Stepwise molecular mechanisms responsible for chemoresistance in bladder cancer cells.

Chemotherapy resistance is an obstacle to cancer therapy and is considered a major cause of recurrence. Thus, understanding the mechanisms of chemoresistance is critical to improving the prognosis of patients. Here, we have established a stepwise gemcitabine-resistant T24 bladder cancer cell line to understand the molecular mechanisms of chemoresistance within cancer cells. The characteristics of the stepwise chemoresistance cell line were divided into 4 phases (parental, early, intermediate, and late phases). These four phase cells showed increasingly aggressive phenotypes in vitro and in vivo experiments with increasing phases and revealed the molecular properties of the biological process from parent cells to phased gemcitabine-resistant cell line (GRC). Taken together, through the analysis of gene expression profile data, we have characterized gene set of each phase indicating the response to anticancer drug treatment. Specifically, we identified a multigene signature (23 genes including GATA3, APOBEC3G, NT5E, MYC, STC1, FOXD1, SMAD9) and developed a chemoresistance score consisting of that could predict eventual responsiveness to gemcitabine treatment. Our data will contribute to predicting chemoresistance and improving the prognosis of bladder cancer patients.

Anti-PTK7 Monoclonal Antibodies Exhibit Anti-Tumor Activity at the Cellular Level and in Mouse Xenograft Models of Esophageal Squamous Cell Carcinoma.

PTK7 is a catalytically defective receptor protein tyrosine kinase upregulated in various cancers, including esophageal squamous cell carcinoma (ESCC). In previous studies, we observed a positive correlation between PTK7 expression levels and tumorigenicity in various ESCC cell lines and xenograft mice with ESCC KYSE-30 cells. In this study, we analyzed the effects of anti-PTK7 monoclonal antibodies (mAbs) on the tumorigenic activity in KYSE-30 cells and in mouse xenograft models. PTK7 mAb-32 and mAb-43 bind with a high affinity to the extracellular domain of PTK7. PTK7 mAbs significantly reduced three-dimensional cell proliferation, adhesion, wound healing, and migration. PTK7 mAbs also reduce chemotactic invasiveness by decreasing MMP-9 secretion. PTK7 mAbs decreased actin cytoskeleton levels in the cortical region of KYSE-30 cells. PTK7 mAbs reduced the phosphorylation of ERK, SRC, and FAK. In a mouse xenograft model of ESCC using KYSE-30 cells, PTK7 mAbs reduced tumor growth in terms of volume, weight, and the number of Ki-67-positive cells. These results demonstrated that PTK7 mAbs can inhibit the tumorigenicity of ESCC at the cellular level and in vivo by blocking the function of PTK7. Considering the anticancer activities of PTK7 mAbs, we propose that PTK7 mAbs can be used in an effective treatment strategy for PTK7-positive malignancies, such as ESCC.

Neutralizing Human Antibodies against Severe Acute Respiratory Syndrome Coronavirus 2 Isolated from a Human Synthetic Fab Phage Display Library.

Since it was first reported in Wuhan, China, in 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic outbreak resulting in a tremendous global threat due to its unprecedented rapid spread and an absence of a prophylactic vaccine or therapeutic drugs treating the virus. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a key player in the viral entry into cells through its interaction with the angiotensin-converting enzyme 2 (ACE2) receptor protein, and the RBD has therefore been crucial as a drug target. In this study, we used phage display to develop human monoclonal antibodies (mAbs) that neutralize SARS-CoV-2. A human synthetic Fab phage display library was panned against the RBD of the SARS-CoV-2 spike protein (SARS-2 RBD), yielding ten unique Fabs with moderate apparent affinities (EC50 = 19-663 nM) for the SARS-2 RBD. All of the Fabs showed no cross-reactivity to the MERS-CoV spike protein, while three Fabs cross-reacted with the SARS-CoV spike protein. Five Fabs showed neutralizing activities in in vitro assays based on the Fabs' activities antagonizing the interaction between the SARS-2 RBD and ACE2. Reformatting the five Fabs into immunoglobulin Gs (IgGs) greatly increased their apparent affinities (KD = 0.08-1.0 nM), presumably due to the effects of avidity, without compromising their non-aggregating properties and thermal stability. Furthermore, two of the mAbs (D12 and C2) significantly showed neutralizing activities on pseudo-typed and authentic SARS-CoV-2. Given their desirable properties and neutralizing activities, we anticipate that these human anti-SARS-CoV-2 mAbs would be suitable reagents to be further developed as antibody therapeutics to treat COVID-19, as well as for diagnostics and research tools.

Selection and Characterization of YKL-40-Targeting Monoclonal Antibodies from Human Synthetic Fab Phage Display Libraries.

YKL-40, also known as chitinase-3-like 1 (CHI3L1), is a glycoprotein that is expressed and secreted by various cell types, including cancers and macrophages. Due to its implications for and upregulation in a variety of diseases, including inflammatory conditions, fibrotic disorders, and tumor growth, YKL-40 has been considered as a significant therapeutic biomarker. Here, we used a phage display to develop novel monoclonal antibodies (mAbs) targeting human YKL-40 (hYKL-40). Human synthetic antibody phage display libraries were panned against a recombinant hYKL-40 protein, yielding seven unique Fabs (Antigen-binding fragment), of which two Fabs (H1 and H2) were non-aggregating and thermally stable (75.5 °C and 76.5 °C, respectively) and had high apparent affinities (KD = 2.3 nM and 4.0 nM, respectively). Reformatting the Fabs into IgGs (Immunoglobulin Gs) increased their apparent affinities (notably, for H1 and H2, KD = 0.5 nM and 0.3 nM, respectively), presumably due to the effects of avidity, with little change to their non-aggregation property. The six anti-hYKL-40 IgGs were analyzed using a trans-well migration assay in vitro, revealing that three clones (H1, H2, and H4) were notably effective in reducing cell migration from both A549 and H460 lung cancer cell lines. The three clones were further analyzed in an in vivo animal test that assessed their anti-cancer activities, demonstrating that the tumor area and the number of tumor nodules were significantly reduced in the lung tissues treated with H1 (IgG). Given its high affinity and desirable properties, we expect that the H1 anti-hYKL-40 mAb will be a suitable candidate for developing anti-cancer therapeutics.

Targeting metastatic breast cancer with peptide epitopes derived from autocatalytic loop of Prss14/ST14 membrane serine protease and with monoclonal antibodies.

BACKGROUND: In order to develop a new immunotherapeutic agent targeting metastatic breast cancers, we chose to utilize autocatalytic feature of the membrane serine protease Prss14/ST14, a specific prognosis marker for ER negative breast cancer as a target molecule. METHODS: The study was conducted using three mouse breast cancer models, 4 T1 and E0771 mouse breast cancer cells into their syngeneic hosts, and an MMTV-PyMT transgenic mouse strain was used. Prss14/ST14 knockdown cells were used to test function in tumor growth and metastasis, peptides derived from the autocatalytic loop for activation were tested as preventive metastasis vaccine, and monoclonal and humanized antibodies to the same epitope were tested as new therapeutic candidates. ELISA, immunoprecipitation, Immunofluorescent staining, and flow cytometry were used to examine antigen binding. The functions of antibodies were tested in vitro for cell migration and in vivo for tumor growth and metastasis. RESULTS: Prss14/ST14 is critically involved in the metastasis of breast cancer and poor survival rather than primary tumor growth in two mouse models. The epitopes derived from the specific autocatalytic loop region of Prss14/ST14, based on structural modeling acted as efficient preventive metastasis vaccines in mice. A new specific monoclonal antibody mAb3F3 generated against the engineered loop structure could reduce cell migration, eliminate metastasis in PyMT mice, and can detect the Prss14/ST14 protein expressed in various human cancer cells. Humanized antibody huAb3F3 maintained the specificity and reduced the migration of human breast cancer cells in vitro. CONCLUSION: Our study demonstrates that Prss14/ST14 is an important target for modulating metastasis. Our newly developed hybridoma mAbs and humanized antibody can be further developed as new promising candidates for the use in diagnosis and in immunotherapy of human metastatic breast cancer.

Thymoquinone-Induced Tristetraprolin Inhibits Tumor Growth and Metastasis through Destabilization of MUC4 mRNA.

Tristetraprolin (TTP), a well-characterized AU-rich element (ARE) binding protein, functions as a tumor suppressor gene. The purpose of this study was to investigate whether a bioactive substance derived from a natural medicinal plant affects the induction of TTP and to elucidate its mechanism. We examined the effects of natural bioactive materials including Resveratrol (RSV), thymoquinone (TQ) and curcumin on the expression of TTP in cancer cell. TQ derived from a natural plant Nigella sativa increased the expression levels of TTP mRNA and proteins in a dose-dependent manner in gastric and breast cancer cells. TQ-induced TTP increased the instability of MUC4 mRNA by direct binding of TTP to ARE in the 3'UTR of MUC4 mRNA. The induction of TTP by TQ also reduced the proliferation, migration and invasion of cancer cells. The expression of the epithelial-mesenchymal (EMT)-related genes, which were target genes of TTP, was also decreased by the TQ treatment. In the in vivo experiments using mouse melanoma cells, TQ-induced TTP inhibited metastasis of tumor cells. We have found that TQ-induced TTP might inhibit metastasis by reducing tumor cell migration and invasion through destabilization of MUC4 mRNA, which suggest the MUC4 as a novel target to TTP.

Tristetraprolin activation by resveratrol inhibits the proliferation and metastasis of colorectal cancer cells.

Resveratrol (RSV) is a polyphenolic compound that naturally occurs in grapes, peanuts and berries. Considerable research has been conducted to determine the benefits of RSV against various human cancer types. Tristetraprolin (TTP) is an AU-rich element-binding protein that regulates mRNA stability and has decreased expression in human cancer. The present study investigated the biological effect of RSV on TTP gene regulation in colon cancer cells. RSV inhibited the proliferation and invasion/metastasis of HCT116 and SNU81 colon cancer cells. Furthermore, RSV induced a dose-dependent increase in TTP expression in HCT116 and SNU81 cells. The microarray experiment revealed that RSV significantly increased TTP expression by downregulating E2F transcription factor 1 (E2F1), a downstream target gene of TTP and regulated genes associated with inflammation, cell proliferation, cell death, angiogenesis and metastasis. Although TTP silencing inhibited TTP mRNA expression, the expression was subsequently restored by RSV. Small interfering RNA-induced TTP inhibition attenuated the effects of RSV on cell growth. In addition, RSV induced the mRNA-decaying activity of TTP and inhibited the relative luciferase activity of baculoviral IAP repeat containing 3 (cIAP2), large tumor suppressor kinase 2 (LATS2), E2F1, and lin­28 homolog A (Lin28) in HCT116 and SNU81 cells. Therefore, RSV enhanced the inhibitory activity of TTP in HCT116 and SNU81 cells by negatively regulating cIAP2, E2F1, LATS2, and Lin28 expression. In conclusion, RSV suppressed the proliferation and invasion/metastasis of colon cancer cells by activating TTP.

Association between the length of the MUC8-minisatellite 5 region and susceptibility to chronic obstructive pulmonary disease (COPD).

In asthma and chronic obstructive pulmonary disease (COPD), mucins display disease-related alterations caused by airway mucus obstruction. MUC5AC, MUC5B and MUC8 are known as the major secretory mucins in human airway epithelial cells. Analysis of mucin genes has identified the presence of several features with a variable number of tandem repeats (VNTR; minisatellites) in the central region of each mucin. In our previous study, six minisatellites in the region of the MUC8 gene were identified, and the MUC8-MS5 minisatellite showed the highest heterozygosity among them. In this study, we evaluated the relationship between MUC8-MS5 and susceptibility to asthma and COPD. A case-control study was performed with 229 controls, 123 COPD cases and 77 asthma cases. A significant association (OR 3.96) between short alleles (2/2 repeats) and the occurrence of COPD was observed [95% confidence interval (CI) 1.32-11.88; p = 0.008]. Hence, the increased frequency of 2/2 homo-short alleles were also found in asthma cases (3.11; CI 0.88-11.05; p = 0.066), though this association was not statistically significant. These results revealed a genetic association between MUC8 and COPD, and that the specific short minisatellite alleles (2/2) of MUC8-MS5 may be a risk factor for COPD.

Drug resistance of bladder cancer cells through activation of ABCG2 by FOXM1.

Recurrence is a serious problem in patients with bladder cancer. The hypothesis for recurrence was that the proliferation of drug-resistant cells was reported, and this study focused on drug resistance due to drug efflux. Previous studies have identified FOXM1 as the key gene for recurrence. We found that FOXM1 inhibition decreased drug efflux activity and increased sensitivity to Doxorubicin. Therefore, we examined whether the expression of ABC transporter gene related to drug efflux is regulated by FOXM1. As a result, ABCG2, one of the genes involved in drug efflux, has been identified as a new target for FOXM1. We also demonstrated direct transcriptional regulation of ABCG2 by FOXM1 using ChIP assay. Consequently, in the presence of the drug, FOXM1 is proposed to directly activate ABCG2 to increase the drug efflux activation and drug resistance, thereby involving chemoresistance of bladder cancer cells. Therefore, we suggest that FOXM1 and ABCG2 may be useful targets and important parameters in the treatment of bladder cancer. [BMB Reports 2018; 51(2): 98-103].

Characterization of VNTRs Within the Entire Region of SLC6A3 and Its Association with Hypertension.

The dopamine transporter SLC6A3 (DAT1) mediates uptake of dopamine into presynaptic terminals. In addition, in previous reports, hypertensive rats were associated with DAT gene, but the genetic association with SLC6A3 and hypertension is still unknown. We examined the distribution of variable number of tandem repeats (VNTRs) and conducted polymorphic analysis of the entire region of SLC6A3. Ten VNTR regions (MS1-10) were revealed throughout the intronic and UTRs; seven VNTR regions were newly isolated and three VNTRs were previously reported. Four VNTR regions (SLC6A3-MS1, -MS4, -MS8 [rs3836790], and -MS9 [rs28363170]) showed polymorphism and these loci were found to be transmitted through meiosis following Mendelian inheritance. These VNTR polymorphisms may be useful markers for paternity mapping and DNA fingerprinting. Furthermore, we also conducted a case-control study between the controls and essential hypertensive cases. Analysis of the genotypes of SLC6A3-MS8 (rs3836790) revealed that having an 8/6-repeat allele, which was only detected in hypertensive cases, was associated with hypertension (p < 0.05). Additional significant association was identified between the short 7-repeat allele of SLC6A3-MS9 (rs28363170) and the occurrence of hypertension (odds ratio 2.02; p < 0.05). These results revealed the genetic association between SLC6A3 and hypertension, and the specific VNTR alleles of SLC6A3 may be a risk factor for hypertension.

Activation of EZH2 and SUZ12 Regulated by E2F1 Predicts the Disease Progression and Aggressive Characteristics of Bladder Cancer.

PURPOSE: Previous study identified E2F1 as a key mediator of non-muscle-invasive bladder cancer (NMIBC) progression. The aim of this study was to identify the E2F1-related genes associated with poor prognosis and aggressive characteristics of bladder cancer. EXPERIMENTAL DESIGN: Microarray analysis was performed to find E2F1-related genes associated with tumor progression and aggressiveness in the gene expression data from 165 primary patients with bladder cancer. The biologic activity of E2F1-related genes in tumor progression and aggressiveness was confirmed with experimental assays using bladder cancer cells and tumor xenograft assay. RESULTS: The expression of E2F1 was significantly associated with EZH2 and SUZ12. The overexpression of E2F1, EZH2, and SUZ12 enhanced cancer progression including cell colony formation, migration, and invasiveness. Knockdown of these genes reduced motility, blocked invasion, and decreased tumor size in vivo. E2F1 bound the proximal EZH2 and SUZ12 promoter to activate transcription, suggesting that E2F1 and its downstream effectors, EZH2 and SUZ12, could be important mediators for the cancer progression. In addition, we confirmed an association between these genes and aggressive characteristics. Interestingly, the treatment of anticancer drugs to the cells overexpressing E2F1, EZH2, and SUZ12 induced the expression of CD44, KLF4, OCT4, and ABCG2 known as cancer stem cell (CSC)-related genes. CONCLUSIONS: The link between E2F1, EZH2, and/or SUZ12 revealed that E2f1 directly regulates transcription of the EZH2 and SUZ12 genes. The signature of E2F1-EZH2-SUZ12 shows a predictive value for prognosis in bladder tumors and the E2F1-EZH2-SUZ12-driven transcriptional events may regulate the cancer aggressiveness and chemo-resistance, which may provide opportunity for development of new treatment modalities.

Tristetraprolin regulates prostate cancer cell growth through suppression of E2F1.

The transcription factor E2F1 is active during G1 to S transition and is involved in the cell cycle and progression. A recent study reported that increased E2F1 is associated with DNA damage and tumor development in several tissues using transgenic models. Here, we show that E2F1 expression is regulated by tristetraprolin (TTP) in prostate cancer. Overexpression of TTP decreased the stability of E2F1 mRNA and the expression level of E2F1. In contrast, inhibition of TTP using siRNA increased the E2F1 expression. E2F1 mRNA contains three AREs within the 3'UTR, and TTP destabilized a luciferase mRNA that contained the E2F1 mRNA 3'UTR. Analyses of point mutants of the E2F1 mRNA 3'UTR demonstrated that ARE2 was mostly responsible for the TTP-mediated destabilization of E2F1 mRNA. RNA EMSA revealed that TTP binds directly to the E2F1 mRNA 3'UTR of ARE2. Moreover, treatment with siRNA against TTP increased the proliferation of PC3 human prostate cancer cells. Taken together, these results demonstrate that E2F1 mRNA is a physiological target of TTP and suggests that TTP controls proliferation as well as migration and invasion through the regulation of E2F1 mRNA stability.

Prognostic discrimination using a 70-gene signature among patients with estrogen receptor-positive breast cancer and an intermediate 21-gene recurrence score.

The Oncotype DX® recurrence score (RS) predictor has been clinically utilized to appropriately select adjuvant chemotherapy for patients with estrogen receptor (ER)-positive early breast cancer. However, the selection of chemotherapy for patients with intermediate RSs remains controversial. We assessed the prognostic value of a 70-gene signature (70GS) among patients with ER-positive breast cancer and intermediate RSs. In addition, we sought to identify genes associated with poor 70GS scores based on gene expression profiling (GEP). GEP was performed using gene expression data from 186 patients with ER-positive breast cancer. The RS and 70GS score were calculated on the basis of GEP. Among 186 patients, 82 ER-positive patients with intermediate RSs were identified. These patients were stratified by 70GS, overall survival (OS) significantly differed according to 70GS (p=0.013). In a supervised hierarchical analysis according to 70GS, the expression of several representative genes for cell proliferation was significantly higher in the poor 70GS cluster than in the good 70GS cluster. Furthermore, among these patients, FOXM1, AURKA, AURKB, and BIRC5 displayed prognostic significance for OS. In conclusion, 70GS can help to discriminate survival differences among ER-positive patients with intermediate RSs. FOXM1, AURKA, AURKB, and BIRC5, are associated with poor 70GS scores.

Association of MUC6-minisatellite variants with susceptibility to rectal carcinoma.

A secreted MUC6 mucin is reported to be expressed highly in the stomach and gall bladder. In previous our study, the five minisatellites were identified and a significant association between MUC6-MS5 alleles and gastric cancer was reported. Because of aberrant MUC6 expression is often found in gastrointestinal diseases, we evaluated a relationship between MUC6-MS5 and susceptibility to colorectal cancers. Case-control study was performed with 1,103 cancer-free controls and 414 rectal cancer cases. A significant association (OR = 2.70) between short rare MUC6-MS5 alleles (7, 9 repeats) and the occurrence of cancer was observed in rectal cancer [95 % confidence interval (CI), 1.12-6.54; p = 0.022]. Furthermore, a comparison by gender showed the differences in the association ratios between rectal cancer and short rare MUC6-MS5 alleles: male, 3.97 (CI: 1.36-11.5; p = 0.006) versus female 0.91 (CI: 0.18-4.75; p = 0.913). We also examined the association according to lymphovascular invasion (LVI). The frequency of LVI positive rectal cancer was increased in short rare allele cases than in the total rectal cases: 16.2 % versus 42.9 %. Therefore, we suggest that the short rare MUC6-MS5 alleles may be related to cancer development in male and these cancer cases may be related the bad prognosis.

Protective effect of [6]-gingerol on the ethanol-induced teratogenesis of cultured mouse embryos.

Excessive ethanol consumption during pregnancy causes fetal alcohol syndrome. We investigated the effect of [6]-gingerol on ethanol-induced embryotoxicity using a whole embryo culture system. The morphological changes of embryos and the gene expression patterns of the antioxidant enzymes cytosolic glutathione peroxidase (cGPx), cytoplasmic Cu/Zn superoxide dismutase (SOD1), and Mn-SOD (SOD2), and SOD activity were examined in the cultured mouse embryos exposed to ethanol (5 µL/3 mL) and/or [6]-gingerol (1×10(-8) or 1×10(-7) µg/mL) for 2 days. In ethanol-exposed embryos, the standard morphological score of embryos was significantly decreased compared with those of the control (vehicle) group. However, cotreatment of embryos with [6]-gingerol and ethanol significantly improved all of the developmental parameters except crownrump length and head length, compared with those of the ethanol alone group. The mRNA expression levels of cGPx and SOD2, not SOD1, were decreased consistently, SOD activity were significantly decreased compared with the control group. However, the decreases in mRNA levels of antioxidant enzymes and SOD activity were significantly restored to the control levels by [6]-gingerol supplement. These results indicate that [6]-gingerol has a protective effect against ethanol-induced teratogenicity during mouse embryogenesis.

Developmental expression of plasma glutathione peroxidase during mouse organogenesis.

Plasma glutathione peroxidase (pGPx) is an extracellular antioxidative selenoenzyme which has been detected in various adult tissues, but little is known about the expression and distribution of pGPx during embryogenesis. To investigate the expression patterns of pGPx during embryogenesis, we performed quantitative real-time PCR, in situ hybridization, Western blot, and immunohistochemistry analyses in whole embryos or each developing organ of mice on embryonic days (E)7.5-18.5. In whole embryos of E7.5-8.5, pGPx mRNA was more typically expressed in extra-embryonic tissues including ectoplacental cone, trophectoderm, and decidual cells than in embryos. However, after E9.5, pGPx mRNA and protein levels were increased in the embryos with differentiation and growth, but trended to gradually decrease in the extra-embryonic tissues until E18.5. In sectioned embryonic tissues on E13.5-18.5, pGPx mRNA and protein were mainly expressed in the developing nervous tissues, the sensory organs, and the epithelia of lung, skin, and intestine, the heart and artery, and the kidney. In particular, pGPx immunoreactivity was very strong in the developing liver. These results indicate that pGPx is spatio-temporally expressed in various embryonic organs as well as extra-embryonic tissues, suggesting that pGPx may function to protect the embryos against endogenous and exogenous reactive oxygen species during organogenesis.

Phospholipid hydroperoxide glutathione peroxidase gene is regulated via an estrogen and estrogen receptor signaling in cultured mouse fetuses.

Although it has been suggested that the transcription of phospholipid hydroperoxide glutathione peroxidase (PHGPx), an essential antioxidant selenoenzyme, may be affected by the estrogen state in mammals, the direct mechanism underlying the regulation of the PHGPx gene by estrogens in mammalian tissues remains to be clearly elucidated. In this study, we evaluated the expression of the PHGPx mRNA in cultured mouse fetuses (embryonic days 8.5-10.5) exposed to 17ß-estradiol (E(2); 0.1, 1, 10, 100, and 1,000 ng/ml); estrogen receptor (ER) agonists [propyl pyrazole triol (PPT, an ERα-selective ligand, 1 µl/ml) and diarylpropionitrile (DPN, an ERß-selective ligand, 1 µl/ml)]; and/or ER antagonist [ICI 182,780 (ICI, 1 µl/ml)] using a whole embryo culture system. E(2)-alone treatment significantly stimulated the expressions of both ERα and ERß mRNAs in all the cultured fetuses (p < 0.05), although the ERß mRNA levels were higher than ERα mRNA. PHGPx mRNA expression was significantly increased in all the fetuses treated with E(2) (1-1,000 ng/ml), PPT, and DPN (p < 0.05). Furthermore, pretreatment with ICI completely blocked the E(2)-induced PHGPx mRNA expression in the fetuses. In addition, the mRNA levels of cytosolic GPx, the other intracellular antioxidant selenoenzyme, did not differ significantly from the controls by an exposure to those agents. These results suggest that the PHGPx gene is regulated via an estrogen and ER signal pathway in the cultured mouse fetus.

Expression profiles of extracellular superoxide dismutase during mouse organogenesis.

Although extracellular superoxide dismutase (EC-SOD), which scavenges the superoxide anion in extracellular spaces, has previously been implicated in the prenatal pulmonary response to oxidative stress in the developing lungs, little is currently known regarding the schematic expression pattern and the roles played by EC-SOD during embryogenesis. In an effort to characterize the pattern of EC-SOD expression during mouse organogenesis, quantitative RT-PCR, Western blotting, and in situ hybridization analyses were conducted in mouse embryos and extraembryonic tissues including placenta on embryonic days (Eds) 7.5-18.5. EC-SOD mRNA and protein were expressed in all the embryos and extraembryonic tissues examined. The mRNA level was higher in the embryos than the extraembryonic tissues on Eds 7.5-10.5, but after Ed 13.5, it evidenced an increasing pattern in the extraembryonic tissues. EC-SOD immunoreactivity also increased in the extraembryonic tissues after Ed 13.5. During organogenesis, EC-SOD mRNA was expressed principally in the ectoplacental cone, amnion, and neural ectoderm on Ed 7.5 and in the neural folds and primitive streak on Ed 8.5. On Eds 9.5-12.5, EC-SOD mRNA was expressed abundantly in the nervous tissues and forelimb and hindlimb buds. On Eds 13.5-18.5, EC-SOD mRNA was observed at high levels in the airway epithelium of lung, liver, the intestinal epithelium, skin, vibrissae, the metanephric corpuscle of kidney, the nasal cavity, and the labyrinth trophoblast, spongiotrophoblast, and blood cells in placenta. Our overall results indicate that EC-SOD is expressed spatiotemporally in developing embryos and surrounding extraembryonic tissues during mouse organogenesis, thus suggesting that EC-SOD may be relevant to organogenesis, playing the role of an antioxidant enzyme against endogenous and exogenous oxygen stresses.

Short rare hTERT-VNTR2-2nd alleles are associated with prostate cancer susceptibility and influence gene expression.

BACKGROUND: The hTERT (human telomerase reverse transcriptase) gene contains five variable number tandem repeats (VNTR) and previous studies have described polymorphisms for hTERT-VNTR2-2nd. We investigated how allelic variation in hTERT-VNTR2-2nd may affect susceptibility to prostate cancer. METHODS: A case-control study was performed using DNA from 421 cancer-free male controls and 329 patients with prostate cancer. In addition, to determine whether the VNTR polymorphisms have a functional consequence, we examined the transcriptional levels of a reporter gene linked to these VNTRs and driven by the hTERT promoter in cell lines. RESULTS: Three new rare alleles were detected from this study, two of which were identified only in cancer subjects. A statistically significant association between rare hTERT-VNTR2-2nd alleles and risk of prostate cancer was observed [OR, 5.17; 95% confidence interval (CI), 1.09-24.43; P = 0.021]. Furthermore, the results indicated that these VNTRs inserted in the enhancer region could influence the expression of hTERT in prostate cancer cell lines. CONCLUSIONS: This is the first study to report that rare hTERT VNTRs are associated with prostate cancer predisposition and that the VNTRs can induce enhanced levels of hTERT promoter activity in prostate cancer cell lines. Thus, the hTERT-VNTR2-2nd locus may function as a modifier of prostate cancer risk by affecting gene expression.