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A sensitive surface enhanced Raman spectroscopy (SERS) substrate based on SiO2@Ag core-shell with a QuEChERs pretreatment method were constructed for the determination of carbendazim residue in tobacco leaves. The SERS substrate was formed by self-assembly of monolayer homogeneous SiO2 nanospheres onto silicon wafer following by vacuum thermal evaporation of silver film. The QuEChERs pretreatment method was used to eliminate the interfering effects of pigments and organic acids in tobacco leaves. The results for carbendazim in tobacco leaves show the limit of detection (LOD) of the SERS substrate was 0.1 mg/kg and the recovery rate was from 82.56% to 95.93%. The proposed low-cost sensitive SERS substrates provide potential application for tobacco safety monitoring. [ABSTRACT FROM AUTHOR]

Simple Summary: The clinical treatment of acute myeloid leukaemia is still dominated by chemotherapy. Clinically used anti-leukaemia drugs have shortcomings such as myelosuppression, toxicity and drug resistance. Therefore, the need to develop other chemotherapeutic drugs to meet more clinical needs is urgent. Ribonucleotide reductase (RNR) consists of a catalytic large subunit M1 (RRM1) and a regulatory small subunit M2 (RRM2), which provides dNTPs for DNA synthesis. The rapid proliferation of cancer cells requires large amounts of dNTPs. Therefore, the use of RNR inhibitors is a promising strategy for the clinical treatment of various malignancies. Monobenzone is an FDA-approved depigmenting agent for vitiligo patients. In this study, we demonstrate that monobenzone is a potent inhibitor of RNR enzyme activity by targeting RRM2 protein, and thus has significant anti-leukaemia efficacy in vitro and in vivo. This finding suggests that monobenzone has the potential to be optimized as a novel anti-AML therapeutic drug in the future. Acute myeloid leukaemia (AML) is one of the most common types of haematopoietic malignancy. Ribonucleotide reductase (RNR) is a key enzyme required for DNA synthesis and cell proliferation, and its small subunit RRM2 plays a key role for the enzymatic activity. We predicted monobenzone (MB) as a potential RRM2 target compound based on the crystal structure of RRM2. In vitro, MB inhibited recombinant RNR activity (IC50 = 0.25 μM). Microscale thermophoresis indicated that MB inhibited RNR activity by binding to RRM2. MB inhibited cell proliferation (MTT IC50 = 6–18 μM) and caused dose-dependent DNA synthesis inhibition, cell cycle arrest, and apoptosis in AML cells. The cell cycle arrest was reversed by the addition of deoxyribonucleoside triphosphates precursors, suggesting that RNR was the intracellular target of the compound. Moreover, MB overcame drug resistance to the common AML drugs cytarabine and doxorubicin, and treatment with the combination of MB and the Bcl-2 inhibitor ABT-737 exerted a synergistic inhibitory effect. Finally, the nude mice xenografts study indicated that MB administration produced a significant inhibitory effect on AML growth with relatively weak toxicity. Thus, we propose that MB has the potential as a novel anti-AML therapeutic agent in the future. [ABSTRACT FROM AUTHOR]

A rapid method for determination of parabens preservatives (methyl paraben, ethyl paraben, isopropyl paraben, propyl paraben, isobutyl paraben, and butyl paraben) in flavors was established by using supercritical fluid chromatography‐tandem mass spectrometry combined with dispersive solid‐phase extraction. After adding methanol and primary secondary amine to the sample simultaneously, high extraction efficiency and good sample cleanup could be obtained by simple shaking. Parabens were well separated on a Chiralpak IG‐3 column in 6 min by gradient elution. Recoveries from spiked blank samples at 0.5, 1.0, and 5.0 mg/kg were determined to be 88.3–106.6%with relative standard deviations less than 8.0%. All analytes achieved good linear relation (r ≥ 0.999 2). The limits of detection for all analytes ranged from 0.03 to 0.09 mg/kg and the limits of quantification from 0.11 to 0.31 mg/kg, respectively. A total of 20 actual samples were successfully analyzed by taking the proposed method. Being simple, rapid, green, and reliable, this method can be taken for the determination of parabens preservatives in flavors. [ABSTRACT FROM AUTHOR]

A novel method has been developed for the simultaneous determination of multiclass pesticide residues in tobacco and cigarette smoke, using a modified QuEChERS (quick, easy, cheap, effective, rugged and safe) procedure and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). Cigarette mainstream smoke particulate was collected on a Cambridge filter pad. Pesticide residues was extracted with an aqueous solution, back extracted into acetonitrile after freezing, purified by dispersive solid phase extraction with primary-secondary amine adsorbents and analyzed by UPLC–MS/MS. The obtained mean recoveries of 16 pesticides commonly used on tobacco at three fortification levels (5.9, 94.1 and 352.9 ng g−1) ranged from 69.3 to 115.9% with relative standard deviations between 2.4 and 11.3%. The limits of detection ranged from 0.14 to 13.28 ng g−1. Finally, the proposed method was applied to study the pesticide smoke transfer ratio in 2 cigarettes with pesticide standard spiked and 51 cigarettes with one or more pesticide residues. The transfer ratio of pesticides residue in tobacco into the smoke might be much less than that from artificially spiked tobacco (<25%) with spiking levels varied from 1.88 to 9.41 μg g−1. The transfer ratio of pesticide from artificially spiked tobacco into cigarette mainstream smoke was from 0.0 to 56.5%, and pesticide residues from tobacco into cigarette smoke were from 0.0 to 26.1% using the ISO smoking method (ISO 3308 2012). [ABSTRACT FROM AUTHOR]

Aims: Alternaria longipes is a causal agent of brown spot of tobacco, which remains a serious threat to tobacco production. Herein, we established a detection method for A. longipes in tobacco samples based on the principle of time‐resolved fluoroimmunoassay, in order to fulfil the requirement of rapid, sensitive and accurate detection in situ. Methods and Results: A monoclonal antibody against A. longipes was generated, and its purity and titration were assessed using western blot and ELISA. The size of europium (III) nanospheres was measured to confirm successful antibody conjugation. The method described here can detect A. longipes protein lysates as low as 0.78 ng ml−1, with recovery rates ranging from 85.96% to 99.67% in spiked tobacco. The specificity was also confirmed using a panel of microorganisms. Conclusions: The fluorescent strips allow rapid and sensitive onsite detection of A. longipes in tobacco samples, with high accuracy, specificity, and repeatability. Significance and Impact of the Study: This novel detection method provides convenience of using crude samples without complex procedures, and therefore allows rapid onsite detection by end users and quick responses towards A. longipes, which is critical for disease control and elimination of phytopathogens. [ABSTRACT FROM AUTHOR]

Polycyclic aromatic hydrocarbons (PAHs), particularly benzo[a]pyrene (B[a]P), found in cigarette smoke and air pollution, is an important carcinogen. Nevertheless, early molecular events and related regulatory effects of B[a]P-mediated cell transformation and tumor initiation remain unclear. This study found that EGR1 was significantly downregulated during human bronchial epithelial cell transformation and mice lung carcinogenesis upon exposure to B[a]P and its active form BPDE, respectively. In contrast, overexpression of EGR1 inhibited the BPDE-induced cell malignant transformation. Moreover, miR-377-3p was strongly enhanced by BPDE/B[a]P exposure and crucial for the inhibition of EGR1 expression by targeting the 3'UTR of EGR1. MiR-377-3p antagomir reversed the effect of EGR1 downregulation in cell malignant transformation and tumor initiation models. Furthermore, the B[a]P-induced molecular changes were evaluated by IHC in clinical lung cancer tissues and examined with a clinic database. Mechanistically, EGR1 inhibition was also involved in the regulation of Wnt/β-catenin transduction, promoting lung tumorigenesis following B[a]P/BPDE exposure. Taken together, the results demonstrated that bBenzo[a]pyrene exposure might induce lung tumorigenesis through miR-377-3p-mediated reduction of EGR1 expression, suggesting an important role of EGR1 in PAHs-induced lung carcinogenesis. [ABSTRACT FROM AUTHOR]

Liver ageing not only damages liver function but also harms systemic metabolism. To better understand the mechanisms underlying liver ageing, we transplanted the livers of young rats to young and old rats and performed untargeted metabolomics to detect changes in the metabolites in the liver tissues and sera. A total of 153 metabolites in the livers and 83 metabolites in the sera were different between the old and young rats that did not undergo liver transplantation; among these metabolites, 7 different metabolites were observed in both the livers and sera. Five weeks after liver transplantation, the levels of 25 metabolites in the young donor livers were similar to those in the old rats, and this result probably occurred due to the effect of the whole‐body environment of the older recipients on the young livers. The 25 altered metabolites included organic acids and derivatives, lipids and lipid‐like molecules, etc. In the sera, the differences in 78 metabolites, which were significant between the young and old rats, were insignificant in the old recipient rats and made the metabolic profile of the old recipients more similar to that of the young recipients. Finally, combining the above metabolomic data with the transcriptomic data from the GEO, we found that the altered metabolites and genes in the liver were enriched in 9 metabolic pathways, including glycerophospholipid, arachidonic acid, histidine and linoleate. Thus, this study revealed important age‐related metabolites and potential pathways as well as the interaction between the liver and the whole‐body environment. [ABSTRACT FROM AUTHOR]

Mining cancer-omics databases deepens our understanding of cancer biology and can lead to potential breakthroughs in cancer treatment. Here, we propose an integrative analytical approach to reveal across-cancer expression patterns and identify potential clinical impacts for genes of interest from five representative public databases. Using ribonucleotide reductase (RR), a key enzyme in DNA synthesis and cancer-therapeutic targeting, as an example, we characterized the mRNA expression profiles and inter-component associations of three RR subunit genes and assess their differing pathological and prognostic significance across over 30-types of cancers and their related subtypes. Findings were validated by immunohistochemistry with clinical tissue samples (n = 211) collected from multiple cancer centers in China and with clinical follow-up. Underlying mechanisms were further explored and discussed using co-expression gene network analyses. This framework represents a simple, efficient, accurate, and comprehensive approach for cancer-omics resource analysis and underlines the necessity to separate the tumors by their histological or pathological subtypes during the clinical evaluation of molecular biomarkers. [ABSTRACT FROM AUTHOR]

Liver metastases develop in more than half of the patients with colorectal cancer (CRC) and are associated with a poor prognosis. The factors influencing liver metastasis of CRC are poorly characterized, but this information is urgently needed. We have now discovered that small extracellular vesicles (sEVs; exosomes) derived from CRC can be specifically targeted to liver tissue and induce liver macrophage polarization toward an interleukin-6 (IL-6)-secreting proinflammatory phenotype. More importantly, we found that microRNA-21-5p (miR-21) was highly enriched in CRC-derived sEVs and was essential for creating a liver proinflammatory phenotype and liver metastasis of CRC. Silencing either miR-21 in CRC-sEVs or Toll-like receptor 7 (TLR7) in macrophages, to which miR-21 binds, abolished CRC-sEVs' induction of proinflammatory macrophages. Furthermore, miR-21 expression in plasma-derived sEVs was positively correlated with liver metastasis in CRC patients. Collectively, our data demonstrate a pivotal role of CRC-sEVs in promoting liver metastasis by inducing an inflammatory premetastatic niche through the miR-21–TLR7–IL-6 axis. Thus, sEVs–miR-21 represents a potential prognostic marker and therapeutic target for CRC patients with liver metastasis. [ABSTRACT FROM AUTHOR]

The histone H3 N-terminal protein domain (N-tail) is regulated by multiple posttranslational modifications, including methylation, acetylation, phosphorylation, and by proteolytic cleavage. However, the mechanism underlying H3 N-tail proteolytic cleavage is largely elusive. Here, we report that JMJD5, a Jumonji C (JmjC) domain-containing protein, is a Cathepsin L-type protease that mediates histone H3 N-tail proteolytic cleavage under stress conditions that cause a DNA damage response. JMJD5 clips the H3 N-tail at the carboxyl side of monomethyl-lysine (Kme1) residues. In vitro H3 peptide digestion reveals that JMJD5 exclusively cleaves Kme1 H3 peptides, while little or no cleavage effect of JMJD5 on dimethyl-lysine (Kme2), trimethyl-lysine (Kme3), or unmethyl-lysine (Kme0) H3 peptides is observed. Although H3 Kme1 peptides of K4, K9, K27, and K36 can all be cleaved by JMJD5 in vitro, K9 of H3 is the major cleavage site in vivo, and H3.3 is the major H3 target of JMJD5 cleavage. Cleavage is enhanced at gene promoters bound and repressed by JMJD5 suggesting a role for H3 N-tail cleavage in gene expression regulation. [ABSTRACT FROM AUTHOR]

Background N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), an alkylating agent and an environmental carcinogen, causes DNA lesions and even carcinomas. DNA damage responses induced by MNNG activate various DNA repair genes and related signaling pathways. The present study aimed to investigate the regulatory mechanisms of human RR small subunit M2 ( h RRM2) in response to MNNG. Results In this study, we demonstrated that the RRM2 gene was transactivated by MNNG exposure more strongly than the other small subunit, p53R2. The upregulated RRM2 translocated to the nucleus for DNA repair. Further study showed that E2F3 transactivated RRM2 expression by directly binding to its promoter after MNNG exposure. The transactivation was enhanced by the upregulation of NFY, which bound to the RRM2 promoter adjacent to the E2F3 binding site and interacted with E2F3. In response to MNNG treatment, E2F3 accumulated mainly through its phosphorylation at S124 and was dependent on ATR–CHK1 signaling. In comparison, p53R2 played a relatively weaker role in the MNNG-induced DNA damage response, and its transcription was regulated by the ATR–CHK2–E2F1/p53 pathway. Conclusions We suggest that MNNG-stimulated ATR/CHK1 signaling stabilizes E2F3 by S124 phosphorylation, and then E2F3 together with NFY co-transactivate RRM2 expression for DNA repair. General significance We propose a new mechanism for RRM2 regulation to maintain genome stability in response to environmental chemical carcinogens. [ABSTRACT FROM AUTHOR]

Discovery of epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) are two milestones in people exploring the nature of malignant tumor in recent decades. Although some studies have presented the potential connections between them, the link details, underneath their superficial correlation, are largely unknown. In this study, we identified a small subpopulation of NANOG-positive colorectal cancer (CRC) cells, and demonstrated that they exhibited characteristics of CSCs and EMT traits simultaneously. Furthermore, we found that NANOG was a core factor in regulating both of EMT and stemness in CRC cells, NANOG modulate EMT and metastasis by binding to Slug promoter and transcriptionally regulate Slug expression. For the first time, we demonstrated that NANOG was regulated by extracellular IGF signaling pathway via STAT3 phosphorylation in CRC. This coincides with that IGF receptor IGF-1R is often increasing expressed in malignant metastasis colon cancer. Taken together, our data define the crucial functions of IGF/STAT3/NANOG/Slug signaling axis in the progression of CRC by operating EMT and CSCs properties, which make them served as potential therapeutic targets for treatment of CRC. S tem C ells 2016;34:820-831 [ABSTRACT FROM AUTHOR]

Ribonucleotide reductase (RR) has been reported to be associated with several types of cancer while the expression and role of RR in thyroid carcinoma (TC) has not been investigated. Here, we first examined the expression level of three RR subunit proteins (RRM1, RRM2, and RRM2B) in papillary thyroid carcinoma (PTC) and undifferentiated thyroid carcinoma (UTC) patient samples by immunohistochemistry. The results showed that RRM1 was higher expressed in 95.2 % cancer tissues compared with their adjacent normal tissues in 146 PTC samples. The expression level of RRM1 was positively correlated with T stage, lymph node metastasis (LNM), extrathyroidal invasion (ETI), and TNM stage in PTC patients. However, in 12 UTC samples, RRM1 expression was negatively expressed in six cases. To further determine the biological role of RRM1 in TC, ectopic expression or siRNA-mediated knockdown of RRM1 were carried out in the high-differentiated thyroid carcinoma cell line TPC-1 and the poor-differentiated thyroid carcinoma cell line SW579, respectively. In TPC-1 and SW579 cells, overexpression and siRNA knockdown of RRM1 demonstrated that RRM1 promoted DNA synthesis and proliferation in both cell lines as shown by EdU incorporation and cell viability assays. However, RRM1 enhanced cell migration and invasion in TPC-1 cells but inhibited that in SW579 cells as shown by wound healing and transwell assays. Moreover, we also found that RRM1 promoted PTEN expression and reduced Akt phosphorylation in a RR-activity-independent manner in the low-differentiated TC cells but not in the high-differentiated TC cells. In contrast, RRM2 expression was higher expressed in both PTC and UTC patient samples, consisting with its oncogenic role in other cancers. Therefore, we suggest that RRM1 promotes thyroid carcinoma proliferation as a component of RR but may play a different role in the invasion and metastasis of differently differentiated thyroid carcinomas through a non-RR pathway, which could be meaningful to precision treatment of thyroid carcinoma with RR inhibitors. [ABSTRACT FROM AUTHOR]

Abstract: (±)-anti-Benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), which causes bulky-adduct DNA damage, is well-characterized as the ultimate carcinogen of benzo[a]pyrene (BaP). In this study, we have employed Affymetrix HG-U133 Plus 2.0 microarray and quantitative real-time RT-PCR methods to investigate a temporal transcriptomic response triggered by a low concentration (0.05μM) of BPDE at 1, 10, and 22h after exposure in normal human cells. The differential gene expression profiles at the three time points varied greatly, and generally reflected a cellular responsive process from initiation to progression and to recovery after the BPDE-caused damage. The dynamic regulation of the genes related with cell cycle progression and cell fate exhibited a tendency from inhibition to survival, which was accordant with the cell cycle arrest and cytotoxicity data induced by the low-dose BPDE exposure. In silico comparison of the genomic data revealed that BPDE and ultraviolet induced a panel of common transcriptional responses, which might be related with a series of similar molecular processes elicited by these two DNA-damaging agents. In conclusion, this whole-genome time-course study has identified a dynamically regulated transcriptional signature after low-dose BPDE exposure, which may help to understand the complex mechanisms of mutagenesis and carcinogenesis induced by BPDE. [Copyright &y& Elsevier]

Abstract: (±)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) is a carcinogen causing bulky-adduct DNA damage. In this study, we investigated early transcriptional signatures induced by various concentrations (0.005, 0.05, and 0.5 μM) of this carcinogen in a normal human cell line (FL human amnion epithelial cells) using the whole-genome Affymetrix HG-U133 Set microarray. The numerous identified genes were involved in multiple functions and higher doses of BPDE elicited more robust expression changes. The disturbance of genes involved in cell cycle regulation, growth and apoptosis was correlated with the S and G2/M phase cell cycle arrest and cytotoxic phenotypes induced by different levels of BPDE. Bioinformatic analysis showed that several transcription factors and their related stress signaling pathways might partly account for the transcriptional signature induced by BPDE. Additionally, gene ontology analysis of the microarray data showed down-regulation of transport, cytoskeleton and DNA repair by 0.5 μM BPDE exposure. In conclusion, this genomic analysis helps to understand the mechanism of cellular response to BPDE. [Copyright &y& Elsevier]

Abstract: The alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces cellular DNA damage and other comprehensive alterations that lead to chromosomal aberrations, mutations, tumor initiation, and cell death. However, the molecular mechanism of MNNG-induced cellular stress remains unclear. We undertook a genome-wide analysis of early transcriptional responses of human FL amnion epithelial cells to three relatively low doses of MNNG (0.2, 1.0, and 10.0μM). Using Affymetrix GeneChip HG-U133 Set oligonucleotide microarrays, a total of 281 genes were found to change their expression after exposure to all three doses of MNNG compared with the vehicle control, DMSO. Of these, 70, 112, and 146 genes showed different expression levels following treatment with low, medium, and high-dose MNNG, respectively. A subset of these genes were selected for further confirmation using quantitative real-time RT-PCR with ABI TaqMan® low-density arrays, and the differential expression of 33 genes was validated. The results demonstrate that low doses of MNNG can induce various changes in gene expression at an early stage of exposure. The responsive genes are involved in multiple cellular biological processes including transcription regulation, signal transduction, cell cycle regulation, cytoskeleton organization, protein synthesis, immune responses, and metabolism. The cell cycle progression was down-regulated, in which several genes were validated involved, including the cell cycle regulators (CDK6, STK6, CENPA, and CCNF), the transcription factor ID-1, and the calcium signaling molecules (CAMK2G and NFAT5). The possible roles of the responsive genes and their related pathways in MNNG-induced cellular responses are discussed. This study helps to complete the picture of how cells respond to environmental chemical exposure via transcriptional regulation. [Copyright &y& Elsevier]

Background N -nitroso compounds (NOC) can cause cancers in a wide variety of animal species, and many of them are also potential human carcinogens. However, their underlying genotoxic mechanisms occurred within the context of chromatin, such as aberrant histone modifications, remained elusive. Methods We investigated the dynamic landscapes of histone modifications after N -nitroso compound N -methyl- N ′-nitro- N -nitrosoguanidine (MNNG) and N -methyl- N -nitroso-urea (MNU) exposure. Among the altered histone modifications, we also investigated the control mechanisms of histone H3 phosphorylation changes and its possible implications on transcriptional repression. Results Significantly, we find a specific biphasic reduction of histone H3 phosphorylation at serine 10 (H3S10ph) and serine 28 (H3S28ph), and a rapid decrease of histone H4 acetylation upon MNNG and MNU exposure. Further investigations reveal that the first hypophosphorylation of H3 occurs in a poly(ADP-ribosyl)ation enzyme PARP-1 (Poly(ADP-Ribose) Polymerase 1) dependent manner, whereas the second decline of H3 phosphorylation is at least partially under the control of histone kinase VRK1 (vaccinia-related kinase 1) and dependent on the tumor suppressor protein p53. In addition, DNA damage induced down-regulation of H3S10/S28 phosphorylation also functions in transcriptional repression of genes, such as cell-cycle regulators. Conclusions Alkylating damage induced by NOC elicits a biphasic reduction of histone H3 phosphorylation with distinct control mechanisms, which is contributing to DNA damage responses such as the repair-facilitated transcriptional repression. General significance Identification of the dynamic changes and underlying mechanisms of histone modifications upon NOC exposure would be of great help in understanding the epigenetic regulations of NOC induced DNA damage responses. [ABSTRACT FROM AUTHOR]

JMJD5 is a Jumonji C domain-containing demethylase/hydroxylase shown to be essential in embryological development, osteoclastic maturation, circadian rhythm regulation and cancer metabolism. However, its role and underlying mechanisms in oncogenesis remain unclear. Here, we demonstrate that JMJD5 forms complex with the tumor suppressor p53 by interacting with p53 DNA-binding domain (DBD), and negatively regulates its activity. Downregulation of JMJD5 resulted in increased expression of multiple p53 downstream genes, such as the cell cycle inhibitor CDKN1A and DNA repair effector P53R2 , only in p53-proficient lung cancer cells. Upon DNA damage, the JMJD5–p53 association decreased, and thereby, promoted p53 recruitment to the target genes and stimulated its transcriptional activity. Furthermore, JMJD5 facilitated the cell cycle progression in a p53-dependent manner under both normal and DNA damage conditions. Depletion of JMJD5 inhibited cell proliferation and enhanced adriamycin-induced cell growth suppression in the presence of p53. Collectively, our results reveal that JMJD5 is a novel binding partner of p53 and it functions as a positive modulator of cell cycle and cell proliferation mainly through the repression of p53 pathway. Our study extends the mechanistic understanding of JMJD5 function in cancer development and implicates JMJD5 as a potential therapeutic target for cancer. [ABSTRACT FROM AUTHOR]

Abstract: JMJD5 has recently been reported to participate in circadian rhythm regulation, embryological development, osteoclastogenesis and tumorigenesis. Although JMJD5 has been found mainly localized in the nucleus of cells, how it enters the nucleus remains unclear. Here we report that JMJD5 contains a functional bipartite nuclear localization signal (NLS) and a chromosome region maintenance 1 (CRM1)-dependent nuclear export signal (NES). Importin α/β and transportin-1 were further identified as JMJD5-associated transport proteins, and different binding regions were determined for the two nuclear import receptors. Additionally, we demonstrate that both the active NLS and the JmjC domain of JMJD5 are necessary for cyclin A1 transcription. Chromatin immunoprecipitation (ChIP) analysis confirmed the alterations of di-methylated lysine 36 of histone H3 (H3K36me2) in the coding region of cyclin A1. These results reveal that the N-terminal domain is essential for the nuclear localization of JMJD5 and its normal enzymatic function towards substrates in the nucleus. [Copyright &y& Elsevier]

Abstract: DNA polymerase κ (Pol κ), a member of Y-family DNA polymerases, can synthesize DNA with moderate fidelity on undamaged DNAs and replicate accurately in vitro thymine glycol, 8-oxo-G and aromatic adducts such as benzo[a]pyrene diol epoxide (BPDE). However, few studies have been done on the transcriptional regulation of Pol κ. In this study, we predicted and cloned the promoter region of the human POLK gene. Through the analysis of deletion constructs of the POLK promoter, we demonstrated that the region −336/−141 contained repressing elements and the region −141/+226 contained positive regulatory elements for transcription of human Pol κ. Furthermore, quantitative RT-PCR showed that human POLK mRNA expression was dysregulated in FL cells treated by BPDE. The transcriptional activities of the POLK promoter regions −336/+437 and +20/+437 were significantly reduced by BPDE treatment, indicating that transcription factors in this two regions, such as HSF1, may regulate the transcription of human POLK gene in response to BPDE. [Copyright &y& Elsevier]

Abstract: Benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) is a carcinogen causing bulky-adduct DNA damage and inducing extensive cell responses regulating cell cycle, cell survival and apoptosis. However, the mechanism of cellular responses to BPDE exposure is not fully understood. In this study, we demonstrated the involvement of the phosphorylation of the α-subunit of the eukaryotic initiation factor-2 (eIF2α) in the cellular response to BPDE exposure and addressed the role of eIF2α phosphorylation in the regulation of the cellular stress. Phosphorylation of eIF2α was induced in a normal human FL amnion epithelial cell line, and the expression of ATF4, a conserved downstream transcriptional factor of eIF2α phosphorylation, was up-regulated after BPDE exposure; however, the four known primary kinases for eIF2α phosphorylation (GCN2, HRI, PKR, and PERK) were not found activated. While BPDE induced severe cell cycle arrest and apoptosis and decreased cell viability in FL cells, salubrinal, a selective inhibitor of eIF2α dephosphorylation, maintained the eIF2α phosphorylation and attenuated cell cycle arrest and apoptosis and promoted cell survival. The findings reveal that when BPDE causes cellular damages, it induces eIF2α phosphorylation as well, which produces a pro-survival and anti-apoptotic effect to alleviate the cellular damages. Thus, the present study proposes a new cellular defensive mechanism during the environmental mutagen and carcinogen attack. [Copyright &y& Elsevier]

Abstract: Human Pol ι is a highly distributed, low-fidelity DNA polymerase lacking intrinsic exonuclease proofreading activity, thus its effects are strictly regulated. We predicted and cloned the promoter region of the human POLI gene. Successively, by transfection of deletion constructs of the POLI promoter, we demonstrated that the regions −848/−408 and −30/+215 contained positive regulatory elements, and the region +215/+335 had proximal promoter activity. Overexpression of Sp1 significantly increased the transcriptional activity of the promoter, and mutation of the Sp1 site reversed Sp1-induced promoter transactivation. Quantitative RT-PCR showed that POLI mRNA expression was up-regulated in human amnion FL cells treated by the carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Reporter gene assays demonstrated that MNNG also significantly increased the transcriptional activity of the predicted promoter (−848/+335) and the proximal promoter (+215/+335). However, the promoter with the Sp1 site mutation had no response to MNNG treatment, suggesting that Sp1 plays an important role in the transcriptional regulation of the POLI gene stimulated by MNNG. Our data suggest that abnormal regulation of Pol ι may be involved in the mutagenesis and carcinogenesis induced by environmental chemicals. [Copyright &y& Elsevier]

Abstract: Ribonucleotide reductase (RR) is a highly regulated enzyme in the deoxyribonucleotide synthesis pathway. RR is responsible for the de novo conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates, which are essential for DNA synthesis and repair. Besides two subunits, hRRM1 and hRRM2, p53R2 is a newly identified member of RR family that is induced by ultraviolet light in a p53-dependent manner. To understand the molecular interaction of RR subunits, we employed a eukaryotic expression system to express and purify all three subunits. After in vitro reconstitution, the results of [3H]CDP reduction assay showed that both eukaryotic recombinant hRRM2 and p53R2 proteins could interact with hRRM1 to form functional RR holoenzyme. The reconstituted RR activity was time-dependent and the reaction rate reached the plateau phase after 40min incubation. No matter the concentration, RR holoenzyme reconstituted from p53R2 and hRRM1 could only achieve about 40–75% kinetic activity of that from hRRM2 and hRRM1. The synthetic C-terminal heptapeptide competition assays confirmed that hRRM2 and p53R2 share the same binding site on hRRM1, but the binding site on hRRM1 demonstrated higher affinity for hRRM2 than for p53R2. In allosteric regulation assay, the effect of activation or inhibition of hRRM1 with ATP or dATP suggested that these effectors could regulate RR activity independent of different RR small subunits. Taken together, the eukaryotic expression system RR holoenzyme will provide a very useful tool to understand the molecular mechanisms of RR activity and the interactions of its subunits. [Copyright &y& Elsevier]

Hepatocellular carcinoma (HCC), one of the most deadly diseases all around the world. HBV infection is a causative factor of HCC and closely associated with HCC development. Ribonucleotide reductase (RR) is a key enzyme for cellular DNA synthesis and RR small subunit M2 (RRM2) is highly upregulated in HCC with poor survival rates. We have previously shown that HBV can activate the expression of RRM2 and the activity of RR enzyme for the viral DNA replication in host liver cells. Thus, RRM2 may be an important therapeutic target for HCC and HBV-related HCC. Pterostilbene, a natural plant component, potently inhibited in vitro RR enzyme activity with the IC 50 of about 0.62 μM through interacting with RRM2 protein, which was much higher than current RRM2 inhibitory drugs. Pterostilbine inhibited cell proliferation with an MTT IC 50 of about 20-40 μM in various HCC cell lines, causing DNA synthesis inhibition, cell cycle arrest at S phase, and accordingly apoptosis. On the other hand, the compound significantly inhibited HBV DNA replication in HBV genome integrated and newly transfected HCC cells, and the EC 50 for inhibiting HBV replication was significantly lower than the IC 50 for inhibiting HCC proliferation. Notably, pterostilbene possessed a similar inhibitory activity in sorafenib and lamivudine resistant HCC cells. Moreover, the inhibitory effects of pterostilbine against HCC proliferation and HBV replication were significantly reversed by addition of dNTP precursors, suggesting that RR was the intracellular target of the compound. Finally, pterostilbine effectively inhibited HCC xenograft growth with a relatively low toxicity in nude mouse experiments. This study demonstrates that pterostilbene is a novel potent RR inhibitor by targeting RRM2. It can simultaneously inhibit HCC proliferation and HBV replication with a potential new use for treatment of HCC and HBV-related HCC., Competing Interests: None., (AJCR Copyright © 2021.)

We previously reported that E2F1 expression is up-regulated and positively correlated with the malignant phenotypes of colorectal cancer (CRC). However, the underlying mechanisms leading to the aberrant up-regulation of E2F1 in CRC have not been clarified. In this study, we observed that miR-526b-3p directly targets the 3'UTR of E2f1 mRNA, leading to reduced E2F1 expression. Overexpression of miR-526b-3p inhibited the proliferation of CRC cells by decreasing the level of E2F1. We also found that the Ying Yang 1 (YY1)-dependent transcriptional suppression of miR-526b-3p is responsible for the up-regulation of E2F1 in CRC, in which YY1 binds to the promoter of miR-526b gene and recruits histone deacetylase (HDAC). Knockdown of YY1 led to cell cycle arrest and diminished colony formation in CRC cells partly through relieving the miR-526b-3p suppression. Clinical analysis showed that YY1 and E2F1 were negatively correlated with miR-526b-3p in CRC tissues. Moreover, a high level of YY1 and E2F1, or a low level of miR-526b-3p , predicted poor survival of CRC patients. In conclusion, our findings highlight the dysregulation of the YY1/ miR-526b-3p /E2F1 axis in CRC development, implicating a novel regulatory pathway for E2F1 as a potential therapeutic target in CRC., Competing Interests: None., (AJCR Copyright © 2019.)

Ribonucleotide reductase (RR) is the rate-limiting enzyme in DNA synthesis, catalyzing the reduction of ribonucleotides to deoxyribonucleotides. During each enzymatic turnover, reduction of the active site disulfide in the catalytic large subunit is performed by a pair of shuttle cysteine residues in its C-terminal tail. Thioredoxin (Trx) and glutaredoxin (Grx) are ubiquitous redox proteins, catalyzing thiol-disulfide exchange reactions. Here, immunohistochemical examination of clinical colorectal cancer (CRC) specimens revealed that human thioredoxin1 (hTrx1), but not human glutaredoxin1 (hGrx1), was up-regulated along with human RR large subunit (RRM1) in cancer tissues, and the expression levels of both proteins were correlated with cancer malignancy stage. Ectopically expressed hTrx1 significantly increased RR activity, DNA synthesis, and cell proliferation and migration. Importantly, inhibition of both hTrx1 and RRM1 produced a synergistic anticancer effect in CRC cells and xenograft mice. Furthermore, hTrx1 rather than hGrx1 was the efficient reductase for RRM1 regeneration. We also observed a direct protein-protein interaction between RRM1 and hTrx1 in CRC cells. Interestingly, besides the known two conserved cysteines, a third cysteine (Cys 779 ) in the RRM1 C terminus was essential for RRM1 regeneration and binding to hTrx1, whereas both Cys 32 and Cys 35 in hTrx1 played a counterpart role. Our findings suggest that the up-regulated RRM1 and hTrx1 in CRC directly interact with each other and promote RR activity, resulting in enhanced DNA synthesis and cancer malignancy. We propose that the RRM1-hTrx1 interaction might be a novel potential therapeutic target for cancer treatment., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside diphosphate substrates (S) to deoxynucleotides with allosteric effectors (e) controlling their relative ratios and amounts, crucial for fidelity of DNA replication and repair. Escherichia coli class Ia RNR is composed of α and β subunits that form a transient, active α2β2 complex. The E. coli RNR is rate-limited by S/e-dependent conformational change(s) that trigger the radical initiation step through a pathway of 35 Å across the subunit (α/β) interface. The weak subunit affinity and complex nucleotide-dependent quaternary structures have precluded a molecular understanding of the kinetic gating mechanism(s) of the RNR machinery. Using a docking model of α2β2 created from X-ray structures of α and β and conserved residues from a new subclassification of the E. coli Ia RNR (Iag), we identified and investigated four residues at the α/β interface (Glu 350 and Glu 52 in β2 and Arg 329 and Arg 639 in α2) of potential interest in kinetic gating. Mutation of each residue resulted in loss of activity and with the exception of E52Q-β2, weakened subunit affinity. An RNR mutant with 2,3,5-trifluorotyrosine radical (F 3 Y 122 • ) replacing the stable Tyr 122 • in WT-β2, a mutation that partly overcomes conformational gating, was placed in the E52Q background. Incubation of this double mutant with His 6 -α2/S/e resulted in an RNR capable of catalyzing pathway-radical formation (Tyr 356• -β2), 0.5 eq of dCDP/F 3 Y 122 • , and formation of an α2β2 complex that is isolable in pulldown assays over 2 h. Negative stain EM images with S/e (GDP/TTP) revealed the uniformity of the α2β2 complex formed., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Escherichia coli class Ia ribonucleotide reductase (RNR) is composed of two subunits that form an active α2β2 complex. The nucleoside diphosphate substrates (NDP) are reduced in α2, 35 Å from the essential diferric-tyrosyl radical (Y 122 • ) cofactor in β2. The Y 122 • -mediated oxidation of C 439 in α2 occurs by a pathway (Y 122 ⇆ [W 48 ] ⇆ Y 356 in β2 to Y 731 ⇆ Y 730 ⇆ C 439 in α2) across the α/β interface. The absence of an α2β2 structure precludes insight into the location of Y 356 and Y 731 at the subunit interface. The proximity in the primary sequence of the conserved E 350 to Y 356 in β2 suggested its importance in catalysis and/or conformational gating. To study its function, pH-rate profiles of wild-type β2/α2 and mutants in which 3,5-difluorotyrosine (F 2 Y) replaces residue 356, 731, or both are reported in the presence of E 350 or E 350 X (X = A, D, or Q) mutants. With E 350 , activity is maintained at the pH extremes, suggesting that protonated and deprotonated states of F 2 Y 356 and F 2 Y 731 are active and that radical transport (RT) can occur across the interface by proton-coupled electron transfer at low pH or electron transfer at high pH. With E 350 X mutants, all RNRs were inactive, suggesting that E 350 could be a proton acceptor during oxidation of the interface Ys. To determine if E 350 plays a role in conformational gating, the strong oxidants, NO 2 Y 122 • -β2 and 2,3,5-F 3 Y 122 • -β2, were reacted with α2, CDP, and ATP in E 350 and E 350 X backgrounds and the reactions were monitored for pathway radicals by rapid freeze-quench electron paramagnetic resonance spectroscopy. Pathway radicals are generated only when E 350 is present, supporting its essential role in gating the conformational change(s) that initiates RT and masking its role as a proton acceptor.