected cells and in anatomical sanctuary sites are major hurdles to HIV eradication. Testicular tissue may represent a significant viral sanctuary site as it constitutes an immunologically privileged compartment. We assessed immunotolerance properties of the testicular tissue in individuals receiving suppressive antiretroviral therapy (ART). Design and methods:Testicular tissue and matched blood samples were collected from six virally suppressed adults and 10 HIV-uninfected controls prior to sex reassignment surgery. T cells were purified from freshly isolated testicular interstitial cell suspensions. T-cell subsets, expression of immune activation markers and HIV DNA were assessed in matched testicular cells and peripheral blood mononuclear cells (PBMCs). Results:When compared with PBMCs, testes were characterized by a lower CD4+ T-cell proportion among total T cells, a decrease in the frequency of naive cells, an increase in the frequency of effector-memory T cells and an increase in CCR5 expression in both the HIV+ and HIV− groups. In HIV-infected individuals on ART, testes displayed higher T-cell immune activation (Coexpression of CD38 and Human Leukocyte Antigen - antigen D Related) than PBMCs. In both groups, testes were characterized by higher frequencies of immunosuppressive CD39+ regulatory T cells and a massive increase in CD73 expression on CD8+ T cells. In addition, a remarkable increase in indoleamine-pyrrole 2,3-dioxygenase immunosuppressive enzyme involved in tryptophan/kynurenine catabolism was observed in testes versus blood. Rare cells harboring HIV DNA were detected in testes from five out six participants. Conclusion:These findings suggest that the adenosine and tryptophan/kynurenine immune-metabolic pathways contribute to immune tolerance in testicular tissue. Our results suggest that testes may represent a distinctive HIV sanctuary site during ART.

Circulating endothelial progenitor cells in HIV infection: A systematic review

Human immunodeficiency virus (HIV)-infected individuals have a cardiovascular disease risk that is almost thrice than that of their HIV-uninfected counterparts. Given the critical role of endothelial progenitor cells (EPCs) in vascular homeostasis and arterial repair postinjury, coupled with their strength as biomarkers predictive of cardiovascular events, interest has arisen in characterizing EPCs in the context of HIV infection. We conducted a systematic review of the literature to determine the current state of knowledge on EPCs in the context of HIV infection. Herein, we summarize the pertinent findings of these studies and discuss important differences in the subpopulations of EPCs examined and the methodologies used for their enumeration which likely contributed to the heterogeneity observed across studies.

IL-7 induces clathrin-mediated endocytosis of CD127 and subsequent degradation by the proteasome in primary human CD8 T cells.

Interleukin‐7 (IL‐7), a key immunoregulatory cytokine, plays an essential role in peripheral T‐cell homeostasis and function. Signaling via the IL‐7 receptor is tightly regulated and we and others have shown IL‐7 provides negative feedback on its own signaling by downregulating expression of the IL‐7 receptor alpha‐chain (CD127) through both suppression of CD127 gene transcription and by internalization of existing CD127 proteins from the cell membrane. We show here for the first time in primary human CD8 T cells that upon stimulation with IL‐7, CD127 is internalized through clathrin‐coated pits, a process dependent on both lipid‐raft formation and the activity of dynamin. As visualized by confocal microscopy, CD127 shows increased co‐localization with clathrin within 5 min of IL‐7 stimulation and within 15–30 min is seen in multiple intracellular punctae co‐localizing with the early endosomal marker EEA1. By 2 h after addition of IL‐7, CD127 staining associates with the late endosomal marker RAB7 and with the proteasomal 20S subunit. By inducing receptor internalization and translocation from early endosomes to the proteasome, IL‐7 directly influences its receptor density on the cell surface and thus regulates the intensity of its own signaling cascades. Given the important role IL‐7 plays in T‐cell development, homeostasis and function, deciphering how expression of its receptor is controlled on the cell surface is essential in understanding how T‐cell activity can be regulated in different microenvironments and in response to different pathogens.

Suppressor of cytokine signaling (SOCS) proteins are induced by IL-7 and target surface CD127 protein for degradation in human CD8 T cells.

Given the essential role interleukin (IL)-7 plays in T-cell survival, homeostasis and function, it is no surprise expression of the IL-7 receptor alpha-chain (CD127) is tightly regulated. We have previously shown IL-7 binding to its receptor on the surface of CD8 T cells leads to both suppression of CD127 gene transcription and loss of existing CD127 protein from the cell membrane. Indeed upon binding IL-7, CD127 is rapidly internalized into early endosomes where phosphorylation by JAK targets the receptor for degradation. We now show that IL-7 induces the expression of suppressor of cytokine signaling (SOCS) proteins CIS, SOCS1 and SOCS2 through the JAK/STAT-5 pathway and that CIS and SOCS2 specifically interact with CD127 in early endosomes and direct the receptor complex to the proteasome for degradation. These results illustrate how expression of the IL-7 receptor and thus IL-7 signaling is modulated in human CD8 T cells by a negative feedback mechanism dependent on members of the SOCS family of proteins.

Distribution and Clustering of Cutaneous T-Cell Lymphoma (CTCL) Cases in Canada During 1992 to 2010

Background: Clustering of patients with cutaneous T-cell lymphoma (CTCL) was reported in several jurisdictions around the world. This rare cancer is known to affect spouses and in some cases multiple members of the same family. These combined results suggest the existence of external disease triggers/promoters. We recently conducted the first comprehensive analysis of CTCL incidence and mortality in Canada, which revealed case clustering in several regions. Objectives: To extend our previous analysis on CTCL incidence across Canada and to provide all the collected data on CTCL patient incidence in Canada during the period of 1992 to 2010. Methods: Clinical parameters for patients with CTCL in Canada were analyzed using 2 independent population-based cancer registries: Canadian Cancer Registry and Le Registre Québécois du Cancer. The CTCL incidence rates were examined on different geographical levels, including provinces/territories, cities, and forward sortation areas. Results: Our findings further corroborate our earlier observations of higher CTCL incidence in Newfoundland and Labrador, maritime provinces (Nova Scotia and New Brunswick), and prairie provinces (Manitoba and Saskatchewan). Also, most cities with high CTCL incidence were located in these provinces. Extensive mapping of high-incidence postal codes supports case clustering in a number of communities that are located in the proximity of industrial centres and seaports. Conclusions: Detailed analysis of CTCL incidence in Canada is critical to fully understand the burden of this disease in our country, to begin the search for a possible external trigger for this lymphoma, and to reform how health care resources are distributed throughout the country to better serve Canadian patients with CTCL.

Analysis of CTCL cell lines reveals important differences between mycosis fungoides/Sézary syndrome vs. HTLV-1 + leukemic cell lines

HTLV-1 is estimated to affect ~20 million people worldwide and in ~5% of carriers it produces Adult T-Cell Leukemia/Lymphoma (ATLL), which can often masquerade and present with classic erythematous pruritic patches and plaques that are typically seen in Mycosis Fungoides (MF) and Sézary Syndrome (SS), the most recognized variants of Cutaneous T-Cell Lymphomas (CTCL). For many years the role of HTLV-1 in the pathogenesis of MF/SS has been hotly debated. In this study we analyzed CTCL vs. HTLV-1+ leukemic cells. We performed G-banding/spectral karyotyping, extensive gene expression analysis, TP53 sequencing in the 11 patient-derived HTLV-1+ (MJ and Hut102) vs. HTLV-1- (Myla, Mac2a, PB2B, HH, H9, Hut78, SZ4, Sez4 and SeAx) CTCL cell lines. We further tested drug sensitivities to commonly used CTCL therapies and studied the ability of these cells to produce subcutaneous xenograft tumors in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Our work demonstrates that unlike classic advanced MF/SS cells that acquire many ongoing balanced and unbalanced chromosomal translocations, HTLV-1+ CTCL leukemia cells are diploid and exhibit only a minimal number of non-specific chromosomal alterations. Our results indicate that HTLV-1 virus is likely not involved in the pathogenesis of classic MF/SS since it drives a very different pathway of lymphomagenesis based on our findings in these cells. This study also provides for the first time a comprehensive characterization of the CTCL cells with respect to gene expression profiling, TP53 mutation status, ability to produce tumors in mice and response to commonly used therapies.

Protocol for adhesion and immunostaining of lymphocytes and other non-adherent cells in culture

Immunostaining of non-adherent cells is commonly performed after adhesion of cells onto microscope slides either using cytocentrifugation or with the help of charged coating substrates. These techniques, however, require either specialized equipment or significant preparation time. Here, we describe a method for immunofluorescent staining of lymphocytes within multi-well culture plates, where cells suspended in phosphate buffered saline (PBS) are adhered to either the plastic well bottom or glass coverslips by gravity sedimentation. This technique requires only common laboratory materials, no coating steps, and allows for densely adherent cell coverage with 1 × 106 cells. Our data show that suspension of cells in PBS, but not serum-containing growth medium, allows for adhesion to plastic or glass after 30 min of gravity sedimentation. We show that this method is applicable for immunofluorescent staining of both primary human lymphocytes and immortalized lymphoma cells, and that it preserves cell morphology.