Design, synthesis and cellular characterization of a new class of IPMK kinase inhibitors.

Many genetic studies have established the kinase activity of inositol phosphate multikinase (IPMK) is required for the synthesis of higher-order inositol phosphate signaling molecules, the regulation of gene expression and control of the cell cycle. These genetic studies await orthogonal validation by specific IPMK inhibitors, but no such inhibitors have been synthesized. Here, we report complete chemical synthesis, cellular characterization, structure-activity relationships and rodent pharmacokinetics of a novel series of highly potent IPMK inhibitors. The first-generation compound 1 (UNC7437) decreased cellular proliferation and tritiated inositol phosphate levels in metabolically labeled human U251-MG glioblastoma cells. Compound 1 also regulated the transcriptome of these cells, selectively regulating genes that are enriched in cancer, inflammatory and viral infection pathways. Further optimization of compound 1 eventually led to compound 15 (UNC9750), which showed improved potency and pharmacokinetics in rodents. Compound 15 specifically inhibited cellular accumulation of InsP 5 , a direct product of IPMK kinase activity, while having no effect on InsP 6 levels, revealing a novel metabolic signature detected for the first time by rapid chemical attenuation of cellular IPMK activity. These studies designed, optimized and synthesized a new series of IPMK inhibitors, which reduces glioblastoma cell growth, induces a novel InsP 5 metabolic signature, and reveals novel aspects inositol phosphate cellular metabolism and signaling.

Overexpression of Bruton Tyrosine Kinase Inhibits the Proliferation, Migration, and Invasion of Non-Small Cell Lung Cancer Cells.

Lung cancer is one of the most lethal malignant tumors in the world. Non-small cell lung cancer (NSCLC) is the most common pathological subtype. However, the molecular mechanism of NSCLC progress is still unclear. We extracted the expression data of the Bruton's tyrosine kinase (BTK) gene in NSCLC tissues from the TCGA database. The results of paired t-test showed that the BTK gene was significantly underexpressed in NSCLC tissues. To further verify the above results, we detected the expression of the BTK gene in NSCLC cell lines A549, H1299, and H1650 at the RNA and protein levels by real-time fluorescent quantitative polymerase chain reaction and Western Blot analysis, respectively. The results showed that BTK was low expressed in NSCLC tissues and cells. More importantly, the expression of the BTK gene is also significantly related to the patient's age, gender, tumor range (T), lymph node invasion (N), tumor stage, and prognosis, and its expression level gradually decreases with the progress of the disease. It is speculated that BTK may be an independent prognostic factor of NSCLC. Our experimental results are consistent with the above clinical correlation analysis results. Overexpression of BTK can significantly inhibit the proliferation, migration, and invasion of NSCLC cells and can block the G0/G1 tumor cell cycle, indicating that overexpression of BTK can inhibit the growth, migration, and invasion of NSCLC cells.

LINC01117 inhibits invasion and migration of lung adenocarcinoma through influencing EMT process.

BACKGROUND: Studying the mechanism of action of LncRNAs in lung adenocarcinoma (LUAD) is of great importance for an in-depth understanding of the molecular mechanism of lung adeno carcinogenesis and development. OBJECTIVE: The aim is to identify a long non-coding RNA LINC01117 that is specifically and highly expressed in LUAD cells and to investigate its biological functions and molecular mechanisms in LUAD cells, providing a new potential target for targeting LUAD therapy. METHODS: This study used publicly available data downloaded from The Cancer Genome Atlas (TCGA) database. Construction of siRNA and overexpression plasmid-packed lentiviral constructs were used to knock down and increase the expression of LINC01117 in LUAD cells. The effect of LINC01117 on LUAD cell migration and invasion was verified by scratch assays and Transwell assays. Western blot assays were performed to verify the effect of knocking down LINC01117 expression on key proteins of the EMT process. The effect of overexpression and knockdown LINC01117 expression on key proteins of the EMT process and the nuclear and cytoplasmic distribution of YAP1, a key effector molecule of the Hippo pathway, was verified by Western blot assays. RESULTS: LINC01117 expression was upregulated in LUAD tissues and cell lines. Clinical correlation and prognostic analyses showed that LINC01117 was associated with poorer clinical features (staging and N classification) and poorer prognosis and could be analyzed as an independent prognostic factor. Cell migration and invasion were significantly inhibited in the knockdown group compared to the control group; in contrast, cell migration and invasion were promoted in the overexpression group. Overexpression of LINC01117 resulted in down-regulation of E-cadherin expression and increased expression levels of N-cadherin, vimentin, ZEB1, snail and slug; in contrast, knockdown of LINC01117 appeared to have the opposite effect. Furthermore, knockdown of LINC01117 increased the enrichment of YAP1 protein in the cytoplasm and reduced its level in the nucleus; overexpression of LINC01117 produced the opposite intracellular distribution results. CONCLUSIONS: LINC01117 was highly expressed in LUAD, and knockdown of LINC01117 significantly inhibited the migration and invasion of LUAD cells, while overexpression of LINC01117 significantly promoted the migration and invasion of LUAD cells, and affected the EMT process, and was able to alter the distribution of YAP1 in the nucleus and cytoplasm. This suggests that LINC01117 may regulate the activity of the Hippo pathway by altering the nuclear and cytoplasmic distribution of YAP1, which in turn induces the EMT process in lung adenocarcinoma cells and thus exerts a pro-cancer effect. It suggests that LINC01117 may play a key role in the occurrence and development of LUAD.

Development of Novel IP6K Inhibitors for the Treatment of Obesity and Obesity-Induced Metabolic Dysfunctions.

Obesity and obesity-induced metabolic dysfunctions are significant risk factors for nonalcoholic fatty liver disease and cardiovascular diseases. Thus, obesity is an economic and social burden in developed countries. Blocking the synthesis of inositol pyrophosphates by inositol hexakisphosphate kinase (IP6K) has been identified as a potential therapeutic strategy for obesity and related diseases. We have developed a novel and potent IP6K inhibitor 20 (UNC7467) (IC50 values: IP6K1 8.9 nM; IP6K2 4.9 nM; IP6K3 1320 nM). Inositol phosphate profiling of the HCT116 colon cancer cell line demonstrates that 20 reduced levels of inositol pyrophosphates by 66-81%, without significantly perturbing levels of other inositol phosphates. Furthermore, intraperitoneal injection of 20 in diet-induced obese mice improved glycemic profiles, ameliorated hepatic steatosis, and reduced weight gain without altering food intake. Thus, inhibitor 20 can be used as an in vivo probe for IP6K-related research. Moreover, it may have therapeutic relevance in treating obesity and related diseases.

The Synthesis and Initial Evaluation of MerTK Targeted PET Agents.

MerTK (Mer tyrosine kinase), a receptor tyrosine kinase, is ectopically or aberrantly expressed in numerous human hematologic and solid malignancies. Although a variety of MerTK targeting therapies are being developed to enhance outcomes for patients with various cancers, the sensitivity of tumors to MerTK suppression may not be uniform due to the heterogeneity of solid tumors and different tumor stages. In this report, we develop a series of radiolabeled agents as potential MerTK PET (positron emission tomography) agents. In our initial in vivo evaluation, [18F]-MerTK-6 showed prominent uptake rate (4.79 ± 0.24%ID/g) in B16F10 tumor-bearing mice. The tumor to muscle ratio reached 1.86 and 3.09 at 0.5 and 2 h post-injection, respectively. In summary, [18F]-MerTK-6 is a promising PET agent for MerTK imaging and is worth further evaluation in future studies.

Development of a novel embryonic germline gene-related prognostic model of lung adenocarcinoma.

BACKGROUND: Emerging evidence implicates the correlation of embryonic germline genes with the tumor progress and patient's outcome. However, the prognostic value of these genes in lung adenocarcinoma (LUAD) has not been fully studied. Here we systematically evaluated this issue, and constructed a novel signature and a nomogram associated with embryonic germline genes for predicting the outcomes of lung adenocarcinoma. METHODS: The LUAD cohorts retrieved from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database were used as training set and testing set, respectively. The embryonic germline genes were downloaded from the website https://venn.lodder.dev. Then, the differentially expressed embryonic germline genes (DEGGs) between the tumor and normal samples were identified by limma package. The functional enrichment and pathway analyses were also performed by clusterProfiler package. The prognostic model was constructed by the least absolute shrinkage and selection operator (LASSO)-Cox regression method. Survival and Receiver Operating Characteristic (ROC) analyses were performed to validate the model using training set and four testing GEO datasets. Finally, a prognostic nomogram based on the signature genes was constructed using multivariate regression method. RESULTS: Among the identified 269 DEGGs, 249 were up-regulated and 20 were down-regulated. GO and KEGG analyses revealed that these DEGGs were mainly enriched in the process of cell proliferation and DNA damage repair. Then, 103 DEGGs with prognostic value were identified by univariate Cox regression and further filtered by LASSO method. The resulting sixteen DEGGs were included in step multivariate Cox regression and an eleven embryonic germline gene related signature (EGRS) was constructed. The model could robustly stratify the LUAD patients into high-risk and low-risk groups in both training and testing sets, and low-risk patients had much better outcomes. The multi-ROC analysis also showed that the EGRS model had the best predictive efficacy compared with other common clinicopathological factors. The EGRS model also showed robust predictive ability in four independent external datasets, and the area under curve (AUC) was 0.726 (GSE30219), 0.764 (GSE50081), 0.657 (GSE37745) and 0.668 (GSE72094). More importantly, the expression level of some genes in EGRS has a significant correlation with the progression of LUAD clinicopathology, suggesting these genes might play an important role in the progression of LUAD. Finally, based on EGRS genes, we built and calibrated a nomogram for conveniently evaluating patients' outcomes.

UNC5293, a potent, orally available and highly MERTK-selective inhibitor.

Inhibition of MER receptor tyrosine kinase (MERTK) causes direct tumor cell killing and stimulation of the innate immune response. Therefore, MERTK has been identified as a therapeutic target in a wide variety of human tumors. Clinical trials targeting MERTK have recently been initiated, however, none of these drugs are MERTK-specific. Herein, we present the discovery of a highly MERTK-selective inhibitor UNC5293 (24). UNC5293 has subnanomolar activity against MERTK with an excellent Ambit selectivity score (S50 (100 nM) = 0.041). It mediated potent and selective inhibition of MERTK in cell-based assays. Furthermore, it has excellent mouse PK properties (7.8 h half-life and 58% oral bioavailability) and was active in bone marrow leukemia cells in a murine model.

In vitro evaluation of the therapeutic effectiveness of EBV-LMP2 recombinant adenovirus vaccine in nasopharyngeal carcinoma.

Immunotherapeutic strategies based on Epstein-Barr virus (EBV) latent membrane protein 2 (LMP2) antigen-specific cytotoxic T lymphocytes (CTLs) have been proven to boost LMP2-specific CTL responses in patients with nasopharyngeal carcinoma (NPC). Such strategies can produce clinical benefits in some patients with NPC. Currently, the major challenge limiting the use of immunotherapy for NPC is its low clinical response rate. The efficacy of immunotherapy based on EBV-LMP2 specific CTLs depends mainly on their cytotoxic activity, but no studies have been conducted to elucidate this activity. In this study, laser confocal scanning microscopy (LCSM) and real-time cell analysis (RTCA) were used to evaluate the killing function and its underlying mechanism of LMP2-specific CTLs. LCSM showed that LMP2-specific CTLs recognize and kill target cells expressing viral escape protein LMP2, and that the killing rate is related to the number of CTLs adhering to the target cells. LMP2-specific CTL-mediated cytotoxicity is rate limited by the time required for effective contact and recognition between CTLs and target cells. RTCA showed that the protective effect of LMP2-specific CTLs required an appropriate effector-to-target ratio, and that LMP2-specific CTLs could not eradicate residual target cells at a low effector-to-target ratio. Moreover, our results revealed that LMP2-specific CTL responses involve two independent but complementary mechanisms: the perforin/granzyme and Fas/FasL pathways. Therefore, we have elucidated, for the first time, the selective cytotoxicity and mechanism by which LMP2-specific CTLs induced by the rAd-LMP2 vaccine kill target cells and have explored the killing mode and several key parameters of killing mediated by LMP2-specific CTLs. Our study will contribute to the knowledge of vaccines targeting EBV-LMP2 and to the improvement of immunotherapeutic strategies.

Identification of prognostic gene signature associated with microenvironment of lung adenocarcinoma.

BACKGROUND: Lung cancer has the highest morbidity and mortality worldwide, and lung adenocarcinoma (LADC) is the most common pathological subtype. Accumulating evidence suggests the tumor microenvironment (TME) is correlated with the tumor progress and the patient's outcome. As the major components of TME, the tumor-infiltrated immune cells and stromal cells have attracted more and more attention. In this study, differentially expressed immune and stromal signature genes were used to construct a TME-related prognostic model for predicting the outcomes of LADC patients. METHODS: The expression profiles of LADC samples with clinical information were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). The differentially expressed genes (DEGs) related to the TME of LADC were identified using TCGA dataset by Wilcoxon rank sum test. The prognostic effects of TME-related DEGs were analyzed using univariate Cox regression. Then, the least absolute shrinkage and selection operator (LASSO) regression was performed to reduce the overfit and the number of genes for further analysis. Next, the prognostic model was constructed by step multivariate Cox regression and risk score of each sample was calculated. Then, survival and Receiver Operating Characteristic (ROC) analyses were conducted to validate the model using TCGA and GEO datasets, respectively. The Kyoto Encyclopedia of Genes and Genomes analysis of gene signature was performed using Gene Set Enrichment Analysis (GSEA). Finally, the overall immune status, tumor purity and the expression profiles of HLA genes of high- and low-risk samples was further analyzed to reveal the potential mechanisms of prognostic effects of the model. RESULTS: A total of 93 TME-related DEGs were identified, of which 23 DEGs were up-regulated and 70 DEGs were down-regulated. The univariate cox analysis indicated that 23 DEGs has the prognostic effects, the hazard ratio ranged from 0.65 to 1.25 (p < 0.05). Then, seven genes were screened out from the 23 DEGs by LASSO regression method and were further analyzed by step multivariate Cox regression. Finally, a three-gene (ADAM12, Bruton Tyrosine Kinase (BTK), ERG) signature was constructed, and ADAM12, BTK can be used as independent prognostic factors. The three-gene signature well stratified the LADC patients in both training (TCGA) and testing (GEO) datasets as high-risk and low-risk groups, the 3-year area under curve (AUC) of ROC curves of three GEO sets were 0.718 (GSE3141), 0.646 (GSE30219) and 0.643 (GSE50081). The GSEA analysis indicated that highly expressed ADAM12, BTK, ERG mainly correlated with the activation of pathways involving in focal adhesion, immune regulation. The immune analysis indicated that the low-risk group has more immune activities and higher expression of HLA genes than that of the high-risk group. In sum, we identified and constructed a three TME-related DEGs signature, which could be used to predict the prognosis of LADC patients.

Bioinformatics analysis of microarray data to identify the candidate biomarkers of lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) is the major subtype of lung cancer and the most lethal malignant disease worldwide. However, the molecular mechanisms underlying LUAD are not fully understood. METHODS: Four datasets (GSE118370, GSE85841, GSE43458 and GSE32863) were obtained from the gene expression omnibus (GEO). Identification of differentially expressed genes (DEGs) and functional enrichment analysis were performed using the limma and clusterProfiler packages, respectively. A protein-protein interaction (PPI) network was constructed via Search Tool for the Retrieval of Interacting Genes (STRING) database, and the module analysis was performed by Cytoscape. Then, overall survival analysis was performed using the Kaplan-Meier curve, and prognostic candidate biomarkers were further analyzed using the Oncomine database. RESULTS: Totally, 349 DEGs were identified, including 275 downregulated and 74 upregulated genes which were significantly enriched in the biological process of extracellular structure organization, leukocyte migration and response to peptide. The mainly enriched pathways were complement and coagulation cascades, malaria and prion diseases. By extracting key modules from the PPI network, 11 hub genes were screened out. Survival analysis showed that except VSIG4, other hub genes may be involved in the development of LUAD, in which MYH10, METTL7A, FCER1G and TMOD1 have not been reported previously to correlated with LUAD. Briefly, novel hub genes identified in this study will help to deepen our understanding of the molecular mechanisms of LUAD carcinogenesis and progression, and to discover candidate targets for early detection and treatment of LUAD.

Multicomponent Catalytic Asymmetric Synthesis of trans-Aziridines.

A multicomponent trans-aziridination of aldehydes, amines, and diazo compounds with BOROX catalysts is developed. The optimal protocol is slightly different for aryl aldehydes than for aliphatic aldehydes. The key to the success with aryl aldehydes was allowing the catalyst, aldehyde, and amine to react for 20 min before addition of the diazo compound. A variety of 11 different electron-poor and electron-rich aryl aldehydes were screened to give trans-aziridines in 73-90% yield with 82-99% ee and trans/cis selectivities of 19:1 to >99:1. The optimal protocol for the trans-aziridination of aliphatic aldehydes did not require prereaction of the catalyst, aldehyde, and amine, and instead, the diazo compound could be added directly. The scope of the reaction is limited to unbranched aliphatic aldehydes and was tolerant of a number of functional groups including ethers, esters, epoxides, carbamates, and phthalimides. A total of 10 aliphatic aldehydes were examined and found to give trans-aziridines in 60-88% yield with 60-98% ee and trans/cis selectivities of 6:1 to >99:1. Alkenyl aldehydes did not react, but an alkynyl aldehyde gave a 71% yield and 95% ee of an aziridine that was found to be the cis- and not the trans-diastereomer. The aryl and aliphatic aldehydes both gave the trans-aziridines with the same absolute configuration with the same catalyst; however, in those cases where cis-aziridines were formed, the configuration was opposite for those formed from aryl versus aliphatic aldehydes.

Multicomponent cis- and trans-Aziridinatons in the Syntheses of All Four Stereoisomers of Sphinganine.

All four stereoisomers of sphinganine can be synthesized by a multicomponent aziridination of an aldehyde, an amine and an α-diazo carbonyl compound mediated by a BOROX catalyst with high asymmetric induction (≥96% ee). The threo isomers are available from ring-opening of cis-aziridines by an oxygen nucleophile with inversion at the C-3 position and the erythro-isomers are likewise available from trans-aziridines.

Catalyst-Controlled Multicomponent Aziridination of Chiral Aldehydes.

A highly diastereoselective and enantioselective method for the multicomponent aziridination of chiral aldehydes has been developed with BOROX catalysts of the VANOL (3,3'-diphenyl-2,2'-bi-1-naphthol) and VAPOL (2,2'-diphenyl-(4-biphenanthrol)) ligands. Very high to perfect catalyst control is observed with most all substrates examined including aldehydes with chiral centers in the α- and ß-positions. High catalyst control was also observed for a number of chiral heterocyclic aldehydes allowing for the preparation of epoxy aziridines, bis(aziridines) and ethylene diaziridines. Application of this reaction in the synthesis of ß3 -homo-d-alloisoleucine and ß3 -homo-l-isoleucine is reported.

Human papillomavirus as a favorable prognostic factor in a subset of head and neck squamous cell carcinomas: A meta-analysis.

Many epidemical and biological studies have proposed that human papillomavirus (HPV), primarily high-risk HPV16/18, is an etiological factor for a subset of head and neck (HN) cancers. On that premise, we systematically reviewed relevant articles and improved the understanding of HPV-related cancers. This article comprehensively described the characteristics of HPV-associated HN tumors according to demography, histopathology, molecular biology, and prognosis. Meta-analyses were conducted to combine the studies that reported the association between HPV status and these variables using Rev Man 5.0. The pooled results showed that HPV-positive tumors were not only poorly differentiated (OR = 2.77, 95% CI: 2.3-3.32) and smaller (OR = 2.21, 95% CI: 1.75-2.8) but were also strongly associated with oropharynx (OR = 5.8, 95% CI: 4.01-8.38) and node involvement (OR = 2.77, 95% CI: 2.3-3.32). HPV-related tumors showed significantly more p16 overexpression (OR = 34.55, 95% CI: 20.91-57.09) and less TP53 mutations (OR = 0.27, 95% CI: 0.18-0.41) than HPV-negative tumors. The patients with HPV-positive cancers had different clinical behaviors, such as a reduced risks of death (HR = 0.32, 95% CI: 0.29-0.36). This study supported the view point that HPV is a favorable indicator of prognosis and that HPV-related HN tumors are distinct from traditional tumors. This etiological relationship could impact future strategies of diagnosis, prevention, therapy, and prognosis for this subset of patients. J. Med. Virol. 89:710-725, 2017. © 2016 Wiley Periodicals, Inc.

Protein transduction domain can enhance the humoral immunity and cross-protection of HPV16L2 peptide vaccines.

Due to type-specificity, commercially available human papillomavirus (HPV) vaccines are only effective against homologous HPV serotypes, providing limited protection. Recent studies have highlighted the role of HPV minor capsid protein (known as L2) in inducing cross-protection. The N-terminal peptides of L2 contain conserved cross-response epitopes that can induce neutralizing antibodies against heterogeneous HPVs. However, when compared with L1, these peptides have lower immunogenicity, which limits the application of these vaccines. The protein transduction domain (PTD), located in the Tat protein of human immunodeficiency virus, facilitates delivery of DNA, peptides, proteins and virus particles into cells by unknown mechanisms, and has been reported to enhance immunogenicity of several antigens. In the present study, two peptides derived from the N-terminal of HPV16L2 were chosen as model antigens and constructed a series of L2 peptide vaccines by either fusing or mixing with PTD. Subsequently their immunogenicity was evaluated. The results indicated that the L2 peptides fused with PTD show considerably enhanced humoral immunity. In particular, they increased the titer of cross-neutralizing antibodies, while L2 peptides that had only been mixed with PTD induced only small cross-protection responses. Overall, the data suggest that fusion of L2 peptides with PTD significantly enhances their cross-protection and may be a promising strategy for the development of broad-spectrum HPV prophylactic vaccines.

miR-100 suppresses the proliferation and tumor growth of esophageal squamous cancer cells via targeting CXCR7.

MicroRNAs are highly conserved non-coding RNAs that regulate gene expression at the post-transcriptional level, and play pivotal roles in cancer development and progression. miR-100 has been reported to be significantly downregulated in a variety of cancers, including esophageal cancer. However, the role of miR-100 in human esophageal cancer has not been fully elucidated. We demonstrated that overexpression of miR-100 in esophageal cancer cells markedly inhibited cell proliferation, migration and invasion as well as tumor growth. We subsequently showed that CXCR7 is a direct target gene of miR-100. Our results indicated that miR-100 plays a tumor-suppressor role in esophageal cancer and suggest its potential application for esophageal cancer treatment.

C3-Luc Cells Are an Excellent Model for Evaluation of Cellular Immunity following HPV16L1 Vaccination.

C3 and TC-1 are the two model cell lines most commonly used in studies of vaccines and drugs against human papillomavirus (HPV) infection. Because C3 cells contain both the HPV16 E and L genes, but TC-1 cells contain only the HPV16 E genes, C3 cells are usually used as the model cell line in studies targeting the HPV16 L protein. However, expression of the L1 protein is difficult to detect in C3 cells using common methods. In our study, Short tandem repeat analysis (STR) was used to demonstrate that C3 cells are indeed derived from mice, PCR results show that HPV16 L1, E6 and E7 genes were detected in C3 genomic DNA, and RT-PCR results demonstrated that L1 transcription had occurred in C3 cells. However, the expression of C3 protein was not found in the results of western blot and immunohistochemistry (IHC). Growth and proliferation of C3 were inhibited by mice spleen lymphocytes that had been immunized with a vaccine against HPV16L1. The luciferase gene was integrated into C3 cells, and it was confirmed that addition of the exogenous gene had no effect on C3 cells by comparing cell growth and tumor formation with untransformed cells. Cells stably expressing luciferase (C3-luc) were screened and subcutaneously injected into the mice. Tumors became established and were observed using a Spectrum Pre-clinical in Vivo Imaging System. Tumor size of mice in the different groups at various time points was calculated by counting photons. The sensitivity of the animals to the vaccine was quantified by statistical comparison. Ten or 30 days following injection of the C3-luc cells, tumor size differed significantly between the PBS and vaccine groups, indicating that C3 cells were susceptible to vaccination even after tumors were formed in vivo.

One-prime multi-boost strategy immunization with recombinant DNA, adenovirus, and MVA vector vaccines expressing HPV16 L1 induces potent, sustained, and specific immune response in mice.

Human papillomavirus (HPV) is associated with various human diseases, including cancer, and developing vaccines is a cost-efficient strategy to prevent HPV-related disease. The major capsid protein L1, which an increasing number of studies have confirmed is typically expressed early in infection, is a promising antigen for such a vaccine, although the E6 and E7 proteins have been characterized more extensively. Thus, the L1 gene from HPV16 was inserted into a recombinant vector, AdHu5, and MVA viral vectors, and administered by prime-boost immunization. Virus-like particles were used as control antigens. Our results indicate that prime-boost immunization with heterologous vaccines induced robust and sustained cellular and humoral response specific to HPV16 L1. In particular, sera obtained from mice immunized with DNA + DNA + Ad + MVA had excellent antitumor activity in vivo. However, the data also confirm that virus-like particles can only elicit low levels cellular immunity and not be long-lasting, and are therefore unsuitable for treatment of existing HPV infections.

Prevalence of human papillomavirus in esophageal carcinoma in Tangshan, China.

AIM: To study the prevalence of human papillomavirus (HPV) in esophageal carcinoma in Tangshan, China, a high-incidence area. METHODS: Formalin-fixed, paraffin-embedded tissue specimens from 198 patients who were pathologically diagnosed with esophageal squamous cell carcinoma from 2011 to 2013 were obtained from a pathology department in Tangshan. DNA was extracted from all 198 specimens to detect HPV by polymerase chain reaction (PCR). ß-globin PCR was performed to check the quality of the DNA extraction procedure. PCR was performed to detect a wide range of HPV types, and type-specific PCR was performed to detect HPV types 16 and 18. Negative and positive controls were used for HPV 16 and 18 detection. RESULTS: The DNA extraction method in this study appeared to be more effective than other previously reported methods. After DNA extraction, more than 98% of the tissue specimens had an acceptable result in the DNA qualification test (ß-globin PCR). The overall prevalence of HPV in tumor tissues by GP6+/GP5+ PCR was 79.79%, and the prevalence of HPV types 16 and 18 was 40.40% and 47.47%, respectively. PCR demonstrated the presence of HPV, and direct sequencing confirmed the HPV genotypes. All HPV-positive PCR products were checked by DNA sequence analysis using DNAman and compared with the known HPV sequences listed in the Basic Local Alignment Search Tool database to evaluate the HPV types. This analysis confirmed the presence of HPV types 16 and 18. CONCLUSION: DNA of high-risk HPV types 16 and 18 is present in esophageal tumors, implicating HPV as a possible etiologic factor for esophageal squamous cell carcinoma.

Integrated HPV genomes tend to integrate in gene desert areas in the CaSki, HeLa, and SiHa cervical cancer cell lines.

AIMS: The integration preferences of human papillomavirus (HPV) have been intensively studied and contested over recent years. To disclose the integration preferences of high-risk HPV in cervical cancer, HPV transcriptional sites and features in different cervical cancer cell lines were identified. MAIN METHODS: In this study, three cervical cancer cell lines (CaSki, HeLa, and SiHa) were subjected for HPV genome status determination by amplification of papillomavirus oncogene transcripts (APOT) assay. The numbers of viral copies in human genomes and numbers of viral-human fusion mRNAs in three HPV-integrated cervical cancer cell lines were measured and analysed. KEY FINDINGS: The results revealed that the gene desert region 8q24 of the HPV type 18 integrated HeLa cell line and the 13q21-22 region of the HPV type 16 integrated CaSki and SiHa cell lines were hotspots for HPV integration, and the numbers of viral copies in the human genomes of the three cell lines that we detected were not in accordance with those reported in previous studies. SIGNIFICANCE: Integration of the HPV genome into the host cell chromosome suggests that persistent HPV infection is vital for malignant cell transformation and carcinogenesis. This study provides information to benefit health care professionals seeking more comprehensive and accurate diagnostics for HPV-related disease"? Please check, and amend as necessary.