This analysis revealed a heterogeneous Ca2+ response of the cell population in a stimulus-dependent manner. Further analysis of T cell receptor (TCR)/CD3 deficient or overexpressing cells showed that CD59-mediated signaling is strongly dependent on TCR/CD3 surface expression. In protein co-patterning and fluorescence recovery after photobleaching experiments no direct physical interaction was observed between CD59 and CD3 at the plasma membrane upon anti-CD59 stimulation. However, siRNA-mediated protein knock-downs of downstream signaling molecules revealed that the Src family kinase Lck and the adaptor molecule linker of activated T cells (LAT) are essential for both signaling pathways. Furthermore, flow cytometry measurements showed that knock-down of Lck accelerates CD3 re-expression at the cell surface after anti-CD59 stimulation similar to what has been observed upon direct TCR/CD3 stimulation. Finally, physically linking Lck to CD3ζ completely abolished CD59-triggered Ca2+ signaling, while signaling was still functional upon direct TCR/CD3 stimulation. Altogether, we demonstrate that Lck mediates signal transmission from CD59 to the TCR/CD3 pathway in Jurkat T cells, and propose that CD59 may act via Lck to modulate T cell responses.The glycosylphosphatidylinositol (GPI)-anchored molecule CD59 has been implicated in the modulation of T cell responses, but the underlying molecular mechanism of CD59 influencing T cell signaling remained unclear. Here we analyzed Jurkat T cells stimulated via anti-CD3ε- or anti-CD59-coated surfaces, using time-resolved single-cell Ca The glycosylphosphatidylinositol (GPI)-anchored molecule CD59 is expressed in almost all cell membranes. It is known to inhibit the complement system by binding to the C8/9 components of the membrane attack complex, thereby preventing its assembly and formation of the lytic pore 2+]i is one of the earliest events upon T cell activation and different types of Ca2+ responses are critical for the differential activation of transcription factors driving T cell proliferation and effector functions 2+ measurements of Jurkat T cells as a read-out for signaling elicited upon Ab-mediated cross-linking of CD59 vs. TCR/CD3 stimulation. It is shown with a series of mutants and siRNA-mediated protein knock-downs that Lck is a key component in coupling CD59-mediated signaling to the TCR/CD3-mediated signaling pathway in Jurkat T cells.Although accumulating data suggest a physiological role for CD59 in T cell activation, the mechanism how signaling via CD59 is transduced through the membrane to modulate the antigen-specific T cell response remains to be explored. In this study we addressed whether and how CD59-mediated signaling is coupled to the TCR/CD3-mediated signaling cascade, using Jurkat cells as a model system. A rise in i in Jurkat cells 2+]i is an early read-out for T cell activation, where type and extent of the Ca2+ response are indicative of stimulation 2+ measurements of Jurkat cells. Cells were stimulated via Ab-coated glass surfaces, an established approach for studying T cell activation 2+]i during stimulation were recorded for 200 s after initial contact of the individual cell with the stimulatory surface, imaging the ratiometric fluorescent Ca2+ indicator Indo-1/AM 2+]i were monitored over time at the single-cell level. For a simplified and unsupervised analysis we implemented cluster analysis of individual Ca2+ time traces. The cluster algorithm is based on affinity propagation 2+ time traces .It has been previously reported that Ab-mediated cross-linking of CD59 results in elevation of [Ca2+]i followed by a sustained elevation of [Ca2+]i, whereas there was a less pronounced increase resulting in lower levels of total [Ca2+]i upon anti-CD59 stimulation  , the observed difference in cluster distribution for anti-CD3 and anti-CD59 stimulation was not due to unsaturated stimulation conditions. Note that using an alternative mAb to CD59 (clone H19) yielded similar results (data not shown). Besides a possible dependence of cluster distribution on stimulus strength, the signaling capacity of a single cell may also be determined by the amount of the targeted surface molecules. Measuring CD59 surface expression by flow cytometry was found homogeneous throughout the Jurkat population. By contrast, CD3 surface expression was heterogeneous in the Jurkat population, uncovering a significant proportion of cells displaying undetectable CD3 surface expression levels . This finding raised the possibility that the heterogeneity in CD59-mediated Ca2+ signaling may be linked to the heterogeneity in CD3 surface expression levels in Jurkat cells.Since all experiments were performed at maximum signaling capacity for the stimuli . The Ca2+ signaling capacity in TCRhigh cells compared to WT Jurkat cells was measured via single-cell Ca2+ measurements for both types of stimulation . Upon anti-CD3 stimulation, there was no change in the number of cells showing Ca2+ release patterns in TCRhigh cells compared to WT cells (92.9±3.9% vs. 91.4±2.1%) .To test the idea of CD59-mediated signaling being dependent on CD3 expression levels, we examined Caterparts D.2+ signaling upon anti-CD59 stimulation, we examined if Ca2+ signaling upon anti-CD59 stimulation could be elicited in a Jurkat mutant lacking the TCR/CD3 complex on the cell surface (TCR-) (for TCR/CD3 surface expression levels see B). Ca2+ signaling was essentially abolished in TCR- cells upon anti-CD59 stimulation (1.6±0.4% Ca2+ release patterns), as well as for anti-CD3 stimulation used as a control (1.9±0.7% Ca2+ release patterns)  and CD59 expression levels were similar in WT and TCR- cells (D). Moreover, when reconstituting ITAM domains in TCR- cells via introducing a construct where the cytoplasmic tail of CD3ζ is fused to CD8 (CD8-ζ), Ca2+ signaling upon anti-CD59 stimulation could be partially restored. The number of cells showing Ca2+ release patterns upon anti-CD59 stimulation raised from 1.6±0.4% in TCR- cells to 13.4±1.6% in CD8-ζ cells, thereby reconstituting 53% of the Ca2+ release patterns observed in WT cells (25.2±6.8%). Upon anti-CD3 stimulation Ca2+ signaling in CD8-ζ cells was scarcely restored (7.4±2.3% Ca2+ release patterns) compared to WT cells (90.1±1.4% Ca2+ release patterns) (E). Altogether, these results demonstrated a critical role of TCR/CD3 for both stimuli. Furthermore, we could show that CD3ζ even without the ligand-binding components of the TCR/CD3 complex is utilized for CD59-mediated signaling pointing to CD59-mediated signaling being linked to the TCR/CD3-mediated route.Since increased CD3 expression levels gave rise to enhanced CaR- cells D. Moreoatterns) E. Altog2+ signaling and served as a negative control. These data revealed no apparent interaction between CD3 and CD59 upon anti-CD59 stimulation on a minute timescale.To unravel the mechanism how the CD59-mediated signaling pathway integrates into the TCR/CD3-mediated signaling pathway, we tested if CD59 and CD3 are physically associated at the plasma membrane upon anti-CD59 stimulation. For this, a live cell protein-protein interaction assay was used that is based on co-patterning of a fluorescent target molecule in the cell with a membrane protein immobilized via capture Abs micropatterned on surfaces. In case of protein-protein interaction, the fluorescent target molecule follows the imposed micropattern in the membrane Further focusing on the plasma membrane, a possible, more dynamic interaction between CD3 and CD59 upon anti-CD59 stimulation was tested by FRAP measurements with CD3ζ-EYFP expressing Jurkat cells. Cells were exposed to surfaces uniformly coated with anti-CD3ε, anti-CD59 or anti-CD71 mAbs. After bleaching EYFP-fluorescence in a defined region of the plasma membrane, recovery of fluorescence intensity was followed for 2 min B. As exSince we could not identify a direct physical interaction between CD3 and CD59 at the plasma membrane, we tested downstream molecules involved in TCR/CD3-mediated signaling as candidates for coupling the two pathways. Molecules tested were the Src family kinases, Lck and Fyn, as the ITAM domains residing on CD3ζ act as a major target for these molecules 2+ technique to siRNA-mediated Lck knock-down cells (siLck), there was no significant difference in the number of cells showing Ca2+ release patterns upon anti-CD3 stimulation compared to cells transfected with negative control siRNA (siNeg) (76.5±10.4% vs. 89.4±10.1%). By contrast, the number of cells showing Ca2+ release patterns did significantly decrease from 36.9±13.2% to 12.7±6.1% when comparing siNeg to siLck cells upon anti-CD59 stimulation   . This cell line exhibited significantly less Ca2+ release patterns compared to siNeg cells upon anti-CD3 stimulation  and showed hardly any Ca2+ signaling upon anti-CD59 stimulation (1.2±1.2% Ca2+ release patterns) (B).When applying our single-cell Caling see C. Thus,atterns) B.C) did not significantly affect Ca2+ signaling upon anti-CD3 stimulation . Furthermore, upon anti-CD59 stimulation the number of cells showing Ca2+ release patterns was comparable, with 36.9±13.2% in siNeg cells and 37.6±14.8% in siFyn cells . By contrast, siRNA-mediated knock-down of LAT (siLAT)  (B). Altogether, these data demonstrated that Fyn is dispensable for anti-CD59 stimulation, whereas loss of Lck or LAT greatly impairs CD59-mediated signaling.siRNA-mediated knock-down of Fyn (siFyn) (for efficiency of knock-down see ctively) B. Altog2+ signaling upon anti-CD59 stimulation    cells were transfected with the CD3ζ-Lck fusion construct, which reconstituted Lck expression to the WT level A. TaggiAccumulating evidence suggests that CD59 is involved in modulating the T cell response. It has been shown that stimulation of Jurkat cells through CD59 leads to protein phosphorylation, proliferation and cytokine release 2+ responses have been observed in T cell populations 2+ response could be related to the extent of T cell activation 2+ responses. Although heterogeneity was detected also in CD3 surface expression levels, it did not affect the threshold for triggering TCR/CD3-mediated Ca2+ signaling. This may be attributed to a reported generally increased signaling capacity of the Jurkat cell line 2+ signaling upon anti-CD59 stimulation, and pointed to CD3ζ being required for CD59-mediated signaling. These findings suggested that the CD59-mediated signaling pathway is coupled into the TCR/CD3 signaling machinery at a very proximal step, utilizing the ITAM domains of the TCR/CD3 complex.Various CaCD59 is considered to be physically separated from the TCR/CD3 complex in the plasma membrane Nevertheless, by testing downstream components with siRNA-mediated protein knock-downs we revealed that the Src family tyrosine kinase Lck and the adaptor molecule LAT are essential for CD59-mediated signaling, as also reported for TCR/CD3 signaling Localization of CD59 to specialized membrane platforms, which are enriched in signaling molecules like Lck, appears to be a prerequisite for the observed signaling capacity of CD59 Uncovering the molecular mechanisms by which accessory molecules expressed on the T cell surface may influence T cell activation, is key for understanding the complex and heterogeneous nature of T cell responses. Here we revealed a link between CD59 and CD3-mediated signaling in Jurkat T cells, where Lck is a key factor of signal transmission from CD59 to the TCR/CD3 complex. It is proposed that CD59 may act via Lck to modulate T cell signaling.Figure S12+ imaging in living Jurkat cells.Ca (A) Schematic drawing of Jurkat cell stimulation. Indo-1/AM loaded cells were flushed onto Ab-coated glass slides and time-lapse imaging was performed for 8 minutes at a frame rate of 30 frames/min. (B) Indo-1/AM was excited at 333 nm and fluorescence emission was measured at 405 nm for the Ca2+-bound and 485 nm for the Ca2+-free form of Indo-1/AM, respectively. The ratio of fluorescence emission intensities at 405 nm and 485 nm reflects changes in [Ca2+]i for each individual cell 2+ time traces of ∼50 cells were recorded simultaneously. For analysis, Ca2+ time traces were followed for 200 s after identification of initial surface contact for each individual cell. Fluorescence and false-color emission ratio images of a representative experiment and a representative Ca2+ time trace are shown.(TIF)Click here for additional data file.Figure S22+ signaling.Stimulatory capacity of antibodies for TCR/CD3- and CD59-mediated Ca (A) Glass slides were coated with increasing concentrations of FITC-conjugated anti-CD3 or anti-CD59 mAb. The amount of surface-bound Ab was measured by fluorescence microscopy. Fluorescence intensities  are shown. (B) Cluster distribution of Ca2+ time traces in WT cells upon stimulation with varying anti-CD3 or anti-CD59 concentrations on the glass substrate. Each color represents the percentage of a certain Ca2+ time trace cluster in the cell population. Clusters representing Ca2+ release patterns are framed in black. Mean results of three technical replicates are shown (n ≥ 86 per stimulatory condition).(TIF)Click here for additional data file.Figure S3-, and TCRhigh cells.Characterization of WT, TCR (A) Individual Ca2+ time traces from single-cell measurements were grouped into 12 clusters by affinity propagation clustering as described in Materials and Methods. Each plot shows the respective Ca2+ time traces for a cluster, an exemplar trace for each cluster is shown in black. Clusters representing Ca2+ release patterns are framed in black. (B) CD3ζ surface expression level in WT, TCR-, and TCRhigh cells tested by flow cytometry. Cells were surface stained with FITC-conjugated anti-CD3ε. Live cells were gated based on the Forward Scatter and Side Scatter profiles and propidium iodide exclusion. Fluorescence values displayed are isotype control corrected . Multiple comparison tests were assessed by one-way ANOVA, significances are shown where applicable, *** p < 0.001. (C) Total CD3ζ levels in WT and TCRhigh cells tested by Western blotting. Cell lysates were probed for CD3ζ expression and the same blot was reprobed using Lck as a loading control. The 43 kDa band represents CD3ζ-EYFP, the 16 kDa represents the endogenous CD3ζ. (D) Total Lck and CD59 levels in WT, TCRhigh, and TCR- cells tested by Western blotting. Cell lysates were probed for CD59 and Lck expression and for β-actin as a loading control. (E) Transfection efficiency of CD8-ζ in TCR- cells tested by flow cytometry. Cells were transiently transfected with control vector (ctrl) or CD8-ζ expression vector, followed by surface staining with FITC-conjugated anti-CD8a. Live cells were gated based on the Forward Scatter and Side Scatter profiles and propidium iodide exclusion. Fluorescence values displayed are isotype control corrected . Multiple comparison tests were assessed by one-way ANOVA, significances are shown where applicable, *** p < 0.001.(TIF)Click here for additional data file.Figure S42+ signaling.Fyn is not essential for TCR/CD3- and CD59-mediated Ca (A) Individual Ca2+ time traces from single-cell measurements were grouped into 11 clusters by affinity propagation clustering as described in Materials and Methods. Each plot shows the respective Ca2+ time traces for a cluster, an exemplar trace for each cluster is shown in black. Clusters representing Ca2+ release patterns are framed in black. (B) Total CD3ε and CD59 levels in WT, J.CaM1.6, and J.CaM2.5 cells tested by Western blotting. Cell lysates were probed for CD59 and Lck expression and the same blot was reprobed using GAPDH as a loading control. (C) Knock-down efficiency of Fyn was tested by Western blotting. 48 h after transfection, cell lysates from cells treated with Fyn-specific siRNA were probed with anti-Fyn and anti-β-actin as a control. (D) Cluster distribution of Ca2+ time traces in Jurkat cells transfected with negative control siRNA (siNeg) or Fyn-specific (siFyn) upon anti-CD3 or anti-CD59 stimulation. Each color represents the percentage of a certain Ca2+ time trace cluster in the cell population. Clusters representing Ca2+ release patterns are framed in black . Mean values from five independent experiments, each with three technical replicates, are shown (n ≥ 288 per cell type and condition). Multiple comparison tests were assessed by one-way ANOVA.(TIF)Click here for additional data file.Figure S52+ signaling.Reconstitution of Lck by forced interaction of CD3ζ and Lck facilitates TCR/CD3- but not CD59-mediated Ca Individual Ca2+ time traces from single-cell measurements were grouped into 10 clusters by affinity propagation clustering as described in Materials and Methods. Each plot shows the respective Ca2+ time traces for a cluster, an exemplar trace for each cluster is shown in black. Clusters representing Ca2+ release patterns are framed in black.(TIF)Click here for additional data file.


Four and a half LIM domain protein 3 (FHL3) is a member of the FHL protein family that plays roles in the regulation of cell survival, cell adhesion and signal transduction. However, the mechanism of action for FHL3 is not yet clear. The aim of present study was to identify novel binding partner of FHL3 and to explore the underlying mechanism. With the use of yeast two-hybrid screening system, FHL3 was used as the bait to screen human fetal hepatic cDNA library for interacting proteins. Methionine-1X was identified as a novel FHL3 binding partner. The interaction between FHL3 and the full length MT-1X was further confirmed by yeast two-hybrid assay, co-immunoprecipitation and GST pull-down assays. Furthermore,the result demonstrated that MT-1X knockdown promoted the FHL3-induced inhibitory effect on HepG2 cells by regulating FHL3-mediated Smad signaling and involving in the modulation the expression of G2/M phase-related proteins through interaction with FHL3. These findings suggest that functional interactions between FHL3 and MT-1X may provide some clues to the mechanisms of FHL3-regulated cell proliferation. FHL3 is a member of the four and half LIM domain (FHL) family, which consists of FHL1, FHL2, FHL3, FHL4, and FHL5/ACT. By Northern analysis, FHL1, FHL2, FHL3, and FHL5 were found to be expressed at high levels in a variety of tumor cell lines, including squamous cell carcinoma, melanoma, and leukemia cell lines in vitro and in vivoFHL3 was initially identified in skeletal muscle and reported to regulate transcription factors including CREB,MZF-1 and MyoD by acting as a transcriptional co-activator or co-repressor in vivo and in vitro. Moreover, MT-1X knockdown promoted the FHL3-induced inhibitory effect on HepG2 cells by regulating FHL3-mediated Smad signaling.More and more functions of FHL3 have been found, but the underlying mechanisms in different carcinogenesis are not quite clear. In order to explore the mechanisms of FHL3-regulated functions, we used FHL3 as the bait to screen human fetal hepatic cDNA library for its novel interacting proteins in yeast two-hybrid system. Methionine-1X subtype (MT-1X), a member of the methionine, was identified as a novel FHL3 binding partner. The specific interaction between FHL3 and the full length MT-1X was further confirmed by yeast two-hybrid assay, co-immunoprecipitation and GST pull-down assays. The results show that direct interaction between FHL3 and MT-1X exists In Vitro protein expression kit for DNA templates were purchased from Thermo scientific. Mouse anti-MT-1X monoclonal antibody was purchased from Novus Biological, CO., Rabbit anti-FHL3 polyclonal antibody was purchased from ProteinTech Group, Inc.Human fetal hepatic MATCHMAKER cDNA library and YEASTMAKER Yeast Transformation system were purchased from CLONTECH Laboratories. Pierce GST Protein Interaction Pull-Down Kit and Pierce Human 2.African green monkey kidney cells (COS-7) (ATCC # CRL-1651), human embryonic kidney cells (HEK 293T) (ATCC # CRL-11268) and human hepatoma cell lines (HepG2) (ATCC # HB-8065) were maintained in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum, at 37°C with 5% CONdeI site (underlined) and a reverse primer (5′?GAAGATCTGAATTCAATGCACTTAGGTGCAAAGAGTTCTCC?3') with an EcoRI site (underlined), respectively. The PCR product was digested with NdeI and EcoRI, and then inserted into the NdeI and EcoRI site of vector pAS2-1 of the yeast two-hybrid system , resulting in an in-frame fusion of human FHL3 mutants with sequential deletion of LIM4 cDNA downstream with GAL4 DNA-binding domain. The correct reading frame was confirmed by sequencing analysis. The expression vector was named as pAS2-1/FHL3d4.We previously demonstrated that the fourth LIM domain (LIM4) of FHL3 has the auto-activation ability It is crucial to test each BD-bait plasmid construct for auto-activation of reporter gene promoters before performing the two-hybrid screen. To check for auto-activation, the yeast strain Y190 was transformed with pAS2-1/FHL3d4 using lithium acetate method as described in protocol. Transformed cells were plated on synthetic dropout medium minus tryptophan . Auto-activation of FHL3d4 was screened by β-galactosidase assay using a filter-based assay.Escherichia coli DH5α cells for further analysis.A human fetal hepatic cDNA library in pACT2 plasmid was screened with bait plasmid pAS2-1/FHL3d4 using a large scale sequential transformation method according to the manufacturer's instructions. Yeast cells of Y190 strain harboring pAS2-1/FHL3d4 were transformed with 50 μg of library cDNA and plated on medium lacking tryptophan, leucine and histidine (SD/-Leu -Trp -His) containing 25 mM 3-amino-1,2,4-triazole(3-AT) and incubated for 4–7 days at 30°C. Then, colonies were assayed for β-galactosidase activity. Following a second round of selection on the same medium and removing the valid AD plasmids, the colonies that activated both reporter genes in Y190 strain were extracted and transformed into After checking AD-cDNA plasmids for auto-activation, AD-cDNA plasmids were transformed into Y190 strain harboring pAS2-1/FHL3d4 plasmid to assay the activation of reporter genes. The positive AD-cDNA clones were further characterized by DNA sequencing and these DNA sequences were analyzed by BLAST search for GenBank database.BamHI site (underlined) and a reverse primer (5′?GCGAATTCTCAGGCACAGCAGC TGCACTT?3') with an EcoRI site (underlined) and then subcloned into pACT2. pAS2-1/FHL3d4 and pACT2/MT-1X were transformed into Y190 strain. Transformation of the plasmids was carried out according to The cDNA coding for MT-1X (GenBank accession no. NM005952) was amplified from the cDNA library of the human fetal hepatic using a forward primer (5′?GCGGATCCCCATGGACCCCAACTGCTCCTGC?3′) with an 6) were co-transfected with pCMV-HA/FHL3 and pCMV-Myc/MT-1X using Lipofectamine 2000 in a 10 cm dish. After 30 h, 293T cells were harvested and lysed in a Triton lysis buffer consisting of 20 mM Tris-HCl, pH7.6, 120 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol with 1 mM Na3VO4, 1 mM DTT, and protease inhibitor cocktail. The cell lysates were incubated at 4°C for 30 min and centrifuged at 12,000 rpm for 10 min. Lysates were immunoprecipitated with anti-Myc antibody  or control anti-IgG antibody  for 4 h at 4°C on a rotor, and then protein A/G–Agarose beads (Santa Cruz) were added and further incubated for 16 h. After incubation, the samples were centrifuged for 5 min at 3000 rpm. The protein A/G–Agarose pellets were then washed and boiled in 2×SDS sample buffer. Immunocomplexes were analyzed by SDS–PAGE and Western blot using anti-myc antibody and anti-HA antibody .The full length cDNA encoding FHL3 was amplified by PCR from pBV220/FHL3 which was previously constructed in our laboratory, and subcloned into pCMV-HA to generate HA-tagged fusion protein expression vector. The cDNA fragment of MT-1X was generated by PCR using pACT2/MT-1X as a template, and inserted into pCMV-Myc to generate Myc-tagged fusion protein expression vector. 293T cells  and tested by Western blot using anti-GST monoclonal antibody . The cDNA for MT-1X was amplified by PCR from pACT2/MT-1X, inserted into pT7CFE1-CHis (Thermo) to generate His-tagged fusion protein expression vector pT7/MT-1X, and then translated into protein. Purified GST–FHL3 and GST were individually incubated with Glutathione Agarose for approximately 4 h. Then the Glutathione Agarose was collected by centrifugation, washed five times with GST pull-down lysis buffer and TBS (1:1). Then, His-MT-1X was added to Glutathione Agarose and incubated at 4 °C for overnight with gentle rocking. Glutathione Agarose was washed five times and resuspended in SDS–PAGE sample buffer. After boiling, the bound proteins were analyzed by 15% SDS–PAGE followed by Western blotting using anti-His antibody. His-MT-1X protein was used as controls.The full length cDNA encoding FHL3 was amplified by PCR from pBV220/FHL3 and subcloned into pGEX-4T-1 . The recombinant protein GST-FHL3 and protein GST were individually expressed in Various concentrations of rhFHL3  were added to 96-well plates containing 5,000 cells/well in transfected with siMT-1X or siNC cells. After incubation for 48 h, cell growth was measured by MTT -2, -diphenyltetrazoliumbromide) proliferation assay. Absorbance at 570 nm was measured using a Bio-Rad model 550 microplate reader. The data represent the means of three independent experiments. The cDNA target sequences of siRNAs for MT-1X were: 5′- GGCAAUAAAUUCAUCUAGATT - 3′ (sense) and 5′- UCUAGAUG AAUUUAUUGCCTT - 3′ (antisense). Negative control (Nc) siRNA was included .2 in air. For transfection, cells were seeded in 96-well or 6-well plates in triplicate containing DMEM supplemented with 10% fetal bovine serum. The cells were transfected with the indicated siRNA or plasmids using Lipofectamine 2000 according to the manufacturer's protocol (Invitrogen). 48 h after treatment with siRNA or plasmids at 37°C , cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in RIPA lysis buffer  for 15 min on ice. Cell lysates were then clarified by microcentrifugation at 12,000×g for 10 min at 4°C. Equal amounts of protein (40 μg) was quantified by Lowry protein assaying kit , and separated by 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes . Membranes were then blocked with PBST (PBS with 0.05% Tween 20) containing 5% non-fat dry milk for 1 h at room temperature and followed by incubation with monoclonal antibodies against pSmad2 (Ser465/467) and Smad2, Smad4, P21, CyclinB1 and CyclinD1 , His and GAPDH   and MT-1X antibodies , polyclonal antibodies against FHL3  overnight at 4°C, respectively. Membranes were then washed with PBST for three times and followed by incubation with HRP-conjugated anti-mouse or HRP-conjugated anti-rabbit immunoglobulin G (IgG)  at room temperature for 1 h. Finally, the membrane was analyzed using the enhanced chemiluminescence detection system .Human hepatoma HepG2 cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in humidified atmosphere of 5% COThe previous results suggested that the fourth LIM domain (LIM4) of FHL3 was the key transactivation domain of FHL3 To identify the results of yeast two-hybrid screening, a 186 bp sequence of MT-1X cDNA was amplified by PCR from a cDNA library of human fetal hepatic and then inserted in pACT2. The recombinant plasmid pACT2/MT-1X , lane4 win vivo, co-immuoprecipitation assays were performed in HEK 293T cells. HA-FHL3 and Myc-MT-1X were co-expressed transiently, then expression of recombinant FHL3 and MT-1X was confirmed by Western blotting using anti-HA and anti-Myc antibody respectively. As a result, anti-HA antibody was able to precipitate Myc-MT-1X protein only in the presence of HA-FHL3. As a control, the anti-IgG antibody did not precipitate Myc-MT-1X. It suggests that MT-1X could physically interact with FHL3 in vivo . The inhibitory effect of FHL3 on the proliferation of HepG2 cells displayed a dose-dependent manner from 10 to 1250 ng/ml (p<0.01), and was saturated above 250 ng/ml. The result was similar to that previously reported The effect of FHL3 on human HepG2 hepatoma cells was examined by MTT assay. FHL3 could inhibit the proliferation of HepG2 cells at 10 ng/ml  . After cTo determine whether the MT-1X influences the growth of HepG2 cells by regulating FHL3-mediated signaling pathways, we primarily focused on the effect of MT-1X on the FHL3-mediated Smad signaling in HepG2 cells. The result of western blotting analysis indicated that knockdown of endogenous MT-1X expression could up-regulate the expression level of FHL3. Moreover, knockdown of MT-1X enhanced phosphorylation of the cytoplasmic Smad2 protein (Ser465/467) and the expression of Smad4 protein, while the protein level of total Smad2 was unchanged. Furthermore, we observed that knockdown of MT-1X induced a up-regulation of tumor suppressor gene p21 in HepG2 cells . In contTo elucidate the molecular mechanism by which MT-1X expression regulated hepatoma cells growth, we investigated the effect of MT-1X on the expression of several cell cycle-related proteins by immunoblotting. Knockdown of endogenous MT-1X in HepG2 cells enhanced the expression of p21 that functions as a negative regulator of cell cycle progression at G1 and G2 FHL3 is a member of the family of LIM proteins and is involved in myogenesis, cytoskeleton reconstruction, cell growth and differentiation. Recently, FHL3 has been shown to play roles in carcinogenesis in vivo and in vitro was verified by co-immunoprecipitation and GST pull-down. Moreover, MT-1X is involved in the FHL3-induced inhibitory effect on HepG2 cells by regulating FHL3-mediated Smad signaling, accompanied by increasing of the cell cycle regulator cyclinB1 and inhibition of the cyclin-dependent kinase inhibitor p21.We started from the interaction proteins of FHL3 to explore the possible mechanism of FHL3 in cell proliferation. Until now, there have been a few proteins known to interact with FHL3, such as Actin, CDC25B2, MZF-1, MyoD, and Sox15 Metallothioneins (MTs) belong to the group of intracellular cysteine-rich, metal-binding proteins that have been found in bacteria, plants, invertebrates and vertebrates A lot of studies have shown that MT proteins are involved in many pathophysiological processes, including metal ion homeostasis and detoxification, protection against oxidative damage, cell proliferation and apoptosis, drug and radiotherapy resistance and several aspects of the carcinogenic process in vitro and in vivoRecently, it has been shown that FHL1, FHL2, and FHL3 physically and functionally interact with Smad2, Smad3, and Smad4, important regulators of cancer development and progression, and inhibit human hepatoma cell growth The cyclin-dependent kinase inhibitor p21, the first identified inhibitor of cyclin/CDK complexes, has been shown to be required for the integrity of G1 and G2 checkpoints There have been several reports on the overexpression and specific staining pattern of MT in human breast cancer. Further studies suggest that MT overexpression is associated with significantly poorer prognosis and increased recurrence in invasive ductal breast carcinomas In summary, by establishing a link between MT-1X and FHL3, this study highlights MT-1X as a potential candidate for investigating the molecular mechanisms of cancer cell anti-apoptotic activity and also novel useful molecular targets for cancer therapy.


During liver regeneration in the rat, hepatocyte cytosolic Ca2+ signalling has been shown on the one hand to be deeply remodelled and on the other hand to enhance progression of hepatocytes through the cell cycle. Mechanisms through which cytosolic Ca2+ signals impact on hepatocyte cell cycle early after PH are not completely understood, but at least they include regulation of immediate early gene transcription and ERK and CREB phosphorylation. In addition to cytosolic Ca2+, there is also evidence that mitochondrial Ca2+ and also nuclear Ca2+ may be critical for the regulation of liver regeneration. Finally, Ca2+ movements in hepatocytes, and possibly in other liver cells, not only impact hepatocyte progression in the cell cycle but more generally may regulate cellular homeostasis after PH.After partial hepatectomy (PH) the initial mass of the organ is restored through a complex network of cellular interactions that orchestrate both proliferative and hepatoprotective signalling cascades. Among agonists involved in this network many of them drive Ca Such agonists include some of the main comitogenic—as noradrenalin i in the cytosol is due to the activity of the Ca2+ ATPases (SERCA pumps), which actively transports Ca2+ from the cytosol into the ER. Ca2+-regulated InsP3Rs and Ca2+ ATPases are together sufficient to generate Ca2+ oscillations —and thus by the rate of Ca2+ entry into the cell through plasma membrane Ca2+ channels. Intracellular Ca2+ waves do not result from a simple diffusion of Ca2+ itself—which is quickly buffered—but from the spreading of InsP3—which is more soluble in the cytosol—that mobilizes Ca2+ from storage compartments throughout the cell. A particular spatial pattern of subcellular InsP3R distribution was reported to support the direction of intracellular Ca2+ waves, starting from the canalicular region containing the most abundant and affine InsP3R isoform (type II InsP3R) and spreading toward the other regions of the cytosol, less sensitive to InsP3 i increase running along hepatocyte plates across the lobules i oscillations i oscillations in calcium-buffered hepatocytes resulted in impaired activation of these pathways. A potential reduction in CaM-kinase activation, as previously reported [2+ oscillation frequency, an attractive hypothesis would be that cytosolic PV expression, by attenuating agonist-generated Ca2+ signals, resulted in impaired phosphorylation of CREB. We found that immediate early gene transcription, early phosphorylation of ERK and CREB, and hepatocyte progression in the cell cycle after PH were inhibited in rats expressing cytosolic PV . These dreported , or redureported , as wellreported  activity2+-mobilizing agonists, suggesting these agonists might be involved in initiating the regeneration process. In particular, EGF and HGF elicit cytosolic Ca2+ oscillations in hepatocytes, the physiological impact of which has never been specifically addressed [2+-mobilizing agonists, have been individually reported to contribute to early phases of liver regeneration. Our study thus suggested that buffering hepatocyte calcium signals, potentially generated by these agonists in the minutes after PH, result in delaying hepatocyte cell cycle progression.Cytosolic calcium signalling impacts most likely the early triggering of hepatocyte progression from G0 to G1 and S phases. In line with this view, a rise in concentration—in the liver and in the plasma—is observed early after PH for several Caddressed . Also, eddressed , argininddressed , and nor3-R. Anti-apoptotic members appeared as reducing calcium transfer from the ER to the mitochondria, whereas proapoptotic factors were reported to enhance this flux [2+, as well as cytosolic Ca2+, may be critical for the regulation of liver regeneration after PH in the rat [in vitro in an hepatoma cell line, as well as in vivo in the rat liver after PH. There is evidence in the literature for the crucial role of mitochondrial calcium in the regulation of apoptotic processes. It is well known in particular, that mitochondrial calcium overload can be one of the pathways leading to the swelling of mitochondria and to the rupture of the outer membrane, in turn releasing proapoptotic molecules in the cytosol. Mechanisms for excessive calcium transfer to mitochondria are debated and include mainly interactions between proteins of the Bcl2 family and the InsPhis flux . In this the rat . The aut3 generation and calcium mobilization from local, intra, or perinuclear Ca2+ stores [3 may again come from the cytosol or be generated in the nucleus [2+ storage, InsP3 production, and calcium release, therefore opening the possibility that local nuclear Ca2+ signals may occur independently from the cytosol [3-sensitive  intranuclear calcium compartment  exists [β, PIP2, and InsP3R have been found in the nucleus, allowing a local InsP3 production and providing the machinery necessary to generate autonomous Ca2+ signals [2+ was necessary for centrosome separation and cell progression through early prophase [3 production and calcium elevation in the nucleus, independently of cytosolic calcium, in a hepatoma cell line [Previous studies have established that growth factors important for liver regeneration such as HGF  and insu+ stores , 65. Nuc cytosol , 65. Alt) exists . PLCβ, P signals , 65. We  signals , 58. Recprophase . Gomes eell line . Very siell line .2+. Therefore, agonist-induced nuclear Ca2+ movements are potentially expected to impact both hepatocyte progression in the cell cycle and more generally cellular homeostasis after PH.Important cellular functions are thought to be regulated by nuclear calcium signals, including nuclear pore permeability, transcription factor activity and protein kinase translocation, thereby controlling gene expression . In part2+ signalling may also regulate physiological processes unrelated directly to cell cycle control, although they may ultimately interfere with cell cycle progression, not only in hepatocytes but also in other liver cell types . Whereas we do not have any direct data about the impact of Ca2+ signals in nonhepatocytic cells after PH, we can easily anticipate about Ca2+-dependent physiological processes that may be crucial in hepatocytes after PH. For example, glucose homeostasis, which regulation is critical for hepatocyte exit from quiescence [2+ signals early after PH. Also, biliary homeostasis, which adaptation after PH is critical for liver regeneration [2+-dependent regulation [It is important to realize that liver regeneration, seen as a process involving the whole organ—and even the entire organism—cannot be restrained to the sole hepatocyte division. In that view, after PH, Caiescence , may depneration , 70, maygulation , 71. An integrated full picture of the “liver calcium signalling”, which is obviously lacking, may improve our knowledge on the interaction network that regulates liver regeneration processes .


Human ASM cells isolated from lung samples incidental to patient surgery were incubated for 24 h (overnight) in medium (control) or 1 nM BDNF in the presence vs. absence of inhibitors of signaling cascades . Measurement of [Ca2+]i responses to acetylcholine (ACh) and histamine using the Ca2+ indicator fluo-4 showed significantly greater responses following BDNF exposure: effects that were blunted by pathway inhibitors. Western analysis of whole cell lysates showed significantly higher expression of CD38, Orai1, STIM1, IP3 and RyR receptors, and SERCA following BDNF exposure, effects inhibited by inhibitors of the above cascades. The functional significance of BDNF effects were verified by siRNA or pharmacological inhibition of proteins that were altered by this NT. Overall, these data demonstrate that NTs activate signaling pathways in human ASM that lead to enhanced [Ca2+]i responses via increased regulatory protein expression, thus enhancing airway contractility.Neurotrophins (NTs), which play an integral role in neuronal development and function, have been found in non-neuronal tissue (including lung), but their role is still under investigation. Recent reports show that NTs such as brain-derived neurotrophic factor (BDNF) as well as NT receptors are expressed in human airway smooth muscle (ASM). However, their function is still under investigation. We hypothesized that NTs regulate ASM intracellular Ca We hypothesized that as a growth factor known to promote protein expression in the nervous system, prolonged BDNF exposure enhances expression and function of [Ca2+]i and force regulatory components in ASM. We tested this hypothesis using primary human ASM cells and determined the role of signaling mechanisms most commonly associated with BDNF.Diseases such as asthma are characterized by both enhanced airway contractility as well as remodeling which in turn may involve ASM cell proliferation. We recently demonstrated that in human ASM, prolonged BDNF exposure enhances cell proliferation The techniques for isolating human ASM cells from lung samples incidental to patient surgery have been previously described 2+. ASM layer was removed, minced and enzymatically dissociated using papain and collagenase with ovomucoid/albumin separation . Cell pellets were re-suspended and seeded into 75 cm2 sterile tissue culture flasks, 100 mm Petri dishes or 96-well clear bottom plates, and maintained under usual culture conditions in phenol red-free DMEM/F-12 (Invitrogen) supplemented with 10% FBS. Prior to experimentation, cells were serum starved for 48 h. Experiments were limited to passages 1–3 of subculture in order to ensure maintenance of ASM phenotype, which was periodically assessed by Western analysis for smooth muscle actin and myosin, and agonist receptors.Samples were placed in cold Hanks’ balanced salt solution  supplemented with 10 mM HEPES and 2 mM Ca2+]i2+]i regulation, whereas 10 nM or higher concentration resulted in significant cell death with prolonged exposure. To determine the signaling mechanisms by which BDNF could affect ASM, additional sets of cells were incubated overnight in 1 nM BDNF in the presence of inhibitors of signaling pathways associated with BDNF: MAP kinase , PI3/Akt  and NFκB  Human ASM cells were incubated for 24 h at 37°C in regular growth media (control), or 1 nM recombinant human BDNF  reconstituted in growth media. The BDNF concentration was selected based on our previous data showing acute effects on i was quantified from fluo-4 levels using previously described empirical calibration procedures ASM cells were incubated in 5 µM fluo-4/AM  for 60 min at room temperature and visualized using a 96-well fluorescence imaging plate reader with robotic pipetting capabilities . Cells were initially perfused with HBSS (2.5 mM Ca2+ at room temperature) for 30 s, and then exposed to 1 µM ACh or 10 µM histamine in HBSS to elicit [Ca2+]i responses. The baseline, peak and plateau values of these responses were measured.For agonist responses, ASM cells exposed to medium alone (control), or 1 nM BDNF in the presence or absence of inhibitors of signaling pathways were initially perfused with HBSS (2.5 mM Ca2+ entry (SOCE), a major [Ca2+]i regulatory mechanism in ASM 2+ removed by perfusion with zero-Ca2+ HBSS (5 mM EGTA). Cells were then exposed to 1 µM nifedipine and 10 mM KCl in the continued absence of extracellular Ca2+ and the sarcoplasmic reticulum passively depleted by 10 µM cyclopiazonic acid (CPA), an inhibitor of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). The intent of adding nifedipine and KCl was to prevent unwanted Ca2+ influx via L-type channels, and to clamp the membrane potential to avoid Ca2+ entry via other voltage-sensitive channels 2+ was rapidly reintroduced in the continued presence of CPA and the observed [Ca2+]i elevation relative to the levels in 0 Ca2+ and CPA measured as an index of SOCE.For examining store-operated CaThe techniques for siRNA transfection in human ASM cells has been previously described 3R, Cell Signaling, 3763), SERCA , and GAPDH .Proteins were separated by SDS-PAGE  for approximately one hour and then transferred to nitrocellulose membranes (Bio-Rad) for 7 min using Trans-Blot Turbo transfer system (Bio-Rad). 5% milk in TBS was used to block the membranes for 1 h. Membranes were then incubated overnight at 4°C with primary antibodies at 1 µg/ml concentration. Following three washes with TBS, proteins were detected using infrared range secondary antibodies developed for the Odyssey Li-Cor Imaging System . Antibodies used included: CD38 , TRPC3 , Orai1 , STIM1, , ryanodine receptor , inositol 1,4,5-trisphosphate receptor  with Bonferroni correction for repeated comparisons. Statistical significance was established at p<0.05. All values are expressed as means ± SE.Bronchial samples from at least 4 patients were used to obtain ASM cells, with biochemistry and molecular biology protocols as well as [Ca2+]i responses involving an initially higher peak followed by a decay to a plateau higher than baseline. Exposure to 1 nM BDNF for 24 h enhanced both peak and plateau [Ca2+]i responses to ACh and histamine compared to vehicle controls , IP3R and RyR channels (Ca2+ release)  ; p<0.05 release) , p<0.05.release) .2+ regulatory protein expression, pharmacological inhibition of signaling pathways thought to be activated by BDNF was used. Inhibition of these pathways generally blunted the enhancing effects of BDNF on expression of all the proteins mentioned above (except TRPC3 obviously), but the contribution of specific pathways differed between the Ca2+ regulatory proteins. There was no significant effect of the MAP kinase inhibitor PD98059 (10 µM) on influx mechanisms: TRPC3, Orai1, or STIM1. However, MAP kinase inhibition blunted BDNF effects on SR Ca2+ regulatory proteins  specifically binds to high-affinity full-length TrkB receptors 2+]i regulation, cell proliferation and migration, and in mediating effects of pro-inflammatory cytokines such as TNFα and IL-13 in asthma 2+]i and force The present study examined signaling mechanisms by which neurotrophins influence [Ca2+]i and contractility 2+]i regulation.We and others have now established that NTs and their receptors are present in different lung components, with BDNF produced by airway epithelium, sensory innervation, ASM itself, and a host of immune cells  for SOCE 2+]i values with agonist stimulation as well as SOCE.The mechanisms by which NTs can influence airway structure or function are still under investigation. NTs can modulate neural influences, thus indirectly increasing airway contractility 2+]i changes, the data showing comparable effects of BDNF and the newly-identified TrkB-specific agonist 7,8-dihydroxyflavone suggest a large role for TrkB signaling in ASM, with a likely small and insignificant role for p75NTR evidenced by the lack of effect of the p75NTR inhibitor TAT-pep5. In cell types other than ASM, BDNF activates a number of pathways including PI3/Akt, ERK1/2 and NFκB. In a recent study 2+]i as well as Ca2+ regulatory protein expression in ASM, evidenced in this study by the effects of different pharmacological inhibitors for various signaling intermediates along this pathway. BDNF activation of ERK1/2 appears to involve TrkB as previously shown using TrkB siRNA 2+]i and protein expression as shown in this study using pharmacological inhibition of both IKK and NFκB. In this regard, the lack of effect of TAT-pep5 on BDNF enhancement of [Ca2+]i is interesting since NFκB activation is typically associated with p75NTR signaling. However, the involvement of PI3/Akt in BDNF effects suggests an alternative route for NFκB activation which may be mediated via TrkB.In terms of [Ca2+]i regulatory proteins, it is interesting that their contributions are complex and appear to be mechanism specific. For example, MAP kinases appear to consistently affecting the intracellular Ca2+ regulatory proteins such as SERCA and release channels, but not influx mechanisms in general. On the other hand, NFκB appears to have a broad and consistent effect on both influx and intracellular Ca2+ mechanisms. The relative contribution of PI3 vs. Akt is even more complex, with both involved for some proteins but not others. These data only highlight the complexity of BDNF signaling in any cell type, and emphasize the need to examine the underlying mechanisms in every cell type of interest rather than extrapolating from the nervous system or other cell types. Indeed, we propose that the differential contribution of different signaling pathways in mediating BDNF effects allows BDNF to have pleiotropic effects on inflammation and other insults that also use these signaling cascades. Accordingly, in diseases such as asthma, BDNF has the potential to enhance one cascade vs. the other to enhance [Ca2+]i or cell proliferation as we have previously shown. While the data in this study using pharmacological inhibitors suggest a role for all of MAP kinase, PI3/Akt and NFκB in mediating the effect of BDNF on expression of i regulation in ASM, the effects of BDNF on CD38 are particularly important. This interesting ectoenzyme is a key molecule for the cyclic ADP ribose-RyR channel second messenger-based Ca2+ regulatory pathway in ASM. CD38 expression is known to be regulated by cascades such as MAP kinases and NFκB, and is considered a key mechanism in mediating the effects of inflammation 2+]i regulatory mechanisms holds in terms of signaling cascades is not yet known. However, the differential effects of inhibiting MAP kinase vs. PI3/Akt vs. NFκB on BDNF enhancement of influx proteins  vs. intracellular proteins (SERCA or RyR) suggest that BDNF can modulate the relative contribution of these Ca2+ pathways in a complex fashion. This pleiotropic effect will facilitate nuanced influences of BDNF on [Ca2+]i regulation via influx vs. intracellular release/reuptake in the setting of inflammation where certain mechanisms may be upregulated, such as influx The present study demonstrates a functional relevance for the altered CaIn conclusion, the present study shows that BDNF upregulates a number of mechanisms important in airway contractility which, in combination with enhancement of ASM cell proliferation and cytokine effects can substantially influence airway hyperresponsiveness. Further in vivo research is required to determine whether interactions between BDNF and inflammation is enhanced in asthmatic airways, setting the stage to investigate the potential for interference with BDNF signaling as a therapeutic avenue.


In a broad pharmacological survey, the relevance of different Ca2+-related mechanisms on methadone-induced DCD was investigated including extracellular calcium, L-type Ca2+ channels, μ-opioid receptor, mitochondrial inner membrane potential, mitochondrial ATP synthesis, mitochondrial Ca2+/2Na+-exchanger, reactive oxygen species, and mitochondrial permeability transition. Only those compounds targeting mitochondria such as oligomycin, FCCP, CGP 37157, and cyclosporine A were able to amend methadone-induced Ca2+ dyshomeostasis suggesting that methadone induces DCD by modulating the ability of mitochondria to handle Ca2+. Consistently, mitochondria became dramatically shorter and rounder in the presence of methadone. Furthermore, analysis of oxygen uptake by isolated rat liver mitochondria suggested that methadone affected mitochondrial Ca2+ uptake in a respiratory substrate-dependent way. We conclude that methadone causes failure of intracellular Ca2+ homeostasis, and this effect is associated with morphological and functional changes of mitochondria. Likely, this mechanism contributes to degenerative side effects associated with methadone treatment.Previously, we have shown that SH-SY5Y cells exposed to high concentrations of methadone died due to a necrotic-like cell death mechanism related to delayed calcium deregulation (DCD). In this study, we show that, in terms of their Ca Methadone  is frequently used in different therapies including opioid addiction , long-la2+]cyt) are involved in control of a large number of cellular and physiological processes including neuronal excitability, synaptic plasticity, and gene transcription [2+ signal can switch to a death signal when the [Ca2+]cyt  increases dramatically. For example, excitotoxic high glutamate concentrations result in an initial transient increase in [Ca2+]cyt  that is followed by a delayed, irreversible rise in [Ca2+]cyt  known as delayed calcium deregulation  (DCD). Although several steps preceding DCD remain to be clarified, there is evidence that DCD is the irreversible end point of a sequence involving mitochondrial Ca2+ overloading. DCD precedes and reliably predicts the necrotic death of cultured neurons cyt [2+]cyt in SH-SY5Y cells in the presence of methadone remained unknown. Therefore, the aim of the present study was to investigate those mechanisms. A clear understanding of the factors that mediate this phenomenon might help to resolve the mechanisms that promote neuronal cell death during methadone-induced cognitive damage.SH-SY5Y cells are considered a suitable model for investigating opioid-mediated responses in neurons. These cells express both eceptors . In prev [2+]cyt , 20. How4 cells/cm2 on μ-Dish 35 mm High IbiTreat  as previously reported [ SH-SY5Y cells (ATCC) were plated at a density of 5.3 × 10reported .2+]cyt in SH-SY5Y cells were measured by loading cells with the calcium probe Fura2/AM and using an inverted fluorescence microscope (Nikon Eclipse TE2000-S) as described elsewhere cyt in the absence and the presence of 0.5 mM methadone in different sets of cells. Cells were pooled according to the dynamics of the rises in [Ca2+].  Changes in cyt in SH-SY5Y cells. Consistent with previous observations from our laboratory [2+]cyt in most of the SH-SY5Y cells. However, the effect of methadone differed considerably across cells. Analysis of the dynamics of the [Ca2+]cyt recordings in the absence and the presence of methadone suggested that four different types of calcium recordings can be observed in SH-SY5Y cells. 2+]cyt was observed during the entire period (30 min) of measurements (type 1). A second group of cells was unable to regulate [Ca2+]cyt homeostasis shortly after the addition of methadone (type 2) and [Ca2+]cyt increased continuously. A third group displayed [Ca2+]cyt  deregulation with a delay of 15–20 minutes (type 3). Finally, the fourth group was able to control [Ca2+]cyt after an initial increase (type 4). Groups 1 and 4 represent, respectively, nonresponsive of cells and cells responding with transient rise in [Ca2+]cyt and amount 20.82 and 31.38% of the cells. However, types 2 and 3 displayed short or delayed continuous rise in [Ca2+]cyt that may be considered as early or delayed Ca2+ deregulation according to the literature cyt response, monitored as changed frequencies of the different types of the responses. We found that in the absence of extracellular calcium (0 Ca) no change in responses to methadone was observed (n = 291 cells). These results suggest that Ca2+ entry does not contribute to the reported changes in [Ca2+]cyt. To confirm that extracellular Ca2+  was not involved in these responses we tested the effect of methadone in the presence of nifedipine, a specific isopropyl L-type Ca2+ channel blocker. Nifedipine (2 μM) did not modify the effects of methadone on the [Ca2+]cyt in SH-SY5Y cells (n = 226 cells). So, we ruled out the involvement of this voltage-dependent channel family in methadone-induced [Ca2+]cyt variations. We asked then whether intracellular Ca2+ stores could contribute to the responses to methadone. Cells were treated with thapsigargin (1–100 μM). To deplete intracellular Ca2+ stores before methadone treatment. As expected, thapsigargin induced a significant release in Ca2+ from the endoplasmic reticulum, causing a transient increase in the [Ca2+]i that failed to returned to the basal level within a 10-minute period. A detailed observation of the methadone induced rise in [Ca2+]i shows that cell responses were partially affected by this treatment, suggesting that the rise in calcium is partially due to release from thapsigargin-sensitive, intracellular Ca2+ stores (n = 146 cells). We found that in untreated cells addition of thapsigargin induced a transient increase in [Ca2+] that has been attributed to a leakage of Ca2+ from the endoplasmic reticulum. This effect of thapsigargin alone interferes the interpretation of the type 2 and 3 responses after methadone addition (data not shown).The mechanisms underlying the observed quantitative changes in the types of CaμM Naloxone), the competitive antagonist of the μ-opioid receptor. We found that this treatment did not modify the relative abundance of any of the four types of cell populations . This result suggests that the changes in [Ca2+]cyt and DCD induced by methadone in SH-SY5Y cells are independent of opioid receptors. In agreement with the lack of μ-opioid receptor participation as found here, it has been reported that methadone-toxic pathways are not mediated by μ receptors  that has been attributed to leakage of Ca2+ from depolarized mitochondria. We found that FCCP induced a 44% decrease in the appearance of type 2 cells whereas increased the relative abundance of cells showing a type 3 response by 2.7-fold in FCCP , an inhibitor of the Ca2+/2Na+-exchanger. A five min exposure of cells to CGP37157 significantly increased the proportion of cells showing no rise in [Ca2+]cyt in response to methadone during the entire [Ca2+]cyt measurement period (type 1) (data not shown) because CGP37157 resulted in a drastic decrease in the population of cells showing either type 2 (74%) or type 3 (85%) responses. In addition, the relative abundance of cells showing a type 1 response returned to the value obtained in untreated cells. These results support data suggesting the relevance of mitochondria in methadone-induced DCD , cells responded to methadone in a different way. Specifically, TEMPOL decreased the number of cells showing a type 2 response by ~70%, whereas the abundance of cells showing a type 3 response was largely increased (3.5 fold). Types 1 and 4 were nearly not present. Consistent with our results, Nicholls et al. [2+ can occur by induction of MPTP formation, which is dependent on the mitochondrial matrix Ca2+ concentration and can be inhibited by cyclosporine A [μM). CsA diminished the occurrence of response type 2 by 36% (n = 89 cells) in cell cultures challenged with 0.5 mM methadone. Moreover, consistent with the hypothesized role of the pore in DCD, CsA induced a 2-fold increase in cells showing type 4 response. However, interpretation of these results is difficult because CsA may also inhibit the mitochondrial Ca2+ uniporter in some instances.To test contribution of reactive oxygen species we used the cell-permeable, small molecule compound TEMPOL  to mimic superoxide dismutase activity. In the presence of TEMPOL , 1.7 ± 0.3 (+FCCP), and 1.7 ± 0.4 (+methadone), and for glutamate/malate 4.8 ± 0.3 (control), 1.8 ± 0.4 (+FCCP), and 1.9 ± 0.5 (+methadone). Thus, the effects of FCCP and methadone on Ca2+ uptake are comparable, although in the presence of glutamate/malate the effect appears to be much more pronounced. This probably results from a stronger uncoupling effect of methadone on glutamate/malate access to the respiratory chain and a consecutive additional impairment of Ca2+ uptake. Therefore, the effect of methadone on Ca2+ uptake by mitochondria may be dependent on the respiratory substrates. On the other hand, when the values of the U/4 ratios calculated in the absence and presence of Ca2+ uptake were compared (Figures 2+ uptake (2+ uptake, leading to a decrease of uncoupling capacity by FCCP and methadone. Finally, the effect of methadone on mitochondrial Ca studied . We usedethadone . The cal Figures . For thi Figures , a disti+ uptake . The cal2+ homeostasis might be important for both early and late stages of the observed side effects and, perhaps account for some of the observed clinical symptoms, for example, memory impairment.The data presented indicate that methadone induces DCD in SH-SY5Y cells by altering the capacity of mitochondria to handle calcium, and correlates with distinct changes of mitochondrial morphology. These morphological changes, in turn, can be associated with mitochondrial damage and cell death. Interestingly, swollen mitochondria have been observed in the context of neurodegenerative diseases , 37. An


In chronic cardiac hypertrophy, however, ANP levels are markedly increased and GC-A/cGMP responses to ANP are blunted due to receptor desensitization.Cardiac atrial natriuretic peptide (ANP) regulates arterial blood pressure, moderates cardiomyocyte growth, and stimulates angiogenesis and metabolism. ANP binds to the transmembrane guanylyl cyclase (GC) receptor, GC-A to exert its diverse functions. This involves a cGMP-dependent signaling pathway preventing pathological [Ca2+ levels ([Ca2+i]). This pathway involves the activation of TRPC3/C6 Ca2+ channels  by GC-A which forms a stable complex with TRPC3/C6 channels. Our results indicate that the resulting TRPC3/C6-mediated Ca2+ entry then stimulates Calmodulin Kinase II (CaMKII) to phosphorylate L-type Ca2+ channels leading to increased L-type Ca2+ channel mediated Ca2+ current and a rise in intracellular Ca2+ levels.Here we show that in this situation ANP binding to GC-A stimulates a novel cGMP-independent signaling pathway in cardiac myocytes, resulting in pathologically elevated intracellular Ca2+i-homeostasis. Under physiological conditions, activation of a cGMP-dependent pathway moderates the Ca2+i-enhancing action of hypertrophic factors such as Angiotensin II. By contrast, a cGMP-independent pathway predominates under pathophysiological conditions, when GC-A is desensitized by high ANP levels. The concomitant rise in [Ca2+i] is likely to increase the propensity to cardiac hypertrophy and arrhythmias.These observations reveal a dual role of the ANP/GC-A signaling pathway in the regulation of cardiac myocyte Ca


Several recent studies suggest they possess additional vascular actions. Although it has been postulated that calcimimetics may have a direct effect on CaSR in the blood vessels, further studies are needed to elucidate their vascular CaSR-dependent versus CaSR-independent effects.Calcimimetics, such as R-568, are thought to activate G protein-linked Ca2+ levels (using a single cell approach and FURA-2AM), in the presence or absence of Calhex-231, a negative modulator of CaSR. To address their potential vascular functions, we also evaluated R- and S-568-stimulated enzymatic release of Nitric Oxide (NO) by DAF-2DA, by Nitric Oxide Synthase (NOS) radiometric assay  and by measuring eNOS-ser1177 phosphorylation levels (Immunoblotting). We show that, although the CaSR protein was expressed in HUVECs, it was mainly distributed in cytoplasm while the functional CaSR dimers, usually localized on the plasma membrane, were absent. In addition, regardless of the presence or absence of Calhex-231, both R- and S-568 significantly increased intracellular Ca2+ levels by mobilization of Ca2+ from intracellular stores, which in turn augmented NO release by a time- and Ca2+-dependent increase in eNOS-ser1177 phosphorylation levels.Focusing on human umbilical vein endothelial cells (HUVECs), we studied the CaSR expression and distribution by Immunofluorescence and Western Blot analysis. CaSR function was evaluated by measuring the potential effect of calcimimetic R-568 and its enantiomer S-568 upon the modulation of intracellular CaTaken together, these data indicate that in human endothelium there is no stereoselectivity in the responses to calcimimetics and that CaSR is probably not involved in the action of R- and S-568. This suggests an additional mechanism in support of the CaSR-independent role of calcimimetics as vasculotrope agents. Calcimimetics represent a new therapeutic opportunity for treating mineral metabolism disorders related to secondary hyperparathyroidism in patients suffering from chronic kidney disease and uremia 2+) levels by virtue of a cell surface calcium sensing receptor (CaSR), which is a low-affinity G protein-coupled receptor consisting of 1078 amino acid residues 2+ itself can be considered the main activator of CaSR, there is a list of known direct (type I agonists) and indirect allosteric (type II agonists) regulators of CaSR functions. Thus, it seems to be a promiscuous receptor that senses changes in multiple physiologic parameters 2+ concentration ([Ca2+]o), subsequently resulting in reduction of parathyroid hormone (PTH) secretion and improvement of calcium phosphate products In humans, the parathyroid gland cells can sense small fluctuations in plasma calcium  that is sensitive to CaThus, although the vascular effect of calcimimetics may depend on calcimimetic-induced suppression of parathyroid hypertensive factors According to this hypothesis, recent studies have demonstrated in vivo hypotensive effects by calcimimetic agents (type II agonists) in both normotensive 2+]i), leading to the production of Nitric Oxide (NO) More recently, it has been demonstrated in vitro that human aortic endothelial cells express a functional CaSR that responds to the endogenous polyamine spermine (CaSR type I agonist) by an increase in intracellular calcium levels enzymatic activity Since it has previously been demonstrated that human aortic endothelial cells express a functional CaSR To determine whether CaSR protein was expressed in HUVECs, immunofluorescence confocal microscopy analysis was performed using a monoclonal antibody against human CaSR. Excited ALEXA-488 emits green fluorescence. Nuclei stained with TO-PRO-3 Iodide display blue fluorescence. As shown in In order to better identify CaSR expression and cellular distribution, Western Blots were also performed. We also performed subcellular fractionation of the HUVECs to ascertain whether CaSR is located in the plasma membrane, in which case it would exert its currently known physiological function. Although we were consistently able to detect the widely described immunoreactive 55–70 kDa polypeptide in both subcellular compartments analyzed, it became evident from the Western Blot analysis that the subfraction containing plasma membranes was poorly enriched in an immunoreactive band of distinctively higher molecular mass , suggesting the absence of the CaSR mature monomers, physiologically relevant in HUVECs. Since it has been reported 2+]i in our cellular model, FURA-2AM-loaded HUVECs were stimulated using different concentrations (1–100 µM) of R-568 or its enantiomer S-568. The evaluation of [Ca2+]i was also performed in the presence or absence of Calhex-231 To determine whether the addition of R- and S-568 might modulate [Ca2+]i. in almost all stimulated cells (responsive cells were 92–100% of the cell population). At concentrations of 1 and 10 µM, R-568 induced a slow and delayed response, while at 100 µM, the compound produced a sustained rapid response. The enantiomer, S-568, had the same qualitative and quantitative effects as R-568 .As shown in as R-568 , D, E, F2+]i. rise was driven only by extracellular Ca2+ or also by Ca2+ release from internal stores. To this end, we performed experiments in an extracellular Ca2+-free condition (0mM Ca2+ + 0.5mM EGTA) and/or in an empty store condition, using thapsigargin (tg) to inhibit Ca2+ re-uptake by intracellular stores and consequently inducing store depletion 2+-free conditions, R-568 and S-568 produced the same qualitative and quantitative effects as shown in complete medium (2mM Ca2+) (2+]i rise was due to ion release from intracellular stores.We tested whether in our cellular model the S- or R-568 triggered o  levels in the presence of 2mM o, both 100 µM R- and S-568 brought a rapid and sustained intracellular NO rise in almost all cultured HUVECs , R-568 and S-568 significantly (p<0.003) stimulated eNOS-ser1177 phosphorylation, which peaked at 0.5 to 1 minute of treatment, as assessed by evaluation of total and Ser1177-phosphorylated forms of eNOS . After 1In order to confirm the calcimimetic role on NO release in HUVECs and to evaluate for the first time the potential effects of R- and S-568 on NO enzymatic release in HAECs we evaluated NOS activity by a highly sensitive method as the 3H-Arginine into 3H-Citrulline conversion assay.As shown in 2+ levels by mobilization of Ca2+ from intracellular stores, which in turn augmented NO release by a time- and Ca2+-dependent increase in eNOS-ser1177 phosphorylation levels. One should note that, in the presence of Calhex-231 (a negative modulator of CaSR), R-568 increased both [Ca2+]i levels and NO release at all concentrations used. Similar data were obtained when S-568 was examined over the same concentration range, showing that there is no stereoselectivity in the responses. Thus, CaSR is unlikely to be involved in the action of R- and S-568.The present study shows for the first time that, although CaSR protein was expressed in HUVECs, it was mainly distributed in cytoplasm while the functional CaSR dimers, usually localized on the plasma membrane, were absent. Nonetheless, both calcimimetics R- and S-568 significantly increased intracellular CaOur interest in this study stemmed from learning that, in addition to the known role of calcimimetics in regulating levels of PTH On the other hand, experimental studies ex vivo as well as on cultured cells support the hypothesis that both R- and S-568 may play a direct role in vascular functions. For example, in in vitro and ex vivo experiments, activation of CaSR seems to have a potential role in the control of vasodilation regulating the myogenic tone in rat subcutaneous arteries, while treatment with the type II CaSR agonist cinacalcet results in a concentration-dependent vasodilatation of isolated precontracted aortae Although these data suggest a direct effect by calcimimetics on the vasculature and in particular on the modulation of NO bioavailability, which is known to play a key role in the regulation of endothelial-mediated vasodilation and vascular homeostasis 2+]i leading to the production of NO Based on recent in vitro evidence from cultured human aortic endothelial cells showing that these cells express CaSR and that the agonist spermine induces an increase in [Ca2+]i  potentially involved in explaining the vasculotropic effect of such calcimimetics, our data show that the proven ability of calcimimetics is unaffected in Ca2+ and closely related to CaSR In support of this hypothesis, recently GPRC6A, a novel G protein-coupled receptor  that is sensitive to CaThus, we may conjecture that in HUVECs R-568 or S-568 might allosterically modulate a member of the GPCR family C or specifically GPRC6A activity and in turn activate various intracellular pathways leading to increased eNOS activity and then NO production.2+]i increase drives both R- and S-568 NO production. Thus, when [Ca2+]i increase is almost completely prevented, the R- and S-568 effects on NO release are totally abolished  and these calcimimetic effects on eNOS phosphorylation disappeared in Ca to rise .From these data it is conceivable that the potential non stereospecific activation of a G protein-coupled receptor by R-or S-568 elicits additional intracellular signals, potentially through interaction with Gαq/11 subunit heterotrimeric G-proteins, resulting in potential activation of a PI3-Kinase/Akt cascade leading to increased eNOS phosphorylation levels Moreover, since the activation of eNOS in endothelial cells involves a complex set of molecular events all acting in concert to increase eNOS activity, further studies will be needed to evaluate the potential role of R-568 or S-568 in the modulation of eNOS activity via intracellular enzyme compartmentalization and/or protein-protein interactions The present study provides further insight into the potential CaSR-independent novel role of calcimimetics in vascular biology, by showing that these compounds increase NO release in cultured HUVECs. It should be noted, however, that, as with the CaSR-independent vasodilatory action of both R-568 and S-568 demonstrated by Nakagawa et al. 2+]o-independent endothelial NO production was increased by calcimimetics, as observed in our study, may support the previous suggestion by Al-Aly Nevertheless, that -arginine into L-[3H]-citrulline.Primary HUVECs were obtained and cultured as previously described 2, pH 7.6) for 5 min at room temperature. Some not permeabilized samples were left in PBS for 5 min at room temperature. For CaSR/nucleus immune-labeling, cells were blocked with 10% Bovine Serum Albumine (BSA) in PBS for 30 min at room temperature, followed by 60 min incubation at 37°C in a 1∶100 dilution in 1% BSA in PBS of anti human CaSR Mouse monoclonal antibody  raised against a synthetic peptide corresponding to residues 214–235 of hCaR protein, at a final concentration of 10 µg/ml. Finally, Alexa-488 (Molecular Probes A11001) goat anti-mouse secondary antibody (1∶1000) in 1% BSA/PBS was incubated for 1 h at room temperature. For nuclear staining, TO-PRO-3 Iodide (Molecular Probes T3605) in a 1∶100 final dilution was added during the last 15 min of incubation. Negative controls were processed as for CaSR staining, but incubated only with Alexa-488 goat anti-mouse secondary antibody. All the slides were mounted with Slowfade (Molecular Probes), and observed with a ZEISS 510META Confocal Microscope. Images were acquired using LSM 510 META confocal microscopy software .For Immunofluorescence Confocal Microscopy experiments, HUVECs were plated onto glass gelatin-coated coverslips in 6-well plates and grown at sub-confluence. Briefly, cells were fixed for 10 min at room temperature with a 3% paraformaldehyde solution in 1X-Dulbecco's phosphate buffered saline (PBS) 2% sucrose (fixation solution pH 7.6). Then, cell membranes were permeabilized . 30 µg of HUVECs, of HAoVSMCs and HAECs (positive controls) total protein lysates, and 15 µg of membrane and cytoplasm extracts were heated at 100°C for 10 min in an SDS sample buffer with 2-Mercaptoethanol (reducing conditions), electrophoretically separated by 4–10% SDS-PAGE under reducing conditions and electroblotted on nitrocellulose membrane. The membranes were blocked in 5% milk solution in TBS-Tween 20 0.1% (TBST) for 1 hour at room temperature and probed with a 1∶2000 dilution of anti-human CaSR mouse monoclonal antibody (MA1-934 ABR) and with a 1∶10000 dilution of anti-human β-Actin mouse monoclonal antibody (Sigma A5441) in 5% milk solution in TBS-Tween 20 0.1% at 4°C overnight. After washing, the membranes were further incubated with a 1∶10000 dilution of Goat Anti-Mouse IgG specific Peroxidase conjugated  in 5% milk solution in TBS-Tween 20 0.1% for 90 min at room temperature. Immunoreactive bands were detected using the ECL system .2+]i measurements HUVECs (6000 cells/well) were plated on black 96-well plates with a clear bottom in complete medium. After 1 day the cultures were serum-starved for 2 hours in EBM  supplemented with 1% platelet-deprived horse serum and 100 µM L-arginine. In the last 45 min of serum-starvation 5 µM FURA-2AM was added to the cells, then rinsed with Standard Medium  and bathed in Ca2+-containing SM. FURA-2AM-loaded cells were sequentially excited at 340 and 380nm by a high-speed wavelength switcher  equipped with 75W-Xenon lamp  and stimulated with R-568 or S-568 (1–10–100 µM). The effect of Calhex was tested on R-568 or S-568 100 µM. To study the role of extra- and intra-cellular Ca2+,the cells were stimulated with R-568 or S-568 100 µM respectively in Ca2+-free SM and after depletion of intracellular Ca2+ stores by thapsigargin. The fluorescence images were acquired every 2 sec by a C6790 cooled CCD camera . The image ratio calculations were carried out on a pair of corresponding 340 and 380 image files. The graphs show the temporal plots calculated from the image ratios (340/380). For each experimental condition, 10 different wells were observed to analyze at least 35–50 cells).For [Ca2+]i variation. In the last 30 minutes of serum-starvation 10 µM DAF-2DA was added to the cells, which were finally rinsed in Standard Medium . DAF-2DA-loaded cells were stimulated with R-568 or S-568 (1–10–100 µM) with or without Calhex 231 (10 µM) in Ca2+-containing SM or Ca2+-free SM plus thapsigargin. The NO donor Sodium Nitroprusside (SNP) was used as a internal control. The fluorescence, read by a microplate-reader  at 490nm excitation/510nm emission, was acquired from each sample (n = 8) and expressed as a mean ± SE of the f/fc ratio .The total NO production was measured in HUVEC populations using a modified version of the method by Wang and Joseph 2+-containing SM or in Ca2+-free SM (CaCl2 was substituted by 2mM MgCl2 plus 0.5mM EGTA) plus 1 µM thapsigargin administration, were excited at 490nm, and during fluorescence recording were stimulated with R-568 or S-568 (1–10–100 µM). The fluorescence images were sampled every 2 sec and traces were calculated as f/f0 . At the end of each experiment, the NO donor SNP (sodium nitroprusside) was used as an internal control. For each experimental condition 10 different wells were observed so as to analyze at least 35–50 cells).NO production was studied in detail in the same culture conditions using the method previously reported by Di Pietro et al., 2006 2+-containing SM and stimulated for 1 minute. To study the effects of R-568 and S-568 (100 µM ) with and without extra- and intracellular Ca2+ conditions on NO production, the cells were treated with R-568 or S-568 (100 µM) time dependently  in Ca2+-containing SM and for 1 minute in Ca2+-free SM. Total protein lisates were recovered and quantified. eNOS-ser1177 phosphorylation levels were determined by Western Blot after immunoprecipitation. Briefly, 500 µg of lysate proteins were incubated overnight with 5 µg of eNOS mouse monoclonal antibody  at 4°C, gently rocking. The next day 50 µl of 50% slurry protein A/G beads (sc-2003 Santa Cruz) was added to the mixture and incubated for 4 hours at 4°C, gently rocking. After 3 washes with IP buffer  immunoprecipitates were resolved on an 8% SDS-PAGE gel and transferred to nitrocellulose. Then, membranes were blocked for 1 hour in 5% BSA in TBST and incubated with a 1∶1500 dilution in 1% BSA of eNOS-Phospho ser1177 rabbit polyclonal primary antibody  overnight at 4°C. After three washes with TBS-Tween 20 0.1%, membranes were incubated with a 1∶2000 dilution in 5% non-fat milk in TBST of anti-rabbit IgG-HRP conjugate secondary antibody (Santa Cruz sc-2004) for 90 minutes at RT. Immunoreactive bands for eNOS-Phospho ser1177 were visualized by ECL Plus detection reagent (Amersham Pharmacia). To determine eNOS total content the membranes were stripped using a Restore Blot Stripping Buffer (Thermo Scientific 21059) following the manufacturer's instructions and then re-probed with a 1∶2000 dilution in 5% non-fat milkof the eNOS mouse monoclonal antibody overnight at 4°C. After three washes with TBST, membranes were incubated with anti-mouse IgG-HRP conjugate secondary antibody  for 90 minutes at RT. Immune complexes were visualized by ECL and quantified by scanning densitometry . The data were elaborated by the Bio-Rad Image Processing and Analysis System. Phospho eNOS was normalized vs eNOS total.HUVECs were grown at sub-confluence on Petri dishes 100mm in complete medium and then serum-starved as described above. To study the effects of different doses of R-568 or S-568 (0.1–1–10–100 µM ) +/- Calhex (10 µM) on HUVECs NO production, the cells were bathed in Ca3H]-arginine into L-[3H]-citrulline as described by +, 0.5 mmol/L EDTA, 1 mmol/L dithiothreitol, pH 7.2) and sonicated on ice. In each test tube the following reagents were added to 100 µl lysate at the final concentrations: 2 mmol/L NADPH, 1.5 mmol/L CaCl2, 0.1 mmol/L BH4 (tetrahydrobiopterin) , 2.5 µCi L-[3H]-arginine ( = 0.4 µM) . After 15 min incubation at 37°C, the reaction was stopped by adding 2 ml Hepes-Na+ pH 6 containing 2 mmol/L EDTA; the entire reaction mixture was applied to 2 ml columns of Dowex AGWX8-200  (Na+ form) and eluted with 4 ml of water. The radioactivity corresponding to L-[3H]-citrulline content in the eluate was measured by a liquid scintillation analyzer . In some experiments, L-NAME  was added 40 min before adding L-[3H]arginine. NOS activity was expressed as pmoles citrulline/min/mg protein.NOS activity was evaluated by measuring the conversion of L-[Data are reported as means ± SE or SD. Statistical comparisons were made using Student's t test for paired and unpaired groups. An analysis of variance was used when multiple comparisons were performed. A difference was considered significant at p<0.05.


Cytogenetic abnormalities in t-AML are similar to those found in p-AML but present more frequent unfavorable karyotypes depending on the inducting agent. Losses of chromosome 5 or 7 are observed after alkylating agents while balanced translocations are found after topoisomerase II inhibitors. This study compared t-AML to p-AML using high resolution array CGH in order to find copy number abnormalities (CNA) at a higher resolution than conventional cytogenetics. More CNAs were observed in 30 t-AML than in 36 p-AML: 104 CNAs were observed with 63 losses and 41 gains (mean number 3.46 per case) in t-AML, while in p-AML, 69 CNAs were observed with 32 losses and 37 gains (mean number of 1.9 per case). In primary leukemia with a previously “normal” karyotype, 18% exhibited a previously undetected CNA, whereas in the (few) t-AML with a normal karyotype, the rate was 50%. Several minimal critical regions (MCRs) were found in t-AML and p-AML. No common MCRs were found in the two groups. In t-AML a 40kb deleted MCR pointed to RUNX1 on 21q22, a gene coding for a transcription factor implicated in frequent rearrangements in leukemia and in familial thrombocytopenia. In de novo AML, a 1Mb MCR harboring ERG and ETS2 was observed from patients with complex aCGH profiles. High resolution cytogenomics obtained by aCGH and similar techniques already published allowed us to characterize numerous non random chromosome abnormalities. This work supports the hypothesis that they can be classified into several categories: abnormalities common to all AML; those more frequently found in t-AML and those specifically found in p-AML.Therapy-related acute leukemia (t-AML), is a severe complication of cytotoxic therapy used for primary cancer treatment. The outcome of these patients is poor, compared to people who develop  Therapy-related myeloblastic leukemia, including therapy-related myelodysplasia, (t-AML), constitutes approximately 10% of AML and has several characteristic features Factors associated with the development of t-AML include prior cytotoxic therapy. Two different types of t-AML are to be distinguished. The first one is due to prior therapy with alkylating agents (AA) or radiotherapy The prognosis is poor in t-AML, excepted in case of t, t and inv(16) which follow the same course as p-AML and the karyotype is more frequently modified with at least 2 abnormalities or more.p-AML are also heterogeneous entities, classified according to bone marrow cell morphology and karyotype with the recent addition of several gene mutations. AML was among the first diseases to be treated and monitored according to somatic acquired chromosomal abnormalities, including the first successful targeted treatment against a pathological gene product in AML3.As t-AML appear to have a particular leukemogenesis pathway 1) to analyze t-AML using high resolution array CGH in order to find genome region or even gene-specific copy number abnormalities (CNA) at a higher resolution than conventional cytogenetics; 2) to delimitate minimal critical regions (MCR) in order to identify potential candidate genes induced by AA or ATII implicated in oncogenesis: 3) to perform a similar analysis for p-AML and 4) to compare these two AML entities in our series and in the literature.The patients were studied according to protocols approved by the Institut Gustave Roussy  Ethics Committee. Between 1995 and 2007, 30 t-AML and 36 p-AML with various karyotype results were collected at disease onset.  and 2.Cytogenetic analyses were performed on metaphase spreads obtained from tumor, bone marrow or blood. In each case, 20 RHG-banded metaphases were analyzed when possible.A set of commercial probes was used to search for abnormalities such as WCP1, WCP2, WCP5, WCP7, WCP8, t RUNX1-ETO fusion probes (fp), tBCR-ABL (fp), MLL dissociation probes, WCP14, t PML-RARA (fp), WCP17  and 2. TMost samples were diagnosis bone marrow cells except for patients t-4, t-9, t-11, t-17, t-19, t-26 and p-33 derived from whole blood. After thawing, all t-AML samples were cultured for 24h  without selecting CD34+ cells. The mononuclear cells were collected and washed before DNA extraction. Bone marrow was cultured in the same flask with the same media, to allow adherent cell proliferation in order to obtain constitutional fibroblasts from the patient. However, no adherent cells were obtained from this procedure. Pooled commercial DNA (Promega®) was thus used for all t-AML and p-AML work in order to maintain a homogenous process for the 64 AML.Tumor genomic DNA was isolated according to Qiagen protocols with modifications http://www.agilent.com). Data were extracted from scanned images using Feature extraction® software . Raw data text files from the latter were then imported for analysis into CGH Analytics® 3.4.40. Aberrations were detected with the ADM2 algorithm and the filtering options of a minimum of 5 probes and abs(log2Ratio) >0.3. Aberration segments were individually reviewed using build 35, hg18 from the UCSC Genome browser Patient leukemia samples were analyzed using 244 K microarrays . Only t-AML were processed as dye-swap pairs. In all experiments, sex-matched DNA from a pooled human female or male individual  was used as the reference. Oligonucleotide aCGH processing was performed as detailed in the manufacturer's protocol  were distinguished from Copy Number Abnormalities (CNA) based on various criteria: i) sequence size below 2 Mb; ii) the presence of repetitive identical breakpoints between patients; iii) the genes involved such as olfactory receptor genes, the NF1P1 locus or GSTT1 (chromosome 22) and iv) consultation of the Database of Genomic Variants Deletions inside immunoglobulin gene (IG) clusters, consecutive to VDJ rearrangement were observed mainly in t-AML. These illegitimate recombinations were considered as acquired CNVs Karyotypes and aCGH were well correlated, with few exceptions. Ninety-six unbalanced chromosomal abnormalities previously undetected by mitotic karyotypes, were detected after high resolution aCGH , 4 and 5In both groups of AML, 25 cytogenetically balanced translocations  and 5 diIn some cases the confrontation between aCGH and the karyotype allowed us to better define the previously diagnosed rearrangements. In patient t-13, an add(1)(q3?) was found in the morphological karyotype. With aCGH, it was concluded that it resulted from an unbalanced translocation der(1)t. The breakpoint was virtually cloned from the arrays: on 1q42.3<231.374>, it was sitting in the middle of PCNXL2, between exons 11 and 12. On 2p16.1<54.943>, it was lying in an EML6 gene in the vicinity of exon 12 of the gene. P-22, a primary AML with a t, had a loss of 10Mb of 12p13 secondary to a additional complex translocation encompassing ETV6 and CDKN1B .All patients  had been treated with multi-agent chemotherapy (AA and ATII) combined or not with radiotherapy. Thus, the type of induction mechanism was most of the time, deduced from the observed chromosomal abnormalities : 9 casesTwenty-five patients (80%) with 104 CNA exhibited 41 gains and 63 losses . The meaThe twelve losses in the immunoglobulin genes (IG) on 2p11 and 14q32.3 were considered as a special category of CNA. Among the 8 patients with IG rearrangements, 2 had bi-phenotypic leukemia and the others had various forms of AML.Among 36 patients, 12 had a normal karyotype, 4 exhibited an anomaly on chromosomes 5 or 7. A t, characterizing AML-M3, was present in 9 cases .In p-AML, 64 CNAs were observed  with 30 A MCR was defined as such if 2 cases or more shared a common genomic location. Twelve MCRs were observed in t-AML and eight in p-AML .The size of MCRs was smaller in t-AML, with a minimum of 0.04 Mb compared to p-AML with a minimum of 0.95 Mb. The longest MCR in t-AML was 36.44 Mb, of the same order of magnitude as in p-AML where it was 45.69 Mb.These single cases were of various sizes, from 0.04Mb to 26.89 Mb. Most of them were small and not known as polymorphisms. They are detailed in As we were obliged to use a pool of normal DNA as a reference, Mendelian CNVs that are present throughout the entire genome vs p-AML), were found to be significantly different, with a p value of <0.002. The fact that a quarter (8/30) of t-AML exhibited a VDJ rearrangement suggests that the transformed cells are often multipotent in these diseases.High resolution aCGH allowed us to study the whole set of IG gene rearrangements The acquired chromosomal abnormalities detected by morphological cytogenetics were quite similar in t-AML and p-AML. The CNA patterns were well correlated between the morphological cytogenetics and aCGH. Their frequency was increased after high resolution aCGH. Losses were more frequent than gains in both series. The number of CNAs was higher in t-AML , than in p-AML . The value found here is of the same order of magnitude than that reported elsewhere de novo leukemias.Twenty-five balanced translocations remained undetected  and 5. NIn the present series, twelve MCRs were isolated in t-AML and 8 in p-AML . The sizWhen we compared the number of losses and gain in the “table” sample to those in the present series, a chi2 test showed that as they were not different, we could include them in the table. In all AML 123 MCR were losses and 51 were gains. In t-AML, 34 MCR were losses and 4 were gains while in p-AML 34 were losses and were 4 gains.We will examine the lost and gained MCR found in our series together with the most specific or frequent MCR found in this table.A small juxta-telomeric MCR loss in -1q44 was not reported in p-AML and does not seem to be very frequent  and 9. ATwo MCR were found. The most telomeric one on 3p21.3 was a 5.18 Mb recurrent loss covering more than 40 genes. It was found in p-AML  but alsoA more centromeric 10Mb loss was found exclusively in t-AML. Both patients in the present work contributing to this MCR had been treated with RT and 5FU. PROK2, a prokineticin 2 isoform A precursor, is highly expressed in bone marrow A specific loss in p-AML was found in 4q24  includinA small loss of 4q31.2, exclusively in 8% of t-AML  and 8 waThe 5q- region was fragmented in more than twelve groups. In p-AML, the most frequent regions were comprised between 86.54 and 114.14 Mb, with proximal and distal “spreading”. This region contains the RASA1 gene which exhibits tumor suppressor activity on the RAS gene. RASA1 has been reported to be a CNA in breast cancers and a putative tumor suppressor gene Two MCR were observed in more than 10% of t-AML . Both weThe second 5.22 Mb MCR in 5q31.3q32, was delineated by the present work . Among tFinding different MCR with a maximum frequency in p-AML and in t-AML suggests different oncogenesis pathways on this chromosome.A 90kb MCR in 7p22.2  and 8 haIn three t-AML cases, a gain of 7p15 concerned the homeogene cluster of HOXA6, HOXA7, HOXA9 and HOXA10 . This arThese genes belong to the HOXA5-11 cluster and are expressed throughout the CD34+ compartment As overexpression of Abd-B HOXA genes has been demonstrated in AML with a rearranged MLL On 7q, the 11 MCR with a specificity for t-AML or p-AML were interlaced. Three MCR were reported in more than 10% of t-AML . The firThe most frequent MCR in t-AML was 7q21.3q22.1  and 8. Tin vitro and in vivo, so that the concomitant overexpression of MCM7 and the miRNA cluster triggers prostatic intraepithelial neoplasia in transgenic mice Three miRNA, mir-25, mir-93 and mir-106b were present in this deleted region , regulatA defect in DNA repair was reported in TRRAP-deficient cells In 7q33q34, a 1.74Mb MCR had a low ratio in a patient (t-2), implying a probable double deletion, while the remaining ratio could have been due to the few normal cells. This MCR was the third in t-AML. It contained miRNA-490 which was probably regulating fewer than a hundred genes, with a maximum context score for the FOS gene. Among several genes , CREB3L2In our series of p-AML, a large 43 Mb MCR was situated between 7q31.3 and the telomere . Table 8The most centromeric p-AML-specific deletion was a 2.2Mb MCR on 7q31.3. Among the 10 genes in the region, only WASL is reported to potentially play a role in the microthrombocytopenia, the characteristic sign of Wiskott-Aldrich syndrome The 7q34 MCR was the most frequent and the most specific in p-AML. It contains 5 genes  that areThey were located between 7q31.3 and 7q36.2. Obviously, there are too many genes in those 5 MCRs, but PTPRZ1, MLL3 and XRCC2 could be candidate leukemogenic genes.A gain of 170 Kb located on 8q24.3 was selected as a MCR from two cases in the present group of t-AML . This MCAlthough no such result was found in our study, 5.6% of the p-AML in other series clearly showed gains or amplifications of this region that contains ETS1 and FLI1 genes. They are members of the ETS gene family that includes genes playing important roles in regulating hematopoiesis, proliferation, differentiation and apoptosis. Interestingly, these gains were found in cases presenting gains of the ETS2 and ERG genes .A 1.48Mb loss was defined as a MCR from three t-AML cases in the present report. The same area is recurrently lost in both types of AML , table 8Although no deletion of this region was found in our series, several reports claimed a loss of this MCR in 12% of t-AML and 3% of p-AML. This region, among several genes, exhibits loss of the TP53 tumor suppressor gene This MCR, that we found in a single case of t-AML , is the Loss of different parts of chromosome 18 were highly recurrent in p-AML . In our This 40kb small MCR contains RUNX1 that was lost in 4 t-AML cases in the present series. Two patients had lost their two alleles either through double losses or deletion and D171N mutation. This mutation has been claimed to result in the proliferation of immature myeloid cells, an enhanced capacity for self-renewal, and the proliferation of primitive progenitors Patient t-29 had a normal karyotype Three deleted cases were exposed to multi-agent chemotherapy with alkylating agents. They exhibited a 7q-/-7 as previously reported RUNX1, the subunit alpha of the Core Binding Factor (CBF), a heterodimeric transcription factor, plays a key role in the regulation of hematopoietic stem cell proliferation and differentiation. It is one of the most frequent targets of chromosome translocations via nearly forty different partners A germline mono-allelic mutation or deletion of RUNX1 Point mutations of RUNXI have been described in radiation-induced MDS Haploinsufficiency of RUNX1 leads to the loss of function of this gene (probably a tumor suppressor function). In the case of t-AML, the various point mutations are localized at the N-terminal, the DNA binding site Distal 21q11.2qter polysomy was observed in three cases , two of The first amplicon  and 6 frThe 1Mb-long second amplicon, observed in cases p-21 and p-24 was found to occur frequently in p-AML  and mostERG and ETS2 were amplified in p-AML with complex karyotypes  and ETS2High resolution cytogenomics obtained by aCGH and similar techniques already published allowed us to characterize numerous untargeted non random chromosome abnormalities. This work supports the hypothesis that they can be classified into several categories: abnormalities common to all AML (e.g. 8q24.3 gain or 17q11.2 deletion involving NF1); those more frequently found in t-AML (e.g. 7q21 or 7q33 deletions or even the specific gain of HOX genes); and those specifically found in p-AML .The genes involved in AML MCRs are often very well known in leukemogenesis but many others need to be explored.