scholarly journals The actin nucleation factor JMY is a negative regulator of neuritogenesis

2011 ◽  
Vol 22 (23) ◽  
pp. 4563-4574 ◽  
Author(s):  
Elif Nur Firat-Karalar ◽  
Peter P. Hsiue ◽  
Matthew D. Welch
Keyword(s):  
Cell Lines ◽  
Regulatory Protein ◽  
Neuronal Cell ◽  
Full Length ◽  
Negative Regulator ◽  
Neurite Formation ◽  
A Minor ◽  
And Function ◽  
In Cells

Junction-mediating and regulatory protein (JMY) is a p53 cofactor that was recently shown to nucleate actin assembly by a hybrid mechanism involving tandem actin monomer binding and Arp2/3 complex activation. However, the regulation and function of JMY remain largely uncharacterized. We examined the activity of JMY in vitro and in cells, its subcellular distribution, and its function in fibroblast and neuronal cell lines. We demonstrated that recombinant full-length JMY and its isolated WASP homology 2 domain, connector, and acidic region (WWWCA) have potent actin-nucleating and Arp2/3-activating abilities in vitro. In contrast, the activity of full-length JMY, but not the isolated WWWCA domain, is suppressed in cells. The WWWCA domain is sufficient to promote actin-based bead motility in cytoplasmic extracts, and this activity depends on its ability to activate the Arp2/3 complex. JMY is expressed at high levels in brain tissue, and in various cell lines JMY is predominantly cytoplasmic, with a minor fraction in the nucleus. Of interest, silencing JMY expression in neuronal cells results in a significant enhancement of the ability of these cells to form neurites, suggesting that JMY functions to suppress neurite formation. This function of JMY requires its actin-nucleating activity. These findings highlight a previously unrecognized function for JMY as a modulator of neuritogenesis.

scholarly journals GFAT1 phosphorylation by AMPK promotes VEGF-induced angiogenesis

2017 ◽  
Vol 474 (6) ◽  
pp. 983-1001 ◽  
Author(s):  
Darya Zibrova ◽  
Franck Vandermoere ◽  
Olga Göransson ◽  
Mark Peggie ◽  
Karina V. Mariño ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Energy Homeostasis ◽  
Vascular Dysfunction ◽  
Negative Regulator ◽  
Label Free ◽  
Hexosamine Biosynthesis ◽  
Underlying Mechanisms ◽  
And Function ◽  
In Cells

Activation of AMP-activated protein kinase (AMPK) in endothelial cells regulates energy homeostasis, stress protection and angiogenesis, but the underlying mechanisms are incompletely understood. Using a label-free phosphoproteomic analysis, we identified glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1) as an AMPK substrate. GFAT1 is the rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP) and as such controls the modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc). In the present study, we tested the hypothesis that AMPK controls O-GlcNAc levels and function of endothelial cells via GFAT1 phosphorylation using biochemical, pharmacological, genetic and in vitro angiogenesis approaches. Activation of AMPK in primary human endothelial cells by 5-aminoimidazole-4-carboxamide riboside (AICAR) or by vascular endothelial growth factor (VEGF) led to GFAT1 phosphorylation at serine 243. This effect was not seen when AMPK was down-regulated by siRNA. Upon AMPK activation, diminished GFAT activity and reduced O-GlcNAc levels were observed in endothelial cells containing wild-type (WT)-GFAT1 but not in cells expressing non-phosphorylatable S243A-GFAT1. Pharmacological inhibition or siRNA-mediated down-regulation of GFAT1 potentiated VEGF-induced sprouting, indicating that GFAT1 acts as a negative regulator of angiogenesis. In cells expressing S243A-GFAT1, VEGF-induced sprouting was reduced, suggesting that VEGF relieves the inhibitory action of GFAT1/HBP on angiogenesis via AMPK-mediated GFAT1 phosphorylation. Activation of GFAT1/HBP by high glucose led to impairment of vascular sprouting, whereas GFAT1 inhibition improved sprouting even if glucose level was high. Our findings provide novel mechanistic insights into the role of HBP in angiogenesis. They suggest that targeting AMPK in endothelium might help to ameliorate hyperglycaemia-induced vascular dysfunction associated with metabolic disorders.

scholarly journals Grafts Derived from an α-Synuclein Triplication Patient Mediate Functional Recovery but Develop Disease-Associated Pathology in the 6-OHDA Model of Parkinson’s Disease

2020 ◽  
pp. 1-14
Author(s):  
Shelby Shrigley ◽  
Fredrik Nilsson ◽  
Bengt Mattsson ◽  
Alessandro Fiorenzano ◽  
Janitha Mudannayake ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Cell Lines ◽  
Functional Recovery ◽  
Embryonic Stem ◽  
Hesc Line ◽  
Alternative Source ◽  
And Function

Background: Human induced pluripotent stem cells (hiPSCs) have been proposed as an alternative source for cell replacement therapy for Parkinson’s disease (PD) and they provide the option of using the patient’s own cells. A few studies have investigated transplantation of patient-derived dopaminergic (DA) neurons in preclinical models; however, little is known about the long-term integrity and function of grafts derived from patients with PD. Objective: To assess the viability and function of DA neuron grafts derived from a patient hiPSC line with an α-synuclein gene triplication (AST18), using a clinical grade human embryonic stem cell (hESC) line (RC17) as a reference control. Methods: Cells were differentiated into ventral mesencephalic (VM)-patterned DA progenitors using an established GMP protocol. The progenitors were then either terminally differentiated to mature DA neurons in vitro or transplanted into 6-hydroxydopamine (6-OHDA) lesioned rats and their survival, maturation, function, and propensity to develop α-synuclein related pathology, were assessed in vivo. Results: Both cell lines generated functional neurons with DA properties in vitro. AST18-derived VM progenitor cells survived transplantation and matured into neuron-rich grafts similar to the RC17 cells. After 24 weeks, both cell lines produced DA-rich grafts that mediated full functional recovery; however, pathological changes were only observed in grafts derived from the α-synuclein triplication patient line. Conclusion: This data shows proof-of-principle for survival and functional recovery with familial PD patient-derived cells in the 6-OHDA model of PD. However, signs of slowly developing pathology warrants further investigation before use of autologous grafts in patients.

Survival and Function of Fibroblasts Stored as Stock Cultures at a Non-freezing Temperature

1993 ◽  
Vol 21 (2) ◽  
pp. 206-209
Author(s):  
Anders H. G. Andrén ◽  
Anders P. Wieslander
Keyword(s):  
Cell Growth ◽  
Cell Line ◽  
Cell Lines ◽  
Fibroblast Cell ◽  
Freezing Temperature ◽  
Mouse Fibroblast ◽  
Toxic Response ◽  
Normal Manner ◽  
And Function

Cytotoxicity, measured as inhibition of cell growth of cultured cell lines, is a widely used method for testing the safety of biomaterials and chemicals. One major technical disadvantage with this method is the continuous routine maintenance of the cell lines. We decided to investigate the possibility of storing stock cultures of fibroblasts (L-929) in an ordinary refrigerator as a means of reducing the routine workload. Stock cultures of the mouse fibroblast cell line L-929 were prepared in plastic vials with Eagle's minimum essential medium. The vials were stored in a refrigerator at 4–10°C for periods of 7–31 days. The condition of the cells after storage was determined as cell viability, cell growth and the toxic response to acrylamide, measured as cell growth inhibition. We found that the L-929 cell line can be stored for 2–3, weeks with a viabilty > 90% and a cell growth of about 95%, compared to L-929 cells grown and subcultured in the normal manner. The results also show that the toxic response to acrylamide, using refrigerator stored L-929 cells, corresponds to that of control L-929 cells. We concluded that it is possible to store L-929 cells in a refrigerator for periods of up to 3 weeks and still use the cells for in vitro cytotoxic assays.

Production of the CYS3 regulator, a bZIP DNA-binding protein, is sufficient to induce sulfur gene expression in Neurospora crassa

1992 ◽  
Vol 12 (4) ◽  
pp. 1568-1577
Author(s):  
J V Paietta
Keyword(s):  
Gene Expression ◽  
Neurospora Crassa ◽  
Structural Gene ◽  
Control Point ◽  
Regulatory Protein ◽  
Negative Regulator ◽  
Temperature Sensitive ◽  
Cys3 Protein

The cys-3+ gene of Neurospora crassa encodes a bZIP (basic region-leucine zipper) regulatory protein that is essential for sulfur structural gene expression (e.g., ars-1+). Nuclear transcription assays confirmed that cys-3+ was under sulfur-regulated transcriptional control and that cys-3+ transcription was constitutive in sulfur controller (scon)-negative regulator mutants. Given these results, I have tested whether expression of cys-3+ under high-sulfur (repressing) conditions was sufficient to induce sulfur gene expression. The N. crassa beta-tubulin (tub) promoter was fused to the cys-3+ coding segment and used to transform a cys-3 deletion mutant. Function of the tub::cys-3 fusion in homokaryotic transformants grown under high-sulfur conditions was confirmed by Northern (RNA) and Western immunoblot analysis. The tub::cys-3 transformants showed arylsulfatase gene expression under normally repressing high-sulfur conditions. A tub::cys-3ts fusion encoding a temperature-sensitive CYS3 protein was used to confirm that the induced structural gene expression was due to CYS3 protein function. Constitutive CYS3 production did not induce scon-2+ expression under repressing conditions. In addition, a cys-3 promoter fusion to lacZ showed that CYS3 production was sufficient to induce its own expression and provides in vivo evidence for autoregulation. Finally, an apparent inhibitory effect observed with a strain carrying a point mutation at the cys-3 locus was examined by in vitro heterodimerization studies. These results support an interpretation of CYS3 as a transcriptional activator whose regulation is a crucial control point in the signal response pathway triggered by sulfur limitation.

scholarly journals Identification of Prostaglandin F2 Receptor Negative Regulator (PTGFRN) as an internalizable target in cancer cells for antibody-drug conjugate development

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0246197
Author(s):  
Jorge Marquez ◽  
Jianping Dong ◽  
Chun Dong ◽  
Changsheng Tian ◽  
Ginette Serrero
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Negative Regulator ◽  
Target Validation ◽  
Antibody Drug

Antibody-drug conjugates (ADC) are effective antibody-based therapeutics for hematopoietic and lymphoid tumors. However, there is need to identify new targets for ADCs, particularly for solid tumors and cancers with unmet needs. From a hybridoma library developed against cancer cells, we selected the mouse monoclonal antibody 33B7, which was able to bind to, and internalize, cancer cell lines. This antibody was used for identification of the target by immunoprecipitation and mass spectrometric analysis, followed by target validation. After target validation, 33B7 binding and target positivity were tested by flow cytometry and western blot analysis in several cancer cell lines. The ability of 33B7 conjugated to saporin to inhibit in vitro proliferation of PTFRN positive cell lines was investigated, as well as the 33B7 ADC in vivo effect on tumor growth in athymic mice. All flow cytometry and in vitro internalization assays were analyzed for statistical significance using a Welsh’s T-test. Animal studies were analyzed using Two-Way Analysis of Variance (ANOVA) utilizing post-hoc Bonferroni analysis, and/or Mixed Effects analysis. The 33B7 cell surface target was identified as Prostaglandin F2 Receptor Negative Regulator (PTGFRN), a transmembrane protein in the Tetraspanin family. This target was confirmed by showing that PTGFRN-expressing cells bound and internalized 33B7, compared to PTGFRN negative cells. Cells able to bind 33B7 were PTGFRN-positive by Western blot analysis. In vitro treatment PTGFRN-positive cancer cell lines with the 33B7-saporin ADC inhibited their proliferation in a dose-dependent fashion. 33B7 conjugated to saporin was also able to block tumor growth in vivo in mouse xenografts when compared to a control ADC. These findings show that screening antibody libraries for internalizing antibodies in cancer cell lines is a good approach to identify new cancer targets for ADC development. These results suggest PTGFRN is a possible therapeutic target via antibody-based approach for certain cancers.

The Expression of CD56 Is Frequently Accompanied with the Expression of Neuronal Cell Markers and Its Down-Regulation Is Induced by IL-6 in Human Myeloma Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3534-3534
Author(s):  
Mohd S. Iqbal ◽  
Ken-ichiro Otsuyama ◽  
Karim Shamsasenjan ◽  
Saeid Abroun ◽  
Jakia Amin ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Cell Lines ◽  
Cell Lineage ◽  
Neuronal Cell ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Cell Markers ◽  
Phenotypic Changes ◽  
Human Myeloma Cells

Abstract Human myeloma cells have the marked phenotypic heterogeneity of surface marker expressions, possibly because of loss of PAX-5 expression. Especially, ectopic expression of CD56, one of non-B cell lineage markers, is frequently detected on primary myeloma cells from more than 80% patients with overt myeloma. However, only 2 (NOP2 and AMO1) out of 10 myeloma cell lines were CD56(+). In primary myeloma cells as well as CD56(−) myeloma cell lines, the treatment with forskolin could induce the expression of CD56 in the in vitro culture. In most CD56(+) primary myeloma cells as well as myeloma cell lines, the expressions of neuronal cell markers such as neuron specific enolase (NSE), nestin, β-tubulin III or chromogranin A were found coincidentally. By gene expression profiling, CD56(+) myeloma cell lines showed the marked expressions of transcription factors involved in neuronal cell lineage. On the other hand, addition of IL-6 down-regulated the expression of CD56 in CD56(+) myeloma cell lines in the in vitro culture. In 13 out of 60 patients with overt myeloma, these myeloma cells showed CD56(−) and their values of plasma CRP were significantly increased and MPC-1(−)CD45(+) immature myeloma cells were also increased compared to those in CD56(+) myeloma cases. Therefore, these results indicate that the expression of CD56 is possibly due to phenotypic changes into neuronal cell lineage, and IL-6 can block these phenotypic changes, keeping PAX-5(−) myeloma cells being uncommitted cells to any lineage.

Latexin: A Potential Tumor Suppressor.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1805-1805
Author(s):  
Ying Liang ◽  
Gary Van Zant
Keyword(s):  
Cell Proliferation ◽  
Stem Cell ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Negative Regulator ◽  
Stem Cell Population ◽  
Western Blots ◽  
Hematopoietic Stem ◽  
Cell Counts

Abstract We recently found that latexin is a negative regulator of the size of the hematopoietic stem cell population in mice. It acts by increasing apoptosis and decreasing cell proliferation. This 29 kD protein bears a strong structural resemblance to tazarotene-induced gene 1 (TIG1), a tumor suppressor down-regulated in a variety of cancers. The structural similarity and close genetic linkage led us to hypothesize that latexin also may have tumor suppressor properties. We found that latexin was down-regulated in a variety of human leukemias and lymphomas as determined by a survey of malignant cell lines and by analysis of CD34+ cells isolated from the blood and marrow of patients diagnosed with these malignancies and presenting with very high white cell counts. Bisulfite sequencing revealed that methylation of CpG dinucleotides in the latexin promoter at least partially accounted for latexin down-regulation. 5-aza-deoxycytidine treatment reinitiated or significantly increased latexin expression in K562, Molt4, CRF-CEM, Jurkat, U937, HL60, KG-1, and Sup B15 cell lines. To test the hypothesis that ectopic latexin expression in tumor cells would inhibit their growth, we developed a retrovirus-based expression vector with which we infected the murine lymphoma cell lines, WEHI231 and A20, neither of which contained significant latexin levels by Western blot. A vector containing GFP, but not latexin, was used to infect control cells. In triplicate experiments, the growth of both cell lines in vitro was inhibited an average 48% by infection with the latexin vector. Western blots revealed that latexin was durably expressed throughout the 2-week culture period at 2- to 4-fold the level expressed in normal T and B cells. As we found in our normal stem cell studies, latexin caused growth inhibition of lymphoma cells by significantly increasing apoptosis by 6-fold, and by suppressing cell proliferation by 2-fold. To test whether tumor inhibition extended to lymphomas in vivo, we injected either control or latexin vector-infected A20 cells subcutaneously in the flanks of BALB/c mice. Three weeks following adoptive transfer of identical numbers of cells, in duplicate experiments the latexin-expressing cells developed tumors only half the volume of those caused by the control cells. These results are consistent with a tumor suppressor role for latexin and suggest that latexin, or molecular mimics thereof, may have clinical efficacy in the treatment of malignancies.

Multiple Myeloma (MM) Highly Expresses CGEN-928 and the Anti-CGEN-928 TM21 Antibody Markedly Inhibits the Growth of MM Cells and Enhances the Anti-Myeloma Effects of Dexamethasone, Melphalan and Bortezomib

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4067-4067
Author(s):  
Haiming Chen ◽  
Mingjie Li ◽  
Cathy Wang ◽  
Jessica Wang ◽  
Eric Sanchez ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Tumor Cells ◽  
Cell Lines ◽  
Polyclonal Antibody ◽  
Flow Cytometric Analysis ◽  
Therapeutic Options ◽  
Treatment Modalities ◽  
In Cells

Abstract Abstract 4067 Although patients with multiple myeloma (MM) initially respond to current treatment modalities, it remains an incurable disease. Many new therapeutic options have become available during the past several years but nearly all patients develop resistance to currently available therapeutic options. In addition, there is no tumor marker that is uniformly expressed in all MM cells although CD138 is considered to be present on the surface of tumor cells in most cases of MM but generally is only present in a subset of the patients' tumor population and may be absent in the most resistant part of the tumor clone. In order to address these unmet clinical needs, we queried Compugen's MED Platform, an expression database which covers over 40,000 microarray experiments, for genes overexpressed in B cell-derived malignancies including MM and that exhibit low expression levels in normal cells and tissues. One of the most prominent candidates was CGEN-928, which was validated as over-expressed in MM at the mRNA level using an independent panel of both hematological malignancies and normal tissues. In this study, we first investigated whether the previously unidentified membrane antigen CGEN-928 is expressed in cells from human MM cell lines, human MM xenografts and fresh bone marrow (BM) aspirates derived from MM patients using flow cytometric analysis and immunohistochemical staining with the anti-CGEN-928 TM21 polyclonal antibody (Compugen Ltd, Tel Aviv, Israel). Using this antibody, we found that CGEN-928 was highly expressed in cells from the MM1s, U266 and RPMI8226 MM cell lines. Next, we examined CGEN-928 antigen expression in fresh tumor cells from BM aspirates from 17 MM patients and also showed high expression of CGEN-928. Notably, expression of this antigen was not only found on CD138+ MM cells but also on MM tumor cells lacking CD138 expression. We also examined the expression of CGEN-928 using our human MM xenograft models LAGκ-1A (bortezomib-sensitive), LAGκ-1B (bortezomib-resistant) and LAGλ-1 (melphalan-resistant). The bortezomib-sensitive MM tumor LAGκ-1A expresses CD138 whereas the bortezomib-resistant version LAGκ-1B developed from the same patient after the patient developed bortezomib reisistance does not express CD138. Cells from all three tumor types showed high levels of reactivity with the TM21 antibody. Similar to the fresh MM BM samples, CGEN-928 expression was not only found on CD138+ MM cells but also on CD138- tumor cells derived from these human MM xenografts. Because this molecule is highly expressed on MM cells, we hypothesized that the anti-CGEN-928 antibody may show anti-MM effects and enhance the anti-MM effects of other anti-MM drugs. To evaluate this, we examined the effect of the TM21 antibody alone and in combination with dexamethasone, melphalan and bortezomib in vitro using cell proliferation MTT assays. Anti-TM21 polyclonal antibody (100 mg/ml) decreased MM tumor cell proliferation and increased apoptosis in cells from the MM1s, RPMI8226 and U266 cell lines. Next, we determined the effects of combining the anti-CGEN-928 antibody with bortezomib, melphalan or dexamethasone on MM1s cells. Cell proliferation assays demonstrated marked enhanced anti-proliferative effects when CGEN-928 antibody at concentrations of 5, 10, 50 and 100 mg/ml was combined with bortezomib, melphalan or dexamethasone. Further investigations are defining the mechanisms and signal transduction pathways that produce the anti-MM effects of CGEN-928. These preliminary studies suggest that the CGEN-928 antigen is highly expressed in MM and treatment with an anti-CGEN-928 polyclonal antibody produces anti-MM effects alone and in combination with other anti-MM agents; and thus, this antigen may be a target for the treatment of multiple myeloma. Currently, a monoclonal anti-CGEN-928 antibody is in development that will be used by our group to evaluate its anti-MM effects both in vitro and in vivo using our SCID-hu models of human MM. Disclosures: Levy: Compugen Ltd.: Employment. Dassa:Compugen Ltd.: Employment. Cojocaru:Compugen Ltd.: Employment. Berenson:Compugen Ltd.: Research Funding. Levine:Compugen Ltd.: Employment.

scholarly journals Peripheral-Type Benzodiazepine Receptor-Mediated Action of Steroidogenic Acute Regulatory Protein on Cholesterol Entry into Leydig Cell Mitochondria

2005 ◽  
Vol 19 (2) ◽  
pp. 540-554 ◽  
Author(s):  
Thierry Hauet ◽  
Zhi-Xing Yao ◽  
Himangshu S. Bose ◽  
Christopher T. Wall ◽  
Zeqiu Han ◽  
...  
Keyword(s):  
Regulatory Protein ◽  
Benzodiazepine Receptor ◽  
Steroidogenic Acute Regulatory Protein ◽  
Rate Determining Step ◽  
Human Choriogonadotropin ◽  
Leydig Tumor Cells ◽  
Peripheral Type ◽  
Steroidogenic Acute Regulatory ◽  
And Function

Abstract Hormone-induced steroid biosynthesis begins with the transfer of cholesterol from intracellular stores into mitochondria. Steroidogenic acute regulatory protein (StAR) and peripheral-type benzodiazepine receptor (PBR) have been implicated in this rate-determining step of steroidogenesis. MA-10 mouse Leydig tumor cells were treated with and without oligodeoxynucleotides (ODNs) antisense to PBR and StAR followed by treatment with saturating concentrations of human choriogonadotropin. Treatment with ODNs antisense but not missense for both proteins inhibited the respective protein expression and the ability of the cells to synthesize steroids in response to human choriogonadotropin. Treatment of the cells with either ODNs antisense to PBR or a transducible peptide antagonist to PBR resulted in inhibition of the accumulation of the mature mitochondrial 30-kDa StAR protein, suggesting that the presence of PBR is required for StAR import into mitochondria. Addition of in vitro transcribed/translated 37-kDa StAR or a fusion protein of Tom20 (translocase of outer membrane) and StAR (Tom/StAR) to mitochondria isolated from control cells increased pregnenolone formation. Mitochondria isolated from cells treated with ODNs antisense, but not missense, to PBR failed to form pregnenolone and respond to either StAR or Tom/StAR proteins. Reincorporation of in vitro transcribed/translated PBR, but not PBR missing the cholesterol-binding domain, into MA-10 mitochondria rescued the ability of the mitochondria to form steroids and the ability of the mitochondria to respond to StAR and Tom/StAR proteins. These data suggest that both StAR and PBR proteins are indispensable elements of the steroidogenic machinery and function in a coordinated manner to transfer cholesterol into mitochondria.

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