Michael adducts of ascorbic acid as inhibitors of protein phosphatase 2A and inducers of apoptosis

Michael adducts of ascorbic acid with alpha,beta-unsaturated carbonyl compounds have been shown to be potent inhibitors of protein phosphatase 1 (PP1) without affecting cell viability at the respective concentrations. Here we were able to show that higher concentrations can partially inhibit PP2A activity and concomitantly induce apoptotic cell death. A nitrostyrene adduct of ascorbic acid proved to be a more potent and effective inhibitor of PP2A as well as a stronger inducer of apoptosis. These adducts only slightly lost their cytotoxic potential in multidrug resistant cells that were 10-fold less sensitive to apoptosis induction by okadaic acid and vinblastine.     

Inhibitory effect of caffeic acid on cancer cell proliferation by oxidative mechanism in human HT-1080 fibrosarcoma cell line

Tyrosine (Y) kinases inhibitors have been approved for targeted treatment of cancer. However, their clinical use is limited to some cancers and the mechanism of their action remains unclear. Previous study has indicated that PP2, a selective inhibitor of the Src family of non-receptor tyrosine kinases (nRTK), efficiently repressed cervical cancer growth in vitro and in vivo. In this regard, our aims are to explore the mechanism of PP2 on cervical cancer cell growth inhibition by investigating the suppressive divergence among PP1, PP2, and a negative control compound PP3. MTT results showed that three compounds had different inhibitory effects on proliferation of two cervical cancer cells, HeLa and SiHa, and PP2 was most efficient in a time- and dose-dependent manner. Moreover, we found 10 μM PP2 down-regulated pSrc-Y416 (P < 0.05), pEGFR-Y845 (P < 0.05), and -Y1173 (P < 0.05) expression levels, while 10 μM PP1 down-regulated pSrc-Y416 (P < 0.05) and pEGFR-Y845 (P < 0.05), but not pEGFR-Y1173; 10 μM PP3 down-regulated only pEGFR-Y1173 (P < 0.05). PP2 could modulate cell cycle arrest by up-regulating p21(Cip1) and p27(Kip1) in both HeLa and SiHa cells and down-regulating expression of cyclin A, and cyclin dependent kinase-2, -4 (Cdk-2, -4) in HeLa and of cyclin B and Cdk-2 in SiHa. Our results indicate that Src pathway and EGFR pathway play different roles in the proliferation of cervical cancer cells and PP2 efficiently reduces cervical cancer cell proliferation by reduction of both Src and EGFR activity.     

Inhibitors of protein phosphatases 1 and 2A differentially prevent intrinsic and extrinsic apoptosis pathways

Inhibitors of serine/threonine protein phosphatases can inhibit apoptosis. We investigated which protein phosphatases are critical for this protection using calyculin A, okadaic acid, and tautomycin. All three phosphatase inhibitors prevented anisomycin-induced apoptosis in leukemia cell models. In vitro, calyculin A does not discriminate between PP1 and PP2A, while okadaic acid and tautomycin are more selective for PP2A and PP1, respectively. Increased phosphorylation of endogenous marker proteins was used to define concentrations that inhibited each phosphatase in cells. Concentrations of each inhibitor that prevented anisomycin-induced apoptosis correlated with inhibition of PP2A. The inhibitors prevented Bax translocation to mitochondria, indicating inhibition upstream of mitochondria. Tautomycin and calyculin A, but not okadaic acid, also prevented apoptosis induced through the CD95/Fas death receptor, and this protection correlated with inhibition of PP1. The inhibitors prevented Fas receptor oligomerization, FADD recruitment, and caspase 8 activation. The differential effects of PP1 and PP2A in protection from death receptor and mitochondrial-mediated pathways of death, respectively, may help one to define critical steps in each pathway, and regulatory roles for serine/threonine phosphatases in apoptosis.     

New pyrazolo[3,4-d]pyrimidines endowed with A431 antiproliferative activity and inhibitory properties of Src phosphorylation

New 4-aminopyrazolo[3,4-d]pyrimidines bearing various substituents at the position 1 and 6, were synthesized. The new compounds showed antiproliferative activity toward A431 cells, were found to be inhibitors of Src phosphorylation, and induced apoptotic cell death. In particular, 2h was a better inhibitor of Src phosphorylation than the reference compound PP2.     

Cdc55p-mediated E4orf4 growth inhibition in Saccharomyces cerevisiae is mediated only in part via the catalytic subunit of protein phosphatase 2A

The adenovirus early region 4 open reading frame 4 (E4orf4) protein specifically induces p53-independent cell death of transformed but not normal human cells, suggesting that elucidation of its mechanism may provide important new avenues for cancer therapy. Wild-type E4orf4 and mutants that retain cancer cell toxicity also induce growth inhibition in Saccharomyces cerevisiae, which provides a genetically tractable system for studying E4orf4 function. Interaction with the protein phosphatase 2A (PP2A) B regulatory subunit is required for E4orf4's effects, suggesting that E4orf4 may function by regulating B subunit-containing heterotrimeric PP2A holoenzymes (PP2A(BAC)), which consist of a B subunit complexed with the PP2A structural (A) and catalytic (C) subunits. However, it is not known whether E4orf4-induced growth inhibition requires interaction with the PP2A C subunit or whether E4orf4 might have PP2A B subunit-dependent effects that are independent of PP2A(BAC) holoenzyme formation. To test these possibilities in S. cerevisiae, we disrupted the stable formation of PP2A(BAC) heterotrimers and thus E4orf4/C subunit association by PP2A C subunit point mutations or by deletion of the gene for the PP2A methyltransferase, Ppm1p, and assayed for effects on E4orf4-induced growth inhibition. Our results support a model in which E4orf4 mediates growth inhibition and cell killing both through PP2A(BAC) heterotrimers and through a B regulatory subunit-dependent pathway(s) that is independent of stable complex formation with the PP2A C subunit. They also indicate that Ppm1p has a function other than regulating the assembly of PP2A heterotrimers and suggest that selective PP2A trimer inhibitors and PP6 inhibitors may be useful as adjuvant anticancer therapies.     

PP2A contributes to endothelial death in high glucose: inhibition by benfotiamine

Endothelial death is critical in diabetic vascular diseases, but regulating factors have been only partially elucidated. Phosphatases play important regulatory roles in cell metabolism, but have not previously been implicated in hyperglycemia-induced cell death. We investigated the role of the phosphatase, type 2A protein phosphatase (PP2A), in hyperglycemia-induced changes in signaling and death in bovine aortic endothelial cells (BAEC). We explored also the influence of benfotiamine on this phosphatase. Activation of PP2A was assessed in BAEC by the extent of methylation and measurement of activity, and the enzyme was inhibited using selective pharmacological (okadaic acid, sodium fostriecin) and molecular (small interfering RNA) approaches. BAECs cultured in 30 mM glucose significantly increased PP2A methylation and activity, and PP2A inhibitors blocked these abnormalities. PP2A activity was increased also in aorta and retina from diabetic rats. NF-κB activity and cell death in BAEC were significantly increased in 30 mM glucose and inhibited by PP2A inhibition. NF-κB played a role in the hyperglycemia-induced death of BAEC, since blocking its translocation with SN50 also inhibited cell death. Inhibition of PP2A blocked the hyperglycemia-induced dephosphorylation of NF-κB and Bad, thus favoring cell survival. Incubation of benfotiamine with BAEC inhibited the high glucose-induced activation of PP2A and NF-κB and cell death, as well as several other metabolic defects, which likewise were inhibited by inhibitors of PP2A. Activation of PP2A contributes to endothelial cell death in high glucose, and beneficial actions of benfotiamine are due, at least in part, to inhibition of PP2A activation.     

Cadmium activates the mitogen-activated protein kinase (MAPK) pathway via induction of reactive oxygen species and inhibition of protein phosphatases 2A and 5

Cadmium (Cd), a highly toxic environmental pollutant, induces neurodegenerative diseases. Recently we have demonstrated that Cd may induce neuronal apoptosis in part through activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (Erk1/2) pathways. However, the underlying mechanism remains enigmatic. Here we show that Cd induced generation of reactive oxygen species (ROS), leading to apoptosis of PC12 and SH-SY5Y cells. Pretreatment with N-acetyl-L-cysteine (NAC) scavenged Cd-induced ROS, and prevented cell death, suggesting that Cd-induced apoptosis is attributed to its induction of ROS. Furthermore, we found that Cd-induced ROS inhibited serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5), leading to activation of Erk1/2 and JNK, which was abrogated by NAC. Overexpression of PP2A or PP5 partially prevented Cd-induced activation of Erk1/2 and JNK, as well as cell death. Cd-induced ROS was also linked to the activation of caspase-3. Pretreatment with inhibitors of JNK (SP600125) and Erk1/2 (U0126) partially blocked Cd-induced cleavage of caspase-3 and prevented cell death. However, zVAD-fmk, a pan caspase inhibitor, only partially prevented Cd-induced apoptosis. The results indicate that Cd induction of ROS inhibits PP2A and PP5, leading to activation of JNK and Erk1/2 pathways, and consequently resulting in caspase-dependent and -independent apoptosis of neuronal cells. The findings strongly suggest that the inhibitors of JNK, Erk1/2, or antioxidants may be exploited for prevention of Cd-induced neurodegenerative diseases.     

Potential involvement of serine/threonine protein phosphatases in apoptosis of HepG2 cells during selenite treatment

Selenium, an essential biological trace element present in both prokaryotic and eukaryotic cells, exerts its regulatory effect in a variety of cellular events, including cell growth, survival, and death. Selenium compunds have been shown in different cell lines to inhibit apoptosis by several mechanisms. Serine/threonine phosphatases (STPs) are potentially important in selenite-induced apoptosis because of their role in regulation of diverse set of cellular processes. In this study, the regulatory role of STPs in selenite-induced apoptosis has been implied by the use of two specific inhibitors: ocadaic acid and calyculin A. Our results show a decrease in cell density in HepG2 cells under selenite treatment. Resulting specific enzyme activities showed a concentration-dependent increase in all three phosphatase activities after 24 h in cells treated with 5 μM selenite and these activities decreased at 48 and 72 h. However, in cells treated with 10μM selenite, PP2A and PP2B decreased at 48 h, whereas PP2C activity did not change at this dose. In cells treated with 25μM, there was not a significant change in PP2C activity. These data suggest that the most specific response to selenite treatment was in PP2A and PP2B activities in a dose-dependent manner. Our results with OA and Cal-A further support the view that PP1 and PP2A might act as negative regulators of growth. With these data, we have first demonstrated the role of serine/threonine protein phosphatases in the signaling pathway of selenite-induced apoptosis and resulting cytotoxicity     

Oxidative stress induces protein phosphatase 2A-dependent dephosphorylation of the pocket proteins pRb,p107, and p130

Oxidative stress induces cell death and growth arrest. In this study, the regulation and the functional role of the retinoblastoma family proteins pRb, p107, and p130 in the cellular response to oxidative stress were investigated. Treatment of endothelial cells with H2O2 induced rapid hypophosphorylation of the retinoblastoma family proteins. This event did not require p53 or p21Waf1/Cip1/Sdi1 and was not associated with cyclin/cyclin-dependent kinase down-modulation. Four lines of evidence indicate that H2O2-induced hypophosphorylation of pRb, p107, and p130 was because of the activity of protein phosphatase 2A (PP2A). First, cell treatment with two phosphatase inhibitors, okadaic acid and calyculin A, prevented the hypophosphorylation of the retinoblastoma family proteins, at concentrations that specifically inhibit PP2A. Second, SV40 small t, which binds and inhibits PP2A, when overexpressed prevented H2O2-induced dephosphorylation of the retinoblastoma family proteins, whereas a SV40 small t mutant unable to bind PP2A was totally inert. Third, PP2A core enzyme physically interacted with pRb and p107, both in H2O2-treated and untreated cells. Fourth, a PP2A phosphatase activity was co-immunoprecipitated with pRb, and the activity of pRb-associated PP2A was positively modulated by cell treatment with H2O2. Because DNA damaging agents inhibit DNA synthesis in a pRb-dependent manner, it was determined whether the PP2A-mediated dephosphorylation of the retinoblastoma family proteins played a role in this S-phase response. Indeed, it was found that inhibition of PP2A by SV40 small t over-expression prevented DNA synthesis inhibition induced by H2O2.     

Development of erlotinib derivatives as CIP2A-ablating agents independent of EGFR activity

Cancerous inhibitor of PP2A (CIP2A) is a novel human oncoprotein that inhibits PP2A, contributing to tumor aggressiveness in various cancers. Several studies have shown that downregulation of CIP2A by small molecules reduces PP2A-dependent phosphorylation of Akt and induces cell death. Here, a series of mono- and di-substituted quinazoline and pyrimidine derivatives based on the skeleton of erlotinib (an EGFR inhibitor) were synthesized and their bioactivities against hepatocellular carcinoma were evaluated. The di-substituted quinazoline and pyrimidine derivatives were more potent inhibitors of cancer-cell proliferation than the mono-substituted derivatives. In particular, compound 1 with chloride at position 2 of quinazoline was as potent as erlotinib in inducing cell death but no inhibition for EGFR activity. Further assays confirmed a correlation between cell death, and CIP2A and Akt inhibition by these derivatives. Among all the derivatives, compounds 19 and 22 showed the most potent antiproliferative activities and the strongest inhibition of CIP2A and p-Akt expression.     

Thyrsiferyl 23-acetate and its derivatives induce apoptosis in various T- and B-leukemia cells

Thyrsiferyl 23-acetate (TF-23A), a cytotoxic compound from marine red alga, induced a rapid cell death in various leukemic T- and B-cell lines. During incubation of Jurkat cells with TF-23A, condensation and fragmentation of nuclei occurred and clusters composed of uneven small cellular particles were formed. Concentration- and time-dependent DNA fragmentation was also induced by the incubation of the cells with TF-23A. These results demonstrate that the TF-23A-induced cell death follows a typical apoptotic process. The TF-23A-induced apoptosis was prevented by fetal calf serum and insulin, but not by EGF or PDGF. TF-23A and its several analogous compounds showed apoptosis-inducing activity. However, only TF-23A out of these compounds showed an inhibitory activity for protein phosphatase 2A, PP2A. These results strongly suggest that a structure of TF-23A involved in induction of apoptosis is different from that involved in the PP2A inhibition.     

Regulation by glucose and calcium of the carboxylmethylation of the catalytic subunit of protein phosphatase 2A in insulin-secreting INS-1 cells

Previously, we reported that the catalytic subunit of protein phosphatase 2A (PP2Ac) undergoes carboxylmethylation (CML) at its COOH-terminal leucine, and that inhibitors of such a posttranslational modification markedly attenuate nutrient-induced insulin secretion from isolated beta-cells. More recent studies have suggested direct inhibitory effects of glucose metabolites on PP2A activity in isolated beta-cells, implying that inhibition of PP2A leads to stimulation of insulin secretion. Because the CML of PP2Ac has been shown to facilitate the holoenzyme assembly and subsequent functional activation of PP2A, we investigated putative regulation by glucose of the CML of PP2Ac in insulin-secreting (INS)-1 cells. Our data indicated a marked inhibition by specific intermediates of glucose metabolism (e.g., citrate and phosphoenolpyruvate) of the CML of PP2Ac in INS-1 cell lysates. Such inhibitory effects were also demonstrable in intact cells by glucose. Mannoheptulose, an inhibitor of glucose metabolism, completely prevented inhibitory effects of glucose on the CML of PP2Ac. Moreover, glucose-mediated inhibition of the CML of PP2Ac was resistant to diazoxide, suggesting that glucose metabolism and the generation of glucose metabolites might control inhibition of the CML of PP2Ac. A membrane-depolarizing concentration of KCl also induced inhibition of the CML of PP2Ac in intact INS cells. On the basis of these data, we propose that glucose metabolism and increase in intracellular calcium facilitate inhibition of the CML of PP2Ac, resulting in functional inactivation of PP2A. This, in turn, might retain the key signaling proteins of the insulin exocytotic cascade in their phosphorylated state, leading to stimulated insulin secretion.     

Combination of mTOR and EGFR Kinase Inhibitors Blocks mTORC1 and mTORC2 Kinase Activity and Suppresses the Progression of Colorectal Carcinoma

Mammalian target of rapamycin complex 1 and 2 (mTORC1/2) are overactive in colorectal carcinomas; however, the first generation of mTOR inhibitors such as rapamycin have failed to show clinical benefits in treating colorectal carcinoma in part due to their effects only on mTORC1. The second generation of mTOR inhibitors such as PP242 targets mTOR kinase; thus, they are capable of inhibiting both mTORC1 and mTORC2. To examine the therapeutic potential of the mTOR kinase inhibitors, we treated a panel of colorectal carcinoma cell lines with PP242. Western blotting showed that the PP242 inhibition of mTORC2-mediated AKT phosphorylation at Ser 473 (AKT^S473) was transient only in the first few hours of the PP242 treatment. Receptor tyrosine kinase arrays further revealed that PP242 treatment increased the phosphorylated epidermal growth factor receptor (EGFR) at Tyr 1068 (EGFR^T1068). The parallel increase of AKT^S473 and EGFR^T1068 in the cells following PP242 treatment raised the possibility that EGFR phosphorylation might contribute to the PP242 incomplete inhibition of mTORC2. To test this notion, we showed that the combination of PP242 with erlotinib, an EGFR small molecule inhibitor, blocked both mTORC1 and mTORC2 kinase activity. In addition, we showed that the combination treatment inhibited colony formation, blocked cell growth and induced apoptotic cell death. A systemic administration of PP242 and erlotinib resulted in the progression suppression of colorectal carcinoma xenografts in mice. This study suggests that the combination of mTOR kinase and EGFR inhibitors may provide an effective treatment of colorectal carcinoma.     

Differential effects on cell motility, embryonic stem cell self-renewal and senescence by diverse Src kinase family inhibitors

The Src family of non-receptor tyrosine kinases (SFKs) has been shown to play an intricate role in embryonic stem (ES) cell maintenance. In the present study we have focused on the underlying molecular mechanisms responsible for the vastly different effects induced by various commonly used SFK inhibitors. We show that several diverse cell types, including fibroblasts completely lacking SFKs, cannot undergo mitosis in response to SU6656 and that this is caused by an unselective inhibition of Aurora kinases. In contrast, PP2 and PD173952 block motility immediately upon exposure and forces cells to grow in dense colonies. The subsequent halt in proliferation of fibroblast and epithelial cells in the center of the colonies approximately 24 h post-treatment appears to be caused by cell-to-cell contact inhibition rather than a direct effect of SFK kinase inhibition. Interestingly, in addition to generating more homogenous and dense ES cell cultures, without any diverse effect on proliferation, PP2 and PD173652 also promote ES cell self-renewal by reducing the small amount of spontaneous differentiation typically observed under standard ES cell culture conditions. These effects could not be mirrored by the use of Gleevec, a potent inhibitor of c-Abl and PDGFR kinases that are also inhibited by PP2.     

Usage of tautomycetin,a novel inhibitor of protein phosphatase 1 (PP1), reveals that PP1 is a positive regulator of Raf-1 in vivo

Protein phosphatase type 1 (PP1), together with protein phosphatase 2A (PP2A), is a major eukaryotic serine/threonine protein phosphatase involved in regulation of numerous cell functions. Although the roles of PP2A have been studied extensively using okadaic acid, a well known inhibitor of PP2A, biological analysis of PP1 has remained restricted because of lack of a specific inhibitor. Recently we reported that tautomycetin (TC) is a highly specific inhibitor of PP1. To elucidate the biological effects of TC, we demonstrated in preliminary experiments that treatment of COS-7 cells with 5 microm TC for 5 h inhibits endogenous PP1 by more than 90% without affecting PP2A activity. Therefore, using TC as a specific PP1 inhibitor, the biological effect of PP1 on MAPK signaling was examined. First, we found that inhibition of PP1 in COS-7 cells by TC specifically suppresses activation of ERK, among three MAPK kinases (ERK, JNK, and p38). TC-mediated inhibition of PP1 also suppressed activation of Raf-1, resulting in the inactivation of the MEK-ERK pathway. To examine the role of PP1 in regulation of Raf-1, we overexpressed the PP1 catalytic subunit (PP1C) in COS-7 cells and found that PP1C enhanced activation of Raf-1 activity, whereas phosphatase-dead PP1C blocked Raf-1 activation. Furthermore, a physical interaction between PP1C and Raf-1 was also observed. These data strongly suggest that PP1 positively regulates Raf-1 in vivo.     

Increased activity of phosphatase PP2A in the presence of the PlA2 polymorphism of alphaIIbbeta3

Polymorphisms in αIIbβ3 are important genetic factors that alter platelet biology and have been associated with susceptibility to thromboembolic disorders. To define the molecular mechanisms that lead to variance in thrombotic diathesis dictated by the β3 polymorphism, we examined regulation of intracellular signaling by αIIbβ3, and studied the effects of a common β subunit PlA2 polymorphism. We found that PP2A regulates αIIbβ3 control of the ERK signaling in a polymorphism specific fashion. In CHO cells, exogenous expression of αIIbβ3 reduced ATP-stimulated ERK phosphorylation and more so for PlA2 than PlA1. Interestingly, reduced level of ERK phosphorylation correlated with an increase in PP2A activity, with higher activity associated with PlA2 than PlA1. We tested the effect of PP2A on αIIbβ3-dependent adhesion, and found that PP2A overexpression increased cell adhesion, while phosphatase inhibitors decreased cell adhesion. We propose that PlA2 alters cell signaling at least in part by increasing β3-associated PP2A activity.     

Protein phosphatase 2A mediates dormancy of glioblastoma multiforme-derived tumor stem-like cells during hypoxia

Purpose

The hypoxic microenvironment of glioblastoma multiforme (GBM) is thought to increase resistance to cancer therapies. Recent evidence suggests that hypoxia induces protein phosphatase 2A (PP2A), a regulator of cell cycle and cell death. The effects of PP2A on GBM tumor cell proliferation and survival during hypoxic conditions have not been studied.

Experimental Design

Expression of PP2A subunits and HIF-α proteins was measured in 65 high-grade astrocytoma and 18 non-neoplastic surgical brain specimens by western blotting. PP2A activity was measured by an immunoprecipitation assay. For in vitro experiments, GBM-derived tumor stem cell-like cells (TSCs) were exposed to severe hypoxia produced by either CoCl₂ or 1% O₂. PP2A activity was inhibited either by okadaic acid or by shRNA depletion of the PP2A C subunit. Effects of PP2A activity on cell cycle progression and cell survival during hypoxic conditions were assessed using flow cytometry.

Results

In our patient cohort, PP2A activity was positively correlated with HIF-1∝ protein expression (P = 0.002). Patients with PP2A activity levels above 160 pMP had significantly worse survival compared to patients with levels below this threshold (P = 0.002). PP2A activity was an independent predictor of survival on multivariable analysis (P = 0.009). In our in vitro experiments, we confirmed that severe hypoxia induces PP2A activity in TSCs 6 hours after onset of exposure. PP2A activity mediated G1/S phase growth inhibition and reduced cellular ATP consumption in hypoxic TSCs. Conversely, inhibition of PP2A activity led to increased cell proliferation, exhaustion of intracellular ATP, and accelerated P53-independent cell death of hypoxic TSCs.

Conclusions

Our results suggest that PP2A activity predicts poor survival in GBM. PP2A appears to reduce the metabolic demand of hypoxic TSCs and enhances tumor cell survival. Modulation of PP2A may be a potential target for cancer therapy.     

Dephosphorylation of tau protein by calcineurin triturated into neural living cells

Alzheimer disease and related dementia are characterized by the presence of hyperphosphorylated tau aggregated into filaments. The role of tau phosphorylation in the fibrillogenesis has not yet been unraveled. Therefore, it is important to know which phosphatases can dephosphorylate tau protein in vivo. The effect of recombinant purified calcineurin (CN(PP2B)) and several calcineurin mutants on tau phosphorylation was studied in two neuronal like cell lines PC12 and SH-SY5Y. The modulation of tau phosphorylation at Ser199/Ser202, Ser396/Ser404, Ser262/Ser356, and Thr181 sites was examined in these cell lines using the phosphorylation state-dependent antitau antibodies Tau 1, PHF1, 12E8, and AT270. The results have shown that CN directly dephosphorylates all of those sites of tau protein. Recombinant calcineurin introduced into cells that have previously been treated with okadaic acid and cyclosporin A, which are inhibitors of phosphatases (PP1/PP2A and PP2B), has a direct effect on the phosphorylation status on all phosphorylation sites studied. We conclude that calcineurin is (besides PP2A) a important modulator of tau phosphorylation in vivo.     

Programmed cell death and the proteasome

A characteristic feature of apoptotic cell death is the activation of a cascade of cytoplasmic proteases that results in the cleavage of a limited number of target proteins. A central role in these proteolytic events has been assigned to members of the capase family. However, the use of low molecular weight proteasomal inhibitors has also demonstrated that protein degradation or processing by the ubiquitin-proteasome system of the cell has a decisive impact on cell survival and death as well, depending on the cell type and/or the proliferative status of the cells studied. Treatment of proliferating cells with proteasome inhibitors leads to cell death, potentially involving an internal signalling conflict between accumulating levels of the cdk inhibitor p27Kip1 and c-myc. In contrast, in terminally differentiated cells the same compounds have the opposite effect of blocking apoptosis, possibly by preventing proteasome-mediated degradation of a capase inhibitor. In this review the role of proteasome-mediated proteolysis in the dying cell is discussed and apparently conflicting results are integrated into a working hypothesis which functionally locates the proteasome upstream of capase3-like enzymes.