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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Human neutrophilic myeloperoxidase (MPO) is involved in the defence mechanism of the body against micro-organisms. The enzyme catalyses the generation of the strong oxidant hypochlorous acid (HOC1) from hydrogen peroxide and chloride ions. In normal neutrophils MPO is present in the dimeric form (140 kDa). The disulphide-linked protomers each consist of a heavy subunit and a light one. Reductive alkylation converts the dimeric enzyme into two protomers, 'hemi-myeloperoxidase'.We studied the initial activities of human dimeric MPO and hemi-MPO at the physiological pH of 7.2 and found no significant differences in chlorinating activity. These results indicate that, at least at neutral pH, the protomers of MPO function independently. The absorption spectra of MPO compounds I1 and 111, both inactive forms concerning HOCl generation, and the rate constants of their formation were the same for dimeric MPO and hemi-MPO, but hemi-MPO required a slightly larger excess of HzOz for complete conversion. Hemi-MPO was less stable at a high temperature (SO'C) as compared to the dimeric enzyme. Furthermore, the resistance of the chlorinating activity of hemi-MPO against its oxidative product hypochlorous acid was somewhat lower (ICso = 32 pM HOC1) compared to dimeric MPO (ICs0 = 50 pM HOCl). The higher stability of dimeric MPO in the presence of its oxidative product compared to that of monomeric MPO might be the reason for the occurrence of MPO as a dimer.The enzyme myeloperoxidase (donor: H 2 0 2 oxidoreductase; MPO) is a major azurophil granule component of normal human ncutrophils and makes up 2 -5% of the total dry mass of these cells [l -41. MPO is involved in the defence mechanism against invading micro-organisms. In the presence of H202, which is generated from 0; produced by NADPH oxidase [5, 61, MPO so-called 'hemi-MPO' [18]. From kinetic studies of the chlorinating and peroxidase (in the absence of CI -) activities in the pH range of 4.4-6.2 of hemi-MPO (generated from normal dimeric MPO [20]), and of monomeric MPO (isolated from HL-60 cells [21]) no significant differences were found between dimeric enzyme, and hemi-or monomeric enzyme. At neutral pH, however, the peroxidase activities of monomeric MPO (79 kDa) [23], both isolated from human leukemia cells, were slightly lower as compared to those of the dimeric or 'large' MPO.Based on the assumption that dimcrization might confer higher catalytic activity or increased stability to the enzyme, we investigated and compared some properties of hemi-MPO with those of dimeric MPO. If the dimer was more stable or more active, this could explain its exclusive occurrence in normal human neutrophils. Besides chlorinating activity at a neutral physiological pH, we also examined compound I1 and compound 111 formation, since both compounds are inactive forms of MPO resulting from side-reactions in the catalytic process [24, 251. Furthermore, we investigated the heat stability and the resistance of both enzyme preparations towards their own product HOCl and the secondary oxid...

Upon agonist binding the heteromeric glucocorticoid receptor complex undergoes a conformational change (receptor activation). This event involves the dissociation of a dimer of 90 kDa heat shock proteins. Whereas receptor activation in cytosolic assays is both rapid and irreversible, less is known about the receptor activation and translocation in intact cells during challenge with an agonist. In this paper we report on the receptor status of glucocorticoid-sensitive murine S49 lymphoma cells during dexamethasone exposure. By three different assays, ligand (re)binding, nuclear translocation and hsp90 co-immunoprecipitation, it was found that the majority of the glucocorticoid receptor protein was in a non-activated conformation. Furthermore, prolonged exposure to dexamethasone did not result in increased levels of activated receptors. By assessing receptor activation in situ we found that physiological temperature was less effective in dissociating hsp90 compared to room temperature. These findings indicate that the physiological temperature negatively controls receptor activation, probably due to a thermolabile interaction between the hormone and its cognate receptor.