Background: Recent studies have shown that a number of cytostatic drugs exert their effect via the induction of apoptosis. Apoptosis, or programmed cell death, results from the activation of proteases that belong to the ICE superfamily which cleave a variety of substrates (such as lamin, actin, poly(ADP)ribose polymerase). This proteolytic process eventually leads to morphologic changes that characterise apoptosis and to DNA fragmentation. Experiments performed in our laboratory have revealed that, dependent on the stimulus that is used to induce apoptosis in cells of the Burkit cell line RAMOS, qualitative differences in the proteolytic activation of a central protease in the ICE cascade, i.e. caspase-3 (forinerly designated CPP32), can be demonstrated.
Purpose: Chronic lymphocytic leukaemia (B-CLL) B cells vary greatly in their ability to undergo apoptosis in response to therapeutic agents. Moreover, patient cells that are resistant to one type of cytostatic treatment may be sensitive to other drugs. At this moment, the mechanism(s) that underlie high and low sensitivity to individual cytostatic drugs is/are not known. Because our preliminary studies have shown that at the level of activation of caspase-3 different apoptotic stimuli are being integrated, we propose to study (non)responsiveness of B-CLL to apoptotic stimuli (i.e. cytostatic drug-induced) using caspase-3 cleavage as a biochemical read-out. Specifically, using the RAMOS model system we will determine whether different cytostatic drugs use identical or distinct pathways to induce caspase-3 activation and investigate if individual agents can synergise in the activation of this protease. Similar biochemical experiments will be performed with freshly isolated cells of B-CLL patients. Moreover, the sensitivity of B-CLL cells to particular cytostatic drugs will be quantitated and be related to the clinical efficacy of these agents.
The plan of investigation can be divided into two parts:
A. Determination of the pathways employed by cytostatic drugs to induce apoptosis. For these experiments the RAMOS cell line will be used. From this cell line variants have already been selected by us that differ in their sensitivity to various pro-apoptotic stimuli (BCR and CD95 ligation). Activation of the executioner caspase-3, and its relative caspase 7, by various cytostatic agents (i.e. fludarabine, chlorambucil, theophylline, prednisolon) will be analysed both quantitatively and qualitatively. Next, mutant cell lines will be selected on basis of their resistance to individual drugs and analysed for cross-resistance to other cytotoxic agents and for sensitivity to physiological apoptosis inducers (BCR and CD95 ligation). In addition, the effect of T-helper signals (i.e. CD40L and TNF-alpha) on the effect of cytotoxic drugs will be evaluated. On basis of these studies we expect to be able to order the intracellular pathways that are induced by individual cytotoxic agents.
B. Cleavage of caspase-3 in B-CLL, sensitivity to cytotoxic drugs in vitro, and relation to clinical efficacy. Biochemical experiments will be performed to assess caspase-3 activation in freshly isolated B-CLL cells. The response of these cells to cytostatic drugs will be quantitated and it will be investigated whether therapeutic drugs can synergise in the induction of apoptosis. Finally, it will be investigated whether the response of B-CLL cells in vitro is correlated with clinical efficacy.
Possible results / relevance for cancer research: Results coming from this study will (1) help to identify patients that will benefit most (or least) from treatment with certain therapeutic agents which is of prime importance in view of the severe side-effects of most drugs; (2) provide a rationale for combination therapy in the treatment for patients with B-CLL; (3) help to establish model systems that can also be applied in the in vitro analyses of cytostatic drugs which are being used lor the treatment of other haematological malignancies. |
