The BCL-2 proto-oncogene encodes a mitochondrial protein that blocks programmed cell death. High amounts of bcl-2 protein are found not only in lymphoid malignancies, but also in normal tissues characterized by apoptotic cell death, including bone marrow. Using a monoclonal antibody to bcl-2 protein, we analyzed 82 samples of newly diagnosed acute myeloid leukemia. The number of bcl-2+ cells in each sample was heterogeneous (range, 0% to 95%), with a mean of 23%. The percentage of bcl-2+ cells was higher in M4 and M5 types, according to French- American-British classification, and in cases with high white blood cell counts. bcl-2 expression was also correlated with that of the stem cell marker CD34. In vitro survival of leukemic cells maintained in liquid culture in the absence of growth factors was significantly longer in cases with a high percentage of bcl-2+ cells. High expression of bcl-2 was associated with a low complete remission rate after intensive chemotherapy (29% in cases with 20% or more positive cells v 85% in cases with less than 20% positive cells, P < 10(-5)) and with a significantly shorter survival. In multivariate analysis, the percentage of bcl-2+ cells (or the blast survival in culture), age, and the percentage of CD34+ cells were independently associated with poor survival.

Excessive apoptosis is implicated in the pathogenesis of myelodysplastic syndromes (MDS). We assessed by flow cytometry the expression of several members of the Bcl-2 family in bone marrow mononuclear cells (BMMNC) of 168 MDS samples at diagnosis. The proteins studied were Bcl-2, Bcl-xL (anti-apoptotic), Bax, Bad, Bak, and Bcl-xS (pro-apoptotic). The percentage of BMMNC expressing Bcl-2 and Bcl-xL was higher in refractory anemia with excess of blasts (RAEB), RAEB in transformation (RAEB-T), and chronic myelomonocytic leukemia (CMML) than in refractory anemia (RA) and RA with ringed sideroblasts (RAS). Conversely pro-apoptotic proteins Bad, Bak, and Bcl-xS were detected in a higher percentage of cells in RA and RAS. RA and RAS were associated with an increased Bcl-xS/Bcl-xL ratio. The expression of anti-apoptotic proteins was also correlated with that of CD34 and P170 and with the percentage of blast cells. Two-color analyses demonstrated that CD34 and Bcl-2 were usually expressed in the same cells. No significant correlation was found with cytogenetic abnormalities. Higher expression of pro-apoptotic Bcl-2-family proteins (Bak, Bad, Bcl-xS) and higher Bcl-xS/Bcl-xL ratio were associated with longer survival and decreased risk of leukemic transformation in univariate analysis, whereas expression of anti-apoptotic proteins was associated with decreased survival. Consequently Bcl-2 proteins expression was well correlated with the International Prognostic Scoring System (IPSS). Our data confirm that the control of apoptosis is deregulated in MDS cells. Moreover, the study of markers such as CD34 (or Bcl-2), Bcl-xL, and Bcl-xS provides additional prognostic information.

The BCL-2 proto-oncogene encodes a mitochondrial protein that blocks programmed cell death. High amounts of bcl-2 protein are found not only in lymphoid malignancies, but also in normal tissues characterized by apoptotic cell death, including bone marrow. Using a monoclonal antibody to bcl-2 protein, we analyzed 82 samples of newly diagnosed acute myeloid leukemia. The number of bcl-2+ cells in each sample was heterogeneous (range, 0% to 95%), with a mean of 23%. The percentage of bcl-2+ cells was higher in M4 and M5 types, according to French- American-British classification, and in cases with high white blood cell counts. bcl-2 expression was also correlated with that of the stem cell marker CD34. In vitro survival of leukemic cells maintained in liquid culture in the absence of growth factors was significantly longer in cases with a high percentage of bcl-2+ cells. High expression of bcl-2 was associated with a low complete remission rate after intensive chemotherapy (29% in cases with 20% or more positive cells v 85% in cases with less than 20% positive cells, P < 10(-5)) and with a significantly shorter survival. In multivariate analysis, the percentage of bcl-2+ cells (or the blast survival in culture), age, and the percentage of CD34+ cells were independently associated with poor survival.

Myelodysplastic syndromes (MDS) are characterized by abnormal growth of committed progenitors in clonogenic assay, with reduced number of colonies and decreased colony/cluster ratio. It has been suggested that excessive apoptosis is the cause of marrow failure in MDS. We studied the expression of caspase-1 (interleukin-1␤-converting enzyme, ICE) and caspase-3 (CPP32/apopain) in marrow mononuclear cells, and the growth pattern of committed progenitors in a series of 83 MDS cases. The percentage of apoptotic cells as detected by TUNEL technique, and the percentage of caspase-3-positive cells were significantly higher in refractory anemia (RA) and RA with ringed sideroblasts (RAS) than in chronic myelomonocytic leukemia (CMML), refractory anemia with excess of blasts (RAEB) and RAEB in transformation (RAEB-T). Spontaneous growth of CFU-GM was associated with a higher percentage of blasts, and with a lower expression of caspase-3 and caspase-1. The yield of CFU-E, BFU-E, and CFU-GM (in the presence of growth factors) was decreased by comparison to normal marrow, but large individual differences were observed in all cytological categories. Inhibition of caspase-1 and caspase-3 activities by specific inhibitors resulted in a significant increase of the production of all types of colonies (up to 50-fold of control). In the presence of caspase-3 inhibitor, the number of BFU-E and CFU-E was in the range of normal values in most cases of RA and RAS. In addition, caspase-1 and -3 protease activities were detectable by fluorogenic assay in all cases studied. Western blot analysis confirmed the expression of caspase-3, including the cleaved (activated)-p17 form in most cases of RA/RAS analyzed. It is concluded that caspase-3 is implicated in the increased apoptosis observed in MDS and that inhibition of its activity can restore at least partially the growth of committed progenitors. Leukemia (2000) 14, 2045-2051.

The expression of Bcl-2 family proteins (Bcl-2, Bcl-X, Bcl-XL, Bcl-Xs, BAX, BAD, MCL-1) and of Interleukin-1 converting enzyme (ICE)-related proteins (ICE, CPP32, ICH- 1) was analyzed in acute leukemia cells by flow cytometry. Most proteins studied were detectable in cell lines such as KG1a, HL60, K562 (myeloblastic), REH, RAJI and MOLT4 (lymphoblastic) and VAL (B-cell lymphoma). However, BCL-Xs and BAK were weakly expressed in K562, as were Bcl-X, BAD and BAK in the VAL line. In acute myeloid leukemia (66 cases studied), the proteins were expressed in most cases in a high percentage of cells, especially BAX and CPP32, without correlation with hematological characteristics. However, Bcl-2 was expressed in a higher percentage of cells in FAB M1 and M5 cases, and in CD34-positive cases, whereas Bcl-Xs was more frequently expressed in M3 cases. No differences were observed regarding fluorescence intensity. Higher percentages of Bcl-2-positive cells were associated with low remission rate, while expression of Bcl-Xs was predictive of high remission rate. In acute lymphoblastic leukemia (36 cases), all proteins studied were expressed in a majority of cases. Bcl-Xs was more frequently detected in T-cell type, and was also associated with a higher remission rate. These results suggest that apoptosis-controlling proteins may have a role in the pathogenesis and response to therapy of acute leukemia.