Do Youn Jun

Nucleotide sequence and differential expression of the human 3-phosphoglycerate dehydrogenase gene ☆

The nucleotide sequence of Hs 3-PGDH gene, encoding human 3-phosphoglycerate dehydrogenase that catalyzes the initiating step in the phosphorylated pathway of serine biosynthesis, has been determined. The 3-PGDH gene has a predicted 533 amino acid open reading frame, encoding a 56.8kDa protein that shares 94.0% similarity with rat-liver 3-PGDH. Two different transcripts corresponding to 3-PGDH mRNA were detected in human normal tissues. A dominant 2.1kb transcript was expressed at high levels in prostate, testis, ovary, brain, liver, kidney, and pancreas, and weakly expressed in thymus, colon, and heart. A 710bp transcript also appeared as a weaker band where the 2.1kb mRNA was expressed, and it was more significant than the 2.1kb mRNA in heart and skeletal muscle. The TPA-induced monocytic differentiation of U937, which also resulted in growth arrest, abruptly downregulated the expression of 3-PGDH. Removal of TPA restored cell growth through the retrodifferentiation process and subsequent expression of 3-PGDH. The 3-PGDH mRNA was markedly expressed in human leukemias, lymphoma Sup-T1, colon adenocarcinoma COLO 320DM, epitheloid carcinoma HeLa S3, and murine lymphoma BW5147.G.1.4, but not in human leukemia K562. This report demonstrates that the human 3-PGDH gene is regulated at the transcriptional level depending on tissue specificty and cellular proliferative status, and its transcriptional regulation mechanism may be a useful target for diagnosis and therapy of cancer.

Characterization of human phosphoserine aminotransferase involved in the phosphorylated pathway of l-serine biosynthesis

In the present study, we first report two forms of human phosphoserine aminotransferase (PSAT) cDNA (HsPSAT alpha and HsPSAT beta). HsPSAT alpha has a predicted open reading frame comprising 324 amino acids, encoding a 35.2 kDa protein (PSAT alpha), whereas HsPSAT beta consists of an open reading frame comprising 370 amino acids that encodes a 40 kDa protein (PSAT beta). PSAT alpha is identical with PSAT beta, except that it lacks 46 amino acids between Val(290) and Ser(337) of PSAT beta, which is encoded by the entire exon 8 (138 bp). Both PSAT alpha and PSAT beta can functionally rescue the deletion mutation of the Saccharomyces cerevisiae counterpart. Reverse transcriptase-PCR analysis revealed that the expression of PSAT beta mRNA was more dominant when compared with PSAT alpha mRNA in all human cell lines tested. PSAT beta was easily detected in proportion to the level of mRNA; however, PSAT alpha was detected only in K562 and HepG2 cells as a very faint band. The relative enzyme activity of glutathione S-transferase (GST)-PSAT beta expressed in Escherichia coli appeared to be 6.8 times higher than that of GST-PSAT alpha. PSAT mRNA was expressed at high levels (approx. 2.2 kb) in the brain, liver, kidney and pancreas, and very weakly expressed in the thymus, prostate, testis and colon. In U937 cells, the levels of PSAT mRNA and protein appeared to be up-regulated to support proliferation. Accumulation of PSAT mRNA reached a maximum in the S-phase of Jurkat T-cells. These results demonstrate that although two isoforms of human PSAT can be produced by alternative splicing, PSAT beta rather than PSAT alpha is the physiologically functional enzyme required for the phosphorylated pathway, and indicate that the human PSAT gene is regulated depending on tissue specificity as well as cellular proliferation status with a maximum level expression in the S-phase.

Mollugin induces apoptosis in human Jurkat T cells through endoplasmic reticulum stress-mediated activation of JNK and caspase-12 and subsequent activation of mitochondria-dependent caspase cascade regulated by Bcl-xL

Exposure of Jurkat T cells to mollugin (15-30 microM), purified from the roots of Rubia cordifolia L., caused cytotoxicity and apoptotic DNA fragmentation along with mitochondrial membrane potential disruption, mitochondrial cytochrome c release, phosphorylation of c-Jun N-terminal kinase (JNK), activation of caspase-12, -9, -7, -3, and -8, cleavage of FLIP and Bid, and PARP degradation, without accompanying necrosis. While these mollugin-induced cytotoxicity and apoptotic events including activation of caspase-8 and mitochondria-dependent activation of caspase cascade were completely prevented by overexpression of Bcl-xL, the activation of JNK and caspase-12 was prevented to much lesser extent. Pretreatment of the cells with the pan-caspase inhibitor (z-VAD-fmk), the caspase-9 inhibitor (z-LEHD-fmk), the caspase-3 inhibitor (z-DEVD-fmk) or the caspase-12 inhibitor (z-ATAD-fmk) at the minimal concentration to prevent mollugin-induced apoptosis appeared to completely block the activation of caspase-7 and -8, and PARP degradation, but failed to block the activation of caspase-9 and -3 with allowing a slight enhancement in the level of JNK phosphorylation. Both FADD-positive wild-type Jurkat clone A3 and FADD-deficient Jurkat clone I2.1 exhibited a similar susceptibility to the cytotoxicity of mollugin, excluding involvement of Fas/FasL system in triggering mollugin-induced apoptosis. Normal peripheral T cells were more refractory to the cytotoxicity of mollugin than were Jurkat T cells. These results demonstrated that mollugin-induced cytotoxicity in Jurkat T cells was mainly attributable to apoptosis provoked via endoplasmic reticulum (ER) stress-mediated activation of JNK and caspase-12, and subsequent mitochondria-dependent activation of caspase-9 and -3, leading to activation of caspase-7 and -8, which could be regulated by Bcl-xL.

CLONING AND EXPRESSION OF HUMAN MITOTIC CENTROMERE-ASSOCIATED KINESIN GENE

The human homologue of the hamster mitotic centromere-associated kinesin (HsMCAK) gene containing a central type motor domain was isolated from a Jurkat T-cell derived cDNA library. The HsMCAK gene has a predicted 723 amino acid open reading frame, encoding a 81 kDa protein that shares 79.2% homology with hamster MCAK. Unstimulated T lymphocytes contained no detectable HsMCAK-specific mRNA. Activation of resting T-cells by immobilized anti-CD3 resulted in the expression of a 2.9-kb transcript during the S phase of the cell cycle. The TPA-induced monocytic differentiation of U937 which also results in growth-arrest abruptly downregulates the expression of HsMCAK. Removal of TPA restored the growth of the cell through the retrodifferentiation process and the subsequent expression of HsMCAK. HsMCAK is expressed in tissues containing dividing cells, such as thymus, testis, small intestine, colon (mucosal lining), and placenta. These results suggest that the expression of HsMCAK is first detected in early S phase to support the proliferative response and is strictly regulated at the transcriptional level.

Arginine antimetabolite l-canavanine induces apoptotic cell death in human Jurkat T cells via caspase-3 activation regulated by Bcl-2 or Bcl-xL

L-Canavanine, a natural L-arginine analog, is known to possess cytotoxicity to tumor cells in culture and experimental tumors in vivo. In this study, we first show that apoptotic cell death is associated with antitumor activity of L-canavanine against human acute leukemia Jurkat T cells. When Jurkat T cells were treated with 1.25-5.0mM L-canavanine for 36 h, apoptotic cell death accompanying several biochemical events such as caspase-3 activation, degradation of poly(ADP-ribose) polymerase (PARP), and apoptotic DNA fragmentation was induced in a dose-dependent manner; however, cytochrome c release from mitochondria was not detected. Under these conditions, the expression of Bcl-2 and its functional homolog Bcl-xL was markedly upregulated. The L-canavanine-induced caspase-3 activation, degradation of PARP, and apoptotic DNA fragmentation were suppressed by ectopic expression of Bcl-2 or Bcl-xL, both of which are known to play roles as anti-apoptotic regulators. These results demonstrate that the cytotoxic effect of L-canavanine on Jurkat T cells is attributable to the induced apoptosis and that L-canavanine-induced apoptosis is mediated by a cytochrome c-independent caspase-3 activation pathway that can be interrupted by Bcl-2 or Bcl-xL.

17α-Estradiol arrests cell cycle progression at G2/M and induces apoptotic cell death in human acute leukemia Jurkat T cells

A pharmacological dose (2.5-10 microM) of 17alpha-estradiol (17alpha-E(2)) exerted a cytotoxic effect on human leukemias Jurkat T and U937 cells, which was not suppressed by the estrogen receptor (ER) antagonist ICI 182,780. Along with cytotoxicity in Jurkat T cells, several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9, -3, and -8, PARP degradation, and DNA fragmentation were induced. The cytotoxicity of 17alpha-E(2) was not blocked by the anti-Fas neutralizing antibody ZB-4. While undergoing apoptosis, there was a remarkable accumulation of G(2)/M cells with the upregulatoin of cdc2 kinase activity, which was reflected in the Thr56 phosphorylation of Bcl-2. Dephosphorylation at Tyr15 and phosphorylation at Thr161 of cdc2, and significant increase in the cyclin B1 level were underlying factors for the cdc2 kinase activation. Whereas the 17alpha-E(2)-induced apoptosis was completely abrogated by overexpression of Bcl-2 or by pretreatment with the pan-caspase inhibitor z-VAD-fmk, the accumulation of G(2)/M cells significantly increased. The caspase-8 inhibitor z-IETD-fmk failed to influence 17alpha-E(2)-mediated caspase-9 activation, but it markedly reduced caspase-3 activation and PARP degradation with the suppression of apoptosis, indicating the contribution of caspase-8; not as an upstream event of the mitochondrial cytochrome c release, but to caspase-3 activation. In the presence of hydroxyurea, which blocked the cell cycle progression at the G(1)/S boundary, 17alpha-E(2) failed to induce the G(2)/M arrest as well as apoptosis. These results demonstrate that the cytotoxicity of 17alpha-E(2) toward Jurkat T cells is attributable to apoptosis mainly induced in G(2)/M-arrested cells, in an ER-independent manner, via a mitochondria-dependent caspase pathway regulated by Bcl-2.

Monoterpenoids from the aerial parts of Aruncus dioicus var. kamtschaticus and their antioxidant and cytotoxic activities

The aerial parts of Aruncus dioicus var. kamtschaticus afforded five new monoterpenoids (1-5): 4-(erythro-6,7-dihydroxy-9-methylpent-8-enyl)furan-2(5H)-one (1, aruncin A), 2-(8-ethoxy-8-methylpropylidene)-5-hydroxy-3,6-dihydro-2H-pyran-4-carboxylic acid (2, aruncin B), 4-(hydroxymethyl)-6-(8-methylprop-7-enyl)-5,6-dihydro-2H-pyran-2-one-11-O-β-D-glucopyranoside (3, aruncide A), (3S,4S,5R,10R)-3-(10-ethoxy-11-hydroxyethyl)-4-(5-hydroxy-7-methylbut-6-enyl)oxetan-2-one-11-O-β-D-glucopyranoside (4, aruncide B), and (3S,4S,5R,7R)-5-(9-methylprop-8-enyl)-1,6-dioxabicyclo[3,2,0]heptan-2-one-7-(hydroxymethyl)-12-O-β-D-glucopyranoside (5, aruncide C). Compound 2 showed potent cytotoxicity against Jurkat T cells with an IC(50) value of 17.15 μg/mL. In addition, compounds 7 and 10 exhibited moderate antioxidant activity with IC(50) values of 46.3 and 11.7 μM, respectively.

Oral administration of Lactococcus lactis expressing Helicobacter pylori Cag7-ct383 protein induces systemic anti-Cag7 immune response in mice

Abstract To express the 3′‐region (1152u2003bp) of the cag7 gene of Helicobacter pylori 51 strain, encoding the C‐terminal 383 amino acid (ct383u2003aa) region of Cag7 protein that is known to cover the needle region of T4SS, in a live delivery vehicle Lactococcus lactis, the cag7‐ct383 gene was amplified by PCR. DNA sequence analysis revealed that the amino acid sequence of Cag7‐ct383 of H. pylori 51 shared 98.4% and 97.4% identity with H. pylori 26695 and J99, respectively. Intramuscular injection of the GST‐Cag7‐ct383 fusion protein into a rat could raise the anti‐Cag7 antibody, indicating the immunogenicity of the Cag7‐ct383 protein. When the cag7‐ct383 gene was cloned in Escherichia coli–L. lactis shuttle vector (pMG36e) and transformed into L. lactis, the transformant could produce the Cag7‐ct383 protein, as evidenced by Western blot analysis. The Cag7‐ct383 protein level in the L. lactis transformant reached a maximum at the early stationary phase without extracellular secretion. The oral administration of the L. lactis transformant into mice generated anti‐Cag7 antibody in serum in five of five mice. These results suggest that L. lactis transformant expressing Cag7‐ct383 protein may be applicable as an oral vaccine to induce mucosal and systemic immunity to H. pylori.

Expression of the apolipoprotein C‐II gene during myelomonocytic differentiation of human leukemic cells

Apolipoprotein C‐II (apoC‐II), which is known to activate lipoprotein lipase (LPL), was identified by ordered differential display (ODD)‐polymerase chain reaction (PCR) as a cDNA fragment exhibiting a distinct increase in expression during 12‐O‐tetradecanoylphorbol 13‐acetate (TPA)‐induced differentiation of promonocytic U937 cells into monocytes and macrophages. The amount of apoC‐II mRNA expression detectable in U937 cells significantly increased and reached a maximum 24–48 h after treatment with 32 nM TPA. apoC‐II mRNA was also detected in monocytic THP‐1 cells but was not detected in promyelocytic HL‐60 cells. In healthy human tissues, the most significant expression of apoC‐II mRNA was in the liver. Although apoC‐II mRNA expression was markedly up‐regulated during the induced differentiation of HL‐60 cells into monocytes and macrophages with 32 nM TPA, such expression was not induced during the differentiation of HL‐60 cells into granulocytes with 1.25% dimethyl sulfoxide. These results suggest that human apoC‐II expression is induced at the transcription level during myelomonocytic differentiation and may confer an important role to macrophages involved in normal lipid metabolism and atherosclerosis.

Nucleotide sequence and cell cycle-associated differential expression of ZF5128, a novel Kruppel type zinc finger protein gene

The nucleotide sequence of the ZF5128 gene, encoding a novel Kruppel type zinc finger protein, has been determined. The ZF5128 gene has a predicted 553-amino acid open reading frame, encoding a putative 61 kDa zinc finger protein. The N-terminus of the ZF5128 coding region has a well-conserved Kruppel-associated box (KRAB) domain that consists of KRAB box A and B, whereas the C-terminus contains a Kruppel type C2H2 zinc finger domain possessing nine C2H2 zinc finger motifs in tandem arrays with the highly conserved space region of the H/C-link. Each C2H2 zinc finger motif has a typical consensus sequence of CX2CX3FX5LX2HX3H. A 3.2 kb transcript specific for ZF5128 was expressed at high levels in the spleen, thymus, and peripheral blood leukocyte, and weakly expressed in the prostate, ovary, small intestine, colon (mucosal lining), placenta, lung, and pancreas. Although there was no detectable ZF5128 mRNA in unstimulated human peripheral T cells, it was first detectable 1.5 h after activation by anti-CD3 plus anti-CD28, and reached a maximum in 25-30 h. During the cell cycle progression of Jurkat T cells, the expression of ZF5128 mRNA appeared to be induced in G1 and reached a maximum in the S phase, but declined as the cells entered the G2/M phase. The 12-O-tetradecanoylphorbol 13-acetate-induced monocytic differentiation of U937, which also resulted in growth arrest, down-regulated the expression of ZF5128 mRNA. Taken together, these results indicate that ZF5128 is a novel gene encoding a Kruppel type C2H2 zinc finger protein and is regulated at the transcriptional level depending on tissue type and the cell cycle status to support cell proliferation.