Downregulation of MALAT1 in triple-negative breast cancer cells.

Background: MALAT1 is one of the most abundant nuclear long non-coding RNAs, which has been found to be elevated in various types of cancers. However, conflicting reports on MALAT1 in breast cancer cell lines challenge understanding of MALAT1's involvement in breast cancer progression. Aim: Measurement of normalized relative quantity (NRQ) of MALAT1 transcripts in cell lines representing triple-negative breast cancer (TNBC) and luminal breast cancer. Materials and methods: The studies were performed using cell lines representing luminal breast cancer (T47D, MCF-7), TNBC (MDA-MB-468, CAL-51, MDA-MB-231), and MCF-10A cell line of normal breast epithelial cells. Total RNA was isolated from six independent cell cultures of each line, treated with DNase I, and used to synthesize complementary DNA, which was used in quantitative real-time PCR (qPCR) assays. Four MALAT1 fragments and reference genes CCSER2, ANKRD17, PUM1, GAPDH were amplified. Results: Geometric means of the NRQ of MALAT1 in breast cancer cell lines had the shortest 95% confidence intervals when CCSER2 was used for normalization. MALAT1 major transcript levels thus estimated in TNBC cell lines were found to be statistically significantly reduced compared to levels in both MCF-10A cells and luminal breast cancer cell lines, while MALAT1 minority splice variants were found to be increased in almost all breast cancer cell lines. Conclusion: CCSER2-normalized qPCR results indicate MALAT1 downregulation in cell lines representing the more aggressive breast cancer subtype compared to both the normal breast epithelial cell line and the estrogen receptor-positive breast cancer cell lines.

MALAT1-miRNAs network regulate thymidylate synthase and affect 5FU-based chemotherapy.

BACKGROUND: The active metabolite of 5-Fluorouracil (5FU), used in the treatment of several types of cancer, acts by inhibiting the thymidylate synthase encoded by the TYMS gene, which catalyzes the rate-limiting step in DNA replication. The major failure of 5FU-based cancer therapy is the development of drug resistance. High levels of TYMS-encoded protein in cancerous tissues are predictive of poor response to 5FU treatment. Expression of TYMS is regulated by various mechanisms, including involving non-coding RNAs, both miRNAs and long non-coding RNAs (lncRNAs). AIM: To delineate the miRNAs and lncRNAs network regulating the level of TYMS-encoded protein. MAIN BODY: Several miRNAs targeting TYMS mRNA have been identified in colon cancers, the levels of which can be regulated to varying degrees by lncRNAs. Due to their regulation by the MALAT1 lncRNA, these miRNAs can be divided into three groups: (1) miR-197-3p, miR-203a-3p, miR-375-3p which are downregulated by MALAT1 as confirmed experimentally and the levels of these miRNAs are actually reduced in colon and gastric cancers; (2) miR-140-3p, miR-330-3p that could potentially interact with MALAT1, but not yet supported by experimental results; (3) miR-192-5p, miR-215-5p whose seed sequences do not recognize complementary response elements within MALAT1. Considering the putative MALAT1-miRNAs interaction network, attention is drawn to the potential positive feedback loop causing increased expression of MALAT1 in colon cancer and hepatocellular carcinoma, where YAP1 acts as a transcriptional co-factor which, by binding to the TCF4 transcription factor/ ß-catenin complex, may increase the activation of the MALAT1 gene whereas the MALAT1 lncRNA can inhibit miR-375-3p which in turn targets YAP1 mRNA. CONCLUSION: The network of non-coding RNAs may reduce the sensitivity of cancer cells to 5FU treatment by upregulating the level of thymidylate synthase.

VDR Agonists Increase Sensitivity of MCF-7 and BT-474 Breast Cancer Cells to 5 FU.

BACKGROUND/AIM: The study aimed to test the potential for increasing the antiproliferative activity of 5-fluorouracil against breast cancer cells of various molecular subtypes by vitamin D receptor (VDR) agonists, calcitriol and tacalcitol, used at a low concentration of 10 nM. MATERIALS AND METHODS: Calcitriol and tacalcitol were used to increase the antiproliferative effect of 5-fluorouracil against the following human breast cancer cell lines: MCF-7, T47D, BT-474 (luminal); JIMT-1, SKBR-3 (HER2-enriched); MDA-MB-231 (triple-negative/basal-B), and non-malignant MCF-10A breast epithelial cells. RESULTS: Both calcitriol and tacalcitol significantly increased the sensitivity of MCF-7 and BT-474 cells to the antiproliferative effect of 5-fluorouracil, while no increase in the sensitivity of MDA-MB-231 cells to 5-fluorouracil treatment was observed. CONCLUSION: The VDR agonist used at the relatively low concentration of 10 nM may increase the sensitivity of breast cancer cells, at least of the luminal subtype, to the antiproliferative effect of 5-fluorouracil.

miR-125b lowers sensitivity to apoptosis following mitotic arrest: Implications for breast cancer therapy.

The process of apoptosis begins when the balance between proapoptotic and antiapoptotic stimuli is disturbed, leading to oligomerization of apoptosis effectors and disruption of the outer mitochondrial membrane. BCL-2 family proteins are the major regulators of mitochondrial pathway of apoptosis. In turn, microRNA-125b (miR-125b) is a member of microRNAs, which are short single-stranded noncoding RNAs that negatively regulate gene expression at the posttranscriptional level. miR-125b targets messenger RNAs encoding proapoptotic (BAK1, PUMA, BMF) and antiapoptotic (MCL1) BCL-2 family proteins. This mini-review briefly describes the involvement of BCL-2 family proteins in triggering apoptosis. Then, attention is paid to the differences in the activation of apoptosis with doxorubicin and paclitaxel, and finally the effect of miR-125b on paclitaxel- and doxorubicin-induced apoptosis in breast cancer cells is considered. It appears that miR-125b downregulates proteins that are significantly involved in the activation of apoptosis after paclitaxel-induced prolonged mitotic arrest or subsequent DNA damage if mitotic slippage occurs. It seems that high levels of miR-125b may not favor paclitaxel therapy, but reduction of miR-125b levels may increase paclitaxel-induced apoptosis, possibly also genotoxic-induced apoptosis, although adverse effects may also occur including decrease in doxorubicin-induced apoptosis.

Steroid hormone calcitriol and its analog tacalcitol inhibit miR-125b expression in a human breast cancer MCF-7 cell line.

MiR-125b belongs to the class of microRNAs, which are short endogenous non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. Recently, it was reported that miR-125b was found to promote migration and invasion of MCF-7 cells and was involved in chemotherapeutic resistance. Decreasing miR-125b expression would have potential therapeutic significance in preventing dissemination of breast cancer cells. The objective of this study was to evaluate miR-125b expression levels in MCF-7 cells following treatment with 1,25-dihydroxyvitamin D3 (calcitriol) and 1,24-dihydroxyvitamin D3 (tacalcitol), active metabolite and synthetic analog of vitamin D3, respectively. We found that treatment with both calcitriol and tacalcitol caused a decrease in miR-125b expression. In addition, treatment with calcitriol and tacalcitol resulted in an increase in the level of pro-apoptotic BAK1 protein encoded by the target gene of miR-125b. We are discussing the putative mechanism of inhibition of the miR-125b expression by vitamin D receptor (VDR) agonists and we suggest that calcitriol and tacalcitol may be used as a miR-125b inhibitor in breast cancer cells expressing VDR.

Sp1 mediates phorbol ester (PMA)-induced expression of membrane-bound guanylyl cyclase GC-A in human monocytic THP-1 cells.

Cyclic guanosine monophosphate (cGMP) is synthesized by two types of enzymes: particulate (membrane-bound) guanylyl cyclases (pGCs) and soluble (cytosolic) guanylyl cyclases (sGCs). sGCs are primarily activated by binding of nitric oxide to their prosthetic heme group while pGCs are activated by binding of peptide ligands to their extracellular domains. One of them, pGC type A (GC-A) is activated by atrial and brain natriuretic peptides (ANP and BNP, respectively). Human monocytes isolated from peripheral blood mononuclear cells have been found to display sGC expression without concomitant expression of GC-A. However, GC-A activity appears in monocytes under certain conditions but a molecular mechanism of GC-A expression is still poorly understood. In this report we show that phorbol ester (PMA) induces transcription of a gene encoding GC-A in human monocytic THP-1 cells. Moreover, we find that PMA-treated THP-1 cells raise cGMP content following treatment with ANP. Studies using pharmacological inhibitors of protein kinases suggest involvement of protein kinase C (PKC), mitogen extracellular kinases (MEK1/2), and extracellular signal-regulated kinases (ERK1/2) in PMA-induced expression of the GC-A encoding gene in THP-1 cells. Finally, we show that PMA stimulates binding of Sp1 transcription factor to GC-rich DNA sequences and mithramycin A (a selective Sp1 inhibitor) inhibits expression of the GC-A mRNA in PMA-treated THP-1 cells. Taken together, our findings suggest that the PMA-stimulated PKC and MEK/ERK signaling pathways induce Sp1-mediated transcription of the GC-A encoding gene in human monocytic THP-1 cells.

SYNTHESIS AND ANTIBACTERIAL ACTIVITY OF NEW SULFONAMIDE ISOXAZOLO[5,4-b]PYRIDINE DERIVATIVES.

A series of novel sulfonamide isoxazolo[5,4-b]pyridines were synthesized. The substrates for their synthesis were 3-aminoisoxazolo[5,4-b]pyridine and selected aryl sulfonic chlorides, chlorosulfonic acid and selected amines. Reactions were carried out using the classical and microwave methods. Selected compounds were tested towards antibacterial and antiproliferative activity. The structure of the obtained new derivatives was determined by elemental analysis and acquired IR and 1H NMR spectra. Among the tested compounds: N- isoxazolo[5,4-b]pyridine-3-yl-benzenesulfonamide (2) and N-isoxazolo[5,4-b]pyridine-3-yl-4-methylbenzene-sulfonamide (5) showed antimicrobial activity towards Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) at doses: 125, 250 and 500 µg. Both compounds showed a 50% inhibition of proliferation of breast carcinoma cell line MCF7 at concentrations of 152.56 µg/mL and 160 161.08 µg/mL, respectively.

[Ubiquitin as a regulator of IFN production in the antiviral response]. / Ubikwityna jako regulator wytwarzania IFN w odpowiedzi przeciwwirusowej.

Type I interferons (IFNs) are important in the immune response. After pathogen detection, host cells rapidly trigger innate immune mechanisms such as inflammatory cytokines production, thus leading to the eradication of the invading virus. Such mechanisms engage signaling cascades which, in the initial phase of infection, lead to the activation of the NF-κB pathway and IFN regulatory factors (IRF-3, IRF-7) which directly control the production of IFNs. Proper regulation of IFN induction takes place by ubiqutination and allows to maintain a balance between the activation and inhibition of the immune system response due to an infection. Studies in recent years indicate that ubiquitination of proteins can affect both proteasomal degradation as well as the non-canonical pathway which results in the regulation of their activity. The type of ubiquitination primarily depends on the attachment of ubiquitin chain to thetarget protein but also on the activity of proteases from DUBs family. The ubiquitin pathway holds many potential therapeutic targets. Thus, the more detailed understanding of the mechanism of ubiquitination and the role of ubiquitin involved in IFNs production pathways may provide a turning point for both antiviral therapy and autoimmune diseases.

Establishment of a cellular model to study TrkC-dependent neuritogenesis.

The rat PC12 cell line has become a widely used research tool for many aspects of neurobiology. Nerve growth factor (NGF)-responsive PC12 cells were engineered to drive expression of doxycycline (Dox)-induced gene of interest in the Tet-On expression system that resulted in obtaining PC12-Tet-On cells. TrkA and TrkC are neurotrophin receptors derived from the tropomyosin-related kinase (Trk) family of receptor tyrosine kinases. TrkA receptor binds and is activated mainly by NGF, while TrkC receptor binds and is activated by neurotrophin 3 (NT3). The purpose of this research was to design and describe PC12-based neuronal cell model to study TrkC-triggered versus TrkA-triggered neurite outgrowth. The second-generation tetracycline-responsive promoter (P tight) was used in order to provide low basal expression in the absence of Dox and high-level Dox-induced expression of TrkC. The main advantage of presented model system is dependence of TrkC level on Dox concentration. It also allows to compare activation of intracellular signaling proteins and neurite outgrowth following activation of TrkA and TrkC receptors by NGF and NT3, respectively, in the context of the same quality and quantity of intracellular adaptor proteins, Ras proteins, protein kinases and phosphatases, and phospholipase Cγ1, as a difference in the activation of intracellular signaling network by these two distinct although related receptor tyrosine kinases is expected. The results of our studies suggest that despite slightly weaker activation of ERK1/2 mitogen-activated protein kinases, NT3-triggered TrkC seems to provide apparently stronger than NGF-triggered TrkA signal for neurite elongation in differentiating PC12 cells.

Phosphodiesterase 2 negatively regulates adenosine-induced transcription of the tyrosine hydroxylase gene in PC12 rat pheochromocytoma cells.

Adenosine induces expression of the tyrosine hydroxylase (TH) gene in PC12 cells. However, it is suggested that atrial natriuretic peptide (ANP) inhibits expression of this gene. Using real-time PCR and luciferase reporter assays we found that ANP significantly decreases the adenosine-induced transcription of the TH gene. Results of measurements of cyclic nucleotide concentrations indicated that ANP-induced accumulation of cGMP inhibits the adenosine-induced increase in cAMP level. Using selective phosphodiesterase 2 (PDE2) inhibitors and a synthetic cGMP analog activating PDE2, we found that PDE2 is involved in coupling the ANP-triggered signal to the cAMP metabolism. We have established that ANP-induced elevated levels of cGMP as well as cGMP analog stimulate hydrolytic activity of PDE2, leading to inhibition of adenosine-induced transcription of the TH gene. We conclude that ANP mediates negative regulation of TH gene expression via stimulation of PDE2-dependent cAMP breakdown in PC12 cells.

[Multilevel maturation of Toll-like receptor 9]. / Wieloetapowy proces dojrzewania receptora Toll-podobnego 9.

Toll-like receptors (TLRs) are essential elements of the innate immune response. TLRs induce expression of inflammatory cytokines or interferons after recognition of microbial or viral structures called pathogen-associated molecular patterns (PAMPs). Two different groups of TLRs can be distinguished: TLRs residing in the plasma membrane or in the endosomal compartment. TLRs localized in endosomes act as sensors for nucleic acids. TLR9, which recognizes unmethylated CpG, belongs to endosomal TLRs. The proper ligand detection by TLR9 depends on its specific subcellular localization and maturation. TLR9 delivery to the endosomes is mediated by two distinct proteins, UNC93B1 and AP-2, and post-early endosome distribution is determined by AP-3. TLR9 localized in the endosome is cleaved by at least two classes of proteases, AEP and cathepsins, which generate the mature form of receptor. Functional C-terminal form of TLR9 is capable of recognition of CpG and activation of signal pathways. Ligand binding to TLR9 causes conformational changes in the structure of this receptor which facilitates recruitment of MyD88 adaptor protein and activation of two distinct cytokine-inducing pathways: IRF-7- and NF-κB-dependent. The specific structure of the synthetic ligand (CpG-A or CpG-B) determines activation of certain transcription factors. Recognition of multimeric CpG-A results in IRF-7-dependent induction of type I interferon production. Monomeric CpG-B activates NF-κB-dependent induction of proinflammatory cytokines, in particular TNFα and IL-6.

[Regulation of Toll-like receptors-dependent inflammatory response]. / Regulacja odpowiedzi zapalnej zaleznej od receptorów Toll-podobnych.

Toll-like receptors (TLRs) are a pivotal part of our innate immune response. They recognize a wide variety of pathogens and instigate an immune response, thus facilitating the removal of the disease-causing agent. Due to the intense nature of this response its strict control is of key importance, as a prolonged inflammatory signal leads to carcinogenesis and autoimmune disorders. The signaling cascade initiated by the activated TLR is complex and consists of multiple stages. It involves a variety of adaptor proteins, protein kinases and effector transcription factors. The number of stages in this process enables many possible checkpoints and ways of regulation. Signal modulation involves differentiated expression of TLRs, splicing variants of their adaptor proteins, enzymes modifying proteins engaged in the cascade and many more. This review focuses on endogenous factors responsible for controlling the TLR-dependent inflammatory response as well as on pharmacological therapies designed for regulating the innate immune response.  

[Phosphodiesterase 3 mediates cross-talk between the protein kinase- and cGMP- dependent pathways and cyclic AMP metabolism]. / Fosfodiesterazy rodziny 3 sprzegaja szlaki sygnalowe zalezne od kinaz bialkowych i cyklicznego GMP z metabolizmem cyklicznego AMP.

PDE3 is a dual-substrate phosphodiesterase responsible for hydrolyzing both cAMP and cGMP whilst being simultaneously inhibited by cGMP. This feature is related to presence of the 44 amino acid insert in the catalytic domain, which determines the mechanism of introduction of the cyclic nucleotide into the catalytic pocket of the enzyme. Once bound in the catalytic site cGMP results in steric hindrance for cAMP to enter the site. The regulatory domain of PDE3 consists of two hydrophobic regions: NHR1 and NHR2. Their presence defines the enzyme's intracellular localization, thus determining its participation in particular signaling cascades. Due to the properties of PDE3 this enzyme has exceptional importance for the cross-talk between cAMP-dependent signaling and other cascades. There are two different mechanisms of action of PDE3 enzymes in cell signaling pathways. In many signaling cascades assembly of a signalosome is necessary for phosphorylation and activation of the PDE3 proteins. In response to certain hormones and growth factors, PDE3 merges the metabolism of cAMP with protein kinase-dependent signaling pathways. PDE3 also controls the level of cAMP with regard to the alternating concentration of cGMP. This effect occurs in signaling cascades activated by natriuretic peptide.

[The mechanism of phospholipase Cγ1 activation]. / Mechanizm aktywacji fosfolipazy Cγ1.

Phospholipase C is an enzyme which catalyzes the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)) into second messengers inositol-1,4,5-triphosphate (Ins(1,4,5)P3) and diacylglycerol (DAG). These messengers then promote the activation of protein kinase C and release of Ca(2+) from intracellular stores, initiating numerous cellular events including proliferation, differentiation, signal transduction, endocytosis, cytoskeletal reorganization or activation of ion channels. There have been identified 14 isozymes of PLC among which PLCγ1 and PLCγ2 are of particular interest. PLCγ contains catalytic region XY and a few regulatory domains: PH, EF and C2. The most unique features of these two enzymes are the Src homology domains (SH2, SH3) and split PH domain within the catalytic barrel. PLCγ1 and PLCγ2 have an identical domain structure, but they differ in their function and occurrence. Phospholipase Cγ1 is expressed ubiquitously, especially in the brain, thymus and lungs. PLCγ1 can be activated by receptor tyrosine kinases (i.e.: PDGFR, EGFR, FGFR, Trk), as well as non-receptor protein kinases (Src, Syk, Tec) or phosphatidic acid, tau protein and its analogue. The molecular mechanism of PLCγ1 activation includes membrane recruitment, phosphorylation, rearrangements and activation in the presence of growth factors. In reference to PLCγ1 regulation, a number of positive and negative modulators have been considered. The most important positive modulator is phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P(2)). Protein kinase A and C, tyrosine phosphatases (SHP-1, PTP-1B) and Cbl, Grb2, Jak2/PTP-1B complex proteins have been described as negative regulators of PLCγ1 activation.

[The roles of orphan nuclear receptors in T-lymphocyte development in the thymus]. / Rola sierocych receptorów jadrowych w rozwoju limfocytów T w grasicy.

T-lymphocyte development in the thymus proceeds through successive steps of differentiation,selection and expansion of thymocytes. Multiple signaling proteins and transcription factors play important roles in mediating the response to signals delivered through the pre-T-cell receptor (pre-TCR) and T-cell receptor (TCR) at two checkpoints during T-cell development. The orphan nuclear receptors RORg and Nur77 are involved in the differentiation and selection processes that are induced following activation of the pre-TCR and TCR, respectively, and they exert opposite effects on the survival of thymocytes. RORg activates the gene encoding the antiapoptotic protein Bcl-X(L) and is required for the survival of CD4+CD8+ thymocytes, while Nur77 is involved in the apoptosis of CD4+CD8+ thymocytes bearing TCRs with a high affinity for self antigens presented in the thymus.

Inducibility of doxycycline-regulated gene in neural and neuroendocrine cells strongly depends on the appropriate choice of a tetracycline-responsive promoter.

Elucidation of the mechanisms underlying specific receptor activation of neural and neuroendocrine cells will require the establishment of cellular systems that permit the regulation of the expression of the protein of interest. In a tetracycline (Tet)-regulated system, the gene encoding the protein of interest is under the control of a Tet promoter and its transcription is activated in the presence of doxycycline (Dox) by the Tet transactivator rtTA. Acceptable inducibility of the gene's expression requires a high level of its expression in the presence of Dox and a minimal basal expression in the absence of Dox. Two Tet promoters are compared here, the original PhCMV*-1 and the second-generation Ptight, with respect to the inducibility of the gene of interest in neuroendocrine and neural cells genetically engineered to express rtTA, namely PC12-Tet-On cells and MB-G-18 cells (mouse brain-derived cells with the phenotype of neuron-restricted precursors). This study demonstrates that the use of Ptight provided a much higher Dox-induced maximal expression in both cell lines, while the basal activities of the two Tet promoters were at similar levels. The additional use of the Tet-controlled silencer (tTS) caused almost complete abrogation of the leakiness of the Ptight promoter and an increase in the inducibility of the regulated gene, but the maximal levels of gene expression driven in the presence of Dox were also markedly reduced.

Transactivation activity of Nur77 discriminates between Ca2+ and cAMP signals.

The orphan nuclear receptors Nur77 and Nurr1 are the members of the Nur77 family of transcription factors. We demonstrate that transcription of the Nur77 family genes was upregulated in PC12 cells following incubation with Ca2+ ionophore as well as cyclic AMP (cAMP) analog. On the other hand, cAMP analog induced strong increase, while Ca2+ ionophore induced weak increase in the transactivation activity of Nur77. We found that Nur77 and Nurr1 proteins were expressed in the nucleus following stimulation with cAMP analog but not after stimulation with Ca2+ ionophore. However, expression of Nur77 protein was increased in the cytoplasm of cells treated with Ca2+ ionophore. In conclusion, our results suggest that cAMP-induced and Ca2+-induced processes may differentially regulate activity of Nur77 at the level of translocation of Nur77 protein from the cytoplasm into the nucleus.

Early neuronal progenitor cell line expressing solely non-catalytic isoform of TrkC.

TrkC is a receptor for neurotrophin-3 that regulates development of neuronal precursors. Transduction of signals into receptor-dependent signaling pathways is mainly due to the activation of the intrinsic tyrosine kinase of the TrkC receptor. Alternative splicing of the trkC transcripts generates catalytic and non-catalytic isoforms. The non-catalytic isoform, denoted as TrkC-NC2, contains unique sequence, instead of deleted entire kinase domain. Here, we report that neural cell line MB-G, derived from brain of embryos of transgenic tsA58-SV40 mice, contains mRNA encoding TrkC-NC2 without concomitant expression of mRNA for catalytic TrkC molecule.

FK506 restores sensitivity of thymic lymphomas to calcium-mediated apoptosis and the inducible expression of Fas ligand.

We have previously described that thymic lymphomas from anti HY-TCR transgenic mice were resistant to TCR-induced (calcium-mediated) apoptosis but sensitive to induction of apoptosis by etoposide. A defect of apoptosis in these cells is located downstream of the nuclear receptor Nur77 induction and upstream from the execution of apoptosis. Furthermore, in contrast to the normal thymocytes, the lymphoma cells do not express the Fas receptor on the cell surface. Here we show that Fas ligand (FasL) induction is also abrogated in thymic lymphomas treated with ionomycin but not with etoposide, which still induced the expression of FasL and Fas receptor as well as apoptosis. It suggested a specific inhibition of the calcium-mediated signaling pathway leading to induction of the FasL expression and apoptosis of lymphomas. Sensitivity to ionomycin-induced apoptosis could be restored by FK506 treatment, which also abolished the abrogation of induction of the FasL expression. Moreover, induction of FasL was always accompanied by increase in expression of Fas receptor. These results indicate that FK506, the agent broadly used as immuno-suppressant, can sensitize resistant tumor cells to induction of apoptosis and it could be considered as a potential agent in the combined therapy of thymic lymphomas. In addition, it appeared that the Fas/FasL system can successfully serve as a molecular target for tumor therapy, even for tumor cells initially lacking expression of Fas.

Nurr1 affects pRL-TK but not phRG-B internal control plasmid in genetic reporter system.

In transcription assays, Renilla luciferase-expressing plasmids (more specifically pRL-TK) are commonly used as an internal control of transfection efficiency. Normalization of the experimental reporter gene transcription to the internal control reporter gene transcription minimizes variability of obtained results caused by differences in transfection efficiency between different samples of transfected cells. It is obvious that co-transfection with other plasmids or applied treatments should not affect the activity of the control reporter. Here we report that expression of the control Renilla luciferase encoded by pRL-TK plasmid was enhanced by co-transfection with vectors expressing orphan nuclear receptors Nur77 family (Nur77, Nurr1, Nor-1), leading to misinterpretation of the assay results. Further, we show that for Nurr1, phRG-B (a promoterless reporter plasmid containing synthetic Renilla luciferase gene) is a better control reporter vector than HSV-TK containing vectors. Finally, we noted the lack of effect of Nurr1 protein on the Fas Ligand promoter-driven transcription.