scholarly journals Effect of selected bisphenol derivatives on nuclear receptor expression in ovarian cell line COV434

2020 ◽  
Vol 54 (4) ◽  
pp. 275-283
Author(s):  
Alzbeta Bujnakova Mlynarcikova ◽  
Sona Scsukova
Keyword(s):  
Cell Viability ◽  
Mrna Expression ◽  
Cell Line ◽  
Nuclear Receptor ◽  
Ovarian Function ◽  
Thyroid Hormone Receptor ◽  
Biological Effects ◽  
Receptor Expression ◽  
Mrna Levels ◽  
Gene Expressions

Abstract Objectives. Bisphenol A (BPA), as an indispensable plastic additive, has also been proven as an endocrine disruptor associated with adverse health effects including impaired ovarian function and cancer. Due to the restrictions of its usage, several analogs have been employed to replace BPA. Although many studies revealed a harmfulness in the biological effects of BPA analogs, their specific targets remain largely unknown. Nuclear receptors (NRs) may be one of the most important targets of bisphenols. Therefore, in this study, our attention was directed to explore the effect of BPA and its analogs, AF and S, on the mRNA expression of selected NRs involved in the steroidogenic and carcinogenic pathways in the human granulosa cell line COV434. The NRs investigated included: thyroid hormone receptor α (THRA), peroxisome proliferator activating receptor β/δ (PPARD), retinoid X receptor α (RXRA), chicken ovalbumin upstream promoter-transcription factor II (COUPTFII), nuclear receptor-related protein 1 (NURR1), and liver receptor homolog-1 (LRH1). Methods. COV434 cells were treated with the bisphenols at the concentrations of 10−9 M, 10−7 M, and 10−5 M, and after 24 and 48 h, cell viability was monitored by the MTS assay and gene expressions were analyzed using RT-qPCR. Results. Bisphenol treatment did not alter the COV434 cell viability. After 24 h, the expression of neither of the NRs was changed. Likewise, after 48 h, the expression of the selected genes was not altered. However, both BPAF and BPS increased, at the highest concentration (10−5 M) used, the mRNA levels of both PPARD and NURR1 NRs after 48 h of the treatment. In the BPA-treated groups, no significant upregulation was observed. Conclusions. In the present study, the effect of bisphenols on COUP-TFII, Nurr1, and LRH-1 NRs was investigated for the first time. Although generally we did not observe that BPs provoked any alterations in the expression of the selected NRs in COV434 cells, at specific concentrations and time points they might alter mRNA expression of certain NRs (NURR1, PPARD).

scholarly journals The Transcription Factor TAp63 Exerts Pro-Survival Effects in Chronic Lymphocytic Leukemia Acting through the BCL2 Pathway

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3110-3110
Author(s):  
Stamatia Laidou ◽  
Stavroula Ntoufa ◽  
Sofia Papanikolaou ◽  
Konstantia Kotta ◽  
Maria Koutroumani ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Viability ◽  
Cell Survival ◽  
Mrna Expression ◽  
Cell Line ◽  
Research Funding ◽  
Lymphocytic Leukemia ◽  
Mrna Levels

Abstract Recent evidence indicates that TAp63, the prevalent isoform of TP63 in Chronic Lymphocytic Leukemia (CLL), is implicated in disease pathogenesis. In CLL, TAp63 expression, modulated by both immune signaling and epigenetic modifications, promotes leukemic cell survival and homing to the bone marrow. In activated normal B cells, the TAp63 transcription factor binds the BCL2 gene, participating in an anti-apoptotic pathway (axis NF-κB/TAp63/BCL2) augmenting cell survival. In this study, we investigated the expression of TAp63 in a large cohort of CLL cases and its potential fluctuation during disease progression. Additionally, in order to further understand the pro-survival role of TAp63 in CLL, we interrogated at the molecular level the interplay betweenΤAp63 and BCL2. Initially, using RT-qPCR we quantified TAp63 mRNA expression in 166 CLL patients, consisting of 89 with unmutated IGHV genes (U-CLL) and 77 with mutated IGHV genes (M-CLL), prior to administration of treatment. Significantly higher TAp63 mRNA levels were observed in U-CLL vs M-CLL (FD=13.83, p<0.0001). However, outliers were identified in both subgroups, prompting us to re-classify all cases into TAp63high and TAp63low subgroups using ROC curve and Youden index statistical procedures. TAp63high patients displayed significantly shorter time-to-first-treatment (TTFT) (TAp63highmedian TTFT: 1.58 years; TAp63lowmedian TTFT: 4.07 years; p=0.03) and shorter overall survival (OS) (TAp63highmedian OS: 7.825 years; TAp63lowmedian OS: not yet reached; p=0.046). Next, we analyzed TAp63 mRNA expression in longitudinal samples of 25 U-CLL cases treated with either FCR or rituximab-chlorambucil. In each case, samples were collected at three 'landmarks'; diagnosis, first progressionand first relapse. Expression analysis by RT-qPCR showed that TAp63 levels significantly increased at disease relapse compared to diagnosis (FD=3.47, p=0.02). We subsequently investigated links between TAp63 and BCL2 by measuring BCL2 mRNA levels in 56 U-CLL cases from the present cohort and found statistically significant correlation with the corresponding TAp63 mRNA levels (spearman rho=0.31, p=0.01). To validate this observation, we undertook functional studies in the MEC1 CLL cell line. Considering that MEC1 cells express high TAp63 mRNA levels, we generated a stable MEC1 cell line to inducibly downregulate TAp63, using CRISPR/dCas9-KRAB upon treatment with doxycycline (Dox). We used 2 different guide RNAs (sgRNAs; sgRNA1, sgRNA2) targeting 2 distinct regions of the endogenous TAp63 promoter. After 5 days of induction, the expression levels of both TAp63 and BCL2 were quantified by one step RT-qPCR in Tet-on-dCas9-KRAB-sgRNA-TAp63 MEC1 cells. Inducible downregulation of TAp63 expression (gRNA1: FD=1.7, gRNA2: FD=1.53) resulted in downregulation of BCL2 expression (gRNA1: FD=1.34, gRNA2: FD=1.12) with strong correlation (rho=0.97, p<0.0001) between TP63 and BCL2 mRNA levels. Furthermore, we also observed correlation between TAp63 and BCL2 protein expression in primary cells of one representative TP63high CLL case (rho=0.94, p=0.01), in which TAp63 was silenced by RNA interference (RNAi) with 3 different siRNAs. Prompted by these results, we additionally assessed ex vivo the effect of the BCL2 inhibitor Venetoclax in primary CLL cells of both TAp63high (n=8) and TAp63low (n=6) cases. Cell viability was measured by flow cytometry at 24 and 48 hours after treatment. TAp63high cases were more resistant to treatment with Venetoclax as they showed no statistically significant reduction in cell viability compared to the respective (DMSO-treated) controls, in contrast to TAp63low cases (24h: FD=3.63, p=0.004; 48h: FD=7.17, p=0.005). In conclusion, we provide evidence suggesting that up-regulated TAp63 expression represents a novel resistance mechanism to chemoimmunotherapy in CLL. The pro-survival role of TAp63 is supported by its strong association with BCL2. Indeed, based on the present findings, TAp63 appears to act as a positive modulator of BCL2 in CLL cells, rendering them less responsive to apoptosis induction with the BCL2 inhibitor Venetoclax. Disclosures Hadzidimitriou: Abbvie: Research Funding; Gilead: Research Funding; Janssen: Honoraria, Research Funding. Stamatopoulos:Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

scholarly journals Relevance of nuclear receptor expression in a Tchreg cell line, HOZOT: RXRα and PPARγ negatively regulate IFN-γ production

2012 ◽  
Vol 2 ◽  
pp. 158-165
Author(s):  
Motoyuki Suzuki ◽  
Makoto Takeuchi ◽  
Kazue Tsuji-Takayama ◽  
Akira Harashima ◽  
Takeshi Otani ◽  
...  
Keyword(s):  
Cell Line ◽  
Nuclear Receptor ◽  
Receptor Expression ◽  
Ifn Γ

Effects of Sex Steroid Hormones on Corticosteroid-Binding Globulin Gene Expression in Human Endometrial Cancer Cell Line Ishikawa

1998 ◽  
Vol 35 (5) ◽  
pp. 637-642 ◽  
Author(s):  
Ryou Misao ◽  
Yoshihito Nakanishi ◽  
Jiro Fujimoto ◽  
Teruhiko Tamaya
Keyword(s):  
Endometrial Cancer ◽  
Mrna Expression ◽  
Cell Line ◽  
Cancer Cell ◽  
Biological Effects ◽  
Cancer Cell Line ◽  
High Dose ◽  
Dependent Manner ◽  
Endometrial Cancer Cell ◽  
Corticosteroid Binding Globulin

The effect of progestins on intracellular corticosteroid-binding globulin (CBG) mRNA expression in an endometrial cancer cell line (Ishikawa) was examined in an attempt to understand the biological effects of high-dose progestins in the treatment of well-differentiated uterine endometrial cancers. Oestradiol-17β (E2) significantly increased CBG mRNA expression in a dose-dependent manner, while a high dose of progesterone with or without E2 suppressed it significantly. Furthermore, a high dose of progesterone or medroxyprogesterone acetate (MPA) suppressed CBG mRNA expression to a greater degree than did chlormadinone acetate or 17 α-hydroxyprogesterone caproate with or without E2. These findings suggest that the effects of high-dose progestins on cancer cells may be mediated via suppression of intracellular CBG.

scholarly journals TNF-α Suppressed FSH-Induced LH Receptor Expression Through Transcriptional Regulation in Rat Granulosa Cells

Endocrinology ◽  
2015 ◽  
Vol 156 (9) ◽  
pp. 3192-3202 ◽  
Author(s):  
Kohshiro Nakao ◽  
Hiroshi Kishi ◽  
Fumiharu Imai ◽  
Hiroto Suwa ◽  
Takashi Hirakawa ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Granulosa Cells ◽  
Promoter Region ◽  
Ovarian Function ◽  
Receptor Expression ◽  
Luciferase Assay ◽  
Pelvic Cavity ◽  
Transcriptional Level ◽  
Lh Receptor ◽  
Tnf Α

Several inflammatory cytokines regulate ovarian function. TNF-α is produced in granulosa cells under physiological conditions and has a reciprocal action on follicle development. In contrast, in pelvic inflammatory diseases, TNF-α is excessively produced in the pelvic cavity and has an adverse effect on reproductive functions. The objective of this study was to elucidate the mechanism of action of TNF-α on the expression of LH receptor (LHR) in immature rat granulosa cells. TNF-α suppressed FSH-induced LHR mRNA and protein expression and was not associated with cAMP accumulation. By using a luciferase assay, the construct containing base pairs −1389 to −1 of the rat Lhcgr promoter revealed that TNF-α decreased FSH-induced promoter activity. In response to TNF-α, nuclear factor (NF)-κB p65 was translocated to the nucleus, and the suppressive effect of TNF-α on LHR mRNA expression was abrogated by an NF-κB inhibitor. In a chromatin immunoprecipitation assay, TNF-α induced the association of NF-κB p65 with the rat Lhcgr transcriptional promoter region. NF-κB p65 and histone deacetylase (HDAC) interact to mediate expression of several genes at a transcriptional level. HDAC activity is thought to induce tight connections within local chromatin structures and repress gene transcription. Furthermore, the TNF-α–induced suppression of LHR mRNA expression was blocked by an HDAC inhibitor. Taken together, these results suggest that the interaction of NF-κB p65 with HDAC in the promoter region of rat Lhcgr might be responsible for TNF-α action on the regulation of LHR.

scholarly journals Influence of chronic undernutrition and leptin on GOAT mRNA levels in rat stomach mucosa

2008 ◽  
Vol 41 (6) ◽  
pp. 415-421 ◽  
Author(s):  
C Ruth González ◽  
María J Vázquez ◽  
Miguel López ◽  
Carlos Diéguez
Keyword(s):  
Energy Balance ◽  
Mrna Expression ◽  
Biological Effects ◽  
Male Rats ◽  
Hydroxyl Group ◽  
Acylated Ghrelin ◽  
Mrna Levels ◽  
Stomach Mucosa ◽  
Chronic Undernutrition ◽  
Fasted Rats

The most unique feature of ghrelin is the acyl-modification of a hydroxyl group of the Ser3 in the N-terminus. The Ser3 is commonly modified by n-octanoic acid in vertebrates being needed for its biological effects, at least in terms of feeding. Therefore, a critical question regarding the role of ghrelin was to characterize the mechanism involved in its acylation. The acyltransferase that catalyzes ghrelin octanoylation has been recently identified and named ghrelin O-acyltransferase (GOAT). The aim of this study was to clarify the physiological implications of GOAT in the regulation of energy balance, by assessing the effect of undernutrition, as well as fasting in adult male rats. We have determined GOAT mRNA expression levels by real time-PCR in the stomach mucosa. Our results show that chronic food restriction led to an increase in GOAT mRNA, particularly following long-term chronic malnutrition (21 days). Furthermore, following 48 h complete fasting, a situation with high-circulating ghrelin levels, we found similar mRNA expression of GOAT in fed and fasted rats; exogenous leptin administration markedly increase GOAT mRNA levels in the stomach mucosa of fasted rats. These findings suggest that increased GOAT mRNA levels may have a role in mediating the physiological responses to chronic undernutrition and could represent an adaptive response to prevent long-lasting alterations in energy balance and body weight homeostasis. Furthermore, our data also offer mechanistic insights into the reason why during fasting acylated ghrelin levels are not increased at a time when a marked increase in an orexigenic signal as important as acylated ghrelin will be expected.

A preprogalanin cDNA from the turtle pituitary and regulation of its gene expression

2007 ◽  
Vol 292 (4) ◽  
pp. R1649-R1656 ◽  
Author(s):  
John Yuh-Lin Yu ◽  
Chin-Hon Pon ◽  
Hui-Chen Ku ◽  
Chih-Ting Wang ◽  
Yung-Hsi Kao
Keyword(s):  
Mrna Expression ◽  
Untranslated Region ◽  
Signal Sequence ◽  
Biological Effects ◽  
In Vitro Study ◽  
The Body ◽  
Mrna Levels ◽  
Coding Region ◽  
Reading Frame

Galanin is a hormone 29 or 30 amino acids (aa) long that is widely distributed within the body and exerts numerous biological effects in vertebrates. To fully understand its physiological roles in reptiles, we analyzed preprogalanin cDNA structure and expression in the turtle pituitary. Using the Chinese soft-shell turtle ( Pelodiscus sinensis order Testudines), we obtained a 672-base pair (bp) cDNA containing a 99-bp 5′-untranslated region, a 324-bp preprogalanin coding region, and a 249-bp 3′-untranslated region. The open-reading frame encoded a 108-aa preprogalanin protein with a putative 23-aa signal sequence at the NH2 terminus. Based on the location of putative Lys-Arg dibasic cleavage sites and an amidation signal of Gly-Lys-Arg, we propose that turtle preprogalanin is processed to yield a 29-aa galanin peptide with Gly1 and Thr29 substitutions and a COOH-terminal amidation. Sequence comparison revealed that turtle preprogalanin and galanin-29 had 48–81% and 76–96% aa identities with those of other vertebrates, respectively, suggesting their conservative nature. Expression of the turtle galanin gene was detected in the pituitary, brain, hypothalamus, stomach, liver, pancreas, testes, ovaries, and intestines, but not in the adipose or muscle tissues, suggesting tissue-dependent differences. An in vitro study that used pituitary tissue culture indicated that treatment with 17β-estradiol, testosterone, or gonadotropin-releasing hormone resulted in increased galanin mRNA expression with dose- or time-dependent differences, whereas leptin and neuropeptide Y reduced galanin mRNA levels. These results suggest a hormone-dependent effect on hypophyseal galanin mRNA expression.

Implication of HIF-1α but Not HIF-2α in the Hypoxic Response of Human Hematopoietic Progenitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4154-4154
Author(s):  
Yanyan Zhang ◽  
Adlen Foudi ◽  
Magali Berthebaud ◽  
Dorothee Buet ◽  
Peggy Jarrier ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Lines ◽  
Hematopoietic Cells ◽  
Receptor Expression ◽  
Metabolic Adaptation ◽  
Protein Accumulation ◽  
Mrna Levels ◽  
Hematopoietic Progenitors ◽  
Cd34 Cells ◽  
Cxcr4 Mrna

Abstract Maturing hematopoietic cells are exposed to hypoxia as they develop and migrate within the bone marrow microenvironment. Previous studies using non hematopoietic cell lines and monocytes showed that CXCR4 is strongly induced by hypoxia but little is known on the regulation of CXCR4 by hypoxia in the other hematopoietic cells and during hematopoietic development. We analyzed the expression and regulation of hypoxia-inducible transcription factor-1a (HIF1a) and 2a (HIF2a), the master regulators of metabolic adaptation to hypoxia, during hematopoiesis. Real time quantitative RT-PCR showed that HIF-1a mRNA was present on all the non hematopoietic and hematopoietic cells lines including HL-60, HEL, TF1, K562, KG1, U937, Jurkat and Mo7e. In contrast, HIF-2a mRNA expression was variable among the cell lines and was detected only at very low level in some cells such as KG1, Jurkat and HEL. Hypoxia exposure rapidly induced VEGF mRNA expression in the cells that expressed HIF-1a mRNA and exhibited HIF-1a protein accumulation. Interestingly, CXCR4 induction was observed only in the cells that exhibit significant expression of HIF-2a mRNA and HIF-2a protein accumulation. A strong correlation between HIF-2a mRNA levels and the induction of CXCR4 mRNA expression by hypoxia was found. Human CD34+ cells also expressed high levels of HIF-1a mRNA, whereas HIF-2a mRNA was barely detected. Interestingly, as observed for several myeloid cell lines, CD34+ cells exhibited a strong induction of VEGF expression in response to hypoxia and hypoxia mimetic agents cobalt chloride and desferrrioxamine whereas CXCR4 receptor expression was not induced suggesting that CXCR4 mRNA induction is related to the expression of HIF-2a. Altogether these data indicated that the hypoxic responses of human hematopoietic progenitors are independent of HIF-2a. Moreover, they establish that CXCR4 regulation by hypoxia is linked to HIF-2a protein expression.

scholarly journals Aminoglutethimide suppresses adrenocorticotropin receptor expression in the NCI-h295 adrenocortical tumor cell line

1998 ◽  
Vol 159 (1) ◽  
pp. 35-42 ◽  
Author(s):  
M Fassnacht ◽  
F Beuschlein ◽  
S Vay ◽  
P Mora ◽  
B Allolio ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Line ◽  
Cdna Probe ◽  
Tumor Cell Line ◽  
Receptor Expression ◽  
Intracellular Camp ◽  
Camp Accumulation ◽  
Transcript Accumulation ◽  
Down Regulation ◽  
Stimulation Of

The adrenostatic compound aminoglutethimide (AG), a potent inhibitor of the P450 side chain cleavage enzyme, is used in the treatment of ACTH-dependent or adrenal Cushing's syndrome. Recently, AG has been shown to inhibit ACTH receptor (ACTH-R) mRNA expression in ovine adrenocortical cells in a time-dependent fashion. To investigate whether ACTH-R down-regulation will also be induced in tumor cells, we studied the effect of AG on ACTH-R expression in the human NCI-h295 adrenocortical carcinoma cell line, which expresses functional ACTH receptors and produces steroids of the glucocorticoid, mineralocorticoid and androgen pathway. The cells were incubated in triplicate with increasing doses of AG (3, 30, 300 microM) which suppressed steroid secretion dose-dependently. After 48 h, cells were harvested, and total RNA was extracted, electrophoresed, blotted and hybridized with a human ACTH-R cDNA probe. In parallel experiments, after preincubation with AG the cells were stimulated with ACTH (10 nM) for 10 min and the intracellular cAMP accumulation was determined by RIA. AG significantly suppressed the baseline ACTH-R mRNA expression in a dose-dependent fashion (300 microM AG, 5+/-1%; 30 microM AG, 64+/-1%; 3 microM AG, 108+/-19% compared with control cells, 100+/-11%). The reduced ACTH-R mRNA expression was paralleled by low ACTH-induced cAMP accumulation indicating reduced expression of the ACTH-R protein. The adrenostatic compound metyrapone, an inhibitor of 11beta-hydroxylase activity, also suppressed ACTH-R mRNA expression in a similar fashion. Stimulation of the protein kinase A pathway by simultaneous incubation of ACTH (10 nM) or forskolin (10 microM) together with AG was not able to overcome the steroid biosynthesis blockade, but reversed the inhibitory effects of AG on the ACTH-R mRNA expression. Also, cortisol (12 microM) reversed the AG-induced ACTH-R mRNA expression. We conclude that AG induces profound ACTH-R down-regulation in the NCI-h295 cell line either by affecting the gene expression or by decreasing transcript accumulation via an effect on RNA stability. This novel action of AG can be reversed by stimulation of the cAMP pathway and of the glucocorticoid-mediated signal transduction cascade. As the down-regulation occurs in vitro at concentrations which are reached during treatment with AG in humans it may contribute to its therapeutic activity in adrenal disease.

NDRG1 Expression Is Inhibited in AML and Its Knockdown Attenuates Neutrophil Differentiation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 927-927
Author(s):  
Mario P. Tschan ◽  
Deborah Shan ◽  
Judith Laedrach ◽  
Marianne Eyholzer ◽  
Elisabeth Oppliger Leibundgut ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cell Line ◽  
Cell Phenotype ◽  
Mrna Levels ◽  
U937 Cells ◽  
Rt Pcr ◽  
Atra Treatment ◽  
Significant Difference ◽  
Blast Cells ◽  
Neutrophil Differentiation

Abstract The N-myc down-regulated gene 1 (NDRG1) is a stressed induced protein whose expression is associated with growth arrest and differentiation of tumor cells. Although the exact function of NDRG1 protein remains unknown, various studies support its role as a suppressor of tumor metastasis. In prostate, colon and breast cancer its expression is associated with a better disease prognosis and patient survival. In hematopoietic cells, NDRG1 was identified in a differential display screen for differentiation-related genes in human myelomonocytic U937 cells. In the present study, we sought to investigate the role of NDRG1 in myeloid differentiation. To this end we first evaluated NDRG1 mRNA expression in acute myeloid leukemia (AML; n=82) patient samples as well as in CD34+ progenitor cells (n=5) and neutrophils (n=6) of healthy donors using quantitative real-time RT-PCR. We found significantly higher NDRG1 mRNA levels in granulocytes as compared to CD34+ (p=0.0043) or AML blast cells (p<0.0001), whereas no significant difference between CD34+ progenitor and AML blast cells was seen (Figure A). Moreover, we found that NDRG1 mRNA levels were increased in 4/5 APL patients upon ATRA therapy. In contrast, the closest relative of NDRG1, NDRG2, did not show significantly different expression in these primary cells, thus indicating a unique role for NDRG1 in granulocyte differentiation. Next we examined NDRG1 expression using quantitative RT-PCR and Western blotting in two different cell line models for ATRA-induced neutrophil differentiation. ATRA treatment of NB4 and HT93 acute promyelocytic leukemia (APL) cells induced NDRG1 mRNA 2.3- and 14.3- fold, respectively. Increased NDRG1 mRNA expression was paralleled by an increase of NDRG1 protein as well as a decrease in c-myc protein. Earlier reports described that NDRG1 is also suppressed by c-myc suggesting that down-regulation of c-myc in our cell line models allowed for an increase of NDRG1. In line with these observations, lentivirus-driven short hairpin (sh)RNA-mediated silencing of NDRG1 diminished ATRA-induced neutrophil differentiation of NB4 and U937 cells as measured by CD11b, CD11c and CD18 surface expression. In NB4 NDRG1 knockdown versus non-targeting shRNA expressing cells mean fluorescent intensities (MFI) for CD11b, CD11c and CD18 upon six days of ATRA-treatment were 99±17 vs 146±7, 20±2 vs 32±10 and 19±2 vs 45±6, respectively (Figure B). Similarly, U937 NDRG1 knockdown versus control cells displayed the following MFIs for CD11b and CD18 upon neutrophil differentiation: 61±1 vs 102±2 and 11±4 vs 33±13, respectively. In conclusion, we report here for the first time an association of low NDRG1 levels with an immature hematopoietic cell phenotype. Using RNAi technology we further provide evidence that NDRG1 is functionally involved in neutrophil maturation. Figure Figure

Association of Asparagine Synthetase Expression and Sensitivity to L-Asparaginase in Cell Lines and Primary Pediatric Acute Lymphoblastic Leukemia Samples.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2738-2738
Author(s):  
Ivana Hermanova ◽  
Jan Trka ◽  
Julia Starkova
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Amino Acid ◽  
Mrna Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Asparagine Synthetase ◽  
Leukemic Cells ◽  
Mrna Levels ◽  
Basal Expression

Abstract Abstract 2738 Poster Board II-714 L-Asparaginase (L-Asp) is an important component in the combined chemotherapy for childhood acute lymphoblastic leukemia (ALL). Administration of L-Asp leads to depletion of plasmatic asparagine and consequently causes loss of intracellular asparagine. As a non-essential amino acid, asparagine is synthesized from aspartate and glutamine by asparagine synthetase (ASNS). Primary ALL cells are believed to have low ASNS expression and therefore to be sensitive to asparagine depletion. Although increased ASNS level was shown to be connected with L-Asp resistance the exact relationship between ASNS expression and L-Asp sensitivity is not clear. We and others have previously shown TEL/AML1[+] ALL blasts express more ASNS mRNA than TEL/AML[-] do although primary TEL/AML[+] cell are in vitro more sensitive to treatment with L-Asp. Hutson et al (1997) showed that amino acid deprivation led to increased expression of ASNS on mRNA and protein level as well as to increased biological activity. On the other hand, Nan Su et al described negative correlation between L-Asp sensitivity and ASNS protein rather than mRNA levels. Therefore, in our studies we concentrated on protein expression of ASNS in patients' samples. So far, there has been no reproducible published data on ASNS protein detection by Western blot in primary patients' samples. Despite using 3 different antibodies and precise optimization we were not able to detect ASNS protein in patients' samples in contrast to cell lines. Transcripts' levels confirmed significantly lower (2 log) expression of ASNS in patients' leukemic cells compared to leukemic cell lines. Therefore, for further studies on gene and protein relation we had to rely on cell lines as a model. We detected ASNS gene expression and ASNS protein content in four ALL cell lines: REH (TEL/AML1[+]), UOCB6 (TEL/AML1[+]), NALM6 (TEL/PDGFRB[+]) and RS4;11 (MLL/AF4[+]). ASNS mRNA levels were in accord with sensitivity to L-Asp. UOCB6 as the most resistant cell line (IC50=0.04U/ml) had the highest expression of ASNS (normalized ASNS, nASNS=4.946), then NALM6 (IC50=0.01U/ml; nASNS=1.8), REH (IC50=0.6.10−4; nASNS=1.176) and RS4;11 (IC50<0.3.10−4; nASNS=0.024). ASNS protein levels significantly differed through passages in REH cells, likely due to rapid turnover. For the remaining three cell lines L-Asp sensitivity correlated also with protein content. We have previously shown that different basal expression levels do not affect short-term dynamics of ASNS expression after L-Asp administration. Here we were interested to see the changes of sensitivity to L-Asp using gradient silencing of ASNS by RNAi in two cell lines with different basal expression: REH cell line with intermediate ASNS mRNA expression and RS4;11 cell line with very low mRNA expression. Gradient silencing revealed that L-Asp sensitivity correlated with ASNS expression till 50% decrease; further silencing did not potentiate the effect. The same response was seen in both cell lines despite different basal ASNS expression and sensitivity to L-Asp. The ASNS is glutamine dependent enzyme therefore we also studied expression of glutamate dehydrogenase (GDH), an enzyme necessary for glutamine synthesis. We found significantly lower GDH mRNA expression in primary TEL/AML1[+] blasts in comparison with TEL/AML[-] blasts (p=0.019), which might lead to deficiency of glutamine in these cells and consequently higher sensitivity to L-Asp. Accordingly, silencing of ASNS in REH tended to increase GDH expression levels. Our data confirm that generally, both ASNS mRNA and protein expression inversely correlate with the sensitivity to L-Asp in the cell lines. However, it may be misleading to draw conclusions for the patients' cells directly from the results obtained in cell line models. The expression patterns of ASNS in primary leukemic cells differ even from those of genotypically identical cell lines. The control of basal levels of ASNS in leukemic cells remains to be elucidated. Our results implicate an important role of GDH and glutamine metabolic pathway in the regulation of ASNS activity. This work was supported by MSM0021620813 and GAUK 7835. Disclosures: No relevant conflicts of interest to declare.

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