The synthetic peptide PrP-(106 -126) has previously been shown to be neurotoxic. Here, for the first time, we report that it induces apoptosis in the human neuroblastoma cell line SH-SY5Y. The earliest detectable apoptotic event in this system is the rapid depolarization of mitochondrial membranes, occurring immediately upon treatment of cells with PrP-(106 -126). Subsequent to this, cytochrome c release and caspase activation were observed. Caspase inhibitors demonstrated that while the peptide activates caspases they are not an absolute requirement for apoptosis. Parallel to caspase activation, PrP-(106 -126) was also observed to trigger a rise in intracellular calcium through release of mitochondrial calcium stores. This leads to the activation of calpains, another family of proteases. A calpain inhibitor demonstrated that while calpains are activated by the peptide they also are not an absolute requirement for apoptosis. Interestingly a combination of caspase and calpain inhibitors significantly inhibited apoptosis. This illustrates alternative pathways leading to apoptosis via caspases and calpains and that blocking both pathways is required to inhibit apoptosis. These results implicate the mitochondrion as a primary site of action of PrP-(106 -126).

Drug resistance remains a serious limiting factor in the treatment of acute myeloid leukenua (AML) either at initial presentation or following primary or subsequent relapses. Using specific kinase inhibitors, this study has investigated the conhibution of the Rks/PI3-kinase regulated survival pathways to drug resistance, and resulting suppression of apoptosis. Inhibition of the Raf/MAP-kinase pathway with Apigenin did not m i t i s e promyelocytic HL60 cells to drug-induced apoptosis, suggeshg a la& of involvement for this pathway in drug resistance. In contrast, the use of two speahc PI3-kinaae inhibitors, LY294002and Wortmarnun. ' did cause a significant increase in apoptosis in annbination with cytotoxic drugs. Following this observation, the contribution of two downstream effectom of PISCinaSe, p7OS6-kinase and PKB/Akt were investigated. Inhibition of p7OS6-kinase with rapamcyin did not alter levels of druginduced apoptosis. in contrast PI3-lanase lnhibition led to significant dephosphorylation of downahPam kmase PKB, suggesting that the PI%kinase/PKB survival pathway may play a mapr role in chemoresistance in AML. This pathway has previoudy been associated with modifications of the apoptotic regulator Bad. However we found no widence of Bad hetendher famation with anti-apoptotic regulators Bcl-2, Bd-& or Md-1, nor of alterations in Bax/Bcl-2 or Bax/Mcl-1 Itetemdimers following PI3-kinase inhibition. This suggests that alternative targets of PI3-kinase/PKB, distinct from the BcI-2 family may be responsible for amhibuting to drug resistance in AML. Thts work may represent a novel strategy for treatment of multidrug resistance in myeloid leukemia by combined use of inhibitors with conventional 13 Stress proteins and the regulation of apoptosis in tumour cells.