Absent in melanoma 2 enhances anti-tumour effects of CAIX promotor controlled conditionally replicative adenovirus in renal cancer.

Conditionally replicative adenoviruses (CRAds) were promising approach for solid tumour treatment, but its oncolytic efficiency and toxicity are still not satisfactory for further clinical application. Here, we developed the CAIX promotor (CAIXpromotor )-controlled CRAd armed with a tumour suppressor absent in melanoma 2 (AIM2) to enhance its oncolytic potency. The CAIXpromotor -AIM2 adenoviruses (Ad-CAIXpromotor -AIM2) could efficiently express E1A and AIM2 in renal cancer cells. Compared with Ad-CAIXpromotor , Ad-CAIXpromotor -AIM2 significantly inhibited cell proliferation and enhanced cell apoptosis and cell killing, thus resulting in the oncolytic efficiency in 786-O cells or OSRC-2 cells. To explore the therapeutic effect, various Ads were intratumourally injected into OSRC-2-xenograft mice. The tumour growth was remarkably inhibited in Ad-CAIXpromotor -AIM2-treated group as demonstrated by reduced tumour volume and weight with a low toxicity. The inflammasome inhibitor YVAD-CMK resulted in the reduction of anti-tumour activity by Ad-CAIXpromotor -AIM2 in vitro or in vivo, suggesting that inflammasome activation response was required for the enhanced therapeutic efficiency. Furthermore, lung metastasis of renal cancer mice was also suppressed by Ad-CAIXpromotor -AIM2 treatment accompanied by the decreased tumour fossil in lung tissues. These results indicated that the tumour-specific Ad-CAIXpromotor -AIM2 could be applied for human renal cancer therapy. The therapeutic strategy of AIM2-based CRAds could be a potential and promising approach for the therapy of primary solid or metastasis tumours.

ACLY and ACC1 Regulate Hypoxia-Induced Apoptosis by Modulating ETV4 via α-ketoglutarate.

In order to propagate a solid tumor, cancer cells must adapt to and survive under various tumor microenvironment (TME) stresses, such as hypoxia or lactic acidosis. To systematically identify genes that modulate cancer cell survival under stresses, we performed genome-wide shRNA screens under hypoxia or lactic acidosis. We discovered that genetic depletion of acetyl-CoA carboxylase (ACACA or ACC1) or ATP citrate lyase (ACLY) protected cancer cells from hypoxia-induced apoptosis. Additionally, the loss of ACLY or ACC1 reduced levels and activities of the oncogenic transcription factor ETV4. Silencing ETV4 also protected cells from hypoxia-induced apoptosis and led to remarkably similar transcriptional responses as with silenced ACLY or ACC1, including an anti-apoptotic program. Metabolomic analysis found that while α-ketoglutarate levels decrease under hypoxia in control cells, α-ketoglutarate is paradoxically increased under hypoxia when ACC1 or ACLY are depleted. Supplementation with α-ketoglutarate rescued the hypoxia-induced apoptosis and recapitulated the decreased expression and activity of ETV4, likely via an epigenetic mechanism. Therefore, ACC1 and ACLY regulate the levels of ETV4 under hypoxia via increased α-ketoglutarate. These results reveal that the ACC1/ACLY-α-ketoglutarate-ETV4 axis is a novel means by which metabolic states regulate transcriptional output for life vs. death decisions under hypoxia. Since many lipogenic inhibitors are under investigation as cancer therapeutics, our findings suggest that the use of these inhibitors will need to be carefully considered with respect to oncogenic drivers, tumor hypoxia, progression and dormancy. More broadly, our screen provides a framework for studying additional tumor cell stress-adaption mechanisms in the future.

The utility of ETV1, ETV4 and ETV5 RNA in-situ hybridization in the diagnosis of CIC-DUX sarcomas.

AIMS: A recently characterized group of undifferentiated small round cell sarcomas harbours fusions of the genes CIC and DUX4. Studies report a distinctive gene expression profile for these sarcomas, including expression of E26 transformation-specific (ETS) family proto-oncogenic transcription factors ETV1, ETV4 and ETV5. To test the utility of an ancillary diagnostic technique for these tumours, we evaluated chromogenic RNA in-situ hybridization assays for ETV1, ETV4 and ETV5 as diagnostic adjuncts for this emerging group of highly malignant sarcomas. METHODS AND RESULTS: We tested six confirmed CIC-DUX4 sarcomas and 105 lesions in the differential, including 48 Ewing sarcomas for expression of ETV1, ETV4 and ETV5, scoring expression utilizing a previously validated scale. ETV1 and ETV4 were positive in five of six cases, while ETV5 was positive in six of six. No Ewing sarcoma or other sarcoma tested showed coexpression of these transcripts, while one ETV1/ETV4/ETV5 triple positive previously unclassified round cell sarcoma was identified as harbouring a CIC rearrangement by break-apart fluorescence in-situ hybridization (FISH). CONCLUSION: We identified overexpression of ETV1, ETV4 and ETV5 transcripts in situ in CIC-DUX4 sarcomas using a robust assay in routine archival sections. One previously unclassified round cell sarcoma showed ETV1/4/5 positivity, and was proved to harbour a CIC rearrangement by break-apart FISH. The sensitivity and specificity observed with our in-situ hybridization assay implies potential utility as an ancillary diagnostic technique, particularly when faced with limited biopsy samples.

ETV4 is a useful marker for the diagnosis of CIC-rearranged undifferentiated round-cell sarcomas: a study of 127 cases including mimicking lesions.

Subsets of primitive round-cell sarcomas remain difficult to diagnose and classify. Among these is a rare round-cell sarcoma that harbors a CIC gene rearrangement known as CIC-rearranged undifferentiated round-cell sarcoma, which is most commonly fused to the DUX4 gene. Owing to its aggressive clinical behavior and potential therapeutic implications, accurate identification of this novel soft tissue sarcoma is necessary. Definitive diagnosis requires molecular confirmation, but only a few centers are as yet able to perform this test. Several studies have shown that PEA3 subfamily genes, notably ETV4 (belonging to the family of ETS transcription factors), are upregulated in CIC-rearranged undifferentiated round-cell sarcomas. We performed a detailed immunohistochemical analysis to investigate ETV4 expression in CIC-rearranged undifferentiated round-cell sarcomas and their potential mimics (especially Ewing sarcomas). The study cohort included 17 cases of CIC-rearranged undifferentiated round-cell sarcomas, and 110 tumors that morphologically mimic CIC-rearranged undifferentiated round-cell sarcomas: 43 Ewing sarcomas, 25 alveolar rhabdomyosarcomas, 20 poorly differentiated round-cell synovial sarcomas, 10 desmoplastic round-cell tumors, 5 BCOR-CCNB3 sarcomas, 5 lymphoblastic lymphomas, and 2 rhabdoid tumors. All CIC-rearranged undifferentiated round-cell sarcomas (on core needle biopsies and open biopsies) were ETV4-positive with a strong diffuse nuclear pattern. Among the other 110 tumors, only six cases (four Ewing sarcomas, one alveolar rhabdomyosarcoma, and one desmoplastic round-cell tumor) showed focal (<5% of tumor cells) and very weak nuclear expression of ETV4; all other tumors were completely negative for ETV4. We conclude that systematic immunohistochemical analysis of ETV4 makes it possible to diagnose undifferentiated round-cell sarcomas (with no molecular markers for sarcoma-associated translocation) such as CIC-rearranged undifferentiated round-cell sarcoma.

Evaluation of ETV4 and WT1 expression in CIC-rearranged sarcomas and histologic mimics.

A distinct subset of round cell sarcomas harbors capicua transcriptional repressor (CIC) rearrangement. Diagnosing these sarcomas can be difficult owing to their resemblance to Ewing sarcoma and other 'small round blue cell tumors'; molecular techniques are generally required. Recent gene expression studies of CIC-rearranged sarcomas identified the upregulation of ETV4. We assessed the sensitivity and specificity of ETV4 and WT1 immunohistochemistry for CIC-rearranged sarcoma. We evaluated whole-tissue sections from 40 CIC-rearranged sarcomas, 40 Ewing sarcomas, 4 BCOR-CCNB3 sarcomas, 6 unclassified round cell sarcomas, and 150 histologic mimics. Moderate-to-strong nuclear immunoreactivity for ETV4 in at least 50% of cells was observed in 36 (90%) CIC-rearranged sarcomas and 10 (5%) other tumors, including 5 unclassified round cell sarcomas, 2 Wilms tumors, and 1 each desmoplastic small round cell tumor, melanoma, and small cell carcinoma. Thirty-eight (95%) CIC-rearranged sarcomas showed nuclear staining for WT1, and 34 (85%) were positive for both ETV4 and WT1. Of 182 other tumors evaluated, 34 (19%) showed nuclear WT1 positivity, including all Wilms tumors and desmoplastic small round cell tumors, 5 unclassified round cell sarcomas, and a subset of lymphoblastic lymphomas, rhabdomyosarcomas, mesenchymal chondrosarcomas, carcinomas, and melanomas. In summary, diffuse moderate-to-strong ETV4 expression is present in most CIC-rearranged sarcomas and unclassified round cell sarcomas. More limited expression is seen in small subsets of various other round cell neoplasms. Nuclear WT1 expression is also present in most CIC-rearranged sarcomas and unclassified round cell sarcomas, along with Wilms tumors and desmoplastic small round cell tumors, and subsets of various histologic mimics. The sensitivity and specificity of diffuse ETV4 expression for CIC-rearranged sarcomas are 90% and 95%, respectively, whereas the sensitivity and specificity of WT1 are 95% and 81%, respectively. Diffuse ETV4 along with at least focal WT1 expression is helpful to distinguish CIC-rearranged sarcoma from Ewing sarcoma and other histologic mimics.

Overexpression of ETV4 is associated with poor prognosis in prostate cancer: involvement of uPA/uPAR and MMPs.

ETS gene fusions involving ERG, ETV1, ETV4, ETV5, and FLI1 define a distinct class of prostate cancer (PCa), and this might have a bearing on diagnosis, prognosis, and rational therapeutic targeting. In the current study, we focused on the clinicopathological significance of ETV4 in Chinese PCa patients and the mechanisms whereby ETV4 overexpression mediates tumor invasion in the prostate. Overall, ETV4 overexpression was identified in 30.4 % (45/148) of PCa cases by immunohistochemistry. Accordingly, ETV4 was rearranged in only 1.6 % (2/128) of PCa patients. Clinically, ETV4 overexpression was significantly correlated with Gleason score (P = 0.045) and pathological tumor stage (P = 0.041). Multivariate Cox regression analysis indicated that ETV4 is an unfavorable independent prognostic factor (P = 0.040). Functional studies further showed that small interfering RNA (siRNA) knockdown of ETV4 significantly decreases proliferation and invasion of PC-3 cell and partially reverses epithelial-mesenchymal transition in vitro. Notably, ETV4 knockdown significantly downregulated expression of urokinase plasminogen activator (uPA) and its receptor (uPAR) at messenger RNA (mRNA) and protein levels. Chromatin immunoprecipitation assay demonstrated that ETV4 regulates uPA expression through direct binding to its promoter region. Additionally, ETV4 knockdown was also observed to significantly inhibit expression of matrix metalloproteinase (MMP)-2 and MMP-9. In conclusion, for the first time, our study suggested that ETV4 is an independent poor prognostic factor in Chinese PCa patients. Silencing of ETV4 suppresses invasion of PCa cells by inhibiting the expression of uPA/uPAR as well as MMPs. Further studies will be needed to determine whether ETV4 could be regarded as a potential target for the management and prevention of PCa.

Experimental studies on treatment of pancreatic cancer with double-regulated duplicative adenovirus AdTPHre-hEndo carrying human endostatin gene.

BACKGROUND: Gene-virus targeted therapy is a promising new method of treating pancreatic cancer. To increase the efficacy and decrease the side-effect, we constructed a conditionally replicative adenovirus (CRAd) expressing human endostatin, with a human Telomoerase Reverse Transcriptase (hTERT) promoter for the regulation of the early stage of adenovirus expression of gene E1a and a Hypoxia Response Element (HRE) promoter to regulate the gene E1b. METHODS: A gene recombination technique was adopted to construct and generate the adenovirus AdTPHre-hEndo. Pancreatic cancer cells were studied both in vitro and in vivo. Western blotting was adopted to observe the expressions of protein E1A and E1B; duplication assay was applied to observe the selective duplication capability of recombinant cells. MTT assay was applied to measure the lethal effects of virus on pancreatic cancer cells, and ELISA was adopted to detect the human endostatin gene expression. A pancreatic cancer transplantation tumor model of nude mice was constructed to observe the antitumor effects of the virus. RESULTS: Double-regulated duplicative adenovirus AdTPHre-hEndo genes were successfully constructed. Duplication and lethal assays proved that AdTPHre-hEndo could replicate specifically in pancreatic cancer cells and kill them. The endostatin expression in a cultured supernatant from tumor cells was significantly higher than that obtained from non-duplicative adenovirus vectors carrying that gene. The animal experiment demonstrated that AdTPHre-hEndo has a high capability to limit pancreatic cancer growth. CONCLUSIONS: AdTPHre-hEndo has a special ability to duplicate and kill pancreatic cancer cells in in vitro and in vivo experiments, thus providing a new gene-virus-based treatment system for pancreatic cancer.

Mechanistic aspects of adenovirus serotype 2 inactivation with free chlorine.

Free chlorine is an effective disinfectant for controlling adenoviruses in drinking water, but little is known about the underlying inactivation mechanisms. The objective of this study was to elucidate the molecular components of adenovirus type 2 (Ad2) targeted by free chlorine during the inactivation process. The effects of free chlorine treatment on several Ad2 molecular components and associated life cycle events were compared to its effect on the ability of adenovirus to complete its life cycle, i.e., viability. Free chlorine treatment of Ad2 virions did not impair their ability to interact with monoclonal antibodies specific for hexon and fiber proteins of the Ad2 capsid, as measured by enzyme-linked immunosorbent assays, nor did it impair their interaction with recombinant, purified Coxsackie-adenovirus receptor (CAR) proteins in vitro. Free chlorine-treated Ad2 virions also retained their ability to bind to CAR receptors on A549 cell monolayers, despite being unable to form plaques, suggesting that free chlorine inactivates Ad2 by inhibiting a postbinding event of the Ad2 life cycle. DNA isolated from Ad2 virions that had been inactivated by free chlorine was able to be amplified by PCR, indicating that genome damage was not the cause of inactivation. However, inactivated Ad2 virions were unable to express E1A viral proteins during infection of A549 host cells, as measured by using immunoblotting. Collectively, these results indicate that free chlorine inactivates adenovirus by damaging proteins that govern life cycle processes occurring after host cell attachment, such as endocytosis, endosomal lysis, or nuclear delivery.

A modified E2F-1 promoter improves the efficacy to toxicity ratio of oncolytic adenoviruses.

The E2F-1 promoter has been used to confer tumor-selective E1A expression in oncolytic adenoviruses. Tumor specificity is mainly conferred by a unique structure of E2F-responsive sites organized in palindromes. Binding of the E2F-pRb complex to these palindromes results in repression of transcription in normal cells. Owing to deregulation of the Rb/p16 pathway in tumor cells, binding of free E2F activates transcription and initiates an autoactivation loop involving E1A and E4-6/7. ICOVIR-7 is a new oncolytic adenovirus designed to increase the E2F dependency of E1A gene expression. It incorporates additional palindromes of E2F-responsive sites in an insulated E2F-1 promoter controlling E1A-Delta24. The E2F palindromes inhibited replication in normal cells, resulting in a low systemic toxicity at high doses in immunocompetent mice. The Delta24 deletion avoids a loop of E2F-mediated self-activation in nontumor cells. Importantly, the additional E2F-binding hairpins boost the positive feedback loop on the basis of E1A-mediated transcriptional regulation of E4-6/7 turned on in cancer cells and increased antitumoral potency as shown in murine subcutaneous xenograft models treated by intravenous injection. These results suggest that the unique genetic combination featured in ICOVIR-7 may be promising for treating disseminated neoplasias.

Construction of an MUC-1 promoter driven, conditionally replicating adenovirus that expresses the sodium iodide symporter for gene therapy of breast cancer.

INTRODUCTION: The sodium iodide symporter (NIS) directs the uptake and concentration of iodide in thyroid cells. This in turn allows radioiodine imaging and therapy for thyroid cancer. To extend the use of NIS-mediated radioiodine therapy to other types of cancer, we successfully transferred and expressed the sodium-iodide symporter (NIS) gene in prostate, colon, and breast cancer cells both in vivo and in vitro by using non-replicating adenoviral vectors. METHODS: To improve virotherapy efficiency, we developed a conditionally replicating adenovirus (CRAd) in which the transcriptional cassette RSV promoter-human NIScDNA-bGH polyA was also inserted at the E3 region. The E1a gene is driven by the tumor-specific promoter MUC-1 in the CRAd Ad5AMUCH_RSV-NIS. RESULTS: In vitro infection of the MUC-1-positive breast cell line T47D resulted in virus replication, cytolysis, and release of infective viral particles. Conversely, the MUC-1-negative breast cancer cell line MDA-MB-231 was refractory to the viral cytopathic effect and did not support viral replication. The data indicate that Ad5AMUCH_RSV-NIS activity is stringently restricted to MUC-1-positive cancer cells. Radioiodine uptake was readily measurable in T47 cells infected with Ad5AMUCH_RSV-NIS 24 hours after infection, thus confirming NIS expression before viral-induced cell death. CONCLUSIONS: This construct may allow multimodal therapy, combining virotherapy with radioiodine therapy to be developed as a novel treatment for breast and other MUC1-overexpressing cancers.

E1a induces the expression of epithelial characteristics.

Cells closely resembling epithelia constitute the first specific cell type in a mammalian embryo. Many other cell types emerge via epithelial-mesenchymal differentiation. The transcription factors and signal transduction pathways involved in this differentiation are being elucidated. I have previously reported (Frisch, 1991) that adenovirus E1a is a tumor suppressor gene in certain human cell lines. In the present report, I demonstrate that E1a expression caused diverse human tumor cells (rhabdomyosarcoma, fibrosarcoma, melanoma, osteosarcoma) and fibroblasts to assume at least two of the following epithelial characteristics: (a) epithelioid morphology; (b) epithelial-type intercellular adhesion proteins localized to newly formed junctional complexes; (c) keratin-containing intermediate filaments; and (d) down-regulation of non-epithelial genes. E1a thus appeared to partially convert diverse human tumor cells into an epithelial phenotype. This provides a new system for molecular analysis of epithelial-mesenchymal interconversions. This effect may also contribute to E1a's tumor suppression activity, possibly through sensitization to anoikis (Frisch, S.M., and H. Francis, 1994. J. Cell Biol. 124:619-626).

Viral proteins E1B19K and p35 protect sympathetic neurons from cell death induced by NGF deprivation.

To study molecular mechanisms underlying neuronal cell death, we have used sympathetic neurons from superior cervical ganglia which undergo programmed cell death when deprived of nerve growth factor. These neurons have been microinjected with expression vectors containing cDNAs encoding selected proteins to test their regulatory influence over cell death. Using this procedure, we have shown previously that sympathetic neurons can be protected from NGF deprivation by the protooncogene Bcl-2. We now report that the E1B19K protein from adenovirus and the p35 protein from baculovirus also rescue neurons. Other adenoviral proteins, E1A and E1B55K, have no effect on neuronal survival. E1B55K, known to block apoptosis mediated by p53 in proliferative cells, failed to rescue sympathetic neurons suggesting that p53 is not involved in neuronal death induced by NGF deprivation. E1B19K and p35 were also coinjected with Bcl-Xs which blocks Bcl-2 function in lymphoid cells. Although Bcl-Xs blocked the ability of Bcl-2 to rescue neurons, it had no effect on survival that was dependent upon expression of E1B19K or p35.

Retargeting improves the efficacy of a telomerase-dependent oncolytic adenovirus for head and neck cancer.

The relative paucity of receptors for the commonly used adenovirus serotype 5 in many cancer types undermines the efficacy of Ad5-based approach for cancer gene therapy. We have previously shown that coxsackie-adenovirus receptor (CAR) is expressed at decreased levels in several primary head and neck squamous cell carcinoma (HNSCC) cell lines established from tumor samples and that retargeting adenoviral infection to the CD46 receptor using the Ad5/35 hybrid virus results in highly efficient transfection of these cells. We sought to examine the effect of this retargeting in the context of the conditionally replicating adenovirus (CRAD) approach. By subjecting the viral E1A gene under the regulation of human telomerase reverse transcriptase promoter we produced Ad5/35-TERT, a transductionally targeted CRAD. The anti-tumor efficacy of this virus was tested in two primary HNSCC cell lines, chosen to represent high (55.2%) and low (3.2%) CAR expression levels. In vitro experiments demonstrated that Ad5/35-TERT is significantly more effective in killing primary HNSCC cells than the non-targeted Ad5-TERT. The difference between the two viruses was clearly more pronounced in HNSCC cells with low CAR expression. In an in vivo experiment using a subcutaneous HNSCC mouse model Ad5/35-TERT was more effective than Ad5-TERT in both high- and low-CAR HNSCC cells. These results demonstrate that enhanced transfection by hybrid virus strategy results in an increased anti-tumor efficacy when using CRADs. The effect is especially distinctive in target cells with low CAR expression.

Modulation of telomerase promoter tumor selectivity in the context of oncolytic adenoviruses.

The telomerase RNA (hTR) and reverse transcriptase (hTERT) promoters are active in most cancer cells, but not in normal cells, and are useful for transcriptional targeting in gene therapy models. Telomerase-specific conditionally replicating adenoviruses (CRAd) are attractive vectors because they should selectively lyse tumor cells. Here, we compare CRAds, in which either the hTR or hTERT promoter controls expression of the adenovirus E1A gene. In replication-defective reporter adenoviruses, the hTR promoter was up to 57-fold stronger in cancer cells than normal cells and up to 49-fold stronger than hTERT. In normal cells, hTERT promoter activity was essentially absent. Doses of telomerase-specific CRAds between 1.8 and 28 infectious units per cell efficiently killed cancer cells, but normal cells required higher doses. However, CRAd DNA replication and E1A expression were detected in both cancer and normal cells. Overall, tumor specificity of the CRAds was limited compared with nonreplicating vectors. Surprisingly, both CRAds expressed similar E1A levels and functional behavior, despite known differentials between hTR and hTERT promoter activities, suggesting that the promoters are deregulated. Rapid amplification of cDNA ends analysis of hTR-/hTERT-E1A transcripts ruled out cryptic transcription from the vector backbone. Blocking E1A translation partially restored the hTR-/hTERT-E1A mRNA differential, evidencing feedback regulation by E1A.

Eradication of therapy-resistant human prostate tumors using a cancer terminator virus.

Terminal prostate cancer is refractory to conventional anticancer treatments because of frequent overexpression of antiapoptotic proteins Bcl-2 and/or Bcl-x(L). Adenovirus-mediated delivery of melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), a secreted cytokine having cancer-selective apoptosis-inducing properties, profoundly inhibits prostate cancer cell growth. However, forced overexpression of Bcl-2 or Bcl-x(L) renders prostate cancer cells resistant to Ad.mda-7. We constructed a conditionally replication-competent adenovirus in which expression of the adenoviral E1A gene, necessary for replication, is driven by the cancer-specific promoter of progression elevated gene-3 (PEG-3) and which simultaneously expresses mda-7/IL-24 in the E3 region of the adenovirus (Ad.PEG-E1A-mda-7), a cancer terminator virus (CTV). This CTV generates large quantities of MDA-7/IL-24 as a function of adenovirus replication uniquely in cancer cells. Infection of Ad.PEG-E1A-mda-7 (CTV) in normal prostate epithelial cells and parental and Bcl-2- or Bcl-x(L)-overexpressing prostate cancer cells confirmed cancer cell-selective adenoviral replication, mda-7/IL-24 expression, growth inhibition, and apoptosis induction. Injecting Ad.PEG-E1A-mda-7 (CTV) into xenografts derived from DU-145-Bcl-x(L) cells in athymic nude mice completely eradicated not only primary tumors but also distant tumors (established in the opposite flank), thereby implementing a cure. These provocative findings advocate potential therapeutic applications of this novel virus for advanced prostate cancer patients with metastatic disease.

[Effect of adenovirus-E1A gene therapy on in vivo radiosensitivity to nasopharyngeal cancer].

OBJECTIVE: To determine the effect of Ad-E1A gene therapy on in vivo radiosensitivity to nasopharyngeal carcinoma. METHODS: CNE-2Z cells (2 x 10(5)) were subcutaneously injected into nude mice to develop tumor (1-3 mm) 6 days later. The tumor-bearing mice were then randomly divided into 6 groups (10 mice per group) for PBS treatment or treatment with radiotherapy, Ad-E1A, or Ad-beta-gal alone or radiotherapy in combination with Ad-E1A or Ad-beta-gal. The mice were treated with Ad-E1A or Ad-beta-gal (5 x 10(9) plaque forming units) by intratumoral injection twice weekly for 2 weeks at beginning of week 2. The mice treated with radiotherapy in combination with Ad-E1A or Ad-beta-gal received 2 Gy radiotherapy daily for 5 days following the first week of treatment with Ad-E1A or Ad-beta-gal. Control mice received PBS therapy or radiotherapy only after tumor cells were injected. When the size of tumor exceeded 2 cm, the mice were killed and the tumors underwent immunohistochemical analysis for VEGF and CD34 expression and TUNEL assay for apoptosis. RESULTS: The growth delay time was longest in the Ad-E1A plus radiotherapy group. Tumors treated with Ad-E1A plus radiotherapy were 4.7-fold smaller than those treated with radiotherapy alone and 5.3-fold smaller than those treated with Ad-E1A alone. The survival rate of tumor-bearing mice treated with Ad-E1A plus radiotherapy was significantly higher than that of other treatment groups. The vessel density and the VEGF expression were significantly lower in tumors treated with Ad-E1A plus radiotherapy than those treated with radiotherapy alone, Ad-E1A alone, Ad-beta-gal alone, or Ad-beta-gal plus radiotherapy (P<0.01). TUNEL staining revealing apoptosis can be detected in the Ad-E1A group, radiotherapy group, Ad-E1A plus radiotherapy group, and more apoptosis can be detected in tumors treated with Ad-E1A plus radiotherapy than those of other treatment groups. CONCLUSION: E1A gene therapy can effectively enhance the nasopharyngeal carcinoma sensitivity to the radiotherapy by down-regulating VEGF expression and inducing apoptosis.

Anisomycin abrogates repression of protooncogene c-fos transcription in E1A + cHa-ras-transformed cells through activation of MEK/ERK kinase cascade.

We have previously shown that transcription of immediate-early c-fos protooncogene is becoming strongly repressed in rat embryo fibroblasts transformed by oncogenes E1A and cHa-ras, so that serum only slightly stimulated c-fos transcription in these cells in contrast to high level of c-fos activation in non-transformed REF52 cells. Here we showed that stress-inducing agent anisomycin was able to override the c-fos repression and to induce c-fos transcription in E1A + ras transformants. In vitro kinase assay data demonstrated that anisomycin increased phosphorylation of transactivation domain of Elk-1 transcription factor--a key regulator of inducible c-fos transcription. Importantly, this activation was mediated through up-regulation of MEK/ERK but not stress-kinase cascades JNK or p38. The activating effect of anisomycin on c-fos transcription could be abrogated by a prior treatment with N-acetyl-L-cysteine. This indicates that anisomycin potentiates generation of reactive oxygen species (ROS), which, in turn, can modulate the activity of MAP kinase-specific phosphatases (MKPs). As anisomycin did not cause acetylation of nucleosome core histones, the present work focuses on the molecular mechanisms mediating the HDAC-independent induction of IEG c-fos by anisomycin in E1A + cHa-ras-transformed fibroblasts.

Effect of exposure to UV-C irradiation and monochloramine on adenovirus serotype 2 early protein expression and DNA replication.

The mechanisms of adenovirus serotype 2 inactivation with either UV light (with a narrow emission spectrum centered at 254 nm) or monochloramine were investigated by assessing the potential inhibition of two key steps of the adenovirus life cycle, namely, E1A protein synthesis and viral genomic replication. E1A early protein synthesis was assayed by using immunoblotting, while the replication of viral DNA was analyzed by using slot blotting. Disinfection experiments were performed in phosphate buffer solutions at pH 8 and room temperature (UV) or 20 degrees C (monochloramine). Experimental results revealed that normalized E1A levels at 12 h postinfection (p.i.) were statistically the same as the corresponding decrease in survival ratio for both UV and monochloramine disinfection. Normalized DNA levels at 24 h p.i. were also found to be statistically the same as the corresponding decrease in survival ratio for monochloramine disinfection. In contrast, for UV disinfection, genomic DNA levels were much lower than E1A or survival ratios, possibly as a result of a delay in DNA replication for UV-treated virions compared to that for controls. Future efforts will determine the pre-E1A synthesis step in the adenovirus life cycle affected by exposure to UV and monochloramine, with the goal of identifying the viral molecular target of these two disinfectants.

Expression of E1AF, an ets-oncogene transcription factor, highly correlates with malignant phenotype of malignant melanoma through up-regulation of the membrane-type-1 matrix metalloproteinase gene.

Matrix metalloproteinase (MMP) is closely involved in the degradation of extracellular matrix and confers invasive and metastatic potential to malignant tumors. MMP-2 is a type-IV collagenase secreted as a proenzyme that is activated on the surface of the tumor cell by membrane-type 1-MMP (MT1-MMP). MT1-MMP plays a critical role during tumor progression and metastasis. We investigated the expression levels of E1AF and MT1-MMP in malignant melanoma cell lines and specimens from patients in order to clarify the mechanisms responsible for the invasion and metastasis of malignant melanoma. High levels of E1AF and MT1-MMP mRNA expression were observed in melanoma cells by Northern blotting and real-time PCR. The expression level was highly correlated with an invasive potential determined by an in vitro invasion assay. The down-regulation of MT1-MMP was identified when E1AF was knocked down by RNA interference. These results suggest that E1AF plays a crucial role in the invasion and metastasis of malignant melanoma through up-regulating the MT1-MMP expression.

TMPRSS2:ETV4 gene fusions define a third molecular subtype of prostate cancer.

Although common in hematologic and mesenchymal malignancies, recurrent gene fusions have not been well characterized in epithelial carcinomas. Recently, using a novel bioinformatic approach, we identified recurrent gene fusions between TMPRSS2 and the ETS family members ERG or ETV1 in the majority of prostate cancers. Here, we interrogated the expression of all ETS family members in prostate cancer profiling studies and identified marked overexpression of ETV4 in 2 of 98 cases. In one such case, we confirmed the overexpression of ETV4 using quantitative PCR, and by rapid amplification of cDNA ends, quantitative PCR, and fluorescence in situ hybridization, we show that the TMPRSS2 (21q22) and ETV4 (17q21) loci are fused in this case. This result defines a third molecular subtype of prostate cancer and supports the hypothesis that dysregulation of ETS family members through fusions with TMRPSS2 may be an initiating event in prostate cancer development.