A Systematic Approach to the Dissection of Apoptotic Blockades in Treatment-Refractory Acute Myeloid Leukemias

Abstract

Therapeutic success in acute myeloid leukemia (AML) relies on the successful initiation and completion of programmed cell death (PCD) in leukemic blasts. However, some AML — esp. those with complex cytogenetic aberrations — often remain treatment-refractory i.e. PCD is not undergone despite the presence of drug-induced DNA damage (genotoxicity). Defects of the apoptotic machinery are a potential common denominator for a wide range of refractoriness which might also include the effector mechanisms of T-cell mediated cytotoxicity (FAS-ligand, Perforin/Granzyme B). Since the relevance of isolated parameters of the redundant PCD network is mostly unclear a prospective analysis was initiated which examines the three distinct phases of PCD — commitment phase (CP), effector phase (EP) and loss of viability (LOV): The initial CP is characterized morphologically by the apoptotic volume decrease (AVD) or blebbing which causes a steep rise in UV absorption of the affected cell suspension in the MicK (microculture kinetic) assay which is performed as a continuous measurement in a heatable thermoreader over 24 hours. As the pivotal phenomenon of the later EP the activation of the central effector caspase (caspase-3) is measured. The final LOV is measured by the loss of mitochondrial activity via a tetrazolium salt based assay (WST). This combination of parallel analyses allows the functional localization of any failure to commit PCD into one of these 3 phases. Analyses following genotoxic signals (VP 16, AraC) and FAS-ligand exposure allow the differentiation of blockades into those involving p53 and caspase-9 (genotoxicity) vs. those involving FADD and caspase-8 (FAS- ligand).

Data so far indicate that genotoxicity by VP 16 and AraC reduces viability significantly at 96 hours whereas Fas-ligand showed only a very moderate effect on AML blasts. No complete block in the induction of cell death following genotoxicity was observed so far. Early cytoreduction at 24 hours following VP 16 was associated (p<0,05) with high caspase activation following VP 16. No such correlation was found for AraC.