Luciferase Expression Vector Research Articles

Effect of Culture Supernatant of Clostridium butyricum TO-A on Human DNA-Repair-Factor-Encoding Gene Promoters.

In this study, Clostridium butyricum TO-A culture supernatant (CBCS) or butyric acid was added to a culture medium of human cervical carcinoma HeLa S3 cells, and changes in DNA-repair-related gene promoter activities were investigated. The HeLa S3 cells were transfected with a luciferase (Luc) expression vector containing approximately 500 bp of the 5'-upstream region of several human DNA-repair-related genes and cultured with a medium containing the CBCS (10%) or butyric acid (2.5 mM). The cells were harvested after 19 to 42 h of incubation. A Luc assay revealed that the human ATM, PARG, PARP1, and RB1 gene promoter activities were significantly increased. A Western blot analysis showed that the amounts of the proteins encoded by these genes markedly increased. Furthermore, 8, 24, and 48 h after the addition of the CBCS (10%), total RNA was extracted and subjected to RNAseq analysis. The results showed that the expression of several inflammation- and DNA-replication/repair-related genes, including NFKB and the MCM gene groups, decreased markedly after 8 h. However, the expression of the histone genes increased after 24 h. Elucidation of the mechanism by which the CBCS and butyrate affect the expression of genes that encode DNA-repair-associated proteins may contribute to the prevention of carcinogenesis, the risk of which rises in accordance with aging.

HIV latency potential may be influenced by intra-subtype genetic differences in the viral long-terminal repeat

IntroductionElucidation of mechanisms that drive HIV latency is essential to identifying cure strategies. While host mechanisms associated with viral persistence on antiretroviral therapy (ART) have been well studied, less is known about the viral properties that influence latency. The viral promoter element, the 5’ long terminal repeat (LTR), has been shown to affect the number of latently infected cells shortly after infection. Here we investigated the role of subtype C LTR genotypic variation on the establishment of latency in a dual reporter HIV-1 infection model.MethodsThe LTR U3 and R regions from 11 women with acute/early subtype C HIV infection were cloned into the dual reporter pRGH plasmid. Latency potential was calculated based on the expression of fluorescent reporter genes in Jurkat E6–1 cells measured by flow cytometry as the proportion of latent (mCherry +ve cells)/proportion of active (eGFP +ve mCherry +ve cells) infection. Reversal of latency was performed using PMA/Ionomycin stimulation 24 hours before fixing of cells. LTR transcriptional capacity, in the presence and absence of a heterologous subtype C Tat, was measured for the same LTRs cloned into a pGL4.10 luciferase expression vector following transfection of HEK293T cells.ResultsThe majority of proviruses were latent at day 8 post-infection, yet the proportion of latently infected cells varied significantly across participants. We observed a median latent:active infection ratio of 1.79 (range 0.86–2.83) across LTRs with the hierarchy of latency potential remaining consistent across repeat experiments. The median latent:active infection ratio decreased by a median of 3-fold following PMA/Ionomycin stimulation to 0.55 (range 0.46–0.78) indicating that a proportion of latently infected cells could produce viral proteins upon activation. Latency potential did not correlate with LTR transcriptional capacity.ConclusionsWe found intra-subtype level differences in the latency potential of LTRs from South African women independent of their transcriptional capacity, suggesting that HIV-1 LTRs have intrinsic properties that influence the proportion of latently infected cells shortly after infection. The inability to reactivate viral expression in all latently infected cells supports the complex nature of mechanisms driving latency and the need for continued advancements in methods used to study these mechanisms.

Induction of the human CDC45 gene promoter activity by natural compound trans‑resveratrol.

GGAA motifs in the human TP53 and HELB gene promoters play a part in responding to trans‑resveratrol (Rsv) in HeLa S3 cells. This sequence is also present in the 5'‑upstream region of the human CDC45 gene, which encodes a component of CMG DNA helicase protein complex. The cells were treated with Rsv (20 µM), then transcripts and the translated protein were analyzed by quantitative RT‑PCR and western blotting, respectively. The results showed that the CDC45 gene and protein expression levels were induced after the treatment. To examine whether they were due to the activation of transcription, a 5'‑upstream 556‑bp of the CDC45 gene was cloned and inserted into a multi‑cloning site of the Luciferase (Luc) expression vector. In the present study, various deletion/point mutation‑introduced Luc expression plasmids were constructed and they were used for the transient transfection assay. The results showed that the GGAA motif, which is included in a putative RELB protein recognizing sequence, plays a part in the promoter activity with response to Rsv in HeLa S3 cells.

Comparative study on estrogen receptor alpha dimerization and transcriptional activity of parabens.

Parabens are used as preservatives in various household products, including oral products, cosmetics, and hair/body washes. In recent years, the widespread use of parabens has raised concerns due to the potential health risks associated with their estrogenic effects. In the present study, we evaluated and compared the estrogenic activity of parabens using two cell-based in vitro tests: (1) bioluminescence resonance energy transfer (BRET)-based estrogen receptor alpha (ERα) dimerization using HEK293 cells that were stably transfected with ERα-fused NanoLuc luciferase (Nluc) and HaloTag (HT) expression vector, and (2) stably transfected transcriptional activation (STTA) assays using ERα-HeLa9903 cells. The following parabens were tested using the BRET-based ERα dimerization assay and showed estrogenic activity (PC20 values): methyl paraben (MP, 5.98 × 10-5M), ethyl paraben (EP, 3.29 × 10-5M), propylparaben (PP, 3.09 × 10-5M), butyl paraben (BP, 2.58 × 10-5M), isopropyl paraben (IsoPP, 1.37 × 10-5M), and isobutyl paraben (IsoBP, 1.43 × 10-5M). Except MP, all other parabens tested using the STTA assay also showed estrogenic activity: EP, 7.57 × 10-6M; PP, 1.18 × 10-6M; BP, 3.02 × 10-7M; IsoPP, 3.58 × 10-7M; and IsoBP, 1.80 × 10-7M. Overall, EP, PP, BP, IsoPP, and IsoBP tested positive for estrogenic activity using both assays. These findings demonstrate that most parabens, albeit not all, induce ERα dimerization and possess estrogenic activity.

Genetic variation of the HIV-1 subtype C transmitted/founder viruses long terminal repeat elements and the impact on transcription activation potential and clinical disease outcomes.

A genetic bottleneck is a hallmark of HIV-1 transmission such that only very few viral strains, termed transmitted/founder (T/F) variants establish infection in a newly infected host. Phenotypic characteristics of these variants may determine the subsequent course of disease. The HIV-1 5' long terminal repeat (LTR) promoter drives viral gene transcription and is genetically identical to the 3' LTR. We hypothesized that HIV-1 subtype C (HIV-1C) T/F virus LTR genetic variation is a determinant of transcriptional activation potential and clinical disease outcome. The 3'LTR was amplified from plasma samples of 41 study participants acutely infected with HIV-1C (Fiebig stages I and V/VI). Paired longitudinal samples were also available at one year post-infection for 31 of the 41 participants. 3' LTR amplicons were cloned into a pGL3-basic luciferase expression vector, and transfected alone or together with Transactivator of transcription (tat) into Jurkat cells in the absence or presence of cell activators (TNF-α, PMA, Prostratin and SAHA). Inter-patient T/F LTR sequence diversity was 5.7% (Renge: 2-12) with subsequent intrahost viral evolution observed in 48.4% of the participants analyzed at 12 months post-infection. T/F LTR variants exhibited differential basal transcriptional activity, with significantly higher Tat-mediated transcriptional activity compared to basal (p<0.001). Basal and Tat-mediated T/F LTR transcriptional activity showed significant positive correlation with contemporaneous viral loads and negative correlation with CD4 T cell counts (p<0.05) during acute infection respectively. Furthermore, Tat-mediated T/F LTR transcriptional activity significanly correlated positively with viral load set point and viral load; and negatively with CD4 T cell counts at one year post infection (all p<0.05). Lastly, PMA, Prostratin, TNF-α and SAHA cell stimulation resulted in enhanced yet heterologous transcriptional activation of different T/F LTR variants. Our data suggest that T/F LTR variants may influence viral transcriptional activity, disease outcomes and sensitivity to cell activation, with potential implications for therapeutic interventions.

The PARP inhibitor Rucaparib synergizes with radiation to attenuate atypical teratoid rhabdoid tumor growth.

Atypical teratoid rhabdoid tumors (ATRT) are highly aggressive pediatric brain tumors. The available treatments rely on toxic chemotherapy and radiotherapy, which themselves can cause poor outcomes in young patients. Poly (ADP-ribose) polymerases (PARP), multifunctional enzymes which play an important role in DNA damage repair and genome stability have emerged as a new target in cancer therapy. An FDA-approved drug screen revealed that Rucaparib, a PARP inhibitor, is important for ATRT cell growth. This study aims to investigate the effect of Rucaparib treatment in ATRT. This study utilized cell viability, colony formation, flow cytometry, western blot, immunofluorescence, and immunohistochemistry assays to investigate Rucaparib's effectiveness in BT16 and MAF737 ATRT cell lines. In vivo, intracranial orthotopic xenograft model of ATRT was used. BT16 cell line was transduced with a luciferase-expressing vector and injected into the cerebellum of athymic nude mice. Animals were treated with Rucaparib by oral gavaging and irradiated with 2 Gy of radiation for 3 consecutive days. Tumor growth was monitored using In Vivo Imaging System. Rucaparib treatment decreased ATRT cell growth, inhibited clonogenic potential of ATRT cells, induced cell cycle arrest and apoptosis, and led to DNA damage accumulation as shown by increased expression of γH2AX. In vivo, Rucaparib treatment decreased tumor growth, sensitized ATRT cells to radiation and significantly increased mice survival. We demonstrated that Rucaparib has potential to be a new therapeutic strategy for ATRT as seen by its ability to decrease ATRT tumor growth both in vitro and in vivo.

Abstract 5989: Brain metastasis mouse models for the evaluation of multikine inhibitors on ROS1-fusion-positive lung cancer

Abstract Lung cancer is the leading cause of cancer-related death worldwide. Nearly 80% of lung cancers are non-small cell lung cancer (NSCLC) and 60% of them are diagnosed at the metastatic stage. Brain metastases affect more than 20% of NSCLC patients with poor prognosis and disabling symptoms. However, few therapies have been approved for the treatment of lung cancer brain metastases. A panel of rapid, predictive and clinically-relevant animal models are urgently needed to study the biology of brain metastases and to identify effective therapeutic approaches.Considering both progression and treatment of lung cancer brain metastases will be limited by the blood-brain barrier (BBB), here we describe methods for efficient establishment of brain metastases mouse models via different injection route (intracranial, intracardiac or intracarotid), to identify a model that permits tumor cell growth in the unique brain microenvironment without compromising BBB. Meanwhile, we established a novel ROS1 positive patient-derived xenograft (PDX) model together with its PDC sub-line, in which the exon 2 of SDC4 was fused to the exon 32 of ROS1 (SDC4ex2-ROS1ex32). The cell has been transduced with firefly luciferase expression vector for in vivo imaging detection. Three ROS1 inhibitors were tested on these brain metastasis and subcutaneous models to compare their efficacies. In summary, we established brain metastases lung cancer PDX models as reliable platforms for interrogation of targets and kinetics related mechanism and for drug screening. The use of such techniques will increase our knowledge of the metastatic process and help identify new targets of cancer metastasis. Citation Format: Hui Qi, Xia Xiong, Huanhuan Liu, Yuying Yang, Chunlei Dai, Haoxia Zhao, Bingrui Han, Xuzhen Tang, Qunsheng Ji. Brain metastasis mouse models for the evaluation of multikine inhibitors on ROS1-fusion-positive lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5989.

Targeting of Marcks By MiR-34a Overcomes Drug Resistance in Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by abnormal proliferation of clonal plasma cells in the bone marrow. Current therapies, such as the proteasome inhibitor bortezomib (BTZ), have improved the outcome of patients. Nevertheless MM remains an incurable disease with a high rate of relapse and development of drug resistance. So far, the pathogenic mechanisms underlying drug resistance of MM are not fully elucidated. Our previous study showed the overexpression of MARCKS (Myristoylated alanine-rich C-kinase substrate) protein in MM cells played an important role in drug resistance. However; the mechanism (s) underlying MARCKS overexpression and its association with drug resistance in MM have not been elucidated.In the current study, to investigate whether miRNAs play a role in aberrant upregulation of MARCKS in drug resistance, several miRNA-target prediction algorithms were exploited to generate a selective miRNA library for subsequent screening. Among 52 miRNA candidates that target MARCKS, the expression level of several miRNAs with higher score was assessed in drug resistant cell lines (8226-R5 and MM1.R) in comparison to the parental cells (8226 and MM1.S). We observed that miR-34a expression was most significantly downregulated in 8226-R5 and MM1.R cell lines relative to their parental lines, 8226 and MM1S. We also identified a negative correlation between MARCKS and miR-34a expression levels in 6 primary patient samples and two healthy donor samples. Next, to confirm direct targeting of MARCKS by miR-34a, two MM resistant cell lines (8226-R5 and MM1R) were transduced with lentiviral vectors harboring miR-34a precursor gene or scramble control and mRNA and protein level of MARCKS were assessed in miR-34a overexpressing cell lines in comparison to scramble control. Overexpression of miR-34a reduced MARCKS expression level in both mRNA and protein levels. In addition, the luciferase reporter assay was performed to validate MARCKS as a direct target of miR-34a. To this end, the 3' UTR sequence of human MARCKS was cloned into the luciferase-expressing vector pEZX-MT01 to the downstream of the firefly luciferase gene. Co-transfection of MARCKS-UTR or negative control luciferase reporter vectors together with miR-34a expressing or control vector showed that miR-34a overexpression significantly reduced the luciferase activity, indicating that miR-34a can directly target the MARCKS 3′ UTR.To examine the functional effect of miR-34a on cell proliferation and apoptosis in MM cells, the miR-34a overexpressing or scramble control cell lines were treated with BTZ. Overexpression of miR-34a in drug resistant cell lines re-sensitized them to BTZ. Furthermore, FACS analysis revealed that overexpression of miR-34a could induce apoptosis in drug resistant MM cell lines as indicated by an increase in the percentage of annexin-V positive cells compared to the control, suggesting that downregulation of miR-34a is associated with acquired BTZ-resistance in MM.To further elucidate the interaction of miR-34a and MARCKS, we knocked down MARCKS expression by transfecting shMARCKS lentiviral vector into 8226-R5 and MM1.R resistant cell lines and examined the miR-34a level in MARCKS-knockdown cells. We found that miR-34a is also regulated by MARCKS as silencing of MARCKS could restore the miR-34a expression level in MM resistant cell lines. In compatible with miR-34a overexpression experiments, following the knockdown of MARCKS, both cell lines had significantly lower viability after treatment with BTZ, in comparison to negative controls. We also showed that knockdown of MARCKS by shMARCKS silencing or suppression of MARCKS by miR-34a overexpression increased the mRNA and protein levels of some pro-apoptotic genes including PUMA which may be involved in induction of apoptosis in drug resistant MM cells.In conclusion, our data indicate that miR-34a is downregulated in drug resistant MM cell lines and primary MM cells, which contributes to the drug resistance of MM through upregulation of MARCKS. Moreover, regulatory interaction between miR-34a and MARCKS provides the rationale that restoration of miR-34a may serve as a promising therapeutic approach by targeting MARCKS in patients with refractory/relapsed MM. DisclosuresNo relevant conflicts of interest to declare.

Effect of Insulin-Regulated FOXC2 Expression in Adipocyte Differentiation and Insulin Resistance.

Objective1) To investigate the effect of FOXC2 on the differentiation of adipose-derived mesenchymal stem cells. 2) To analyze the mechanism between FOXC2 expression regulation in adipose differentiation and insulin resistance (IR).MethodsWe first amplified the FOXC2 promoter region-512 and cloned it into the luciferase expression vector. The reporter gene system was transfected into the adipose tissue-derived mesenchymal stem cell to study insulin-mediated FOXC2 expression. We also manipulated FOXC2 protein expression by either siRNA or overexpression and studied the differentiation capability of adipose tissue-derived mesenchymal stem cell into adipocytes, as well as the influence on several IR-related genes: GLUT4, PPARγ, UCP1 and PAI-1.Results1) Insulin effectively induced the expression of FOXC2 protein in adipose tissue-derived mesenchymal stem cells under differentiation (P<0.01). Insulin also induced FOXC2-pro-512T promoter activity significantly (P<0.01). 2) The stem cell adipose differentiation decreased in the FOXC2 overexpression group. 3) When FOXC2 was overexpressed, the expression of GLUT4, PAI-1 and UCP1 was higher than control groups (p<0.001). When FOXC2 was down-regulated by siRNA, both GLUT4 and PAI-1’s protein expression were decreased (p<0.001), and the protein expression of PPARγ was increased (p<0.001). In the presence of insulin induction, overexpression of FOXC2 led to significantly higher UCP-1 expression (p<0.001) and lower PAI-1 expression (p<0.001). The protein expression of GLUT4, PAI-1 (p<0.001) and UCP-1 (p<0.05) was decreased in cells transfected with FOXC2 siRNA.ConclusionInsulin effectively induced the expression of FOXC2 protein in adipose tissue-derived mesenchymal stem cells under differentiation, possibly through the regulation of the FOXC2-pro-512T promoter activity. The different protein expression of FOXC2 has regulatory effects on several genes related to insulin resistance. FOXC2 is an important regulatory factor in adipocyte differentiation and insulin resistance.

Abstract PR09: MicroRNA-1205 regulation of FRYL and aggressive prostate cancer in men of African ancestry

Abstract Men of African ancestry (moAA) have increased likelihood for development of aggressive prostate cancer (PCa). PCa deaths are often associated with castration-resistant prostate cancer (CRPC) due to maintenance of androgen receptor (AR) signaling in PCa cells following androgen ablation therapy (ADT). The 8q24 chromosomal locus is a highly susceptible PCa region that carries risk genetic variants associated with increased incidence of aggressive PCa in moAA. This region also carries frequent amplifications of the PVT1 gene, a nonprotein coding gene that encodes microRNA-1205 whose function was previously unknown. We examined miR-1205 mRNA expression in a cohort of normal (n=22), benign prostatic hyperplasia (n=42), and PCa (n=26) histologically confirmed prostatic tissues obtained from prostatectomy or transrectal biopsies of moAA in Ibadan, Nigeria. A Tukey post hoc test revealed decreased miR-1205 expression in benign prostatic hyperplasia (4.61 ± 7.5) and PCa (3.39 ± 3.53) when compared to normal tissues (6.55 ± 9.5), suggesting that miR-1205 may function as a tumor suppressor and loss of miR-1205 is characteristic of PCa in moAA. In vitro studies revealed decreased miR-1205 expression in CRPC (C4-2B and 22RV1) cells when compared to non-CRPC (LNCaP) cells, suggesting a role for miR-1205 in CRPC. Furthermore, we observed significant inhibition of tumor growth in NOD/SCID gamma mice implanted with C4-2B cells that were administered our novel synthetic analog of miR-1205 (patent pending) when compared to mice treated with a scramble oligonucleotide, indicating that miR-1205 can suppress CRPC tumors. We identified Fry-like (FRYL) as a putative target of miR-1205 using a miSVR computer algorithm and subsequently observed FRYL and AR overexpression in prostate tumors compared to normal tissue from fourteen PCa patients when whole-transcriptome analysis was performed using the Galaxy web platform. Moreover, FRYL was overexpressed in CRPC cells when compared with non-CRPC cells, further suggesting a role in CRPC development. C4-2B cells transfected with miR-1205 and the 3′ UTR of FRYL in a luciferase expressing vector revealed a significant decrease in luciferase activity when compared to control cells, indicating direct binding of miR-1205 to the 3′ UTR of FRYL. These observations strongly suggest that miR-1205 acts as a tumor suppressor by directly targeting FRYL mRNA in PCa cells. FRYL is predicted to regulate dendritic branching, leading to our hypothesis that FRYL plays a role in the development of PCa with neuroendocrine differentiation (PCND), a resulting mechanism due to ADT resistance. We observed that FRYL mRNA was overexpressed and miR-1205 was significantly underexpressed after fourteen days of inducing PCND in LNCaP cells, suggesting that miR-1205 regulation of FRYL mRNA may play a role in PCND development. Further understanding miR-1205 regulation of FRYL may provide novel insights into the molecular mechanisms of aggressive PCa. This abstract is also being presented as Poster B068. Citation Format: Michelle K. Naidoo, Fayola Levine, Tamara Gillot, Thahmina Ali, Konstantinos Krampis, Akintunde Orunmuyi, E.O. Olapade-Olaopa, Olorunseun O. Ogunwobi. MicroRNA-1205 regulation of FRYL and aggressive prostate cancer in men of African ancestry [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr PR09.

Effect of the natural compound trans‑resveratrol on human MCM4 gene transcription.

trans‑Resveratrol (Rsv) is a natural compound contained in red wine and grape skins that has various beneficial effects for organisms such as lengthening of their life span. Rsv induces expression of the human TP53 and HELB genes, which are involved in the regulation of DNA maintenance. In the present study, a luciferase expression vector containing 309bp of the 5' upstream end of the human MCM4 gene was transfected into HeLa S3 cells. A luciferase assay revealed that Rsv treatment increased the minichromosome maintenance4 (MCM4) gene promoter activity by GC‑box and GGAA (TTCC) motifs. Electro phoretic mobility shift assay revealed that the specific binding factor (complex) contains PU.1 (SPI1). Quantitative reverse transcription‑polymerase chain reaction analysis indicated that MCM4 gene expression was transiently induced by Rsv. Moreover, western blotting revealed that the SP1/PU.1 ratio markedly increased after Rsv treatment, indicating that a balance or profile of these transcription factors may control Rsv‑inducible initiation of transcription. These observations indicated that the beneficial effects of Rsv can be attributed to induction of the chromosomal DNA maintenance factor encoding gene expression.

The 3ʹ-untranslated region contributes to the pregnane X receptor (PXR) expression down-regulation by PXR ligands and up-regulation by glucocorticoids

Pregnane X receptor (PXR) is the major regulator of xenobiotic metabolism. PXR itself is controlled by various signaling molecules including glucocorticoids. Moreover, negative feed-back regulation has been proposed at the transcriptional level. We examined the involvement of the 3′-untranslated region (3′-UTR) of NR1I2 mRNA and microRNAs in PXR- and glucocorticoid receptor (GR)-mediated regulation of NR1I2 gene expression. PXR ligands were found to significantly downregulate NR1I2 mRNA expression in a set of 14 human hepatocyte cultures. Similarly, PXR was downregulated by PCN in the C57/BL6 mice liver. In mechanistic studies with the full-length 3′-UTR cloned into luciferase reporter or expression vectors, we showed that the 3′-UTR reduces PXR expression. From the miRNAs tested, miR-18a-5p inhibited both NR1I2 expression and CYP3A4 gene induction. Importantly, we observed significant upregulation of miR-18a-5p expression 6 h after treatment with the PXR ligand rifampicin, which indicates a putative mechanism underlying NR1I2 negative feed-back regulation in hepatic cells. Additionally, glucocorticoids upregulated NR1I2 expression not only through the promoter region but also via 3′-UTR regulation, which likely involves downregulation of miR-18a-5p. We conclude that miR-18a-5p is involved in the down-regulation of NR1I2 expression by its ligands and in the upregulation of NR1I2 mRNA expression by glucocorticoids in hepatic cells.

Repression of Death Receptor–Mediated Apoptosis of Hepatocytes by Hepatitis B Virus e Antigen

Hepatitis B virus (HBV) e antigen (HBeAg) is associated with viral persistence and pathogenesis. Resistance of HBV-infected hepatocytes to apoptosis is seen as one of the primary promotors for HBV chronicity and malignancy. Fas receptor/ligand (Fas/FasL) and the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) system plays a key role in hepatic death during HBV infection. We found that HBeAg mediates resistance of hepatocytes to FasL or TRAIL-induced apoptosis. Introduction of HBeAg into human hepatocytes rendered resistance to FasL or TRAIL cytotoxicity in a p53-dependent manner. HBeAg further inhibited the expression of p53, total Fas, membrane-bound Fas, TNF receptor superfamily member 10a, and TNF receptor superfamily member 10b at both mRNA and protein levels. Incontrast, HBeAg enhanced the expression of soluble forms of Fas through facilitation of Fas alternative mRNA splicing. In a mouse model, expression of HBeAg in mice injected with recombinant adenovirus-associated virus 8 inhibited agonistic anti-Fas antibody-induced hepatic apoptosis. Xenograft tumorigenicity assay also found that HBeAg-induced carcinogenesis was resistant to the proapoptotic effect of TRAIL and chemotherapeutic drugs. These results indicate that HBeAg may prevent hepatocytes from FasL and TRAIL-induced apoptosis by regulating the expression of the proapoptotic and antiapoptotic forms of death receptors, which may contribute to the survival and persistence of infected hepatocytes during HBV infection.

Characterization of the human E2F4 promoter region and its response to 12-O-tetradecanoylphorbol-13-acetate.

The E2F transcription factors (TFs), which control the progression of the cell cycle in response to DNA-damage and various stresses, are known to interact with a tumor suppressor, Retinoblastoma 1 (RB1). We previously showed that the response of the human RB1 promoter to a 12-O-tetradecanoylphorbol-13-acetate (TPA) in HL-60 cells is mediated by a duplicated GGAA-motif, which is also present in the 5'-upstream of the E2F family genes. The motifs are especially rich in the 5'-upstream of the E2F4 gene. In the present study, we constructed Luciferase (Luc) expression vectors containing a 466-bp of the 5'-upstream of the human E2F4 gene. The transfection of this plasmid and deletion/mutation-introduced derivatives into HL-60 cells and a Luc reporter assay showed that duplicated and triplicated GGAA (TTCC) motifs in the E2F4 promoter respond to TPA. As expected, electrophoretic mobility shift assay (EMSA) indicated that SPI1 (PU.1) binds to the GGAA-motif containing element. A quantitative RT-PCR and Western blotting showed that the E2F4 transcripts and its encoding proteins accumulate during the differentiation of HL-60 into macrophage-like cells. In contrast, the expression of the E2F1 gene and the protein, which possibly acts as a cell cycle accelerator, was greatly diminished.

Abstract 503: MicroRNA-1205 as a tumor suppressor in castration resistant prostate cancer

Abstract Prostate cancer (PCa) is the second most common cancer and third deadliest cancer in American men, yet the only established risk factors are familial genetics, age and race. Men of African Ancestry (moAA) have higher PCa incidence rates when compared to Caucasian men in the United States, which are due to differences in genetic susceptibility variants. Moreover, high mortality rates of PCa are associated with castration-resistant prostate cancer (CRPC) due to maintenance of androgen receptor (AR) signaling in PCa cells following androgen ablation therapy. The 8q24 chromosomal locus is a highly susceptible PCa region that has frequent amplifications of the c-MYC oncogene and downstream PVT1 gene. PVT1 is a long non-protein coding gene that encodes six annotated microRNAs (miRNAs), including microRNA-1205 (miR-1205), yet the function of these miRNAs are poorly understood. To elucidate the role of miR-1205 in PCa, we examined miR-1205 mRNA expression in a cohort of normal (n=22), benign (n=42), and malignant (n=26) histologically confirmed prostatic tissues obtained from prostatectomy or transrectal biopsies of moAA men in Ibadan, Nigeria. One-way ANOVA analysis determined changes in the relative expression of miR-1205 between groups (F(2,87) = 1.153) and a Tukey post hoc test revealed decreased miR-1205 expression in benign (4.61 ± 7.5) and malignant tumors (3.39 ± 3.53) when compared to normal tissues (6.55 ± 9.5). These data suggest that miR-1205 may function as a miRNA tumor suppressor and is characteristic to moAA-associated PCa. To elucidate the molecular mechanism of miR-1205, we examined AR and c-MYC mRNA expression using androgen-dependent LNCaP and CRPC C4-2B and 22RV1 cells. We observed a two-fold decrease of miR-1205 expression and overexpression of AR and c-MYC in C4-2B and 22RV1 cells when compared to LNCaP, suggesting that miR-1205 may regulate AR and c-MYC signaling in CRPC. Next, we identified Fry-like (FRYL) as a putative target using a miSVR computer algorithm and subsequently performed whole transcriptome analysis on prostate tumors and adjacent normal tissue from fourteen PCa patients using the Galaxy web platform. FRYL and AR overexpression was observed in diseased patients suggesting that FRYL may function as an oncogene. Moreover, FRYL was overexpressed in C4-2B and 22RV1 cells when compared to LNCaP, further suggesting a role in CRPC development. C4-2B cells transfected with miR-1205 and the 3'UTR of FRYL in a luciferase expressing vector revealed a significant decrease in luciferase activity when compared to control cells, indicating direct binding of miR-1205 to the 3'UTR of FRYL. These observations strongly suggest that miR-1205 acts as a tumor suppressor that may regulate AR and c-MYC expression, and directly targets the 3'UTR of FRYL in PCa cells. Further understanding the role of miR-1205 regulation of FRYL, AR, and c-MYC signaling may provide novel insights into the molecular mechanisms of CRPC. Citation Format: Michelle K. Naidoo, Dibash K. Das, Adeodat Ilboudo, Akintunde Orunmuyi, Gabriel O. Ogun, S. A. Adebayo, E. O. Olapade-Olaopa, Olorunseun Ogunwobi. MicroRNA-1205 as a tumor suppressor in castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 503.

Porphyromonas endodontalis reactivates latent Epstein-Barr virus.

To determine whether Porphyromonas endodontalis can reactivate latent Epstein-Barr virus (EBV). The concentrations of short-chain fatty acids (SCFAs) in P.endodontalis culture supernatants were determined using high-performance liquid chromatography. A promoter region of BamHI fragment Z leftward open reading frame 1 (BZLF-1), which is a transcription factor that controls the EBV lytic cycle, was cloned into luciferase expression vectors. Then, the luciferase assay was performed using P.endodontalis culture supernatants. Histone acetylation using Daudi cells treated with P.endodontalis culture supernatants was examined using Western blotting. BZLF-1 mRNA and BamHI fragment Z EB replication activator (ZEBRA) protein were also detected quantitatively using real-time polymerase chain reaction (PCR) and Western blotting. Surgically removed periapical granulomas were examined to detect P.endodontalis, EBV DNA, and BZLF-1 mRNA expression using quantitative real-time PCR. Statistical analysis using Steel tests was performed. The concentrations of n-butyric acid in P.endodontalis culture supernatants were significantly higher than those of other SCFAs (P=0.0173). Using B-95-8-221 Luc cells treated with P.endodontalis culture supernatants, the luciferase assay demonstrated that P.endodontalis induced BZLF-1 expression. Hyperacetylation of histones was also observed with the culture supernatants. BZLF-1 mRNA and ZEBRA protein were expressed by Daudi cells in a dose-dependent manner after the treatment with P.endodontalis culture supernatants. P.endodontalis and BZLF-1 in periapical granulomas were also detected. The expression levels of BZLF-1 mRNA were similar to the numbers of P.endodontalis cells in each specimen. n-butyric acid produced by P.endodontalis reactivated latent EBV.

Treatment of collagenase-induced osteoarthritis with a viral vector encoding TSG-6 results in ectopic bone formation.

ObjectiveTumor necrosis factor-inducible gene 6 (TSG-6) has anti-inflammatory and chondroprotective effects in mouse models of inflammatory arthritis. Because cartilage damage and inflammation are also observed in osteoarthritis (OA), we determined the effect of viral overexpression of TSG-6 in experimental osteoarthritis.MethodsBone marrow-derived cells were differentiated to multinucleated osteoclasts in the presence of recombinant TSG-6 or after transduction with a lentiviral TSG-6 expression vector. Multi-nucleated osteoclasts were analyzed after tartrate resistant acid phosphatase staining and resorption activity was determined on dentin slices. Collagenase-induced osteoarthritis (CIOA) was induced in C57BL/6 mice after intra-articular injection of an adenoviral TSG-6 or control luciferase expression vector. Inflammation-related protease activity was measured using bioluminescent Prosense probes. After a second adenovirus injection, cartilage damage was assessed in histological sections stained with Safranin-O. Ectopic bone formation was scored in X-ray images of the affected knees.ResultsTSG-6 did not inhibit the formation of multi-nucleated osteoclasts, but caused a significant reduction in the resorption activity on dentin slices. Adenoviral TSG-6 gene therapy in CIOA could not reduce the cartilage damage compared to the luciferase control virus and no significant difference in inflammation-related protease activity was noted between the TSG-6 and control treated group. Instead, X-ray analysis and histological analysis revealed the presence of ectopic bone formation in the TSG-6 treated group.ConclusionGene therapy based on the expression of TSG-6 could not provide cartilage protection in experimental osteoarthritis, but instead resulted in increased ectopic bone formation.

A New Cellular Model System for Study of Regulatory Mechanisms of Human Ribosomal Gene Transcription

The rate of protein synthesis strictly correlates with the rRNA quantity in a cell. The rRNA synthesis is the first stage in the ribosomal synthesis process. It is actively regulated by such external factors as type of nutrition, growth factor, and cell stress. Thus, pre-rRNA transcription and maturation play a central role in processes of cell growth and proliferation. In recent years, numerous data have been obtained that make it possible to suggest that quantitative and qualitative changes in the rRNA synthesis are the most important molecular indicators of cell malignancy. The principle of suppressing activity of rRNA genes at various stages of their expression is used today in chemotherapeutic methods of treatment of oncological diseases. A number of data indicate that the adaptation of the nucleoli precedes the malignant transformation and is not the result of it. These data offer new possibilities for studying the relationship between the transcription of ribosomal DNA (rDNA) and cell proliferation. Understanding these subtle connections is necessary for the development of new approaches to the molecular characterization and subsequent therapy of neoplastic diseases. The aim of this work was to obtain a cellular model system that makes it possible to adequately estimate the activity of RNA polymerase I in vivo by measuring the expression level of the luciferase reporter gene. For this purpose, the regulatory region of the 6.7 kb rRNA gene preceding the transcriptional start point was recloned into the luciferase expression vector pGL4.77. The obtained construct was used to transfect the line HEK293, which resulted in the line pqA77.HEK293. The new model system opens wide prospects for studying transcription regulation mechanisms of human ribosomal genes.

Isolation and characterization of the 5′-flanking region of the human PDXK gene

Pyridoxal kinase is a key enzyme for the biosynthesis of pyridoxal 5′-phosphate. Pyridoxal 5′-phosphate is the catalytically active form of vitamin B6, and acts as a cofactor in >140 different enzyme reactions. It is still unknown how the kinase synthesis is regulated in the cells, and nothing has been reported about the gene promoter. In the present study, based on the bioinformatics analysis of the 5′-flanking region of the human PDXK gene, we cloned the promoter region by PCR. Through the construction of a series of luciferase expression vectors containing the human PDXK promoter region, we characterized the promoter in terms of its structure and function. The transcription start site is at 198bp upstream of the ATG translation initiation site. An important regulatory region is located at −665/−433bp upstream of the transcription start site. The promoter lacks the canonical TATA box, but contains three GC-boxes and one E-box. A deletion and mutation experiment revealed that the transcription factor Sp1 binding site C (−553/−543) is critical in maintaining the robust promoter activity. Knockdown of Sp1 by RNA interference and chromatin immunoprecipitation analysis further proved that the Sp1 is involved in the regulation of the PDXK gene expression.

Unbiased identification of signal-activated transcription factors by barcoded synthetic tandem repeat promoter screening (BC-STAR-PROM).

The discovery of transcription factors (TFs) controlling pathways in health and disease is of paramount interest. We designed a widely applicable method, dubbed barcorded synthetic tandem repeat promoter screening (BC-STAR-PROM), to identify signal-activated TFs without any a priori knowledge about their properties. The BC-STAR-PROM library consists of ∼3000 luciferase expression vectors, each harboring a promoter (composed of six tandem repeats of synthetic random DNA) and an associated barcode of 20 base pairs (bp) within the 3' untranslated mRNA region. Together, the promoter sequences encompass >400,000 bp of random DNA, a sequence complexity sufficient to capture most TFs. Cells transfected with the library are exposed to a signal, and the mRNAs that it encodes are counted by next-generation sequencing of the barcodes. This allows the simultaneous activity tracking of each of the ∼3000 synthetic promoters in a single experiment. Here we establish proof of concept for BC-STAR-PROM by applying it to the identification of TFs induced by drugs affecting actin and tubulin cytoskeleton dynamics. BC-STAR-PROM revealed that serum response factor (SRF) is the only immediate early TF induced by both actin polymerization and microtubule depolymerization. Such changes in cytoskeleton dynamics are known to occur during the cell division cycle, and real-time bioluminescence microscopy indeed revealed cell-autonomous SRF-myocardin-related TF (MRTF) activity bouts in proliferating cells.