Defects in apoptosis underpin both tumorigenesis and drug resistance, and because of these defects chemotherapy often fails. Understanding the molecular events that contribute to drug-induced apoptosis, and how tumors evade apoptotic death, provides a paradigm to explain the relationship between cancer genetics and treatment sensitivity and should enable a more rational approach to anticancer drug design and therapy.

Many chemotherapeutic agents induce mitochondrial-membrane disruption to initiate apoptosis. However, the upstream events leading to drug-induced mitochondrial perturbation have remained poorly defined. We have used a variety of physiological and pharmacological inhibitors of distinct apoptotic pathways to analyze the manner by which suberoylanilide hydroxamic acid (SAHA), a chemotherapeutic agent and histone deacetylase inhibitor, induces cell death. We demonstrate that SAHA initiates cell death by inducing mitochondria-mediated death pathways characterized by cytochrome c release and the production of reactive oxygen species, and does not require the activation of key caspases such as caspase-8 or -3. We provide evidence that mitochondrial disruption is achieved by means of the cleavage of the BH3-only proapoptotic Bcl-2 family member Bid. SAHA-induced Bid cleavage was not blocked by caspase inhibitors or the overexpression of Bcl-2 but did require the transcriptional regulatory activity of SAHA. These data provide evidence of a mechanism of cell death mediated by transcriptional events that result in the cleavage of Bid, disruption of the mitochondrial membrane, and production of reactive oxygen species to induce cell death.

The essential upstream steps in granzyme B–mediated apoptosis remain undefined. Herein, we show that granzyme B triggers the mitochondrial apoptotic pathway through direct cleavage of Bid; however, cleavage of procaspases was stalled when mitochondrial disruption was blocked by Bcl-2. The sensitivity of granzyme B–resistant Bcl-2–overexpressing FDC-P1 cells was restored by coexpression of wild-type Bid, or Bid with a mutation of its caspase-8 cleavage site, and both types of Bid were cleaved. However, Bid with a mutated granzyme B cleavage site remained intact and did not restore apoptosis. Bid with a mutation preventing its interaction with Bcl-2 was cleaved but also failed to restore apoptosis. Rapid Bid cleavage by granzyme B (<2 min) was not delayed by Bcl-2 overexpression. These results clearly placed Bid cleavage upstream of mitochondrial Bcl-2. In granzyme B–treated Jurkat cells, endogenous Bid cleavage and loss of mitochondrial membrane depolarization occurred despite caspase inactivation with z-Val-Ala-Asp-fluoromethylketone or Asp-Glu-Val-Asp-fluoromethylketone. Initial partial processing of procaspase-3 and -8 was observed irrespective of Bcl-2 overexpression; however, later processing was completely abolished by Bcl-2. Overall, our results indicate that mitochondrial perturbation by Bid is necessary to achieve a lethal threshold of caspase activity and cell death due to granzyme B.

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Various first messengers linked to phospholipase C, including acetylcholine and interleukin 1, regulate the production both of the secreted form of the amyloid protein precursor (APP) and of amyloid a-protein. We have now identified intracellular signals which are responsible for mediating these effects. We show that activation of phospholipase C may affect APP processing by either of two pathways, one involving an increase in protein kinase C and the other an increase in cytoplasmic calcium levels. The effects ofcalcium on APP processing appear to be independent of protein kinase C activation. The observed effects of calcium on APP processing may be of therapeutic utility.Alzheimer disease is characterized by distinct neuropathological lesions, including intracellular neurofibrillary tangles, extracellular parenchymal and cerebrovascular amyloid deposits; and selective cell death that particularly affects cholinergic neurons in the basal forebrain (1). The principal component of parenchymal amyloid plaque cores and cerebrovascular amyloid is the amyloid (3-protein (A(3) (2-4). It has been shown that this -4-kDa protein is produced by various cultured cells (5-7), including transfected cells stably expressing the amyloid protein precursor (APP), from which AB is derived (8)(9)(10)(11)(12)(13)(14).During the past few years, a variety of evidence has emerged indicating that the processing of APP is regulated by signal transduction pathways. Thus, phorbol esters (activators of protein kinase C) and okadaic acid (an inhibitor of protein phosphatases 1 and 2A) increase APP metabolism and secretion (15-18). More recently, it has been shown that first messengers known to activate the phospholipase C/protein kinase C cascade increase the secretion of APP (17,19). It was also shown that the formation of a peptide with properties similar to those of Af was decreased by phorbol esters, by okadaic acid, by direct activators of phospholipase C, and by first messengers that activate phospholipase C (20-22). However, activation of phospholipase C not only activates protein kinase C (through the formation ofdiacylglycerol) but also increases cytoplasmic calcium (through the action of inositol 1,4,5-trisphosphate, IP3). For this reason, we undertook an investigation to determine whether the IP3/calcium limb of this pathway might, like the diacylglycerol/protein kinase C limb, affect APP processing. MATERIALS AND METHODSCell culture conditions and the sources of analytical reagents have been described (17,20 Pulse-chase labeling of cells was carried out on confluent cell monolayers in six-well culture dishes (Corning) with 1 ml of methionine-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 1 mCi (37 MBq) of [35S]methionine/cysteine (EXPRE35S35S; NEN). Metabolic labeling was carried out for 2 hr, followed by a chase period of 2 hr. The chase was initiated by replacing the labeling medium with DMEM containing 0.2 mM unlabeled methionine. Two minutes after the start of the chase, the indicated test compounds ...

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Multidrug resistance (MDR) mediated by the ATP-dependent efflux protein P-glycoprotein (P-gp) is a major obstacle to the successful treatment of many cancers. In addition to effluxing toxins, P-gp has been shown to protect tumor cells against caspase-dependent apoptosis mediated by Fas and tumor necrosis factor receptor (TNFR) ligation, serum starvation and ultraviolet (UV) irradiation. However, P-gp does not protect against caspase-independent cell death mediated by granzyme B or pore-forming proteins (perforin, pneumolysin and activated complement). We examined the effects of the chemotherapeutic hybrid polar compound suberoylanilide hydroxamic acid (SAHA) on P-gp-expressing MDR human tumor cell lines. In the CEM T-cell line, SAHA, a histone deacetylase inhibitor, induced equivalent death in P-gp-positive cells compared with P-gp-negative cells. Cell death was marked by the caspase-independent release of cytochrome c, reactive oxygen species (ROS) production and Bid cleavage that was not affected by P-gp expression. However, consistent with our previous findings, SAHA-induced caspase activation was inhibited in P-gp-expressing cells. These data provide evidence that P-gp inhibits caspase activation after chemotherapeutic drug treatment and demonstrates that SAHA may be of value for the treatment of P-gp-expressing MDR cancers. © 2002 Wiley-Liss, Inc. Key words: histone deacetylase; P-glycoprotein; multidrug resistance; apoptosis; chemotherapyMany chemotherapeutic drugs induce death of their target cells by activating physiologic apoptotic pathways. Two functionally separable, yet molecularly linked intracellular death pathways requiring a family of cysteine aspases (caspases) have been identified. 1,2 One pathway requires ligation and oligomerization of death receptors such as Fas and tumor necrosis factor (TNF) receptor I to initiate the activation of membrane-proximal caspases such as caspase-8 and -10, which in turn cleave and activate effector caspases such as caspase-3 and -7. The other pathway requires disruption of the mitochondrial membrane, usually by pro-apoptotic Bcl-2 proteins, the dissipation of transmembrane potential (⌬⌿ m ) and the release of mitochondrial proteins including cytochrome c. Cytochrome c functions with Apaf-1 to induce activation of caspase-9, thereby initiating the apoptotic caspase cascade. Many commonly used chemotherapeutic agents such as doxorubicin, vincristine and cisplatin can induce death of their tumor targets by initiating the mitochondrial apoptotic pathway. 3,4 Multidrug resistance (MDR) is a major obstacle to the successful treatment of many human cancers. A hallmark of MDR is the expression of the ABC transporter molecule P-glycoprotein (P-gp). Typically, P-gp functions to confer MDR by actively effluxing chemotoxins from cells, thereby preventing intracellular accumulation and subsequent apoptotic cell death. 5 In addition, we and others have recently shown that P-gp can inhibit the activation of caspases and prevent apoptosis induced by diverse stimuli such as ser...

Previous studies by our laboratory have shown that the drug transporter protein P-glycoprotein, P-gp, can specifically inhibit Fas-induced caspase-3 activation and apoptosis. Importantly, inhibition of both caspase-3 activation and cell death could be reversed by pharmacological and antibody inhibitors of P-gp function. However, the molecular mechanisms underpinning P-gp-mediated resistance to Fas-induced cell death and caspase activation remained unknown. We therefore sought to identify the point(s) within the death receptor pathway at which P-gp exerted its inhibitory effect and to determine whether the ATPase activity of P-gp was required. Structure-function analysis determined that ATP hydrolysis was necessary for P-gp to confer resistance to Fasinduced caspase activation and cell death. Importantly, although both FADD and caspase-8 were recruited to the Death Inducing Signal Complex (DISC) in wild-type P-gp expressing cells following Fas ligation, subsequent activation of caspase-8 at the DISC was inhibited. The ability of P-gp to inhibit caspase-8 activation was also ATP dependent. These studies demonstrate that P-gp inhibits Fas-induced caspase-8 activation but not formation of the DISC and that this activity of P-gp is dependent on ATP hydrolysis.