Cyclophosphamide stimulates endoplasmic reticulum stress and induces apoptotic cell death in human glioblastoma cell lines.

Cyclophosphamide (CP) is an alkylating chemotherapeutic agent commonly used in cancer treatments. In this study, we aimed to investigate the effects of 4-Hydroperoxy cyclophosphamide (4-HC), which is active form of CP, on glucose-regulated protein 78 (GRP78), activating transcription factor 6 (ATF6), phospho-protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (p-PERK), phospho-inositol-requiring enzyme 1 alpha (p-IRE1α), eukaryotic translation initiation factor 2 alpha (eIF2α), and caspase-3 messenger ribonucleic acids (mRNAs) and proteins that play roles in the ER stress pathway and apoptosis in U87 and T98 human glioblastoma cell lines. U87 and T98 human glioblastoma cell lines were divided into control and 4-HC-treated groups. Cell viability assay was used to detect the half maximal inhibitory concentration (IC50) for 24 hours of 4-HC. Immunocytochemistry and quantitative polymerase chain reaction (qPCR) methods were used to evaluate the levels of proteins and their mRNAs. The IC50 values of U87 and T98 cells were calculated as 15.67±0.58 µM and 19.92±1 µM, respectively. The levels of GRP78, ATF6, p-PERK, p-IRE1α, eIF2α, and caspase-3 protein expressions in the 4-HC-treated group compared to that in the control group. These increased protein expressions also were correlated with the mRNA levels. The ER stress signal pathway could be active in 4-HC-induced cell death. Further studies of ER-related stress mechanisms in anticancer treatment would be important for effective therapeutic strategies.

[Co-exposure of carbon black and cadmium induces autophagy and inflammation in human bronchial epithelial cells via PERK pathway].

Objective: To investigate the effects of carbon black and cadmium (Cd) combined exposure on autophagy and inflammatory response mediated by protein kinase R-like endoplasmic reticulum kinase (PERK) pathway in human bronchial epithelial (16HBE) cells. Methods: In January 2022, human bronchial epithelial (16HBE) cells were resuscitated and cultured. Carbon black nanoparticles (CBNPs) were oxidized to adsorb Cd ions to construct "CBNPs-Cd" complexes. CCK-8 assay was used to detect the effects of different concentrations and time combinations of CBNPs and Cd on the viability of 16HBE cells. The subsequent dose groups were exposed to 2 µg/ml Cd, 100 µg/ml CBNPs, 100 µg/ml CBNPs+2 µg/ml Cd for 24 h. The number of autophagosomes and autolysosomes was detected by transmission electron microscopy. Western blotting was used to detect the protein expressions of PERK, eukaryotic initiation factor 2α (eIf2α), activating transcription factor 4 (ATF4), sequestosome 1 (SQSTM1/P62), and microtubule-associated protein 1 light chain 3 (LC3). After PERK gene was silenced by siRNA technology, the changes of autophagy marker proteins P62 and LC3 were detected, and the expressions of inflammatory factors interleukin-6 (IL6) and interleukin-8 (IL8) were detected by fluorescence quantitative PCR technique. One-way ANOVA analysis was used to compare three groups or more. LSD test was used for comparison between two groups. Factorial analysis was used for multivariate component analysis. Results: There was no significant change in cell viability of 16HBE after 24 h exposure to CBNPs and Cd alone or combined (P>0.05). Compared with the control group, the expressions of P62 and LC3 in 16HBE cells were significantly increased in the CBNPs and Cd alone/combined exposure group (P<0.05), and the number of autophagosomes and autophagolysosomes in the combined exposure group was increased compared with other groups. Compared with the control group, CBNPs and Cd alone exposure group had no significant effects on p-PERK/PERK and p-eIf2α/eIf2α protein expression (P>0.05). However, the protein expressions of p-PERK/PERK and p-eIf2α/eIf2α and ATF4 were all increased in the combined exposure group (P<0.05), and the levels of IL6 and IL8 in 16HBE cells in the combined exposure group of CBNPs and Cd were significantly higher than those in the control group (P<0.05). The levels of LC3 protein, IL6 and IL8 were decreased in the CBNPs-Cd combined exposure group after knockdown of PERK gene (P<0.05). The results of factorial analysis showed that exposure to CBNPs and Cd had significant effects on the expression of P62, LC3 and IL6 (P<0.05), but the interaction between the two chemicals had no statistical significance (P>0.05) . Conclusion: CBNPs-Cd combined exposure may inhibit autophagy and increase inflammation in human bronchial epithelial cells through activation of PERK-eIf2α-ATF4 pathway.

Mannose-doped metal-organic frameworks induce tumor cell pyroptosis via the PERK pathway.

BACKGROUND: The implementation of pyroptosis exhibits significant potential as a tactic to enhance tumor immune microenvironments. Previous applications of pyroptosis inducers have encountered various limitations, such as the development of drug resistance, manifestation of toxic side effects, and a deficiency in targeting capabilities. As a result, there is a growing demand for tumor therapeutic molecules that can overcome these obstacles. Therefore, the objective of this study is to develop a multifunctional nanospheres that addresses these challenges by enabling high-precision targeting of tumor cells and inducing effective pyroptosis. RESULTS: We prepared a mannose-modified MOF called mannose-doped Fe3O4@NH2-MIL-100 (M-FNM). M-FNM could enter CAL27 cells through MR-mediated endocytosis, which caused in a significant increase in the level of intracellular ROS. This increase subsequently triggered ER stress and activated the PERK-eIF2α-ATF4-CHOP signaling pathway. CHOP then mediated the downstream cascade of Caspase-1, inducing pyroptosis. In in vivo experiments, M-FNM demonstrated excellent targeting ability and exhibited anti-tumor effects. Additionally, M-FNM reshaped the immune microenvironment by promoting the infiltration of anti-tumor immune cells, primarily T lymphocytes. CONCLUSIONS: M-FNM significantly decreased tumor growth. This novel approach to induce pyroptosis in tumor cells using M-FNM may offer new avenues for the development of effective immunotherapies against cancer.

Endoplasmic reticulum stress: a novel targeted approach to repair bone defects by regulating osteogenesis and angiogenesis.

Bone regeneration therapy is clinically important, and targeted regulation of endoplasmic reticulum (ER) stress is important in regenerative medicine. The processing of proteins in the ER controls cell fate. The accumulation of misfolded and unfolded proteins occurs in pathological states, triggering ER stress. ER stress restores homeostasis through three main mechanisms, including protein kinase-R-like ER kinase (PERK), inositol-requiring enzyme 1ɑ (IRE1ɑ) and activating transcription factor 6 (ATF6), collectively known as the unfolded protein response (UPR). However, the UPR has both adaptive and apoptotic effects. Modulation of ER stress has therapeutic potential for numerous diseases. Repair of bone defects involves both angiogenesis and bone regeneration. Here, we review the effects of ER stress on osteogenesis and angiogenesis, with emphasis on ER stress under high glucose (HG) and inflammatory conditions, and the use of ER stress inducers or inhibitors to regulate osteogenesis and angiogenesis. In addition, we highlight the ability for exosomes to regulate ER stress. Recent advances in the regulation of ER stress mediated osteogenesis and angiogenesis suggest novel therapeutic options for bone defects.

Folic acid depletion along with inhibition of the PERK arm of endoplasmic reticulum stress pathway promotes a less aggressive phenotype of hepatocellular carcinoma cells.

Folate is a vital vitamin involved in one-carbon metabolism and any changes in folate status may lead to epigenetic alterations. It is already known that stages and liver cancer progression are negatively correlated with folate levels. Nevertheless, mechanisms involved in folate deficiency in HCC (Hepatocellular carcinoma) are still not completely understood. So, this study tests the hypothesis that due to the increased demand for ER (endoplasmic reticulum) proteins, folate deficiency might lead to the induction of UPR (unfolded protein response), which is further correlated with HCC outcomes. HCC cells were cultured in both folate normal (FN) and folate deficient (FD) conditions and the expression of genes of ER stress pathway was investigated. The results demonstrated activation of UPR via induction of PERK, ATF4, and LAMP3. Besides this, FD reduced the migratory capacity and the invasiveness of HCC cells along with the reduction in mesenchymal markers like vimentin but increased apoptosis. Treatment with GSK2606414 (PERK inhibitor) decreased the FD induced expression of PERK, ATF4, and LAMP3 in FD cells. Also, GSK2606414 was found to increase apoptotic cell death and to further reduce the cancer hallmarks selectively in FD cells but not in FN cells. Altogether, our data suggest that targeting the ER stress pathway along with folate deficiency may provide a more promising elimination of the metastatic potential of HCC cells contributing to more effective therapeutic agents.

Promotion of osteogenesis in BMSC under hypoxia by ATF4 via the PERK-eIF2α signaling pathway.

Mandibular distraction osteogenesis (MDO) is an endogenous tissue engineering technology in which bone marrow mesenchymal stem cells (BMSC) play a key role in MDO-related osteogenesis. Activating transcription factor 4 (ATF4) is involved in osteogenesis through activation of PERK (Protein kinase R-like endoplasmic reticulum kinase) in endoplasmic reticulum stress (ERS) condition under hypoxia. However, the specific role of ATF4 in MDO with BMSC remains unknown. The aim of this study was to explore the effects of ATF4 in MDO with BMSC under hypoxia. Briefly, canine BMSCs were cultured in a hypoxic chamber, and effects of hypoxia were evaluated using cell migration assay and Alizarin Red S staining. Expression levels of protein kinase R-like endoplasmic reticulum kinase, eukaryotic translation initiation factor 2α, ATF4, osteocalcin, and bone sialoprotein were evaluated using quantitative polymerase chain reaction and western blotting. BMSCs were transduced with the ATF4-small interfering RNA lentivirus. The effects were evaluated using all the aforementioned experiments. The results showed that hypoxia promoted migration, osteoblast differentiation, and ATF4 expression in BMSC. ATF4 knockdown in BMSC significantly inhibited migration and osteoblast differentiation abilities, while hypoxia reversed these effects to some extent. In addition, the molecular mechanism partly depended on the ERS signaling pathway, with ATF4 as the key factor. In summary, we presented a novel mechanism of ATF4-mediated regulation of BMSC under hypoxia.

Insulin-like growth factor-1 reduces hyperoxia-induced lung inflammation and oxidative stress and inhibits cell apoptosis through PERK/eIF2α/ATF4/CHOP signaling.

Background: Insulin-like growth factor-1 (IGF-1), a member of the insulin family, has a high degree of homology with insulin and exhibits anti-inflammatory and anti-oxidative stress properties. However, the potential protective effect of IGF-1 on hyperoxia-induced lung injury remains unknown. In this study, we aimed to explore the effects and mechanism of action of IGF-1 in hyperoxia-induced lung injury in neonatal rats. Materials and Methods: Hematoxylin-eosin staining was used to observe pathological changes in lung tissue; transmission electron microscopy was used to examine the ultrastructure, and ELISA was used to detect the level of pro-inflammatory cytokines in bronchoalveolar lavage fluid. Further, malondialdehyde, glutathione, and superoxide dismutase activities in lung tissue were evaluated. TUNEL staining was used to detect cell apoptosis, and western blot analysis was used to detect the expression of Bax, Bcl-2, Caspase-3, p-PERK, p-eIF2α, ATF4, and CHOP in the lung tissue. Moreover, the wet/dry weight ratio of lung tissue was determined. Results: Intraperitoneal injection of IGF-1 effectively reduced lung tissue damage induced by hyperoxia; production of inflammatory cells and release of pro-inflammatory cytokines, oxidative stress, and cell apoptosis. Further, IGF-1 down-regulated the expression of ATF4, CHOP, and Bax/Bcl-2, and inhibited the phosphorylation of PERK and eIF2α. Conclusion: The results suggest that IGF-1 reduces hyperoxia-induced lung inflammation and oxidative stress in neonatal rats through the PERK/eIF2α/ATF4/CHOP signaling pathway and inhibits cell apoptosis.

Gestational exposure to BDE-209 induces placental injury via the endoplasmic reticulum stress-mediated PERK/ATF4/CHOP signaling pathway.

Several epidemiological studies have reported significant associations between prenatal polybrominated diphenyl ethers (PBDEs) exposure and adverse birth outcomes. Placental injury is thought to mediate these associations. However, few study has investigated the adverse effects of PBDEs exposure on placental growth and development. We examined the impacts of gestational exposure to BDE-209, the most abundant PBDE conger detected in human samples, on placental structure and function, and its model of action in vivo and in vitro. Pregnant mice were exposed to 0, 2, 20, 200 mg/kg/day of BDE-209 by gavages from gestational day (GD) 0 to GD18. Results showed that gestational BDE-209 exposure significantly reduced placental weight, impaired placental vascular development and induced placental cell apoptosis. In addition, gestational BDE-209 exposure impaired placental transport and endocrine function as demonstrated by markedly downregulated expression of Glut1, Znt1, Pgf and Igf2 in BDE-209-treated placentas. Mechanistically, gestational exposure to BDE-209 upregulated the expression of GRP78, and 3 downstream proteins (p-eIF2α, ATF4 and CHOP) of the PERK signaling, suggesting the activation of endoplasmic reticulum (ER) stress and PERK signaling pathway in mouse placentas. Further in vitro study showed that PERK siRNA pretreatment markedly reversed BDE-209-induced cell apoptosis in human JEG-3 cells. Collectively, our results suggest that the activation of the ER stress-mediated PERK/ATF4/CHOP signaling pathway played a role in BDE-209-induced placental injury. Our findings provide new insight into the mechanisms of BDE-209 induced reproductive and developmental toxicity.

Artesunate Restrains Maturation of Dendritic Cells and Ameliorates Heart Transplantation-Induced Acute Rejection in Mice through the PERK/ATF4/CHOP Signaling Pathway.

BACKGROUND: Heart transplantation (HT) is the only effective treatment for end-stage heart failure because it can effectively improve the survival rate and quality of life of patients with heart failure. Artesunate (ART) is an artemisinin derivative, with good water solubility and higher oral bioavailability. The main aim of this study was to determine the role of ART in HT mice. METHODS: In animal experiments, mice were divided into the control group, HT group, low ART+HT group, and high ART+HT group. Next, inflammatory cell infiltration, oxidative stress injury, and myocardial cell apoptosis were determined in heart tissue. The proportion of multiple lymphocytes in spleen and lymph nodes was then determined using flow cytometry. In addition, cell experiments were conducted to determine the changes in expression of surface maturation markers of BMDC and changes in intracellular reactive oxygen species after LPS stimulation. Finally, western blot analysis was performed to determine the levels of endoplasmic reticulum stress-related proteins (CHOP/ATF4/PERK). RESULTS: The survival time of mice in the ART treatment group was significantly prolonged and was positively correlated with the dose. In animal experiments, ART significantly reduced inflammatory cell infiltration in heart tissue and the proportion of CD4+CD8+ T cells in spleens and lymph nodes. Moreover, ART treatment lowered the 8-OHdg in hearts and myocardial apoptosis. In cell experiments, ART treatment slowed down the development and maturation of BMDCs by inhibiting the expression of endoplasmic reticulum stress-related proteins. Furthermore, the treatment alleviated the oxidative stress damage of BMDCs. CONCLUSION: ART can inhibit maturation of dendritic cells through the endoplasmic reticulum stress signaling pathway, thereby alleviating acute rejection in mice after heart transplantation.

ER stress modulates apoptosis in A431 cell subjected to EtNBSe-PDT via the PERK pathway.

Photodynamic therapy (PDT) is a promising modality against various cancers including squamous cell carcinoma (SCC) with which the induction of apoptosis is an effective mechanism. Here, we initially describe the preclinical activity of 5-ethylamino-9-diethylaminobenzo [a] phenoselenazinium(EtNBSe)-mediated PDT treatment in SCC. Results of our studies suggest that EtNBSe-PDT provokes a cellular state of endoplasmic reticulum (ER) stress triggering the PERK/ eIF2α signaling pathway and induces the appearance of apoptosis in A431 cells at the meantime. With ER stress inhibitor 4-PBA or eIF2α inhibitor ISRIB, suppressing the EtNBSe-PDT induced ER stress substantially promotes apoptosis of A431 cells. Furthermore, we demonstrate that ATF4, whose expression is ER-stress-inducible and elevated in response to the PERK/eIF2α signaling pathway activation, contributes to cytoprotection against EtNBSe-PDT induced apoptosis. In a mouse model bearing A431 cells, EtNBSe shows intense phototoxicity and when associated with decreased ER stress, EtNBSe-PDT ameliorates tumor growth. Taken together, our study reveals an antagonistic activity of ER stress against EtNBSe-PDT treatment via inhibiting apoptosis in A431 cells. With further development, these results provide a proof-of-concept that downregulation of ER stress response has a therapeutic potential to improve EtNBSe-PDT sensitivity in SCC patients via the promotion of induced apoptosis.

Identification of Goose PKR Gene: Structure, Expression Profiling, and Antiviral Activity Against Newcastle Disease Virus.

Double-stranded RNA-dependent protein kinase (PKR) is an important antiviral IFN-stimulated gene (ISGs) that recognizes double-stranded RNA (dsRNA) and mediates inhibition of translation initiation and protein synthesis in various types of viral infection. In this study, the complete coding sequence (CDS) of goose PKR (goPKR) is identified and characterized. The open reading frame (ORF) of goPKR is 1668 bp, which encodes a polypeptide of 555 amino acids. The sequence identity results demonstrate that the goose PKR is most closely related to duck PKR gene, with nucleotide identities of 91.6%, whereas nucleotide identity of the goose PKR to chicken, human, and mouse PKR is 76.4%, 51.9%, and 52.0%, respectively. Interestingly, the deduced amino acid sequence of goose PKR contains 3 main structure domains, including 2 double-strand RNA-binding motif (dsRBM) domains and one serine/threonine protein kinase domain. This is similar to the chicken and mammals, whereas it is different from duck PKR protein, which contains only one dsRBM1 domain and one serine/threonine protein kinase domain. Quantitative real-time PCR analysis indicates that goose PKR mRNA is widely expressed in all sampled tissues. It is highly expressed in the blood, spleen, lung, and bursa of Fabricius and jejunum and is slightly expressed in heart, muscle, trachea, and brain. The results of confocal microscopy suggest that PKR-EGFP is mainly localized in the cytoplasm, and overexpression of goPKR protein significantly reduces Newcastle disease virus (NDV) replication (viral copies and viral titer) in goose embryo fibroblasts. These findings show that goose PKR is an important antiviral ISG, involved in the antiviral innate immune defense to NDV in geese.

Inhibition of PERK-dependent pro-adaptive signaling pathway as a promising approach for cancer treatment.

Endoplasmic Reticulum (ER) is an organelle that is vital for cell growth and maintenance of homeostasis. Recent studies have reported that numerous human diseases, including cancer, are strictly connected to disruption of ER homeostasis. In order to counteract adverse intracellular conditions, cancer cells induce protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)-dependent, pro-adaptive unfolded protein response (UPR) signaling branches. If ER stress is severe or prolonged, pro-adaptive signaling networks are insufficient, resulting in apoptotic cell death of cancer cells. The main aim: of the study was to evaluate the biological activity of a small-molecule PERK inhibitor GSK2606414 in two cancer cell lines - human neuroblastoma (SH-SY5Y) and human colorectal adenocarcinoma (HT-29) cell lines. We analyzed the level of phosphorylation of the eukaryotic initiation factor 2 (eIF2), which is the main substrate of PERK and a subsequent activator of UPR, which under long-term ER stress may evoke apoptotic death of cancer cells. MATERIAL AND METHODS: In the study, we utilized commercially available cell lines of human colorectal adenocarcinoma HT-29 and human neuroblastoma SH-SY5Y. Cells were exposed to the tested PERK-dependent signaling inhibitor GSK2606414 in suitable culture media with addition of thapsigargin (500 nM) to induce ER stress. To identify the protein, Western blot with specific antibodies was used. Detection of immune complexes was performed using chemiluminescence. RESULTS: We found a complete inhibition of p-eIF2α expression due to the GSK2606414 inhibitor in both cell lines, SH-SY5Y and HT-29. CONCLUSIONS: Currently available cancer treatments are insufficient and cause various side effects. It has been assumed that utilization of small-molecule inhibitors of the PERK-dependent signaling pathway, like GSK2606414, may switch the pro-adaptive branch of UPR to its pro-apoptotic branch. It is believed that the tested inhibitor GSK2606414 may become a promising treatment for many cancer types.

PERK-mediated Autophagy in Osteosarcoma Cells Resists ER Stress-induced Cell Apoptosis.

Osteosarcoma is a bone cancer that develops commonly in children and adolescents. However, osteosarcoma treatments often fail by the development of chemoresistance to apoptosis, and the molecular mechanisms remain unclear. In this study, we propose that autophagy is responsible for osteosarcomatous resistance to apoptosis. We implicate PERK-mediated autophagy as a significant contributor to apoptosis resistance due to ER stress in osteosarcoma cells. By immunostainings and western blots, we identified that PERK activated osteosarcomatous autophagy via inhibiting mTORC1 pathway, thereby preventing cell apoptosis. While using RNAi, we knocked down PERK and found that autophagy was suppressed, result in osteosarcomatous apoptosis. Our results identify a novel role of PERK-mediated autophagy as a significant mechanism for osteosarcoma cell survival. These results will help to understand the mechanism of chemoresistance in osteosarcoma cells, and indicate a novel target for improving osteosarcoma therapy.

Sensing endoplasmic reticulum stress by protein kinase RNA-like endoplasmic reticulum kinase promotes adaptive mitochondrial DNA biogenesis and cell survival via heme oxygenase-1/carbon monoxide activity.

Endoplasmic reticulum (ER) stress activates the adaptive unfolded protein response, allowing cells to recover folding capacity in the organelle. However, the overwhelming response to severe damage results in apoptotic cell death. Because of the physical proximity between ER and mitochondria, a functional interrelationship between these two organelles, including mitochondrial ATP production and apoptosis, has been suggested. The adaptive response to ER stress includes the maintenance of cellular energetics, which eventually determines cell fate. We previously demonstrated that heme oxygenase-1 (HO-1) activity protects cells against ER stress in a protein kinase RNA-like endoplasmic reticulum kinase (PERK)-dependent pathway. Here, we provide evidence that PERK-mediated induction of HO-1 in murine macrophages, RAW264.7, relays ER stress to mitochondrial DNA (mtDNA) replication and function. ER stress induced by thapsigargin treatments (10-100 nM) resulted in a 2-fold increase in mtDNA contents compared with that in the untreated control. HO-1 activity on ER stress is proven to be critical for mitochondrial integrity because chemical inhibition (zinc protoporphyrin, 5-20 µM) and genetic depletion of HO-1 by small interference RNA transfection suppress the activation of transcription factors for mitochondrial biogenesis. Carbon monoxide (CO), an enzymatic by-product of HO-1 activity is responsible for the function of HO-1. Limited bioavailability of CO by hemoglobin treatment triggers cell death with a concomitant decline in ATP production. Approximately 78.1% of RAW264.7 cells were damaged in the presence of hemoglobin compared with the percentage of injured cells (26.9%) under ER stress alone. Mitochondrial generation of ATP levels significantly declined when CO availability was limited under prolonged ER stress. Taken together, these results suggest that the cellular HO-1/CO system conveys ER stress to cell survival signals from mitochondria via both the activation of transcriptional factors and functional integrity of mtDNA.

Necrosis is associated with IL-6 production but apoptosis is not.

Due to loss of cell membrane integrity, necrotic cells passively release several cytosolic factors that can activate antigen presenting cells and other immune cells. In contrast, cells dying by apoptosis do not induce an inflammatory response. Here we show that necrotic cell death induced by several stimuli, such as TNF, anti-Fas or dsRNA, coincides with NF-kappaB-and p38MAPK-mediated upregulation and secretion of the pro-inflammatory cytokine IL-6. This event is greatly reduced or absent in conditions of apoptotic cell death induced by the same stimuli. This demonstrates that besides the capacity of necrotic cells to induce an inflammatory response due to leakage of cellular contents, necrotic dying cells themselves are involved in the expression and secretion of inflammatory cytokines. Moreover, inhibition of NF-kappaB and p38MAPK activation does not affect necrotic cell death in all conditions tested. This suggests that the activation of inflammatory pathways is distinct from the activation of necrotic cell death sensu strictu.

A cellular screening assay to test the ability of PKR to induce cell death in mammalian cells.

Long double-stranded RNA (>30 bp), usually expressed in cells infected with RNA viruses, triggers antiviral responses that induce apoptosis of the infected cells. PKR can be selectively activated in glioblastoma cells by in situ generation of dsRNA following introduction of antisense RNA complementary to an RNA expressed specifically in these cells. Harnessing PKR for the selective killing of cancer cells is potentially a powerful strategy for treating cancer, but we were unable to induce apoptosis by this approach in a T cell lymphoma. We therefore established a cellular screening assay to test the ability of PKR to induce death in cell lines, especially those originating from human cancers. This "PKR killing screen" is based on the infection of cells with an adenoviral vector encoding GyrB-PKR, followed by coumermycin treatment. Cancers represented by cell lines in which PKR activation leads to cell death are good candidates for the dsRNA killing approach, using antisense to RNA molecules specifically expressed in these cells. The PKR killing screen may also serve as a tool for exploring PKR signaling and other related pathways, by identifying new cases in which PKR signaling is inhibited or impaired.

Identification by electron microscopy of the maturation steps in vaccinia virus morphogenesis inhibited by the interferon-induced enzymes, protein kinase (PKR), 2-5A synthetase, and nitric oxide synthase (iNOS).

Interferons (IFN) play a major role as a first-line host defense mechanism against viral infections. As treatment of animal cells with IFN induces a large number of genes, it has been difficult to assign the role of these genes in the antiviral action of IFN. Vaccinia virus (VV) is an ideally suited system to study IFN action because all steps in viral morphogenesis can be followed easily by electron microscopy (EM) of ultrathin sections from infected cells. To define the role of IFN-induced genes in viral morphogenesis, we have independently expressed from VV recombinants in primary chicken embryo fibroblast (CEF) cells each of the three IFN-induced genes encoding protein kinase (PKR), 2-5A synthetase, and inducible nitric oxide synthase (iNOS). By EM analysis, we have identified the steps in VV morphogenesis that are affected by each of the IFN-induced enzymes in comparison with untreated and IFN-treated cells. We found that in cells pretreated with IFN and infected with VV, immature virus (IV) is formed, but further stages of maturation are blocked. In cells infected with a VV recombinant expressing PKR (VV-PKR), there is severe inhibition on virus factories, and only few IV are formed. In cells infected with a VV recombinant expressing 2-5A synthetase (VV-2-5A), VV assembly is inhibited at or after IV formation. In cells infected with a VV recombinant expressing iNOS (VV-iNOS), all stages in VV morphogenesis are observed but with aberrant forms. In addition to the effects on viral assembly, in cells infected with either VV-PKR, VV-2-5AS, or VV-iNOS, there is nucleus condensation characteristic of apoptosis. Our findings have identified the steps in VV morphogenesis inhibited by PKR, 2-5A, and iNOS, provided a distinction between these effects, and highlighted a functional redundancy of the IFN system to block viral infection and to induce apoptosis.

Induction of programmed cell death in Kaposi's sarcoma cells by preparations of human chorionic gonadotropin.

BACKGROUND: Isolation of the first neoplastic acquired immunodeficiency syndrome-related Kaposi's sarcoma (KS) cell line (KS Y-1) has furthered understanding of the pathogenesis of KS. Studies with KS Y-1 cells have indicated that inhibition of KS cell proliferation occurs in early pregnancy in mice and after treatment with certain commercial preparations of human chorionic gonadotropin (hCG, a pregnancy hormone purified from urine). The activity of the commercial preparations has been attributed to an hCG-associated factor(s) (HAF). While several clinical benefits of HAF are clearly evident, the basis for its anti-KS properties remains unknown. We investigated the apoptosis-inducing effects of HAF and the expression of apoptosis-related proteins in KS cells. METHODS: KS Y-1 and KS SLK cells were treated with clinical-grade crude preparations of hCG, recombinant hCG, or urine fractions exhibiting anti-KS activity and then examined for features of apoptosis. Levels of proteins associated with apoptosis were monitored by western blot analysis, and cell DNA content was assessed by flow cytometry. Tumors induced in mice by inoculation of KS Y-1 cells were treated with preparations of hCG, and the tumors were examined for cell morphology and also for DNA fragmentation by use of the terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine triphosphate nick-end-labeling (TUNEL) assay. RESULTS: The HAF present in some preparations of hCG and in urine fractions has the ability to induce apoptosis in KS cells in vitro and in vivo. HAF-triggered apoptosis was preceded by increased levels of the apoptosis-related proteins c-Myc and c-Rel and cell accumulation in Go/G1 phase of the cell cycle. KS Y-1 cells transfected with a c-Myc complementary DNA showed elevated rates of apoptosis. CONCLUSION: The anti-KS activity of HAF appears to induce apoptosis. Such activity suggests a role for HAF in pregnancy-related regulation of cell death.