Caspase activation in MCF7 cells responding to etoposide treatment

Studies of the biochemical mechanisms evoked by conventional treatments for neoplastic diseases point to apoptosis as a key process for elimination of unwanted cells. Although the pathways through which chemotherapeutics promote cell death remain largely unknown, caspase proteases play a central role in the induction of apoptosis in response to a variety of stimuli including tumor necrosis factor, fas ligand, and growth factor deprivation. In this article, we demonstrate the induction of caspase protease activity in MCF7 human breast carcinoma cells exposed to the topoisomerase inhibitor, etoposide. Caspase protease activity was assessed by incubating cell lysates with the known caspase substrates, acetyl-L-aspartic-L-glutamic-L-valyl-L-aspartic acid 4-methyl-7-aminocoumarin or acetyl-L-tyrosyl-L-valyl-L-aspartic acid 4-methyl-7-aminocoumarin. We observed maximal cleavage of acetyl-L-aspartic-L-glutamic-L-valyl-L-aspartic acid 4-methyl-7-aminocoumarin within 6 hr following etoposide addition, a time that precedes cell death. In contrast, acetyl-L-tyrosyl-L-valyl-L-aspartic acid 4-methyl-7-aminocoumarin was resistant to cleavage activity. This substrate cleavage specificity implies that a caspase-3-like protease is activated in response to DNA damage. Consistent with the lysate protease activity, an intracellular marker of caspase activation, poly-ADP ribose polymerase (PARP), was cleaved in a concentration- and time-dependent manner after etoposide-treatment. PARP cleavage followed caspase activation and reached maximum cleavage between 12 and 16 hr. Incubation of the cells with the peptidic caspase inhibitor z-valine-alanine-asparagine-CH2F prevented caspase activation, inhibited PARP cleavage, and inhibited cell death. Thus, etoposide killing of MCF7 cells requires a caspase-3-like protease.     

Alphaviruses induce apoptosis in Bcl-2-overexpressing cells: evidence for a caspase-mediated, proteolytic inactivation of Bcl-2

Bcl-2 oncogene expression plays a role in the establishment of persistent viral infection by blocking virus-induced apoptosis. This might be achieved by preventing virus-induced activation of caspase-3, an IL-1beta-converting enzyme (ICE)-like cysteine protease that has been implicated in the death effector phase of apoptosis. Contrary to this model, we show that three cell types highly overexpressing functional Bcl-2 displayed caspase-3 activation and underwent apoptosis in response to infection with alphaviruses Semliki Forest and Sindbis as efficiently as vector control counterparts. In all three cell types, overexpressed 26 kDa Bcl-2 was cleaved into a 23 kDa protein. Antibody epitope mapping revealed that cleavage occurred at one or two target sites for caspases within the amino acid region YEWD31 (downward arrow) AGD34 (downward arrow) A, removing the N-terminal BH4 region known to be essential for the death-protective activity of Bcl-2. Preincubation of cells with the caspase inhibitor Z-VAD prevented Bcl-2 cleavage and partially restored the protective activity of Bcl-2 against virus-induced apoptosis. Moreover, a murine Bcl-2 mutant having Asp31, Asp34 and Asp36 substituted by Glu was resistant to proteolytic cleavage and abrogated apoptosis following virus infection. These findings indicate that alphaviruses can trigger a caspase-mediated inactivation of Bcl-2 in order to evade the death protection imposed by this survival factor.     

An induced proximity model for caspase-8 activation

The assembly of the CD-95 (Fas/Apo-1) receptor death-inducing signaling complex occurs in a hierarchical manner; the death domain of CD-95 binds to the corresponding domain in the adapter molecule Fas-associated death domain (FADD) Mort-1, which in turn recruits the zymogen form of the death protease caspase-8 (FLICE/Mach-1) by a homophilic interaction involving the death effector domains. Immediately after recruitment, the single polypeptide FLICE zymogen is proteolytically processed to the active dimeric species composed of large and small catalytic subunits. Since all caspases cleave their substrates after Asp residues and are themselves processed from the single-chain zymogen to the two-chain active enzyme by cleavage at internal Asp residues, it follows that an upstream caspase can process a downstream zymogen. However, since FLICE represents the most apical caspase in the Fas pathway, its mode of activation has been enigmatic. We hypothesized that the FLICE zymogen possesses intrinsic enzymatic activity such that when approximated, it autoprocesses to the active protease. Support for this was provided by (i) the synthesis of chimeric Fpk3FLICE molecules that can be oligomerized in vivo by the synthetic cell-permeable dimerizer FK1012H2. Cells transfected with Fpk3FLICE underwent apoptosis after exposure to FK1012H2; (ii) the creation of a nonprocessable zymogen form of FLICE that retained low but detectable protease activity.     

Dissection of pathways leading to antigen receptor-induced and Fas/CD95-induced apoptosis in human B cells

To dissect intracellular pathways involved in B cell Ag receptor (BCR)-mediated and Fas-induced human B cell death, we isolated clones of the Burkitt lymphoma cell line Ramos with different apoptosis sensitivities. Selection for sensitivity to Fas-induced apoptosis also selected for clones with enhanced BCR death sensitivity and vice versa. In contrast, clones resistant to Fas-mediated apoptosis could still undergo BCR-induced cell death. Based on the functional phenotypes of these clones, we hypothesized that both receptor-induced apoptosis pathways are initially distinct but may eventually converge. Indeed, ligation of both Fas and BCR resulted in cleavage of the IL-1beta-converting enzyme/Ced-3-like protease caspase 3 and its substrates Ac-Asp-Glu-Val-Asp-aldehyde and poly(ADP-ribose) polymerase. Markedly, qualitative differences in the caspase 3 cleavage pattern induced by Fas or BCR ligation were observed; whereas Fas ligation generated caspase 3 cleavage products of 19/20 and 17 kDa, only the latter cleavage product was found upon BCR cross-linking. The caspase inhibitor Val-Ala-Asp-fluoromethylketone blocked both Fas- and BCR-mediated apoptosis, but differentially affected caspase 3 cleavage induced by either stimulus. Finally, overexpression of a Fas-associated death domain (FADD) dominant-negative mutant protein was found to inhibit Fas-induced apoptosis but not BCR-induced apoptosis. Together our findings imply that Fas and BCR couple, via FADD-dependent and FADD-independent mechanisms, respectively, to distinct proteases upstream of caspase 3.     

Novel sodium binding properties of some cyclopentapeptide endothelin A selective receptor antagonists: electrospray and fast-atom-bombardment mass spectrometric studies

Due to their growth arrest- and apoptosis-inducing ability, glucocorticoids (GC) are widely used in the therapy of various lymphoid malignancies. Cell death is associated with activation of members of the interleukin-1beta-converting enzyme (ICE) protease/caspase family and, is presumably prevented by the anti-apoptotic protein Bcl-2. To further address the role of Bcl-2 in GC-mediated cytotoxicity, we generated subclones of the GC-sensitive human T-cell acute lymphoblastic leukemia line CCRF-CEM, in which transgenic Bcl-2 expression is regulated by tetracycline. Up to about 48 h, exogenous Bcl-2 almost completely protected these cells from apoptosis, digestion of poly-ADP ribose polymerase (PARP) and generation of Asp-Glu-Val-Asp cleaving (DEVDase) activity. However, when the cells were cultured for another 24 h in the continuous presence of GC, they underwent massive apoptosis that was associated with DEVDase activity and PARP cleavage. Bcl-2 did not markedly affect GC-mediated growth arrest, thereby separating the anti-proliferative from the apoptosis-inducing effect of GC. Moreover, Bcl-2 did not prevent the dramatic reduction in the levels of several mRNAs observed during GC treatment, including the transgenic Bcl-2 mRNA. Thus, Bcl-2 can be placed upstream of effector caspase activation, but downstream of other GC-regulated events, such as growth arrest and the potentially critical repression of steady state levels of multiple mRNA.     

Huntington's disease: trinucleotide disease or polyglutamine disease?

Huntington's disease (HD) is associated with expansion of an unstable CAG repeat. Using antibodies against the synthetic peptide corresponding to the sequence of HD gene IT15, we have identified the HD gene product in normal lymphocytes as a approximately 350kDa protein by immunoblot analysis. Moreover, when a modified SDS-PAGE using a low concentration of methylenbisacrylamide was run longer, abnormal immunoreactive bands larger than normal ones were found exclusively in HD samples. We also found double bands in HD brain homogenate samples. Recently on the other CAG repeat diseases, such as SCA1 and DRPLA, abnormal gene products were also reported. These results demonstrate the existence of the expanded CAG repeat gene products and open a possibility that the expanded polyglutamine stretch may really participate in the pathological process of the CAG repeat diseases.     

Inhibition of the protein kinase C pathway promotes anti-CD95-induced apoptosis in Jurkat T cells

The role of the basal activity of the serine/threonine protein kinase, protein kinase C (PKC) in the regulation of anti-CD95-induced apoptosis in Jurkat T cells was investigated. The PKC-specific inhibitor GF 109203X and the proposed cPKC-specific inhibitor Go 6976, in a concentration-dependent manner, increased the percentage of cells undergoing apoptosis induced by anti-CD95 mAb as demonstrated by propidium iodide (PI) staining, TUNEL assay and DNA fragmentation by gel electrophoresis. Furthermore, Go 6976 and GF 109203X abrogated phorbol myristate acetate-induced inhibition of anti-CD95-induced apoptosis. To examine the molecular mechanism by which PKC modulates anti-CD95-induced apoptosis, the effects of Go 6976 on known effector and regulatory molecules of cell death were studied. Increased recruitment of cells undergoing apoptosis was associated with enhanced anti-CD95-induced proteolytic cleavage of the most receptor-proximal cysteine protease caspase-8, subsequent cleavage and activation of the machinery protease caspase-3, and cleavage of the caspase substrates DNA-dependent protein kinase catalytic subunit, poly-(ADP-ribose) polymerase and lamin B1. CD95 and FADD protein levels in Jurkat T cells were not altered by Go 6976 treatment. In addition, Go 6976 did not alter protein levels and subcellular distribution of the anti-apoptotic molecules Bcl-2 and Bcl-xL. These data suggest indirectly that basal PKC activity acts at an early stage in the anti-CD95-induced caspase pathway to attenuate subsequent activation of downstream effector molecules and associated apoptosis in Jurkat T cells.     

Kennedy's disease: clinical and molecular study of two Italian families

Kennedy's disease, or spinal and bulbar muscular atrophy (SBMA), is a rare X-linked motoneuron disorder with variable signs of androgen insensitivity. It is associated with the expansion of a trinucleotide CAG repeat within the androgen receptor (AR) gene. We here report our clinical and molecular findings in two Italian families with Kennedy's disease. The increased size of the CAG repeat was demonstrated in four affected males and seven carrier females.     

Oral Neospora caninum inoculation of neonatal calves

The bcl-2 protein plays an essential role in preventing cell death. Its activity is regulated through association with bcl-2 homologous and nonhomologous proteins and also by serine phosphorylation. We now report that bcl-2 can be proteolytically cleaved towards its N-terminus by a cysteine proteinase present in RL-7 lymphoma cell lysates, yielding a major product of apparent MW 20 kDa, different from the products of bcl-2 cleavage by HIV protease. Moreover, bcl-2 proteins mutated for Asp residues at positions 31 and 34 were efficiently cleaved by RL-7 cell lysates, indicating that this proteolytic activity is distinct from the caspase-3 that cleaves bcl-2 at Asp 34. This bcl-2 cleaving activity is inhibited by E-64 and is therefore distinct from the proteinases of the ICE/Ced-3 family (caspases), whereas reciprocally, ICE (caspase-1) is unable to cleave bcl-2. It is optimally active at pH 5, a feature distinguishing it from calpain, another non-ICE cysteine proteinase which has been associated with apoptosis. This novel bcl-2 cleaving protease, although constitutively present in RL-7 cells and resting peripheral blood lymphocytes (PBL) was upregulated following induction of apoptosis in RL-7 cells or mitogen activation in PBL. The N-terminus of bcl-2 which contains the BH4 domain that binds the kinase Raf-1 and the phosphatase calcineurin is essential for anti-apoptotic activity. Its cleavage might provide a novel post-translational mechanism for regulating bcl-2 function and could amplify ongoing programmed cell death.     

The complex pathology of trinucleotide repeats

The expansion of trinucleotide repeat sequences has now been shown to be the underlying cause of at least ten human disorders. Unifying features among these diseases include the unstable behavior of the triplet repeat during germline transmission when the length of the repeat exceeds a critical value. However, the trinucleotide repeat disorders can be divided into two distinct groups. Type I disorders involve the expansion of CAG repeats, which encode an expanded polyglutamine, inserted into the open-reading frame of a gene that is usually quite broadly expressed. Recently, mouse models for type I disorders have been developed and the basis of pathology is under study, both in these models and through biochemical and cell biological approaches. The type II disorders involve repeat expansions in noncoding regions of genes. The mechanisms by which these repeat expansions lead to pathology may be quite diverse.     

Activation of CPP32-like proteases is not sufficient to trigger apoptosis: inhibition of apoptosis by agents that suppress activation of AP24, but not CPP32-like activity

The 24-kD apoptotic protease (AP24) is a serine protease that is activated during apoptosis and has the capacity to activate internucleosomal DNA fragmentation in isolated nuclei. This study examined the following: (a) the functional relationship between AP24 and the CPP32-like proteases of the caspase family; and (b) whether activation of CPP32-like proteases is sufficient to commit irreversibly a cell to apoptotic death. In three different leukemia cell lines, we showed that agents that directly (carbobenzoxy-Ala-Ala-borophe (DK120) or indirectly inhibit activation of AP24 (protein kinase inhibitors, basic fibroblast growth factor, tosylphenylalaninechloromethylketone, and caspase inhibitors) protected cells from apoptosis induced by TNF or UV light. Only the caspase inhibitors, however, prevented activation of CPP32-like activity as revealed by cleavage of the synthetic substrate, DEVD-pNa, by cell cytosols, and also by in vivo cleavage of poly (ADP-ribosyl) polymerase, a known substrate of CPP32. Activation of DEVD-pNa cleaving activity without apoptosis was also demonstrated in two variants derived from the U937 monocytic leukemia in the absence of exogenous inhibitors. Cell-permeable peptide inhibitors selective for CPP32-like proteases suppressed AP24 activation and apoptotic death. These findings indicate that CPP32-like activity is one of several upstream signals required for AP24 activation. Furthermore, activation of CPP32-like proteases alone is not sufficient to commit irreversibly a cell to apoptotic death under conditions where activation of AP24 is inhibited.     

Inhibition of caspase activity prevents anti-IgM induced apoptosis but not ceramide generation in WEHI 231 B cells

In apoptosis induced by Reaper in Drosophila, as well as in a number of other systems, it has been suggested that the increased synthesis of ceramide might be a consequence of the activation of the caspase/ICE (Interleukin-1beta converting enzyme) protease pathway involved in cell death, implying that ceramide generation might often be the result rather than the cause of apoptosis. WEHI 231 B cells have previously been shown to undergo apoptosis following exposure to exogenous ceramide and to produce increased amounts of ceramide in response to anti-IgM crosslinking. We show here that in WEHI 231 cells a peptide inhibitor of caspase activity blocks cell death in response to both anti-IgM and exogenous ceramide. However, the induction of ceramide synthesis by WEHI 231 cells in response to anti-IgM crosslinking is not blocked by this peptide. These results indicate that antigen receptor induced ceramide generation in WEHI 231 cells does not require caspase activation, and support the view that ceramide generation in immature B cells may be the cause rather than the consequence of activation of the caspase dependent death pathway.     

Expression of polyglutamine-expanded Huntingtin activates the SEK1-JNK pathway and induces apoptosis in a hippocampal neuronal cell line

Huntington's disease is one of a growing number of hereditary neurodegenerative disorders caused by expansion of a polyglutamine stretch at the NH2 terminus of huntingtin. To explore whether polyglutamine-expanded huntingtin induces neuronal toxicity, I examined the expression of the full-length of huntingtin with 16, 48, or 89 polyglutamine repeats in a rat hippocampal neuronal cell (HN33). Expression of mutated huntingtin with 48 or 89 polyglutamine repeats stimulated c-Jun amino-terminal kinases (JNKs) activity and induced apoptotic cell death in HN33 cells while expression of normal huntingtin with 16 polyglutamine repeats had no toxic effect. The JNK activation precedes apoptotic cell death and co-expression of a dominant negative mutant form of stress-signaling kinase (SEK1) nearly completely blocked activation of JNKs and neuronal apoptosis mediated by mutated huntingtin. Taken together, my studies demonstrate that expression of polyglutamine-expanded huntingtin induces neuronal apoptosis via activation of the SEK1-JNK pathway.     

Reconstructive surgery for ischemic-type lesions at the bile duct bifurcation after liver transplantation

Cholestatic liver injury appears to result from the induction of hepatocyte apoptosis by toxic bile salts such as glycochenodeoxycholate (GCDC). Previous studies from this laboratory indicate that cathepsin B is a downstream effector protease during the hepatocyte apoptotic process. Because caspases can initiate apoptosis, the present studies were undertaken to determine the role of caspases in cathepsin B activation. Immunoblotting of GCDC-treated McNtcp.24 hepatoma cells demonstrated cleavage of poly(ADP-ribose) polymerase and lamin B1 to fragments that indicate activation of effector caspases. Transfection with CrmA, an inhibitor of caspase 8, prevented GCDC-induced cathepsin B activation and apoptosis. Consistent with these results, an increase in caspase 8-like activity was observed in GCDC-treated cells. Examination of the mechanism of GCDC-induced caspase 8 activation revealed that dominant-negative FADD inhibited apoptosis and that hepatocytes isolated from Fas-deficient lymphoproliferative mice were resistant to GCDC-induced apoptosis. After GCDC treatment, immunoprecipitation experiments demonstrated Fas oligomerization, and confocal microscopy demonstrated DeltaFADD-GFP (Fas-associated death domain-green fluorescent protein, aggregation in the absence of detectable Fas ligand mRNA. Collectively, these data suggest that GCDC-induced hepatocyte apoptosis involves ligand-independent oligomerization of Fas, recruitment of FADD, activation of caspase 8, and subsequent activation of effector proteases, including downstream caspases and cathepsin B.     

An isoform of ataxin-3 accumulates in the nucleus of neuronal cells in affected brain regions of SCA3 patients

Autosomal dominant spinocerebellar ataxias (SCA) form a group of clinically and genetically heterogeneous neurodegenerative disorders. The defect responsible for SCA3/Machado-Joseph disease (MJD) has been identified as an unstable and expanded (CAG)n trinucleotide repeat in the coding region of a novel gene of unknown function. The MJD1 gene product, ataxin-3, exists in several isoforms. We generated polyclonal antisera against an alternate carboxy terminus of ataxin-3. This isoform, ataxin-3c, is expressed as a protein of approximately 42 kDa in normal individuals but is significantly enlarged in affected patients confirming that the CAG repeat is part of the ataxin-3c isoform and is translated into a polyglutamine stretch, a feature common to all known CAG repeat disorders. Ataxin-3 like immunoreactivity was observed in all human brain regions and peripheral organs studied. In neuronal cells of control individuals, ataxin-3c was expressed cytoplasmatically and had a somatodendritic and axonal distribution. In SCA3 patients, however, C-terminal ataxin-3c antibodies as well as anti-ataxin-3 monoclonal antibodies (1 H9) and anti-ubiquitin antibodies detected intranuclear inclusions (NIs) in neuronal cells of affected brain regions. A monoclonal antibody, 2B6, directed against an internal part of the protein, barely detected these NIs implying proteolytic cleavage of ataxin-3 prior to its transport into the nucleus. These findings provide evidence that the alternate isoform of ataxin-3 is involved in the pathogenesis of SCA3/MJD. Intranuclear protein aggregates appear as a common feature of neurodegenerative polyglutamine disorders.     

Inhibition of apoptosis and clonogenic survival of cells expressing crmA variants: optimal caspase substrates are not necessarily optimal inhibitors

To study the role of various caspases during apoptosis, we have designed a series of caspase inhibitors based on the cowpox virus cytokine response modifier A (crmA) protein. Wild-type crmA inhibits caspases 1 and 8 and thereby protects cells from apoptosis triggered by ligation of CD95 or tumour necrosis factor (TNF) receptors, but it does not protect against death mediated by other caspases. By replacing the tetrapeptide pseudosubstrate region of crmA (LVAD) with tetrapeptides that are optimal substrates for the different families of caspases, or with the four residues from the cleavage site of the baculovirus protein p35 (DQMD), we have generated a family of caspase inhibitors that show altered ability to protect against cell death. Although DEVD is the optimal substrate for caspase 3, crmA DEVD was degraded rapidly and was a weaker inhibitor than crmA DQMD, which was not degraded. Unlike wild-type crmA and crmA DEVD, crmA DQMD was able to inhibit apoptosis caused by direct activation of caspase 3 and protected lymphoid cells from death induced by radiation and dexamethasone. Significantly, the protected cells were capable of sustained growth.     

Activation of caspase-1 in the nucleus requires nuclear translocation of pro-caspase-1 mediated by its prodomain

The interleukin-1beta-converting enzyme-like protease precursor, pro-caspase-1, has an N-terminal prodomain that is removed during cleavage activation of the protease. Here we show that tumor necrosis factor treatment of HeLa cells induced apoptosis without detectable proteolytic activation of caspase-1 in the cytosol. Instead, tumor necrosis factor induced the translocation of pro-caspase-1 to the nucleus where it was proteolytically activated, releasing the intact prodomain. We identified a nuclear localization signal in the prodomain, which was required for translocation of both pro-caspase-1 as well as its prodomain to the nucleus. Surprisingly, transfected MCF-7 carcinoma or embryonic kidney 293T cells expressing the prodomain alone underwent apoptosis. These results show that death signal-induced nuclear targeting is a novel activity of a caspase prodomain and indicate that caspase-1 and its prodomain may have hitherto unsuspected nuclear functions in apoptosis.     

Phosphorylation of PITSLRE p110 isoforms accompanies their processing by caspases during Fas-mediated cell death

A number of cellular proteins have been identified as caspase targets during cell death, including the PITSLRE protein kinases. These targets generally fall into one of three possible categories: 1) other caspases, 2) proteins that are inactivated during apoptosis, and 3) proteins that are required for execution of the cell death program. However, not all proteins are cleaved by caspases during apoptosis. Why only specific proteins are destined to be processed by caspases during cell death is currently not clear. Here we show that multiple caspase-like activities are involved in the processing of the PITSLRE p110 isoforms during Fas-induced apoptosis in Jurkat T-cells. Three p110 caspase cleavage sites have been mapped to the amino-terminal domain of p110 and verified by site-directed mutagenesis. Curiously, the mutagenesis studies revealed that cleavage of two juxtaposed caspase sites is necessary for the complete processing of this protein during cell death in vivo. Finally, we demonstrate that the PITSLRE p110 protein is rapidly phosphorylated during Fas-induced apoptosis in Jurkat cells and that phosphorylation of an amino-terminal portion of the protein may enhance caspase cleavage in this region.