This finding suggests that NPM1 mutation protein may regulate Bax/Bcl-2 expression and increase chemosensitivity of leukemia cells

This finding suggests that NPM1 mutation protein may regulate Bax/Bcl-2 expression and increase chemosensitivity of leukemia cells. DNR-and Ara-C-induced apoptosis. Interestingly, expression of NPM1mA could upregulate Bax and downregulate Bcl-2 at mRNA and protein levels in THP-1 cells when treated with DNR or Ara-C. We also demonstrated that restoration of NF-B activity via TNF- pre-treatment reversed the effect of NPM1mA on the Bax/Bcl-2 expression. Furthermore, evaluation of gene expression data from The Cancer Genome Atlas (TCGA) dataset revealed that NPM1-mutated patients showed a higher expression of Bax and a lower expression of Bcl-2. These results suggest that the NPM1 gene mutations could confer increased sensitivity to chemotherapeutic brokers, at least in part, by suppressing NF-B activity and regulating Bax/Bcl-2 expression. Keywords: nucleophosmin, gene mutation; chemosensitivity; NF-B; Bax; Bcl-2; acute myeloid leukemia. == Intro == Nucleophosmin (NPM1), also named B23, is a multifunctional nucleolar LXS196 protein that primarily localized in nucleoli but able to shuttle between nucleus and cytoplasm1. NPM1 plays crucial roles in ribosome maturation and export, centrosome duplication, cell cycle progression, histone assembly and response to a variety of stress stimuli2-3. NPM1gene heterozygous mutations are present in roughly a third of patients with acute myeloid leukemia (AML), making it one of the most frequent genomic alterations in these patients4Mutated NPM1 protein disrupts the C-terminal nucleolar localization signal of nucleophosmin and produces a new nuclear export signal, which alters the normal balance in nuclear-cytoplasmic NPM1 shuttling and causes the characteristic cytoplasmic localization5, 6. In 2005, Falini Wager al. 7first observed cytoplasmic nucleophosmin (NPMc+) in AML and reported that the type A NPM1 mutation (NPM1 mA, 4 base TCTG insertion) was the most frequent in Rabbit polyclonal to HPX adults (75-80% of cases). NPMc+ AML present with specific clinical, phenotypical, and genetic features8, 9. Strikingly, several clinical studies indicated a higher rate of total remission after chemotherapy in patients carrying theNPM1mutations when compared to wild type10-13. However , the molecular basis of this observation remains at present unclear. Nuclear factor-B (NF-B) is a member of the Rel family members proteins, which regulates protein expression mediating cell cycle/proliferation, anti-apoptosis, and cytokine secretion14. NF-B transcription factors are involved in disparate processes such as inflammation, growth and development, and drug resistance15, 16. NF-B has been demonstrated to be abnormally activated in blast cells from a significant number of AML patients17. Indeed, it has been demonstrated that many types of AMLs produce tumor necrosis factor (TNF)-, and this TNF- activates NF-B transcription factors18. Additionally , recent studies have established that NF-B activation is a key response of leukemia cells to chemotherapy19, 20. It is well known that inhibition of NF-B increases sensitivity to many chemotherapeutical agents21-23. Activated NF-B seems to trigger a series of molecular reactions24, 25. For instance, NF-B can inhibit pro-apoptotic protein Bax and induce the expression of anti-apoptotic protein Bcl-2, which involved LXS196 in chemotherapy induced apoptosis26, 27. In this study, we found that enforced expression of NPM1 mA increased THP-1 leukemia cells sensitivity to chemotherapeutic agents and reduced the NF-B transcription activity of THP-1 cells upon drug treatment. In addition , expression of LXS196 NPM1mA upregulated Bax expression and downregulated Bcl-2 levels. Notably, the potentiating effect of NPM1 mA on chemosensitivity was rescued by pre-treatment with TNF-. Furthermore, NPM1-mutated patients showed a higher expression of Bax and a lower expression of Bcl-2. The results presented here demonstrate that the NPM1 gene mutations may confer increased sensitivity to chemotherapeutic agents, at least in part, by regulating NF-B activity and the apoptosis related proteins expression. == Materials and methods == == Cell line and reagents == Human monocytic leukemia THP-1 cells (purchased from Shanghai Institutes intended for Biological Sciences) were managed in RPMI-1640 medium (Gibco, MD, USA), which was supplemented with 10% fetal bovine serum (FBS, Gibco, MD, USA) and 100 U of penicillin and streptomycin (Sangon Biotech, Shanghai, China) in a 5% CO2-humidified incubator at 37C. Daunorubicin (DNR) and cytarabine (Ara-C) were purchased from Sigma. TNF- was purchased from Beyotime. == Plasmids and cell infection == The pEGFPC1-NPM1mA and empty pEGFPC1 vectors were.