Biomedical subjects

Kum-Joo Shin

Publications and source records attributed to Kum-Joo Shin.

8 recordsLinked to original sources

PubMed2006

A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression.

RNAi is proving to be a powerful experimental tool for the functional annotation of mammalian genomes. The full potential of this technology will be realized through development of approaches permitting regulated manipulation of endogenous gene expression with coordinated reexpression of exogenous transgenes. We describe the development of a lentiviral vector platform, pSLIK (single lentivector for inducible knockdown), which permits tetracycline-regulated expression of microRNA-like short hairpin RNAs from a single viral infection of any naïve cell system. In mouse embryonic fibroblasts, the pSLIK platform was used to conditionally deplete the expression of the heterotrimeric G proteins Galpha12 and Galpha13 both singly and in combination, demonstrating the Galpha13 dependence of serum response element-mediated transcription. In RAW264.7 macrophages, regulated knockdown of Gbeta2 correlated with a reduced Ca(2+) response to C5a. Insertion of a GFP transgene upstream of the Gbeta2 microRNA-like short hairpin RNA allowed concomitant reexpression of a heterologous mRNA during tetracycline-dependent target gene knockdown, significantly enhancing the experimental applicability of the pSLIK system.

Animals↗

PubMed2005

Molecular cloning and characterization of a novel phospholipase C, PLC-eta.

PLC (phospholipase C) plays an important role in intracellular signal transduction by hydrolysing phosphatidylinositol 4,5-bisphosphate, a membrane phospholipid. To date, 12 members of the mammalian PLC isoforms have been identified and classified into five isotypes beta, gamma, delta, epsilon and zeta, which are regulated by distinct mechanisms. In the present study, we describe the identification of a novel PLC isoform in the brains of human and mouse, named PLC-eta, which contains the conserved pleckstrin homology domain, X and Y domains for catalytic activity and the C2 domain. The first identified gene encoded 1002 (human) or 1003 (mouse) amino acids with an estimated molecular mass of 115 kDa. The purified recombinant PLC-eta exhibited Ca2+-dependent catalytic activity on phosphatidylinositol 4,5-bisphosphate. Furthermore, molecular biological analysis revealed that the PLC-eta gene was transcribed to several splicing variants. Although some transcripts were detected in most of the tissues we examined, the transcript encoding 115 kDa was restricted to the brain and lung. In addition, the expression of the 115 kDa protein was defined in only nerve tissues such as the brain and spinal cord. In situ hybridization analysis with brain revealed that PLC-eta was abundantly expressed in various regions including cerebral cortex, hippocampus, zona incerta and cerebellar Purkinje cell layer, which are neuronal cell-enriched regions. These results suggest that PLC-eta may perform fundamental roles in the brain.

Amino Acid Sequence↗

PubMed2005

Phospholipase C-beta3 mediates the thrombin-induced Ca2+ response in glial cells.

Phospholipase C-beta (PLC-beta) hydrolyses phosphatidylinositol 4,5-bisphosphate and generates inositol 1,4,5-trisphosphate in response to activation of various G protein-coupled receptors (GPCRs). Using glial cells from knock-out mice lacking either PLC-beta1 [PLC-beta1 (-/-)] or PLC-beta3 [PLC-beta3 (-/-)], we examined which isotype of PLC-beta participated in the cellular signaling events triggered by thrombin. Generation of inositol phosphates (IPs) was enhanced by thrombin in PLC-beta1 (-/-) cells, but was negligible in PLC-beta3 (-/-) cells. Expression of PLC-beta3 in PLC-beta3 (-/-) cells resulted in an increase in pertussis toxin (PTx)-sensitive IPs in response to thrombin as well as to PAR1-specific peptide, while expression of PLC-beta1 in PLC-beta1 (-/-) cells did not have any effect on IP generation. The thrombin-induced [Ca2+]i increase was delayed and attenuated in PLC-beta3 (-/-) cells, but normal in PLC-beta1 (-/-) cells. Pertussis toxin evoked a delayed [Ca2+]i increase in PLC-beta3 (-/-) cells as well as in PLC-beta1 (-/-) cells. These results suggest that activation of PLC-beta3 by pertussis toxin-sensitive G proteins is responsible for the transient [Ca2+]i increase in response to thrombin, whereas the delayed [Ca2+]i increase may be due to activation of some other PLC, such as PLC-beta4, acting via PTx-insensitive G proteins.

Animals↗

PubMed2005

G2 arrest and apoptosis by 2-amino-N-quinoline-8-yl-benzenesulfonamide (QBS), a novel cytotoxic compound.

We screened a library of 11,000 small molecular weight chemicals, looking for compounds that affect cell viability. We have identified 2-amino-N-quinoline-8-yl-benzenesulfonamide (QBS) as a potent cytotoxic compound that induces cell cycle arrest and apoptosis. Treatment of Jurkat T cells with QBS increased the levels of cyclin B1 as well as phosphorylated-cdc2, which was accompanied by reduced activity of cdc2 kinase, suggesting that QBS may induce cell cycle arrest at G2 phase. Structural analogues of QBS also exhibited similar effects on cell cycle progression and cell viability. Long-term treatment with QBS resulted in DNA fragmentation, cytochrome C release, and PARP cleavage, and an increase in the number of subdiploidy cells, indicative of cellular apoptosis. Moreover, QBS-induced apoptosis was blocked by z-VAD-fmk, a pan-caspase inhibitor. These results suggest that QBS is a novel and potent compound that induces G2 arrest and subsequent apoptosis, implicating it as a putative candidate for chemotherapy.

Apoptosis↗

PubMed2004

2,2',4,6,6'-Pentachlorobiphenyl-induced apoptosis is limited by cyclooxygenase-2 induction.

Polychlorinated biphenyls (PCBs), a group of persistent and widespread environmental pollutants, are considered to be immunotoxic, carcinogenic, and to induce apoptosis. However, the cellular mechanisms underlying the action of PCBs have not been established. Here, we investigated the effects of PCBs on the induction of cyclooxygenase-2 (COX-2). Among the several congeners examined, only 2,2',4,6,6'-pentachlorobiphenyl (PeCB) specifically increased the COX-2 promoter activity, and the levels of COX-2 mRNA and protein, and thereby enhanced prostaglandin E2 (PGE2) synthesis in Rat-1 cells. By conducting mutation analyses of the COX-2 promoter and its transcription factor, we found that the CRE site in COX-2 promoter and c-Jun are important for increased COX-2 promoter activity induced by 2,2',4,6,6'-PeCB. In addition, 2,2',4,6,6'-PeCB-stimulated COX-2 induction was reduced by the specific MAPK kinase (MEK) inhibitor, PD98059, and in p53-deficient cells, implying that COX-2 induction requires the activation of ERK1/2 MAPK and p53. The selective COX-2 inhibitor, NS-398, potentiated the 2,2',4,6,6'-PeCB-induced mitochondrial apoptotic pathway involved in Bcl-xL attenuation, cytochrome c release and the subsequent activation of caspase-3. Furthermore, the cell death was prevented by PGE2 treatment, suggesting that 2,2',4,6,6'-PeCB-induced apoptosis is restricted by prostaglandin upregulation by COX-2. Taken together, these results demonstrate that 2,2',4,6,6'-PeCB-induced COX-2 expression may be an important compensatory mechanism for abating 2,2',4,6,6'-PeCB toxicity.

Animals↗

PubMed2004

2,2',4,6,6'-Pentachlorobiphenyl induces mitotic arrest and p53 activation.

Polychlorinated biphenyls (PCBs), a class of persistent organic pollutants (POPs), have been considered to be involved in cancers, but the underlying mechanisms are not known well. Various cancers are closely related to genetic alteration; therefore, we investigated the effect of PCBs on genetic stability, through p53, a guardian of genome, in NIH 3T3 fibroblasts. Among several congeners examined, 2,2',4,6,6'-pentachlorobiphenyl (PeCB) specifically activated p53-dependent transcription. It also induced p53 nuclear accumulation, but did not cause DNA strand breakage. On the other hand, cell cycle progression that is closely connected to p53 was affected by 2,2',4,6,6'-PeCB, resulting in mitotic arrest. In the arrested cells, mitotic spindle damage was detected. Moreover, in the absence of functional p53, polyploidy was caused by 2,2',4,6,6'-PeCB. These results imply that 2,2',4,6,6'-PeCB induces mitotic arrest by interfering with mitotic spindle assembly, followed by genetic instability which triggers p53-activating signals to prevent further polyploidization. Taking these findings together, we suggest that 2,2',4,6,6'-PeCB could be involved in cancer development by causing genetic instability through mitotic spindle damage, which brings about aneuploidy in p53-deficient tumor cells.

Animals↗

PubMed2003

Luteolin inhibits the nuclear factor-kappa B transcriptional activity in Rat-1 fibroblasts.

Flavonoids are natural polyphenolic compounds that have anti-inflammatory, cytoprotective and anticarcinogenic effects. In this study, we investigated the effects of several flavonoids on nuclear factor-kappa B (NF-kappa B) activation by using luciferase reporter gene assay. Among the flavonoids examined, luteolin showed the most potent inhibition on lipopolysaccharide (LPS)-stimulated NF-kappa B transcriptional activity in Rat-1 fibroblasts. Luteolin did not inhibit either I kappa B alpha degradation or NF-kappa B nuclear translocation, DNA binding or phosphorylation by LPS. However, luteolin prevented LPS-stimulated interaction between the p65 subunit of NF-kappa B and the transcriptional coactivator CBP. In addition, a specific PKA inhibitor that blocked the phosphorylation of CREB and c-Jun by luteolin partially reversed the inhibitory effect of luteolin on NF-kappa B.CBP complex formation and NF-kappa B transcriptional activity by LPS. These data imply that inhibition of NF-kappa B transcriptional activity by luteolin may occur through competition with transcription factors for coactivator that is available in limited amounts. Taken together, this study provides a molecular basis for the understanding of the anti-inflammatory effects of luteolin.

Activating Transcription Factor 1↗

PubMed2002

Phospholipase A2-mediated Ca2+ influx by 2,2',4,6-tetrachlorobiphenyl in PC12 cells.

Polychlorinated biphenyls (PCBs) are a group of persistent and widespread environmental pollutants, and known to affect signaling molecules. Phospholipase A2 (PLA2) mediates cellular destructive processes as well as normal physiological responses in neuronal cells. In this study, we examined whether PLA2 can be activated by PCBs in PC12 cells. Of the congeners tested, ortho-substituted PCBs were found to induce PLA2 activation. PLA2 activation by 2,2',4,6-tetrachlorobiphenyl (TeCB), the most potent congener, was inhibited by bromoenol lactone (BEL), a calcium-independent PLA2 (iPLA2) inhibitor, and methyl arachidonyl fluorophosphonate (MAFP), a cytosolic PLA2 and iPLA2 inhibitor. In the case of Ca2+, although 2,2',4,6-TeCB increased [Ca2+]i in the presence of extracellular Ca2+, PLA2 activation was not inhibited by EGTA and 1,2-bis (o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxy-methyl) ester, an extracellular and an intracellular Ca2+ chelator, respectively. On the other hand, 2,2',4,6-TeCB-induced Ca2+ increase was partially inhibited by BEL and MAFP. In addition, 2,2',4,6-TeCB induced apoptotic cell death in these cells. Taken together, our results suggest that ortho-substituted PCBs might induce apoptosis through PLA2-mediated Ca2+ influx, which provides a clue to understand the mechanism of neurotoxic effects of ortho-substituted PCBs.

Animals↗