Methotrexate induces CerS6-dependent formation of ER membrane aggregates in A549 non-small cell lung carcinoma cells.

<p>(A) Treatment with MTX (10 nM) resulted in the increase of autophagosome number, but CerS6 was not associated with these organelles. (B) MTX (10 nM) induced aggregation of ER membranes and the accumulation of CerS6 in the aggregates. Arrows indicate ER membrane aggregates. Note the lack of such aggregates after lometrexol (1.0 μM) treatment. (C) MTX (10 nM) induced ER aggregation (detected by monitoring CALR-RFP) in the absence of exogenous CerS6. (D) A549 cells lacking CerS6 (siRNA silencing) did not form ER aggregates in response to MTX. Cells were co-transfected with GFP-CerS6 and LC3-RFP (panel A) or CALR-RFP (panel B), MTX was added 6 h after transfection and live cell images were captured 48 h later. In experiments with endogenous CerS6, cells were co-transfected with GFP and CALR-RFP. Six hours before co-transfection, cells were transfected with scrambled siRNA (<i>panel C</i>) or CerS6 siRNA (<i>panel D</i>). MTX was added 6 h after the second transfection and images were captured 48 h later. Pearson’s coefficient for co-localization (calculated using Fiji software) is indicated for each panel.</p

Proposed two-step mechanism for CerS6-dependent MTX cytotoxicity.

<p>Step 1: MTX induces p53, which functions as the transcriptional activator of CerS6 and increases levels of the protein in ER. Step 2: MTX induces ER stress leading to aggregation of CerS6-containing ER membranes. Enhanced generation of C₁₆-ceramide by CerS6 further promotes cytotoxic effect.</p

Substance abuse as compensation for stress factors involved in the performance of helping professions

THE ABSTRACT It has been shown recently that workload, stress, and burnout syndrome among the staff of the medical rescue service may be major risk factors in terms of triggering the use of psychoactive substances. Representing what is understandably a delicate issue, substance use among emergency medical staff has not been thoroughly studied in our country. Emergency medical workers' difficult working conditions and the chronic stress they are exposed to, in combination with a lack of support and care on the part of their employers, result in exhaustion and general distress, accompanied by the development of symptoms associated with both physical and mental disorders. This condition may lead to the use of psychoactive substances as a negative coping strategy. Consisting of both theoretical background and case studies, the paper points out the relationship between the chronic effect of stressors pertaining to the job of emergency medical workers and the use of psychoactive substances as a way of coping with and compensating for the implications of work-related stress and fatigue. Thorough case studies are presented to demonstrate the onset and development of addictive behaviour within a wider context, with special emphasis being placed on its association with coping with both acute and chronic occupational..

CerS6 is a target of MTX in mediation of antiproliferative effect.

<p>(A) Silencing CerS6 by siRNA rescues A549 cells from cytotoxic effect of MTX while silencing CerS4 does not change the drug toxicity. Cells were transfected with scrambled siRNA, CerS6 siRNA or CerS4 siRNA (25 pmol), MTX was added 6 h later and live cells were assessed by MTT assay 48 h after the beginning of MTX treatment. Data represent an average of two independent experiments, each performed in quadruplicates with error bars representing SD. Student’s <i>t</i> test was performed for statistical analysis (difference in cell number between control and CerS6-silenced cells were statistically significant with <i>p<</i>0.005; there were no significant differences in cell number between control and CerS4-silenced cells). Panels on the right show the efficiency of CerS6 and CerS4 silencing (evaluated at 48 h of MTX treatment). (B) Levels of ceramide in control A549 cells (<i>Cntr</i>, transfected with scrambled siRNA), MTX-treated cells transfected with scrambled siRNA (<i>MTX</i>) and MTX-treated cells after CerS6 silencing (<i>CerS6 siRNA/MTX</i>, transfected with CerS6 siRNA). Differences between the groups were analyzed by one-way ANOVA and asterisks indicate statistically significant changes (p<0.05). (C) Silencing of CerS6 by siRNA protects cancer cells from MTX cytotoxic effect. Student’s <i>t</i>-test was performed and the differences between MTX effect in control and CerS6-silenced cells were statistically significant (p<0.01). (D) MTX (10 nM) leads to elevation of CerS6 but not of CerS4 in A549 cells.</p

Effects of MTX on CerS6, ceramide levels and cellular proliferation.

<p>(A) Levels of CerS6 (Western blot) in cancer cells with and without MTX treatment. Cells were collected 48 h after MTX was added. (B) Changes in CerS6 levels after treatment with 10 nM MTX for 48 h (calculated from A). The intensity of bands was quantified using ImageJ software. In all cases, normalization for levels of actin was performed. (C) Changes in levels of selected ceramides in A549 cells after treatment with 10 nM MTX; P_i, total phospholipid inorganic phosphate present in cell extracts. Student’s <i>t</i>-test was performed for statistical analysis of the changes in ceramide levels and asterisks indicate statistically significant differences (p<0.05). (D) Antiproliferative effect of MTX (10 and 100 nM) in a panel of cancer cell lines. Live cells were assessed by MTT assay. Error bars show SD of four measurements. Differences between control and drug treated groups were statistically significant (calculated by one-way ANOVA) with <i>p</i> value below 0.0007 in all cases with the exception of A549 p53^-/- cells (p = 0.0357). (E) Scatter plot of CerS6 protein levels (from panel B) <i>versus</i> cell survival after MTX treatment (from panel D) for ten cell lines indicated in the above panels. Pearson’s coefficient (<i>r</i> = −0.83; n = 10; <i>p</i><0.01) indicates strong negative correlation between changes in CerS6 levels and cell survival upon MTX treatment.</p

Targeting of CerS6 by MTX is p53-dependent.

<p>(A) MTX (10 nM) treatment strongly elevates CerS6 in p53^+/+ but not p53^-/- cells. (B) Antifolate lometrexol (LTX) bypasses p53 and does not affect CerS6: <i>left panel</i>, LTX inhibits both p53^+/+ and p53^-/- A549 cells (Experiments were performed three times with six wells per concentration point in each experiment; error bars, SD) no statistically significant differences between p53^+/+ and p53^-/- cells were observed (p>0.1); <i>right panel</i>, levels of p53 and CerS6 (Western blot) in the cells after LTX treatment. Cells were exposed to LTX for 48 h.</p

CHIP E3 ligase mediates proteasomal degradation of the proliferation regulatory protein ALDH1L1 during the transition of NIH3T3 fibroblasts from G₀/G₁ to S-phase

<div><p>ALDH1L1 is a folate-metabolizing enzyme abundant in liver and several other tissues. In human cancers and cell lines derived from malignant tumors, the <i>ALDH1L1</i> gene is commonly silenced through the promoter methylation. It was suggested that ALDH1L1 limits proliferation capacity of the cell and thus functions as putative tumor suppressor. In contrast to cancer cells, mouse cell lines NIH3T3 and AML12 do express the ALDH1L1 protein. In the present study, we show that the levels of ALDH1L1 in these cell lines fluctuate throughout the cell cycle. During S-phase, ALDH1L1 is markedly down regulated at the protein level. As the cell cultures become confluent and cells experience increased contact inhibition, ALDH1L1 accumulates in the cells. In agreement with this finding, NIH3T3 cells arrested in G₁/S-phase by a thymidine block completely lose the ALDH1L1 protein. Treatment with the proteasome inhibitor MG-132 prevents such loss in proliferating NIH3T3 cells, suggesting the proteasomal degradation of the ALDH1L1 protein. The co-localization of ALDH1L1 with proteasomes, demonstrated by confocal microscopy, supports this mechanism. We further show that ALDH1L1 interacts with the chaperone-dependent E3 ligase CHIP, which plays a key role in the ALDH1L1 ubiquitination and degradation. In NIH3T3 cells, silencing of CHIP by siRNA halts, while transient expression of CHIP promotes, the ALDH1L1 loss. The downregulation of ALDH1L1 is associated with the accumulation of the ALDH1L1 substrate 10-formyltetrahydrofolate, which is required for <i>de novo</i> purine biosynthesis, a key pathway activated in S-phase. Overall, our data indicate that CHIP-mediated proteasomal degradation of ALDH1L1 facilitates cellular proliferation.</p></div

Levels of ALDH1L1 protein in NIH3T3 cells arrested at difference phases.

<p><b>A</b>, NIH3T3 cells arrested in G₀/G₁ (serum starvation), S-phase (double thymidine block) or G₂/M (double thymidine block and nocodazole treatment) phase. Asynchronous cells shown as a control. Numbers on the panels indicate distribution of cells between cell cycle phases. Fitted peaks are: <i>Blue</i>, calculated G₀/G₁ phase; <i>yellow</i>, S phase; <i>green</i>, G₂/M phase. Cell cycle data were analyzed using FlowJo software. <b>B</b>, Western blot assay of ALDH1L1 in NIH3T3 cells arrested in indicated phase (20 μg of total cell lysate was loaded in each lane). Actin is shown as loading control. Arrows indicate molecular weight standards (St). Numbers show ALDH1L1 band intensity (arbitrary densitometry units) normalized to actin. Experiments were performed three times.</p

Omitting components of ubiquitination machinery prevents <i>in vitro</i> ALDH1L1 ubiquitination.

<p>Purified ALDH1L1 (1.1 μg) was incubated for 1 h with the <i>in vitro</i> ubiquitination kit that included all components or with omission of ATP, E1, E2 or E3 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199699#sec002" target="_blank">Materials and methods</a>). Reaction products were resolved by 7.5% SDS-PAGE followed by Western blot assay with ALDH1L1-specific antibody and Ubiquitin (Ub)-specific antibody. <i>St</i>, pre-stained molecular mass protein standards (numbers on the right indicates standard molecular masses, kDa). Experiment was performed three times with the same outcome.</p

ALDH1L1 is ubiquitinated in NIH3T3 cells.

<p><b>A</b>, ALDH1L1 pulled-down from NIH3T3 cell lysates using ALDH1L1-specific antibody and protein A beads; elution with glycine buffer (<i>lane 1</i>), followed by elution with SDS-PAGE loading buffer (<i>lane 2</i>). Proteins were resolved on a 7.5% SDS-PAGE gel followed by Western blot assay with ubiquitin-specific antibody (<i>left panel</i>) or ALDH1L1-specific antibody (<i>right panel</i>). Lane <i>St</i> is purified recombinant ALDH1L1. <b>B</b>, ALDH1L1 was immunoprecipitated from NIH3T3 cell lysates using an ALDH1L1-specific antibody and Protein A Magnetic beads; samples were resolved on a 7.5% SDS-PAGE followed by Western blot assay with anti-ubiquitin monoclonal antibody. Cells were harvested at different time points after splitting (as indicated); lysates were treated with deubiquitinase inhibitor (4.0 μM recombinant human ubiquitin aldehyde C-terminal derivative) prior to immunoprecipitation. After immunoprecipitation, eluates were treated with deubiquitinase (200 nM of recombinant human USP2 catalytic domain); <i>control</i>, untreated lysates. <b>C</b>, ALDH1L1 was immunoprecipitated from NIH3T3 cells as in <b>B</b> and treated with USP7. Cells were treated with 10 μM MG-132 for 4 h before the pull-down. After treatment with USP7, we have repeated the pull-down with ALDH1L1-specific antibody and detected ubiquitinated species as in <b>B</b>.</p