Bcl-2 family members in childhood acute lymphoblastic leukemia: relationships with features at presentation, in vitro and in vivo drug response and long-term clinical outcome.

We have found that, in addition to Bcl-2 and Bax, the expression levels of apoptosis inducers (Bad, Bak) and inhibitors (Bcl-xL, Mcl-1) were highly variable in blasts from 78 children with newly diagnosed acute lymphoblastic leukemia (ALL). The patients were enrolled in the national study ALL-7 of the Dutch Childhood Leukemia Study Group. In contrast to Bcl-2 that inversely correlated with %S-phase cells and WBC, and was lower in T than in B-lineage ALL, the Bcl-2 family members were not found to be associated with features at presentation. These expression levels were also compared with drug resistance in in vitro MTT (methyl-thiazol-tetrazolium) assays for prednisolone, vincristine and asparaginase in 46 children. Protein expression levels of the Bcl-2 family were not found to correlate with in vitroresistance to the individual drugs or the combined drug resistance profile. In addition, neither peripheral blast reduction after 1 week of prednisone monotherapy nor long-term disease-free interval or survival showed a correlation with protein expression. Our results indicate that the anti-proliferative function of Bcl-2 dominates its anti-apoptotic function in ALL, but neither Bcl-2 nor the Bcl-2 family members gained prognostic information in the risk-adapted protocol ALL-7.

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

Bcl-2 over-expression has been shown to inhibit apoptosis induced by a variety of stimuli, whereas a predominance of Bax alpha 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 Bax alpha-to-Bcl-2 ratio as assessed by Western-blot analysis correlated with sensitivity to dexamethasone treatment. In addition, in HAbax alpha-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 = 48; AML, n = 8), the Bcl-2 and Bax alpha levels were highly variable, but well within the range found in the Bax alpha 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, Bax alpha:Bcl-2 was independent of any presenting feature and was largely dependent on Bax alpha levels. Results suggest that Bax alpha:Bcl-2, rather than Bcl-2 alone is important for the survival of drug-induced apoptosis in leukemic cell lines and ALL.

BCL-2 expression and mitochondrial activity in leukemic cells with different sensitivity to glucocorticoid-induced apoptosis.

The present study investigates the relationship between mitochondrial activity and the expression of the BCL-2 gene in a panel of six human and murine leukemia/lymphoma cell lines. The cell lines all contained normal glucocorticoid receptors but differed widely in sensitivity to dexamethasone, ranging from very sensitive S49 lymphoma to completely resistant HL-60 acute leukemia cells. In this panel, 10- to 15-fold differences in basal adenosine triphosphate (ATP) content and adenosine diphosphate (ADP)/ATP ratio were correlated with up to fivefold differences in bcl-2 protein (in human cells) and approximately 25-fold difference in bcl-2 mRNA content (all cell lines). Moreover, ATP content and BCL-2 gene expression were inversely correlated with glucocorticoid sensitivity and cell cycle length. In resistant cell lines, sensitivity to dexamethasone was restored by the mitochondrial inhibitors rotenone and meta-iodobenzylguanidine. This sensitization was not accompanied by detectable reductions in bcl-2 mRNA or protein content, suggesting that the inhibitors were capable of overriding BCL-2-mediated inhibition of apoptosis. Increased mitochondrial activity and (overexpressed) BCL-2 appeared closely related properties of glucocorticoid-resistant cells, sharing common cellular targets in hormone-induced apoptosis.

Uptake of the neuron-blocking agent meta-iodobenzylguanidine and serotonin by human platelets and neuro-adrenergic tumour cells.

The adrenomedulla-imaging agent meta-iodobenzylguanidine (MIBG) is concentrated by various tumours of neuroectodermal origin. Radio-iodinated [131I]MIBG is therefore increasingly used for diagnosis and therapy of these disorders. To study the cause of thrombocytopenia associated with [131I]MIBG therapy, we investigated the uptake of MIBG in human platelets in comparison with that of serotonin. Specific imipramine-sensitive uptake of [131I]MIBG was much slower than of [3H]serotonin, but after prolonged incubation high and serotonin-equivalent uptake levels were observed. Accumulation of MIBG saturated at 10- to 100-fold higher concentration than serotonin, and the affinity for uptake and intracellular storage in platelets was much higher for serotonin than for MIBG. Conversely, serotonin was not detectably concentrated by neuroadrenergic Uptake-I in SK-N-SH neuroblastoma and PC12 pheochromocytoma cells. Fluvoxamine inhibited the uptake of norepinephrine and MIBG in PC12 cells, similarly to that of serotonin in platelets. However, the drug was 100-fold more effective in inhibiting platelet transport of MIBG than of serotonin. The results indicate that MIBG uptake in platelets is not mediated by a neuro-adrenergic Uptake-I, but probably proceeds via the serotonin transport system. MIBG concentration by platelets was at least as efficient as in neuro-adrenergic tumour cells and has therefore (radio)biological potential for injuring these cells or precursor megakaryocytes. Platelet uptake of MIBG could be selectively blocked by fluvoxamine in concentrations which minimally affected its accumulation in neuro-adrenergic target cells.