Transforming growth factor-beta(1) induces apoptosis in CD34(+)CD38(-/low) cells that express Bcl-2 at a low level.
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.


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.
Xiao
M
M
Pacific Northwest Regional Blood Services, American Red Cross, Portland, OR 97208, USA. xiaom@usa,redcross.org
Oppenlander
B K
BK
Dooley
D C
DC
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D015703:Antigens, CD; D018952:Antigens, CD34 / Q000097:blood*; D000943:Antigens, Differentiation / Q000097:blood*; D017209:Apoptosis / Q000187:drug effects*; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000378:metabolism / Q000494:pharmacology; D009244:NAD+ Nucleosidase / Q000097:blood*; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000187:drug effects / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein
D015703:Antigens, CD; D018952:Antigens, CD34; D000943:Antigens, Differentiation; C494819:BAD protein, human; C494826:BAX protein, human; C494811:BCL2L1 protein, human; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein; C084881:flt3 ligand protein; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1