Objective: Transforming growth factor-beta(1) (TGF-beta(1)) strongly inhibits the proliferation and differentiation of primitive CD34(+)CD38(-) hematopoietic cells. In contrast, Flt3 ligand (FL) is a positive effector of CD34(+)CD38(-/low) cell proliferation. Because apoptosis plays a critical role in hematopoietic development, TGF-beta(1) and FL were analyzed as possible modulators of apoptosis. Specifically, this report examined expression of apoptotic promoters Bax and Bad and apoptotic inhibitors Bcl-2 and Bcl-x (all members of the Bcl-2 protein family). Protein levels were determined in fresh and cultured CD34(+)CD38(+) cells and CD34(+)CD38(-/low) cells with and without treatment with TGF-beta(1) and FL.
Materials and methods: Cells fractions were purified by sorting CD34(+)-enriched mononuclear cells from mobilized peripheral blood. Expression of Bcl-2, Bcl-x, Bax, and Bad and the extent of apoptosis were determined by flow cytometric analysis of freshly isolated cells and cells cultured with TGF-beta(1) and FL effectors.
Results: TGF-beta(1) reduced CD34(+)CD38(+) cell expansion and arrested cell division. Inhibition of growth was not accompanied by an increase in apoptosis. In CD34(+)CD38(-)(/low) cells, serum TGF-beta(1) and added TGF-beta(1) inhibited cell growth and significantly increased apoptotic cell death. Freshly isolated CD34(+)CD38(+) and CD34(+)CD38(-/low) cells expressed Bcl-2 at similar low levels. However, after 3 days, Bcl-2 expression was markedly higher in cultured CD34(+)CD38(+) cells. TGF-beta(1) significantly increased Bax expression in both fractions after 3 days cultivation (p = 0.0034). Thus, addition of TGF beta-1 further reduced the already low Bcl-2:Bax ratio in CD34(+)CD38(-/low) cells.
Conclusions: Compared to CD34(+)CD38(+) cells, CD34(+)CD38(-/low) cells were slow to up-regulate expression of Bcl-2 during ex vivo culture. TGF-beta(1) up-regulated Bax expression by both CD34(+)CD38(+) and CD34(+)CD38(-)(/low) cells and promoted apoptosis in the latter fraction. This suggests that the preferential induction of apoptosis in primitive cells by TGF-beta(1) may be due to its further reduction of the Bcl-2:Bax ratio.