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Endostatin (ES, ENDO) has been reported to suppress the growth of tumors while inducing the proliferation of lung cancer stem cells (LCSCs), causing a poor prognosis for lung cancer. In this study, we aimed to clarify whether BRM270 can inhibit the proliferation of cancer stem cells (CSCs). Endostatin + BRM270 showed anti-tumor effects by reducing tumor volume and increasing survival. Administration of BRM270 reduced the number of aldehyde dehydrogenase-positive (ALDH+) cells and the level of ALDH1A1 expression in tumors by increasing the level of miR-128 while decreasing the levels of BMI-1, ABCC-5, E2F3, and c-MET. The luciferase activity of miR-128 promoter was increased by an increasing concentration of BRM270. In addition, BMI-1, ABCC-5, E2F3, and c-MET were identified as candidate targets of miR-128, and the overexpression of miR-128 significantly reduced mRNA/protein levels of BMI-1, ABCC-5, E2F3, and c-MET in A549 and H460 cells. Administration of BRM270 inhibited the expression of BMI-1, ABCC-5, E2F3, and c-MET in a dose-dependent manner. In this study, we showed for the first time that the combined administration of endostatin and BRM270 achieved anti-tumor effects while suppressing the proliferation of stem cells., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Objective: To explore the role of lung dendritic cells (DCs) and Th17/regulatory T cells (Treg) pathway in the pathogenesis of chronic obstructive pulmonary disease(COPD). Methods: COPD patients who received lobectomy from Sep. 2015 to Mar. 2016 in our hospital were enrolled and classified into non-smoking non-COPD group, smoking without COPD group and COPD group. The expression of CD(80), chemokine recepter-6 (CCR6), interleukin-17A (IL-17A) and fork-head transcription factor P3 (FoxP3) were detected by immunohistochemistry (IHC) in lung tissue. Mature DCs (mDCs), immature DCs (imDCs), Th17 cells and Treg cells in lung tissue were detected by flow cytometry (FCM) and the correlation between Th17/Treg cells with lung function was analyzed. Results: (1) The expression of CD(80) and FoxP3 in COPD group was decreased, while the expression of CCR6 and IL-17A was increased ( P< 0.05). (2) The percentage of mDCs and Treg in lung tissue of COPD group was significantly decreased. In contrast, the proportion of imDCs and Th17 cells in COPD group was significantly increased ( P< 0.05). (3) The imbalance of Th17/Treg ratio in lung tissue was seen in patients with COPD, suggesting the potential mechanism of Th17 cell-mediated proinflammatory response. (4) The percentage of Th17 cells and Th17/Treg ratio in COPD patients was negatively correlated with forced expiratory volume in the first second (FEV(1)) as a percentage of predicted value (FEV(1)% pred), forced vital capacity(FVC) as a percentage of predicted value (FVC% pred), FEV(1)/FVC. On the other hand, the percentage of Treg cells was positively correlated with FEV(1)% pred, FVC% pred, FEV(1)/FVC. Conclusions: The data in this study demonstrate the maturation disorder of dendritic cells in lung tissue of COPD patients. The imbalance of Th17/Treg ratio suggests that Th17 cell-mediated proinflammatory response may be involved in the pathogenesis of COPD.

Autophagy is a process of cell self-renewal that is dependent on the degradation of the cytoplasmic proteins or organelles of lysosomes. Many diseases, such as metabolic diseases, cancer, neurodegenerative diseases, and lung diseases, have been confirmed to be associated with elevated or impaired levels of autophagy. At present, studies have found that autophagy participates in the regulation of chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, pulmonary hypertension, acute lung injury, lung cancer, and other pulmonary diseases. Using recent literature on the signal transduction mechanisms of autophagy and the effects of autophagy signalling on lung diseases, this review intends to clarify the mechanisms of lung disease to guide the treatment of related diseases. The reviews of this paper are available via the supplemental material section.

Oncolytic adenoviruses (Ads) are cancer selective tumoricidal agents; however their mechanism of Ad-mediated cancer cell lysis, or oncolysis, remains undefined. This report focuses upon the autophagy mediator c-JUN n-terminal kinase (JNK) and its effects upon Ad oncolysis and replication. Previously, E1b-deleted Ads have been used to treat several hundred cancer patients with limited clinical efficacy. We hypothesize that by studying the potential interactions between E1b and JNK, mechanisms to improve oncolytic Ad design and cancer therapeutic efficacy may be elucidated. To test this hypothesis, E1b was selectively deleted from the Ad genome. These studies indicated that Ads encoding E1b induced JNK phosphorylation predominately occurred via E1b-19K. The expression of another crucial Ad gene E1a was then overexpressed by the CMV promoter via the replication competent Ad vector Adhz69; these data indicated that E1A also induced JNK phosphorylation. To assess the effects of host cell JNK expression upon Ad oncolysis and replication, siRNA targeting JNK1 and JNK2 (JNK1/2) were utilized. The oncolysis and replication of the E1b-19K wild-type Ads Ad5 and Adhz63 were significantly attenuated following JNK1/2 siRNA transfection. However the oncolytic effects and replication of the E1b-19K deleted Ad Adhz60 were not altered by JNK1/2 siRNA transfection, further implicating the crucial role of E1b-19K for Ad oncolysis and replication via JNK phosphorylation. This study has demonstrated for the first time that JNK is an intriguing molecular marker associated with enhanced Ad virotherapy efficacy, influencing future Ad vector design.

Adenoviruses (Ads) have been extensively manipulated for the development of cancer selective replication, leading to cancer cell death or oncolysis. Clinical studies using E1 -modified oncolytic Ads have shown that this therapeutic platform was safe, but with limited efficacy, indicating the necessity of targeting other viral genes for manipulation. To improve the therapeutic efficacy of oncolytic Ads, we treated the entire Ad genome repeatedly with UV-light and have isolated AdUV which efficiently lyses cancer cells as reported previously (Wechman, S. L. et al. Development of an Oncolytic Adenovirus with Enhanced Spread Ability through Repeated UV Irradiation and Cancer Selection. Viruses 2016 , 8 , 6). In this report, we show that no mutations were observed in the early genes ( E1 or E4 ) of AdUV while several mutations were observed within the Ad late genes which have structural or viral DNA packaging functions. This study also reported the increased release of AdUV from cancer cells. In this study, we found that AdUV inhibits tumor growth following intratumoral injection. These results indicate the potentially significant role of the viral late genes, in particular the DNA packaging genes, to enhance Ad oncolysis., Competing Interests: The authors declare no conflict of interest.

Oncolytic adenoviruses (Ads) have been shown to be safe and have great potential for the treatment of solid tumors. However, the therapeutic efficacy of Ads is antagonized by limited spread within solid tumors. To develop Ads with enhanced spread, viral particles of an E1-wildtype Ad5 dl309 was repeatedly treated with UV type C irradiation and selected for the efficient replication and release from cancer cells. After 72 cycles of treatment and cancer selection, AdUV was isolated. This vector has displayed many favorable characteristics for oncolytic therapy. AdUV was shown to lyse cancer cells more effectively than both E1-deleted and E1-wildtype Ads. This enhanced cancer cell lysis appeared to be related to increased AdUV replication in and release from infected cancer cells. AdUV-treated A549 cells displayed greater expression of the autophagy marker LC3-II during oncolysis and formed larger viral plaques upon cancer cell monolayers, indicating increased virus spread among cancer cells. This study indicates the potential of this approach of irradiation of entire viral particles for the development of oncolytic viruses with designated therapeutic properties.

Background: Clinical trials have indicated that preclinical results obtained with human tumor xenografts in mouse models may overstate the potential of adenovirus (Ad)-mediated oncolytic therapies. We have previously demonstrated that the replication of human Ads depends on cyclin E dysregulation or overexpression in cancer cells. ED-1 cell derived from mouse lung adenocarcinomas triggered by transgenic overexpression of human cyclin E may be applied to investigate the antitumor efficacy of oncolytic Ads., Methods: Ad-cycE was used to target cyclin E overexpression in ED-1 cells and repress tumor growth in a syngeneic mouse model for investigation of oncolytic virotherapies., Results: Murine ED-1 cells were permissive for human Ad replication and Ad-cycE repressed ED-1 tumor growth in immunocompetent FVB mice. ED-1 cells destroyed by oncolytic Ads in tumors were encircled in capsule-like structures, while cells outside the capsules were not infected and survived the treatment., Conclusion: Ad-cycE can target cyclin E overexpression in cancer cells and repress tumor growth in syngeneic mouse models. The capsule structures formed after Ad intratumoral injection may prevent viral particles from spreading to the entire tumor.

Oncolytic virotherapy can selectively destroy cancer cells and is a potential approach in cancer treatment. A strategy to increase tumor-specific selectivity is to control the expression of a key regulatory viral gene with a tumor-specific promoter. We have previously found that cyclin E expression is augmented in cancer cells after adenovirus (Ad) infection. Thus, the cyclin E promoter that is further activated by Ad in cancer cells may have unique properties for enhancing oncolytic viral replication. We have shown that high levels of viral E1a gene expression are achieved in cancer cells infected with Ad-cycE, in which the endogenous Ad E1a promoter was replaced with the cyclin E promoter. Ad-cycE shows markedly selective oncolytic efficacy in vitro and destroys various types of cancer cells, including those resistant to ONYX-015/dl1520. Furthermore, Ad-cycE shows a strong capacity to repress A549 xenograft tumor growth in nude mice and significantly prolongs survival. This study suggests the potential of Ad-cycE in cancer therapy and indicates the advantages of using promoters that can be upregulated by virus infection in cancer cells in development of oncolytic viruses. Key messages: Cyclin E promoter activity is high in cancer cells and enhanced by adenovirus infection. Cyclin E promoter is used to control the E1a gene of a tumor-specific oncolytic adenovirus. Ad-cycE efficiently targets cancer cells and induces oncolysis. Ad-cycE significantly repressed xenograft tumor and prolonged survival.

The aim of the present study was to characterize and quantify the numbers and expression levels of cells markers associated with dendritic cell (DC) maturation in small airways in current smokers and non-smokers with or without chronic obstructive pulmonary disease (COPD). Lung tissues from the following 32 patients were obtained during resection for lung cancer: Eight smokers with COPD, eight non-smokers with COPD, eight current smokers without COPD and eight non-smokers without COPD, serving as a control. The tissue sections were immunostained for cluster of differentiation (CD)83+ and CD1a+ to delineate mature and immature DCs, and chemokine receptor type 7 (CCR7+) to detect DC migratory ability. Myeloid DCs were collected from the lung tissues, and subsequently the CD83+ and CCR7+ expression levels in the lung myeloid DCs were detected using flow cytometry. The expression levels of CD83+, CD1a+ and CCR7+ mRNA in total lung RNA were evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Evident chronic bronchitis and emphysema pathological changes were observed in the lung tissues of patients with COPD. The results revealed that the numbers of CD83+ and CCR7+ DCs were reduced but the numbers of CD1a+ DCs were significantly increased in the COPD group as compared with the control group (P<0.05, respectively). Using RT-qPCR, the expression levels of CCR7+ and CD83+ mRNA were found to be reduced in the smokers with COPD as compared with the non-smokers without COPD group (P<0.05, respectively). Excessive local adaptive immune responses are key elements in the pathogenesis of COPD. Cigarette smoke may stimulate immune responses by impairing the homing of airway DCs to the lymph nodes and reduce the migratory potential of DCs. The present study revealed that COPD is associated with reduced numbers of mature CD83+ DCs and lower CCR7+ expression levels in small airways.

Epidemiological studies suggest that high intake of cruciferous vegetables is associated with a lower risk of cancer. Experiments have shown that indole-3-carbinol (I3C), a naturally occurring compound derived from cruciferous vegetables, exhibits potent anticarcinogenic properties in a wide range of cancers. In this study, we showed that higher doses of I3C (≥400 μM) induced apoptotic cancer cell death and lower doses of I3C (≤200 μM) repressed cancer cell growth concurrently with suppressed expression of cyclin E and its partner CDK2. Notably, we found that pretreatment with low doses of I3C enhanced Ad-mediated oncolysis and cytotoxicity of human carcinoma cells by synergistic upregulation of apoptosis. Thus, the vegetable compound I3C as a dietary supplement may benefit cancer prevention and improve Ad oncolytic therapies.

Background: Combination of oncolytic adenoviruses (Ads) and chemotherapy drugs has shown promising therapeutic results and is considered as a potential approach for cancer therapy. We previously have shown that autophagy may generate decomposed cellular molecules that can be used as nutrition to support virus replication in cancer cells. In this study, we evaluated a unique combination of the novel oncolytic Ad-cycE with rapamycin, an autophagy inducer and first-line chemotherapeutic drug., Methods: The combination of oncolytic Ad-cycE and the autophagy inducer rapamycin was assessed for enhanced antitumor effect. We also evaluated the combined effects of rapamycin and Ad-cycE on cancer cell viability. The interaction between Ad-cycE and rapamycin was analyzed with Calcusyn (Biosoft, Ferguson, MO)., Results: We show that rapamycin induces autophagy, enhances Ad E1A expression and increases Ad oncolytic replication. Combination of rapamycin and Ad-cycE elicits stronger cytotoxicity than single treatment alone. The analyzed data indicates that the Ad-cycE and rapamycin combination has a significantly synergistic antitumor effect., Conclusions: Our study provides a new insight into vector development and demonstrates the novel roles of autophagy in adenovirus replication. The combination of autophagy-induced chemotherapy and oncolytic virotherapy may be a new approach to improve future cancer treatment.

Adenoviruses (Ads) with deletion of E1b55K preferentially replicate in cancer cells and have been used in cancer therapies. We have previously shown that Ad E1B55K protein is involved in induction of cyclin E for Ad replication, but this E1B55K function is not required in cancer cells in which deregulation of cyclin E is frequently observed. In this study, we investigated the interaction of cyclin E and CDK2 in Ad-infected cells. Ad infection significantly increased the large form of cyclin E (cyclin EL), promoted cyclin E/CDK2 complex formation and increased CDK2 phosphorylation at the T160 site. Activated CDK2 caused pRb phosphorylation at the S612 site. Repression of CDK2 activity with the chemical inhibitor roscovitine or with specific small interfering RNAs significantly decreased pRb phosphorylation, with concomitant repression of viral replication. Our results suggest that Ad-induced cyclin E activates CDK2 that targets the transcriptional repressor pRb to generate a cellular environment for viral productive replication. This study reveals a new molecular basis for oncolytic replication of E1b-deleted Ads and will aid in the development of new strategies for Ad oncolytic virotherapies.

Adenovirus-mediated gene transfer into a tumor mass can be improved by combining it with conditionally-replicating adenovirus (CRAd) when both vectors co-infect the same cancer cell. We investigated the efficiency of enhancing transgene expression and effectiveness of cancer killing of two advenoviruses (Ads), one expressing E2F-1 (AdE2F-1) and another expressing a truncated form of E2F-1 that lacks the transactivation domain (AdE2Ftr), when combined with oncolytic Adhz60. We found that AdE2F-1 with Adhz60 actually decreased E2F-1 expression and viral replication through a mechanism apparently involving repression of the cyclin-E promoter and decreased expression of early and late structural proteins necessary for viral replication. In contrast, AdE2Ftr with Adhz60 resulted in increased E2Ftr expression, AdE2Ftr replication, and cancer cell death both in vitro and in vivo. These results indicate that AdE2Ftr coupled with a CRAd enhances AdE2Ftr-mediated cancer cell death., (Copyright © 2012 Elsevier Inc. All rights reserved.)

E2F-1-deleted mutant, 'truncated E2F' (E2Ftr, E2F-1[1-375]), lacking the carboxy-terminal transactivation domain, was shown to be more potent at inducing cancer cell apoptosis than wild-type E2F-1 (wtE2F-1; full-length E2F-1). Mechanisms by which wtE2F-1 and E2Ftr induce apoptosis, however, are not fully elucidated. Our study demonstrates molecular effects of pro-apoptotic BH3-only Bcl-2 family member Harakiri (Hrk) in wtE2F-1- and E2Ftr-induced melanoma cell apoptosis. We found that Hrk mRNA and Harakiri (HRK) protein expression was highly up-regulated in melanoma cells in response to wtE2F-1 and E2Ftr overexpression. HRK up-regulation did not require the E2F-1 transactivation domain. In addition, Hrk gene up-regulation and HRK protein expression did not require p53 in cancer cells. Hrk knockdown by Hrk siRNA was associated with significantly reduced wtE2F-1- and E2Ftr-induced apoptosis. We also found that an upstream factor, 'downstream regulatory element antagonist modulator' (DREAM), may be involved in HRK-mediated apoptosis in response to wtE2F-1 and E2Ftr overexpression. DREAM expression levels increased following wtE2F-1 and E2Ftr overexpression. Western blotting detected increased DREAM primarily in dimeric form. The homodimerization of DREAM resulting from wtE2F-1 and E2Ftr overexpression may contribute to the decreased binding activity of DREAM to the 3'-untranslated region of the Hrk gene as shown by electromobility shift assay. Results showed wtE2F-1- and E2Ftr-induced apoptosis is partially mediated by HRK. HRK function is regulated in response to DREAM. Our findings contribute to understanding the mechanisms that regulate wtE2F-1- and E2Ftr-induced apoptosis and provide insights into the further evaluation of how E2Ftr-induced apoptosis may be used for therapeutic gain., (© 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Adenoviruses with deletion of E1b have been used in clinical trials to treat cancers that are resistant to conventional therapies. The efficacy of viral replication within cancer cells determines the results of oncolytic therapy, which remains poorly understood and requires further improvement. In this report, we show that adenoviruses induce autophagy by increasing the conversion of LC3-I to LC3-II and the formation of the Atg12-Atg5 complex. Inhibition of autophagy with 3-methyladenine (3MA) resulted in a decreased synthesis of adenovirus structural proteins, and thereby a poor viral replication; promotion of autophagy with rapamycin increased adenovirus yield. This study indicates that adenovirus-induced autophagy correlates positively with virus replication and oncolytic cell death, and that autophagy may generate nutrients that can be used for building viral progeny particles. These results further suggest that chemotherapeutic agents that increase cancer cell autophagy may improve the efficacy of oncolytic virotherapy., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Adenovirus (Ad)-mediated E2F-1 gene transfer induces apoptosis in cancer cells in vitro and in vivo, but clinical application of E2F-1 in cancer gene therapy remains controversial because of the oncogenic potential of E2F-1. This barrier can be circumvented by using the truncated form of the E2F-1 gene (E2Ftr) (amino acids 1 through 375), which lacks the E2F-1 transactivation domain and cell cycle-promoting effects., Methods: The authors constructed 3 adenoviral vectors that expressed E2Ftr under regulation of the tetracycline (Tet)-off system (AdTet-E2Ftr1, AdTet-E2Ftr2, and AdTet-E2Ftr3). These vectors were compared for E2Ftr expression and apoptosis induction in cancer cells and normal cells. E2Ftr antitumor activity in vivo also was assessed in a melanoma xenograft model., Results: One of the 3 vectors, AdTet-E2Ftr3, had the highest E2Ftr protein expression levels, which were correlated with the greatest induction of apoptosis and inhibition of cancer cell growth. E2Ftr induced apoptosis in a variety of cancer cell lines independent of p53 status with little cytotoxicity in normal cell lines. In a mouse melanoma xenograft model, AdTet-E2Ftr3 exhibited an approximately 80% decrease in tumor size compared with controls in vivo., Conclusions: The current results indicated that AdTet-E2Ftr3 is a novel anticancer agent that has significant therapeutic activity in vitro and in vivo., (© 2010 American Cancer Society.)

Adenoviral vectors are highly efficient at transferring genes into cells and are broadly used in cancer gene therapy. However, many therapeutic genes are toxic to vector host cells and thus inhibit vector production. The truncated form of E2F-1 (E2Ftr), which lacks the transactivation domain, can significantly induce cancer cell apoptosis, but is also toxic to HEK-293 cells and inhibits adenovirus replication. To overcome this, we have developed binary- and single-vector systems with a modified tetracycline-off inducible promoter to control E2Ftr expression. We compared several vectors and found that the structure of expression cassettes in vectors significantly affects E2Ftr expression. One construct expresses high levels of inducible E2Ftr and efficiently causes apoptotic cancer cell death by activation of caspase-3. The approach developed in this study may be applied in other viral vectors for encoding therapeutic genes that are toxic to their host cells and/or inhibit vector propagation., (Copyright 2009 Elsevier Inc. All rights reserved.)

Adenoviruses (Ads) with E1B55K mutations can selectively replicate in and destroy cancer cells. However, the mechanism of Ad-selective replication in tumor cells is not well characterized. We have shown previously that expression of several cell cycle-regulating genes is markedly affected by the Ad E1b gene in WI-38 human lung fibroblast cells (X. Rao, et al., Virology 350:418-428, 2006). In the current study, we show that the Ad E1B55K region is required to enhance cyclin E expression and that the failure to induce cyclin E overexpression due to E1B55K mutations prevents viral DNA from undergoing efficient replication in WI-38 cells, especially when the cells are arrested in the G(0) phase of the cell cycle by serum starvation. In contrast, cyclin E induction is less dependent on the function encoded in the E1B55K region in A549 and other cancer cells that are permissive for replication of E1B55K-mutated viruses, whether the cells are in the S phase or G(0) phase. The small interfering RNA that specifically inhibits cyclin E expression partially decreased viral replication. Our study provides evidence suggesting that E1B55K may be involved in cell cycle regulation that is important for efficient viral DNA replication and that cyclin E overexpression in cancer cells may be associated with the oncolytic replication of E1B55K-mutated viruses.

Adenoviruses with deletion of E1b gene can selectively replicate in cancer cells. The underlying mechanisms in tumor-selective replication of E1b-deleted adenoviruses are insufficiently understood. Identifying genes with altered expression patterns caused by the E1B proteins in virus-infected cells will further increase our understanding of E1B functions and provide insight into the tumor-selective replication of E1b-mutated adenoviruses on the molecular level. An approach based on large-scale gene array was applied to analyze molecular changes affected by viral E1B. We identified a total of 345 genes with expression changes of two-fold or greater affected by wild-type adenovirus compared with its E1b-deleted counterpart. The gene array data were confirmed by quantitative real-time PCR and Western blot. E1B proteins affect the expression of a diverse range of genes involved in cell cycle regulation, apoptosis, stress responses and angiogenesis. This is the first study of the global profile of gene expression altered by the viral E1B proteins in human lung cells, and the majority of the genes were previously not known to be affected by the viral proteins. The data presented in this study will lead to more detailed analysis of E1B functions and may also lead to development of new agents and approaches for oncolytic therapy.

E1B55K-deleted dl1520 could selectively replicate in cancer cells and has been used in clinical trials as an antitumor agent. The mechanism of virus selective replication in cancer cells, including a possible role of p53, is unclear. Studies with established cancer cell lines have demonstrated that some cancer cells are resistant to dl1520 replication, regardless of the p53 status. Hep3B cells supported the E1b-deleted adenoviruses to replicate, whereas Saos2 cells were resistant to viral replication. We applied p53-null Hep3B and Saos2 cells as models to clarify the replication ability of E1B55K-deleted adenoviruses with different expression levels of E1a. We show that lower E1A expression in Saos2 may be the reason for the poor replication in some cancer cells due to the fact that E1a promoter was less activated in Saos2 than in Hep3B. We also demonstrate that the E1B55K protein can increase E1A expression in Saos2 cells for efficient virus replication. In addition, the upstream regions of the E1a promoter have transcriptional activity in Hep3B cells but not in Saos2 cells. The viral E1B55K protein may activate cancer cellular factor(s) that targets the upstream regions of the E1a gene to increase its expression. This is the first study demonstrating that E1B55K protein affects the E1A production levels that is related to cancer selective replication. Our studies have suggested that increase of E1A expression from E1b-deleted adenoviruses may enhance killing cancer cells that otherwise are resistant to viral replication.

The structural and functional properties of arginine kinase (AK) in alkaline conditions in the absence or presence of salt have been investigated. The conformational changes of AK during alkaline unfolding and salt-induced folding at alkaline pH were monitored using intrinsic fluorescence emission, binding of the fluorescence probe 1-anilino-8-naphthalenesulfonate and circular dichroism. The results for the alkaline unfolded enzyme showed that much lower pH (11.0) was required to cause the complete loss of AK activity than was required to cause an obvious conformational change of the enzyme. Compared with the completely unfolded state in 5M urea, the high pH denatured enzyme had some residual secondary and tertiary structure even at pH 13.0. Increasing the ionic strength by adding salt at pH 12.75 resulted in the formation of a relatively compact tertiary structure and a little new secondary structure with hydrophobic surface enhancement. These results indicate that the partially folded state formed under alkaline conditions may have similarities to the molten globule state which is compact, but it has a poorly defined tertiary structure and a native-like secondary structure.

One of the promising strategies for targeting replication of oncolytic adenovirus in tumor cells is to regulate the expression of essential viral genes such as E1a by using tumor- or tissue-specific promoters that are preferentially active in cancer cells. However, this approach may lead to some degree of viral replication in normal cells other than in cancer cells if the viral gene also expresses in normal cells. In this study, we investigated the effect of E1a expression levels on the virus replication ability in human cells. Three vectors, all with mutated E1B55K, were created, one without any promoter controlling the E1a gene and two vectors with the E1a gene being controlled by either its endogenous promoter or a strong CMV promoter. We observed that the CMV promoter-mediated high levels of E1A expression could increase virus replication, resulting in the titers of the E1B55K-mutated virus being even higher than the wild-type virus in some cancer cells. However, the strong CMV promoter could not always enhance virus replication, such as in cancer cells OE33 and OsACL. The results suggest that whether increased E1A levels would enhance E1B55K-mutated virus replication may be also depended on cellular factors or pathways in cancer cells. We also observed that the virus without any promoter for the E1a gene could still express leaky levels of E1A which can lead to viral replication in normal and cancer cells. Future efforts in the development of transcription-controlled oncolytic adenoviruses should focus on how to completely block E1a expression in normal cells.

Strong aggregation occurred in the refolding route of arginine kinase (AK) denatured with 3 mol GdnHCl/L (GdnHCl, guanidine hydrochloride). The activity recovery of GdnHCl-denatured AK was very low and dependent on the protein concentration in the process of refolding. For denatured AK at 1.2 micromol/L concentration, the recovered activity yield was about 45.2% of the native enzyme, whereas at 5.2 micromol/L the activity recovery yield was only 20% of native activity. The nonionic detergent Triton X-100 and Tween 20 (< or = 100 mmol/L concentration) not only effectively blocked the aggregation but also enabled the denatured AK to recover most of its native activity. The kinetics of aggregate solubilization showed that there was an induction phase dependent on the detergent, but there was no dependency when detergent was absent. The apparent activity recovery had a cooperative relation with detergents in the process of refolding, which suggested the existence of some interaction between the detergent and the refolding intermediate. On the basis of the study results, a scheme of refolding was proposed.

Introduction: Once metastasis has occurred, the possibility of completely curing breast cancer is unlikely, particularly for the 30 to 40% of cancers overexpressing the gene for HER2/neu. A vaccine targeting p185, the protein product of the HER2/neu gene, could have therapeutic application by controlling the growth and metastasis of highly aggressive HER2/neu+ cells. The purpose of this study was to determine the effectiveness of two gene vaccines targeting HER2/neu in preventive and therapeutic tumor models., Methods: The mouse breast cancer cell line A2L2, which expresses the gene for rat HER2/neu and hence p185, was injected into the mammary fat pad of mice as a model of solid tumor growth or was injected intravenously as a model of lung metastasis. SINCP-neu, a plasmid containing Sindbis virus genes and the gene for rat HER2/neu, and Adeno-neu, an E1,E2a-deleted adenovirus also containing the gene for rat HER2/neu, were tested as preventive and therapeutic vaccines., Results: Vaccination with SINCP-neu or Adeno-neu before tumor challenge with A2L2 cells significantly inhibited the growth of the cells injected into the mammary fat or intravenously. Vaccination 2 days after tumor challenge with either vaccine was ineffective in both tumor models. However, therapeutic vaccination in a prime-boost protocol with SINCP-neu followed by Adeno-neu significantly prolonged the overall survival rate of mice injected intravenously with the tumor cells. Naive mice vaccinated using the same prime-boost protocol demonstrated a strong serum immunoglobulin G response and p185-specific cellular immunity, as shown by the results of ELISPOT (enzyme-linked immunospot) analysis for IFNgamma., Conclusion: We report herein that vaccination of mice with a plasmid gene vaccine and an adenovirus gene vaccine, each containing the gene for HER2/neu, prevented growth of a HER2/neu-expressing breast cancer cell line injected into the mammary fat pad or intravenously. Sequential administration of the vaccines in a prime-boost protocol was therapeutically effective when tumor cells were injected intravenously before the vaccination. The vaccines induced high levels of both cellular and humoral immunity as determined by in vitro assessment. These findings indicate that clinical evaluation of these vaccines, particularly when used sequentially in a prime-boost protocol, is justified.

Apoptotic pathways are initiated as a cellular defense mechanism to eliminate adenovirus-infected cells. We have investigated how E1A-induced apoptosis interferes with viral replication in cancer cells. We found that E1B19K alone can efficiently suppress E1A-induced apoptosis in cancer cells. Viruses deleted for both E1B19K and E1B55K resulted in cellular DNA degradation. However, less than 20% of human lung cancer cells infected with a virus deleted for both E1B19K and E1B55 K had evidence of chromatin condensation and multiple-micronuclei formation (apoptotic hallmarks); these cells could not produce infectious viral particles. The majority of cancer cells infected with viruses deleted for the entire E1b gene did not undergo extended apoptosis and produced abundant viral progeny. Thus, only a fraction of cancer cells underwent apoptosis and did not allow E1b-deleted viruses to replicate, while the majority of cancer cells were resistant to E1A-induced apoptosis and could support virus-selective replication. The results of this study imply that, in addition to inhibiting E1A-induced apoptosis, E1B proteins may contribute other important roles in the viral life cycle. Our results also suggest that combining virus-induced apoptosis and selective viral replication into one vector will be a novel approach to destroy cancer cells.

The refolding process and the equilibrium intermediates of urea-denatured arginine kinase (AK) were investigated by 1-anilino-8-naphthalenesulfonate (ANS) intrinsic fluorescence, far-UV circular dichroism (CD), size-exclusion chromatography (SEC), and enzymatic activity. In dilute denaturant, two equilibrium refolding intermediates (I and N') were discovered, and a refolding scheme of urea-denatured AK was proposed. During the refolding of urea-denatured AK, the fluorescence intensity increased remarkably, accompanied by a significant blue shift of the emission maximum and a pronounced increase in molar ellipticity of CD at 222 nm. The first folding intermediate (I) was inactive in urea solution ranging between 2.4 and 3.0 M. The second (N') existed between a 0.4- and 0.8-M urea solution, with slightly increased activity. Neither the blue shift emission maximum nor the molar ellipticity of CD at 222 nm showed significant changes in these two regions. The two intermediates were characterized by monitoring the ANS binding ability in various residual urea solutions, and two peaks of the emission intensity were observed in urea solutions of 0.6 and 2.8 M, respectively. The SEC results indicated that a distribution coefficient (K(D)) platform existed in urea solutions ranging between 2.4 and 3.0 M urea, suggesting that there was a similarly apparent protein profile and size in the urea solution region. The refolding kinetics showed that the urea-denatured AK was in two-phase refolding. Proline isomerization occurred in the unfolding process of AK, which blocked the slow phase of refolding. These results suggested that the refolding process of urea-denatured AK contained at the least two equilibrium refolding intermediates.

The adenovirus E1A proteins are involved in the transcriptional activation of viral and cellular genes needed for controlling cell cycle and virus replication. Undifferentiated embryonic carcinoma cells have the ability to produce an E1A-like activity that can induce the expression of E1A-targeted adenoviral and cellular genes in the absence of the E1A products. Differentiated embryonic carcinoma cells lose the ability to produce the E1A-like activity. In this study, we investigated the E1A-like activity in cancer cells with an adenovirus having a mutated E1a gene. The mutation is generated by the insertion of a large DNA fragment in the E1a gene and interrupts the COOH-terminal region of both the E1A 12S and 13S proteins. The E1a-mutated virus can efficiently replicate in HepG2 and Hep3B liver cancer cells and produce high titers of virus. Replication of the E1a-mutated virus inhibits tumor formation and destroys tumors in vivo. The results obtained in this study imply that cancer cells may produce an E1A-like activity to support the selective replication of mutated virus in cancer cells. In addition, we found that although the E1a-mutated virus could not replicate in Huh1.cl2 liver cells, the viral DNA could amplify in the cells. This result suggests that replication of adenoviral DNA is necessary, but not sufficient, for generating infectious viral progeny and destroying tumor cells.

Background: The helper-dependent (HD) adenoviral (Ad) vector relies on a helper virus to provide viral proteins for vector amplification. HD-Ad vectors can significantly increase therapeutic gene expression and improve safety. However, the yield of an HD-Ad vector is generally lower than that of an E1-deleted first-generation vector, likely due to the alterations in viral E3 or packaging regions of a helper virus that attenuate its replication and complementing for an HD-Ad vector., Methods: To study this question and improve HD-Ad vector production, we have generated four different helper viruses with a wild-type or deleted E3 region, and with a relocated loxP. We have also constructed a first-generation vector with a wild-type E3 region and without the loxP site. We compared the replication of these viruses in Cre-positive and -negative cells and studied their complementing for HD-Ad vector production., Results: Viruses with deleted E3 formed smaller plaques and produced lower titer compared with viruses containing the E3 region. The site where a loxP is inserted can also affect virus replication. Higher yield of HD-Ad vector was obtained when a helper virus with wild-type E3 was used. We also showed that deletion of the packaging signal in a helper virus through loxP/Cre interaction decreased the viral DNA complementing ability., Conclusions: Although the E3 region is not essential for adenovirus replication in vivo, deletion of this region attenuates virus replication. Production of HD-Ad vector can be further improved by modifications in helper virus structure., (Copyright 2002 John Wiley & Sons, Ltd.)

Adenoviral vectors are attractive for the delivery of transgenes into mammalian cells because of their efficient transduction, high titer, and stability. The major concerns with using E1-deleted adenoviral vectors in gene therapy are the pathogenic potential of the virus backbone and the leaky viral protein synthesis that leads to host immune responses and a short duration of transgene expression. Helper-dependent (HD) adenoviral vectors that are devoid of all viral protein-coding sequences have significantly increased the safety and reduced the immunogenicity of these vectors. Currently available HD vectors depend on an E1-deleted adenovirus as a helper to provide viral proteins in trans. As a consequence, contamination with helper virus cannot be avoided in the HD vector preparation though it can be decreased to 0.01% using a Cre/loxP mechanism. Since the presence of E1-deleted helper virus may have substantial unwanted effects, we have developed a new Cre-expressing cell line based on an E1- and E2a-complementing cell. This new cell line can efficiently cleave the packaging region in the helper virus genome. We have also developed an E1 and E2a double-deleted helper virus. By using the CreE cell with the helper virus deleted in both the E1 and the E2a genes it may be possible to further improve the safety of the vectors.

The gene for HER2/neu is overexpressed in 30-40% of breast and ovarian cancers, and this overexpression correlates with increased metastasis and poor prognosis. The HER2/neu gene product, a transmembrane protein kinase member of the EGF receptor family, has significant potential as a tumor antigen for vaccination. We inserted the sequence for neu into a novel plasmid called ELVIS containing a Sindbis virus replicon that reproduces multiple copies of mRNA. Mice vaccinated one time intramuscularly demonstrated a strong antibody response against A2L2, a murine breast cancer cell line transfected to express neu. Vaccinated mice challenged in the mammary fatpad with A2L2 had reduced tumor incidence and reduced tumor mass compared to mice challenged with tumor cells lacking the neu insert. Intradermal vaccination was also protective and required 80% less plasmid for a similar level of protection. Vaccination reduced the incidence of lung metastasis from mammary fatpad tumors and reduced the number of lung metastases resulting from intravenous injection of A2L2 cells. Cytotoxic T lymphocytes cultures of immune spleen cells with P815-neu cells produced high levels of interferon-gamma indicating an antigen-specific Th1-type immune response resulting from the vaccination. In a spontaneous breast tumor model using neu transgenic mice, vaccination with ELVIS-neu protected against development of spontaneous breast tumors. Our preclinical data indicate that therapeutic vaccination of patients with ELVIS-neu may reduce metastasis from HER2/neu-expressing breast and ovarian tumors.

Vaccination against Helicobacter pylori using DNA sequences encoding Urease A and B subunits was compared to immunization with urease antigen and MTP-PE in a liposome formulation. To determine the effectiveness of a vaccine against H. pylori in a mouse model it is essential to quantify the number of H. pylori remaining in the stomachs following challenge with an inoculum of live bacteria. Culture assays and enzymatic assays produce inconsistent results often unsuitable to conclude if vaccine candidates are protective. To overcome this problem, we developed two assays: 1) a competitive quantitative PCR using a colorimetric readout and 2) a non-competitive direct quantitative PCR using a highly sensitive bioluminescent readout. The competitive PCR requires coamplification of a segment of the urease C sequence and an internal control standard in a competitive manner using a single set of primers. PCR products were quantified colorimetrically by an enzyme-linked immunosorbent assay and compared with known quantities of the internal control standard added to the PCR reaction. The highly sensitive, bioluminescent assay measures the amplified DNA directly using a flash-type luminescent tag and a specific probe. The Sydney strain of H. pylori was used for the mouse infection model. Quantification of H. pylori by either the bioluminescent assay or the competitive PCR was reliable, specific and sensitive compared to quantitative growth assays which often gave false results. The bioluminescent assay was much more sensitive and less labor/time intensive than the competitive PCR. The bioluminescent assay was able to quantitate as few as 100 bacteria, while the competitive assay could not detect less than 10(3) bacteria per mouse stomach. Quantification of H. pylori by bioluminescent assay was superior to the competitive assay and may be used for research applications, such as the development of vaccines, pathogenesis of gastric disease and monitoring of antibiotic treatment.

Background: The density of Helicobacter pylori is thought to correlate with the degree of inflammation and thus indirectly with the outcome of the infection. Rapid quantitative assays of H. pylori in gastric or duodenal mucosa are lacking. The aim was to develop quantitative assays using the polymerase chain reaction to assess the quantity of H. pylori in the gastric mucosa., Methods: Competitive PCR was based on coamplification of a segment of the ureC sequence and an internal control using a single set of primers. PCR products were quantified colorimetrically by an enzyme-linked immunosorbent assay and compared with known quantities of the internal control standard added to the PCR reaction. The highly sensitive, noncompetitive PCR assay does not use coamplification and measures the amplified DNA sequence using a flash-type luminescent tag and a specific probe. The mouse infected model using H. pylori strain SS-1 was used to develop the assays., Results: Quantification of H. pylori using either the competitive or noncompetitive PCR was reliable, highly sensitive and specific., Conclusions: The ability to rapidly quantitate H. pylori from gastric mucosa should be useful to investigate the role of H. pylori density and infection outcome, as well as to monitor the effectiveness of antibiotic treatment or vaccines against H. pylori.

Immunoliposomes containing monoclonal antibodies (MAbs) to the costimulatory molecules CD28 and CTLA4 and their counterreceptors B7-1 (CD80) and B7-2 (CD86) were evaluated for the ability to increase the immune response to recombinant envelope protein rgp120 of the MN strain of human immunodeficiency virus type 1 (HIV-1) during vaccination. MAbs were attached to rgp120-containing liposomes via a biotin-avidin-biotin bridge. Mice vaccinated with immunoliposomes were found to have a strong delayed-type hypersensitivity (DTH) response to the weakly immunogenic gp120 that was dependent on the presence of the MAbs. However, this vaccination protocol did not induce humoral immunity. The DTH response was not accompanied by increased production of interferon gamma (IFN-gamma) or interleukin 4 (IL-4), implying that the primary cellular interaction was between the immunoliposomes and cells of the reticuloendothelial system and not helper T (Th) cells. This strategy of incorporating antibodies to costimulatory molecules on the surface of antigen-containing particulates, such as liposomes or microspheres, can be used to increase DTH immune responses to protein or peptide vaccines.

Background: A murine model for Helicobacter pylori infection could facilitate vaccine development. This study was designed to determine the effect of various conditions of dose, frequency of administration, and fasting on H. pylori infection of mice., Materials and Methods: Balb/c and C3H/HeN mice were inoculated orogastrically with clinical isolates of H. pylori grown in liquid culture. At 2-week intervals, the stomachs were removed for secondary culture on horse blood agar and for histological analysis. H. pylori from secondary cultures or homogenized stomach tissue from infected mice was inoculated a second time in naïve animals., Results: H. pylori was cultured with high frequency only from the stomachs of C3H/HeN mice. Fasting the mice and increasing the number of organisms inoculated did not increase the rate of infection. Histological analysis detected no inflammation, but mucus depletion and erosion were present in the stomachs of C3H/HeN mice. H. pylori organisms were not observed. Secondary cultures of H. pylori or homogenized infected stomach tissue did not cause infection when inoculated in naïve mice., Conclusions: Clinical isolates of H. pylori transiently infect C3H/HeN mice. This murine model is suitable for testing oral vaccines. Effective vaccination against H. pylori could prevent transient infection and reduce subsequent gastritis.

The ability of cytokines to regulate and augment an immune response makes them likely agents to be included in vaccines. The systemic use of cytokines as vaccine adjuvants has been hampered by toxicity at effective doses. More precise delivery of cytokines and immunogen can be achieved through the use of microparticle carriers such as liposomes. Several cytokines, including IL-2, IL-7, IL-6 and IFN-gamma have been shown to increase the adjuvant activity of liposomes. It may be possible to use certain cytokines to induce immunoglobulin production, others to induce cytotoxic T lymphocytes (CTL) and still others to promote IgA production at mucosal surfaces. An alternative to liposomes containing cytokines may be liposomes containing cytokine-inducing adjuvants such as MTPPE, MPL and QS21. This approach may produce undesirable immunomodulators as well as beneficial cytokines.

Dehydration-rehydration liposome vesicles (DRVs) containing various cytokines were evaluated for their ability to induce delayed-type hypersensitivity (DTH) and humoral immunity to the recombinant envelope protein rgp120 of the MN strain of human immunodeficiency virus type 1 (HIV-1). The DRVs trapped approximately 25% of the radiolabeled cytokines and approximately 17% of the radiolabeled rgp120 that were added. The level of trapping was greater than the aqueous volume of the DRVs, indicating association of the proteins with the lipid bilayer. Flow cytometric analysis using antibody to rgp120 or the V3 loop of rgp120 showed the diameter of the DRVs to be 2-7.5 microns. Transmission electron microscopy confirmed the heterogeneity in size of the DRVs and revealed morphological heterogeneity. Transmission electron microscopy with immunogold labeling also revealed the presence of rgp120 on the surface of the DRVs. In vitro bioassays demonstrated slow leakage of biologically active cytokines from DRVs soaked in tissue culture medium containing serum. Mice injected subcutaneously three times at 14-day intervals with DRVs containing 15 micrograms of rgp120 plus interleukin 6 (IL-6) or interferon gamma (IFN-gamma) produced significantly greater DTH responses than mice injected with DRVs containing rgp120 alone. Soluble rgp120 plus soluble IFN-gamma produced DTH in some experiments, but of lower magnitude than the comparable DRVs. Interleukin 6, but not IFN-gamma, increased the antibody titer to rgp120 when included in the DRVs. The mice did not develop antibodies to IFN-gamma or IL-6. Induction of DTH by vaccines may increase protection from viral pathogens such as HIV. Cytokine-containing liposomes may be an effective adjuvant for the induction of a DTH response to envelope-antigen subunit vaccines.

Conjugal transfer of plasmid R1162 is initiated and terminated at a 38-bp origin of transfer (oriT). Plasmids containing two directly repeated copies of oriT were used to determine the actual frequency of termination at this site. This frequency was calculated both for oriTnic, a mutated origin that cannot act as the initiation site of transfer, and for an unmutated oriT where transfer had been initiated. In both cases, the termination frequency decreased as the distance between the initiation and termination sites became greater and was significantly less than one for plasmids the size of R1162. A substantial proportion of recipient cells received more than one plasmid copy during transfer. Our results indicate that termination is inefficient but that this is partly compensated for by the transmission of multiple plasmid copies.

Conjugal transfer of the broad-host-range plasmid R1162 is initiated and terminated at the nic site within the 38-bp origin of transfer (oriT). Termination involves ligation of the transferred single strand by the plasmid-encoded MobA protein. Several different assays were used to identify the oriT DNA required for termination. For plasmids containing two oriTs, with transfer initiated at one and terminated at the other, the inverted repeat within oriT is important for termination. Deletion of the outer arm reduces the termination frequency; those terminations that do occur probably depend upon nicking at this oriT prior to transfer. The locations of second-site suppressor mutations indicate that base pairing between the arms of the inverted repeat is important for termination. In vitro, the inverted repeat is not required for specific cleavage of single-stranded DNA at nic, but competition experiments indicate that oriTs with the inverted repeat are preferentially cleaved. We propose that the function of the oriT inverted repeat is to trap the plasmid-encoded MobA protein at the end of a round of strand transfer, thus ensuring that the protein is available for the ligation step.