E1A Transfectants Research Articles

Coordinate Inhibition of Cytokine-mediated Induction of Ferritin H, Manganese Superoxide Dismutase, and Interleukin-6 by the Adenovirus E1A Oncogene

Adenovirus E1A sensitizes cells to the cytotoxic action of tumor necrosis factor alpha (TNF-alpha). This effect has been attributed to direct blockade of NF-kappaB activation, as well as to increased activation of components of the apoptotic pathway and decreases in inhibitors of apoptosis. In this report we evaluated the mechanism by which E1A modulates the expression of the cytokine-inducible cytoprotective genes manganese superoxide dismutase (MnSOD), interleukin-6 (IL-6), and ferritin heavy chain (FH). We observed that E1A blocks induction of MnSOD, IL-6, and FH by TNF-alpha or IL-1alpha. Because NF-kappaB plays a role in cytokine-dependent induction of MnSOD, IL-6, and FH, we assessed the effect of E1A on NF-kappaB in cells treated with TNF. IkappaB, the inhibitor of NF-kappaB, was degraded similarly in the presence and absence of E1A. TNF induced a quantitatively and temporally equivalent activation of NF-kappaB in control and E1A-transfected cells. However, TNF-dependent acetylation of NF-kappaB was diminished in cells expressing E1A. E1A mutants unable to bind p400 or the Rb family proteins were still capable of repressing TNF-dependent induction of FH. However, mutants of E1A that abrogated binding of p300/CBP blocked the ability of E1A to repress TNF-dependent induction of FH. These results suggest that p300/CBP is a critical control point in NF-kappaB-dependent transcriptional regulation of cytoprotective genes by cytokines.

Involvement of Bcl-XL deamidation in E1A-mediated cisplatin sensitization of ovarian cancer cells

The adenovirus E1A protein has been shown to be involved in the potentiation of apoptosis induced by chemotherapeutic agents, yet the molecular events of E1A-mediated apoptosis are not very clear. A recent report has suggested that deamidation of the Bcl-X(L) protein inhibits its antiapoptotic ability and leads to apoptosis induced by alkylating agents in Rb-deficient tumor cells. Since Rb is known to interact with E1A, which interrupts Rb's normal function, we examined Bcl-X(L) deamidation and cell death induced by cisplatin in E1A transfectants. We found that the E1A transfectants became sensitive to cisplatin-induced apoptosis compared to the parental cells, SKOV3.ip1. Our data show that cisplatin treatment induced the modification of Bcl-X(L) in the E1A transfectants in dosage and time-dependent manner. Furthermore, phosphatase treatment had no effect on the level of Bcl-X(L) modification, whereas alkaline lysis buffer appeared to induce the same modification of Bcl-X(L). Ectopic expression of the deamidated forms of Bcl- X(L) in SKOV3.ip1 cells revealed that the modification to the Bcl- X(L) protein molecules was deamidation. Expression of the E1A mutant (dl1108) which contains deletion at CR2 domain suppressed Bcl-X(L) deamidation and apoptosis induced by cisplatin. We also found that expression of the nondeamidated Bcl-X(L) protected E1A transfectants from apoptosis. These findings suggest that Bcl-X(L) deamidation contributes to E1A-mediated cisplatin sensitization in SKOV3.ip1 cells.

Antitumor effect of E1A in ovarian cancer by cytoplasmic sequestration of activated ERK by PEA15

The adenovirus type 5 gene E1A is known to suppress tumorigenicity by transcriptionally downregulating HER-2/neu (HER2) or by inducing apoptosis. We show here that E1A also suppressed the tumorigenicity of the low-HER2-expressing ovarian cancer cell line OVCAR-3 by decreasing cell proliferation. We further found that the mechanism responsible for this reduced proliferation is the presence of PEA15 (phosphoprotein enriched in astrocytes), which is upregulated by E1A in ovarian cancer; PEA15 promotes translocation of ERK from the nucleus to the cytoplasm, leading to inhibition of ERK-dependent transcription and proliferation. Indeed, siRNA-mediated knockdown of PEA15 expression in OVCAR-3 stable E1A transfectants resulted in a nuclear accumulation of the active form of ERK, followed by an increase in Elk-1 activity, DNA synthesis, and anchorage-independent growth. Finally, PEA15 by itself suppressed colony formation in breast and ovarian cancer cell lines, in which E1A is known to have antitumor activity. We conclude that part of the antitumor effect of E1A in ovarian cancer results from cytoplasmic sequestration of the activated form of ERK by PEA15.

Effect of the adenovirus E1A gene on nitric oxide production in alveolar epithelial cells

This study determined the effect of the adenovirus E1A gene on nitric oxide (NO) production in alveolar epithelial (A549) cells. E1A-positive A549 cells (E1A transfectants), E1A-negative A549 cells (control transfectants) and untransfected A549 cells were placed in 96-well tissue culture plates. After stimulation with lipopolysaccharide (LPS) or cytokine mixture (CM), the biochemical reaction products of NO (nitrite and nitrate) in the culture medium were measured by chemiluminescence. The inducible (iNOS) and the endothelial (eNOS) isoforms of nitric oxide synthase (NOS) protein expression were examined by Western blotting. iNOS mRNA expression was examined by Northern blotting and RT-PCR. CM-induced NO production by E1A-positive A549 cells was significantly lower than that of E1A-negative cells (p < 0.0001). LPS stimulation failed to enhance NO production in both cell types. CM induced iNOS protein expression in E1A-negative A549 cells, but not in E1A-positive cells. eNOS protein expression was constitutive and was not affected by CM stimulation, LPS stimulation or E1A. CM induced iNOS mRNA expression in E1A-negative A549 cells, but not in E1A-positive cells. In conclusion, the adenovirus E1A gene suppressed NO production through transcriptional control of the iNOS gene in A549 cells. This inhibition of NO production may enable the virus to persist in human tissue, since NO is an antiviral effector of the innate immune system.

E1A Sensitizes Cancer Cells to TRAIL-Induced Apoptosis through Enhancement of Caspase Activation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis of cancer cells. Sensitization of cancer cells to TRAIL, particularly TRAIL-resistant cancer cells, could improve the effectiveness of TRAIL as an anticancer agent. The adenovirus type 5 E1A that associates with anticancer activities including sensitization to apoptosis induced by tumor necrosis factor is currently being tested in clinical trials. In this study, we investigated the sensitivity to TRAIL in the E1A transfectants ip1-E1A2 and 231-E1A cells and the parental TRAIL-resistant human ovarian cancer SKOV3.ip1 and TRAIL-sensitive human breast cancer MDA-MB-231 cells. The results indicated that the percentage of TRAIL-induced apoptotic cells was significantly higher in the E1A transfectants of both cell lines than it was in the parental cell lines. To further investigate the cellular mechanism of this effect, we found that E1A enhances TRAIL-induced activation of caspase-8, caspase-9, and caspase-3. Inhibition of caspase-3 activity by a specific inhibitor, Z-DEVD-fmk, abolished TRAIL-induced apoptosis. In addition, E1A enhanced TRAIL expression in ip1-E1A2 cells, but not in 231-E1A cells, and the anti-TRAIL neutralizing antibody N2B2 blocked the E1A-mediated bystander effect in vitro. Taken together, these results suggest that E1A sensitizes both TRAIL-sensitive and TRAIL-resistant cancer cells to TRAIL-induced apoptosis, which occurs through the enhancement of caspase activation; activation of caspase-3 is required for TRAIL-induced apoptosis; and E1A-induced TRAIL expression is involved in the E1A-mediated bystander effect. Combination of E1A and TRAIL could be an effective treatment for cancer.

Inflammatory mediator mRNA expression by adenovirus E1A-transfected bronchial epithelial cells.

Lung tissue from patients with emphysema and airway obstruction carries excess adenoviral E1A DNA that is expressed as protein in airway surface epithelium and is associated with an increased inflammatory response. To examine mechanisms by which latent adenoviral infection might amplify the inflammatory process, we transfected primary human bronchial epithelial (HBE) cells from three separate patients undergoing lung resection so that they stably expressed adenovirus E1A. Lipopolysaccharide stimulation of the E1A-transfected HBE cells increased intercellular adhesion molecule-1 and interleukin-8 mRNA and protein expression compared with control cells from the same patient. It also induced greater intercellular adhesion molecule-1 promoter activity and greater nuclear factor-kappa B binding activity of nuclear extracts in E1A transfectants than controls. E1A-positive transfectants constitutively expressed transforming growth factor-beta 1 mRNA and protein, whereas this expression was either very low or not detected in control cells. We conclude that adenoviral E1A transfection transforms primary HBE cells and upregulates their production of mediators that are clinically relevant to the pathogenesis of chronic obstructive pulmonary disease.

The E1A Gene Therapy: A Two Way Translational Research

Adenovirus type 5 E1A is associated with multiple anti-tumor activities and transcriptionally represses HER2/mu. E1A gene therapy induced regression of HER-2/neu-overexpressing cancers in nude mice. To evaluate the feasibility of E1A gene therapy for patient with HER-2/neuoverexpressing cancer, we conducted a phase 1 clinical trial. An E1A/DC-Chol complex was injected weekly into the thoracic or peritoneal cavity of 18 patients with advanced breast and/or ovarian cancers. The most common toxicities were fever, nausea, vomiting, and/or discomfort at the injection sites. After E1A/DC-Chol complex delivery, E1A gene expression was detectable in tumor cells with HER-2/neu downregulation, increased apoptosis, and reduced proliferation. Thus intracavitary E1A gene therapy is a feasible approach resulting m clear proof of preclinical concept. Phase II trials on ovarian cancer are recently initiated under the support of M. D. Anderson Ovarian Spore. In addition to downregulation of HER-2/neu overexpression, we have recently identified molecular mechanisms that provide scientific basis for E1A-mediated antitumor activities independent of HER-2/neu status. These laboratory findings are helpful to understand E1A-associated anti-tumor activity and will be incorporated to the future clinical trials: 1.E1A sensitized TNF α – and ionizing γ – irradiation-induced cell death by inhibition of NF-κB activity. Activation of the transcription factor NF-κB has been shown to play a key role in the anti-apoptotic pathway of TNF α and radiation-induced apoptosis. We have found that TNF α- or radiation-induced activation of NF κB occurred m the cancer cells but was blocked when E1A was expressed. We further showed that this inhibition was through suppression of IκB kinase (IKK) activity and IκB phosphorylation. Since IκB phosphorylation results in degradation of IκB, suppression of IκB phosphorylation leads to increased IκB level that then in turn binds to NF-κB to inbibit NF-κB anti-apoptotic activity. These results open an interesting possibility to combine E1A gene therapy with radation therapy or TNF α therapy. It should also be mentioned that TNF α expression was found to be increased in the body fluid from the treated patients in the Phase 1 trail. The increased TNF α expression may account for the increased apoptosis of cancer cells in the treated patients. In this regards, the E1A/DC-Chol-induced TNF α expression in patients may contribute to its anti-tumor activity. 2.E1A induced bystander effect by inhibiting angiogenesis and inducing apoptosis. To investigate the E1A mediated bystander effect and the mechanisms that may be associated with it. We inoculated nude mice subcutaneously with a mixture of E1A transfectants and parental cells, we found that the E1A transfectants exhibited a bystander effect on inhibition of tumor growth. We further showed that E1A mediated suppression of angiogenesis and induction of apoptosis in the tumors, likely contributing to the bystander effect. 3. E1A inhibits expression of another transforming tyrosine kinase, Axl. To study the mechanism underlying the E1A-mediated tumor suppressing function, we exploited a modified tyrosine kinase profile assay to identify potential tyrosine kinase regulated by E1A. We identified that E1A negatively regulated the expression of the transforming receptor tyrosine kinase Ax1 at the transcriptional level and downregulation of Aχ1 by E1A is required for E1A-mediated growth suppression and induction of apoptosis. These results indicated that downregulation of Ax1 receptor by E1A is involved in E1A-mediated growth suppression and E1A-induced apoptosis and thereby may contribute to E1A’s antitumor activities. Although the clinical trial of E1A gene therapy was initiated by the known mechanism of HER-2/neu downregulation, the above results together with other studies in the literature, indicate that E1A actually associate with multiple mechanisms that may contribute to anti-tumor activity. These results provide scientific basis to open the E1A gene therapy clinical trials for patients without HER-2/neu-verexpressing cancer.

Akt is required for Axl-Gas6 signaling to protect cells from E1A-mediated apoptosis.

Adenovirus type 5 E1A protein (E1A) associates with anti-tumor activities by reversing the transformed phenotype, inhibiting metastasis, and inducing apoptosis. We have previously identified that E1A suppresses the expression of Axl, a transforming tyrosine kinase and that Axl-Gas6 receptor-ligand interaction prevents E1A transfectants from apoptosis induced by serum deprivation. To determine how the Axl-Gas6 interaction prevents E1A-mediated apoptosis, we analysed the expression of anti-apoptotic molecules and found that the activated form of Akt was suppressed in the E1A transfectant ip 1-E1A and that Gas6 was able to activate Akt in ip 1-E1A cells reexpressing Axl (ip 1-E1A-Axl). To determine whether activated Akt is required to prevent E1A-mediated apoptosis, ip 1-E1A-Axl cells were treated with the phosphatidylinositol-3'-OH kinase inhibitor wortmannin or transfected with a dominant negative Akt mutant. In both cases, Gas6 no longer protected the cells from serum deprivation-induced apoptosis. Thus, we conclude that activated Akt is required for Axl-Gas6 signaling to prevent E1A-mediated apoptosis induced by serum deprivation. Downstream molecules of Akt, including NF-kappaB, Fas ligand, and BAD were examined, among which phosphorylation of BAD by Axl-Gas6 signaling is associated with the anti-apoptotic activity of Akt in our study.

Endotoxin-specific NF-kappaB activation in pulmonary epithelial cells harboring adenovirus E1A.

Adenovirus E1A DNA and proteins are detected in lung epithelial cells of patients with chronic obstructive pulmonary disease. In investigating E1A regulation of inflammatory mediator expression in human lung epithelial cells, we found increased intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 expression after lipopolysaccharide (LPS) stimulation of A549 cells stably transfected with adenovirus 5 E1A. We now show that E1A-dependent induction of interleukin-8 expression is specific to LPS, superinduced by cycloheximide, and not observed after tumor necrosis factor or phorbol 12-myristate 13-acetate stimulation. Electrophoretic mobility shift assays revealed that tumor necrosis factor or phorbol 12-myristate 13-acetate induced nuclear factor-kappaB binding complexes of Rel A and p50 in E1A and control transfectants, whereas LPS was effective only in E1A transfectants. Similarly, LPS-induced nuclear translocation of nuclear factor-kappaB was observed only in E1A transfectants. CCAAT-enhancer binding protein binding was undetected and activator protein-1 binding was unaffected by LPS in either cell type, whereas basal mRNA levels of c-jun were unchanged by E1A. We conclude that E1A enhances the expression of these inflammatory mediator genes by modulating events specific to LPS-triggered nuclear factor-kappaB induction in these cells.

E1A Sensitizes Cells to Tumor Necrosis Factor-induced Apoptosis through Inhibition of IκB Kinases and Nuclear Factor κB Activities

The adenovirus E1A protein has been implicated in increasing cellular susceptibility to apoptosis induced by tumor necrosis factor (TNF); however, its mechanism of action is still unknown. Since activation of nuclear factor kappaB (NF-kappaB) has been shown to play an anti-apoptotic role in TNF-induced apoptosis, we examined apoptotic susceptibility and NF-kappaB activation induced by TNF in the E1A transfectants and their parental cells. Here, we reported that E1A inhibited activation of NF-kappaB and rendered cells more sensitive to TNF-induced apoptosis. We further showed that this inhibition was through suppression of IkappaB kinase (IKK) activity and IkappaB phosphorylation. Moreover, deletion of the p300 and Rb binding domains of E1A abolished its function in blocking IKK activity and IkappaB phosphorylation, suggesting that these domains are essential for the E1A function in down-regulating IKK activity and NF-kappaB signaling. However, the role of E1A in inhibiting IKK activity might be indirect. Nevertheless, our results suggest that inhibition of IKK activity by E1A is an important mechanism for the E1A-mediated sensitization of TNF-induced apoptosis.

Inhibition of Nuclear Factor-κB Activity Is Involved in E1A-mediated Sensitization of Radiation-induced Apoptosis

The adenoviral E1A protein has been implicated in the potentiation of apoptosis induced by various external stimuli, but the exact mechanism of that potentiation is not clear. In this study, we compared the sensitivity to ionizing gamma-irradiation of E1A transfectants with that of parental cells in a human ovarian cancer cell line (SKOV3.ip1); we found that the E1A transfectants became sensitive to radiation-induced apoptosis. Recently, activation of the transcription factor nuclear factor-kappaB (NF-kappaB) has been shown to play a key role in the anti-apoptotic pathway of radiation-induced apoptosis. In an attempt to determine whether NF-kappaB was involved in the E1A-mediated sensitization of radiation-induced apoptosis, we found that radiation-induced activation of NF-kappaB occurred in the parental cells but was blocked in the E1A transfectants. Furthermore, parental cells cotransfected with NF-kappaB and E1A were better protected from undergoing apoptosis upon irradiation than those transfected with E1A alone. Thus, our results suggest that inhibition of NF-kappaB activation by E1A is a plausible mechanism for E1A-mediated sensitization of radiation-induced apoptosis.

Adenovirus E1A gene dysregulates ICAM-1 expression in transformed pulmonary epithelial cells.

Previous studies from our laboratory demonstrated that adenovirus E1A DNA and proteins are detected in lungs of patients with chronic obstructive pulmonary disease (COPD). Since adenovirus E1A gene products are known to regulate the expression of many genes by interacting with cellular transcription factors, we postulate that E1A enhances the production of inflammatory mediators and exacerbates the inflammatory process in smokers' lungs. To examine this possibility, we transfected A549 human pulmonary epithelial cells with a plasmid carrying the adenoviral E1A gene and isolated stable transfectants expressing E1A proteins. These E1A-producing clones were tested for intercellular adhesion molecule-1 (ICAM-1) expression. As compared with parental cells or cells transfected with control plasmid, ICAM-1 expression was suppressed after IFN-gamma stimulation but markedly increased by LPS stimulation of E1A-positive cells. This LPS-mediated ICAM-1 induction was serum-dependent but the LPS receptor, CD14, was not detected on the surface of the E1A transfectants. We conclude that E1A proteins modulate ICAM-1 induction by inflammatory stimuli and render lung epithelial cells sensitive to LPS, and suggest that dysregulation of inflammatory mediator expression by adenoviral E1A could amplify the inflammatory process present in airways of smokers to produce COPD.

Preferential repression of the H subunit of ferritin by adenovirus E1A in NIH-3T3 mouse fibroblasts.

Ferritin is the major intracellular iron-storage protein in eucaryotic cells and plays a prominent role in maintaining intracellular iron homeostasis. We observed that transfection of NIH-3T3 mouse fibroblasts with the adenovirus E1A oncogene specifically repressed the mRNA for one of the subunits of ferritin, ferritin H. This occurred in the absence of any effect of E1A on the mRNA for the L subunit of ferritin. The repression of ferritin H was not a general feature of oncogene expression since transfection of NIH-3T3 cells with H-ras did not affect ferritin composition. Deletion of the conserved regions of E1A responsible for immortalization and transcriptional repression impaired the ability of E1A to repress ferritin H. Immunoprecipitation of ferritin in E1A transfectants demonstrated that the decrease in the ferritin H/L ratio observed at the mRNA level was also exhibited at the protein level. The E1A-dependent inhibition of ferritin H was also observed in a chimeric gene containing the ferritin H promoter ligated to the chloramphenicol acetyltransferase reporter gene, but was not observed in control genes in which chloramphenicol acetyltransferase activity was dependent on promoters derived from SV40 or the interleukin-3 gene. This suggests that E1A may repress ferritin H at the transcriptional level. These results demonstrate that the adenovirus E1A oncogene specifically modulates ferritin H expression. They also suggest that alterations in cellular iron metabolism may be among the diverse array of cellular responses induced by E1A.

Malignant conversion of murine squamous papilloma cell lines by transfection with the fos oncogene

Murine papilloma cell lines 308 and SP-1 have been used as recipients for transfected oncogenes to investigate malignant conversion. These cell lines express an activated c-rasHa gene with a codon 61 mutation and produce squamous papillomas when transplanted as skin grafts onto nude mice. They are not tumorigenic by subcutaneous injection. Both papilloma cell lines were stably transfected with plasmid DNA containing either a rearranged murine plasmacytoma-derived c-myc (minus exon 1), adenovirus 5 E1A, FBJ v-fos or a human c-fos/FBJ v-fos chimera, using cotransfection with the neomycin resistance gene contained in pSV2neo to select for transformants. Southern and northern blotting analysis confirmed the uptake and expression of exogenous DNA in both G418-selected cell lines and in the derived tumors. Unlike the E1A- and myc-containing plasmids, both fos constructs caused malignant conversion in either cell line, as defined by the squamous cell carcinoma histology of tumors from grafted cells and the development of carcinomas after subcutaneous injection into athymic nude mice. Immunofluorescence analysis for specific keratin gene expression indicated that tumors derived by introduction of either of the fos oncogenes were devoid of staining for K1, a 67 kDa epidermal keratin that is expressed in papillomas but not in squamous carcinomas. Tumors from E1A, myc, or pSV2neo transfectants expressed K1, although in a focal distribution. The malignant phenotype induced by the fos oncogene constructs was not associated with the ability to form agar colonies in vitro or to express gamma-glutamyl transpeptidase in the tumors. Since both 308 and SP-1 were sensitive to the fos oncogene for malignant conversion and insensitive to E1A or myc, it is possible that fos may cooperate with the endogenous-activated c-rasHa gene to convert these cells to malignancy. However, since gamma-glutamyl transpeptidase activity is found in the majority of chemically induced mouse skin carcinomas that possess an activated c-rasHa gene, fos activation may not be a common pathway for spontaneous malignant conversion.