Biomedical subjects

I C Ho

Publications and source records attributed to I C Ho.

At least 37 records · Page 2Linked to original sources

PubMed1995

Sodium arsenite induces chromosome endoreduplication and inhibits protein phosphatase activity in human fibroblasts.

Arsenic, strongly associated with increased risks of human cancers, is a potent clastogen in a variety of mammalian cell systems. The effect of sodium arsenite (a trivalent arsenic compound) on chromatid separation was studied in human skin fibroblasts (HFW). Human fibroblasts were arrested in S phase by the aid of serum starvation and aphidicolin blocking and then these cells were allowed to synchronously progress into G2 phase. Treatment of the G2-enriched HFW cells with sodium arsenite (0-200 microM) resulted in arrest of cells in the G2 phase, interference with mitotic division, inhibition of spindle assembly, and induction of chromosome endoreduplication in their second mitosis. Sodium arsenite treatment also inhibited the activities of serine/threonine protein phosphatases and enhanced phosphorylation levels of a small heat shock protein (HSP27). These results suggest that sodium arsenite may mimic okadaic acid to induce chromosome endoreduplication through its inhibitory effect on protein phosphatase activity.

Arsenites↗

PubMed1995

Modulation of cellular antioxidant defense activities by sodium arsenite in human fibroblasts.

Many studies have shown that oxygen radicals can be produced during arsenic metabolism. We report here that in human fibroblasts (HFW cells) sodium arsenite exposure caused increased formation of fluorescent dichlorofluorescein (DCF) by oxidation of the nonfluorescent form. The enhanced DCF fluorescence was inhibited by a radical scavenger, butylated hydroxytoluene. The effects of sodium arsenite treatment on cellular antioxidant activities were then examined. Treatment of HFW cells with sodium arsenite resulted in a significant increase in heme oxygenase activity and ferritin level. Sodium arsenite-enhanced heme oxygenase synthesis was inhibited by co-treatment of cells with the antioxidants sodium azide and dimethyl sulfoxide. Furthermore, sodium arsenite treatment did not apparently affect glucose-6-phosphate dehydrogenase activity, but resulted in significantly increased glutathione levels and superoxide dismutase activity, slightly decreased glutathione peroxidase activity, and significantly decreased catalase activity. Sodium arsenite toxicity was partly reduced by addition of catalase to the culture medium. These results imply that arsenite can enhance oxidative stress in HFW cells.

Arsenites↗

PubMed1994

Expression of heme oxygenase in arsenic-resistant human lung adenocarcinoma cells.

We have established arsenic-resistant cells (CL3R) and their subclones from a human lung adenocarcinoma cell line (CL3). CL3R cells and their subclones were maintained in the presence of 4 microM sodium arsenite. They were 6-fold more resistant than CL3 cells to arsenite. Heme oxygenase was expressed in CL3R cells and their subclones, as demonstrated by electrophoretic analysis, Northern blotting, and enzyme activity assay. When CL3R15 cells were grown in arsenite-free medium, their arsenite resistance declined in parallel with their decreasing heme oxygenase activity. Tin-protoporphyrin, a heme oxygenase inhibitor, was found to increase the toxicity of arsenic to CL3R cells. Expression of heme oxygenase might therefore be involved in the mechanism of arsenic resistance. CL3R cells were also shown to be cross-resistant to oxygen-radical generating agents, such as menadione and Adriamycin. Furthermore, sodium arsenite treatment dose-dependently increased the dichlorofluorescein fluorescence in CL3 cells but not in CL3R15 cells. These results suggest that heme oxygenase plays an important role in reducing cellular oxidants that are enhanced by sodium arsenite treatment.

Adenocarcinoma↗

PubMed1994

Differential cytotoxic effects of arsenic on human and animal cells.

Human fibroblasts (HFW) were 10-fold more susceptible than Chinese hamster ovary (CHO-K1) cells to sodium arsenite. Comparison of cellular antioxidant enzyme activities showed that CHO-K1 cells contained 3- and 8-fold more glutathione-peroxidase and catalase activities, respectively, than HFW cells. Since vitamin E, methylamine, and benzyl alcohol could prevent, in part, the arsenite-induced killing of HFW cells, we suggest that arsenite can induce oxidative damage in HFW cells. We have also established arsenic-resistant cells, SA7 and CL3R, from CHO cells and from a human lung adenocarcinoma cell line (CL3), respectively. The arsenic resistance of SA7 cells was attributed mainly to elevation of glutathione S-transferase pi levels, and that of CL3R cells was possibly due to an increase in heme oxygenase activity. Since induction of heme oxygenase is a general response to oxidative stress, we suspect that the differential toxicity of arsenic to human and animal cells could be due to arsenic's more efficient induction of oxidative damage in human cells.

Animals↗

PubMed1993

Protection of a rat tracheal epithelial cell line from paraquat toxicity by inhibition of glucose-6-phosphate dehydrogenase.

Transformed rat tracheal epithelial cells (U2) were found to be 3.6-fold more sensitive than lung fibroblasts (RLF) to paraquat. Although the toxic effects of paraquat are associated with the generation of very active superoxides, U2 cells contained higher levels of superoxide dismutase and catalase than RLF cells. On the other hand, the specific activities of both NADPH-cytochrome c reductase and glucose-6-phosphate dehydrogenase (G6DP) were 3- to 4-fold higher in U2 cells than in RLF cells. Treatment with dehydroepiandrosterone (DHEA) and epiandrosterone (EPI), G6PD inhibitors, significantly decreased the intracellular NADPH and protected U2 cells from paraquat toxicity. Since DHEA and EPI treatment did not affect the uptake of paraquat, our results suggest that paraquat sensitivity may depend on the redox cycling-associated activities of paraquat.

Androsterone↗

PubMed1993

Induction of chromatid breaks and tetraploidy in Chinese hamster ovary cells by treatment with sodium arsenite during the G2 phase.

Treatment of Chinese hamster ovary (CHO) cells with sodium arsenite during the G2 phase induced poorly condensed chromosomes and chromatid breaks. The induction of chromatid breaks was confirmed by the appearance of micronucleated cells after arsenite-treated G2 cells were allowed to re-enter interphase. When the duration of the G2 phase was artificially divided into 4 periods, more chromatid breaks were induced by treatment with arsenite during the very early G2 phase (or G2/S boundary). In addition to the induction of chromatid breaks, arsenite treatment also remarkably retarded the re-entry of mitotic cells into interphase. By replating and incubating arsenite-treated G2 cells in drug-free medium, we subsequently observed the appearance of a population of cells whose DNA content was between 4C and 8C, and metaphase cells with near-tetraploid chromosome numbers in the next mitotic division.

Animals↗

PubMed1992

Evolutionarily conserved Ets family members display distinct DNA binding specificities.

Members of the Ets family of proto-oncogenes encode sequence-specific transcription factors that bind to a purine-rich motif centered around a conserved GGA trinucleotide. Ets binding sites have been identified in the transcriptional regulatory regions of multiple T cell genes including the T cell receptor alpha and beta (TCR-alpha and -beta) enhancers and the IL-2 enhancer, as well as in the enhancers of several T cell-trophic viruses including Maloney sarcoma virus, human leukemia virus type 1, and human immunodeficiency virus-2. T cells express multiple members of the Ets gene family including Ets-1, Ets-2, GABP alpha, Elf-1, and Fli-1. The different patterns of expression and protein-protein interactions of these different Ets family members undoubtedly contribute to their ability to specifically regulate distinct sets of T cell genes. However, previous studies have suggested that different Ets family members might also display distinct DNA binding specificities. In this report, we have examined the DNA binding characteristics of two Ets family members, Ets-1 and Elf-1, that are highly expressed in T cells. The results demonstrate that the minimal DNA binding domain of these proteins consists of adjacent basic and putative alpha-helical regions that are conserved in all of the known Ets family members. Both regions are required for DNA binding activity. In vitro binding studies demonstrated that Ets-1 and Elf-1 display distinct DNA binding specificities, and, thereby interact preferentially with different naturally occurring Ets binding sites. A comparison of known Ets binding sites identified three nucleotides at the 3' end of these sequences that control the differential binding of the Ets-1 and Elf-1 proteins. These results are consistent with a model in which different Ets family members regulate the expression of different T cell genes by binding preferentially to purine-rich sequences that share a GGA core motif, but contain distinct flanking sequences.

Amino Acid Sequence↗

PubMed1992

cis-acting sequences required for inducible interleukin-2 enhancer function bind a novel Ets-related protein, Elf-1.

The recent definition of a consensus DNA binding sequence for the Ets family of transcription factors has allowed the identification of potential Ets binding sites in the promoters and enhancers of many inducible T-cell genes. In the studies described in this report, we have identified two potential Ets binding sites, EBS1 and EBS2, which are conserved in both the human and murine interleukin-2 enhancers. Within the human enhancer, these two sites are located within the previously defined DNase I footprints, NFAT-1 and NFIL-2B, respectively. Electrophoretic mobility shift and methylation interference analyses demonstrated that EBS1 and EBS2 are essential for the formation of the NFAT-1 and NFIL-2B nuclear protein complexes. Furthermore, in vitro mutagenesis experiments demonstrated that inducible interleukin-2 enhancer function requires the presence of either EBS1 or EBS2. Two well-characterized Ets family members, Ets-1 and Ets-2, are reciprocally expressed during T-cell activation. Surprisingly, however, neither of these proteins bound in vitro to EBS1 or EBS2. We therefore screened a T-cell cDNA library under low-stringency conditions with a probe from the DNA binding domain of Ets-1 and isolated a novel Ets family member, Elf-1. Elf-1 contains a DNA binding domain that is nearly identical to that of E74, the ecdysone-inducible Drosophila transcription factor required for metamorphosis (hence the name Elf-1, for E74-like factor 1). Elf-1 bound specifically to both EBS1 and EBS2 in electrophoretic mobility shift assays. It also bound to the purine-rich CD3R element from the human immunodeficiency virus type 2 long terminal repeat, which is required for inducible virus expression in response to signalling through the T-cell receptor. Taken together, these results demonstrate that multiple Ets family members with apparently distinct DNA binding specificities regulate differential gene expression in resting and activated T cells.

Adult↗

PubMed1991

Human GATA-3: a lineage-restricted transcription factor that regulates the expression of the T cell receptor alpha gene.

In addition to its role in the recognition of foreign antigens, the T cell receptor (TCR) alpha gene serves as a model system for studies of developmentally-regulated, lineage-specific gene expression in T cells. TCR alpha gene expression is restricted to cells of the TCR alpha/beta+ lineage, and is controlled by a T cell-specific transcriptional enhancer located 4.5 kb 3' to the C alpha gene segment. The TCR alpha enhancer contains four nuclear protein binding sites called T alpha 1-T alpha 4. In this report we describe the identification and characterization of a novel human cDNA, hGATA-3 that binds to the T alpha 3 element of the human TCR alpha enhancer. hGATA-3 contains a zinc finger domain that is highly related to the DNA-binding domain of the erythroid-specific transcription factor, GATA-1, and binds to a region of T alpha 3 that contains a consensus GATA binding site (AGATAG). Northern blot analyses of hematopoietic cell lines demonstrate that hGATA-3 is expressed exclusively in T cells. Overexpression of hGATA-3 in HeLa cells or human B cells specifically activated transcription from a co-transfected reporter plasmid containing two copies of the T alpha 3 binding site located upstream of the minimal SV40 promoter. Taken together these results demonstrate that hGATA-3 is a novel lineage-specific hematopoietic transcription factor that appears to play an important role in regulating the T cell-specific expression of the TCR alpha gene.

Amino Acid Sequence↗

PubMed1990

Sequence-specific binding of human Ets-1 to the T cell receptor alpha gene enhancer.

Expression of the human T cell receptor (TCR) alpha gene is regulated by a T cell-specific transcriptional enhancer that is located 4.5 kilobases (kb) 3' to the C alpha gene segment. The core enhancer contains two nuclear protein binding sites, T alpha 1 and T alpha 2, which are essential for full enhancer activity. T alpha 1 contains a consensus cyclic adenosine monophosphate (cAMP) response element (CRE) and binds a set of ubiquitously expressed CRE binding proteins. In contrast, the transcription factors that interact with the T alpha 2 site have not been defined. In this report, a lambda gt11 expression protocol was used to isolate a complementary DNA (cDNA) that programs the expression of a T alpha 2 binding protein. DNA sequence analysis demonstrated that this clone encodes the human ets-1 proto-oncogene. Lysogen extracts produced with this cDNA clone contained a beta-galactosidase-Ets-1 fusion protein that bound specifically to a synthetic T alpha 2 oligonucleotide. The Ets-1 binding site was localized to a 17-base pair (bp) region from the 3' end of T alpha 2. Mutation of five nucleotides within this sequence abolished both Ets-1 binding and the activity of the TCR alpha enhancer in T cells. These results demonstrate that Ets-1 binds in a sequence-specific fashion to the human TCR alpha enhancer and suggest that this developmentally regulated proto-oncogene functions in regulating TCR alpha gene expression.

Base Sequence↗

PubMed1990

The T alpha 2 nuclear protein binding site from the human T cell receptor alpha enhancer functions as both a T cell-specific transcriptional activator and repressor.

T cell-specific expression of the human T cell receptor alpha (TCR-alpha) gene is regulated by the interaction of variable region promoter elements with a transcriptional enhancer that is located 4.5 kb 3' of the TCR-alpha constant region (C alpha) gene segment. The minimal TCR-alpha enhancer is composed of two nuclear protein binding sites, T alpha 1 and T alpha 2, that are both required for the T cell-specific activity of the enhancer. The T alpha 1 binding site contains a consensus cAMP response element (CRE), and binds a set of ubiquitous nuclear proteins. The T alpha 2 binding site does not contain known transcriptional enhancer motifs. However, it binds at least two nuclear protein complexes, one of which is T cell specific. We now report that although the T alpha 2 nuclear protein binding site displays transcriptional activator activity in the context of the TCR-alpha enhancer, this site alone can function as a potent, T cell-specific transcriptional repressor when positioned either upstream, or downstream of several heterologous promoter and enhancer elements. These results demonstrate that a single nuclear protein binding site can function as a T cell-specific transcriptional activator or repressor element, depending upon the context in which it is located.

Base Sequence↗

PubMed1990

Effects of sodium arsenite on the cytotoxicity of bleomycin.

Our present data show that posttreatment with sodium arsenite has no effect on the cytotoxicity of bleomycin (BLM), a radiomimetic agent, in Chinese hamster ovary (CHO) cells, human skin fibroblasts, and HeLa cells. However, pretreatment with sodium arsenite potentiated the cytotoxic effects of BLM in CHO cells. This effect decreased with increasing time interval between the treatments with sodium arsenite and BLM. BLM-inactivating activity was markedly reduced in cells pretreated with sodium arsenite. Furthermore, both arsenite-potentiated BLM cytotoxicity and arsenite-reduced BLM-inactivating activity were abolished by cycloheximide. These results suggest that the potentiation effect of sodium arsenite on BLM cytotoxicity may be due to the decrease of BLM-inactivating activity. In addition, only a slight increase in G2 phase population and no apparent change in intracellular glutathione levels were observed in CHO cells pretreated with sodium arsenite.

Animals↗

PubMed1990

Paraquat-resistant cell lines derived from Chinese hamster ovary cells.

Two paraquat-resistant clones, PR-1 and PR-2, were selected from CHO K1 cells pretreated with ethyl methanesulfonate. PR-1 and PR-2, routinely cultured in a normal medium without paraquat, were six fold more resistant to paraquat than the parental CHO K1 cells. There was no difference in the uptake of [3H]paraquat among PR-1, PR-2, and CHO K1 cells. Both PR-1 and PR-2 cells showed no cross resistance to free radical generating agents and no increase in total activity of superoxide dismutase. The activities of paraquat-dependent NADPH oxidase and glucose-6-phosphate dehydrogenase were significantly reduced in PR-1 and PR-2 cells, hence the rate of paraquat radical formation will be limited. In addition, an elevation of glutathione levels in PR-1 cells or an increase in glutathione S-transferase activity in PR-2 cells may also play a certain role in protective mechanisms against the toxicity of paraquat.

Animals↗

PubMed1990

Regulation of the human T-cell receptor alpha gene enhancer: multiple ubiquitous and T-cell-specific nuclear proteins interact with four hypomethylated enhancer elements.

Transcription of human T-cell receptor (TCR) alpha genes is regulated by a T-cell-specific transcriptional enhancer that is located 4.5 kilobases 3' of the C alpha gene segment. Previous studies have demonstrated that this enhancer contains at least five nuclear protein-binding sites called T alpha 1 to T alpha 5. In the studies described in this report, we have determined the molecular requirements for human TCR alpha enhancer function. In vitro mutagenesis and deletion analyses demonstrated that full enhancer activity is retained in a 116-base-pair fragment containing the T alpha 1 and T alpha 2 nuclear protein-binding sites and that both of these sites are required for full enhancer function. Functional enhancer activity requires that the T alpha 1 and T alpha 2 binding sites be separated by more than 15 and fewer than 85 base pairs. However, the sequence of this spacer region and the relative phase of the two binding sites on the DNA helix do not affect enhancer function. Deletion and mutation analyses demonstrated that the T alpha 3 and T alpha 4 nuclear protein-binding sites are not necessary or sufficient for TCR alpha enhancer activity. However, a fragment containing these two sites was able to compensate for T alpha 1 and T alpha 2 mutations that otherwise abolished enhancer activity. Electrophoretic mobility shift analyses of the TCR alpha enhancer binding proteins revealed that the T alpha 1, T alpha 3, and T alpha 4 binding proteins are expressed in a variety of T-cell and non-T-cell tumor cell lines. In contrast, one of the two T alpha 2 binding activities was detected only in T-cell nuclear extracts. The activity of the TCR alpha enhancer does not appear to be regulated solely at the level of DNA methylation on that the enhancer sequences were found to be identically hypomethylated in B and T cells as compared with fibroblasts. Taken together, these results suggest that TCR alpha enhancer activity is regulated by the interaction of multiple T-cell-specific and ubiquitous nuclear proteins with partially redundant cis-acting enhancer elements that are hypomethylated in cells of the lymphoid lineage.

Base Sequence↗

PubMed1989

Elevation of glutathione levels and glutathione S-transferase activity in arsenic-resistant Chinese hamster ovary cells.

Arsenic-resistant Chinese hamster ovary (CHO) cells were established by progressively increasing the concentration of sodium arsenite in culture medium. One of the resistant clones, SA7, was also cross-resistant to As(V), Zn, Fe(II), Co, and Hg. The susceptibilities to sodium arsenite in parental CHO cells, revertant SA7N cells, and resistant SA7 cells were correlated with their intracellular glutathione (GSH) levels and glutathione S-transferase (GST) activity. The resistance in SA7 cells was diminished by depletion of GSH in cells after treatment with buthionine sulfoximine. Furthermore, after reexposure of revertant SA7N cells to sodium arsenite, the intracellular GSH levels, GST activity, and resistance to sodium arsenite were raised to the same levels as SA7 cells. These data indicate that the elevation of intracellular GSH levels and GST activity in SA7 cells may be responsible for the resistance to arsenite. A p25 protein, which could be a monomer subunit of GST, accumulated in SA7 cells. In addition, an outward transport inhibitor, verapamil, indiscriminately increased the arsenite toxicity in resistant and parental cells.

Animals↗

PubMed1989

Ethanol and acetaldehyde potentiate the clastogenicity of ultraviolet light, methyl methanesulfonate, mitomycin C and bleomycin in Chinese hamster ovary cells.

Ethanol itself did not induce any apparent chromosome aberrations in Chinese hamster ovary cells. However, posttreatment with ethanol potentiated the chromosome aberrations induced by ultraviolet light (UV), methyl methanesulfonate (MMS), mitomycin C (MMC) or bleomycin (BLM). Chromatid exchanges were predominantly increased in cultures treated with UV, MMS or MMC and then with ethanol, whereas chromosome breaks and chromatid exchange were the major types of aberrations increased in the cultures treated with BLM and ethanol. Posttreatment with acetaldehyde, the major metabolite of ethanol, also potentiated the chromosome aberrations induced by UV, MMS, MMC or BLM. The main types of aberrations potentiated by posttreatment with acetaldehyde were similar to those by posttreatment with ethanol.

Acetaldehyde↗

PubMed1989

A T-cell-specific transcriptional enhancer element 3' of C alpha in the human T-cell receptor alpha locus.

A transcriptional enhancer element has been identified 4.5 kilobases 3' of C alpha (constant region alpha chain) in the human T-cell receptor (TCR) alpha-chain locus. This enhancer is active on both a TCR V alpha (variable region alpha chain) promoter and the minimal simian virus 40 promotor in TCR alpha/beta Jurkat and EL4 cells but is inactive on a V alpha promoter in human TCR gamma/delta PEER and Molt-13 cells, clone 13 B cells, and HeLa fibroblasts. The enhancer has been localized to a 116-base-pair BstXI/Dra I restriction enzyme fragment, which lacks immunoglobulin octamer and kappa B enhancer motifs but does contain a consensus cAMP-response element (CRE). DNase I footprint analyses demonstrated that the minimal enhancer contains two binding sites for Jurkat nuclear proteins. One of these sites corresponds to the CRE, while the other does not correspond to a known transcriptional enhancer motif. These data support a model in which TCR alpha gene transcription is regulated by a unique set of cis-acting sequences and trans-acting factors, which are differentially active in cells of the TCR alpha/beta lineage. In addition, the TCR alpha enhancer may play a role in activating oncogene expression in T-lymphoblastoid tumors that have previously been shown to display chromosomal translocations into the human TCR alpha locus.

Base Sequence↗

PubMed1989

Clostridium perfringens septicemia with massive hemolysis.

Massive hemolysis with acute renal failure occurred in a previously healthy 69-year-old patient as a complication of Clostridium perfringens septicemia secondary to gall bladder empyema. To our knowledge, this is one of the few patients with C. perfringens septicemia and massive intravascular hemolysis who survived the episode and regained a normal renal function.

Acute Kidney Injury↗