Expression Of Several Gene Products Research Articles

Expression of PRL-3 regulates proliferation and invasion of breast cancer cells in vitro.

The protein tyrosine phosphatase PRL-3 plays an important role in cancer cell migration, invasion and metastasis. In breast cancer, PRL-3 is overexpressed in 70-75% of tumors and even more frequently in lymph node metastases. Moreover, PRL-3 overexpression in breast cancer is associated with an adverse disease outcome. Aim of this study was to determine the role of PRL-3 in breast cancer cell proliferation, migration and invasion in vitro. PRL-3 mRNA expression was evaluated in 6 breast cancer cell lines by quantitative real-time PCR. To investigate the effect of PRL-3 expression in breast cancer cells in vitro we both up- and downregulated PRL-3 expression in breast cancer cells and performed in vitro wound repair cell motility assays and invasion assays. The influence of PRL-3 knockdown in MCF-7 cells on the expression of several gene products involved in cell invasion and cytoskeletal function was evaluated with real-time PCR. PRL-3 mRNA expression was demonstrated in all breast cancer cell lines evaluated. Knockdown of PRL-3 in MCF-7 cells resulted in decreased proliferation, wound healing and invasion. PRL-3 knockdown in MCF-7 cells resulted in a significant reduction of heparanase, MMP-9, actin gamma-2 and Myosin 9 expression, and significant elevation of E-cadherin. We conclude that PRL-3 is an important regulatory factor for breast cancer cell proliferation and invasion. Loss of PRL-3 function induces an antimetastatic gene expression profile in breast cancer cells. Due to its role in tumor growth and metastasis, PRL-3 emerges as a new therapeutic target in breast cancer therapy.

5-Aminoisoquinolin-1-one (5-AIQ), a Water-Soluble Inhibitor of the Poly(ADP-Ribose)Polymerases (PARPs)

5-Aminoisoquinolin-1-one (5-AIQ) is a water-soluble inhibitor of the poly(ADPribose) polymerases (PARPs), lacking isoform-selectivity. Although of only moderate potency in vitro against PARP-1, it is highly active in many assays in cells and in models in vivo, indicating excellent uptake. Optimisation of the several synthetic sequences to 5-AIQ has led to development of a short and efficient route from 1-chloroisoquinoline. It has been used widely as a biochemical and pharmacological tool to study the effects of inhibition of the PARPs. It ameliorates the damage to cells and tissues following reperfusion of ischaemic tissue, showing significant protective activity in a rodent model of haemorrhagic shock at the remarkably low dose of 30 µg Kg(-1). Protection is also seen in models of myocardial infarction, ischaemic kidney and liver disorders, stroke and organ transplantation. Inhibition of PARP-1 by 5-AIQ causes down-regulation of the activity of NF-κB, which then down-regulates the expression of several gene products. Thus 5-AIQ has anti-inflammatory activity in vivo, through modulating the expression of cytokines and adhesion molecules. This indirect inhibition of expression is relevant in the activity of 5-AIQ in models of arthritis, Parkinson's disease, multiple sclerosis, spinal cord injury, periodontitis and inflammatory conditions of the lung. Inhibition of expression of matrix metalloproteinases and other factors gives rise to anti-angiogenic activity and to remarkable anti-metastatic activity in a mouse model. Thus, although it has been overtaken by other PARP-inhibiting drugs in the oncological clinic, 5-AIQ remains a valuable tool to study the roles of PARPs in health and in diverse diseases.

Beclin-1 Deficiency Alters Autophagosome Formation, Lysosome Biogenesis and Enhances Neuronal Vulnerability of HT22 Hippocampal Cells.

Beclin-1 is assumed to be a critical component participating in autophagosome formation in mammals; however, the exact role of Beclin-1 in autophagy remains controversial. Here (1) we created a HT22-Beclin-1-knockdown cell line using the Q-techBECN1 technique, (2) examined the potential role of Beclin-1 in an autophagic response in hippocampal HT22 neurons challenged with rapamycin, (3) investigated the expression of several gene products involved in the autophagic pathway, and (4) checked the effects of Beclin-1 knockdown on neuronal death induced by AAS. Rapamycin induced and altered the expression of autophagy signature proteins in wild-type cultures as well as in HT22-Beclin-1-knockdown cells. However, among the examined markers, only two factors exhibited dramatic changes when comparing controls to HT22-Beclin-1-knockdown cells. The amount of LC3, an important protein for the initiation of autophagosome formation and LAMP-1, a major constituent of the lysosomal membrane, underwent a dramatic and highly significant increase in control cultures challenged with rapamycin. In contrast, rapamycin was not able to induce any significant changes in LC3 and LAMP-1 levels in HT22-Beclin-1-knockdown cells. In addition, the knockdown of Beclin-1 enhanced neuronal susceptibility to death signals induced by AAS. Our data demonstrate the essential role of Beclin-1 in the formation of autophagosomes and lysosome biogenesis and underline that deletion of this key system is deleterious for cell viability.

Can an oral antidiabetic (rosiglitazone) be of benefit in leukemia treatment?

PPARs are ligand-regulated transcription factors and regulate expression of several gene products. Therefore, PPARs are being studied for their possible contribution to the treatment of cancer, atherosclerosis, inflammation, infertility and demyelinating diseases. Primary AML patients were observed to have significantly elevated PPARγ mRNA expression compared to normal peripheral blood or bone marrow mononuclear cells. This study investigated the cytotoxic effects of rosiglitazone maleate, a pure PPARγ agonist, in vitro in HL-60 cell line.This study obtained results which can provide guidance for future studies. Whether the PPARy agonist rosiglitazone maleate may provide additive effects in refractory or relapsing cases of acute leukemia may be set as an objective for the future studies.

The Novel Role of Yin Yang 1 in the Regulation of Epithelial to Mesenchymal Transition in Cancer Via the Dysregulated NF-κB/Snail/YY1/RKIP/PTEN Circuitry

The majority of cancers develop genetic and molecular strategies to resist conventional cytotoxic therapies as well as escape from the host's immune surveillance. These events lead to tumor persistence and spread through activation of the epithelial to mesenchymal transition (EMT) program and metastasis. Expression profiling analysis has revealed various tumor metastasis-inducing and metastasis-suppressing genes that regulate the metastatic process and maintain the microenvironment of the tumor cells. EMT in cancer entails the molecular reprogramming and phenotypic changes that characterize the conversion of immobile cancer epithelial cells to motile mesenchymal cells. A hallmark of EMT is the loss of E-cadherin, the epithelial homotypic adhesion molecule, and gain of mesenchymal markers such as vimentin and fibronectin. The molecular mechanisms underlying the initiation of EMT consist, in part, in the constitutive activation of survival signaling pathways such as the nuclear factor (NF)-κB pathway. The NF-κB pathway has been implicated directly in the regulation of EMT and indirectly through the transcription and expression of several gene products that participate in the EMT cascade, such as Snail, the metastasis-inducer and E-cadherin suppressor transcription factor. In turn, Snail represses the metastasis-suppressor gene product Raf-kinase inhibitor protein (RKIP) that inhibits both the Raf-1/MEK/ERK and NF-κB survival pathways implicated in EMT. Consequently, tumor cells normally exhibit a dysregulated NF-κB/Snail/RKIP circuitry that is intimately involved in the initiation of EMT and maintenance of drug resistance. Additional deregulated gene products in this circuit, such as the metastasis-suppressor phosphatase and tensin homologue (PTEN; repressed by Snail) and the putative-metastasis inducer Yin Yang (YY) 1 (target of NF-κB) also have been associated in the regulation of EMT. We recently have investigated the direct role of YY1 in the regulation of EMT. This review discusses the molecular regulation of EMT in cancer cells through the activity of the dysregulated NF-κB/Snail/ YY1/PTEN/RKIP circuitry. In addition, we propose YY1 as a potential novel prognostic biomarker for EMT and a therapeutic target for both the prevention of metastasis and the reversal of resistance.

Comparison of Gene Expression Profiles in Tubulocystic Carcinoma and Collecting Duct Carcinoma of the Kidney

The relationship between tubulocystic carcinoma and collecting duct carcinoma of the kidney remains controversial. Some experts are of the opinion that the tumors are related, considering tubulocystic carcinoma to be synonymous with low-grade collecting duct carcinoma. However, others maintain that the 2 are distinct, unrelated entities on the basis of morphologic features and clinical outcome. To explore the relationship between tubulocystic carcinoma and collecting duct carcinoma, we compared the expression of several gene products at the mRNA level in cohorts of each tumor subtype. Seven cases of tubulocystic carcinoma and 8 cases of collecting duct carcinoma were identified. Total RNA was isolated from formalin-fixed paraffin-embedded tissue from each case. Relative expression levels of vimentin, alpha methylacyl CoA racemase, E-cadherin, p53, CD10 antigen, parvalbumin, cytokeratin 7, and cytokeratin 19 were assessed by quantitative reverse transcription polymerase chain reaction. Tubulocystic carcinoma was characterized by relative overexpression of vimentin, p53, and alpha methylacyl CoA racemase, compared with collecting duct carcinoma (P<0.05 for each gene, t test). In general, tubulocystic carcinoma expressed higher levels of E-cadherin and CD10, whereas collecting duct carcinoma expressed higher levels of cytokeratin 19; however, these trends did not reach statistical significance in this study cohort. Tubulocystic carcinoma and collecting duct carcinoma did not express cytokeratin 7 differentially. Case-to-case variability of gene expression limited the effectiveness of any one marker to distinguish the tumor types. Our study demonstrates that tubulocystic carcinoma and collecting duct carcinoma have different expression profiles of selected genes, including vimentin, p53, and alpha methylacyl CoA racemase. Further analysis of additional cases, using quantitative reverse transcription polymerase chain reaction and immunohistochemistry, will be useful to test the reproducibility of these findings. In addition, larger studies may establish statistical differences in expression of other genes analyzed in this study. Overall, these findings support the view that tubulocystic carcinoma and collecting duct carcinoma should be considered as 2 distinct entities at the molecular level.

Glycogen Synthase Kinase-3 regulates IGFBP-1 gene transcription through the Thymine-rich Insulin Response Element

BackgroundHepatic expression of several gene products involved in glucose metabolism, including phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase) and insulin-like growth factor binding protein-1 (IGFBP-1), is rapidly and completely inhibited by insulin. This inhibition is mediated through the regulation of a DNA element present in each of these gene promoters, that we call the Thymine-rich Insulin Response Element (TIRE). The insulin signalling pathway that results in the inhibition of these gene promoters requires the activation of phosphatidylinositol 3-kinase (PI 3-kinase). However, the molecules that connect PI 3-kinase to these gene promoters are not yet fully defined. Glycogen Synthase Kinase 3 (GSK-3) is inhibited following activation of PI 3-kinase. We have shown previously that inhibitors of GSK-3 reduce the activity of two TIRE-containing gene promoters (PEPCK and G6Pase), whose products are required for gluconeogenesis.ResultsIn this report we demonstrate that in H4IIE-C3 cells, four distinct classes of GSK-3 inhibitor mimic the effect of insulin on a third TIRE-containing gene, IGFBP-1. We identify the TIRE as the minimum requirement for inhibition by these agents, and demonstrate that the target of GSK-3 is unlikely to be the postulated TIRE-binding protein FOXO-1. Importantly, overexpression of GSK-3 in cells reduces the insulin regulation of TIRE activity as well as endogenous IGFBP-1 expression.ConclusionsThese results implicate GSK-3 as an intermediate in the pathway from the insulin receptor to the TIRE. Indeed, this is the first demonstration of an absolute requirement for GSK-3 inhibition in insulin regulation of gene transcription. These data support the potential use of GSK-3 inhibitors in the treatment of insulin resistant states such as Type 2 diabetes mellitus, but suggest that it will be important to identify all TIRE-containing genes to assess potential side effects of these agents.

Expression of interleukin-5- and granulocyte macrophage-colony-stimulating factor-responsive genes in blood and airway eosinophils.

Because interleukin (IL)-5 family cytokines are critical regulators of eosinophil development, recruitment, and activation, this study was initiated to identify proteins induced by these cytokines in eosinophils. Using oligonucleotide microarrays, numerous transcripts were identified as responsive to both IL-5 and granulocyte macrophage-colony-stimulating factor (GM-CSF), but no transcripts were markedly affected by one cytokine and not the other. Expression of several gene products were seen to be increased following in vitro stimulation of human blood eosinophils, including the IL-3 receptor alpha subunit, lymphotoxin beta, Pim-1, and cyclin D3. Given that eosinophils recovered from the bronchoalveolar lavage fluid of allergic patients after antigen challenge are exposed to IL-5 or GM-CSF in the airway prior to isolation, the hypothesis was tested that selected IL-5- and GM-CSF-responsive genes are upregulated in airway eosinophils relative to the expression in blood cells. Airway eosinophils displayed greater cell surface expression of the IL-3 receptor alpha subunit, CD44, CD25, and CD66e, suggesting that these proteins may be markers of eosinophil activation by IL-5 family cytokines in airway eosinophils. Other genes that were induced by both IL-5 and GM-CSF showed protein expression at similar or decreased levels in airway eosinophils relative to their circulating counterparts (i.e., lymphotoxin beta and CD24). These studies have identified several transcriptional targets of IL-5 and GM-CSF in human eosinophils and suggest that a number of protein products are critical to the responsiveness of airway eosinophils.

Enhanced expression of enterocyte P-glycoprotein depresses cyclosporine bioavailability in a recipient of living donor liver transplantation

We evaluated levels of intestinal expression of absorptive-barrier P-glycoprotein (PGP) and cytochrome P-450 IIIA4 (CYP3A4) and immunosuppressant therapy in a patient who underwent living donor liver transplantation (LDLT) and received a second living donor liver transplant after chronic rejection of the first. PGP and CYP3A4 expression were measured using part of a Roux-en-Y limb. After the first LDLT, the concentration-dose ratio of orally administered tacrolimus was 159.8 ± 125.3 (average ± SD of 32 different days), similar to the average for 46 recipients of living donor liver transplants in our hospital (161.3 ± 88.1). However, the recipient required very large oral doses of cyclosporine (703.9 ± 385.4 mg/d, average ± SD of 13 different days) after the second LDLT. Although intestinal PGP level was increased markedly at the second LDLT, CYP3A4 level was decreased. In addition, levels of messenger RNA expression of several gene products related to the local inflammation, such as cyclooxygenase 2, interleukin-1β (IL-1β), IL-2, IL-6, IL-8, IL-10, and tumor necrosis factor-α, were increased. These results suggest that hepatic failure after LDLT, including chronic rejection and/or cholangitis, was accompanied by upregulation of intestinal PGP expression, which could depress the bioavailability of the immunosuppressant. (Liver Transpl 2003;9:1108-1113.)

Pleomorphic carcinomas of the lung show a selective distribution of gene products involved in cell differentiation, cell cycle control, tumor growth, and tumor cell motility: a clinicopathologic and immunohistochemical study of 31 cases.

We investigated 31 cases of pleomorphic carcinomas of the lung, with a double component of neoplastic epithelial cells and of spindle and/or giant cells. To correlate the morphologic diversity of these two cell components with their immunophenotype, we evaluated the expression of several gene products involved in cell differentiation (cytokeratins, epithelial membrane antigen, carcinoembryonic antigen, vimentin, S-100 protein, smooth muscle actin, desmin), cell cycle control and apoptosis (p53, p21Waf1, p27Kip1, FHIT), tumor growth (proliferative fraction, assessed by Ki-67 antigen, and microvascular density, assessed by CD34 immunostaining), and tumor cell motility (fascin). We found the epithelial component to be significantly more immunoreactive for cytokeratins, epithelial membrane antigen, carcinoembryonic antigen, cell cycle inhibitors p21Waf1, p27Kip1 and tumor suppressor gene FHIT, whereas the sarcomatoid component, independent of tumor stage and size, was more immunoreactive for vimentin, fascin, and microvascular density. Accordingly, we suggest a model of tumorigenesis whereby the mesenchymal phenotype of pleomorphic cells is likely induced by the selective activation and segregation of several molecules involved in cell differentiation, cell cycle control, and tumor cell growth and motility. Whether pleomorphic carcinomas of the lung are tumors with a dismal prognosis still remains an unsettled issue. In our series, however, stage I pleomorphic carcinomas have the same clinical behavior as ordinary non-small cell lung cancer, and only a high proliferative index (Ki-67 labeling index >35%) is associated with a worse prognosis in these tumors.

Gene product immunophenotyping of neuroendocrine lung tumors. No linking evidence between carcinoids and small-cell lung carcinomas suggested by multivariate statistical analysis.

Fifty-three neuroendocrine lung tumors (24 carcinoids, one atypical carcinoid, five large-cell neuroendocrine carcinomas, and 23 small-cell lung carcinomas) were investigated for immunocytochemical expression of several gene products, i.e., p53, Rb, bcl-2, c-kit, mdm-2, cdk-4, p21 proteins, and proliferation index as assessed by MIB-1. The goal of the study was to explore the relationships between histotypes in light of their own gene product-based immunophenotypical profiles. To this aim we applied the multiple correspondence analysis, which is an exploratory statistical multivariate technique that converts a data matrix into a particular type of graphic display in which the rows and columns are depicted as points. Such statistical analysis displayed that some categories of the gene product-based immunophenotyping variables are grouped in the plot identifying three groups: the first group related to carcinoids, the second to small-cell carcinomas, and the third to large-cell neuroendocrine carcinomas. These data support the evidence that carcinoids and small-cell carcinomas are two distinct, apparently immunogenotypically unrelated entities among neuroendocrine lung tumors and that atypical carcinoids and large-cell neuroendocrine carcinomas seem not to represent intermediate steps between them.

A nutrient-sensing pathway regulates leptin gene expression in muscle and fat.

Leptin, the protein encoded by the obese (ob) gene, is synthesized and released in response to increased energy storage in adipose tissue. However, it is still not known how incoming energy is sensed and transduced into increased expression of the ob gene. The hexosamine biosynthetic pathway is a cellular 'sensor' of energy availability and mediates the effects of glucose on the expression of several gene products. Here we provide evidence for rapid activation of ob gene expression in skeletal muscle by glucosamine. Increased tissue concentrations of the end product of the hexosamine biosynthetic pathway, UDP-N-acetylglucosamine (UDP-GlcNAc), result in rapid and marked increases in leptin messenger RNA and protein levels (although these levels were much lower than those in fat). Plasma leptin levels and leptin mRNA and protein levels in adipose tissue also increase. Most important, stimulation of leptin synthesis is reproduced by either hyperglycaemia or hyperlipidaemia, which also increase tissue levels of UDP-N-acetylglucosamine in conscious rodents. Finally, incubation of 3T3-L1 pre-adipocytes and L6 myocytes with glucosamine rapidly induces ob gene expression. Our findings are the first evidence of inducible leptin expression in skeletal muscle and unveil an important biochemical link between increased availability of nutrients and leptin expression.

Development of a Nonradioactive, Time-Resolved Fluorescence Assay for the Measurement of Jun N-Terminal Kinase Activity

Activated transcription factor AP-1 is composed of c-Jun homodimers or c-Jun/c-Fos heterodimers and mediates expression of several gene products that have been implicated in disease pathogenesis. Activation of AP-1 is dependent on phosphorylation of c-Jun by Jun N-terminal kinase (JNK). Therefore, identification of inhibitors of JNK-mediated phosphorylation of c-Jun may lead to a novel class of therapeutics. A nonradioactive, high-through-put, time-resolved fluorescence assay was developed to measure and identify inhibitors of JNK activity. This assay utilized a lanthanide (europium)-labeled antibody that was specific for N-terminally phosphorylated c-Jun. The optimized europium-based assay was approximately 15-fold more sensitive compared to a similar 32P-based JNK assay. Compounds that were identified as inhibitors of JNK using the europium-based assay also inhibited JNK activity in the 32P-based assay with similar IC50 values. The europium-based JNK assay eliminates the contamination problems associated with the use of radioactivity. The sensitivity and safety of the europium-based assay make it amenable to robotics that will significantly increase screening throughput.

Camptothecin Sensitivity Is Mediated by the Pleiotropic Drug Resistance Network in Yeast

The antineoplastic alkaloid camptothecin interferes with the catalytic cycle of DNA topoisomerase I rendering it a cellular poison. Camptothecin stabilizes a covalent enzyme-DNA intermediate that is converted into lethal double strand DNA lesions during S phase of the cell cycle. Yeast SCT1 mutants were isolated in a screen for mutations in genes other than TOP1 that result in camptothecin resistance. Here we report SCT1 is allelic to PDR1 and that a Thr-879 to Met substitution in the PDR1-101 transcription factor confers multiple drug resistance. PDR1 regulates the expression of several gene products including the ATP-binding cassette transmembrane transport proteins PDR5, YOR1, and SNQ2. The PDR1 T879M mutant increased PDR5 transcription compared with wild-type PDR1 strains. Deletion of PDR1 or the downstream effector SNQ2 increased cell sensitivity to camptothecin, whereas deletion of YOR1 or PDR5 had little effect on camptothecin sensitivity. However, the camptothecin resistance accompanying GAL1-promoted overexpression of PDR5 suggests some substrate promiscuity among the ATP-binding cassette transporters. These data underscore the role of the pleiotropic drug resistance network in regulating camptothecin toxicity and are consistent with a model of decreased intracellular concentrations of camptothecin resulting from the increased expression of the SNQ2 transporter.

Recent insights into the regulation of cardiac Ca2+ flux during perinatal development and in cardiac failure.

Systolic and diastolic cardiac function improve during the transition from fetus to newborn to adult. This perinatal maturation is temporally correlated with and at least partially dependent on subcellular changes in the expression of several gene products that regulate cytosolic Ca2+ concentration. Expression of the Na(+)-Ca2+ exchanger of the sarcolemma is highest in the fetus, whereas expression of the sarcoplasmic reticulum Ca2+ pump and the voltage-dependent Ca2+ channel of the sarcolemma increase in conjunction with the perinatal maturation of cardiac function. Whereas cardiac relaxation in the normal mature heart depends primarily on the sarcoplasmic reticulum Ca2+ pump, relaxation in the fetal heart appears to be more dependent on transsarcolemmal Ca2+ flux. Like the fetal heart during prolonged relaxation, the failing human heart exhibits impaired relaxation and abnormal Ca2+ flux regulation. Although altered expression of several gene products has been demonstrated in the failing heart, the responsible factor(s) remains unknown.