Abstract
The transcription factor C/EBPalpha plays a critical role in the process of granulocytic differentiation. Recently, mutations that abrogated transcriptional activation of C/EBPalpha were detected in acute myeloid leukemia patient samples. Moreover, the progression of chronic myelogenous leukemia (CML) to blast crisis in patients was correlated with down-modulation of C/EBPalpha. The KCL22 cell line, derived from BCR-ABL+ CML in blast crisis, expressed wild-type C/EBPepsilon protein but not a functional C/EBPalpha, -beta, and -gamma. Restoration of C/EBPalpha expression in KCL22 cells triggered a profound proliferative arrest, a block in the G2/M phase of the cell cycle and a gradual increase in apoptosis. Within 3 days of inducing expression of C/EBPalpha, a remarkable neutrophilic differentiation of the KCL22 blast cells occurred as shown by morphologic changes, induction of expression of CD11b, primary, secondary, and tertiary granule proteins, and granulocyte colony-stimulating factor receptor. Using high density oligonucleotide microarrays, the gene expression profile of KCL22 cells stably transfected with C/EBPalpha was compared with that of empty vector, and we identified genes not previously known to be regulated by C/EBPalpha. These included the up-regulation of those genes important for regulation of hematopoietic stem cell homing, granulocytic differentiation, and cell cycle, whereas down-regulation occurred for genes coding for signaling molecules and transcription factors that are implicated in regulation of proliferation and differentiation of hematopoietic cells. Our study showed that restoration of C/EBPalpha expression in BCR-ABL+ leukemic cells in blast crisis is sufficient for rapid neutrophil differentiation suggesting a potential therapeutic role for ectopic transfer of C/EBPalpha in acute phase of CML.
Highlights
The transcription factor CCAAT/enhancer-binding protein ␣ (C/EBP␣)1 has been implicated as an inhibitor of cell prolifer
Using high density oligonucleotide microarrays, the gene expression profile of KCL22 cells stably transfected with C/EBP␣ was compared with that of empty vector, and we identified genes not previously known to be regulated by C/EBP␣
These included the up-regulation of those genes important for regulation of hematopoietic stem cell homing, granulocytic differentiation, and cell cycle, whereas down-regulation occurred for genes coding for signaling molecules and transcription factors that are implicated in regulation of proliferation and differentiation of hematopoietic cells
Summary
Chemicals—All-trans-retinoic acid (ATRA) and 9-cis-retinoic acid (RA) (Sigma, St. Louis, MO) were dissolved in 95% ethanol to a stock concentration of 10Ϫ2 mol/liter and stored at Ϫ70 °C. G-CSF receptor; ATRA, all-trans-retinoic acid; RA, 9-cis-retinoic acid; HMBA, hexamethylene bisacetamide; RT, reverse transcription; FACS, fluorescence-activated cell sorting; MPO, myeloperoxidase. KCL22-pMT␣ cells (5 ϫ 104) were grown in RPMI with 10% fetal bovine serum either with or without ZnSO4 (100 M), and the mean number of viable cells in triplicate experiments was determined on days 0, 1, 3, and 5 using trypan blue exclusion. Oligonucleotide Microarray Expression Analysis—KCL22-pMT and KCL22-pMT␣ cells were cultured in media containing 100 M ZnSO4 (Sigma) for 12 h for the induction of C/EBP␣. Cells were washed in staining buffer, fixed with 2% paraformaldehyde, and analyzed by flow cytometry
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
