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1.  Genome-wide Association Studies of Maximum Number of Drinks 
Journal of psychiatric research  2013;47(11):1717-1724.
Maximum number of drinks (MaxDrinks) defined as “Maximum number of alcoholic drinks consumed in a 24-hour period” is an intermediate phenotype that is closely related to alcohol dependence (AD). Family, twin and adoption studies have shown that the heritability of MaxDrinks is approximately 0.5. We conducted the first genome-wide association (GWA) study and meta-analysis of MaxDrinks as a continuous phenotype. 1059 individuals were from the Collaborative Study on the Genetics of Alcoholism (COGA) sample and 1628 individuals were from the Study of Addiction – Genetics and Environment (SAGE) sample. Family sample with 3137 individuals was from the Australian twin-family study of alcohol use disorder (OZALC). Two population-based Caucasian samples (COGA and SAGE) with 1 million single-nucleotide polymorphisms (SNPs) were used for gene discovery and one family-based Caucasian sample was used for replication. Through meta-analysis we identified 162 SNPs associated with MaxDirnks (p < 10−4). The most significant association with MaxDrinks was observed with SNP rs11128951 (p = 4.27×10−8) near SGOL1 gene at 3p24.3. Furthermore, several SNPs (rs17144687 near DTWD2, rs12108602 near NDST4, and rs2128158 in KCNB2) showed significant associations with MaxDrinks (p < 5×10−7) in the meta-analysis. Especially, 8 SNPs in DDC gene showed significant associations with MaxDrinks (p< 5×10−7) in the SAGE sample. Several flanking SNPs in above genes/regions were confirmed in the OZALC family sample. In conclusions, we identified several genes/regions associated with MaxDrinks. These findings can improve the understanding about the pathogenesis of alcohol consumption phenotypes and alcohol-related disorders.
doi:10.1016/j.jpsychires.2013.07.013
PMCID: PMC4286179  PMID: 23953852
Maximum number of drinks; Genome-wide association; Meta-analysis; SGOL1; DTWD2; NDST4; KCNB2; DDC
2.  Trp63 is regulated by STAT5 in mammary tissue and subject to differentiation in cancer 
Endocrine-related cancer  2014;21(3):443-457.
Trp63, founding member of the Trp53 family, contributes to epithelial differentiation and is expressed in breast neoplasia. Trp63 features two distinct promoters yielding specific mRNAs encoding two major TRP63 isoforms, a transactivating transcription factor and a dominant negative isoform. Specific TRP63 isoforms are linked to cell cycle arrest, apoptosis, survival and epithelial mesenchymal transition. Although TRP63 overexpression in cultured cells is used to elucidate functions, little is known about Trp63 regulation in normal and cancerous mammary tissue. This study used ChIP-seq to interrogate transcription factor binding and histone modifications of the Trp63 locus in mammary tissue and RNA-seq and immunohistochemistry to gauge gene expression. H3K4me2 and H3K4me3 marks coincided only with the proximal promoter, supporting RNA-seq data showing the predominance of the dominant negative isoform. STAT5 bound specifically to the Trp63 proximal promoter and Trp63 mRNA levels were elevated upon deleting STAT5 from mammary tissue, suggesting its role as a negative regulator. The dominant negative TRP63 isoform was localized to nuclei of basal mammary epithelial cells throughout reproductive cycles, and retained in a majority of the triple negative cancers generated from loss of full-length BRCA1. Increased expression of dominant negative isoforms was correlated with developmental windows of increased progesterone receptor binding to the proximal Trp63 promoter and decreased expression during lactation was correlated with STAT5 binding to the same region. TRP63 is present in the majority of triple negative cancers resulting from loss of BRCA1 but diminished in less differentiated cancer subtypes and in cancer cells undergoing epithelial mesenchymal transition.
doi:10.1530/ERC-14-0032
PMCID: PMC4073690  PMID: 24692510
mammary gland; gene regulation; molecular genetics; oncogene; neoplasia
3.  Ethanol extract of Portulaca Oleracea L. reduced the carbon tetrachloride induced liver injury in mice involving enhancement of NF-κB activity 
Acute hepatic injury causes high morbidity and mortality world-wide. Management of severe acute hepatic failure continues to be one of the most challenging problems in clinical medicine. In present study, carbon tetrachloride (CCl4) was used to induce acute liver damage in mice and the protective effects of ethanol extract of Portulaca Oleracea L. (PO) were examined. The aminotransferase activities were biochemical estimated and the liver damage was tested by morphological histological analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. The role of PO on the activity of NF-κB was determined by luciferase reporter gene assay and immunohistochemistry. The level of p-p65 was tested by western blot. Our results showed that PO administration on mice would decrease the serum aminotransferase level and reduced the liver histological damage. We also found that nuclear translocation of p65 was enhanced in liver tissues of mice treated with PO compared with control animals. In addition, in cultured hepatic cells, PO increased the NF-κB luciferase reporter gene activity and upregulated the level of phosphorylation of p65, but had no effects on mice liver SOD activity and MDA level. Collectively, PO attenuated CCl4 induced mice liver damage by enhancement of NF-κB activity.
PMCID: PMC4297342  PMID: 25628785
Carbon tetrachloride (CCl4); ethanol extract of Portulaca Oleracea L (PO); liver injury; NF-κB
4.  DACT2 is frequently methylated in human gastric cancer and methylation of DACT2 activated Wnt signaling 
Dapper, Dishevelled-associated antagonist of β-catenin (DACT), is a key regulator of Wnt signaling pathway. The purpose of this study is to explore the epigenetic changes and the function ofDACT2 in human gastric cancer (GC). Eight human gastric cancer cell lines, 167 cases of primary gastric cancer and 8 cases of normal gastric mucosa were involved in this study. In addition, methylation Specific PCR (MSP), semi-quantitative RT-PCR, colony formation assay, flow cytometry assay, siRNA, immunofluorescence techniques and xenograft mice models were employed. The results indicate that DACT2 is frequently methylated in human primary gastric cancer (55.7%), and that methylation of DACT2 is associated with lost or reduction in its expression (X2 test, P<0.01). We found that DACT2 expression was regulated by promoter region hypermethylation. Methylation of DACT2 is associated with tumor differentiation, invasion and intravascular cancerous emboli (X2 test, P<0.05, P<0.05 and P<0.05). In gastric cancer patients treated with 5-FU and cisplatin, the five-year survival rates are higher in DACT2 methylated cases. DACT2 inhibits cell proliferation, migration and invasion in gastric cancer cells and suppresses gastric cancer xenografts in mice. Restoration of DACT2 expression inhibits both canonical and noncanonical WNT signaling in SGC7901 cells. Restoration of DACT2 expression sensitized gastric cancer cells to paclitaxel and 5-FU. In conclusion, DACT2 is frequently methylated in human gastric cancer and DACT2 expression is silenced by promoter region hypermethylation. DACT2 suppressed gastric cancer proliferation, invasion and metastasis by inhibiting Wnt signaling both in vitro and in vivo.
PMCID: PMC4266706  PMID: 25520862
Gastric cancer; DACT2; DNA methylation; Wnt signaling pathway; chemo-sensitivity
5.  The induction of the p53 tumor suppressor protein bridges the apoptotic and autophagic signaling pathways to regulate cell death in prostate cancer cells 
Oncotarget  2014;5(21):10678-10691.
The p53 tumor suppressor protein plays a crucial role in influencing cell fate decisions in response to cellular stress. As p53 elicits cell cycle arrest, senescence or apoptosis, the integrity of the p53 pathway is considered a key determinant of anti-tumor responses. p53 can also promote autophagy, however the role of p53-dependent autophagy in chemosensitivity is poorly understood. VMY-1-103 (VMY), a dansylated analog of purvalanol B, displays rapid and potent anti-tumor activities, however the pathways by which VMY works are not fully defined. Using established prostate cancer cell lines and novel conditionally reprogrammed cells (CRCs) derived from prostate cancer patients; we have defined the mechanisms of VMY-induced prostate cancer cell death. Herein, we show that the cytotoxic effects of VMY required a p53-dependent induction of autophagy, and that inhibition of autophagy abrogated VMY-induced cell death. Cancer cell lines harboring p53 missense mutations evaded VMY toxicity and treatment with a small molecule compound that restores p53 activity re-established VMY-induced cell death. The elucidation of the molecular mechanisms governing VMY-dependent cell death in cell lines, and importantly in CRCs, provides the rationale for clinical studies of VMY, alone or in combination with p53 reactivating compounds, in human prostate cancer.
PMCID: PMC4279402  PMID: 25296977
p53; apoptosis; autophagy; primary cells; prostate
6.  Comparison of the operation of arthroscopic tibial inlay and traditional tibial inlay for posterior cruciate ligament reconstruction 
Objective: To perform dual-bundle reconstruction of posterior cruciate ligament using full arthroscopic tibial inlay technology with self-designed tibia tunnel drilling system and to compare the effect of arthroscopic tibial inlay versus traditional technique for posterior cruciate ligament reconstruction. Material and methods: 32 patients were randomly divided into experiment group (improved tibial inlay, n = 17) and control group (traditional tibial inlay, n = 15). Self-designed tibia tunnel drill system was used to produce intraoperative deep-limited bone tunnel. During follow-up, the location of the bone block and the healing situation were checked by knee X-ray and spiral CT scan. Blood loss, operation time and nerve vascular injuries were evaluated. Results: Mean intraoperative blood loss was 123.53 ± 74.05 ml in the improved tibial inlay group compared with 332 ± 114.26 ml in the traditional tibial inlay group (t = 6.12, P < 0.05). Mean operation time was 235.27 ± 58.88 min in the improved tibial inlay group compared with 346.37 ± 59.67 min in the traditional tibial inlay group (t = 5.19, P < 0.05). Posterior drawer test were negative in 15 cases, slight positive in 2 with improved tibial inlay technique compared with 14 negative cases and 2 positive cases of traditional tibial Inlay technique. The X-ray and spiral CT scan showed the location of the bone block were perfect and healed well with the patent who received improved tibial inlay technology after 12 weeks postoperatively. Conclusion: Accurate depth-limited bone tunnel can be produced by the tibia tunnel drill system with minor trauma, less bleeding and reducing of nerves or vessels and the recent clinical effects of PCL reconstruction were pretty good.
PMCID: PMC4238543  PMID: 25419349
Posterior cruciate ligament; inlays; arthroscopy
7.  Severe Sepsis Facilitates Intestinal Colonization by Extended-Spectrum-β-Lactamase-Producing Klebsiella pneumoniae and Transfer of the SHV-18 Resistance Gene to Escherichia coli during Antimicrobial Treatment 
Infections caused by multidrug-resistant pathogens are frequent and life threatening in critically ill patients. To investigate whether severe sepsis affects gut colonization by resistant pathogens and genetic exchange between opportunistic pathogens, we tested the intestinal-colonization ability of an extended-spectrum beta-lactamase-producing Klebsiella pneumoniae strain carrying the SHV-18 resistance gene and the transfer ability of the resistance gene to endogenous Escherichia coli under ceftriaxone treatment in rats with burn injury only or severe sepsis induced by burns plus endotoxin exposure. Without ceftriaxone treatment, the K. pneumoniae strain colonized the intestine in both septic and burned rats for a short time, with clearance occurring earlier in burn-only rats but never in sham burn rats. In both burned and septic rats, the colonization level of the challenge strain dropped at the beginning and then later increased during ceftriaxone treatment, after which it declined gradually. This pattern coincided with the change in resistance of K. pneumoniae to ceftriaxone during and after ceftriaxone treatment. Compared with burn-only injury, severe sepsis had a more significant effect on the change in antimicrobial resistance to ceftriaxone. Only in septic rats was the resistance gene successfully transferred from the challenge strain to endogenous E. coli during ceftriaxone treatment; the gene persisted for at least 4 weeks after ceftriaxone treatment. We concluded that severe sepsis can facilitate intestinal colonization by an exogenous resistant pathogen and the transfer of the resistance gene to a potential endogenous pathogen during antimicrobial treatment.
doi:10.1128/AAC.01632-13
PMCID: PMC3910833  PMID: 24277046
8.  Coherent Longitudinal Acoustic Phonon Approaching THz Frequency in Multilayer Molybdenum Disulphide 
Scientific Reports  2014;4:5722.
Coherent longitudinal acoustic phonon is generated and detected in multilayer Molybdenum Disulphide (MoS2) with number of layers ranging from 10 to over 1300 by femtosecond laser pulse. For thin MoS2, the excited phonon frequency exhibits a standing wave nature and shows linear dependence on the sample thickness. The frequency varies from 40 GHz to 0.2 THz (10 layers), which promises possible application in THz frequency mechanical resonators. This linear thickness dependence gradually disappears in thicker samples above about 150 layers, and the oscillation period shows linear dependence on the probe wavelength. From both the oscillation period of the coherent phonon and the delay time of acoustic echo, we can deduce a consistent sound velocity of 7.11*103 m/s in MoS2. The generation mechanisms of the coherent acoustic phonon are also discussed through pump power dependent measurement.
doi:10.1038/srep05722
PMCID: PMC4101472  PMID: 25031087
9.  Who Sits Where? Infrastructure-Free In-Vehicle Cooperative Positioning via Smartphones 
Sensors (Basel, Switzerland)  2014;14(7):11605-11628.
Seat-level positioning of a smartphone in a vehicle can provide a fine-grained context for many interesting in-vehicle applications, including driver distraction prevention, driving behavior estimation, in-vehicle services customization, etc. However, most of the existing work on in-vehicle positioning relies on special infrastructures, such as the stereo, cigarette lighter adapter or OBD (on-board diagnostic) adapter. In this work, we propose iLoc, an infrastructure-free, in-vehicle, cooperative positioning system via smartphones. iLoc does not require any extra devices and uses only embedded sensors in smartphones to determine the phones' seat-level locations in a car. In iLoc, in-vehicle smartphones automatically collect data during certain kinds of events and cooperatively determine the relative left/right and front/back locations. In addition, iLoc is tolerant to noisy data and possible sensor errors. We evaluate the performance of iLoc using experiments conducted in real driving scenarios. Results show that the positioning accuracy can reach 90% in the majority of cases and around 70% even in the worst-cases.
doi:10.3390/s140711605
PMCID: PMC4168505  PMID: 24984062
in-vehicle positioning; smartphone sensing; opportunistic sensing; signal processing
10.  Increasing effect of Tangzhiqing formula on IRS-1-dependent PI3K/AKT signaling in muscle 
Background
Tangzhiqing fomula (TZQ-F), the mixture of Red Paeony root, Mulberry leaf, Lotus leaf, Danshen root and Hawthorn leaf, regulates the abnormal glucose and lipids in prediabetic patients. In this study, we focus on the mechanism of TZQ-F and its fractions on glucose metabolism.
Methods
After orally administration of TZQ-F for 4 weeks in KK-Ay mice, we dissected out the liver and muscle, and employed PCR and western blotting to screening the PI3K/AKT pathway. The following PI3K/AKT signaling pathway were performed in L-6 myotube and HepG2 cells.
Results
In the liver of KK-Ay mice, no significance was observed on PI3K, AKT and their phosphorylation between TZQ-F and controls , while, in the muscle, up-regulation of PI3K, AKT, Glycogen synthase (GYS) and their phosphorylation type, as well as GluT4, was deteced in TZQ-F. In HepG2 cells, TZQ-F increased IRS-2 by 10 folds, without interrupting AKT, IRS-1 and GluT4. In L-6 myotube cells, TZQ-F and its fractions treatment significantly increased IRS-1 and AKT at mRNA level.
Conclusion
TZQ-F prevents pre-diabetes through increasing effect on IRS-1-dependent PI3K/AKT signaling pathway in muscle.
doi:10.1186/1472-6882-14-198
PMCID: PMC4096524  PMID: 24952587
IRS-1-dependent PI3K signaling pathway; Insulin resistance; Paeonia lactiflora; Morus alba; Nelumbo nucifera; Salvia miltiorrhiza; Crataegus pinnatifida
11.  Use of Reprogrammed Cells to Identify Therapy for Respiratory Papillomatosis 
The New England journal of medicine  2012;367(13):1220-1227.
SUMMARY
A patient with a 20-year history of recurrent respiratory papillomatosis had progressive, bilateral tumor invasion of the lung parenchyma. We used conditional reprogramming to generate cell cultures from the patient’s normal and tumorous lung tissue. Analysis revealed that the laryngeal tumor cells contained a wild-type 7.9-kb human papillomavirus virus type 11 (HPV-11) genome, whereas the pulmonary tumor cells contained a 10.4-kb genome. The increased size of the latter viral genome was due to duplication of the promoter and oncogene regions. Chemosensitivity testing identified vorinostat as a potential therapeutic agent. At 3 months after treatment initiation, tumor sizes had stabilized, with durable effects at 15 months.
doi:10.1056/NEJMoa1203055
PMCID: PMC4030597  PMID: 23013073
12.  Conditionally Reprogrammed Normal and Transformed Mouse Mammary Epithelial Cells Display a Progenitor-Cell–Like Phenotype 
PLoS ONE  2014;9(5):e97666.
Mammary epithelial (ME) cells cultured under conventional conditions senesce after several passages. Here, we demonstrate that mouse ME cells isolated from normal mammary glands or from mouse mammary tumor virus (MMTV)-Neu–induced mammary tumors, can be cultured indefinitely as conditionally reprogrammed cells (CRCs) on irradiated fibroblasts in the presence of the Rho kinase inhibitor Y-27632. Cell surface progenitor-associated markers are rapidly induced in normal mouse ME-CRCs relative to ME cells. However, the expression of certain mammary progenitor subpopulations, such as CD49f+ ESA+ CD44+, drops significantly in later passages. Nevertheless, mouse ME-CRCs grown in a three-dimensional extracellular matrix gave rise to mammary acinar structures. ME-CRCs isolated from MMTV-Neu transgenic mouse mammary tumors express high levels of HER2/neu, as well as tumor-initiating cell markers, such as CD44+, CD49f+, and ESA+ (EpCam). These patterns of expression are sustained in later CRC passages. Early and late passage ME-CRCs from MMTV-Neu tumors that were implanted in the mammary fat pads of syngeneic or nude mice developed vascular tumors that metastasized within 6 weeks of transplantation. Importantly, the histopathology of these tumors was indistinguishable from that of the parental tumors that develop in the MMTV-Neu mice. Application of the CRC system to mouse mammary epithelial cells provides an attractive model system to study the genetics and phenotype of normal and transformed mouse epithelium in a defined culture environment and in vivo transplant studies.
doi:10.1371/journal.pone.0097666
PMCID: PMC4022745  PMID: 24831228
13.  Epigenetic regulation of the Wnt signaling inhibitor DACT2 in human hepatocellular carcinoma 
Epigenetics  2013;8(4):373-382.
DACT2 (Dapper, Dishevelled-associated antagonist of β-catenin homolog 2) is a member of the DACT family involved in the regulation of embryonic development. Human DACT2 is localized on 6q27, a region of frequent loss of heterozygosity in human cancers. However, the regulation of DACT2 expression and function in hepatocellular carcinoma (HCC) remains unclear. In this study, genetic and epigenetic changes of DACT2 were analyzed in HCC cell lines and primary cancer. We found no single-nucleotide polymorphism (SNP) associated with HCC. Promoter region methylation was correlated with loss or reduction of DACT2 expression, and restoration of DACT2 expression was induced by 5-aza-2’-deoxycytidine (5-AZA) in HCC cell lines. Promoter region methylation was found in 54.84% of primary HCC. Reduction of DACT2 expression was associated with promoter hypermethylation, and expression of DACT2 was inversely related to β-catenin expression in primary HCC. DACT2 suppressed cell proliferation, induced G2-M arrest in cell lines and inhibited tumor growth in xenograft nude mice. The transcriptional activity of TCF-4 and the expression of Wnt signaling downstream genes were suppressed by DACT2 re-expression and reactivated by depletion of DACT2. In conclusion, DACT2 is frequently methylated in HCC and its expression is regulated by promoter hypermethylation. DACT2 suppresses HCC by inhibiting Wnt signaling in human HCC.
doi:10.4161/epi.24113
PMCID: PMC3674046  PMID: 23449122
DACT2; hepatocellular carcinoma; epigenetics; DNA methylation; Wnt signaling
14.  Discovery of a Strongly-Interrelated Gene Network in Corals under Constant Darkness by Correlation Analysis after Wavelet Transform on Complex Network Model 
PLoS ONE  2014;9(3):e92434.
Coral reefs occupy a relatively small portion of sea area, yet serve as a crucial source of biodiversity by establishing harmonious ecosystems with marine plants and animals. Previous researches mainly focused on screening several key genes induced by stress. Here we proposed a novel method—correlation analysis after wavelet transform of complex network model, to explore the effect of light on gene expression in the coral Acropora millepora based on microarray data. In this method, wavelet transform and the conception of complex network were adopted, and 50 key genes with large differences were finally captured, including both annotated genes and novel genes without accurate annotation. These results shed light on our understanding of coral's response toward light changes and the genome-wide interaction among genes under the control of biorhythm, and hence help us to better protect the coral reef ecosystems. Further studies are needed to explore how functional connections are related to structural connections, and how connectivity arises from the interactions within and between different systems. The method introduced in this study for analyzing microarray data will allow researchers to explore genome-wide interaction network with their own dataset and understand the relevant biological processes.
doi:10.1371/journal.pone.0092434
PMCID: PMC3961355  PMID: 24651851
15.  Regulation of Autophagy Via PERK-eIF2α Effectively Relieve the Radiation Myelitis Induced by Iodine-125 
PLoS ONE  2013;8(11):e76819.
Radiation myelitis is the most serious complication in clinical radiotherapy for spinal metastases. We previously showed that 125I brachytherapy induced apoptosis of spinal cord neurons accompanied by autophagy. In this study, we further investigated the mechanism by which 125I radiation triggered autophagy in neural cells. We found that autophagy induced by 125I radiation was involved in endoplasmic reticulum (ER) stress and mainly dependent on PERK-eIF2α pathway. The expressions of LC3II, ATG12 and PI3K were significantly suppressed in PERK knockout neural cells. Meanwhile, the expressions of phosphorylated-Akt s473 and caspase3/8 all significantly increased in neural cells transfected with a PERK siRNA and which enhanced apoptosis of neurons after 125I radiation. The results were consistent with that by MTT and Annexin-FITC/PT staining. In annimal model of banna pigs with radiation myelitis caused by 125I brachytherapy, we have successfully decreased PERK expression by intrathecal administration of the lentivirus vector. The apoptosis rate was significantly higher than that in control group and which deteriorated radiation myelitis of banna pigs. Thus, autophagy caused by 125I radiation was mainly as an attempt of cell survival at an early stage, but it would be a self-destructive process and promoted the process of apoptosis and necrosis radiated by 125I for more than 72 hours. The study would be useful and helpful to maximize efficiency of radiation therapy in clinical therapy.
doi:10.1371/journal.pone.0076819
PMCID: PMC3818370  PMID: 24223705
16.  Genome-Wide Identification of Bone Metastasis-Related MicroRNAs in Lung Adenocarcinoma by High-Throughput Sequencing 
PLoS ONE  2013;8(4):e61212.
Background
MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression at the post-transcriptional level. They participate in a wide variety of biological processes, including apoptosis, proliferation and metastasis. The aberrant expression of miRNAs has been found to play an important role in many cancers.
Results
To understand the roles of miRNAs in the bone metastasis of lung adenocarcinoma, we constructed two small RNA libraries from blood of lung adenocarcinoma patients with and without bone metastasis. High-throughput sequencing combined with differential expression analysis identified that 7 microRNAs were down-regulated and 21 microRNAs were up-regulated in lung adenocarcinoma with bone metastasis. A total of 797 target genes of the differentially expressed microRNAs were identified using a bioinformatics approach. Functional annotation analysis indicated that a number of pathways might be involved in bone metastasis, survival of the primary origin and metastatic angiogenesis of lung adenocarcinoma. These include the MAPK, Wnt, and NF-kappaB signaling pathways, as well as pathways involving the matrix metalloproteinase, cytoskeletal protein and angiogenesis factors.
Conclusions
This study provides some insights into the molecular mechanisms that underlie lung adenocarcinoma development, thereby aiding the diagnosis and treatment of the disease.
doi:10.1371/journal.pone.0061212
PMCID: PMC3620207  PMID: 23593434
17.  HPV16 E7 Protein and hTERT Proteins Defective for Telomere Maintenance Cooperate to Immortalize Human Keratinocytes 
PLoS Pathogens  2013;9(4):e1003284.
Previous studies have shown that wild-type human telomerase reverse transcriptase (hTERT) protein can functionally replace the human papillomavirus type 16 (HPV-16) E6 protein, which cooperates with the viral E7 protein in the immortalization of primary keratinocytes. In the current study, we made the surprising finding that catalytically inactive hTERT (hTERT-D868A), elongation-defective hTERT (hTERT-HA), and telomere recruitment-defective hTERT (hTERT N+T) also cooperate with E7 in mediating bypass of the senescence blockade and effecting cell immortalization. This suggests that hTERT has activities independent of its telomere maintenance functions that mediate transit across this restriction point. Since hTERT has been shown to have a role in gene activation, we performed microarray studies and discovered that E6, hTERT and mutant hTERT proteins altered the expression of highly overlapping sets of cellular genes. Most important, the E6 and hTERT proteins induced mRNA and protein levels of Bmi1, the core subunit of the Polycomb Group (PcG) complex 1. We show further that Bmi1 substitutes for E6 or hTERT in cell immortalization. Finally, tissue array studies demonstrated that expression of Bmi1 increased with the severity of cervical dysplasia, suggesting a potential role in the progression of cervical cancer. Together, these data demonstrate that hTERT has extra-telomeric activities that facilitate cell immortalization and that its induction of Bmi1 is one potential mechanism for mediating this activity.
Author Summary
The human papillomaviruses (HPVs) are critical elements in the etiology of cervical cancer, as well as several other human cancers. The E6 protein, in combination with the E7 protein of these viruses, immortalizes epithelial cells and increases the expression of the hTERT protein. In the current study we show that the enzymatic activity of hTERT is not required for cooperating in cell immortalization. We further demonstrate that hTERT proteins increase the expression of the Bmi1 protein, which is also capable of cooperating in cell immortalization. We anticipate that these findings will stimulate new studies of telomerase in HPV biology, cancer etiology, and stem cell reprogramming.
doi:10.1371/journal.ppat.1003284
PMCID: PMC3617164  PMID: 23592995
18.  Quantitative Measurement of Human Papillomavirus Type 16 E5 Oncoprotein Levels in Epithelial Cell Lines by Mass Spectrometry 
Journal of Virology  2012;86(17):9465-9473.
The high-risk human papillomavirus type 16 (HPV-16) E5 protein (16E5) induces tumors in a transgenic mouse model and may contribute to early stages of cervical carcinogenesis. Although high-risk E5 expression is generally thought to be lost during the progression to cervical carcinoma following integration of HPV DNA into the host genome, episomal viral DNA has been documented in a subset of HPV-16-positive malignant lesions. Numerous studies have shown that transcripts that could potentially encode 16E5 are present in cervical biopsy specimens and cervical cancer cell lines, but the presence of E5 protein has been demonstrated in only two reports that have not been corroborated. In the present study, we show that trypsin cleavage of 16E5 generates a unique four-amino-acid C-terminal peptide (FLIT) that serves as a marker for E5 expression in transfected cells and epithelial cell lines containing integrated and episomal HPV-16 DNA. Following trypsin cleavage, reversed-phase chromatography and mass spectrometry (MS) were used to detect FLIT. Immunoprecipitation assays using a newly generated anti-16E5 antibody confirmed that 16E5 was solely responsible for the FLIT signal, and deuterated FLIT peptide provided an internal standard that enabled us to quantify the number of 16E5 molecules per cell. We show that 16E5 is expressed in the Caski but not in the SiHa cervical cancer cell line, suggesting that 16E5 may contribute to the malignant phenotype of some cervical cancers, even in cells exclusively containing an integrated HPV genome.
doi:10.1128/JVI.01032-12
PMCID: PMC3416135  PMID: 22740411
19.  Percutaneous vertebroplasty combined with interstitial implantation of 125I seeds in banna mini-pigs 
Background
Although brachytherapy is one of the most effective ways to treat metastatic spinal tumor with little damage to surrounding healthy tissue, it may cause radiation myelopathy if an overdose occurs. Establishing a valuable animal model can help to find a method to overcome its complications. In the current study, we set up a banna mini-pig model to mimic percutaneous vertebroplasty with 125I seed implantation.
Methods
Percutaneous vertebroplasty (PVP) combined with interstitial implantation of 125I seeds, 125I seeds were transplanted into the vertebral body at the T13 level of the spine in banna mini-pigs. After raising them for up to eight months, the spinal cord and vertebral body were collected for pathological analysis.
Results
A potential animal model had been successfully established, no case of radiation myelopathy was found in any of the treated banna pigs, and no significant cellular impairment was noted by pathological analysis.
Conclusions
It proves that PVP with 125I brachytherapy is an effective method to treat metastasis spinal tumor, and that the banna mini-pig can be a suitable model to investigate the mechanism of brachytherapy complications.
doi:10.1186/1477-7819-11-46
PMCID: PMC3598515  PMID: 23442259
Percutaneous vertebroplasty; Brachytherapy; 125I seeds; Radiation myelopathy
20.  Immortalization and transformation of human mammary epithelial cells by a tumor-derived Myc mutant 
The Myc transcription factor is commonly dysregulated in many human cancers, including breast carcinomas. However, the precise role of Myc in the initiation and maintenance of malignancy is unclear. In this study we compared the ability of wild-type Myc (wt Myc) or Myc phosphorylation deficient mutants (T58A, S62A or T58A/S62A) to immortalize and transform human mammary epithelial cells (HMECs). All Myc constructs promoted cellular immortalization. As previously reported in other cells, the Myc T58A mutant tempered apoptotic responses and increased Myc protein stability in HMEC cells. More importantly, we now show that HMECs overexpressing the Myc T58A mutant acquire a unique cellular phenotype characterized by cell aggregation, detachment from the substrate and growth in liquid suspension. Coincident with these changes, the cells become anchorage-independent for growth in agarose. Previous studies have shown that wt Myc can collaborate with hTERT in inducing HMEC anchorage-independent growth. We have verified this observation and further shown that Myc T58A was a stronger facilitator of such co-transformation. Thus, our findings indicate that differences in Myc protein phosphorylation modulate its biological activity in human breast epithelial cells and specifically that the T58A mutation can facilitate both cellular immortalization and transformation. Finally, we used the isogenic cell lines generated in this study to identify a subset of genes whose expression is greatly altered during the transition from the immortal to the anchorage-independent states.
doi:10.1007/s10549-008-0127-x
PMCID: PMC3576819  PMID: 18642118
Mammary cells; Myc; immortalization; anchorage-independent growth; phosphorylation
21.  The E6 Oncoprotein from HPV16 Enhances the Canonical Wnt/β-catenin Pathway in Skin Epidermis in vivo 
Molecular Cancer Research  2011;10(2):250-258.
The contribution of the Wnt signaling pathway to HPV-induced carcinogenesis is poorly understood. In high-grade dysplastic lesions that are caused by high-risk human papilloma viruses (HR-HPVs), β-catenin is often located in the cell nucleus, which suggests that Wnt pathway may be involved in the development of HPV-related carcinomas. Most of the oncogenic potential of HR-HPVs resides on the E6 protein’s PDZ-binding domain. We hypothesized that the PDZ-binding domain of the HPV16-E6 oncoprotein induces the nuclear accumulation of β-catenin due to its capacity to degrade PDZ-containing cellular targets. To test this hypothesis, we evaluated the staining pattern of β-catenin in the skin epidermis of transgenic mice expressing the full-length E6 oncoprotein (K14E6 mice) and measured LacZ gene expression in K14E6 mice that were crossed with a strain expressing LacZ that was knocked into the Axin2 locus (Axin2+/LacZ mice). Here, we show that the E6 oncoprotein enhances the nuclear accumulation of β-catenin, the accumulation of cellularβ-catenin-responsive genes and the expression of LacZ. None of these effects were observed when a truncated E6 oncoprotein that lacks the PDZ-binding domain was expressed alone (K14E6ΔPDZ mice) or in combination with Axin2+/LacZ. Conversely, co-transfection with either E6 or E6ΔPDZ similarly enhanced canonical Wnt signaling in short-term in vitro assays that utilized a luciferase Wnt/β-catenin/TCF-dependent promoter. We propose that the activation of canonical Wnt signaling could be induced by the HPV16-E6 oncoprotein; however, the participation of the E6 PDZ-binding domain seems to be important in in vivo models only.
doi:10.1158/1541-7786.MCR-11-0287
PMCID: PMC3332097  PMID: 22160870
HPV; Wnt/β-catenin; K14E6; K14E6-ΔPDZ; Axin2+/LacZ
22.  Two-dimensional ultrathin gold film composed of steadily linked dense nanoparticle with surface plasmon resonance 
Nanoscale Research Letters  2012;7(1):683.
Background
Noble metallic nanoparticles have prominent optical local-field enhancement and light trapping properties in the visible light region resulting from surface plasmon resonances.
Results
We investigate the optical spectral properties and the surface-enhanced Raman spectroscopy of two-dimensional distinctive continuous ultrathin gold nanofilms. Experimental results show that the one- or two-layer nanofilm obviously increases absorbance in PEDOT:PSS and P3HT:PCBM layers and the gold nanofilm acquires high Raman-enhancing capability.
Conclusions
The fabricated novel structure of the continuous ultrathin gold nanofilms possesses high surface plasmon resonance properties and boasts a high surface-enhanced Raman scattering (SERS) enhancement factor, which can be a robust and cost-efficient SERS substrate. Interestingly, owing to the distinctive morphology and high light transmittance, the peculiar nanofilm can be used in multilayer photovoltaic devices to trap light without affecting the physical thickness of solar photovoltaic absorber layers and yielding new options for solar cell design.
doi:10.1186/1556-276X-7-683
PMCID: PMC3552831  PMID: 23259927
Ultrathin gold film; Surface plasmon resonance; SERS
23.  Gold nanoparticle thin films fabricated by electrophoretic deposition method for highly sensitive SERS application 
Nanoscale Research Letters  2012;7(1):613.
We report an electrophoretic deposition method for the fabrication of gold nanoparticle (GNP) thin films as sensitive surface-enhanced Raman scattering (SERS) substrates. In this method, GNP sol, synthesized by a seed-mediated growth approach, and indium tin oxide (ITO) glass substrates were utilized as an electrophoretic solution and electrodes, respectively. From the scanning electron microscopy analysis, we found that the density of GNPs deposited on ITO glass substrates increases with prolonged electrophoresis time. The films possess high mechanical adhesion strength and exhibit strong localized surface plasmon resonance (LSPR) effect by showing high SERS sensitivity to detect 1 × 10−7 M rhodamine 6 G in methanol solution. Finally, the relationship between Raman signal amplification capability and GNP deposition density has been further investigated. The results of our experiment indicate that the high-density GNP film shows relatively higher signal amplification capability due to the strong LSPR effect in narrow gap regions between the neighboring particles on the film.
doi:10.1186/1556-276X-7-613
PMCID: PMC3502474  PMID: 23130848
Gold nanoparticle; Electrophoretic deposition; SERS
24.  Fabrication and spectroscopic investigation of branched silver nanowires and nanomeshworks 
Nanoscale Research Letters  2012;7(1):596.
Wide wavelength ranges of light localization and scattering characteristics can be attributed to shape-dependent longitude surface plasmon resonance in complicated nanostructures. We have studied this phenomenon by spectroscopic measurement and a three-dimensional numerical simulation, for the first time, on the high-density branched silver nanowires and nanomeshworks at room temperature. These nanostructures were fabricated with simple light-induced colloidal method. In the range from the visible to the near-infrared wavelengths, light has been found effectively trapped in those trapping sites which were randomly distributed at the corners, the branches, and the junctions of the nanostructures in those nanostructures in three dimensions. The broadened bandwidth electromagnetic field enhancement property makes these branched nanostructures useful in optical processing and photovoltaic applications.
doi:10.1186/1556-276X-7-596
PMCID: PMC3577568  PMID: 23101991
Silver Nanowires; Nanomeshworks; Branched nanostructures; Localized surface plasmon resonance; Hot spots; Bandwidth
25.  Intron Definition and a Branch Site Adenosine at nt 385 Control RNA Splicing of HPV16 E6*I and E7 Expression 
PLoS ONE  2012;7(10):e46412.
HPV16 E6 and E7, two viral oncogenes, are expressed from a single bicistronic pre-mRNA. In this report, we provide the evidence that the bicistronic pre-mRNA intron 1 contains three 5′ splice sites (5′ ss) and three 3′ splice sites (3′ ss) normally used in HPV16+ cervical cancer and its derived cell lines. The choice of two novel alternative 5′ ss (nt 221 5′ ss and nt 191 5′ ss) produces two novel isoforms of E6E7 mRNAs (E6*V and E6*VI). The nt 226 5′ ss and nt 409 3′ ss is preferentially selected over the other splice sites crossing over the intron to excise a minimal length of the intron in RNA splicing. We identified AACAAAC as the preferred branch point sequence (BPS) and an adenosine at nt 385 (underlined) in the BPS as a branch site to dictate the selection of the nt 409 3′ ss for E6*I splicing and E7 expression. Introduction of point mutations into the mapped BPS led to reduced U2 binding to the BPS and thereby inhibition of the second step of E6E7 splicing at the nt 409 3′ ss. Importantly, the E6E7 bicistronic RNA with a mutant BPS and inefficient splicing makes little or no E7 and the resulted E6 with mutations of 91QYNK94 to 91PSFW94 displays attenuate activity on p53 degradation. Together, our data provide structural basis of the E6E7 intron 1 for better understanding of how viral E6 and E7 expression is regulated by alternative RNA splicing. This study elucidates for the first time a mapped branch point in HPV16 genome involved in viral oncogene expression.
doi:10.1371/journal.pone.0046412
PMCID: PMC3464268  PMID: 23056301

Results 1-25 (38)