Expression In Primary Cells Research Articles

In memoriam: Professor Brian Maurice John Foxwell, 1956 to 2008

In memoriam: Professor Brian Maurice John Foxwell, 1956 to 2008

Opposite Expression of the Antioxidant Heme Oxygenase-1 in Primary Cells and Tumor Cells: Regulation by Interaction of USF-2 and Fra-1

Heme oxygenase-1 is the rate-limiting enzyme for the degradation of the prooxidant heme. Previously, we showed that an E-box within the HO-1 promoter is crucial for the regulation of HO-1 expression in primary hepatocytes. Further to investigate the importance of this E-box, we determined the regulatory capacity of the E-box-binding factor USF-2 in primary cells in comparison with transformed cell lines. We found that HO-1 expression was inhibited by USF-2 in primary cells, whereas it was induced in tumor cell lines. Mutation of either the E-box or the AP-1 site within the HO-1 promoter only partially affected the USF-dependent regulation. However, this regulation was dramatically reduced in tumor cells and completely abolished in primary cells transfected with an HO-1 promoter construct containing mutations in both the E-box and the AP-1 site, suggesting that AP-1 factors and USF-2 may act in a cooperative manner. Indeed, protein-protein interaction studies revealed that USF proteins interacted with Fra-1. Further, the USF-dependent HO-1 promoter activity was not detectable with an USF-2 mutant lacking residues of the USF-specific region (USR) or the transactivation domain encoded by exon 4. Together, these data suggest that USF-2 has opposite regulatory roles for HO-1 gene expression in primary cells and tumor cell lines.

Tristetraprolin Down-Regulates IL-2 Gene Expression through AU-Rich Element-Mediated mRNA Decay

Posttranscriptional regulation of IL-2 gene expression at the level of mRNA decay is mediated by an AU-rich element (ARE) found in the 3'-untranslated region. We hypothesized that the ARE-binding protein tristetraprolin (TTP) regulates T lymphocyte IL-2 mRNA decay by interacting with the IL-2 ARE and targeting the transcript for decay. rTTP protein expressed in HeLa cells bound specifically to the IL-2 ARE with high affinity in a gel shift assay. In primary human T lymphocytes, TTP mRNA and protein expression were induced by TCR and CD28 coreceptor stimulation. Using a gel shift assay, we identified a cytoplasmic RNA-binding activity that was induced by TCR and CD28 coreceptor stimulation and bound specifically to the IL-2 ARE sequence. Using anti-TTP Abs, we showed by supershift that this inducible activity contained TTP. We also showed that insertion of the IL-2 ARE sequence into the 3'-untranslated region of a beta-globin reporter construct conferred TTP-dependent mRNA destabilization on the beta-globin reporter. To determine whether TTP also regulates IL-2 gene expression in vivo, we examined IL-2 expression in primary cells from wild-type and TTP knockout mice. Compared with their wild-type counterparts, TCR- and CD28-activated splenocytes and T cells from TTP knockout mice overexpressed IL-2 mRNA and protein. Also, IL-2 mRNA was more stable in activated splenocytes from TTP knockout mice compared with wild-type mice. Taken together, these data suggest that TTP functions to down-regulate IL-2 gene expression through ARE-mediated mRNA decay.

Conditional Expression of K- ras in an Epithelial Compartment that Includes the Stem Cells Is Sufficient to Promote Squamous Cell Carcinogenesis

Ras genes are the most frequently mutated oncogenes in human cancer. However, the contribution of ras to tumor initiation still is unclear because ras expression in primary cells can cause cell cycle arrest and even cell death by apoptosis. Furthermore, when expressed in the epidermis of mice, mutant ras promotes the formation of benign papillomas, only few of which will progress into carcinomas. However, in these cases, ras-transgene expression often is restricted to suprabasal or follicular epithelial cells that may lack self-renewal capacity. Thus, it still is conceivable that expression of active ras in other epithelial compartments may exert a distinct ability to promote malignant progression. To address this possibility, transgenic mice carrying the tetracycline-inducible system (tet-on receptor) targeted to the basal layer of stratified epithelium, which includes the epithelial stem cells, were engineered and crossed with mice expressing the K-ras(G12D) oncogene under the control of tet-regulated responsive elements. On doxycycline administration, proliferative lesions ranging from hyperplasias, papillomas, and dysplasias to metastatic carcinomas developed in squamous epithelia of the skin, oral mucosa, salivary glands, tongue, esophagus, forestomach, and uterine cervix within just 10 to 20 days. The most noticeable lesions were invasive squamous carcinomas of the skin and oral mucosa. These findings suggest that the expression of oncogenes in an epithelial compartment that includes the stem cells may be sufficient to promote squamous carcinogenesis. They also provide a molecularly defined conditional animal model system in which the mechanisms responsible for cancer initiation, maintenance, and metastatic spread can be readily investigated.

Potential role of APRIL as autocrine growth factor for megakaryocytopoiesis

AbstractA proliferation-inducing ligand (APRIL) is a new tumor necrosis factor family member implicated in tumor cell proliferation. We investigated the role of APRIL in megakaryocytopoiesis, a developmental hematopoietic process responsible for progenitor cell differentiation to megakaryoblasts and megakaryocytes, leading to platelet formation. APRIL is not expressed in CD34+ progenitor cells from healthy donors, but it is massively up-regulated during the proliferative phase of megakaryocytic cell differentiation. Exogenous APRIL expression in primary cells increases megakaryocytic cell growth, suggesting that APRIL acts as a proliferative factor in megakaryocytopoiesis. More importantly, neutralization of endogenous APRIL was able to dramatically reduce megakaryocyte expansion and platelet production. Thus, our data provide evidence that APRIL acts as a growth factor for terminal megakaryocytopoiesis and may promote physiologic platelet production.

P53 and p21 Form an Inducible Barrier that Protects Cells against Cyclin E-cdk2 Deregulation

Background: Cyclin E, in conjunction with its catalytic partner cdk2, is rate limiting for entry into the S phase of the cell cycle. Cancer cells frequently contain mutations within the cyclin D-Retinoblastoma protein pathway that lead to inappropriate cyclin E-cdk2 activation. Although deregulated cyclin E-cdk2 activity is believed to directly contribute to the neoplastic progression of these cancers, the mechanism of cyclin E-induced neoplasia is unknown.Results: We studied the consequences of deregulated cyclin E expression in primary cells and found that cyclin E initiated a p53-dependent response that prevented excess cdk2 activity by inducing expression of the p21Cip1 cdk inhibitor. The increased p53 activity was not associated with increased expression of the p14ARF tumor suppressor. Instead, cyclin E led to increased p53 serine15 phosphorylation that was sensitive to inhibitors of the ATM/ATR family. When either p53 or p21cip1 was rendered nonfunctional, then the excess cyclin E became catalytically active and caused defects in S phase progression, increased ploidy, and genetic instability.Conclusions: We conclude that p53 and p21 form an inducible barrier that protects cells against the deleterious consequences of cyclin E-cdk2 deregulation. A response that restrains cyclin E deregulation is likely to be a general protective mechanism against neoplastic transformation. Loss of this response may thus be required before deregulated cyclin E can become fully oncogenic in cancer cells. Furthermore, the combination of excess cyclin E and p53 loss may be particularly genotoxic, because cells cannot appropriately respond to the cell cycle anomalies caused by excess cyclin E-cdk2 activity.

The oncogenic potential of Kaposi's sarcoma-associated herpesvirus cyclin is exposed by p53 loss in vitro and in vivo.

Expression of the Kaposi's sarcoma-associated herpesvirus (KSHV) cyclin D homolog, K cyclin, is thought to contribute to viral oncogenesis. We show that K cyclin expression in primary cells sensitizes to apoptosis and induces growth arrest, both of which are dependent on p53 but independent of E2F1 or p19(ARF). DNA synthesis, but not cytokinesis, continues in K cyclin-expressing cells, leading to multinucleation and polyploidy. Such polyploid cells exhibit pronounced centrosome amplification and consequent aneuploidy. Our data suggest that K cyclin expression leads to cytokinesis defects and polyploidy, which activates p53. However, in the absence of p53, such cells survive and expand as an aneuploid population. Corroborating these findings, in vivo Emu; K cyclin expression cooperates with p53 loss in the induction of lymphomas.

Macrolide-based transgene control in mammalian cells and mice.

Heterologous mammalian gene regulation systems for adjustable expression of multiple transgenes are necessary for advanced human gene therapy and tissue engineering, and for sophisticated in vivo gene-function analyses, drug discovery, and biopharmaceutical manufacturing. The antibiotic-dependent interaction between the repressor (E) and operator (ETR) derived from an Escherichia coli erythromycin-resistance regulon was used to design repressible (E(OFF)) and inducible (E(ON)) mammalian gene regulation systems (E.REX) responsive to clinically licensed macrolide antibiotics (erythromycin, clarithromycin, and roxithromycin). The E(OFF) system consists of a chimeric erythromycin-dependent transactivator (ET), constructed by fusing the prokaryotic repressor E to a eukaryotic transactivation domain that binds and activates transcription from ETR-containing synthetic eukaryotic promoters (P(ETR)). Addition of macrolide antibiotic results in repression of transgene expression. The E(ON) system is based on E binding to artificial ETR-derived operators cloned adjacent to constitutive promoters, resulting in repression of transgene expression. In the presence of macrolides, gene expression is induced. Control of transgene expression in primary cells, cell lines, and microencapsulated human cells transplanted into mice was demonstrated using the E.REX (E(OFF) and E(ON)) systems. The macrolide-responsive E.REX technology was functionally compatible with the streptogramin (PIP-regulated and tetracycline (TET-regulated expression systems, and therefore may be combined for multiregulated multigene therapeutic interventions in mammalian cells and tissues.

Retroviral expression in embryonic stem cells and hematopoietic stem cells.

Achieving long-term retroviral expression in primary cells has been problematic. De novo DNA methylation of infecting proviruses has been proposed as a major cause of this transcriptional repression. Here we report the development of a mouse stem cell virus (MSCV) long terminal repeat-based retroviral vector that is expressed in both embryonic stem (ES) cells and hematopoietic stem (HS) cells. Infected HS cells and their differentiated descendants maintained long-term and stable retroviral expression after serial adoptive transfers. In addition, retrovirally infected ES cells showed detectable expression level of the green fluorescent protein (GFP). Moreover, GFP expression of integrated proviruses was maintained after in vitro differentiation of infected ES cells. Long-term passage of infected ES cells resulted in methylation-mediated silencing, while short-term expression was methylation independent. Tissues of transgenic animals, which we derived from ES cells carrying the MSCV-based provirus, did not express GFP. However, treatment with the demethylating agent 5-azadeoxycytidine reactivated the silent provirus, demonstrating that DNA methylation is involved in the maintenance of retroviral repression. Our results indicate that retroviral expression in ES cells is repressed by methylation-dependent as well as methylation-independent mechanisms.

Transfection of cultured myoblasts in high serum concentration with DODAC:DOPE liposomes

The inhibitory effect of serum is one of the main obstacles to the in vivo use of cationic liposomes as a DNA delivery system. We have found that a novel liposome formulation, DODAC:DOPE (1:1) is totally resistant to the inhibitory effects of serum for transfection of cultured myoblasts and myotubes. Transfection with a lacZ reporter gene in the presence of 95% fetal bovine serum gave up to 25% beta-gal-positive cells in C2C12 myoblasts and about six-fold less in primary human myoblasts. The lower transgene expression in primary cells does not appear to be a result of less DNA uptake but might result from differences in intracellular trafficking of the complexes. DODAC-based liposomes are unique in their resistance to serum inhibition and may therefore be valuable for the systemic delivery of genetic information to muscle and other tissues.

Cytokine Control of Interstitial Collagenase and Collagenase-3 Gene Expression in Human Chondrocytes

Human collagenase-3 expression, previously seen only in a breast tumor tissue, is here shown to be expressed in primary human chondrocytes derived from the joint tissue and in transformed human chondrocytes. Its mRNA is inducible by the inflammatory cytokines interleukin-1beta plus tumor necrosis factor-alpha, but not by phorbol 12-myristate 13-acetate and only slightly by the growth factors platelet-derived growth factor and epidermal growth factor. Human synovial fibroblasts, another prominent cell type in the joint tissue, do not produce collagenase-3 message. Expression of the murine collagenase, which is possibly the counterpart of human collagenase-3, is induced by interleukin-1beta plus tumor necrosis factor-alpha, and its full induction requires the presence of the transcription factor, c-FOS. This family of transcription factors also plays a role in induction of human collagenase-3, since it binds to the AP-1 site of this matrix metalloproteinase.

Distinct Patterns of Cyclin D1 Regulation in Models of Liver Regeneration and Human Liver

The expression of cyclin D1 and associated cdks was examined in models of liver regeneration and human liver specimens. In mouse liver after 70% partial hepatectomy, there was >20-fold induction of cyclin D1 mRNA and protein, beginning prior to peak. DNA synthesis. In normal rat liver, basal levels of cyclin D1 protein were significantly higher than in the mouse. After hepatectomy in the rat, cyclin D1 mRNA was induced 6- to 10-fold, while the protein levels changed <2-fold and did not parallel changes in the mRNA. Cyclin D1 protein was detected in freshly isolated rat hepatocytes, but this diminished within 6 hours in culture. After growth stimulation with HGF, cyclin D1 mRNA was induced 3- to 5-fold and its protein >20-fold in rat hepatocytes. Immunoprecipitation of cyclin D1 demonstrated its association with cdk4 but not cdk5 in regenerating liver. In human liver biopsy specimens, cyclin D1 protein was detectable in normal liver and induced 2- to 10-fold in mitotically active liver following transplantation. These results suggest that the regulation of cyclin D1 protein in human liver may more closely parallel the mouse than the rat hepatectomy model. Furthermore, cyclin expression in primary cells in culture may differ significantly from that observed in vivo.

Molecular and Cellular Approaches to the Treatment of Neurological Disease

Introduction: molecular and cellular organization of the central nervous system - implications for new therapeutics. Part 1 Mechanisms of neuronal injury and death: calcium and glutamate-induced cortical neuronal death glutamate receptors and excitotoxicity cellular physiology of hypoxia of the mammalian central nervous system metrochondrial dysfunction as a mechanism of CNS injury free radicals in CNS injury. Part 2 Neuronal injury in specific disease states: strokes involving gray matter - studies on in situ models of cerebral anoxia anoxic injury of central myelinated axons - ionic mechanisms and pharmacology the ischemic penumbra in stroke - prospects for analysis by nuclear magnetic resonance spectroscopy role of excitotoxins in heredito-degenerative neurologic diseases the therapeutic window for methylprednisolone treatment of acute spinal cord injury - implications for cell injury mechanisms. Part 3 Therapeutic strategies: emerging strategies for the treatment of ischemic brain injury the design and appraisal of randomized clinical trials in cerebrovascular disease and CNS trauma pharmacological modification of axon membrane molecules and cell transplantation as approaches to the restoration of conduction in demyelinated axons current anti-sense nucleic acid strategies for manipulating neuronal and glial cells. Part 4 Cellular and molecular replacement: factors affecting proviral expression in primary cells grafted into the CNS cellular replacement of dopamine deficit in Parkinson's disease using human foetal mesencephalic tissue - preliminary results in four patients intracerebral transplantation - prospects for neuronal replacement in neurodegenerative diseases.