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Blood, Vol. 92 No. 6 (September 15), 1998:
pp. 2041-2052
Apoptotic Regulation in Primitive Hematopoietic Precursors
Rowayda Peters,
Serge Leyvraz, and
Lucien Perey
From the Centre Pluridisciplinaire d'Oncologie, University Hospital
(CHUV), Lausanne, Switzerland.
Bcl-2 and bcl-xL function as suppressors of programmed
cell death. The expression of bcl-2 protein in vivo is associated with long-lived hematopoietic cells such as mature lymphocytes and early
myeloid progenitors. Bcl-xL, a homologue of bcl-2, is also expressed in lymphocytes and thymocytes. In contrast, the bcl-2-related proteins (bax, bad, and bak) act by promoting apoptotic cell death as
shown from their expression in hematopoietic cell lines. We analyzed
the expression of bcl-2 and bcl-x proteins in hematopoietic precursors
obtained from various cell sources in adult mobilized peripheral blood
collected from 13 patients with solid tumors, 8 adult bone marrow, and
12 umbilical cord blood. The analysis was based on the expression of
the proliferation and activation specific antigens, CD38 and class II
(HLA-DR). Similarly, we analyzed the expression of bcl-2-related
proteins bcl-xL, bax, bad, and bak before and during
ex-vivo expansion. Hematopoietic precursors expressing strongly the
CD34 antigen (CD34s+) and lacking CD38 or HLA-DR
expression were analyzed by using three-color immunofluorescence
staining. The majority of CD34+ cells expressed bcl-2 and
unexpectedly showed a bimodal distribution of low and high expression.
More cells that lacked or expressed low density CD38 expressed low
bcl-2 than the more differentiated counterparts (those with high
density CD38). Immaturity (ie, little or no HLA-DR) is associated with
the expression of low bcl-2 compared with HLA-DR+.
However, HLA-DR /low population contained a lower number
of cells expressing low bcl-2 (30% to 40%) than
CD38/low in comparable samples. The hematopoietic
precursors with bcl-2low and bcl-2high formed a
homogeneous population of undifferentiated lymphoid-like cells having a
similar forward scatter. These cells expressed strongly the
bcl-xL protein (>95%) but were bax low (4% to 12%), bad low (0% to 0.8%), and bak low (0% to 3%). The expression of apoptosis specific protein (ASP) was also low (3.4% ± 3.1%) as was
Annexin V. In addition, the CD34+/CD38
showed low cell cycle activity (<2.2%). Induction of apoptosis by
overnight incubation of CD34 cells in serum-deprived medium resulted in
the upregulation of bcl-2 as a single population histogram. Thus, these
results suggest that in quiescent hematopoietic precursors, the bcl-2
protein plays a less prominent role as a survival promoter than
bcl-xL and that the low bcl-2 expression did not promote apoptosis. During day 10 of ex vivo expansion of CD34+
cells in liquid culture containing stem cell factor, interleukin-3 (IL-3), IL-6, IL-1 , and erythropoietin, the
CD34+/CD38 cells expressed high bcl-2 as a
single population histogram, and greater than 90% were
bcl-xL high. However, the expression of pro- and apoptotic
antigens increased: bax (10% to 15%), bad (5% to 8%), bak (6% to
14%), and ASP (6% to 10%). These results show the importance of
monitoring the expression of these proteins when defining the culture
conditions for ex vivo expansion.
© 1998 by The American Society of Hematology.
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