The Prohibitin Protein Complex Promotes Mitochondrial Stabilization and Cell Survival In Hematologic Malignancies

Lymphocyte proliferation and differentiation is coordinated with high precision in healthy humans and is vital to maintaining a normal immune system. Imbalance of these events can result in the development of autoimmune diseases, immunodeficiencies and hematopoietic malignancies. These pathologies, specifically leukemia and lymphoma have a high incidence of relapse and mortality due to limited treatment options. Therefore, there is a critical need to characterize the signal transduction pathways and understand molecular hallmarks that mediate T cell activation in order to develop new strategies for diagnosis and treatments of these diseases. Prohibitins (PHB1 and PHB2) have been proposed to play important roles in cancer development and disease progression. In this study we used immunoprecipitation coupled to mass spectrometry to identify new forms of PHB regulation. We report four novel interleukin 2 (IL-2) inducible phosphosites in PHB2. To elucidate the potential regulatory role of these sites we generated phosphospecific polyclonal antibodies against one key phosphoresidue Threonine-62, to characterize its function. In addition, evidence is provided for PHB1 and PHB2 upregulation in tumor cell lines and localization mainly at the mitochondria. These proteins were also upregulated during reactive oxygen species (ROS)-mediated apoptosis. Similarity, PHB1 and PHB2 protein levels were significantly higher in tumor cells isolated from leukemia and lymphoma patients and determined to mainly localize to the mitochondria, possibly to maintain mitochondria integrity, which may facilitate the energy requirements of these tumor cells. Therefore, prohibitins may serve not only as biomarkers for cancer, but also act as molecular target for therapeutic intervention in hematopoietic malignancies

The Genomic Landscape of a Restricted ALL Cohort from Patients Residing on the U.S./Mexico Border

Next-generation sequencing (NGS) has identified unique biomarkers yielding new strategies in precision medicine for the treatment of Acute lymphoblastic leukemia (ALL). Hispanics show marked health disparities in ALL, often absent in clinical trials or cancer research. Thus, it is unknown whether Hispanics would benefit equally from curated data currently guiding precision oncology. Using whole-exome sequencing, nine ALL patients were screened for mutations within genes known to possess diagnostic, prognostic and therapeutic value. Genes mutated in Hispanic ALL patients from the borderland were mined for potentially pathogenic variants within clinically relevant genes. KRAS G12A was detected in this unique cohort and its frequency in Hispanics from the TARGET-ALL Phase II database was three-fold greater than that of non-Hispanics. STAT5B N642H was also detected with low frequency in Hispanic and non-Hispanic individuals within TARGET. Its detection within this small cohort may reflect a common event in this demographic. Such variants occurring in the MAPK and JAK/STAT pathways may be contributing to Hispanic health disparities in ALL. Notable variants in ROS1, WT1, and NOTCH2 were observed in the ALL borderland cohort, with NOTCH2 C19W occurring most frequently. Further investigations on the pathogenicity of these variants are needed to assess their relevance in ALL

PKC-Dependent GlyT1 Ubiquitination Occurs Independent of Phosphorylation: Inespecificity in Lysine Selection for Ubiquitination

<div><p>Neurotransmitter transporter ubiquitination is emerging as the main mechanism for endocytosis and sorting of cargo into lysosomes. In this study, we demonstrate PKC-dependent ubiquitination of three different isoforms of the glycine transporter 1 (GlyT1). Incubation of cells expressing transporter with the PKC activator phorbol ester induced a dramatic, time-dependent increase in GlyT1 ubiquitination, followed by accumulation of GlyT1 in EEA1 positive early endosomes. This occurred via a mechanism that was abolished by inhibition of PKC. GlyT1 endocytosis was confirmed in both retinal sections and primary cultures of mouse amacrine neurons. Replacement of only all lysines in the <i>N</i>-and <i>C</i>-termini to arginines prevented ubiquitination and endocytosis, displaying redundancy in the mechanism of ubiquitination. Interestingly, a 40–50% reduction in glycine uptake was detected in phorbol-ester stimulated cells expressing the WT-GlyT1, whereas no significant change was for the mutant protein, demonstrating that endocytosis participates in the reduction of uptake. Consistent with previous findings for the dopamine transporter DAT, ubiquitination of GlyT1 tails functions as sorting signal to deliver transporter into the lysosome and removal of ubiquitination sites dramatically attenuated the rate of GlyT1 degradation. Finally, we showed for the first time that PKC-dependent GlyT1 phosphorylation was not affected by removal of ubiquitination sites, suggesting separate PKC-dependent signaling events for these posttranslational modifications.</p></div

GlyT1 phosphorylation and glycine uptake.

<p><b>A)</b> PAE cells stably expressing FH-GlyT1 were labeled with 50 μCi ³²P-orthophosphate/ml followed by incubation with DMSO or 1 μM PMA for 0 to 120 min. Labeled GlyT1 was purified by tandem affinity chromatography and analyzed by autoradiography and Western blotting with GlyT1 antibodies. <b>B)</b> PAE cells expressing WT FH-GlyT1c, or the mutants NTK-1c, CTK-1c, and NTK-CTK-1c were labeled with 50 μCi ³²P-orthophosphate/ml followed by incubation with DMSO or 1 μM PMA for 60 min and treated as described in A. The autoradiography and GlyT1 blots were subjected to densitometry analysis and the resulting values are expressed as mean ± SEM, n = 3, <b>C)</b> For uptake experiments, cells were incubated with vehicle (DMSO) or 1μM PMA for 30 min followed by a 10 min incubation with 400 μM of [³H]-Gly at 37°C. Values are represented as % of control DMSO for each cell line, calculated from the following average specific activities in nmol/min/mg of protein: WT-1c, 41.3+/-3; NTK-1c,51.2+/-6; CTK-1c 39.4+/-3: NTK-CTK-1c, 56.8+/- 4;. Error bars represent the mean ± SE, <i>n</i> = 3, *<i>p</i> = <i>0</i>.<i>002</i>, **<i>p</i> <0.001. <b>D)</b> PAE cells expressing WT-DAT, and the mutant DAT were labeled with 50 μCi ³²P-orthophosphate/ml followed by incubation with DMSO or 1 μM PMA for 60 min. Total DAT was purified by tandem affinity chromatography and analyzed by autoradiography and Western blotting with DAT antibodies. Values are expressed as mean + SEM, n = 3. A value of <i>p</i><0.05 was obtained when each experimental sample was compared with untreated control cells <i>via</i> one-way analysis of variance (ANOVA) and Student’s <i>t</i>-test.</p

Time course of PKC-induced ubiquitination of GlyT1 isoforms.

<p><b>A)</b> PAE cells stably expressing FH-GlyT1a were incubated with PMA (1μM) for 0 to 120 min. After incubation, the proteins were solubilized and GlyT1 purified by tandem affinity chromatography using Ni-NTA-agarose and FLAG M2 gel. Purified GlyT1 transporter was subjected to SDS-PAGE and western blotting using ubiquitin and GlyT1 antibodies. <b>B)</b> FH-GlyT1b isoform. <b>C)</b> FH-GlyT1c isoform. Blots were subjected to densitometry analysis using image J software and the relative amount of ubiquitinated GlyT1 was normalized to the total GlyT1 transporter. The Y axes represent the relative amount of ubiquitinated GlyT1. Data are expressed as the mean ± SEM, n = 4. ** A value of <i>p</i><0.05 (*) was obtained when each experimental sample was compared with untreated control cells <i>via</i> one-way analysis of variance (ANOVA) and Student’s <i>t</i>-test. <i>GlyT1-Ub</i>, ubiquitinated glycine transporter; <i>FH-GlyT1</i>, Flag, His-tagged glycosylated glycine transporter; <i>ng-GlyT1</i>, non-glycosylated glycine transporter.</p

Localization of GlyT1 in mouse amacrine neurons.

<p><b>A</b>) Vertical sections from adult C57BL/6J mouse retinas were stained for GlyT1, DAPI and EEA1, and analyzed by confocal microscopy. DAPI staining depicts the nuclei in cell bodies of the retina layers. Outer Nuclear Layer, ONL; Outer Plexiform Layer, OPL; Inner Nuclear Layer, INL; Inner Plexiform Layer, INL and Ganglion cell layer, GCL. <b>B)</b> Retinas from neonatal mouse were isolated, the tissue digested with papain and the cells plated on poly-L lysine and laminin- coated glass coverslips. Primary cultures were incubated with DMSO or 1 μM PMA for 1 h followed by detection of glycinergic amacrine neurons by immunostaining with GlyT1 and co-localization with EEA1. Single 0.65 μm optical sections were acquired by confocal microscopy and analyzed with ZEN 2009 software, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138897#pone.0138897.g001" target="_blank">Fig 1D</a>. <i>Scale bars</i>, 10 μm. <b>C)</b> Co-localization was measured for 10 EEA1- and 10 EEA1/GlyT1-positive endosomes from retinal sections depicted in panel A. Data is represented as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138897#pone.0138897.g001" target="_blank">Fig 1D</a> and analyzed by Student’s <i>t</i>-test.</p

GB caused perturbation of the cell-cycle profile of BJAB cells.

<p>After 96 h GB induced apoptotic DNA fragmentation and G2/M phase arrest in a dose-dependent modality. Cells were harvested, permeabilized and stained and analyzed <i>via</i> flow cytometry. Each bar represents the average of three independent replicates, and the error bars represent the corresponding standard deviation. (A–D) The percentage of cell frequency is graphed along the <i>y</i>-axis, and the different treatments are plotted along the <i>x</i>-axis. (E–I) Representative single parameter histograms where four gates are annotated exhibiting the percentage of cell frequency in each phase of the cell cycle. Gates from left to right: sub-G0/G1 (hypodiploid; counted as an apoptotic subpopulation), G0/G1 (diploid), S (hyperdiploid) and G2/M (tetraploid). This series of experiments included several controls: two compounds provoking cell-cycle alteration, (G) 80 nM etoposide (ETO) and (H) 1 mg/ml G418; (F) the GB diluent, PBS, as contained in the experimental samples; and (I) untreated (Unt) cells, as a negative control. The significance of the differences between 50 µl GB-treated cells as compared to 50 µl PBS-treated cells, and also, with untreated cells, is of <i>P</i> < 0.03 (*) and <i>P</i> < 0.04 (‡), respectively. GB 10 µl = 0.3 ± 0.009 mg/ml, and GB 50 µl = 1.5 ± 0.048 mg/ml lyophilized powder.</p

Ubiquitination and endocytosis of multi-lysine GlyT1c mutants.

<p><b>A)</b> PAE cells expressing WT FH-GlyT1c, NTK-1c, CTK-1c, and NTK-CTK-1c were incubated with DMSO or PMA (1μM) for 30 min. After incubation, the proteins were solubilized and GlyT1 purified by tandem affinity chromatography using Ni-NTA-agarose and FLAG M2 gel. Purified transporter was subjected to SDS-PAGE and western blot with ubiquitin and GlyT1 antibodies. Densitometry analysis was performed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138897#pone.0138897.g003" target="_blank">Fig 3</a> and expressed as a mean ± SEM, n = 4. <b>B)</b> PAE cells expressing WT FH-GlyT1c, NTK-1c, CTK-1c, and NTK-CTK-1c were incubated with DMSO or PMA (1 μM) for 30 min. at 37°C, fixed and immunostained with anti-GlyT1 and anti-EAA1 antibodies followed by incubation with a CY-3 and Alexa 488 labeled secondary antibodies, respectively. A z-stack of optical sections was acquired through YFP (green) and CY3 (red) filter channels. Single optical sections through the middle of the cells are shown. ‘Yellow’ in the merged images signifies co-localization of CY3 (GlyT1) and YFP (EEA1). Images were selected to represent the cell population. <i>Scale bars</i>, 10 μm. C) Manders’ overlap coefficient of merged images captured from doubly-labeled PAE cells with anti-GlyT1 (red) and anti-EEA1 (green) antibodies; a value of 1 represents 100% of both fluorescence signals co-localized in all the pixels involved in the regions of interest (ROIs). Values are presented for 15 randomly selected endosomes in different cells from wild-type and mutants. Statistical analysis was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138897#pone.0138897.g001" target="_blank">Fig 1</a>.</p

Searching in Mother Nature for Anti-Cancer Activity: Anti-Proliferative and Pro-Apoptotic Effect Elicited by Green Barley on Leukemia/Lymphoma Cells

<div><p>Green barley extract (GB) was investigated for possible anti-cancer activity by examining its anti-proliferative and pro-apoptotic properties on human leukemia/lymphoma cell lines. Our results indicate that GB exhibits selective anti-proliferative activity on a panel of leukemia/lymphoma cells in comparison to non-cancerous cells. Specifically, GB disrupted the cell-cycle progression within BJAB cells, as manifested by G2/M phase arrest and DNA fragmentation, and induced apoptosis, as evidenced by phosphatidylserine (PS) translocation to the outer cytoplasmic membrane in two B-lineage leukemia/lymphoma cell lines. The pro-apoptotic effect of GB was found to be independent of mitochondrial depolarization, thus implicating extrinsic cell death pathways to exert its cytotoxicity. Indeed, GB elicited an increase of TNF-α production, caspase-8 and caspase-3 activation, and PARP-1 cleavage within pre-B acute lymphoblastic leukemia Nalm-6 cells. Moreover, caspase-8 and caspase-3 activation and PARP-1 cleavage were strongly inhibited/blocked by the addition of the specific caspase inhibitors Z-VAD-FMK and Ac-DEVD-CHO. Furthermore, intracellular signaling analyses determined that GB treatment enhanced constitutive activation of Lck and Src tyrosine kinases in Nalm-6 cells. Taken together, these findings indicate that GB induced preferential anti-proliferative and pro-apoptotic signals within B-lineage leukemia/lymphoma cells, as determined by the following biochemical hallmarks of apoptosis: PS externalization, enhanced release of TNF-α, caspase-8 and caspase-3 activation, PARP-1 cleavage and DNA fragmentation Our observations reveal that GB has potential as an anti-leukemia/lymphoma agent alone or in combination with standard cancer therapies and thus warrants further evaluation <i>in vivo</i> to support these findings.</p> </div