Interaction between arsenic trioxide (ATO) and human neutrophils

The cytotoxic effect of arsenic trioxide (ATO) is known to be mediated by its ability to induce cell apoptosis in a variety of cells, including neutrophils. More recently, we demonstrated that ATO induced several parameters involved in endoplasmic reticulum (ER) stress-induced neutrophil apoptosis but that caspase-4 was not involved. The aim of this study was to better understand how neutrophils are activated by ATO and to further demonstrate that ATO is an ER stressor. Human neutrophils were isolated from healthy blood donors and incubated in vitro in the presence or absence of ATO and several parameters were investigated. We found that ATO induced the expression of the proapoptotic GADD153 protein, a key player involved in ER stress-induced apoptosis, activated nuclear nuclear factor κB (NF-κB) DNA binding activities, and increased prostaglandine E2 (PGE2) production. Using an antibody array approach, we found that ATO increased the production of several cytokines, with interleukin 8 (IL-8) being the predominant one. We confirmed that ATO increased the production of IL-8 by enzyme-linked-immunosorbent assay (ELISA). Treatment with a caspase-4 inhibitor did not inhibit IL-8 production. The results of the present study further support the notion that ATO is an ER stressor and that, although its toxic effect is mediated by induction of apoptosis, this chemical also induced, in parallel, NF-κB activation, the production of PGE2 and several cytokines probably involved in other cell functions. Also, we conclude that the production of IL-8 is not induced by a caspase-4-dependent mechanism, suggesting that ATO-induced caspase-4 activation is involved in other as yet unidentified functions in human neutrophils.

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iPLA2β Contributes to ER Stress-Induced Apoptosis during Myocardial Ischemia/Reperfusion Injury

Cells ◽

10.3390/cells10061446 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1446

Author(s):

Tingting Jin ◽

Jun Lin ◽

Yingchao Gong ◽

Xukun Bi ◽

Shasha Hu ◽

...

Keyword(s):

Cell Death ◽

Heart Disease ◽

Myocardial Ischemia ◽

Ischemic Heart Disease ◽

Er Stress ◽

Ischemia Reperfusion ◽

Myocardial Ischemia Reperfusion ◽

Induced Apoptosis

Both calcium-independent phospholipase A2 beta (iPLA2β) and endoplasmic reticulum (ER) stress regulate important pathophysiological processes including inflammation, calcium homeostasis and apoptosis. However, their roles in ischemic heart disease are poorly understood. Here, we show that the expression of iPLA2β is increased during myocardial ischemia/reperfusion (I/R) injury, concomitant with the induction of ER stress and the upregulation of cell death. We further show that the levels of iPLA2β in serum collected from acute myocardial infarction (AMI) patients and in samples collected from both in vivo and in vitro I/R injury models are significantly elevated. Further, iPLA2β knockout mice and siRNA mediated iPLA2β knockdown are employed to evaluate the ER stress and cell apoptosis during I/R injury. Additionally, cell surface protein biotinylation and immunofluorescence assays are used to trace and locate iPLA2β. Our data demonstrate the increase of iPLA2β augments ER stress and enhances cardiomyocyte apoptosis during I/R injury in vitro and in vivo. Inhibition of iPLA2β ameliorates ER stress and decreases cell death. Mechanistically, iPLA2β promotes ER stress and apoptosis by translocating to ER upon myocardial I/R injury. Together, our study suggests iPLA2β contributes to ER stress-induced apoptosis during myocardial I/R injury, which may serve as a potential therapeutic target against ischemic heart disease.

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Differential activation of ER stress and apoptosis in response to chronically elevated free fatty acids in pancreatic β-cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00478.2007 ◽

2008 ◽

Vol 294 (3) ◽

pp. E540-E550 ◽

Cited By ~ 108

Author(s):

Elida Lai ◽

George Bikopoulos ◽

Michael B. Wheeler ◽

Maria Rozakis-Adcock ◽

Allen Volchuk

Keyword(s):

Er Stress ◽

Cell Lines ◽

Cell Apoptosis ◽

Human Islets ◽

Pancreatic Β Cell ◽

Β Cell ◽

Min6 Cells ◽

Relative Contribution ◽

Induced Apoptosis

Chronic exposure to elevated saturated free fatty acid (FFA) levels has been shown to induce endoplasmic reticulum (ER) stress that may contribute to promoting pancreatic β-cell apoptosis. Here, we compared the effects of FFAs on apoptosis and ER stress in human islets and two pancreatic β-cell lines, rat INS-1 and mouse MIN6 cells. Isolated human islets cultured in vitro underwent apoptosis, and markers of ER stress pathways were elevated by chronic palmitate exposure. Palmitate also induced apoptosis in MIN6 and INS-1 cells, although the former were more resistant to both apoptosis and ER stress. MIN6 cells were found to express significantly higher levels of ER chaperone proteins than INS-1 cells, which likely accounts for the ER stress resistance. We attempted to determine the relative contribution that ER stress plays in palmitate-induced β-cell apoptosis. Although overexpressing GRP78 in INS-1 cells partially reduced susceptibility to thapsigargin, this failed to reduce palmitate-induced ER stress or apoptosis. In INS-1 cells, palmitate induced apoptosis at concentrations that did not result in significant ER stress. Finally, MIN6 cells depleted of GRP78 were more susceptible to tunicamycin-induced apoptosis but not to palmitate-induced apoptosis compared with control cells. These results suggest that ER stress is likely not the main mechanism involved in palmitate-induced apoptosis in β-cell lines. Human islets and MIN6 cells were found to express high levels of stearoyl-CoA desaturase-1 compared with INS-1 cells, which may account for the decreased susceptibility of these cells to the cytotoxic effects of palmitate.

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Regulation of PDGFR-α in rat pulmonary myofibroblasts by staurosporine

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2001.280.2.l354 ◽

2001 ◽

Vol 280 (2) ◽

pp. L354-L362 ◽

Cited By ~ 7

Author(s):

Pamela M. Lindroos ◽

Yi-Zhe Wang ◽

Annette B. Rice ◽

James C. Bonner

Keyword(s):

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Protein A ◽

Nuclear Factor Κb ◽

Mitogen Activated Protein Kinase ◽

Specific Gene ◽

Autocrine Loop ◽

P38 Inhibitor ◽

Mitogen Activated Protein

Upregulation of the platelet-derived growth factor (PDGF) receptor-α (PDGFR-α) is a mechanism of myofibroblast hyperplasia during pulmonary fibrosis. We previously identified interleukin (IL)-1β as a major inducer of the PDGFR-α in rat pulmonary myofibroblasts in vitro. In this study, we report that staurosporine, a broad-spectrum kinase inhibitor, upregulates PDGFR-α gene expression and protein. A variety of other kinase inhibitors did not induce PDGFR-α expression. Staurosporine did not act via an IL-1β autocrine loop because the IL-1 receptor antagonist protein did not block staurosporine-induced PDGFR-α expression. Furthermore, staurosporine did not activate a variety of signaling molecules that were activated by IL-1β, including nuclear factor-κB, extracellular signal-regulated kinase, and c-Jun NH2-terminal kinase. However, both staurosporine- and IL-1β-induced phosphorylation of p38 mitogen-activated protein kinase and upregulation of PDGFR-α by these two agents was inhibited by the p38 inhibitor SB-203580. Finally, staurosporine inhibited basal and PDGF-stimulated mitogenesis over the same concentration range that induced PDGFR-α expression. Collectively, these data demonstrate that staurosporine is a useful tool for elucidating the signaling mechanisms that regulate PDGFR expression in lung connective tissue cells and possibly for evaluating the role of the PDGFR-α as a growth arrest-specific gene.

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N6-(2-hydroxyethyl)-Adenosine Induces Apoptosis via ER Stress and Autophagy of Gastric Carcinoma Cells In Vitro and In Vivo

International Journal of Molecular Sciences ◽

10.3390/ijms21165815 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5815

Author(s):

Hongqing Xie ◽

Xiaotong Li ◽

Weiwei Yang ◽

Liping Yu ◽

Xiasen Jiang ◽

...

Keyword(s):

Gastric Carcinoma ◽

Er Stress ◽

Carcinoma Cells ◽

Dependent Manner ◽

Antineoplastic Effect ◽

Tumor Tissues ◽

Induced Apoptosis ◽

Species Production

Gastric cancer is the most common malignant tumor of the digestive tract and is great challenge in clinical treatment. N6-(2-Hydroxyethyl)-adenosine (HEA), widely present in various fungi, is a natural adenosine derivative with many biological and pharmacological activities. Here, we assessed the antineoplastic effect of HEA on gastric carcinoma. HEA exerted cytotoxic effects against gastric carcinoma cells (SGC-7901 and AGS) in a dose and time-dependent manner. Additionally, we found that HEA induced reactive oxygen species production and mitochondrial membrane potential depolarization. Moreover, it could trigger caspase-dependent apoptosis, promoting intracellular Ca2+-related endoplasmic reticulum (ER) stress and autophagy. On the other hand, HEA could significantly inhibit the growth of transplanted tumors in nude mice and induce apoptosis of tumor tissues cells in vivo. In conclusion, HEA induced apoptosis of gastric carcinoma cells in vitro and in vivo, demonstrating that HEA is a potential chemotherapeutic agent for gastric carcinoma.

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Sulfate import in Salmonella Typhimurium impacts bacterial aggregation and the respiratory burst in human neutrophils

Infection and Immunity ◽

10.1128/iai.00701-20 ◽

2021 ◽

Author(s):

T. L. Westerman ◽

M. K. Sheats ◽

J. R. Elfenbein

Keyword(s):

Respiratory Burst ◽

Gene Deletion ◽

Single Gene ◽

Protein A ◽

Human Neutrophils ◽

Deletion Mutants ◽

Flagellar Motility ◽

Neutrophil Respiratory Burst ◽

Single Gene Deletion

During enteric salmonellosis, neutrophil generated reactive oxygen species alter the gut microenvironment favoring survival of Salmonella Typhimurium. While the type-3 secretion system-1 (T3SS-1) and flagellar motility are potent Salmonella Typhimurium agonists of the neutrophil respiratory burst in vitro, neither of these pathways alone are responsible for stimulation of a maximal respiratory burst. In order to identify Salmonella Typhimurium genes that impact the magnitude of the neutrophil respiratory burst, we performed a two-step screen of defined mutant libraries in co-culture with human neutrophils. We first screened Salmonella Typhimurium mutants lacking defined genomic regions and then tested single gene deletion mutants representing particular regions under selection. A subset of single gene deletion mutants were selected for further investigation. Mutants in four genes, STM1696 (sapF), STM2201 (yeiE), STM2112 (wcaD), and STM2441 (cysA), induced an attenuated respiratory burst. We linked the altered respiratory burst to reduced T3SS-1 expression and/or altered flagellar motility for two mutants (ΔSTM1696 and ΔSTM2201). The ΔSTM2441 mutant, defective for sulfate transport, formed aggregates in minimal media and adhered to surfaces in rich media, suggesting a role for sulfur homeostasis in regulation of aggregation/adherence. We linked the aggregation/adherence phenotype of the ΔSTM2441 mutant to biofilm-associated protein A and flagellins and hypothesize that aggregation caused the observed reduction in the magnitude of the neutrophil respiratory burst. Our data demonstrate that Salmonella Typhimurium has numerous mechanisms to limit the magnitude of the neutrophil respiratory burst. These data further inform our understanding of how Salmonella may alter human neutrophil antimicrobial defenses.

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Down-Regulation of miR-212-5p Facilitates Homocysteine-Induced Hepatocytes Apoptosis Via Targeting PSMD10

10.21203/rs.3.rs-136080/v1 ◽

2020 ◽

Author(s):

Huiping Zhang ◽

Kun Xiao ◽

Shengchao Ma ◽

Long Xu ◽

Ning Ding ◽

...

Keyword(s):

Liver Injury ◽

Er Stress ◽

Underlying Mechanism ◽

Luciferase Reporter ◽

Down Regulation ◽

Over Expression ◽

Direct Binding ◽

Induced Apoptosis ◽

Insight Into

Abstract Background: Increasing evidences supported that elevated homocysteine (Hcy) levels contribute to cell apoptosis is implicated in the pathogenesis of liver injury, it correlates with liver disease severity. However, the underlying mechanism of apoptosis in Hcy-mediated liver injury remains obscure. Results: In this study, we found that homocysteine increases ER stress-mediated apoptosis and aggravates liver injury through up-regulation of PSMD10 expression in cbs+/- mice mice fed with high methionine diet and hepatocytes treated with homocysteine in vitro. Knockdown of PSMD10 expression remarkably reduced ER stress or apoptosis-associated protein in hepatocytes exposed to homocysteine. Moreover, bioinformatics analysis revealed that PSMD10 is a potential target gene of miR-212-5p, and luciferase reporter assay also confirmed that miR-212-5p negatively regulated PSMD10 expression by direct binding to its 3’-UTR regions. Subsequently, over-expression of miR-212-5p inhibited ER stress-mediated hepatocytes apoptosis though targeting PSMD10, all of which were abrogated by knockdown of miR-212-5p expression. Further study showed that the interaction between PSMD10 and GRP78 accelerated ER stress-mediated hepatic apoptosis induced by homocysteine. Conclusion: Taken together, these results demonstrated that down-regulation of miR-212-5p facilitates homocysteine-induced hepatocytes apoptosis via targeting PSMD10, which provides novel insight into the mechanism of homocysteine induced apoptosis in liver injury.

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Treatment of Acute Myelogenous Leukemia (non-APL) with Intravenous Trisenox® (Arsenic Trioxide) and Ascorbic Acid: Preliminary Results.

Blood ◽

10.1182/blood.v104.11.1815.1815 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1815-1815 ◽

Cited By ~ 1

Author(s):

Dan Douer ◽

Kristy Watkins ◽

Robert Louie ◽

Ilene Weitz ◽

Ann Mohrbacher ◽

...

Keyword(s):

Multiple Myeloma ◽

Ascorbic Acid ◽

Arsenic Trioxide ◽

Peripheral Blood ◽

Sensory Neuropathy ◽

Myelogenous Leukemia ◽

Clinical Activity ◽

Preliminary Results ◽

Induced Apoptosis

Abstract Introduction: Arsenic trioxide (ATO) is an exceptionally active drug in acute promyelocytic leukemia (APL), inducing complete remissions in 85% of relapsed patients. ATO also has clinical activity in myelodysplastic syndrome (MDS). In non-APL AML cells lines, ATO induces apoptosis in vitro; however, in a small study of 11 non-APL AML patients, ATO showed no activity (Parmer et al Leuk Res28:090, 2004). In some types of cancer cells, ATO-induced apoptosis has been shown to correlate inversely with the level of intracellular reduced glutathione (GSH) via generation of reactive oxygen species; cells with high concentrations of GSH are more resistant to ATO. Ascorbic acid (AA) increases apoptosis and overcomes resistance to ATO in multiple myeloma, non-APL AML and other cell lines by reducing intracellular GSH levels. AA alone has no activity in these cells. We therefore conducted a clinical trial in patients with non-APL AML combining ATO and AA. Methods: ATO at a dose of 0.25 mg/kg is administrated intravenously over 1–3 hour with 1 gram of intravenous AA given within 30 minutes daily for five days a week (five days on/2 days off) for five weeks (25 doses - one cycle). These doses were based on a phase I/II trial of ATO+AA in patients with multiple myeloma (Behalis et al Clin Cancer Res8:3658, 2002)). Responding patients receive an additional consolidation cycle of 25 doses followed by maintenance of two weeks of every month of ATO+ AA for 4 cycles. Patients who fail to respond after two cycles are considered treatment failures. Results: Seven patients have so far enrolled: three (aged 36,52,59) had relapsed after chemotherapy, and four aged 66–84 (median 70), never received chemotherapy. For these untreated patients ATO+AA was given as front line treatment. In three of the four previously untreated patients the number of bone marrow blasts dropped from > 40% to < 5% (2 pts. after 1 cycle; 1 pt. after 2 cycles) At the time of this report only one of the responding patients received more than one cycle and had improvement in the peripheral blood counts. The three patients, who failed chemotherapy, did not respond to ATO+AA (one patient received only one cycle). Despite the high doses of ATO, higher than used in APL and MDS, the combination was very well tolerated with grade 3 toxicity in one patient only (sensory neuropathy). One responding patient developed shortness of breath with severe hypoxemia, reminiscent of the APL differentiation syndrome, which responded immediately to dexamethasone. Conclusion: These preliminary results in patients with non-APL AML suggest that: (1) AA +ATO has anti-leukemia activity in untreated non-APL AML patients with minimal toxicity; (2) more than one cycle is probably needed to achieve a response in the peripheral blood counts; (3) in non-APL AML, ATO can cause the so called “differentiation syndrome” which should be anticipated and treated early. If confirmed in additional patients, ATO+AA might be a less toxic alternative upfront approach to intensive chemotherapy in elderly patients with non-APL AML.

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Arsenic Trioxide in Mantle Cell Lymphoma.

Blood ◽

10.1182/blood.v106.11.4814.4814 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4814-4814 ◽

Cited By ~ 1

Author(s):

Yok Lam Kwong ◽

Chit Chow ◽

Cyrus R. Kumana ◽

Gopesh Srivastava ◽

Wing Yan Au

Keyword(s):

Cyclin D1 ◽

Mantle Cell Lymphoma ◽

Arsenic Trioxide ◽

Cell Lymphoma ◽

Down Regulation ◽

Synergistic Interactions ◽

Mantle Cell ◽

Induced Apoptosis ◽

Mtt Assays

Abstract Background. Mantle cell lymphoma (MCL) is incurable for many patients. Arsenic trioxide (As2O3) has activity in vitro against lymphoid malignancies. The effects of As2O3 on MCL in vitro and in patients with refractory disease were investigated. Materials and methods. Mantle cell lymphoma (MCL) lines (Jeko-1, Granta-519) were treated with As2O3, in combination with mitoxantrone (MTZ) and ascorbic acid (AA). Consenting patients with refractory MCL were treated with oral-As2O3 (10 mg/day), AA (1 g/day) and chlorambucil (4 mg/day) as outpatients until maximum response or the disease judged refractory. Responses were defined by standard NCI criteria. In patients showing an initial response, vincristine (2 mg intravenously) and prednisolone (30 mg/day) might also be added. After achievement of maximum response, patients were maintained with As2O3 (10 mg/day) and AA (1 g/day) for two weeks every month, for a planned two years. Results. As2O3 and MTZ but not AA induced a dose dependent apoptosis in the MCL lines, as shown by flow cytometry and MTT assays. As2O3, MTZ and AA were tested in various combinations in MTT assays. Synergistic interactions were observed only in the combinations As2O3 (1 uM) + MTZ (0.2 mg/L), and As2O3 (1 uM) + MTZ (0.2 mg/L) + AA (100 uM). Western blotting showed that As2O3-induced apoptosis was associated with a dose and time dependent down-regulation of cyclin D1. However, quantitative polymerase chain reaction showed no change in cyclin D1 gene transcription during As2O3-induced apoptosis. Eleven patients (10 men, 1 women) at a median age of 69 (51–70) years with refractory MCL were studied, at a median of 33 (8–85) months from diagnosis. At the time of As2O3 treatment, they had already had a median of 2 (1 – 4) relapses managed with a median of 2 (1 – 6) previous chemotherapeutic regimens. Eight of eleven patients were evaluable (for the other three, one died after 4 days of treatment, and the two were still receiving therapy). At a median follow up of 9 (4 – 20) months, 4 patients had reached complete remission (CR) or probable CR (CRu), two were in good partial remission, and two had died with progressive disease. Conclusion. As2O3 induced apoptosis of MCL cells by post-transcription down-regulation of cyclin D1. Synergistic interactions were observed with AA and cytotoxics. Oral-As2O3, AA and chlorambucil were an active regimen for relapsed and therapy-refractory MCL, and treatment results compared favorably with other salvage regimens. This entirely oral regimen has several attractions, including outpatient treatment, low toxicity and cost.

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Evaluation of Mechanisms of Resistance to Arsenic Trioxide In Patients with Acute Promyelocytic Leukemia

Blood ◽

10.1182/blood.v116.21.2746.2746 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2746-2746

Author(s):

Ezhilarasi Chendamarai ◽

Ansu Abu Alex ◽

Saravanan Ganesh ◽

Kavitha M Lakshmi ◽

Sachin Jadhav ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Arsenic Trioxide ◽

Acute Promyelocytic Leukemia ◽

Promyelocytic Leukemia ◽

Newly Diagnosed ◽

Mechanisms Of Resistance ◽

Induced Apoptosis ◽

The Impact

Abstract Abstract 2746 Introduction: Newly diagnosed and relapsed acute promyelocytic leukemia (APL) patients respond to therapy with arsenic trioxide (ATO) based regimens. Significantly more patients with relapsed APL have disease recurrence after ATO based therapy than newly diagnosed cases. We undertook a series of experiments to evaluate the potential mechanisms to explain this increased recurrence rate in patients with relapsed APL. Patients and methods: From April 2007 to March 2009 bone marrow samples from newly diagnosed and relapsed cases admitted at our center were utilized for these studies. If required the bone marrow blasts and promyelocyte component was enriched to above 90% using a lineage depletion cocktail and VarioMACS (Miltenyi Biotec, Germany). For in-vitro intracellular ATO concentration measurement, 2 × 107 cells were washed and suspended in RPMI media with 0.5 μM concentration of ATO and incubated for 24 hours. Cells were then washed, made into a pellet and solubilized with HNO3 and H2O2 and ATO content measured using an atomic absorption spectrophotometer. An in-vitro sensitivity assay of malignant cells as previously reported was standardized using an MTT assay system. The impact of co-culture of mesenchymal stromal cells (MSC) and malignant promyelocytes on ATO induced apoptosis was studied with 7AAD and Annexin staining using a flowcytometer. A gene expression array using 44k human microarray chip analysis (Agilent technologies) was done on 8 newly diagnosed and 8 relapsed cases. Results: Sixty five patients were included in this study. Of these 47 (72%) were newly diagnosed and 18 (28%) were relapsed cases. On immunophenotyping, CD34 was positive (>20%) in 3.6% of newly diagnosed and 50% of relapsed cases (P=0.001). The mean MFI of the relapsed cases for expression of CD38, VLA-5 and CD13 was significantly lower in the relapsed group. The ability of both newly diagnosed and relapsed primary APL cells to concentrate ATO intracellular was not significantly different (15.2±9 nG/107 cell Vs. 16.3±9.7 nG/107 cell). Similarly the in-vitro IC-50 assay was not significantly different between the two groups (5.5±3.8 Vs. 4.7±4.5 μM). Neither of these assays correlate with clinical parameters such as relapse, event free (EFS) or overall survival (OS). Evaluation of the impact of MSC on ATO induced apoptosis demonstrates a protective effect in newly diagnosed and relapsed cases (Fig 1). This effect was mediated partly by the MSC conditioning media and could not be overcome by addition of VLA-4 or VLA-5 blocking antibodies (data not shown). Gene expression studies comparing the two cohorts revealed 1744 genes that were differentially expressed (>2 fold) between samples at diagnosis and at relapse. Quantitative Real-time RT-PCR using SYBR- Green detection system was done to confirm the gene expression results obtained from microarray analysis. Using ΔΔCT method the fold difference was calculated for five selected genes which validated the microarray data. Conclusion: Relapsed patients have significant immuno-phenotypic differences from newly diagnosed cases. Mechanisms of resistance to ATO are probably multi-factorial but are unlikely to be related to intra-cellular ATO concentration. In-vitro IC-50 does not appear to predict clinical outcomes. Stromal interaction protects malignant promyelocytes from the apoptotic action of ATO which appears more pronounced in relapsed than in newly diagnosed cases. Further evaluation of parameters that enhance such stromal interaction and protect malignant promyelocytes from the apoptotic effect of ATO along with evaluation of dysregulated genes and pathways are required. Disclosure: No relevant conflicts of interest to declare.

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The Canine Papillomavirus E5 Protein Signals from the Endoplasmic Reticulum

Journal of Virology ◽

10.1128/jvi.01003-09 ◽

2009 ◽

Vol 83 (24) ◽

pp. 12833-12841 ◽

Cited By ~ 8

Author(s):

Rachel Condjella ◽

Xuefeng Liu ◽

Frank Suprynowicz ◽

Hang Yuan ◽

Sawali Sudarshan ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Growth Inhibition ◽

Er Stress ◽

Protein A ◽

Keratinocyte Differentiation ◽

Keratinocyte Proliferation ◽

Reading Frame ◽

E5 Protein

ABSTRACT The recently discovered Canis familiaris papillomavirus (PV) type 2 (CfPV2) provides a unique opportunity to study PV gene functions in vitro and in vivo. Unlike the previously characterized canine oral PV, CfPV2 contains an E5 open reading frame and is associated with progression to squamous cell carcinoma. In the current study, we have expressed and characterized the CfPV2-encoded E5 protein, a small, hydrophobic, 41-amino-acid polypeptide. We demonstrate that, similar to the E5 protein from high-risk human PV type 16, the CfPV2 E5 protein is localized in the endoplasmic reticulum (ER) and that its expression decreases keratinocyte proliferation and cell life span. E5 expression also increases the percentage of cells in the G1 phase of the cell cycle, with a concomitant decrease in the percentage of cells in S phase. To identify a potential mechanism for E5-mediated growth inhibition from the ER, we developed a real-time PCR method to quantify the splicing of XBP1 mRNA as a measure of ER stress. We found that the CfPV2 E5 protein induced ER stress and that this, as well as the observed growth inhibition, is tempered significantly by coexpression of the CfPV2 E6 and E7 genes. It is possible that the spatial/temporal regulation of E6/E7 gene expression during keratinocyte differentiation might therefore modulate E5 activity and ER stress.

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