Dong-Chul Kang

Identification and cloning of human astrocyte genes displaying elevated expression after infection with HIV-1 or exposure to HIV-1 envelope glycoprotein by rapid subtraction hybridization, RaSH.

Neurodegeneration and dementia are common complications of AIDS caused by human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system. HIV-1 target cells in the brain include microglia, infiltrating macrophages and astrocytes, but rarely neurons. Astrocytes play an important role in the maintenance of the synaptic micro-environment and in neuronal signal transmission. To investigate potential changes in cellular gene expression associated with HIV-1 infection of astrocytes, we employed an efficient and sensitive rapid subtraction hybridization approach, RaSH. Primary human astrocytes were isolated from abortus brain tissue and low-passage cells were infected with HIV-1. To identify genes that display both early and late expression modifications after HIV-1 infection and to avoid cloning genes displaying normal cell cycle fluctuations in astrocytes, RNAs were isolated and pooled from 6, 12, 24 h and 3 and 7 day uninfected and infected cells and used for RaSH. Temporal cDNA libraries were prepared from double-stranded cDNAs that were enzymatically digested into small fragments, ligated to adapters, PCR amplified, and hybridized by incubation of tester and driver PCR fragments. By subtracting temporal cDNAs derived from uninfected astrocytes from temporal cDNAs made from HIV-1 infected cells, genes displaying elevated expression in virus infected cells, termed astrocyte elevated genes (AEGs), were identified. Both known and novel AEGs, not reported in current DNA databases, are described that display early or late expression kinetics following HIV-1 infection or treatment with recombinant HIV-1 envelope glycoprotein (gp120). For selected AEGs, expression of their protein products was also tested by Western blotting and found to display elevated expression following HIV-1 infection. The comparable pattern of regulation of the AEGs following HIV-1 infection or gp120 treatment suggest that HIV-1 exposure of astrocytes, even in the absence of productive infection, can induce changes in cellular gene expression.

Genomic structure, chromosomal localization and expression profile of a novel melanoma differentiation associated ( mda -7) gene with cancer specific growth suppressing and apoptosis inducing properties

Abnormalities in cellular differentiation are frequent occurrences in human cancers. Treatment of human melanoma cells with recombinant fibroblast interferon (IFN-β) and the protein kinase C activator mezerein (MEZ) results in an irreversible loss in growth potential, suppression of tumorigenic properties and induction of terminal cell differentiation. Subtraction hybridization identified melanoma differentiation associated gene-7 (mda-7), as a gene induced during these physiological changes in human melanoma cells. Ectopic expression of mda-7 by means of a replication defective adenovirus results in growth suppression and induction of apoptosis in a broad spectrum of additional cancers, including melanoma, glioblastoma multiforme, osteosarcoma and carcinomas of the breast, cervix, colon, lung, nasopharynx and prostate. In contrast, no apparent harmful effects occur when mda-7 is expressed in normal epithelial or fibroblast cells. Human clones of mda-7 were isolated and its organization resolved in terms of intron/exon structure and chromosomal localization. Hu-mda-7 encompasses seven exons and six introns and encodes a protein with a predicted size of 23.8 kDa, consisting of 206 amino acids. Hu-mda-7 mRNA is stably expressed in the thymus, spleen and peripheral blood leukocytes. De novo mda-7 mRNA expression is also detected in human melanocytes and expression is inducible in cells of melanocyte/melanoma lineage and in certain normal and cancer cell types following treatment with a combination of IFN-β plus MEZ. Mda-7 expression is also induced during megakaryocyte differentiation induced in human hematopoietic cells by treatment with TPA (12-O-tetradecanoyl phorbol-13-acetate). In contrast, de novo expression of mda-7 is not detected nor is it inducible by IFN-β+MEZ in a spectrum of additional normal and cancer cells. No correlation was observed between induction of mda-7 mRNA expression and growth suppression following treatment with IFN-β+MEZ and induction of endogenous mda-7 mRNA by combination treatment did not result in significant intracellular MDA-7 protein. Radiation hybrid mapping assigned the mda-7 gene to human chromosome 1q, at 1q 32.2 to 1q41, an area containing a cluster of genes associated with the IL-10 family of cytokines. Mda-7 represents a differentiation, growth and apoptosis associated gene with potential utility for the gene-based therapy of diverse human cancers.

Expression analysis and genomic characterization of human melanoma differentiation associated gene-5, mda-5: a novel type I interferon-responsive apoptosis-inducing gene

Melanoma differentiation associated gene-5 (mda-5) was identified by subtraction hybridization as a novel upregulated gene in HO-1 human melanoma cells induced to terminally differentiate by treatment with IFN-β+MEZ. Considering its unique structure, consisting of a caspase recruitment domain (CARD) and an RNA helicase domain, it was hypothesized that mda-5 contributes to apoptosis occurring during terminal differentiation. We have currently examined the expression pattern of mda-5 in normal tissues, during induction of terminal differentiation and after treatment with type I IFNs. In addition, we have defined its genomic structure and chromosomal location. IFN-β, a type I IFN, induces mda-5 expression in a biphasic and dose-dependent manner. Based on its temporal kinetics of induction and lack of requirement for prior protein synthesis mda-5 is an early type I IFN-responsive gene. The level of mda-5 mRNA is in low abundance in normal tissues, whereas expression is induced in a spectrum of normal and cancer cells by IFN-β. Expression of mda-5 by means of a replication incompetent adenovirus, Ad.mda-5, induces apoptosis in HO-1 cells as confirmed by morphologic, biochemical and molecular assays. Additionally, the combination of Ad.mda-5+MEZ further augments apoptosis as observed in Ad.null or uninfected HO-1 cells induced to terminally differentiate by treatment with IFN-β+MEZ. The mda-5 gene is located on human chromosome 2q24 and consists of 16 exons, without pseudogenes, and is conserved in the mouse genome. Present data documents that mda-5 is a novel type I IFN-inducible gene, which may contribute to apoptosis induction during terminal differentiation and during IFN treatment. The conserved genomic and protein structure of mda-5 in human and mouse will permit analysis of the evolution and developmental aspects of this gene.

Insights into glutamate transport regulation in human astrocytes: Cloning of the promoter for excitatory amino acid transporter 2 (EAAT2)

Glutamate transport is central to neurotransmitter functions in the brain. Impaired glutamate transport induces neurotoxicity associated with numerous pathological processes, including stroke/ischemia, temporal lobe epilepsy, Alzheimers disease, amyotrophic lateral sclerosis, Huntingtons disease, HIV-1-associated dementia, and growth of malignant gliomas. Excitatory amino acid transporter-2 (EAAT2) is a major glutamate transporter in the brain expressed primarily in astrocytes. We presently describe the cloning and characterization of the human EAAT2 promoter, demonstrating elevated expression in astrocytes. Regulators of EAAT2 transport, both positive and negative, alter EAAT2 transcription, promoter activity, mRNA, and protein. These findings imply that transcriptional processes can regulate EAAT2 expression. Moreover, they raise the intriguing possibility that the EAAT2 promoter may be useful for targeting gene expression in the brain and for identifying molecules capable of modulating glutamate transport that could potentially inhibit, ameliorate, or prevent various neurodegenerative diseases.

Identification and cloning of human polynucleotide phosphorylase, hPNPaseold-35, in the context of terminal differentiation and cellular senescence

Terminal differentiation and cellular senescence display common properties including irreversible growth arrest. To define the molecular and ultimately the biochemical basis of the complex physiological changes associated with terminal differentiation and senescence, an overlapping-pathway screen was used to identify genes displaying coordinated expression as a consequence of both processes. This approach involved screening of a subtracted cDNA library prepared from human melanoma cells induced to terminally differentiate by treatment with fibroblast IFN and mezerein with mRNA derived from senescent human progeria cells. This strategy identified old-35, which encodes an evolutionary conserved gene, human polynucleotide phosphorylase (hPNPaseold-35), that is regulated predominantly by type I IFNs. The hPNPaseOLD-35 protein localizes in the cytoplasm of human cells and induces RNA degradation in vitro, as does its purified bacterial protein homologue. Ectopic expression of hPNPaseold-35 in human melanoma cells reduces colony formation, confirming inhibitory activity of this RNA-degradation enzyme. Identification of hPNPaseold-35, an IFN-inducible 3′-5′ RNA exonuclease, provides additional support for a relationship between IFN action and RNA processing and suggests an important role for this gene in growth control associated with terminal differentiation and cellular senescence.

Mda-7/IL-24 induces apoptosis of diverse cancer cell lines through JAK/STAT-independent pathways.

Experimental evidence documents that the MDA‐7/IL‐24 protein (an IL‐10 family cytokine) binds to IL‐20 and IL‐22 receptor complexes resulting in the activation of JAK/STAT signaling pathways. Recent published reports utilizing human blood derived primary lymphocytes have provided additional confirmatory evidence relating to the cytokine properties of this molecule. A notable attribute of mda‐7/IL‐24 is its cancer cell‐specific apoptosis inducing capacity, which currently remains incompletely understood. Treatment with distinctive tyrosine kinase inhibitors (Genistein and AG18) or a JAK‐selective inhibitor (AG490) did not prevent Ad.mda‐7 induced apoptosis in diverse cell lines. In addition, there is no apparent correlation between patterns of expression of IL‐20R1, IL‐20R2, and IL‐22R mRNA and susceptibility to Ad.mda‐7 in different cell lines. Furthermore, Ad.mda‐7 is able to induce killing in STAT/JAK deficient cells. In contrast, treatment with the p38MAPK selective inhibitor SB203580, partially inhibited apoptosis induced by Ad.mda‐7 in different cell lines. These results demonstrate for the first time that signaling events leading to susceptibility to Ad.mda‐7 induced apoptosis, might be tyrosine kinase independent and can thus be distinguished from its cytokine function related properties mediated by the IL‐20/IL‐22 receptor complexes that require JAK/STAT kinase activity. J. Cell. Physiol. 196: 334–345, 2003.

Oncogene AEG-1 Promotes Glioma-Induced Neurodegeneration by Increasing Glutamate Excitotoxicity

Aggressive tumor growth, diffuse tissue invasion, and neurodegeneration are hallmarks of malignant glioma. Although glutamate excitotoxicity is considered to play a key role in glioma-induced neurodegeneration, the mechanism(s) controlling this process is poorly understood. Astrocyte elevated gene-1 (AEG-1) is an oncogene that is overexpressed in several types of human cancers, including more than 90% of brain tumors. In addition, AEG-1 promotes gliomagenesis, particularly in the context of tumor growth and invasion, 2 primary characteristics of glioma. In the present study, we investigated the contribution of AEG-1 to glioma-induced neurodegeneration. Pearson correlation coefficient analysis in normal brain tissues and samples from glioma patients indicated a strong negative correlation between expression of AEG-1 and a primary glutamate transporter of astrocytes EAAT2. Gain- and loss-of-function studies in normal primary human fetal astrocytes and T98G glioblastoma multiforme cells revealed that AEG-1 repressed EAAT2 expression at a transcriptional level by inducing YY1 activity to inhibit CBP function as a coactivator on the EAAT2 promoter. In addition, AEG-1-mediated EAAT2 repression caused a reduction of glutamate uptake by glial cells, resulting in induction of neuronal cell death. These findings were also confirmed in samples from glioma patients showing that AEG-1 expression negatively correlated with NeuN expression. Taken together, our findings suggest that AEG-1 contributes to glioma-induced neurodegeneration, a hallmark of this fatal tumor, through regulation of EAAT2 expression.

Molecular characterization of prostate carcinoma tumor antigen-1, PCTA-1, a human Galectin-8 related gene

The galectin family of proteins has been associated with several diverse cellular processes. More than 30 years since the discovery of the first member, precise biological functions for the family as a whole, or for individual members has proven elusive. The isolation of Prostate Carcinoma Tumor Antigen-1 (PCTA-1), a cDNA closely related to rat and human Galectin-8, as a surface marker associated with prostate cancer was achieved using a previously described immunological subtraction approach, Surface Epitope Masking (SEM) approach, in combination with expression screening. It appears that PCTA-1 expression is almost ubiquitous in normal human tissues and could alter in specific contexts such as transformation or metastasis. Multiple expression isoforms of PCTA-1 at the mRNA level are observed. PCTA-1 maps to 1q42-43, a locus associated with predisposition to prostate cancer. We have determined the genomic structure of PCTA-1 to account for the several observed isoforms, performed expression analysis to determine distribution in normal and transformed contexts at the RNA and protein level and conducted over-expression studies to determine effects on cellular phenotype.

5 - Identification of Differentially Expressed Genes Using Rapid Subtraction Hybridization: Detailed Methodology for Performing Rapid Subtraction Hybridization

This chapter presents a protocol for subtracted complementary DNA (cDNA) library construction by rapid subtraction hybridization (RaSH). Additional methods, including reverse Northern and Northern Blot hybridization, which are part of the RaSH approach, are described in the chapter. Proof-of-principle for the RaSH approach was obtained by analyzing the temporal changes of gene expression occurring during the induction of terminal differentiation in human melanoma cells treated with interferon-beta plus mezerein. In RaSH, complementary DNA (cDNA) samples are restriction-digested with frequent cutters, such as DpnII , to an average size of 256 bp for improving hybridization efficiency. Moreover, the restriction digestion of tester cDNA only prior to hybridization enables the selection and cloning of tester-unique cDNAs by a direct ligation of hybridization mixtures to corresponding sites in the plasmid vector. Thus, RaSH reduces the amount of starting materials required for conventional subtraction hybridization by one tenth by using polymerase chain reaction (PCR) and obviates the need to separate and clone subtracted cDNA species into plasmid vector or bacteriophage, a nontrivial, technically demanding step in subtractive cDNA library construction.