The influence of antisense oligonucleotides against STAT5 on the regulation of normal haematopoiesis in a bone marrow model.
Cytokines and growth factors that take part in the regulation of haematopoietic cell development activate many signalling pathways in target cells. The STAT5 (signal transducers and activators of transcription) proteins are members of a family of signal transducers and activators of transcription that can be activated after cytokine stimulation. Their binding to promoters of different genes influences cell proliferation, differentiation and survival. It is suggested that they play an important role in haematopoiesis, however, the question of the real function of STAT5 proteins requires further examination. The aim of our study was to investigate the role of STAT5 in the proliferation and apoptosis of normal haematopoietic bone marrow cells derived from heparinized cadaveric organ donors (HCOD). We applied antisense oligodeoxynucleotides (ODNs) to block STAT5A and STAT5B at the mRNA level and the reverse transcription polymerase chain reaction method to study STAT5 mRNA expression in the cells after incubation with ODNs. Moreover, we performed Western blot analysis of the STAT5A protein after exposure to antisense STAT5A. We analysed the clonogenicity of the colony-forming unit of granulocytes-macrophages and the burst-forming unit of erythrocytes in methylcellulose cultures according to the type and the dose of ODNs. We also examined apoptosis induced in bone marrow mononuclear and CD34(+) cells by employing annexin V staining and the TUNEL method using flow cytometry (FACScan). We found that the perturbation of STAT5 expression decreased the clonogenicity of bone marrow haematopoietic cells. However, we did not observe any significant increase in the percentage of apoptotic cells after incubation with antisense ODNs. It was concluded that the STAT5 proteins play a significant role in the proliferation of human bone marrow cells harvested from HCOD. These proteins might be critical in the regulation of haematopoiesis, especially under stress conditions.
Baśkiewicz-Masiuk
M
M
Department of General Pathology, Pomeranian Medical University, Szcecin, Poland. poziomka@med.pam.szczecin.pl
Paczkowski
M
M
Machaliński
B
B
D000328:Adult; D017304:Annexin A5 / Q000378:metabolism; D018952:Antigens, CD34 / Q000276:immunology; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D002999:Clone Cells / Q000187:drug effects / Q000502:physiology; D003114:Colony-Forming Units Assay; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005260:Female; D006410:Hematopoiesis / Q000235:genetics*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects; D008297:Male; D008875:Middle Aged; D008894:Milk Proteins; D008954:Models, Biological; D020320:Oligoribonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000378:metabolism; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D013312:Stress, Physiological / Q000378:metabolism / Q000503:physiopathology; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D025521:Tumor Suppressor Proteins
D017304:Annexin A5; D018952:Antigens, CD34; D004268:DNA-Binding Proteins; D008894:Milk Proteins; D020320:Oligoribonucleotides, Antisense; D012333:RNA, Messenger; D050799:STAT5 Transcription Factor; C494092:STAT5A protein, human; C494095:STAT5B protein, human; D015534:Trans-Activators; D025521:Tumor Suppressor Proteins