NTAL Potentiates Glucocorticosteroid-Induced Apoptosis in T-ALL – in Vivo Observations and Experimental Model

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5043-5043
Author(s):  
Karel Svojgr ◽  
Tatiana Burjanivova ◽  
Martina Vaskova ◽  
Tomas Kalina ◽  
Tomas Brdicka ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Line ◽  
Jurkat Cell ◽  
Annexin V ◽  
Signal Propagation ◽  
Living Cells ◽  
Negative Control ◽  
Jurkat Cells ◽  
Wild Type ◽  
Jurkat Cell Line

Abstract PAG, LAT, NTAL and LIME belong to category of transmembrane adaptor proteins (TRAPs). They do not possess an enzymatic or kinase function, but they are involved in mediation of signal transmission from surface receptors to cell nucleus. We propose that some of them are engaged in development or maintenance of leukaemia. We have previously demonstrated that expression status of TRAPs at mRNA level is specific in some subgroups of childhood ALL, particularly in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) with the TEL/AML1 fusion gene. Furthermore, we have described that variable expression of NTAL mRNA is related to the response to initial glucocorticosteroid pre-phase in the treatment of childhood T-ALL; patients with high NTAL levels show better treatment response compared to the low-NTAL cases. In the current study, we aimed to prove experimentally that different levels of NTAL protein influence response of leukaemic T cells to glucocorticosteroids. In the wild type Jurkat cells (human T-cell leukaemia cell line) the NTAL protein is undetectable. For the in-vitro experiments we created a derivative Jurkat cell line transfected with the NTAL construct. In the derivative cell line, the NTAL positivity at both mRNA and protein level was verified using RT-PCR and Western blotting. The derivative cell line in a cell culture behaves similar as wild type Jurkat cell line. Transfectants and wild type Jurkat cells were incubated with methylprednisolone, dexamethasone or with solvent alone (H2O) as a negative control. Using flow cytometry we determined a percentage of surviving cells after 24, 48 and 72 hours of treatment. Cells were stained with Annexin V and DAPI and living cells were defined as Annexin V, DAPI negative. At each time point, the number of living cells in the negative control was set to 100%. After 48 hours of dexamethasone treatment the number of surviving cells in the Jurkat wild-type was higher by 12% compared to the Jurkat cells expressing NTAL. After 72 hours this difference was even more prominent reaching 46%. The same effect of methylprednisolone treatment was less pronounced (non-significant difference at 48 hours and 5% difference at 72 hours). Based on our experimental data we propose that NTAL acts in T-cells as a putative tumour suppressor. A plausible mechanism of its function is that NTAL competitively inhibits another adaptor LAT, which is required for signal propagation to the nucleus in T-cells. NTAL and LAT can compete for its localisation in proper “lipid rafts” or for palmitoylation. Our data can be used for subsequent functional analysis of signaling pathways in leukaemic blasts as well as in physiological lymphoid cells.

scholarly journals Validation of a method evaluating T cell metabolic potential in compliance with ICH Q2 (R1)

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Patricia Mercier-Letondal ◽  
Chrystel Marton ◽  
Yann Godet ◽  
Jeanne Galaine
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Jurkat Cell ◽  
Cellular Therapy ◽  
Adoptive Cell Therapy ◽  
Metabolic Potential ◽  
Experimental Conditions ◽  
Jurkat Cell Line ◽  
Set Up

Abstract Background Metabolic cell features are able to give reliable information on cell functional state. Thus, metabolic potential assessment of T cells in malignancy setting represents a promising area, especially in adoptive cell therapy procedures. Easy to set up and convenient Seahorse technology have recently been proposed by Agilent Technologies and it could be used to monitor T cells metabolic potential. However, this method demonstrates an inter-assay variability and lacks practices standardization. Results We aimed to overcome these shortcomings thanks to a lymphoblastic derived JURKAT cell line seeding in each experiment to standardize the Seahorse process. We used an adapted XF Cell MitoStress Kit protocol, consisting in the evaluation of basal, stressed and maximal glycolysis and oxidative phosphorylation related parameters, through sequential addition of oligomycin and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) to a glucose containing medium. Data were acquired and analyzed through Agilent Seahorse XFe96 analyzer. Indeed, we validated this method in the light of ICH Q2 (R1) guidelines. We were able to confirm the specificity and accuracy of the method. We also demonstrated the precision, linearity and range of the method in our experimental conditions. Conclusion The validation of the method consisting in a JURKAT cell line experimental incorporation as a control material contributes to improve the Seahorse technology’s robustness. These results lay the groundwork for the implementation of this technology to optimize T cell based cellular therapy products production process and monitoring.

scholarly journals Validation of a method evaluating T cell metabolic potential in compliance with ICH Q2 (R1)

2020 ◽  
Author(s):  
Patricia Letondal ◽  
Chrystel Marton ◽  
Yann Godet ◽  
Jeanne Galaine
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Internal Control ◽  
Jurkat Cell ◽  
Cellular Therapy ◽  
Adoptive Cell Therapy ◽  
Metabolic Potential ◽  
Experimental Conditions ◽  
Jurkat Cell Line

Abstract Background Metabolic cell features are able to give reliable information on cell functional state. Thus, metabolic potential assessment of T cells in malignancy setting represents a promising area, especially in adoptive cell therapy procedures. Easy to set up and convenient Seahorse technology have recently been proposed by Agilent Technologies and it could be used to monitor T cells metabolic potential. However, this method demonstrates an inter-assay variability and lacks practices standardization. Results We aimed to overcome these shortcomings thanks to a lymphoblastic derived JURKAT cell line seeding in each experiment to standardize the Seahorse process. We used an adapted XF Cell MitoStress Kit protocol, consisting in the evaluation of basal, stressed and maximal glycolysis and oxidative phosphorylation related parameters, through sequential addition of oligomycin and FCCP to a glucose containing medium. Data were acquired and analyzed through Agilent Seahorse XFe96 analyzer. Indeed, we validated this method in the light of ICH Q2 (R1) guidelines. We were able to confirm the specificity and accuracy of the method. We also demonstrated the precision, linearity and range of the method in our experimental conditions. Conclusion The validation of the method consisting in a JURKAT cell line experimental incorporation as internal control contributes to improve the Seahorse technology’s robustness. These results lay the groundwork for the implementation of this technology to optimize T cell based cellular therapy products production process and monitoring.

scholarly journals Association of proliferating cell nuclear antigen with cyclin-dependent kinases and cyclins in normal and transformed human T lymphocytes

Blood ◽  
1994 ◽  
Vol 84 (10) ◽  
pp. 3413-3421 ◽  
Author(s):  
A Szepesi ◽  
EW Gelfand ◽  
JJ Lucas
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Cell Line ◽  
Proliferating Cell Nuclear Antigen ◽  
Kinase Activity ◽  
Jurkat Cells ◽  
Nuclear Antigen ◽  
Human T Lymphocytes ◽  
Normal Human ◽  
Proliferating Cell

Abstract The proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase delta and appears to be needed for both DNA synthesis and DNA repair. It is present in low amount in resting normal human T lymphocytes and, upon mitogenic stimulation with phorbol dibutyrate and ionomycin, begins to increase in mid-G1 phase, approximately 12 to 15 hours before entry into S phase. PCNA continues to increase in amount throughout the cell cycle and remains high in proliferating cultures. PCNA was extracted from activated normal T cells and from the transformed T-lymphoblastoid cell line Jurkat by a method that recovered approximately 98% of total cellular PCNA but yet retained its associations with other proteins. PCNA immunoprecipitates possessed H1 histone kinase activity, which increased in parallel with increasing cellular content of PCNA. Both the cdc2 and cdk2 kinases were found associated with PCNA in normal T cells, in amounts consistent with detected kinase activity. The results indicate that PCNA is not an inhibitory molecule of cdk/cyclin activity. Both normal and transformed T cells contained PCNA in association with cdk2, cdk4, cdk5, and cdk6, with the amount of PCNA associated with these molecules increasing in the order listed. Relatively high amounts of PCNA were also found associated with cyclins D2 and D3, the major cyclin partners of cdk6 in T cells. Though detected in normal cells, PCNA/cdc2 complexes were present in exceedingly low amount, if at all, in Jurkat cells. This cell line appeared to contain more of nearly all of the cdk and cyclin molecules analyzed, but there seemed to be little difference in the patterns of association of these molecules with PCNA in the cell line as compared with normal human T cells.

C1GALT1C1 (COSMC) Mutations in Four Individuals with Tn+ Phenotype.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1727-1727
Author(s):  
Vanja Karamatic Crew ◽  
Carole Green ◽  
Stephen Parsons ◽  
Belinda K. Singleton ◽  
Geoff Daniels ◽  
...  
Keyword(s):  
Cell Line ◽  
Dna Analysis ◽  
Single Point ◽  
Jurkat Cells ◽  
Erythroid Cell ◽  
Single Point Mutation ◽  
Wild Type ◽  
Membrane Glycoproteins ◽  
Hemopoietic Cells ◽  
Elevated Expression

Abstract Tn is a cryptantigen located on O-linked oligosaccharides of cell membrane glycoproteins and is composed of N-acetylgalactosamine a -linked to serine or threonine of the protein backbone. Tn is expressed on hemopoietic cells of individuals with the rare idiopathic Tn syndrome, characterized by a variable pattern of Tn expression suggestive of its somatic and clonal origin. Tn is also associated with overexpression in several autoimmune diseases and malignancies. Tn is a bioprecursor of the T cryptantigen and subsequently the disialotetrasaccharide units typical of O-linked oligosaccharides of RBC sialoglycoproteins. Tn results from defective oligosaccharide biosynthesis caused by the malfunction of T-synthase, a b1,3-galactosyltransferase. Recent evidence suggested that the activity of T-synthase is dependent on a molecular chaperone, Cosmc (Ju and Cummings, PNAS2002;99:16613–18). In view of this evidence, we investigated whether Cosmc is required for T-synthase activity and ultimately for Tn phenotype by obtaining material from 4 apparently healthy, unrelated Caucasian individuals with Tn phenotype. Case 1 was 93.9% Tn+ on an EBV-transformed lymphoblastoma cell line. Analysis of the Cosmc gene, C1GALT1C1, showed a homozygous 428C>T, Ala143Val mutation and Case 1 showed a complete lack of expression of C1GALT1C1 cDNA. Case 2 revealed no C1GALT1C1 mutations in DNA extracted from plasma, but in DNA of lymphocyte origin an apparent heterozygous 454G>A, Glu152Lys change was observed, in agreement with Ju and Cummings (Nature2005, 437:1252). When Case 2 hemopoietic progenitor cells were expanded into the erythroid cell line, the mutation appeared homozygous. Tn expression varied from 76.1% in lymphocytes, 90.7% in RBCs to 96.9% in erythroblasts. Case 3 showed 19.4% Tn+ lymphocytes, compared to 97.0% Tn+ RBCs. In Case 3 we found a single point mutation 577T>C, Ser193Pro. Case 4 exhibited 46.5% Tn+ lymphocytes, 96.0% Tn+ RBCs and 90.2% Tn+ erythroblasts. DNA analysis revealed 3G>C, converting the translation-initiating methionine to isoleucine and predicting the loss of first 12 amino acids of the protein, potentially altering its morphology. The mutations found in Cases 2–4 reflected the clonal nature of Tn, appearing heterozygous in DNA of lymphocyte origin and homozygous in DNA of erythroid origin. To confirm that the observed mutations are indeed responsible for Tn phenotype, pBabe puro vector with Tn or wild type C1GALT1C1 inserts was transfected into Jurkat cells. Untransfected cells and cells transfected with bare vector expressed Tn. Jurkat cells transfected with wild-type C1GALT1C1 were Tn-negative while those transfected with C1GALT1C1 from Cases 2–4 expressed Tn. From this evidence we postulate that Cosmc is directly involved in the expression of Tn phenotype. To investigate the involvement of other genes, we performed expression profiling of 3 Tn and 4 control samples hybridized to HG-U133A arrays. A list of 100 up-regulated and 173 down-regulated genes, with 1.5× fold difference in expression, was obtained. Some genes, relating to erythrocyte development/heme biosynthesis were upregulated, while down-regulated genes were related to cholesterol/lipid metabolism. Real-time Q-PCR on six differentially expressed genes of interest, down-regulated FABP5, CYP1B1 and LRP8 and up-regulated AQP1, AQP3 and EPB42, confirmed the microarray results, and elevated expression of AQP3 on Tn + RBCs was detected serologically. The effects of C1GALT1C1 mutations are wider than Tn expression on hemopoietic cells.

Inhibition of DLEU1 Following siRNA Transfection in Burkett’s Lymphoma (BL): Implication for Tumor Repressor Role of DLEU1 in C-Myc-Activated BL Lymphomagenesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5335-5335
Author(s):  
Nancy Day ◽  
Janet Ayello ◽  
Ian Waxman ◽  
Evan Shereck ◽  
Catherine McGuinn ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Promoter Region ◽  
Transfection Efficiency ◽  
Gc Content ◽  
Annexin V ◽  
Suppressor Gene ◽  
Negative Control ◽  
13Q Deletion

Abstract Background: The progress of childhood BL and DLBCL has improved dramatically in the past three decades; however, patients with a 13q-deletion have a significantly poorer outcome (Cairo/Patte et al Blood, 2007 and Patte/Cairo et al Blood, 2007; Poirel/Cairo et al Leukemia 2008). DLEU1, a potential tumor suppressor gene, is located within the 13q-deletion. DLEU1 was reported to be a key gene in the Burkitt classifier genes (Dave/Staudt et al NEJM, 2006) and c-myc binds to the promoter region of DLEU1. DLEU1-network proteins include, among others, E3 ubiquitin-protein ligase (UBR1), Tubulin beta-2C (TUBB2C) and RASSF1A. We previously demonstrated that UBR1, TUBB2C, and RASSF1A, were differentially expressed in BL vs DLBCL patients and cell lines by global gene profiles and real time RT-PCR studies (Day/Cairo et al AACR 2008; Day/Cairo et al ICML 2008). We further demonstrated decreased expression of UBR1 (33.2±4.5% reduction compared to control (p<0.02)) and TUBB2C (30.0±3.5% reduction compared to control (p<0.001)) by DLEU1 gene siRNA knock down, while expression of RASSF1A was not changed (Day/Cairo, et al SIOP 2008). Taken together, these data suggest the hypothesis that DLEU1 interacting with UBR1 may interfere with microtubule function, and therefore act as a tumor repressor in c-myc-activated BL lymphomagenesis, by arrest of the cell cycle at G2/M and subsequent inducion of apoptosis. Objective: In this study, we investigated the role of DLEU1 in regulation of apoptosis in BL by inhibition of DLEU1 gene expression by a DLEU1 siRNA and evaluated it effects on the apoptotic rate in a BL cell line. Methods: The Ramos BL cell line was transiently transfected with a 25-nucleotide modified DLEU1 siRNA (5′-AUACUUGGCAUGAAUGAACUUAUGU-3′ and 3′-UAUGAACCGUACUUACUUGAAUACA-5′). Stealth RNAi whose GC content is similar to that of this DLEU1 siRNA was used as negative control. The transient transfection of DLEU1 siRNA (10 – 20 nM) was achieved using Lipofectamine RNAiMAX. The transfection efficiency of siRNA was evaluated using Alexa Fluor Red Fluorescent Oligo. DLEU1 contents were measured by qRT-PCR with ddCt relative quantitative determination. GAPDH was used as endogenous control. Statistical analysis was conducted by one-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparisons test. To determine the early and late stages of apoptosis, we transfected Ramos BL cells with DLEU1 siRNA, and then incubated cells with Annexin V-FITC and Propidium Iodide for 15 minutes, respectively (BD Pharmingen), followed by FACS using BD LSRII with FACSDiva. Results: The DLEU1 siRNA decreased the expression of DLEU1 RNA (52±13%; p<0.0006). The transfection efficiency of siRNA was 85 – 90%. Comparing to untreated cells, DLEU1 siRNA treatment significantly reduced early apoptosis (16.90±0.37%; p<0.001) and late stage apoptosis (14.70±0.27%; p<0.0001). Conclusion: These results suggest that when DLEU1 gene expression is decreased in BL cells, there is a significant reduction in both early and late apoptosis. The results strongly support a relationship between DLEU1 gene and regulation of BL apoptotic mechanisms. In concert with previous investigations, this data suggests that DLEU1 may function as a tumor growth repressor via UBR1 and TUBB2C-regulated mechanism in the cellular apoptotic process. Since c-myc binds promoter region of DLEU1 and these two genes are a part of the c-myc signaling network, this further underscores the importance of DLEU1 and its network proteins may play in c-myc-activated BL lymphomagenesis.

Effect of a Novel Nucleoside Analogue, Triciribine Phosphate (TCN-P) on Murine Acute Graft-Vrs-Host Disease (aGvHD)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2977-2977
Author(s):  
Edward Dela Ziga ◽  
Jaebok Choi ◽  
Mark Needles ◽  
Julie Ritchey ◽  
John F. DiPersio
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Nucleoside Analogue ◽  
Foxp3 Expression ◽  
Negative Control ◽  
Jurkat Cells ◽  
Acute Graft

Abstract Abstract 2977 BACKGROUND: The successful establishment of donor registries and development of improved conditioning regimens among others, has led to the increased use of hematopoietic stem cell transplant (HSCT) as a key component in the treatment of some malignant and benign hematopoietic/lymphoid disorders as well as some metabolic disorders. Although a potential curative therapy for many hematologic diseases, allogeneic stem cell transplantation is associated with considerable morbidity and mortality primarily from acute graft-versus-host disease (aGvHD). Furthermore, graft-versus-leukemia (GVL) mediated by donor T cells can be abrogated with T cell depletion or suppression in vivo resulting in disease relapse with treatment of aGvHD. Moreso, modern therapies for aGvHD are limited and often toxic, thus there is a need for novel treatments and approaches that control aGvHD without compromising GVL. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor has been shown to decrease the severity of aGvHD (Reddy et al, PNAS 2004) through its effect on pro-inflammatory cytokines while maintaining GVL in a murine GvHD model. Also, previous work from our lab demonstrated that treatment of mice with the hypomethylating agent azacitidine (AzaC) after allogeneic HSCT mitigates aGvHD while preserving GVL by inducing FOXP3 expression in activated non-T regulatory cells (Choi et al, Blood 2010). However, the myelosuppression mediated by AzaC is a potential limitation that results in delayed donor engraftment. This led us to explore alternate options for single or combination drug therapy in the treatment of aGvHD. We screened a library of 2000 chemical agents obtained from the National Institutes of Health. Screening resulted in a single hit identified as Triciribine phosphate (TCN-P), an Akt inhibitor with structural similarity to the nucleoside analogue AzaC. In this experiment, a Foxp3 promoter-luciferase construct was designed and transfected into Jurkat cells. Cells were incubated for 2 days and then treated with three concentrations (0.1uM, 1umM and 10uM) of each chemical agent in the library. Bioluminescence imaging (BLI) was done on day 4 with AzaC as positive control (Choi et al, Blood 2010) and PBS as negative control. Only wells treated with TCN-P 10uM showed a signal, suggesting luciferase activity secondary to the Foxp3-promoter activation. We therefore hypothesized that TCN-P as a single agent or in combination with SAHA and or AzaC would mitigate GvHD by inducing FOXP3 without interfering with engraftment or immune reconstitution. METHODS: Using a C57BL/6(H2b) into Balb/c (H2d) murine MHC mismatch bone marrow transplant (BMT) model, we transplanted 5 × 106 T cell-depleted (TCD) bone marrow cells obtained from C57BL/6 (H2b, CD45.1+) mice into Balb/c (H2d, CD45.2+) mice after 900cGy of TBI. Delayed donor infusions of 2 × 106 pan-T cells/mouse obtained from FOXP3/GFP KI: B6 CD45.2+ H2b mice were infused on Day +11 in order to induce GvHD. Azacitidine 2mg/kg, SAHA 35mg/kg and TCN-P 10mg/kg were injected intraperitoneally every other day from Day +15 to Day +21(total of 4 doses). Acute GvHD was assessed by a standardized scoring developed by Cooke and Ferrara. (Blood, 1996) RESULTS : 1. Using our Foxp3-reporter system, both AzaC and TCN-P induced significant luciferase expression in Jurkat cells. SAHA had no effect. 2. Only AzaC but neither SAHA nor TCN-P induced significant Foxp3 expression in WT bead activated T cells. 3. In vivo, both AzaC 2mg/kg and TCN-P 10mg/kg but not SAHA 35mg/kg significantly improved survival of mice with less weight loss and clinical signs of aGvHD in a MHC mismatched aGvHD model. CONCLUSION: A novel nucleoside analogue TCN-P that was previously FDA approved for treatment of multiple myeloma and structurally related to AzaC, induces Foxp3 using a luciferase reporter construct in Jurkat cells and improves survival in mice after MHC mismatched allogeneic transplant. Though the 100 day survival between TCN-P and PBS (as negative control) in our murine aGvHD model was not quite statistically significant, the findings suggest a therapeutic potential for TCN-P and possibly other Akt inhibitors in the mitigation of aGvHD. Disclosures: No relevant conflicts of interest to declare.

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