scholarly journals Unscheduled Akt-Triggered Activation of Cyclin-Dependent Kinase 2 as a Key Effector Mechanism of Apoptin's Anticancer Toxicity

2008 ◽  
Vol 29 (5) ◽  
pp. 1235-1248 ◽  
Author(s):  
Subbareddy Maddika ◽  
Soumya Panigrahi ◽  
Emilia Wiechec ◽  
Sebastian Wesselborg ◽  
Ute Fischer ◽  
...  
Keyword(s):  
Cell Death ◽  
Tumor Cells ◽  
Pi3 Kinase ◽  
Dominant Negative ◽  
Chicken Anemia Virus ◽  
Cyclin Dependent Kinase ◽  
Normal Cells ◽  
Tumor Selectivity ◽  
Induced Apoptosis ◽  
Anemia Virus

ABSTRACT Apoptin, a protein from the chicken anemia virus, has attracted attention because it specifically kills tumor cells while leaving normal cells unharmed. The reason for this tumor selectivity is unclear and depends on subcellular localization, as apoptin resides in the cytoplasm of normal cells but in the nuclei of transformed cells. It was shown that nuclear localization and tumor-specific killing crucially require apoptin's phosphorylation by an as yet unknown kinase. Here we elucidate the pathway of apoptin-induced apoptosis and show that it essentially depends on abnormal phosphatidylinositol 3-kinase (PI3-kinase)/Akt activation, resulting in the activation of the cyclin-dependent kinase CDK2. Inhibitors as well as dominant-negative mutants of PI3-kinase and Akt not only inhibited CDK2 activation but also protected cells from apoptin-induced cell death. Akt activated CDK2 by direct phosphorylation as well as by the phosphorylation-induced degradation of the inhibitor p27Kip1. Importantly, we also identified CDK2 as the principal kinase that phosphorylates apoptin and is crucially required for apoptin-induced cell death. Immortalized CDK2-deficient fibroblasts and CDK2 knockdown cells were markedly protected against apoptin. Thus, our results not only decipher the pathway of apoptin-induced cell death but also provide mechanistic insights for the selective killing of tumor cells.

scholarly journals Activation of the Chicken Anemia Virus Apoptin Protein by Chk1/2 Phosphorylation Is Required for Apoptotic Activity and Efficient Viral Replication

2016 ◽  
Vol 90 (20) ◽  
pp. 9433-9445 ◽  
Author(s):  
Thomas J. Kucharski ◽  
Timothy F. Ng ◽  
David M. Sharon ◽  
Pedram Navid-Azarbaijani ◽  
Mahvash Tavassoli ◽  
...  
Keyword(s):  
Dna Damage ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Chicken Anemia Virus ◽  
Transformed Cells ◽  
Normal Cells ◽  
Checkpoint Kinase ◽  
Checkpoint Kinase 1 ◽  
Dna Damage Signaling ◽  
Anemia Virus

ABSTRACTChicken anemia virus (CAV) is a single-stranded circular DNA virus that carries 3 genes, the most studied of which is the gene encoding VP3, also known as apoptin. This protein has been demonstrated to specifically kill transformed cells while leaving normal cells unharmed in a manner that is independent of p53 status. Although the mechanistic basis for this differential activity is unclear, it is evident that the subcellular localization of the protein is important for the difference. In normal cells, apoptin exists in filamentous networks in the cytoplasm, whereas in transformed cells, apoptin is present in the nucleus and appears as distinct foci. We have previously demonstrated that DNA damage signaling through the ataxia telangiectasia mutated (ATM) pathway induces the translocation of apoptin from the cytoplasm to the nucleus, where it induces apoptosis. We found that apoptin contains four checkpoint kinase consensus sites and that mutation of either threonine 56 or 61 to alanine restricts apoptin to the cytoplasm. Furthermore, treatment of tumor cells expressing apoptin with inhibitors of checkpoint kinase 1 (Chk1) and Chk2 causes apoptin to localize to the cytoplasm. Importantly, silencing of Chk2 rescues cancer cells from the cytotoxic effects of apoptin. Finally, treatment of virus-producing cells with Chk inhibitor protects them from virus-mediated toxicity and reduces the titer of progeny virus. Taken together, our results indicate that apoptin is a sensor of DNA damage signaling through the ATM-Chk2 pathway, which induces it to migrate to the nucleus during viral replication.IMPORTANCEThe chicken anemia virus (CAV) protein apoptin is known to induce tumor cell-specific death when expressed. Therefore, understanding its regulation and mechanism of action could provide new insights into tumor cell biology. We have determined that checkpoint kinase 1 and 2 signaling is important for apoptin regulation and is a likely feature of both tumor cells and host cells producing virus progeny. Inhibition of checkpoint signaling prevents apoptin toxicity in tumor cells and attenuates CAV replication, suggesting it may be a future target for antiviral therapy.

scholarly journals PLASMID FOR EXPRESSION OF APOPTIN IN Escherichia coli

2015 ◽  
Vol 2 (1) ◽  
pp. 487
Author(s):  
Suyatmi . ◽  
Tri Agusti Sholikah ◽  
Yoga Mulia Pratama ◽  
Afiono Agung Prasetyo
Keyword(s):  
Escherichia Coli ◽  
Tumor Cells ◽  
Cancer Gene Therapy ◽  
Chicken Anemia Virus ◽  
Wild Type ◽  
Expression Plasmid ◽  
Reading Frame ◽  
Pcr Product ◽  
Induced Apoptosis ◽  
Anemia Virus

<p>The chicken anemia virus (CAV) Apoptin can induce apoptosis specifically in tumor cells and not in normal cells. Therefore, this shows a promising use for cancer gene therapy in the future. However, the mechanism of apoptin-induced apoptosis in tumor cells has not been well characterized. We tried to construct p-Ec-Apo (Apoptin expression plasmid for Escherichia coli) for further use, especially to explore the molecular aspect and therapeutically strategy of Apoptin. Aim of this study is to construct plasmid for expression of Apoptin in Escherichia coli. A complete open reading frame of the Apoptin gene of CAV/wild type was amplified by PCR. The PCR product was then purified from the agarose using QIAquick Gel Extraction Kit (Qiagen), subcloned into the respective sites of pETBlue-1 (Novagen). The molecular clone was transformed into Escherichia coli, purified, and sequenced. The nucleotide sequence was analyzed by CLC Main Workbench (CLC Bio). Results: The clone thus obtained, p-Ec-Apo, was successfully constructed.</p><p><br /><strong>Keywords</strong>: Apoptin, Escherichia coli</p>

A Putative NES Mediates Cytoplasmic Localization of Apoptin in Normal Cells

2004 ◽  
Vol 36 (12) ◽  
pp. 817-823 ◽  
Author(s):  
Qing-Ming Wang ◽  
Guo-Cai Fan ◽  
Ji-Zhong Chen ◽  
Hui-Peng Chen ◽  
Fu-Chu He
Keyword(s):  
Tumor Cells ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Chicken Anemia Virus ◽  
Nuclear Accumulation ◽  
Cytoplasmic Localization ◽  
Normal Cells ◽  
Localization Signal ◽  
Anemia Virus ◽  
Export Signal

Abstract Apoptin, a protein expressed by chicken anemia virus, is found predominantly in the cytoplasm in normal cells, whereas it localizes in the nucleus in transformed and malignant cells. However, the mechanisms that regulate the different subcellular localization of Apoptin in normal and tumor cells have not been fully clarified. In this work, a putative nuclear export signal (NES) in Apoptin was predicted. It was testified that the putative NES (pNES) of Apoptin was not a functional NES, but actually acted as a cytoplasmic retention signal. Deletion of the pNES led to the nuclear accumulation of Apoptin in normal cells. In addition, when a strong nuclear localization signal was introduced into Apoptin, it exclusively translocated to the nucleus in normal cells. These observations indicated that the cytoplasmic localization of Apoptin in normal cells results from the balance between cytoplasmic retention and nuclear import. On the other hand, the pNES was also proved to be necessary for Apoptin multimerization. Mutants lacking the pNES did not form obviously visible globular aggregates in normal or tumor cells.

Cell death provoked by loss of interleukin-3 signaling is independent of Bad, Bim, and PI3 kinase, but depends in part on Puma

Blood ◽  
2006 ◽  
Vol 108 (5) ◽  
pp. 1461-1468 ◽  
Author(s):  
Paul G. Ekert ◽  
Anissa M. Jabbour ◽  
Anand Manoharan ◽  
Jacki E. Heraud ◽  
Jai Yu ◽  
...  
Keyword(s):  
Cell Death ◽  
Family Members ◽  
Pi3 Kinase ◽  
Minor Role ◽  
Growth And Survival ◽  
Interleukin 3 ◽  
Dependent Cell ◽  
Induced Apoptosis ◽  
A Minor ◽  
Survival Signals

Growth and survival of hematopoietic cells is regulated by growth factors and cytokines, such as interleukin 3 (IL-3). When cytokine is removed, cells dependent on IL-3 kill themselves by a mechanism that is inhibited by overexpression of Bcl-2 and is likely to be mediated by proapoptotic Bcl-2 family members. Bad and Bim are 2 such BH3-only Bcl-2 family members that have been implicated as key initiators in apoptosis following growth factor withdrawal, particularly in IL-3-dependent cells. To test the role of Bad, Bim, and other proapoptotic Bcl-2 family members in IL-3 withdrawal-induced apoptosis, we generated IL-3-dependent cell lines from mice lacking the genes for Bad, Bim, Puma, both Bad and Bim, and both Bax and Bak. Surprisingly, Bad was not required for cell death following IL-3 withdrawal, suggesting changes to phosphorylation of Bad play only a minor role in apoptosis in this system. Deletion of Bim also had no effect, but cells lacking Puma survived and formed colonies when IL-3 was restored. Inhibition of the PI3 kinase pathway promoted apoptosis in the presence or absence of IL-3 and did not require Bad, Bim, or Puma, suggesting IL-3 receptor survival signals and PI3 kinase survival signals are independent.

scholarly journals Cross-Resistance of CD95- and Drug-Induced Apoptosis as a Consequence of Deficient Activation of Caspases (ICE/Ced-3 Proteases)

Blood ◽  
1997 ◽  
Vol 90 (8) ◽  
pp. 3118-3129 ◽  
Author(s):  
Marek Los ◽  
Ingrid Herr ◽  
Claudia Friesen ◽  
Simone Fulda ◽  
Klaus Schulze-Osthoff ◽  
...  
Keyword(s):  
Cell Death ◽  
Drug Treatment ◽  
Tumor Cells ◽  
Anticancer Drugs ◽  
Ex Vivo ◽  
Resistant Cell ◽  
Cross Resistance ◽  
Strong Increase ◽  
Drug Induced ◽  
Induced Apoptosis

Abstract The cytotoxic effect of anticancer drugs has been shown to involve induction of apoptosis. We report here that tumor cells resistant to CD95 (APO-1/Fas) -mediated apoptosis were cross-resistant to apoptosis-induced by anticancer drugs. Apoptosis induced in tumor cells by cytarabine, doxorubicin, and methotrexate required the activation of ICE/Ced-3 proteases (caspases), similarly to the CD95 system. After drug treatment, a strong increase of caspase activity was found that preceded cell death. Drug-induced activation of caspases was also found in ex vivo-derived T-cell leukemia cells. Resistance to cell death was conferred by a peptide caspase inhibitor and CrmA, a poxvirus-derived serpin. The peptide inhibitor was effective even if added several hours after drug treatment, indicating a direct involvement of caspases in the execution and not in the trigger phase of drug action. Drug-induced apoptosis was also strongly inhibited by antisense approaches targeting caspase-1 and -3, indicating that several members of this protease family were involved. CD95-resistant cell lines that failed to activate caspases upon CD95 triggering were cross-resistant to drug-mediated apoptosis. Our data strongly support the concept that sensitivity for drug-induced cell death depends on intact apoptosis pathways leading to activation of caspases. The identification of defects in caspase activation may provide molecular targets to overcome drug resistance in tumor cells.

Autophagy is associated with apoptosis in cisplatin injury to renal tubular epithelial cells

2008 ◽  
Vol 294 (4) ◽  
pp. F777-F787 ◽  
Author(s):  
Cheng Yang ◽  
Varsha Kaushal ◽  
Sudhir V. Shah ◽  
Gur P. Kaushal
Keyword(s):  
Cell Death ◽  
Epithelial Cells ◽  
Beclin 1 ◽  
Lag Phase ◽  
Tubular Epithelial Cells ◽  
Renal Tubular Epithelial Cells ◽  
Normal Cells ◽  
Life And Death ◽  
Renal Tubular ◽  
Induced Apoptosis

Autophagy has emerged as another major “programmed” mechanism to control life and death much like “programmed cell death” is for apoptosis in eukaryotes. We examined the expression of autophagic proteins and formation of autophagosomes during progression of cisplatin injury to renal tubular epithelial cells (RTEC). Autophagy was detected as early as 2–4 h after cisplatin exposure as indicated by induction of LC3-I, conversion of LC3-I to LC3-II protein, and upregulation of Beclin 1 and Atg5, essential markers of autophagy. The appearance of cisplatin-induced punctated staining of autophagosome-associated LC3-II upon GFP-LC3 transfection in RTEC provided further evidence for autophagy. The autophagy inhibitor 3-methyladenine blocked punctated staining of autophagosomes. The staining of normal cells with acridine orange displayed green fluorescence with cytoplasmic and nuclear components in normal cells but displayed considerable red fluorescence in cisplatin-treated cells, suggesting formation of numerous acidic autophagolysosomal vacuoles. Autophagy inhibitors LY294002 or 3-methyladenine or wortmannin inhibited the formation of autophagosomes but induced apoptosis after 2–4 h of cisplatin treatment as indicated by caspase-3/7 and -6 activation, nuclear fragmentation, and cell death. This switch from autophagy to apoptosis by autophagic inhibitors further suggests that the preapoptotic lag phase after treatment with cisplatin is mediated by autophagy. At later stages of cisplatin injury, apoptosis was also found to be associated with autophagy, as autophagic inhibitors and inactivation of autophagy proteins Beclin 1 and Atg5 enhanced activation of caspases and apoptosis. Our results demonstrate that induction of autophagy mounts an adaptive response, suppresses cisplatin-induced apoptosis, and prolongs survival of RTEC.

Inhibition of the PI3 kinase/Akt pathway enhances doxorubicin-induced apoptotic cell death in tumor cells in a p53-dependent manner

2006 ◽  
Vol 340 (2) ◽  
pp. 560-566 ◽  
Author(s):  
Yusuke Fujiwara ◽  
Kei Kawada ◽  
Daiki Takano ◽  
Susumu Tanimura ◽  
Kei-ichi Ozaki ◽  
...  
Keyword(s):  
Cell Death ◽  
Tumor Cells ◽  
Apoptotic Cell ◽  
Pi3 Kinase ◽  
Apoptotic Cell Death ◽  
Akt Pathway ◽  
Dependent Manner

scholarly journals Doxorubicin induces cardiomyocyte apoptosis and atrophy through cyclin-dependent kinase 2–mediated activation of forkhead box O1

2020 ◽  
Vol 295 (13) ◽  
pp. 4265-4276 ◽  
Author(s):  
Peng Xia ◽  
Jingrui Chen ◽  
Yuening Liu ◽  
Maya Fletcher ◽  
Brian C. Jensen ◽  
...  
Keyword(s):  
Cell Death ◽  
Target Gene ◽  
Ring Finger ◽  
Cell Number ◽  
Cardiomyocyte Apoptosis ◽  
Cyclin Dependent Kinase ◽  
Cardiac Atrophy ◽  
Forkhead Box ◽  
Heart Mass ◽  
Induced Apoptosis

Recent clinical investigations indicate that anthracycline-based chemotherapies induce early decline in heart mass in cancer patients. Heart mass decline may be caused by a decrease in cardiac cell number because of increased cell death or by a reduction in cell size because of atrophy. We previously reported that an anthracycline, doxorubicin (DOX), induces apoptotic death of cardiomyocytes by activating cyclin-dependent kinase 2 (CDK2). However, the signaling pathway downstream of CDK2 remains to be characterized, and it is also unclear whether the same pathway mediates cardiac atrophy. Here we demonstrate that DOX exposure induces CDK2-dependent phosphorylation of the transcription factor forkhead box O1 (FOXO1) at Ser-249, leading to transcription of its proapoptotic target gene, Bcl-2–interacting mediator of cell death (Bim). In cultured cardiomyocytes, treatment with the FOXO1 inhibitor AS1842856 or transfection with FOXO1-specific siRNAs protected against DOX-induced apoptosis and mitochondrial damage. Oral administration of AS1842856 in mice abrogated apoptosis and prevented DOX-induced cardiac dysfunction. Intriguingly, pharmacological FOXO1 inhibition also attenuated DOX-induced cardiac atrophy, likely because of repression of muscle RING finger 1 (MuRF1), a proatrophic FOXO1 target gene. In conclusion, DOX exposure induces CDK2-dependent FOXO1 activation, resulting in cardiomyocyte apoptosis and atrophy. Our results identify FOXO1 as a promising drug target for managing DOX-induced cardiotoxicity. We propose that FOXO1 inhibitors may have potential as cardioprotective therapeutic agents during cancer chemotherapy.

scholarly journals The Chicken Anemia Virus-Derived Protein Apoptin Requires Activation of Caspases for Induction of Apoptosis in Human Tumor Cells

2000 ◽  
Vol 74 (15) ◽  
pp. 7072-7078 ◽  
Author(s):  
A. A. A. M. Danen-van Oorschot ◽  
A. J. van der Eb ◽  
M. H. M. Noteborn
Keyword(s):  
Mammalian Cells ◽  
Caspase 3 ◽  
Human Tumor ◽  
Chemotherapeutic Agents ◽  
Chicken Anemia Virus ◽  
Virus Protein ◽  
Nuclear Morphology ◽  
Induction Of Apoptosis ◽  
Induced Apoptosis ◽  
Anemia Virus

ABSTRACT The chicken anemia virus protein Apoptin has been shown to induce apoptosis in a large number of transformed and tumor cell lines, but not in primary cells. Whereas many other apoptotic stimuli (e.g., many chemotherapeutic agents and radiation) require functional p53 and are inhibited by Bcl-2, Apoptin acts independently of p53, and its activity is enhanced by Bcl-2. Here we study the involvement of caspases, an important component of the apoptotic machinery present in mammalian cells. Using a specific antibody, active caspase-3 was detected in cells expressing Apoptin and undergoing apoptosis. Although Apoptin activity was not affected by CrmA, p35 did inhibit Apoptin-induced apoptosis, as determined by nuclear morphology. Cells expressing both Apoptin and p35 showed only a slight change in nuclear morphology. However, in most of these cells, cytochrome c is still released and the mitochondria are not stained by CMX-Ros, indicating a drop in mitochondrial membrane potential. These results imply that although the final apoptotic events are blocked by p35, parts of the upstream apoptotic pathway that affect mitochondria are already activated by Apoptin. Taken together, these data show that the viral protein Apoptin employs cellular apoptotic factors for induction of apoptosis. Although activation of upstream caspases is not required, activation of caspase-3 and possibly also other downstream caspases is essential for rapid Apoptin-induced apoptosis.

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