Leon Strzadala

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.

Non-genomic effects of the NR4A1/Nur77/TR3/NGFIB orphan nuclear receptor

The orphan nuclear receptor NR4A1/Nur77/TR3/NGFIB acts primarily as a transcription factor to regulate the expression of multiple genes. However, increasing research attention has recently been given to non-genomic activities of NR4A1. The first description of a non-genomic action of NR4A1 referred to the conversion of anti-apoptotic Bcl-2 into a pro-apoptotic protein by direct interaction with NR4A1. In response to certain apoptotic stimuli, NR4A1 translocates from the nucleus to the mitochondrial outer membrane (MOM) where it associates with Bcl-2 and thereby causes apoptosis. Afterwards, it appeared that NR4A1 could also bind and convert other anti-apoptotic Bcl-2 family members. The latest studies indicate a significant role of NR4A1 in the process of autophagy. For example, a new NR4A1-mediated pathway specific for melanoma cells has been described where NR4A1 interacts with the adenine nucleotide translocase 1 (ANT1) on the mitochondrial inner membrane (MIM) leading to induction of the autophagy pathway. Moreover, NR4A1 interaction with cytoplasmic p53 may also contribute to the induction of autophagy. In addition to mitochondria, NR4A1 could be translocated to the outer membrane of the endoplasmic reticulum (ER) and associate with Bcl-2 or translocon-associated protein subunit γ (TRAPγ) causing ER stress-induced apoptosis. NR4A1 also contributes to the proteasomal degradation of β-catenin in colon cancer cells in vitro and in vivo, as well as to the stabilization of hypoxia-inducible factor-1α (HIF-1α) under non-hypoxic conditions. This review summarizes research findings on non-genomic effects of NR4A1 in normal and cancer cells.

Inhibition of MEK induces fas expression and apoptosis in lymphomas overexpressing Ras.

Published results implicate PI3 kinase as a target of oncogenic Ras activity leading to the suppression of Fas but whether other Ras targets (e.g. Raf-1) are also involved is unclear. Here we report that thymic lymphomas overexpressing Ras and Raf-1 exhibit low expression of Fas. We show that expression of Fas in these lymphomas can be increased not only in the presence of a specific inhibitor (LY294002) of PI3 kinase, but also in the presence of specific inhibitor (PD98059) of MEK, downstream target of Raf-1. Both treatments result in accumulation of ERK in cytosol of lymphoma cells suggesting cross-talk between these two pathways regulating Fas expression. Treatment with PD98059 also results in apoptosis of the lymphoma cells but not of normal thymocytes expressing low Raf-1 levels. These observations provide evidence for involvement of Raf-1/MEK/ERK pathway in Ras-mediated inhibition of Fas expression and in selective promotion of survival of lymphoma cells.

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.

Overexpression of Ras, Raf and L-myc but not Bcl-2 family proteins is linked with resistance to TCR-mediated apoptosis and tumorigenesis in thymic lymphomas from TCR transgenic mice

Mice with transgenic TCR anti H-Y/Db develop spontaneous thymic tumors with a high frequency (up to 50%). Oncogenicity of TCR transgenes could depend on the deregulated expression of oncoproteins engaged in transduction pathways leading to proliferation or apoptosis. In agreement with this possibility we have found that cells of thymic lymphomas from TCR transgenic mice were largely resistant to TCR-dependent Ca++-mediated apoptosis but not to TCR-independent, p53-mediated (etoposide) apoptosis. Here we show raised expression of Bcl-2 protein in some but not in all thymic lymphoma cell lines. It suggests that the antiapoptotic function of Bcl-2 is not necessary for the process of tumorigenesis and the resistance of these lymphomas to Ca++-mediated apoptosis. On the other hand we show that all thymic lymphomas overexpressed Ras/Raf and L-myc proteins. Stimulation of the Ras/Raf pathway was reported to be required to maintain cell viability by preventing programmed cell death in thymic tumors derived from lck transgenic mice. Similarly, in TCR transgenic lymphomas overexpression of Ras, Raf and L-myc but not Bcl-2 family proteins may be responsible for the resistance of these lymphomas to TCR-mediated apoptosis but not affect p53-mediated apoptosis.

HA1004, an inhibitor of serine/threonine protein kinases, restores the sensitivity of thymic lymphomas to Ca2+-mediated apoptosis through a protein kinase A-independent mechanism.

Our previous reports showed that thymic lymphomas arising in TCR transgenic mice are resistant to Ca2+-mediated apoptosis. Here we show that induction of apoptosis in thymic lymphomas involves a process that is cAMP-mediated and which depends on the activation of protein kinase A (PKA) despite the lower level of PKA type I in these lymphomas compared to thymocytes. Further, we show that treatment of the lymphomas with HA1004, a serine/threonine protein kinase inhibitor, restores their susceptibility to ionomycin-induced apoptosis. Results indicate that HA1004-induced restoration of sensitivity to ionomycin proceeds through a PKA-independent mechanism. Moreover, activation of PKA instead of its inhibition induces apoptosis of lymphoma cells.

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 genes 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.

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.

Apoptosis of lymphoma cells is abolished due to blockade of cytochrome c release despite Nur77 mitochondrial targeting

Nur77 is reported to undergo translocation to mitochondria in response to apoptotic signaling in a variety of cancer cell lines. It was shown that on the mitochondrial membrane, Nur77 interacts with Bcl-2, leading to the conversion of this protein from a protector to a killer with subsequent release of cytochrome c to the cytosol. Here it is shown that in thymic lymphoma cells resistant to calcium-mediated apoptosis, cytochrome c release is abolished despite of Nur77 mitochondrial targeting. However, cytochrome c release and apoptosis can be restored by treatment with FK506. Hence, the molecular target regulation of the sensitivity of lymphoma cells to calcium signaling is associated with cytochrome c release and is FK506 sensitive. These results provide new insight into the role of FK506-sensitive factors as a critical link between calcium signaling and resistance of lymphoma cells to death.

Synergistic activity of sorafenib and betulinic acid against clonogenic activity of non‐small cell lung cancer cells

The highly selective multi‐targeted agent sorafenib is an inhibitor of a number of intracellular signaling kinases with anti‐proliferative, anti‐angiogenic and pro‐apoptotic effects in various types of tumors, including human non‐small cell lung cancer (NSCLC). Betulin displays a broad spectrum of biological and pharmacological properties, including anticancer and chemopreventive activity. Combination of drugs with different targets is a logical approach to overcome multilevel cross‐stimulation among key signaling pathways in NSCLC progression. NSCLC cell lines, A549, H358 and A427, with different KRAS mutations, and normal human peripheral blood lymphocyte cells, were treated with sorafenib and betulinic acid alone and in combination. We examined the effect of different combined treatments on viability (MTS test), proliferation and apoptotic susceptibility based on flow cytometry, alterations in signaling pathways by western blotting and colony‐forming ability. The combination of sorafenib with betulinic acid had a strong effect on the induction of apoptosis of different NSCLC cell lines. In addition, this combination was not toxic for human peripheral blood lymphocytes. Combination treatment changed the expression of proteins involved in the mitochondrial apoptosis pathway and induced apoptotic death by caspase activation. Importantly, combination treatment with low drug concentrations tremendously reduced the colony‐forming ability of A549, H358 and A427 cells, as compared to both compounds alone. In this study, we showed that combination therapy with low concentrations of sorafenib and betulinic acid had the capacity to induce high levels of cell death and abolish clonogenic activity in some NSCLC cell lines regardless of KRAS mutations.