Publication Date:
2013-11-15
Description:
Background Resistance against the glucocorticoid prednisolone still remains an obstacle for treatment of pediatric precursor B acute lymphoblastic leukemia (BCP-ALL) at initial diagnosis and even more after relapse. The molecular mechanisms behind prednisolone resistance in pediatric BCP-ALL is poorly defined. The NR4A family, consisting of NR4A1 (Nur77), NR4A2 (Nurr1) and NR4A3 (Nor1), are orphan nuclear receptors, which antagonize the glucocorticoid receptor. We hypothesized that upregulated NR4A family expression is responsible for prednisolone resistance in BCP-ALL. Methods Newly diagnosed pediatric acute lymphoblastic leukemia patients’ cells were isolated from bone marrow aspirates and only samples with ≥ 90% leukemic blasts were used in the present study. Gene expression microarrays of 178 BCP-ALL patients tested for in vitro prednisolone resistance were analyzed with Limma R Package in the statistical environment R, version 2.15.0. Microarray expression levels were confirmed using qRT-PCR. Nur77, Nurr1 and Nor1 protein expression in primary BCP-ALL patients’ were assessed with reverse phase protein array. Leukemic patients’ cells were transfected with labeled siRNA against NR4A1, NR4A2, and NR4A3, simultaneously, or with labeled siScrl, using the transfection reagent Dharmafect 4. Hereafter, cytotoxicity to prednisolone was determined by the in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) drug-resistance assay. Furthermore, viability of transfected cells was counted by trypan blue exclusion assay and cells were harvested after 72 hours of culture for RNA and protein isolation. Knockdown was confirmed with qRT-PCR and Western blot. Results In this study of 178 precursor BCP-ALL patients we discovered a 3.0-fold (p=0.007) raise in NR4A1, NR4A2, and NR4A3 microarray mRNA expression in in vitro prednisolone resistant compared to sensitive BCP-ALL patients’ cells, which was confirmed by qRT-PCR. In addition, reverse phase protein array identified a 2.7-fold (p
Print ISSN:
0006-4971
Electronic ISSN:
1528-0020
Topics:
Biology
,
Medicine
Permalink