Cleavage of zetaPKC but not lambda/iotaPKC by caspase-3 during UV-induced apoptosis

The stimulation of caspases is a critical event in apoptotic cell death. Several kinases critically involved in cell proliferation pathways have been shown to be cleaved by caspase-mediated mechanisms. Thus, the degradation of delta protein kinase C (PKC) and MEKK-1 by caspase-3 generates activated fragments corresponding to their catalytic domains, consistent with the observations that both enzymes are important for apoptosis. In contrast, other kinases reported to have anti-apoptotic properties, such as Raf-1 and Akt, are inactivated by proteolytic degradation by the caspase system. Since the atypical PKCs have been shown to play critical roles in cell survival, in the study reported here we have addressed the potential degradation of these PKCs by the caspase system in UV-irradiated HeLa cells. Herein we show that although zetaPKC and lambda/iotaPKC are both inhibited in UV-treated cells, only zetaPKC but not lambda/iotaPKC is cleaved by a caspase-mediated process. This cleavage generates a fragment that corresponds to its catalytic domain that is enzymatically inactive. The sequence where caspase-3 cleaves zetaPKC was mapped, and a mutant resistant to degradation was shown to protect cells from apoptosis more efficiently than the wild-type enzyme.     

Early secreted antigenic target of 6-kDa of Mycobacterium tuberculosis promotes caspase-9/caspase-3-mediated apoptosis in macrophages

Molecular and Cellular Biochemistry - Mycoplasma pneumoniae pneumonia (MPP) is the most common respiratory infection in young children and its incidence has increased worldwide. In this study, high...     

ATR (AT mutated Rad3 related) activity stabilizes Cdc6 and delays G2/M-phase entry during hydroxyurea-induced S-phase arrest of HeLa cells

The Cdc6 protein, a key DNA replication initiation factor, contributes to the long-term maintenance of the S-phase checkpoint by anchoring the Rad3–Rad26 complex to chromatin. Here, we demonstrate that ATR (AT mutated and Rad3 related) activity is essential for maintaining high chromatin levels of the Cdc6 protein, thereby delaying entry into mitosis during hydroxyurea (HU)-induced S-phase arrest of HeLa cells. Downregulation of ATR (AT mutated and Rad3 related) (i.e., using ATR-siRNA) reduced the protein levels of chromatin Cdc6 and significantly increased the cellular levels of phospho-histone H3 (Ser 10), an index of mitosis. Downregulation of Cdc6 was completely restored by pretreatment with MG132, a proteasome inhibitor. Moreover, mitotic entry of MG132-pretreated cells was significantly downregulated. Our results also show that ATR (AT mutated and Rad3 related) kinase phosphorylates Cdc6 at serine residue 6. Thus, this ATR (AT mutated and Rad3 related)-mediated phosphorylation of Cdc6 is likely associated with stabilization of Cdc6 protein, thereby maintaining high levels of chromatin Cdc6 and delaying premature mitotic entry. This novel mechanism likely contributes to the functional regulation of chromatin Cdc6, which delays the cell cycle of hydroxyurea-induced cells to enter mitosis at the S-phase checkpoint.     

Induction of apoptosis by Centaurea nerimaniae extract in HeLa and MDA-MB-231 cells by a caspase-3 pathway

We investigated the cytotoxic and apoptotic effects of a methanol extract of Centaurea nerimaniae, a plant endemic in Turkey, on HeLa and MDA-MB-231 cells. Eight concentrations of C. nerimaniae extract were applied to cells, and cytotoxic effects were measured using the xCELLigence system. The TUNEL assay was used to assess apoptotic cell death and immunohistochemistry was used to determine active caspase-3 using the effective cytotoxic doses of the extract. Doses of 1.42 mg/ml C. nerimaniae inhibited the growth of HeLa cells and 3.67 mg/ml C. nerimaniae inhibited the growth of MDA-MB-231 cells in a dose- and time-dependent manner. The apoptotic indexes for HeLa and MDA-MB-231 cells were increased significantly compared to control groups. Immunohistochemistry showed that the number of caspase-3 immunostained cells increased in the extract treatment groups for both HeLa and MDA-MB-231 cells. In the MDA-MB-231 cell line, caspase-3 immunostaining was observed in nuclei and/or cytoplasm in the extract treated group. Caspase-3 activation was greater in HeLa cells than in MDA-MB-231 cells. We found that the extract of C. nerimaniae had a strong antiproliferative effect and induced apoptosis via caspase-3; MDA-MB-231 cancer cells were more resistant than HeLa cells.     

Beclin 1 cleavage by caspase-3 inactivates autophagy and promotes apoptosis

Autophagy and apoptosis are both highly regulated biological processes that play essential roles in tissue homeostasis, development and diseases. Autophagy is also described as a mechanism of death pathways, however, the precise mechanism of how autophagy links to cell death remains to be fully understood. Beclin 1 is a dual regulator for both autophagy and apoptosis. In this study we found that Beclin 1 was a substrate of caspase-3 with two cleavage sites at positions 124 and 149, respectively. Furthermore, the autophagosome formation occurred, followed by the appearance of morphological hallmarks of apoptosis after staurosporine treatment. The cleavage products of Beclin 1 reduced autophagy and promoted apoptosis in HeLa cells and the cells in which Beclin 1 was stably knocked down by specific shRNA. In addition, the cleavage of Beclin 1 resulted in abrogating the interaction between Bcl-2 with Beclin 1, which could be blocked by z-VAD-fmk. Thus, our results suggest that the cleavage of Beclin 1 by caspase-3 may contribute to inactivate autophagy leading towards augmented apoptosis.     

TRAIL-induced apoptosis proceeding from caspase-3-dependent and -independent pathways in distinct HeLa cells

The apoptotic pathway in higher eukaryotes remains controversial with respect to the necessity of activation of caspase-3 in TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-treated cells. In this study, a fluorescence resonance energy transfer (FRET) probe was developed to image the activation of caspase-3 and the related apoptotic pathway in TRAIL-treated cells in real time. Both kinds of apoptotic pathways were observed simultaneously in the same experiment proceeding from activation and non-activation of caspase-3. The total apoptotic rate was 56.08%, the apoptotic rates for activation and non-activation of caspase-3 pathways were 21.5% and 34.58%, respectively, which were examined later for Hoechst 33258 staining and morphological characteristics. The apoptotic rate due to the activation of caspase-3 pathways in TRAIL-treated cells has been independently measured to be around 25.11% by capillary electrophoresis (CE) analysis, which confirmed the apoptotic rate due to activation of caspase-3 pathways as found by FRET analysis. This result also suggests that rest apoptosis is preceded by caspase-3-independent pathways, as CE has the ability to quantitatively detect caspase-dependent apoptosis. The observation of the coexistence of caspase-3-dependent and caspase-3-independent apoptotic pathways in the TRAIL-treated cells was unusual in comparison with the previous reports.     

Deregulation of Cdc2 kinase induces caspase-3 activation and apoptosis

Progression of the cell cycle and control of apoptosis are tightly linked processes. It has been reported that manifestation of apoptosis requires cdc2 kinase activity yet the mechanism(s) of which is largely unclear. In an attempt to study the role of human MDM2 (HDM2) in interphase and mitosis, we employed the Xenopus cell-free system to study HDM2 protein stability. Interestingly, HDM2 is specifically cleaved in Xenopus mitotic extracts but not in the interphase extracts. We demonstrate that HDM2 cleavage is dependent on caspase-3 and that activation of cdc2 kinase results in caspase-3 activation in the Xenopus cell-free system. Furthermore, expression of cdc2 kinase in mammalian cells leads to activation of caspase-3 and apoptosis. Taken together, these data indicate that deregulation of cdc2 kinase activity can trigger apoptotic machinery that leads to caspase-3 activation and apoptosis.     

Function of the ATR N-terminal domain revealed by an ATM/ATR chimera

The ATM and ATR kinases function at the apex of checkpoint signaling pathways. These kinases share significant sequence similarity, phosphorylate many of the same substrates, and have overlapping roles in initiating cell cycle checkpoints. However, they sense DNA damage through distinct mechanisms. ATR primarily senses single stranded DNA (ssDNA) through its interaction with ATRIP, and ATM senses double strand breaks through its interaction with Nbs1. We determined that the N-terminus of ATR contains a domain that binds ATRIP. Attaching this domain to ATM allowed the fusion protein (ATM*) to bind ATRIP and associate with RPA-coated ssDNA. ATM* also gained the ability to localize efficiently to stalled replication forks as well as double strand breaks. Despite having normal kinase activity when tested in vitro and being phosphorylated on S1981 in vivo, ATM* is defective in checkpoint signaling and does not complement cellular deficiencies in either ATM or ATR. These data indicate that the N-terminus of ATR is sufficient to bind ATRIP and to promote localization to sites of replication stress.     

Cdc42 promotes G1 progression through p70 S6 kinase-mediated induction of cyclin E expression

The Rho family GTPase Cdc42 is recognized for its role in cellular proliferation and transformation. However, the mechanism by which it promotes cell cycle progression has remained undefined. Using an inducible expression system, we show that constitutively active Cdc42 (Cdc42V12) is sufficient by itself to induce anchorage-independent but not mitogen-independent growth in NIH3T3 cells. However, Cdc42V12 markedly accelerates activation of cyclin E-Cdk2 in response to mitogen. These effects were highly specific, as the kinetics of cyclin D-Cdk4 activation was unaltered. Cdc42V12 promotes Cdk2 activation by selectively inducing cyclin E expression without affecting other regulatory proteins such as the p27 Cdk inhibitor or Cdc25A. Furthermore, Cdc42V12 was able to activate a reporter gene driven by the cyclin E promoter in the absence of exogenous mitogen or adhesion. Cyclin E induction was sensitive to rapamycin but not inhibitors of mitogen-activated protein kinases, implicating p70 S6 kinase (p70S6k) as the relevant mediator. Consistent with this notion, wild type and constitutively active alleles of p70S6k were sufficient to activate the cyclin E promoter. In sum, these studies provide novel insights into the mechanism by which Cdc42 promotes G1 progression.     

Spirooxindole-pyrrolidine heterocyclic hybrids promotes apoptosis through activation of caspase-3

A small library of spirooxindole-pyrrolidine hybrids have been synthesized for the first time in an ionic liquid, [bmim]Br in good to excellent yields employing a new class of non-stabilized azomethine ylides derived from isatin and tyrosine, a combination that has been rarely employed for the in situ generation of azomethine ylides using [3+2] cycloaddition strategy. Following the synthesis and characterization of the spirooxindole-pyrrolidine heterocyclic hybrids, they were tested for their anticancer activity as against the changes in the concentrations and time periods with different in vitro cell cultures containing cancer and non-cancer cells, where the results revealed for a potential therapeutic activity. Further analysis for the mechanism of cell death by the cancer cells indicated for the caspase-dependent apoptotic pathway, specifically mediated by caspase-3. Based on these results, it can be demonstrated that the synthesized spirooxindole-pyrrolidine hybrids may serve as one of the better therapeutic agents used for the treatment of malignant tumors.     

ATR/ATM targets are phosphorylated by ATR in response to hypoxia and ATM in response to reoxygenation

The ATR kinase phosphorylates both p53 and Chk1 in response to extreme hypoxia (oxygen concentrations of less than 0.02%). In contrast to ATR, loss of ATM does not affect the phosphorylation of these or other targets in response to hypoxia. However, hypoxia within tumors is often transient and is inevitably followed by reoxygenation. We hypothesized that ATR activity is induced under hypoxic conditions because of growth arrest and ATM activity increases in response to the oxidative stress of reoxygenation. Using the comet assay to detect DNA damage, we find that reoxygenation induced significant amounts of DNA damage. Two ATR/ATM targets, p53 serine 15 and histone H2AX, were both phosphorylated in response to hypoxia in an ATR-dependent manner. These phosphorylations were then maintained in response to reoxygenation-induced DNA damage in an ATM-dependent manner. The reoxygenation-induced p53 serine 15 phosphorylation was inhibited by the addition of N-acetyl-l-cysteine (NAC), indicating that free radical-induced DNA damage was mediated by reactive oxygen species. Taken together these data implicate both ATR and ATM as critical roles in the response of hypoxia and reperfusion in solid tumors.     

Homeostatic regulation of meiotic DSB formation by ATM/ATR

Ataxia–telangiectasia mutated (ATM) and RAD3-related (ATR) are widely known as being central players in the mitotic DNA damage response (DDR), mounting responses to DNA double-strand breaks (DSBs) and single-stranded DNA (ssDNA) respectively. The DDR signalling cascade couples cell cycle control to damage-sensing and repair processes in order to prevent untimely cell cycle progression while damage still persists [1]. Both ATM/ATR are, however, also emerging as essential factors in the process of meiosis; a specialised cell cycle programme responsible for the formation of haploid gametes via two sequential nuclear divisions. Central to achieving accurate meiotic chromosome segregation is the introduction of numerous DSBs spread across the genome by the evolutionarily conserved enzyme, Spo11. This review seeks to explore and address how cells utilise ATM/ATR pathways to regulate Spo11-DSB formation, establish DSB homeostasis and ensure meiosis is completed unperturbed.     

Cleavage of Ku80 by caspase-2 promotes non-homologous end joining-mediated DNA repair

Non-homologous end-joining (NHEJ)-mediated repair of DNA double-strand breaks (DSBs) requires the formation of a Ku70/Ku80/DNA-PKcs complex at the DSB sites. A previous study has revealed Ku80 cleavage by caspase-3 during apoptosis. However, it remains largely unknown whether and how Ku80 cleavage affects its function in mediating NHEJ-mediated DNA repair. Here we report that Ku80 can be cleaved by caspases-2 at D726 upon a transient etoposide treatment. Caspase-2-mediated Ku80 cleavage promotes Ku80/DNA-PKcs interaction as the D726A mutation diminished Ku80 interaction with DNA-PKcs, while a Ku80 truncate (Ku80 ΔC6) lacking all the 6 residues following D726 rescued the weakened Ku80/DNA-PKcs interaction caused by caspase-2 knockdown. As a result, depletion or inhibition of caspase-2 impairs NHEJ-mediated DNA repair, and such impairment can be reversed by Ku80 ΔC6 overexpression. Taken together, our current study provides a novel mechanism for regulating NHEJ-mediated DNA repair, and sheds light on the function of caspase-2 in genomic stability maintenance.     

Proteolysis of the mismatch repair protein MLH1 by caspase-3 promotes DNA damage-induced apoptosis

Caspases are critical proapoptotic proteases that execute cell death signals by selectively cleaving proteins at Asp residues to alter their function. Caspases trigger apoptotic chromatin degradation by activating caspase-activated DNase and by inactivating a number of enzymes that sense or repair DNA damage. We have identified the mismatch repair protein MLH1 as a novel caspase-3 substrate by screening small pools of a human prostate adenocarcinoma cDNA library for cDNAs encoding caspase substrates. In this report, we demonstrate that human MLH1 is specifically cleaved by caspase-3 at Asp(418) in vitro. Furthermore, MLH1 is rapidly proteolyzed by caspase-3 in cancer cells induced to undergo apoptosis by treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the topoisomerase II inhibitor etoposide, which damages DNA. Importantly, proteolysis of MLH1 by caspase-3 triggers its partial redistribution from the nucleus to the cytoplasm and generates a proapoptotic carboxyl-terminal product. In addition, we demonstrate that a caspase-3 cleavage-resistant D418E MLH1 mutant inhibits etoposide-induced apoptosis but has little effect on TRAIL-induced apoptosis. These results indicate that the proteolysis of MLH1 by caspase-3 plays a functionally important and previously unrecognized role in the execution of DNA damage-induced apoptosis.     

Cleavage of claspin by caspase-7 during apoptosis inhibits the Chk1 pathway

Claspin is required for the phosphorylation and activation of the Chk1 protein kinase by ATR during DNA replication and in response to DNA damage. This checkpoint pathway plays a critical role in the resistance of cells to genotoxic stress. Here, we show that human Claspin is cleaved by caspase-7 during the initiation of apoptosis. In cells, induction of DNA damage by etoposide at first produced rapid phosphorylation of Chk1 at a site targeted by ATR. Subsequently, etoposide caused activation of caspase-7, cleavage of Claspin, and dephosphorylation of Chk1. In apoptotic cell extracts, Claspin was cleaved by caspase-7 at a single aspartate residue into a large N-terminal fragment and a smaller C-terminal fragment that contain different functional domains. The large N-terminal fragment was heavily phosphorylated in a human cell-free system in response to double-stranded DNA oligonucleotides, and this fragment retained Chk1 binding activity. In contrast, the smaller C-terminal fragment did not bind Chk1, but did associate with DNA and inhibited the DNA-dependent phosphorylation of Chk1 associated with its activation. These results indicate that cleavage of Claspin by caspase-7 inactivates the Chk1 signaling pathway. This mechanism may regulate the balance between cell cycle arrest and induction of apoptosis during the response to genotoxic stress.     

Human monocytes undergo excessive apoptosis following temozolomide activating the ATM/ATR pathway while dendritic cells and macrophages are resistant

Immunodeficiency is a severe therapy-limiting side effect of anticancer chemotherapy resulting from sensitivity of immunocompetent cells to DNA damaging agents. A central role in the immune system is played by monocytes that differentiate into macrophages and dendritic cells (DCs). In this study we compared human monocytes isolated from peripheral blood and cytokine matured macrophages and DCs derived from them and assessed the mechanism of toxicity of the DNA methylating anticancer drug temozolomide (TMZ) in these cell populations. We observed that monocytes, but not DCs and macrophages, were highly sensitive to the killing effect of TMZ. Studies on DNA damage and repair revealed that the initial DNA incision was efficient in monocytes while the re-ligation step of base excision repair (BER) can not be accomplished, resulting in an accumulation of DNA single-strand breaks (SSBs). Furthermore, monocytes accumulated DNA double-strand breaks (DSBs) following TMZ treatment, while DCs and macrophages were able to repair DSBs. Monocytes lack the DNA repair proteins XRCC1, ligase IIIα and PARP-1 whose expression is restored during differentiation into macrophages and DCs following treatment with GM-CSF and GM-CSF plus IL-4, respectively. These proteins play a key role both in BER and DSB repair by B-NHEJ, which explains the accumulation of DNA breaks in monocytes following TMZ treatment. Although TMZ provoked an upregulation of XRCC1 and ligase IIIα, BER was not enhanced likely because PARP-1 was not upregulated. Accordingly, inhibition of PARP-1 did not sensitize monocytes, but monocyte-derived DCs in which strong PARP activation was observed. TMZ induced in monocytes the DNA damage response pathways ATM-Chk2 and ATR-Chk1 resulting in p53 activation. Finally, upon activation of the Fas-receptor and the mitochondrial pathway apoptosis was executed in a caspase-dependent manner. The downregulation of DNA repair in monocytes, resulting in their selective killing by TMZ, might impact on the immune response during cancer chemotherapy.     

Cleavage of 14-3-3 protein by caspase-3 facilitates bad interaction with Bcl-x(L) during apoptosis

The 14-3-3 epsilon protein was identified as one of the caspase-3 substrates by the modified yeast two-hybrid system. The cellular 14-3-3 epsilon protein was also cleaved in response to the treatment of apoptosis inducers in cultured mammalian cells. Asp238 of the 14-3-3 epsilon protein was determined as the site of cleavage by caspase-3. The affinity of the cleaved 14-3-3 mutant protein (D238) to Bad, a death-promoting Bcl-2 family protein, was lower than that of wild type or the uncleavable mutant 14-3-3 epsilon protein (D238A). However, Bad associated with the cellular Bcl-x(L) more effectively in human 293T cells co-expressing Bad with the truncated form of the 14-3-3 epsilon protein (D238) than in control cells co-expressing Bad with wild type or the uncleavable mutant 14-3-3 epsilon protein (D238A). The present study suggests that the cleavage of 14-3-3 protein during apoptosis promotes cell death by releasing the associated Bad from the 14-3-3 protein and facilitates Bad translocation to the mitochondria and its interaction with Bcl-x(L).     

Human replication protein Cdc6 is selectively cleaved by caspase 3 during apoptosis

In eukaryotes, the initiation of DNA replication involves the ordered assembly on chromatin of pre-replicative complexes (pre-RCs), including the origin recognition complex (ORC), Cdc6, Cdt1 and the minichromosome maintenance proteins (MCMs). In light of its indispensable role in the formation of pre-RCs, Cdc6 binding to chromatin represents a key step in the regulation of DNA replication and cell proliferation. Here, we study the human Cdc6 (HuCdc6) protein during programmed cell death (apoptosis). We find that HuCdc6, but not HuOrc2 (a member of the ORC) or HuMcm5 (one of the MCMs), is specifically cleaved in several human cell lines induced to undergo apoptosis by a variety of stimuli. Expression of caspase-uncleavable mutant HuCdc6 attenuates apoptosis, delaying cell death. Therefore, an important function for cleavage of HuCdc6 is to prevent a wounded cell from replicating and to facilitate death.     

Caspase-3-mediated cleavage of Cdc6 induces nuclear localization of p49-truncated Cdc6 and apoptosis

We show that Cdc6, an essential initiation factor for DNA replication, undergoes caspase-3-mediated cleavage in the early stages of apoptosis in HeLa cells and SK-HEP-1 cells induced by etoposide, paclitaxel, ginsenoside Rh2, or tumor necrosis factor-related apoptosis-inducing ligand. The cleavage occurs at the SEVD442/G motif and generates an N-terminal truncated Cdc6 fragment (p49-tCdc6) that lacks the carboxy-terminal nuclear export sequence. Cdc6 is known to be phosphorylated by cyclin A-cyclin dependent kinase 2 (Cdk2), an event that promotes its exit from the nucleus and probably blocks it from initiating inappropriate DNA replication. In contrast, p49-tCdc6 translocation to the cytoplasm is markedly reduced under the up-regulated conditions of Cdk2 activity, which is possibly due to the loss of nuclear export sequence. Thus, truncation of Cdc6 results in an increased nuclear retention of p49-tCdc6 that could act as a dominant negative inhibitor of DNA replication and its accumulation in the nucleus could promote apoptosis. Supporting this is that the ectopic expression of p49-tCdc6 not only promotes apoptosis of etoposide-induced HeLa cells but also induces apoptosis in untreated cells. Thus, the caspase-mediated cleavage of Cdc6 creates a truncated Cdc6 fragment that is retained in the nucleus and induces apoptosis.