The Baxα:Bcl-2 ratio modulates the response to dexamethasone in leukaemic cells and is highly variable in childhood acute leukaemia

Abstract

Bcl-2 over-expression has been shown to inhibit apoptosis induced by a variety of stimuli, whereas a predominance of Baxa to Bcl-2 accelerates apoptosis upon apoptotic stimuli. We sought to study the relevance of these apoptotic regulating gene products in leukaemia. In a panel of leukaemia and lymphoma cell lines (HL60, DoHH2, CEM C7, L1210 and S49), the Baxa-to-Bcl-2 ratio as assessed by Western-blot analysis correlated with sensitivity to dexamethasone treatment. In addition, in HAbaxa-transfected CEM C7 clones, a similar correlation was found for dexamethasone and thapsigargin sensitivity. In bone-marrow aspirates from patients with childhood acute lymphoblastic or myelocytic leukaemia (ALL, n 5 48; AML, n 5 8), the Bcl-2 and Baxa levels were highly variable, but well within the range found in the Baxa transfectants and in the established cell lines. Bcl-2 levels were lower in T-than in B-lineage ALL, which could be ascribed to simultaneous inverse relation between Bcl-2 and WBC. By contrast, Baxa:Bcl-2 was independent of any presenting feature and was largely dependent on Baxa levels. Results suggest that Baxa:Bcl-2, rather than Bcl-2 alone is important for the survival of drug-induced apoptosis in leukemic cell lines and ALL. Int. J. Cancer 71:959-965, 1997.r 1997 Wiley-Liss, Inc.Bcl-2 was initially identified because of its involvement in the chromosomal translocation t(14;18), resulting in high levels of the 26-kDa Bcl-2 protein. Bcl-2 over-expression was subsequently shown to inhibit apoptosis (for review, see Reed, 1995). Moreover, over-expression of Bcl-2 has been implicated in the insensitivity of cells to a variety of anti-cancer drugs, especially those used in the chemotherapy of childhood ALL, notably glucocorticoids (GC) (Miyashita and Reed, 1992;Sentman et al., 1991;Smets et al., 1994). Bcl-2 may, therefore, define a new category of drugresistance genes regulating the physiological cell-death pathway (Reed, 1995). Accordingly, increased levels of Bcl-2 may be associated with poor prognosis in leukaemia by a dual action. First, it could expand the population of circulating blasts by promoting their survival and, second, it could antagonize response to chemotherapy by inhibiting steroid hormone-and cytostatic-drug-induced apoptosis. Attesting to this view, high expression of Bcl-2 has been associated with high WBC and poor prognosis in adult AML (Campos et al., 1993).In childhood ALL, the role of Bcl-2 is not clear yet. A number of studies (Campana et al., 1993;Gala et al., 1994;Coustan-Smith et al., 1996) agree that high Bcl-2 expression is the rule in ALL blasts, compared with their normal precursors, while no study found an association between Bcl-2 levels and the presenting features immunophenotype, ploidy and WBC. Increased expression of Bcl-2 was not found to correlate with prognosis in one study (Gala et al., 1994), but correlated with poor initial response to chemotherapy in another report (Maung et al., 1994). By contrast, CoustanSmith et al. (1996) found that elevated levels of Bcl-2 we...

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“…between Bcl-2 and immunophenotype and WBC. 12,14 However, this study is the first to reveal in ALL a significant inverse correlation between Bcl-2 levels and the percentage S-phase cells in both PB (P Ͻ 0.0001, r = −0.52; Figure 2, Table 3) and BM blasts (P = 0.0044, r = −0.34). In PB the determination of %S may be complicated by contaminating normal cells.…”

“…We confirmed an inverse correlation between Bcl-2 and WBC, as well as a relation with immunophenotype. 12,14 In addition a weak correlation was found between Bcl-2 and high RF and hyperdiploidy. More importantly, this is the first study that reveals an inverse relationship in ALL between Bcl-2 and %S phase cells, suggesting that Bcl-2 has an adverse effect on the rate of proliferation of leukemic blasts.…”

“…Patient characteristics and cell biological variables are detailed in Table 1. Mononuclear cells were isolated as described previously 14 at the DCLSG. Analysis of immunophenotype was centrally determined by the DCLSG.…”

“…The membranes were dried at room temperature and cut into two sections. The section containing proteins Ͻ35 kDa was either used to detect Bcl-2/Bax (DAKO: clone 124, Santa Cruz Biotechnology: sc-493) as described previously 14 or used to detect either Bcl-x L (sc-634), Bak (sc-832) or Bad (Transduction Laboratories: 36420), while the section containing proteins of Ͼ35 kDa was used to detect actin (Boehringer Mannheim Biochemica, Mannheim, Ger- Table 1 Characteristics of study patients (n = 86) …”

“…4,11 Bcl-2 is highly variable among cases with newly diagnosed childhood acute lymphoblastic leukemia (ALL). [12][13][14] Despite the fact that Bcl-2 expression levels in cell lines modulate the sensitivity to most clinically relevant drugs, the pretreatment Bcl-2 levels do not predict 2.5-year survival rates in ALL or the in vitro response to cytostatic treatment. 12,13 It has been speculated that the balance of apoptosis-inducers vs inhibitors decides whether or not a cell survives upon apoptotic stimuli.…”