Biomedical subjects

Dong-Chul Kang

Publications and source records attributed to Dong-Chul Kang.

11 recordsLinked to original sources

PubMed2005

Complete open reading frame (C-ORF) technology: simple and efficient technique for cloning full-length protein-coding sequences.

Technical difficulties in full-length cDNA cloning hinder successful characterization of many unknown and potentially novel expressed sequence tags (ESTs). We presently describe improved methods for cDNA cloning. This scheme is based on the polymerase chain reaction and utilizes a degenerate stem-loop annealing primer (dSLAP), consisting of a stem-loop structure followed by 12 random nucleotides, and is called the C-ORF (complete open reading frame) technique. The dSLAP is designed to anneal to first-strand cDNA, while suppressing second-strand synthesis from internal sites because of its bulky stem-loop structure. The C-ORF technique consists of three steps: reverse transcription, dSLAP annealing plus the second-strand synthesis, and PCR amplification. Applications of dSLAP to both known and previously unknown cDNA targets resulted in cloning of their complete open reading frames, in most cases after a single application of the C-ORF method. The currently described protocol is simple and does not require unusual molecular biology reagents, except for reverse transcriptase, Taq polymerase and a DNA primer, which makes it readily amenable for cloning purposes in individual laboratories. Moreover, this approach has wide applicability and in principle can be used to identify the protein-coding region of virtually any gene in which limited or incomplete sequence information is available.

Base Sequence↗

PubMed2005

Cloning and characterization of HIV-1-inducible astrocyte elevated gene-1, AEG-1.

We presently describe the full-length cloning and functional characterization of an HIV-1-inducible gene, astrocyte elevated gene (AEG)-1. Additionally, a novel method is outlined for producing tag-free recombinant protein in a baculovirus system and its use in producing AEG-1 protein. AEG-1 mRNA is expressed ubiquitously with higher expression in tissues containing muscular actin and its expression is increased in astrocytes infected with HIV-1 or treated with gp120 or tumor necrosis factor (TNF)-alpha. The mRNA encodes a single pass transmembrane protein of predicted molecular mass of 64-kDa and pI 9.3 that predominantly localizes in the endoplasmic reticulum and perinuclear region. Ectopic expression of AEG-1 inhibits excitatory amino acid transporter 2 (EAAT2) promoter activity with the potential to promote glutamate excitotoxicity and consequently HIV-1-associated dementia (HAD). AEG-1 expression is elevated in subsets of breast carcinomas, malignant gliomas and melanomas and it synergizes with oncogenic Ha-ras to enhance soft agar colony forming ability of non-tumorigenic immortalized melanocytes, documenting its tumor promoting activity. AEG-1 may affect tumor progression in multiple cell lineages by augmenting expression of the transformed phenotype and/or by inducing glutamate excitotoxicity in malignant glioma. In these contexts, an HIV-1-inducible gene, AEG-1, may contribute to multiple brain abnormalities, including HAD and tumor formation, by both common and distinct mechanisms.

Amino Acid Transport System X-AG↗

PubMed2004

Identification and cloning of genes displaying elevated expression as a consequence of metastatic progression in human melanoma cells by rapid subtraction hybridization.

Although extensively investigated, the complete repertoire of genes associated with and causative of metastasis remain largely unknown. We developed an efficient approach for identifying differentially expressed genes that involves rapid subtraction hybridization (RaSH) of cDNA clones prepared from two cell populations, a driver and a tester. This RaSH approach has previously documented high sensitivity and effectiveness in identifying genes that are differentially expressed as a function of induction of terminal differentiation in human melanoma cells, resistance or sensitivity to human immunodeficiency virus-1 (HIV-1) infection of human T cells and perturbation in gene expression in normal human fetal astrocytes infected with HIV-1 or treated with HIV-1 gp120 viral envelope glycoprotein or tumor necrosis factor-alpha (TNF-alpha). In the present study, RaSH has been applied to a metastatic melanoma model, which mimics the early events of metastasis in humans, comprising weakly metastatic vs. immunosuppressed newborn rat-selected highly metastatic variants. This has now resulted in the identification of eight genes displaying elevated expression in the high metastatic variants vs. normal immortal melanocytes or weakly metastatic parental clones. These include six known genes, 67-kDa laminin receptor (67LR), endothelin receptor B (ENDRB), Na+/K+-ATPase, Ku antigen, interleukin-receptor-associated kinase-1 (IRAK-1) and ribosomal protein RPLA, which may contribute to the complex process of melanoma metastasis. Additionally, two unknown genes (not reported in current databases) that may also impact on the metastatic phenotype have also been identified. These studies provide additional support of the use of the RaSH approach, in this application in the context of closely related variant cell lines with different metastatic potential, for effective differential gene identification and elucidate eight previously unrecognized genes whose role in melanoma progression to metastatic competence can now be scrutinized.

Animals↗

PubMed2004

Human polynucleotide phosphorylase (hPNPaseold-35): a potential link between aging and inflammation.

Chronic inflammation is a characteristic feature of aging, and the relationship between cellular senescence and inflammation, although extensively studied, is not well understood. An overlapping pathway screen identified human polynucleotide phosphorylase (hPNPase(old-35)), an evolutionary conserved 3',5'-exoribonuclease, as a gene up-regulated during both terminal differentiation and cellular senescence. Enhanced expression of hPNPase(old-35) via a replication-incompetent adenovirus (Ad.hPNPase(old-35)) in human melanoma cells and normal human melanocytes results in a characteristic senescence-like phenotype. Reactive oxygen species (ROS) play a key role in the induction of both in vitro and in vivo senescence. We now document that overexpression of hPNPase(old-35) results in increased production of ROS, leading to activation of the nuclear factor (NF)-kappaB pathway. Ad.hPNPase(old-35) infection promotes degradation of IkappaBalpha and nuclear translocation of NF-kappaB and markedly increases binding of the transcriptional activator p50/p65. The generation of ROS and activation of NF-kappaB by hPNPase(old-35) are prevented by treatment with a cell-permeable antioxidant, N-acetyl-l-cysteine. Infection with Ad.hPNPase(old-35) enhances the production of interleukin (IL)-6 and IL-8, two classical NF-kappaB-responsive cytokines, and this induction is inhibited by N-acetyl-l-cysteine. A cytokine array reveals that Ad.hPNPase(old-35) infection specifically induces the expression of proinflammatory cytokines, such as IL-6, IL-8, RANTES, and matrix metalloproteinase (MMP)-3. We hypothesize that hPNPase(old-35) might play a significant role in producing pathological changes associated with aging by generating proinflammatory cytokines via ROS and NF-kappaB. Understanding the relationship between hPNPase(old-35) and inflammation and aging provides a unique opportunity to mechanistically comprehend and potentially intervene in these physiologically important processes.

Adenoviridae↗

PubMed2004

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-beta+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-beta, 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-beta. 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-beta+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.

Animals↗

PubMed2004

The origin of polynucleotide phosphorylase domains.

In this report, we document the presence of polynucleotide phosphorylase (PNPase) in the animal eukaryotes. These proteins contain several domains, including 2 RNase PH domains (PNPase 1 and PNPase 2) which are closely related functionally and in sequence similarity to ribonuclease PH (RPH) protein. Phylogenetic analysis of the gene genealogy of these three domains suggests that PNPase was formed via a duplication event that also produced the RNase PH protein. Given the current distribution of these domains in the tree of life, these duplication events most likely occurred in the common ancestor of the three organismal superkingdoms, Archaea, Eukarya, and Bacteria. In particular, PNPase 2 and RPH are more closely related to each other than either one is to PNPase 1, suggesting a deeper differentiation of PNPase 1 in the common organismal ancestor. In addition, while PNPase 1 and PNPase 2 appear to have the same evolutionary signal as determined by the incongruence length difference (ILD) test, RPH appears to have an incongruent signal with both of the PNPase domains. This result suggests that RPH experienced different evolutionary divergence patterns than the PNPase domains, consistent with the linked nature of the two PNPase domains.

5' Untranslated Regions↗

PubMed2004

Effects of human immunodeficiency virus type 1 on astrocyte gene expression and function: potential role in neuropathogenesis.

Neurodegeneration and dementia caused by human immunodeficiency virus type 1 (HIV-1) infection of the brain are common complications of acquired immunodeficiency syndrome (AIDS). Introduction of highly active antiretroviral therapy (HAART) reduced the incidence of HIV-1-associated dementia, but so far had no effect on the high frequency of milder neurological disorders caused by HIV-1. This indicates that some neuropathogenic processes persist during limited HIV-1 replication in the central nervous system (CNS). The authors are evaluating the hypothesis that interaction of HIV-1 with astrocytes, which bind HIV-1 but support limited productive HIV-1 infection, may contribute to these processes by disrupting astrocyte functions that are important for neuronal activity or survival. Using laser-capture microdissection on brain tissue samples from HIV-1-infected individuals, we found that HIV-1 DNA can be detected in up to 1% of cortical and basal ganglia astrocytes, thus confirming HIV-1 infection in astrocytes from symptomatic patients. Using rapid subtraction hybridization, the authors cloned and identified 25 messenger RNAs in primary human fetal astrocytes either up-regulated or down-regulated by native HIV-1 infection or exposure to gp120 in vitro. Extending this approach to gene microarray analysis using Affymetrix U133A/B gene chips, the authors determined that HIV-1 alters globally and significantly the overall program of gene expression in astrocytes, including changes in transcripts coding for cytokines, G-coupled protein receptors, transcription factors, and others. Focusing on a specific astrocyte function relevant to neuropathogenesis, the authors showed that exposure of astrocytes to HIV-1 or gp120 in vitro impairs the ability of the cells to transport L-glutamate and the authors related this defect to transcriptional inhibition of the EAAT2 glutamate transporter gene. These findings define new pathways through which HIV-1 may contribute to neuropathogenesis under conditions of limited virus replication in the brain.

AIDS Dementia Complex↗

PubMed2003

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 p38(MAPK) 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.

Adenoviridae↗

PubMed2003

Identification of gene products suppressed by human immunodeficiency virus type 1 infection or gp120 exposure of primary human astrocytes by rapid subtraction hybridization.

Neurodegeneration and human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) are the major disease manifestations of HIV-1 colonization of the central nervous system (CNS). In the brain, HIV-1 replicates in microglial cells and infiltrating macrophages and it persists in a low-productive, noncytolytic state in astrocytes. Astrocytes play critical roles in the maintenance of the brain microenvironment, responses to injury, and in neuronal signal transmission, and disruption of these functions by HIV-1 could contribute to HAD. To better understand the potential effects of HIV-1 on astrocyte biology, the authors investigated changes in gene expression using an efficient and sensitive rapid subtraction hybridization approach, RaSH. Primary human astrocytes were isolated from abortus brain tissue, low-passage cells were infected with HIV-1 or mock infected, and total cellular RNAs were isolated at multiple time points over a period of 1 week. This approach is designed to identify gene products modulated early and late after HIV-1 infection and limits the cloning of genes displaying normal cell-cycle fluctuations in astrocytes. By subtracting temporal cDNAs derived from HIV-1-infected astrocytes from temporal cDNAs made from uninfected cells, 10 genes displaying reduced expression in infected cells, termed astrocyte suppressed genes (ASGs), were identified and their suppression was confirmed by Northern blot hybridization. Both known and novel ASGs, not reported in current DNA databases, that are down-regulated by HIV-1 infection are described. Northern blotting confirms suppression of the same panel of ASGs by treatment of astrocytes with recombinant HIV-1 envelope glycoprotein, gp120. These results extend our previous analysis of astrocyte genes induced or enhanced by HIV-1 infection and together they suggest that HIV-1 and viral proteins have profound effects on astrocyte physiology, which may influence their function in the CNS.

AIDS Dementia Complex↗

PubMed2002

Identification and cloning of human polynucleotide phosphorylase, hPNPase old-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 (hPNPase(old-35)), that is regulated predominantly by type I IFNs. The hPNPase(OLD-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 hPNPase(old-35) in human melanoma cells reduces colony formation, confirming inhibitory activity of this RNA-degradation enzyme. Identification of hPNPase(old-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.

Amino Acid Sequence↗

PubMed2002

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.

Actinin↗