Shuwei Jiang

Expression of Bcl-2-related genes in normal and AML progenitors: changes induced by chemotherapy and retinoic acid

The expression of Bcl-2 family members was examined in normal and leukemic hematopoietic cells. Immature hematopoietic progenitor cells (CD34+/33−/13−) did not express Bcl-2 but Bcl-XL, the majority of CD34 cells expressed Bcl-2, Bcl-XL and BAD, and normal promyelocytes (CD34−/33+) lacked expression of both Bcl-2 and Bcl-XL, while leukemic CD34+progenitors and promyelocytes expressed these anti-apoptotic proteins. In AML, Bcl-2 expression was higher on CD34+ than on all AML cells, however, expression of Bcl-2 or Bcl-XL did not predict achievement of complete remission. Surprisingly, low Bcl-2 content was associated with poor survival in a group of patients with poor prognosis cytogenetics. The anti-apoptotic BAD protein was found to be expressed in AML, but was phosphorylated in 41/42 samples. Phosphorylation was found at both sites, Ser 112 and Ser 136. During induction chemotherapy, Bcl-2 levels of CD34 cells increased significantly. In the context of evidence for small numbers of leukemic CD34+ cells expressing very high levels of Bcl-2 prior to therapy, this finding is interpreted as a survival advantage of Bcl-2 overexpressing progenitors and rapid elimination of cells with low Bcl-2. Bcl-2 and Bcl-XL were both expressed in minimal residual disease cells. Downregulation of Bcl-2 mRNA and protein was observed by ATRA and the combination of Ara-C, followed by ATRA, resulted in markedly increased cytotoxicity in HL-60 cells, as compared to Ara-C alone or ATRA followed by Ara-C. Implications of these findings for the development of new therapeutic strategies for AML are discussed.

The anti-apoptotic genes Bcl-XL and Bcl-2 are over-expressed and contribute to chemoresistance of non-proliferating leukaemic CD34+ cells

Summary. In acute myeloid leukaemia (AML), cell kinetic quiescence has been postulated to contribute to drug resistance. As the anti‐apoptotic genes Bcl‐2 and Bcl‐XL have been implicated in cell cycle regulation, we investigated the expression of these genes in non‐proliferating (Q) and proliferating (P) AML and normal CD34+ progenitor cells. Using reverse transcription polymerase chain reaction, Bcl‐XL and Bcl‐2 were overexpressed in Q versus P AML cells, whereas no difference in Bcl‐XS and Bax expression was found. Furthermore, the Bcl‐XL/XS but not the Bcl‐2/Bax ratio was higher in Q AML compared with normal CD34+ Q cells (P = 0·001). An inverse correlation between Bcl‐2 expression of leukaemic Q cells and their ability to enter the cell cycle was found. Treatment with all‐trans retinoic acid (ATRA) reduced Bcl‐2 and Bcl‐XL expression in the leukaemic Q cells, and enhanced their chemosensitivity to cytosine arabinoside (ara‐C). These findings demonstrate overexpression of the anti‐apoptotic proteins Bcl‐XL and Bcl‐2 in quiescent CD34+ AML cells and suggest their involvement in the chemoresistance. The observed inverse correlation between Bcl‐2 and proliferation suggests a role for Bcl‐2 in the cell cycle regulation of AML. These findings could be used in the development of therapies that selectively induce apoptosis in quiescent leukaemic progenitor cells.

Cell-surface exposure of phosphatidylserine correlates with the stage of fludarabine-induced apoptosis in chronic lymphocytic leukemia and expression of apoptosis-regulating genes.

BACKGROUND Programmed cell death (PCD) is characterized by a sequence of tightly regulated events that result in the activation of caspases and in internucleosomal DNA cleavage. Late apoptotic events such as DNA-strand breaks can be assayed by in situ end labeling (ISEL) and DNA measurement (sub G1) using flow cytometry. Phosphatidylserine (PS) redistribution from the inner plasma membrane leaflet to the outer leaflet, an early event in PCD, can be detected by annexin V (AxV) binding to PS. AxV-fluorescein isothiocyanate (FITC) fluorescence intensity is variable and characterizes different cell populations, denoted here as AxV-negative (AxV(neg)), AxV-low-positive (AxV(lo)), and AxV-high-positive (AxV(hi)). METHODS We investigate the correlation of three methods (ISEL, sub G1 DNA content, and AxV assay) for detecting apoptosis with focus on differences between populations with different levels of PS. We also examined the expression of PCD-regulating Bcl-2 family members in these cell populations by reverse transcription-polymerase chain reaction (RT-PCR). Chronic lymphocytic leukemia (CLL) cells exposed to fludarabine (FAMP) were used as an in vitro model. Cells with different PS/AxV levels were separated using fluorescence-activated cell sorting (FACS). RESULTS Only purified AxV(hi) cells had high positivity in the ISEL and sub G1 assays (94 +/- 0.6%, 88.6 +/- 6.6%, and 98.6 +/- 0.6%, respectively), indicating that late apoptotic cells are detected equally by all three methods. In the AxV(lo) population, ISEL was positive in 21% +/- 13% and DNA sub G1 in 20% +/- 6.6% of cells, suggesting that AxV identifies early apoptotic cells better than the other assays. Anti-apoptotic Bcl-2 and Bcl-X(L) were upregulated by FAMP when cells entered apoptosis (AxV(lo)), as was pro-apo- ptotic Bcl-X(S), which was undetectable in nonapoptotic AxV(neg) cells. Pro-apoptotic Bax was only expressed in AxV(neg) and AxV(lo) cells. Late apoptotic AxV(hi) cells did not express Bcl-X(S) or Bax. RESULTS (1) AxV staining is more sensitive than sub G1 or ISEL in detecting early apoptotic cells; (2) only late apoptotic cells are equally detected by all assays; (3) AxV is a valuable tool in the detection and isolation of apoptotic cells at different stages of PCD; and (4) pro-apoptotic Bcl-X(S) and Bax are expressed at early, not late, stages of apoptosis.

Quantitation of minimal residual disease in acute myelogenous leukemia and myelodysplastic syndromes in complete remission by molecular cytogenetics of progenitor cells

Detection of karyotypic clonal abnormalities are prognostically useful in patients with acute myelogenous leukemia (AML) and myelodysplastic syndromes (MDS), but cytogenetic methods are not sensitive enough to detect low numbers of residual leukemic cells in patients who have achieved complete remission (CR). Fluorescence in situ hybridization (FISH) and fluorescence activated cell sorting (FACS) were used to investigate the frequency and presence of minimal residual disease (MRD) in AML and MDS patients (n = 28) with monosomy of chromosomes 7, 17 and 18 and trisomy of chromosomes 6, 8, 9 and 10 in CR. MRD was detected in all patients with monosomy 7 (n = 10) and followed by relapse in eight patients after 4.8 ± 3.1 months. In contrast, persistent leukemic cells occurred in 11/12 patients with trisomy 8, but only three of them relapsed after 7.7 ± 4.0 months. Cox regression analysis showed that cytogenetic class and levels of clonal cells at CR were related to time to relapse (P = 0.001). The level of MRD identified patients at high and low risk of relapse. High absolute levels of proliferating residual leukemic cells correlated with monosomy 7 and high risk of relapse.

Minimal residual disease in acute myelogenous leukaemia and myelodysplastic syndromes: a follow‐up of patients in clinical remission

The majority of patients with acute myelogenous leukaemia (AML) and myelodysplastic syndromes (MDS) relapse, especially those with unfavourable cytogenetics.