Dataset Information

Apoptosis Induction by dsRNA-Dependent Protein Kinase R (PKR) in EPC Cells via Caspase 8 and 9 Pathways.

ABSTRACT: dsRNA-dependent protein kinase R (PKR) is an interferon-inducible protein that mediates antiviral effects and induces apoptosis. We studied PKR-related apoptosis mechanisms by transfecting wild type pcDNA-carp-wtPKR, a catalytically inactive mutant pcDNA-mut-carpPKR, and empty plasmid in Epithelioma papulosum cyprini (EPC) cells, designated wtPKR, mutPKR, and pcDNA3.1, respectively. PKR was inefficiently expressed from wtPKR unlike mutPKR that produced high PKR levels detected by western blot. eIF2? phosphorylation increased in wtPKR-transfected cells, while for mutPKR, phosphorylation was not different from non-transfected controls. Flow-cytometry revealed high level of apoptosis in wtPKR transfected cells, corresponding with high cytopathic effect. mutPKR and pcDNA3.1 transfection gave significantly less apoptosis and were not different from each other. Caspase-8 and -9 were activated for wtPKR, suggesting death receptor-caspase-8 and mitochondrion-dependent caspase-9 activated pathways, similar to mammalian cells. These findings suggest that the induction of apoptosis via the caspase-8 and -9 pathways are conserved in vertebrate taxa and likely play a role in viral infections of lower vertebrates.

SUBMITTER: Xu C

PROVIDER: S-EPMC6213184 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Apoptosis Induction by dsRNA-Dependent Protein Kinase R (PKR) in EPC Cells via Caspase 8 and 9 Pathways.

Xu Cheng C Gamil Amr A A AAA Munang'andu Hetron Mweemba HM Evensen Øystein Ø

Viruses 20180927 10

dsRNA-dependent protein kinase R (PKR) is an interferon-inducible protein that mediates antiviral effects and induces apoptosis. We studied PKR-related apoptosis mechanisms by transfecting wild type pcDNA-carp-wtPKR, a catalytically inactive mutant pcDNA-mut-carpPKR, and empty plasmid in <i>Epithelioma papulosum cyprini</i> (EPC) cells, designated wtPKR, mutPKR, and pcDNA3.1, respectively. PKR was inefficiently expressed from wtPKR unlike mutPKR that produced high PKR levels detected by western ...[more]

PMID: 30261686

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Project description:Apoptosis is an essential strategy of host defense responses and is used by viruses to maintain their life cycles. However, the apoptotic signals involved in virus replication are poorly known. In the present study, we report the molecular mechanism of apoptotic induction by the viral protein ORF4, a newly identified viral protein of porcine circovirus type 2 (PCV2). Apoptosis detection revealed not only that the activity of caspase-3 and -9 is increased in PCV2-infected and ORF4-transfected cells but also that cytochrome c release from the mitochondria to the cytosol is upregulated. Subsequently, ORF4 protein colocalization with adenine nucleotide translocase 3 (ANT3) was observed using structured illumination microscopy. Moreover, coimmunoprecipitation and pulldown analyses confirmed that the ORF4 protein interacts directly with mitochondrial ANT3 (mtANT3). Binding domain analysis further confirmed that N-terminal residues 1 to 30 of the ORF4 protein, comprising a mitochondrial targeting signal, are essential for the interaction with ANT3. Knockdown of ANT3 markedly inhibited the apoptotic induction of both ORF4 protein and PCV2, indicating that ANT3 plays an important role in ORF4 protein-induced apoptosis during PCV2 infection. Taken together, these data indicate that the ORF4 protein is a mitochondrial targeting protein that induces apoptosis by interacting with ANT3 through the mitochondrial pathway.IMPORTANCE The porcine circovirus type 2 (PCV2) protein ORF4 is a newly identified viral protein; however, little is known about its functions. Apoptosis is an essential strategy of the host defense response and is used by viruses to maintain their life cycles. In the present study, we report the molecular mechanism of the apoptosis induced by the ORF4 protein. The ORF4 protein contains a mitochondrial targeting signal and is an unstable protein that is degraded by the proteasome-dependent pathway. Viral protein ORF4 triggers caspase-3- and -9-dependent cellular apoptosis in mitochondria by directly binding to ANT3. We conclude that the ORF4 protein is a mitochondrial targeting protein and reveal a mechanism whereby circovirus recruits ANT3 to induce apoptosis.

Project description:It has been reported that the expression and activity of the interferon-inducible, dsRNA-dependent protein kinase, PKR, is increased in mammary carcinoma cell lines and primary tumor samples. To extend these findings and determine how PKR signaling may affect breast cancer cell sensitivity to chemotherapy, we measured PKR expression by immunohistochemical staining of 538 cases of primary breast cancer and normal tissues. Significantly, PKR expression was elevated in ductal, lobular and squamous cell carcinomas or lymph node metastases but not in either benign tumor specimens or cases of inflammation compared to normal tissues. Furthermore, PKR expression was increased in precancerous stages of mammary cell hyperplasia and dysplasia compared to normal tissues, indicating that PKR expression may be upregulated by the process of tumorigenesis. To test the function of PKR in breast cancer, we generated MCF7, T-47D and MDA-MB-231 breast cancer cell lines with significantly reduced PKR expression by siRNA knockdown. Importantly, while knockdown of PKR expression had no effect on cell proliferation under normal growth conditions, MCF7, T-47D or MDA-MB-231 cells with reduced PKR expression or treated with a small molecule PKR inhibitor were significantly less sensitive to doxorubicin or H(2)O(2)-induced toxicity compared to control cells. In addition, the rate of eIF2α phosphorylation following treatment with doxorubicin was delayed in breast cancer cell lines with decreased PKR expression. Significantly, treatment of breast cancer lines with reduced PKR expression with either interferon-α, which increases PKR expression, or salubrinal, which increases eIF2α phosphorylation, restored doxorubicin sensitivity to normal levels. Taken together these results indicate that increased PKR expression in primary breast cancer tissues may serve as a biomarker for response to doxorubicin-containing chemotherapy and that future therapeutic approaches to promote PKR expression/activation and eIF2α phosphorylation may be beneficial for the treatment of breast cancer.

Dataset Information

Apoptosis Induction by dsRNA-Dependent Protein Kinase R (PKR) in EPC Cells via Caspase 8 and 9 Pathways.

Publications

Apoptosis Induction by dsRNA-Dependent Protein Kinase R (PKR) in EPC Cells via Caspase 8 and 9 Pathways.

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