Transforming growth factor β3 inhibits chronic myelogenous leukemia hematopoiesis by inducing Fas-independent apoptosis

Objective Transforming growth factor β3 (TGF-β3) is a potent suppressor of human hematopoietic progenitor cells. In this article, we compare the activity of TGF-β3 on highly purified CD34+ cells and more immature CD34+DR− cells from chronic myelogenous leukemia (CML) patients in chronic phase and normal donors. Materials and Methods Primitive hematopoietic progenitors were stimulated in liquid cultures and clonogenic assays by early-acting growth factors such as stem cell factor (SCF) and interleukin 11 (IL-11) and the intermediate-late–acting stimulating factors IL-3, granulocyte-macrophage colony-stimulating factor, and erythropoietin. Molecular analysis of bcr/abl mRNA was performed on single CML colonies by nested reverse transcriptase polymerase chain reaction. Moreover, cell cycle analysis and assessment of apoptosis of normal and leukemic CD34+ cells were performed by propidium iodide (PI) alone and simultaneous staining with annexin V and PI, respectively. Results The colony-forming efficiency of CML CD34+ cells was generally inhibited by more than 90% regardless of whether the colony-stimulating factors were used alone or combined. When compared to normal CD34+ cells, leukemic cells were significantly more suppressed in 6 of 8 culture conditions. The inhibitory effect of TGF-β3 on CD34+ cells was exerted within the first 24 hours of incubation as demonstrated by short-term preincubation followed by IL-3– and SCF-stimulated colony assays. Evaluation of bcr/abl transcript on residual CML colonies incubated with TGF-β3 demonstrated a small subset of neoplastic CD34+ cells unresponsive to the inhibitory effect of the study cytokine. TGF-β3 demonstrated a greater inhibitory activity on primitive CD34+DR− cells than on more mature CD34+ cells. Again, CML CD34+DR− cells were significantly more inhibited by TGF-β3 than their normal counterparts in 3 of 8 culture conditions. Kinetic analysis performed on CD34+ cells showed that TGF-β induces cell cycle arrest in G1 phase. However, this mechanism of action is shared by normal and leukemic cells. Conversely, TGF-β3 preferentially triggered the programmed cell death of CML CD34+ cells without increasing the proportion of leukemic cells coexpressing CD95 (Fas receptor), and this effect was not reversed by functional blockade of Fas receptor. Conclusion We demonstrate that TGF-β3 exerts a potent suppressive effect on CML cells that is partly mediated by Fas-independent apoptosis.