Biomedical subjects

H R Kim

Publications and source records attributed to H R Kim.

At least 91 records · Page 5Linked to original sources

PubMed2000

Biological properties and expression of mucins in 5-fluorouracil resistant HT29 human colon cancer cells.

We have previously reported that HT29 human colon cancer cells selected by adaptation to 5-fluorouracil (5FU) (HT29-5FU cells) express increased levels of a major intestinal mucin MUC2 mRNA compared with parental HT29 cells. In this study, we examined in detail the changes in synthesis and secretion of mucin that occur in these cells and accompanying changes in the expression of cancer associated mucin related carbohydrate antigens and cell lineage associated biochemical markers. We further investigated their relationship to biological properties of cells. Northern blot analysis revealed a markedly increased level of MUC2 mRNA but no significant change in the mRNA levels of other mucins in HT29-5FU cells compared with parental HT29 cells. Labeling with radiolabeled precursors demonstrated increased synthesis and secretion of mucin glycoproteins by HT29-5FU cells. Immunoblot analysis showed a higher expression of mucin associated carbohydrate antigens such as T, Tn, sialyl Tn, sialyl Lea, sialyl Lex and non-O-acetylated sialic acid concomitant with significant increases in the expression of goblet cell lineage marker, MUC2 apomucin and a panepithelial cell marker, carcinoembryonic antigen. HT29-5FU cells showed significantly higher adhesion to E-selectin and to matrigel and in vitro invasive properties and significantly increased liver colonization capacity in nude mice following splenic vein injection. Nude mouse xenograft tumors produced by HT29-5FU cells showed a greater degree of differentiation, consisting of mucin secreting glands than those produced by parental HT29 cells. These results indicate that predominantly colonic type mucin, MUC2, has been selectively induced in HT29-5FU cells and that altered regulation of mucin genes associated with altered synthesis and secretion of mucin glycoproteins and the degree of differentiation in cancer cells may be responsible for the altered biological properties of these cells.

Animals↗

PubMed2000

Phorbol 12-myristate 13-acetate induces alteration in mucin gene expression and biological properties of colon cancer cells.

Phorbol esters such as phorbol 12-myristate 13-acetate (PMA) have been reported to modulate diverse cellular responses through signal transduction pathways including the protein kinase C (PKC) pathway. In the present study, we sought to determine the effect of PMA on mucin gene expression and on the biological properties of a human colon cancer cell line, HM3. The cells were treated for 8 and 24 h with various concentrations of PMA and total RNA was extracted and Northern and slot blot analyses were carried out using MUC2, MUC3 and MUC5AC mucin cDNA probes to assess the steady state levels of mRNA. Spent media were collected and the level of cancer associated carbohydrate antigens (T, Tn, sialyl Tn, sialyl Lex, and sialyl Lea) and matrix-degrading metalloproteinase (MMPs) activity were examined. Trypsinized cells were used for assessing in vitro invasion, motility and adhesion to matrigel. Our results showed that PMA caused upregulation of steady state mRNA levels of MUC2, MUC3 and MUC5AC which was inhibited after treatment with protein synthesis inhibitors. Calphostin C, a highly specific inhibitor of protein kinase C significantly inhibited the PMA induced induction of mRNA levels of MUC2, MUC3, and MUC5AC. The levels of all cancer-associated mucin carbohydrate antigens examined in the media were increased by PMA treatment. PMA also caused an increase in MMPs activity and in in vitro invasion and motility properties, but did not affect adhesion of HM3 cells to matrigel. Thus, PMA caused a significant increase in the expression of all three mucin genes through signaling pathways involving protein kinase C and increased secretion of mucin associated carbohydrate antigens. These changes were associated with increases in MMP activity as well as by increases in the invasive and motility properties of HM3 colon cancer cells. These data suggest that protein kinase C signaling pathways may be involved in mucin gene regulation and in modulating the invasive and metastatic properties of colon cancer cells.

Adenocarcinoma↗

PubMed2000

Neutron radiation enhances cisplatin cytotoxicity independently of apoptosis in human head and neck carcinoma cells.

Recent advances in combined modality treatment of locally advanced head and neck cancer have improved local and regional disease control and survival with better functional outcome. However, the local and regional failure rate after radiation therapy is still high for tumors that respond poorly to cisplatin-based neoadjuvant chemotherapy. This clinical observation suggests a common biological mechanism for resistance to cisplatin and photon irradiation. In this report, we investigated the molecular basis underlying cisplatin resistance in head and neck squamous carcinoma (HNSCC) cells and asked if fast neutron radiation enhances cisplatin cytotoxicity in cisplatin-resistant cells. We found that cisplatin sensitivity correlates with caspase induction, a cysteine proteinase family known to initiate the apoptotic cell death pathway, suggesting that apoptosis may be a critical determinant for cisplatin cytotoxicity. Neutron radiation effectively enhanced cisplatin cytotoxicity in HNSCCs including cisplatin-resistant cells, whereas photon radiation had little effect on cisplatin cytotoxicity. Interestingly, neutron-enhanced cisplatin cytotoxicity was associated neither with apoptosis nor with cell cycle regulation, as determined by caspase activity assay, annexin V staining, and flow cytometric analysis. Taken together, the present study provides a molecular insight into cisplatin resistance and may also provide a basis for more effective multimodality protocols involving neutron radiation for patients with locally advanced head and neck cancer.

Annexin A5↗

PubMed2000

LPS induces direct death of IFN-gamma primed murine embryonic hepatocyte, BNL CL2 cells in a TNF-alpha independent manner.

Although it has been well known that the role of LPS on liver damage is mediated through TNF-alpha, the mechanism by which LPS modulates the cytotoxicity of IFN-gamma on hepatocytes has not yet been clearly demonstrated. Here, we demonstrate that IFN-gamma mediated apoptosis in murine embryonic hepatocyte BNL CL2 cells is potentiated by the addition of LPS (0.5 microg/ml). Consistently, LPS markedly increases the catalytic activity of caspase 3-like protease but not caspase 1-like protease in IFN-gamma treated cells. In addition, TNF-alpha alone does not affect cell viability but rather it potentiates the cytotoxic effect of IFN-gamma on BNL CL2 cells. However, the cell viability of IFN-gamma/LPS treated cells is affected by the addition of polymyxin B but not by TNF binding protein I (TNF-BPI). These data suggest that the lipid moiety of LPS may mediate direct cytotoxicity of BNL CL2 cells in a TNF-alpha independent manner.

Animals↗

PubMed1999

Molecular genetic relationships between Bombycidae and Saturniidae based on the mitochondria DNA encoding of large and small rRNA.

The phylogenetic relationships between Bombycidae (Bombyx mori and Bombyx mandarina) and Saturniidae (Antheraea yamamai and Antheraea pernyi) were investigated based on large and small mitochondiral rRNA genes. About 430 bp of four kinds of PCR-amplified fragments were sequenced and aligned. For the 16S rRNA gene, B. mori shared a 98, 87 and 86% sequence homology with B. mandarina, A. yamamai and A. pernyi, and for the 12S rRNA gene, B. mori shared a 99, 89 and 88% sequence homology with B. mandarina, A. yamamai and A. pernyi, respectively. DNA sequence data were also used for a phylogenetic analysis. All of the trees showed monophyly for both Bombycidae and Saturniidae. The monophyly confidence limits of these trees were estimated using bootstrapping tests and measured more than 99% for all trees for both Bombycidae and Saturniidae.

Animals↗

PubMed1999

Tissue inhibitor of metalloproteinase-1 inhibits apoptosis of human breast epithelial cells.

The signaling pathways critical for cell survival are mediated in part by the composition and integrity of the extracellular matrix and the action of its components on specific cell adhesion receptors. Withdrawal of anchorage-dependent epithelial cells from their association with ECM results in apoptotic cell death. Consistently, the matrix metalloproteinases (MMPs) or their inhibitors (TIMPs) have been suggested to regulate apoptosis. In this report, we investigated whether bcl-2 inhibition of apoptosis involves regulation of TIMP expression. We have found that bcl-2 overexpression induces TIMP-1 expression in breast epithelial cell lines (MCF10A, MCF10AneoT.TG3B, and MCF-7), whereas it has no effect on TIMP-2 expression. We demonstrated that TIMP-1 inhibits cell death induced by hydrogen peroxide, Adriamycin, or X-ray irradiation. In addition, TIMP-1 overexpression inhibits apoptosis after the loss of cell adhesion (anoikis) in MCF10A cells, suggesting that the antiapoptotic activity of TIMP-1 does not depend on its ability to stabilize cell-matrix interactions. We also showed that TIMP-1 overexpression is associated with constitutive activation of focal adhesion kinase, a signaling molecule known to be critical for the cell survival pathway.

Apoptosis↗

PubMed1999

Cell cycle arrest and inhibition of anoikis by galectin-3 in human breast epithelial cells.

Galectin-3 is a member of a growing family of animal beta-galactoside-binding proteins shown to be involved in cell growth, differentiation, apoptosis resistance, and tumor progression. In the present study, we investigated whether galectin-3 can protect against apoptosis induced by the loss of cell anchorage (anoikis). Because studies suggest that cellular sensitivity to anoikis is associated with cell cycle regulation, we examined the role of galectin-3 on cell cycle regulation. Although BT549 cells (human breast epithelial cells) undergo anoikis, galectin-3-overexpressing BT549 cells respond to the loss of cell adhesion by inducing G1 arrest without detectable cell death. Galectin-3-mediated G1 arrest involves down-regulation of G1-S cyclin levels (cyclin E and cyclin A) and up-regulation of their inhibitory protein levels (p21(WAF1/CIP1) and p27KIP1). After the loss of cell anchorage, Rb protein becomes hypophosphorylated in galectin-3-overexpressing cells, as predicted from the flow cytometric analysis and immunoblot analysis of cyclins and their inhibitors. Interestingly, galectin-3 induces cyclin D1 expression (an early G1 cyclin) and its associated kinase activity in the absence of cell anchorage. On the basis of these results, we propose that galectin-3 inhibition of anoikis involves cell cycle arrest at an anoikis-insensitive point (late G1) through modulation of gene expression and activities of cell cycle regulators. The present study suggests that galectin-3 may be a critical determinant for anchorage-independent cell survival of disseminating cancer cells in the circulation during metastasis.

Antigens, Differentiation↗

PubMed1999

Enhanced apoptotic response to photodynamic therapy after bcl-2 transfection.

Apoptosis is a cellular death process involving the sequential activation of a series of caspases, endonucleases, and other enzymes. The initiation of apoptosis can be inhibited by overexpression of bcl-2 and certain other members of a related family of proteins. We examined the effects of bcl-2 overexpression on the apoptotic response to photodynamic therapy (PDT), using aluminum phthalocyanine as the photosensitizing agent. In this study, we compared the immortalized human breast epithelial cell line MCF10A with a subline (MCF10A/bcl-2) transfected with the human bcl-2 gene. The latter was approximately 2-fold more sensitive to the phototoxic effects of PDT. At a 50 mJ/cm2 light dose, photodamage to MCF-10A/bcl-2 resulted in a greater loss of the mitochondrial membrane potential (delta(psi)m), enhanced release of mitochondrial cytochrome c, a more rapid and greater activation of caspase-3, and a greater apoptotic response. Western blot analysis revealed that the transfected cell line showed overexpression of both bcl-2 and bax, and that PDT caused selective destruction of bcl-2, leaving bax unaffected. The greater apoptotic response by the transfected line is, therefore, attributed to the higher bax:bcl-2 ratio after photodamage.

Apoptosis↗

PubMed1999

Expression, purification, characterization and crystallization of flap endonuclease-1 from Methanococcus jannaschii.

A gene coding for a protein homologous to the flap endonuclease-1 (FEN-1) was cloned from Methanococcus jannaschii, overexpressed, purified and characterized. The gene product from M. jannaschii shows 5' endo-/exonuclease and 5' pseudo-Y-endonuclease activities as observed in the FEN-1 in eukaryotes. In addition, Methanococcus jannaschii FEN-1 functions effectively at high concentrations of salt, unlike eukaryotic FEN-1. We have crystallized Methanococcus jannaschii FEN-1 and analyzed its preliminary character. The crystal belongs to the space group of P2(1) with unit cell dimensions of a = 58.93 A, b = 42.53 A, c = 62.62 A and beta = 92.250. A complete data set has been collected at 2.0 A resolution using a frozen crystal.

Blotting, Western↗

PubMed1999

Cecropin D-like antibacterial peptides from the sphingid moth, Agrius convolvuli.

Two major antibacterial peptides were isolated and purified from immunized larval hemolymph of Agrius convolvuli. Acid extraction, gel filtration, ultrafiltration, and reversed-phase FPLC were used for purification of peptides. These peptides had similar molecular mass and amino acid composition. Moreover, 21 of the first 23 N terminal residues were identical. The peptides were highly homologous with cecropin D in size and primary sequence, and named Agrius cecropin D1 and D2. The molecular masses of Agrius cecropin D1 and D2 were 3,879.39 and 3,839.27, respectively. In antibacterial and hemolytic assays, Agrius cecropin D showed potent antibacterial activities against a panel of Gram positive and negative bacteria without hemolytic activity against human red blood cells. Notably, our antibacterial assay revealed Agrius cecropin D possessed stronger or at least equivalent activities against B. megaterium than cecropin A. It suggests that Agrius cecropin D, which has an alternative structure from cecropin D, could be the model for the development of peptide antibiotics. Arch.

Amino Acid Sequence↗

PubMed1999

Neurotoxic and neuroprotective actions of catecholamines in cortical neurons.

We examined the possibility that catecholamines (CA) could act as endogenous modulators of neuronal death. Exposure to high doses (>100 microM) of dopamine (DA) caused widespread neuronal death within 24 h in mouse cortical cell cultures and was accompanied by cell body shrinkage, aggregation and condensation of nuclear chromatin, and prominent internucleosomal DNA fragmentation. Epinephrine, but not norepinephrine (NE), was slightly toxic to neurons at doses higher than 1 mM. DA-induced death was attenuated by the addition of three different anti-apoptosis agents, 1 microgram/ml cycloheximide, 25 mM K(+), or 100 ng/ml brain-derived neurotrophic factor (BDNF). While treatment with 100 microM N-acetyl-l-cysteine attenuated DA neurotoxicity, neither the glutamate antagonists (10 microM MK-801 plus 50 microM CNQX) nor several antioxidants [trolox, 100 microM; Mn (III) tetrakis (4-benzoic acid) porphyrin chloride, 100 microM; Mn (III) tetrakis (1-methyl-4-pyridyl) prophyrin pentachloride, 100 microM; N-tert-butyl-alpha-phenylnitrone, 3 mM] prevented the CA-induced apoptosis. Interestingly, all CA at 1-30 microM attenuated free radical-mediated neuronal necrosis following exposure to 30 microM Fe(2+) or 200 microM H(2)O(2), which was insensitive to DA or NE antagonists. Like trolox, CA reduced levels of the stable free radical 1,1-diphenyl-2-picrylhydrazyl under cell-free conditions, raising the possibility that CA as an antioxidant protects neurons. We also found that the neuroprotective effect of CA prolonged the protective effects of BDNF against serum deprivation. The present findings suggest that CA induces apoptosis at high doses but prevents free radical-mediated neurotoxicity as an anti-oxidant without being coupled to the receptors.

Acetylcysteine↗

PubMed1999

Purification and characterization of chitin-binding proteins from the hemolymph of sweet potato hornworm, Agrius convolvuli.

Three chitin-binding proteins (CBPs: CBP9, CBP15, CBP66) were identified from the larval hemolymph of sweet potato hornworm, Agrius convolvuli. Two (CBP9 and CBP15) of them have been isolated and purified by gel filtration (Superdex HR 75), cation-exchange chromatography (Mono S), and reverse-phase chromatography (muRPC PC 2.1/3). In experiments to detect CBPs in hemolymph, we examined whether ionic strength and existence of bovine serum albumin in the incubation solution influenced binding affinity of CBPs to chitin. The N-terminal sequences of three CBPs were determined by the automated Edman degradation and showed the sequence homology in basic local alignment search tool search CBP15 and CBP66 were quite similar to lysozymes and bovine serum albumins, respectively. In contrast, CBP9 is not similar to any other known protein, as judged from databank comparisons. Therefore, we concluded that CBP9 is a novel protein with binding capacity to chitin that is a component of the fungal cell wall. CBP9 has no antibacterial activity against Escherichia coli and Micrococcus luteus, and also showed negative response in hemagglutination assay. CBP9 is confirmed as a monomer with a molecular mass of 9.14 kDa by electron spray ionization and matrix-assisted laser desorption ionization mass spectrometry.

Amino Acid Sequence↗

PubMed1999

Acute inflammation of the proliferative zone of gastric mucosa in Helicobacter pylori gastritis.

The neutrophilic infiltration has been regarded to represent the activity of Helicobacter pylori gastritis. It may involve the epithelium and/or lamina propria. The incidence and degree of the two types of infiltration do not correlate with each other frequently. We correlated the two types of neutrophilic infiltration with H. pylori infection and other pathologic parameters respectively in 300 randomly selected gastric biopsies as well as serial biopsies from a separate group of 95 patients who were treated for H. pylori infection. The "random biopsies" had chronic gastritis of various degrees, and the organisms were identified in 239 cases (79.7%); in the "treated group," the organisms disappeared completely in 62 cases (65.3%). Characteristically, the intraepithelial neutrophilic infiltration was predominantly localized to the proliferative zone of the gastric mucosa (zone 2) where the density of H. pylori was considerably lower than the surface epithelium. In the "random biopsies," both acute epithelial and interstitial neutrophilic infiltration correlated significantly (p < 0.01) with the H. pylori infection. In the "treated group," however, only acute epithelial inflammation correlated significantly (p < 0.01) with the eradication of infection while acute interstitial inflammation did not. Acute epithelial inflammation was no less frequently present in advanced chronic gastritis than in early chronic gastritis. Acute epithelial inflammation of the proliferative zone is a characteristic pathologic finding of H. pylori gastritis, and appears to be directly associated with the pathogenesis of H. pylori gastritis and its progression.

Biopsy↗

PubMed1999

Signal transduction of thapsigargin-induced apoptosis in osteoblast.

The toxicity of thapsigargin, a selective inhibitor of endoplasmic reticular Ca2+-ATPase, was investigated in osteoblasts. We induced apoptosis in murine osteoblastic MC3T3E1 cells by exposure to the thapsigargin. Thapsigargin transiently increased the phosphotransferase activity of c-Jun N-terminal kinases1 (JNK1), which might in turn activate transcriptional activity of activation protein-1 (AP-1). We then prepared extracts from thapsigargin-treated MC3T3E1 cells and monitored cleavage of acetyl-YVAD-AMC and acetyl-DEVD-AMC, fluorogenic substrates for caspase 1-like and caspase 3-like proteases, respectively. Thapsigargin significantly increased the proteolytic activity of caspase 3-like proteases, but not the activity of caspase 1-like proteases. Furthermore, thapsigargin increased the transcriptional activity of nuclear factor-kappaB (NF-kappaB). These data suggest that thapsigargin-induced apoptosis in osteoblasts may be via activation of JNK1, caspase 3-like family proteases, and transcriptional factors including AP-1 and NF-kappaB.

Animals↗

PubMed1999

Geldanamycin induces heat shock protein expression through activation of HSF1 in K562 erythroleukemic cells.

HSP90 is one of the most abundant heat shock proteins (HSPs) in eukaryotic cells and is found in complex with several regulatory proteins such as kinases and transcription factors. Geldanamycin (GA), a benzoquinone ansamycin, specifically binds to HSP90 and disrupts the interaction of HSP90 and target proteins. Thus, GA has been used as a specific inhibitor of HSP90. In this study, we examined whether GA could affect protein synthesis and gene expression in the human erythroleukemic cell line K562. Treatment with GA, but not herbimycin A (another benzoquinone ansamycin), highly induced a 70-kDa protein, which was revealed to be HSP70 by immunoblotting and immunoprecipitation with anti-HSP70 antibody. The expression of HSP28 was also enhanced by GA. Furthermore, GA induced the activation of heat shock factor 1 (HSF1), but not HSF2, as determined by electromobility shift and electromobility supershift assay. In addition, similar to heat shock treatment, GA induced the phosphorylation of HSF1. Heat shock element-binding activity and phosphorylation of HSF1 were attenuated 3 h after GA treatment. These results indicate that the functional inactivation of HSP90 by GA potentially stimulates the expression of heat shock proteins through activation of HSF1.

Antibiotics, Antineoplastic↗

PubMed1999

Geldanamycin induces cell cycle arrest in K562 erythroleukemic cells.

Geldanamycin (GA), a benzoquinone ansamycin, is one of the specific inhibitors of 90-kDa heat shock protein and induces growth inhibition and apoptosis in certain cancer cell lines. We have investigated the mechanism of GA-induced growth inhibition in K562 erythroleukemic cells. DNA flow-cytometric analysis indicated that GA-induced growth arrest was associated with G2/M phase arrest of the cell cycle. GA treatment down-regulated the expression of cyclin B1 and inhibited phosphorylation of Cdc2 protein, both key regulatory proteins at the G2/M boundary. GA also markedly inhibited the Cdc2 kinase activity, which may be in part a result of up-regulation of p27KIP1 by GA. The present results suggest a novel mechanism that p27KIP1 could be involved in the regulation of G2 to M phase transition.

Antibiotics, Antineoplastic↗

PubMed1999

Identification of heat shock protein 90-associated 84-kDa phosphoprotein.

In eukaryotic cells, HSP90 is associated with several protein kinases and regulates their activities. HSP90 was also reported to possess an autophosphorylase activity. In this study, we examined in vitro autophosphorylation of HSP90, which was purified from chick muscle. We show that HSP90 was not phosphorylated in vitro, but an 84-kDa protein (p84) was highly phosphorylated. P84 was neither HSP90 nor its degradative product, as it was not detected by an antibody (BF4) specific to HSP90 in denaturing immunoprecipitation and Western blot analysis. Phosphorylation of a protein similar to p84 was also detected with purified human brain and HeLa HSP90, indicating that p84 is present in many different types of cells. P84 appeared to exist as large complexes, as determined by HPLC and native gel electrophoresis. Native immunoprecipitation using anti-HSP90 (BF4)-conjugated Affi-gel revealed that this phosphoprotein is specifically associated with HSP90. The interaction of p84 and HSP90 was not affected by p84 phosphorylation. In addition, p84 phosphorylation was prevented by the presence of divalent cations such as Mg(2+) and Mn(2+). In contrast, p84 phosphorylation was significantly activated by addition of exogenous Ca(2+) between 100 and 500 microM, although it was blocked by higher concentrations (>1 mM) of Ca(2+). HSP90, but not p84, could be phosphorylated by casein kinase II. Finally, p84 phosphorylation was specifically prevented by hemin, but not by other kinase inhibitors, indicating that p84 phosphorylation may be regulated by heme-regulated protein kinase.

Animals↗

PubMed1999

Shini-san inhibits mast cell-dependent immediate-type allergic reactions.

Shini-San has been used for treatment of allergic disease in Korea. However, its effect in experimental models remains unknown. The mast cell plays a pivotal role in initiating allergic response by secreting intracytoplasmic granular mediators such as histamine. The present report describes an inhibitory effect of Shini-San on mast cell-mediated immediate-type allergic reactions. Topical application of compound 48/80 can induce an ear swelling response in normal (WBB6F1(-)+/+) mice but not in congenic mast cell-deficient WBB6F1-W/WV mice. Shini-San inhibited concentration-dependent mast cell-dependent ear swelling response induced by compound 48/80 in normal mice. Shini-San inhibited concentration-dependent passive cutaneous anaphylaxis induced by anti-dinitrophenyl (DNP) immunoglobulin E (IgE) in rats by topical application. Shini-San also inhibited in concentration-dependent fashion the histamine release from the rat peritoneal mast cells by compound 48/80 or anti-DNP IgE. Moreover, Shini-San had a significant inhibitory effect on compound 48/80-induced systemic anaphylactic reaction. These results indicate that Shini-San inhibits immediate type allergic reactions by inhibition of mast cell degranulation in vivo and in vitro.

Animals↗