Post-transcriptional regulation of glutathione peroxidase gene expression by selenium in the HL-60 human myeloid cell line

We have used a cloned cDNA for the major human selenoprotein, glutathione peroxidase (GPx), to assess the mode of regulation of human GPx gene (GPX-1) expression by selenium. When the HL-60 human myeloid cell line is grown in a selenium-deficient medium, GPx enzymatic activity decreases 30-fold compared with selenium-replete cells. Upon return to a medium containing selenium in the form of selenite, GPx activity in the cells starts to increase within 48 hours and reaches maximal (selenium-replete) levels at 7 days. Steady-state immunoreactive protein levels correlate with enzymatic activity. Cycloheximide inhibits the rise in GPx activity that accompanies selenium replenishment, indicating that protein synthesis is required for the increase. However, GPx mRNA levels and the rate of transcription of the human GPx gene change very little and thus appear to be independent of the selenium supply. Thus the human GPx gene appears to be regulated post-transcriptionally, probably cotranslationally, in response to selenium availability

MDA-7/IL-24 suppresses human ovarian carcinoma growth in vitro and in vivo

BACKGROUND: Previous studies showed that the human melanoma differentiation-associated gene-7 (mda-7), also known as interleukin-24 (IL-24), has potent antitumor activity against human and murine cancer cells. However, the majority of these studies were limited to in vitro testing. In the present study, we investigated the antitumor activity of mda-7/IL-24 against human ovarian cancer cells both in vitro and in vivo. RESULTS: In vitro, treatment of ovarian cancer cells with an adenoviral vector carrying the mda-7 gene (Ad-mda7) resulted in inhibition of cell proliferation and induction of cell cycle arrest, leading to apoptosis. We did not observe inhibitory activity in Ad-mda7-treated normal cells. In vivo, treatment of subcutaneous tumor xenografts with Ad-mda7 resulted in significant tumor growth inhibition when compared with that in control groups (p < 0.001). Molecular analysis of ovarian tumor tissue lysates treated with Ad-mda7 showed that MDA-7 protein expression was associated with activation of the caspase cascade. CONCLUSION: Our results show that treatment of ovarian cancer cells with mda-7/IL-24 results in growth suppression both in vitro and in vivo

mda-7/IL-24, novel anticancer cytokine: Focus on bystander antitumor, radiosensitization and antiangiogenic properties and overview of the phase I clinical experience (Review)

Subtraction hybridization applied to a ‘differentiation therapy’ model of cancer employing human melanoma cells resulted in the cloning of melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24). Initial studies confirm an inverse correlation between mda-7 expression and melanoma development and progression. Forced expression of mda-7 by means of a plasmid or via a replication incompetent adenovirus (Ad.mda-7) promotes growth suppression and induces apoptosis in a broad array of human cancers. In contrast, mda-7 does not induce growth suppressive or toxic effects in normal cells. Based on structure (containing an IL-10 signature motif), secretion by cells (including subsets of T-cells) and location on chromosome 1q (in an area containing IL-10- family genes), mda-7 has now been renamed mda-7/IL-24. Studies by several laboratories have uncovered many of mda-7/ IL-24\u27s unique properties, including cancer-specific apoptosisinduction, cell cycle regulation, an ability to inhibit angiogenesis, potent ‘bystander antitumor activity’ and a capacity to enhance the sensitivity of tumor cells to radiation, chemo- therapy and monoclonal antibody therapy. Moreover, based on its profound cancer tropism, substantiated by in vivo human xenograft studies in nude mice, mda-7/IL-24 (administered as Ad.mda-7) was evaluated in a phase I clinical trial in patients with melanomas and solid cancers. These studies document that mda-7/IL-24 is well tolerated and demonstrates evidence of significant clinical activity. In these contexts, mda-7/IL-24 represents a unique cytokine gene with potential for therapy of human cancers. The present review focuses on three unique properties of mda-7/IL-24, namely its potent ‘bystander antitumor activity’, ability to sensitize tumor cells to radiation, and its antiangiogenesis properties. Additionally, an overview of the phase I clinical trial is provided. These studies affirm that mda-7/IL-24 has promise for the management of diverse cancers

A Molecular Analysis of Selenium Incorporation into Glutathione Peroxidase: Stop Is Not the End: A Thesis

Selenium is toxic at high doses, yet metabolically essential in trace amounts, and therefore provides an excellent illustration of the rule of paracelsus that the dose alone determines the poison . The only mammalian selenoprotein of known function is glutathione peroxidase (GPx). This enzyme is expressed ubiquitously, and is responsible for detoxifying peroxides and hydroperoxides which, if left unchecked, may damage important biomolecules such as DNA and membrane fatty-acids. GPx is a homotetramer; each subunit contains one mole atom of selenium incorporated as a selenocysteine residue in the active site of the enzyme. Using oligonucleotides generated against the known bovine GPx amino-acid sequence, cDNA clones were isolated corresponding to the human GPx mRNA. Sequence analysis indicated that the selenocysteine in the active site of the enzyme was incorporated at an opal terminator (UGA) codon. Therefore the glutathione peroxidase mRNA constitutes the first example of natural suppression of a terminator codon in human cells. Regulation of human GPx gene expression by selenium was examined. Selenium replete HL-60 cells possessed approximately 30-fold more enzymatic GPx activity than selenium deficient cells. However steady-state GPx mRNA levels and rate of transcription of the GPx gene differed by less than 1.5-fold. Cycloheximide abolished the increase in enzymatic activity observed upon selenium replenishment. Cellular immunoreactive GPx protein levels correlated with enzymatic activity, indicating that the human GPx gene is regulated post-transcriptionally by selenium. The mechanism of this post-transcriptional regulation was investigated. A selenium labelled tRNA species was identified which exhibited features in common with a previously characterized tRNAUGA. This data suggested that selenium may be incorporated into GPx via a co-translational mechanism using a selenocysteinyl tRNA intermediate. Selenium did not alter cytoplasmic levels of the tRNAUGA, indicating that accumulation of cytoplasmic suppressor tRNA was not the point of regulation of GPx by selenium. A model is proposed for the co-translational insertion of selenocysteine into GPx mediated by a charged tRNA species present in selenium replete but absent from selenium deficient cells. Models are also proposed to explain the discrimination between the selenocysteine UGA codon and authentic UGA terminator codons. The regulation of the GPx gene was examined during mono-myelocytic differentiation of HL-60 cells in vitro and also during interferon-gamma activation of human peripheral blood macrophages and PMN. During phagocyte cell differentiation or activation, the ability to generate peroxide developed, however the peroxide-destroying capacities of GPx did not increase concomitantly. Complex regulatory patterns involving both transcriptional and translational controls were observed. The association of GPx gene expression with chronic granulomatous disease was explored. No correlation was found with either the autosomal or X-linked forms of the disease, a finding contradictory to previously published material

Regulation of the human cellular glutathione peroxidase gene during in vitro myeloid and monocytic differentiation

We have used the HL-60 and PLB-985 myeloid leukemia cell lines to examine the regulation of expression of the important intracellular antioxidant enzyme, glutathione peroxidase (GSH-Px), during phagocytic cell differentiation in vitro. Induction of differentiation along the monocytic pathway by phorbol ester results in an approximately twofold rise in enzyme activity and a parallel increase in the rate of 75Se incorporation into immunoprecipitable GSH-Px protein. Induction along the granulocytic pathway by dimethyl formamide (DMF) results in similar changes in steady-state enzyme levels and rates of GSH-Px protein synthesis. Steady-state levels of GSH-Px gene transcripts also increase more than twofold, approximately in parallel with the enzyme levels. Nuclear run-on transcription assays of GSH-Px mRNA synthesis show ratios of induced to uninduced transcript levels of 2.24 and 1.59 with phorbol myristate acetate (PMA) induction and DMF, respectively, in HL-60 cells, and ratios of 1.34 and 3.46 with PMA and DMF, respectively, in PLB-985 cells. Half lives of GSH-Px mRNA are unchanged or slightly shorter after differentiation of HL-60 cells, and slightly longer after induction of PLB-985. Overall, the present studies show that GSH-Px activity rises during in vitro-induced monocytic or granulocytic differentiation of myeloid cell lines and that the increased expression of the cellular GSH-Px gene occurs through complex mechanisms that include transcriptional up-regulation. This pattern contrasts with the nearly complete cotranslational regulation of GSH-Px expression by exogenous selenium

P53 vaccines for the treatment of cancers

The present invention relates to immunotherapy methods for treating hyperproliferative disease in humans, particularly to hyperproliferative disease that is refractory to therapy. More specifically, the invention is directed, in one embodiment, to methods for treating a subject with a hyperproliferative disease in which the expression of a self gene is upregulated in therapy-resistant hyperproliferative cells. In another embodiment, an adenoviral expression construct comprising a self gene under the control of a promoter operable in eukaryotic cells is administered to the therapy-resistant hyperproliferative cells. The present invention thus provides immunotherapies for treating therapy-resistant hyperproliferative disease by attenuating the natural immune system\u27s CTL response against hyperproliferative cells or overexpressing mutant p53 antigens, for example

Isolation and chromosomal localization of the human glutathione peroxidase gene

We have isolated cDNA clones for the gene, termed GPX1, encoding the major human selenoprotein, glutathione peroxidase. Sequence analysis confirmed previous findings that the unusual amino acid seleno-cysteine is encoded by the opal terminator codon UGA. Southern blot analysis of human genomic DNA with the GPX1 cDNA showed that restriction endonucleases without sites in the probe sequence produced three hybridizing bands at standard stringency, diminishing to one strongly and one weakly hybridizing band at high stringency. In situ hybridization localized the human GPX1 gene to a single site on chromosome 3, at region 3q11-13.1. Thus, three genomic sites bear sequence homology to the GPX1 cDNA, and the one most homologous maps to 3q11-13.1

Killing of human melanoma cells induced by activation of class I interferon-regulated signaling pathways via MDA-7/IL-24

Restoration of the tumor-suppression function by gene transfer of the melanoma differentiation-associated gene 7 (MDA7)/interleukin 24 (IL-24) successfully induces apoptosis in melanoma tumors in vivo. To address the molecular mechanisms involved, we previously revealed that MDA7/IL-24 treatment of melanoma cells down-regulates interferon regulatory factor (IRF)-1 expression and concomitantly up-regulates IRF-2 expression, which competes with the activity of IRF-1 and reverses the induction of IRF-1-regulated inducible nitric oxide synthase (iNOS). Interferons (IFNs) influence melanoma cell survival by modulating apoptosis. A class I IFN (IFN-alpha) has been approved for the treatment of advanced melanoma with some limited success. A class II IFN (IFN-gamma), on the other hand, supports melanoma cell survival, possibly through constitutive activation of iNOS expression. We therefore conducted this study to explore the molecular pathways of MDA7/IL-24 regulation of apoptosis via the intracellular induction of IFNs in melanoma. We hypothesized that the restoration of the MDA7/IL-24 axis leads to upregulation of class I IFNs and induction of the apoptotic cascade. We found that MDA7/IL-24 induces the secretion of endogenous IFN-beta, another class I IFN, leading to the arrest of melanoma cell growth and apoptosis. We also identified a series of apoptotic markers that play a role in this pathway, including the regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas-FasL. In summary, we described a novel pathway of MDA7/IL-24 regulation of apoptosis in melanoma tumors via endogenous IFN-beta induction followed by IRF regulation and TRAIL/FasL system activation

Adenoviral melanoma differentiation-associated gene 7 induces apoptosis in lung cancer cells through mitochondrial permeability transition-independent cytochrome c release

Objective: Melanoma differentiation-associated gene 7 is a novel tumor suppressor gene that induces apoptosis in lung cancer cells when delivered by adenoviral gene transfer as Ad-mda7. The mechanisms of action are not well defined but may involve release of cytochrome c from the mitochondria with subsequent caspase activation. Methods: The lung cancer cell lines A549 and H1299 were transduced with Ad-mda7, adenovirus containing the gene for p53 (Ad-p53), and control adenoviral luciferase vectors. Staurosporine was used as a positive control to induce cytochrome c release through mitochondrial permeability transition-dependent pores, whereas cyclosporine (INN: ciclosporin) was used to specifically inhibit these mitochondrial permeability transition-dependent pores. Apoptosis was evaluated with fluorescence-activated cell sorting analysis of subdiploid populations and mitochondrial membrane potential changes with tetramethylrhodamine ethylester perchlorate. Results: Melanoma differentiation-associated gene 7, transduced by Ad-mda7 into H1299 and A549 lung cancer cells, resulted in sharp increases in cytosolic cytochrome c levels followed by induction of apoptosis and cellular death. The release of cytochrome c from the mitochondria occurred without changes in the mitochondrial membrane potential. Unlike staurosporine treatment, transduction with Ad-p53 and Ad-mda7 caused releases of cytochrome c and apoptosis that were not blocked by cyclosporine, suggesting a mitochondrial permeability transition pore-independent pathway. Conclusions: Ad-mda7 induces apoptosis in lung cancer cells through mitochondrial cytochrome c release in a process that is not dependent on mitochondrial membrane potential changes and occurs through mitochondrial permeability transition-independent pores. This unique mechanism of action may allow treatment of patients with lung cancer resistant to mitochondrial permeability transition-dependent cell death processes

Development of Ad-mda7/IL-24-resistant lung cancer cell lines

Many cancers can become resistant to repeated administration of even the most effective therapeutic agents. In developing adenoviral mda-7/IL-24 (Ad-mda-7/IL-24) therapy for lung cancer, we have anticipated this potential clinical problem by attempting to identify the molecular mechanisms of Ad-mda7/IL-24 resistance in several Ad-mda7/IL-24-resistant lung cancer cell lines that we have developed. For the present study, we established four Admda7- resistant cell lines by repeated selection of resistant clones of parental Ad-mda7-sensitive A549 cells: two lines (A549R1 and A549R2) resistant to both adenoviral vector and the mda-7 gene and two (A549R3 and A549R4) resistant to the therapeutic mda-7 gene only. As shown by western blot analysis of several known anti-apoptotic proteins, parental A549 and resistant A549R3 cells expressed similar levels of AKT and phosphorylated AKT (p-AKT), whereas resistant A549R3 and A549R4 cells expressed higher levels of bcl-2 and lower levels of bcl-xL than did their parental cells. As shown by flow-cytometric analysis, treating resistant A549R3 and A549R4 cells with a combination of Ad-mda7 and 17-allyl-amino-17-demethoxygeldanamycin (17AAG) (50 nM) for 48 hours enhanced apoptosis. Together, these in vitro findings indicate that an antiapoptotic mechanism may underlie Ad-mda7 resistance and that such resistance can be overcome by addition of 17AAG. Further investigations along these lines are warranted