APOPTOTIC REGULATION IN PRIMITIVE HEMATOPOIETIC PRECURSORS

Bcl-2 and bcl-x(L) function as suppressors of programmed cell death. T he expression of bcl-2 protein in vivo is associated with long-lived h ematopoietic cells such as mature lymphocytes and early myeloid progen itors. Bcl-x(L), a homologue of bcl-2, is also expressed in lymphocyte s and thymocytes. In contrast, the bcl-2-related proteins (bax, bad, a nd bag() act by promoting apoptotic cell death as shown from their exp ression in hematopoietic cell lines. We analyzed the expression of bcl -2 and bcl-x proteins in hematopoietic precursors obtained from variou s cell sources in adult mobilized peripheral blood collected from 13 p atients with solid tumors, 8 adult bone marrow, and 12 umbilical cord blood. The analysis was based on the expression of the proliferation a nd activation specific antigens, CD38 and class II (HLA-DR). Similarly , we analyzed the expression of bcl-2-related proteins bcl-x(L), bar, had, and bak before and during ex-vivo expansion. Hematopoietic precur sors expressing strongly the CD34 antigen (CD34(s+)) and lacking CD38 or HLA-DR expression were analyzed by using three-color immunofluoresc ence staining. The majority of CD34(+) cells expressed bcl-2 and unexp ectedly showed a bimodal distribution of low and high expression. More cells that lacked or expressed low density CD38 expressed low bcl-2 t han the more differentiated counterparts (those with high density CD38 ). Immaturity tie, little or no HLA-DR) is associated with the express ion of low bcl-2 compared with HLA-DR+. However, HLA-DR-/low populatio n contained a lower number of cells expressing low bcl-2 (30% to 40%) than CD38(-/low) in comparable samples. The hematopoietic precursors w ith bcl-2(low) and bcl-2(high) formed a homogeneous population of undi fferentiated lymphoid-like cells having a similar forward scatter. The se cells expressed strongly the bcl-x(L) protein (>95%) but were bar l ow (4% to 12%), bad low (0% to 0.8%), and bak low (0% to 3%). The expr ession of apoptosis specific protein (ASP) was also low (3.4% +/- 3.1% ) as was Annexin V. In addition, the CD34(+)/CD38(-) showed low cell c ycle 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 t hat in quiescent hematopoietic precursors, the bcl-2 protein plays a l ess prominent role as a survival promoter than bcl-x(L) and that the l ow bcl-2 expression did not promote apoptosis. During day 10 of ex viv o expansion of CD34+ cells in liquid culture containing stem cell fact or, interleukin-1 (IL-3), IL-6, IL-1 beta, and erythropoietin, the CD3 4(+)/CD38(-) cells expressed high bcl-2 as a single population histogr am, and greater than 90% were bcl-x(L) high. However, the expression o f pro- and apoptotic antigens increased: bar (10% to 15%), bad (5% to 8%), bak (6% to 14%), and ASP (6% to 10%). These results show the impo rtance of monitoring the expression of these proteins when defining th e culture conditions for ex vivo expansion. (C) 9998 by The American S ociety of Hematology..