Apoptotic Effects of Corticosteroids on Pure CD34+ Hematopoietic Stem Cells and Modulation of This Effect with Cytokines

Hematopoietic stem cells which can self-renew, are very important for regeneration of hematopoietic system. Because, in chemotherapy applications, malign cells are killed but on the other hand normal cells are also damaged. It is known that corticosteroids, which is essential drug for all chemotherapy protocols, efficiently induce apoptosis not only in malign cells also in normal hematopoietic cells. But, there are no comprehensive studies about the apoptotic effects of glucocorticoids on pure CD34⁺ hematopoietic cell population. In several studies, it has been showed that hematopoietic cytokines can prevent apoptosis caused by chemotherapy and decrease toxic effects of this drugs. In this study, for the first time, we investigated apoptotic effects of corticosteroids on CD34+ hematopoietic stem cell population collected from human peripheral blood buffy coats by using immunomagnetic positive selection method and protective effects of hematopoietic cytokines. Buffy coats were obtained from standard blood collection bags of voluntary blood donors. Two control groups are composed that is not including cytokines in serum free medium (SFC) and induced with cytokine coctail that including interleukin 3, thrombopoietin, stem cell factor and flt3/flk2 ligand (CC). Test groups are dexamethasone group (D), prednisolone group (P) and groups induced with cytokines before drug application (CD, CP). High concentrations of dexamethasone (10^-3 M) and prednisolone (10^-3 M) were used in vitro in concordance with clinical treatment doses. To determine apoptotic mechanisms, immunohistochemical staining was carried out by using monoclonal antibodies against fas, caspase 3, sitocrom c, bax and bcl2. Then cells were counting and determining degree of peroxidase reactions were quantified using H-score. For statistical analysis paired t-test and one sample t-test were used. As a result, both dexamethasone and prednisolon induce apoptosis in pure CD34+ cells. When compared with control groups, corticosteroids caused significant increase for apoptotic fas, caspase 3, sitocrom c and bax (p<0,05), and a significant decrease for antiapoptotic bcl2 (p<0,05). In control that is not including cytokines (SFC) apoptosis induced and same results were determined. When cytokines were added before, it was observed a significant decrease for all apoptotic markers (p<0,05) and a significant increase for antiapoptotic bcl2 (p<0,05) (Figure 1). So that, corticosteroids induce apoptosis in human pure CD34⁺ hematopoietic stem cells and cytokines decrease apoptotic effects of this drugs and prevent apoptosis in vitro. As a results, our findings demonstrated that may be useful cytokine treatment in order to minimize hematopoietic toxicity and to protect CD34+ cells from high-dose corticosteroid damage.