The role of STAT5 proteins in the regulation of normal hematopoiesis in a cord blood model.
The signal transducers and activators of transcription - STAT5A and STAT5B - are responsible for the control of proliferation, differentiation and apoptosis, via their effect on gene expression. They are activated by the binding of many cytokines, growth factors and hormones to their receptors on the cell surface. Many of these cytokines regulate hematopoietic cell development; therefore, STAT5 proteins are suggested to play an important role in hematopoiesis. There are numerous contradictory reports available in the literature on the role of STAT5 in normal hematopoietic cell development; hence, the question of the real function of STAT5 proteins clearly requires further studies. The aim of our study was to evaluate the role of STAT5 in normal hematopoiesis using oligodeoxynucleotide (ODN) strategy against STAT5 mRNA. We employed the RT-PCR method to study STAT5 mRNA expression in cells after their incubation with ODNs. We analyzed the effect of blocking STAT5 proteins on the viability and clonogenecity of the CFU-GM (Colony Forming Unit of Granulocyte-Macrophages) and the BFU-E (Burst Forming Unit of Erythrocytes) obtained from human cord blood (CB). The clonogenic growth of the cells was assessed in methylcellulose cultures according to the type of oligodeoxynucleotides. We also attempted to estimate the level of apoptosis induced in cord blood mononuclear and CD34+ cells by employing different assays: i) Annexin V staining using flow cytometry (FACSCalibur); ii) terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL); iii) analysis of Bax and Bcl-X(L) gene expression by RT-PCR. Perturbation of STAT5 expression with antisense oligodeoxynucleotides had no impact on the viability, clonogenecity and apoptosis of CB hematopoietic cells. Our results showed that STAT5 proteins do not play a significant role in the regulation of proliferation of normal hematopoietic cells derived from cord blood.
Baśkiewicz-Masiuk
Magdalena
M
Department of General Pathology, Pomeranian Medical University, Al. Powstańców Wlkp.72, 70-111 Szczecin, Poland. poziomka@med.pam.szczecin.pl
Masiuk
Marek
M
Czajka
Ryszard
R
Machaliński
Bogusław
B
D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005312:Fetal Blood / Q000378:metabolism*; D006410:Hematopoiesis / Q000502:physiology*; D006801:Humans; D020287:In Situ Nick-End Labeling; D008894:Milk Proteins; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050799:STAT5 Transcription Factor; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins
D017304:Annexin A5; D004268:DNA-Binding Proteins; D008894:Milk Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D050799:STAT5 Transcription Factor; C494092:STAT5A protein, human; C494095:STAT5B protein, human; D015534:Trans-Activators; D025521:Tumor Suppressor Proteins