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Biomedical subjects

K Yoon

Publications and source records attributed to K Yoon.

At least 19 recordsLinked to original sources
PubMed2006

Decreased survival of C/EBP beta-deficient keratinocytes is due to aberrant regulation of p53 levels and function.

Recent studies have identified roles for C/EBPbeta in cellular survival and tumorigenesis, however, the mechanisms through which C/EBPbeta regulates these processes are not fully understood. Previously, we demonstrated that C/EBPbeta(-/-) mice are resistant to carcinogen-induced skin tumorigenesis and in response to topical carcinogen treatment display a 17-fold increase in keratinocyte apoptosis compared to wild-type mice. Here, we have investigated the mechanisms through which C/EBPbeta regulates apoptosis in response to carcinogenic stress. Analysis of carcinogen-treated C/EBPbeta(-/-) mouse skin revealed a striking increase in the number of p53 immunopositive keratinocytes in the epidermis of C/EBPbeta(-/-) mice compared to wild-type mice and this increase was temporally associated with a concomitant anomalous increase in apoptosis. The increased levels of p53 were functional as Mdm2, Bcl-2, C/EBPalpha and p21 were differentially regulated in the epidermis of carcinogen-treated C/EBPbeta(-/-) mice. The increase in p53 protein was not associated with an increase in p53 mRNA levels. To determine whether p53 is required for the increased apoptosis in C/EBPbeta(-/-) mice, C/EBPbeta/p53 compound knockout mice were generated. Carcinogen-treated C/EBPbeta/p53 compound knockout mice did not display increased apoptosis demonstrating p53 is required for the proapoptotic phenotype in C/EBPbeta(-/-) mice. Our results demonstrate that altered keratinocyte survival in C/EBPbeta(-/-) mice results from aberrant regulation of p53 protein and function and indicate C/EBPbeta has a role in the negative regulation of p53 protein levels in response to carcinogen-induced stress.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2006

Differential cellular responses to exogenous DNA in mammalian cells and its effect on oligonucleotide-directed gene modification.

Transient transfection has been widely used in many biological applications including gene regulation and DNA repair, but, so far, little attention has been paid to cellular responses induced by the transfected DNA. Here, we report that double-stranded (ds) DNA introduced into mammalian cells induced expression of a variety of genes involved in DNA damage signaling and DNA repair. The expression profile of the induced genes was highly dependent on the cell type, suggesting interactions between exogenous dsDNA and cellular proteins. Moreover, each cell line elicited a markedly different level of intrinsic cellular responses to the introduced dsDNA. Furthermore, the presence of single-stranded oligonucleotides or short duplexes consisting of two complementary oligonucleotides did not affect cellular response, indicating that the induction was highly dependent on the structure and length of exogenous DNA. The extent of induction of DNA damage, signaling and DNA repair activities correlated to episomal and chromosomal gene correction frequencies. In addition, the presented data indicate that the presence of exogenous dsDNA triggered a DNA damage response by activation of ATR (ataxia telangiectasia-Rad3-related) but not ATM (ataxia telangiectasia mutated) pathway.

Animals↗
PubMed2003

Oligonucleotide-directed single-base DNA alterations in mouse embryonic stem cells.

We have investigated the use of single-stranded oligodeoxy-nucleotides (ssODN) to produce specific single-base alterations in episomal and chromosomal DNA in mouse embryonic stem (ES) cells. Two different reporter genes, EGFP and LacZ, each with a single point mutation that inactivates reporter activity, were used. ssODN homologous to the target sequence, except for a single mismatch at the mutant base, were used to correct the mutant reporter genes. When tested in CHO-K1 cells, the ssODN showed correction rates of 0.5-1.0%, consistent with prior reports. ssODN in the antisense orientation provided higher rates of gene conversion than those in the sense orientation for both reporter genes. Nuclear extracts from mouse ES cells exhibited nearly the same correction activity as extracts from CHO-K1 cells. ssODN corrected the mutant bases of both episomal and chromosomal mutant reporter genes in mouse ES cells. Although the efficiency of gene correction observed in ES cells is low, approximately 10(-4), these results demonstrate that ssODN can produce single-base alterations in the genomic DNA of mouse ES cells. As conversion efficiency is improved by the continued development of oligonucleotide structure and DNA delivery methods, ssODN could be used to produce ES cells with specific mutations in any gene in a single step. The targeted ES cells could in turn be used to create accurate mouse models of inherited diseases.

Animals↗
PubMed2003

An unusual cause of hyperinsulinaemic hypoglycaemia syndrome.

Endogenous hyperinsulinism as a cause for hypoglycaemia can be attributed to a number of different causes including insulinoma, sulphonylurea drugs and the newly described disorder non-insulinoma pancreatogenous hypoglycaemia (NIPH). The calcium stimulation test is increasingly used as a method for not only localizing insulinoma but also for distinguishing the above entities. We describe a case in which felonious sulphonylurea administration was used to mimic either an insulinoma or NIPH. Importantly, this case demonstrates that, contrary to previous reports, the insulin response to calcium stimulation in such cases may be uniformly positive and should alert the physician to possible surreptitious sulphonylurea ingestion.

Calcium↗
PubMed2003

In vivo alteration of the keratin 17 gene in hair follicles by oligonucleotide-directed gene targeting.

Using intradermal injection of a chimeric RNA-DNA oligonucleotide (RDO) or a single-stranded oligonucleotide (ssODN) into murine skin, we attempted to make a dominant mutation (R94p) in the conserve alpha-helical domain of keratin 17 (K17), the same mutation found in pachyononychia congenichia type 2 (PC-2) patients with phenotypes ranging from twisted hair and multiple pilosebaceous cysts. Both K17A-RDO and -ssODN contained a single base mismatch (CGC to CCC) to alter the normal K17 sequence to cause an amino acid substitution (R94P). The complexes consisting of oligonucleotides and cationic liposomes were injected to C57B1/6 murine skin at 2 and 5 day after birth. Histological examination of skin biopsies at postnatal day 8 from several mice showed consistent twisted hair shafts or broken hair follicles at the sebaceous gland level and occasional rupture of the hair bulb or epidermal cyst-like changes. In the injected area, the number of full anagen hair follicles decrease by 50%. Injection of the control oligonucleotide, identical to K17A-RDO but containing no mismatch to the normal sequence, did not result in any detectable abnormality. The frequency of gene alteration was lower than 3%, according to the restriction fragment length polymorphism (RFLP) analysis of the genomic DNA isolated by dissection of hair follicles from slides. Although intradermal injection of K17A-RDO or K17-ssODN caused a dominant mutation in K17 affecting hair growth and morphology, these phenotypic changes were transient either due to the compensation of K17 by other keratins or the replacement of the mutated cells by normal surrounding cells during hair growth.

Animals↗
PubMed2002

Multilineage differentiation of adult human bone marrow progenitor cells transduced with human papilloma virus type 16 E6/E7 genes.

We have established a new adult human bone marrow-derived cell line hMPC 32F, stably transduced with human papilloma virus type 16 E6/E7 genes, that displays mesenchymal multilineage differentiation ability in vitro. The hMPC 32F cells exhibited a population doubling time of 22 h and have been maintained in culture for about 20 passages. When cultured in conditions promoting osteogenic, adipogenic, or chondrogenic differentiation, hMPC 32F cells expressed mature differentiated phenotypes. These include (1) osteoblastic phenotype characterized by upregulated alkaline phosphatase (ALP) expression and extracellular matrix mineralization, (2) adipocytic phenotype with the presence of intracellular lipid droplets, and (3) chondrocytic phenotype of round cells surrounded by a sulfated proteoglycan-rich matrix. In addition, the hMPC 32F cells expressed differentiation lineage-specific genes, as detected by RT-PCR. Furthermore, osteogenic and adipogenic cultures responded to regulatory factors such as transforming growth factor-beta1 (TGF-beta1) and 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3). Thus, continuous treatment of osteogenic cultures for 2 weeks with TGF-beta1 decreased ALP activity and mRNA expression and inhibited osteocalcin mRNA expression and matrix mineralization, whereas l,25(OH)2D3 had an additive, stimulatory effect. In adipogenic cultures, treatment with TGF-beta1 for 2 weeks markedly inhibited adipogenesis whereas 1,25(OH)2D3 had no obvious effect. Finally, clonal analysis of hMPC 32F cells revealed a high percentage of multipotent clones, although clones of more restricted differentiation potential were also present. These characteristics of the hMPC 32F cell line suggest their pluripotent, progenitor, and nontransformed nature and indicate their potential application for studying the mechanisms governing developmental potential of adult human bone marrow mesenchymal progenitor cells.

Adipocytes↗
PubMed2002

Simultaneous targeted alteration of the tyrosinase and c-kit genes by single-stranded oligonucleotides.

We have shown that various forms of oligonucleotides, chimeric RNA-DNA oligonucleotide (RDO) and single-stranded oligodeoxynucleotide (ODN), are capable of chromosomal gene alterations in mammalian cells. Using two ODNs we corrected an inactivating mutation in the tyrosinase gene and introduced an activating mutation into the c-kit gene in a single albino mouse melanocyte. Relying on a pigmentation change caused by tyrosinase gene correction, we determined the frequency of gene targeting events ranging from 2 x 10(-4) to 1 x 10(-3), which is comparable to our previously published data using RDO. However, ODN showed more reproducible gene correction than RDO and produced pigmented cells among 60% of experiments, in comparison with 10% by RDO. DNA sequence analysis of the converted cells revealed that two out of eight individual pigmented clones harbored the mutated c-kit gene. Targeted modification of both genes resulted in the ability of the tyrosinase to convert tyrosine to melanin, and in the constitutive activation of the Kit receptor kinase. Thus, for the first time, we demonstrate the feasibility of simultaneous targeting of two genes in a single cell and show that a selection strategy to identify cells that have undergone a gene modification can enrich the targeted cells with the desired gene alteration.

Amino Acid Sequence↗
PubMed2002

Inhibitory effects of N-(substituted benzoylamino)-4-ethyl-1,2,3,6-tetrahydropyridines on nitric oxide generation in stimulated raw 264.7 macrophages.

There has been great interest in reactive nitrogen intermediates and nitric oxide production in macrophages, particularly because of their contributory role in several pathophysiological conditions during acute and chronic inflammation. Several N-(substituted benzoylamino)-4-ethyl-1,2,3,6-tetrahydropyridines were previously synthesized as potential antiinflammatory agents. In the present study, the effects of four previously synthesized tetrahydropyridines (THPs) on cyclooxygenase (COX)-1 and COX-2 were screened and the effects of these compounds on lipopolysaccharide (LPS)-induced (2 micrograms/ml) nitric oxide and inducible nitric oxide synthase (iNOS) activity in RAW 264.7 macrophages were examined. 4-Bromo THP showed 9.4 microM of IC50 as the most potent derivative among the tested THPs followed by 4-nbuthyl, 4-fuoro, and 4-methyl THP with IC50 values of 30.9, 38.9 and 80.3 microM, respectively (indomethacin IC50 = 53.8 microM). None of the tested compounds showed cytotoxic effects to the RAW 264.7 macrophages. All of the tested THPs exhibited COX-1 and COX-2 nonselective inhibition. These results suggest that previously synthesized THP derivatives may have dual effects through inhibiting both COX and nitric oxide by inhibiting iNOS.

Animals↗
PubMed2001

Differential effects of primary human immunodeficiency virus type 1 nef sequences on downregulation of CD4 and MHC class I.

We analyzed two primary nef sequences, KS2 (subtype B) and K306 (subtype D), each directly isolated from patients. Cell lines constitutively expressing respective Nef proteins were constructed using a retroviral vector. There were significant differences in the ability to downregulate surface CD4 and MHC class I proteins between different nef sequences. When the nef sequence from NL432 was used as a reference, KS2 Nef demonstrated the highest ability to downregulate MHC class I, whereas it appeared to lack the ability to downregulate CD4. On the contrary, Nef from K306 decreased the level of surface CD4 to a greater extent, but was less effective on downregulation of MHC class I. These results showed that the levels of downregulation of CD4 and MHC class I could significantly vary among HIV strains and that two well-known functions of Nef, downregulation of CD4 and MHC class I, would be separated.

Base Sequence↗
PubMed2001

Stable expression of human immunodeficiency virus type 1 Nef confers resistance against Fas-mediated apoptosis.

To investigate the effect of Nef on Fas-mediated apoptosis, we compared T cells, both population and subclones stably expressing Nef from HIV-1(NL432), with Nef(-) control cells. Fas-mediated apoptosis was significantly delayed in Nef(+) cells as determined by annexin V staining and the percentage of apoptotic cells was lower in all Nef-expressing cells than in the control cells by a maximum of 10-fold. Next we measured cell surface levels of Fas to test whether the delayed apoptosis in Nef(+) cells was due to reduced cell surface expression of Fas. We found that there was no significant difference in the surface level of Fas between the Nef(+) and Nef(-) cells. To further define the steps affected by Nef in the Fas signaling pathway, the activation of caspase-3 and caspase-8 was investigated. A reasonable correlation was found between the magnitude of apoptosis measured by annexin V staining and the enzymatic activity of caspase-3. The overall level of caspase-8 activity in Nef(+) cells was also lower than in Nef(-) cells, although the extent of inhibition was not as significant as seen for caspase-3. Overall, our results indicate that long-term stable expression of Nef, which mimicks persistent or latent infection in vivo, confers resistance against anti-Fas Ab-induced apoptosis through inhibition of caspase-3 and caspase-8 activation.

Annexin A5↗
PubMed2001

Differential activation of wild-type and variant forms of estrogen receptor alpha by synthetic and natural estrogenic compounds using a promoter containing three estrogen-responsive elements.

Structure-dependent estrogen receptor alpha (ER alpha) agonist and antagonist activities of synthetic and natural estrogenic compounds were investigated in human HepG2, MDA-MB-231 and U2 cancer cell lines. Compounds used in this study include 4'-hydroxytamoxifen, ICI 182,780, bisphenol-A (BPA), 2',4',6'-trichloro-4-biphenylol (3Cl-PCB-OH), 2',3',4',5'-tetrachloro-4-biphenylol (4Cl-PCB-OH), p-t-octylphenol, p-nonylphenol, naringenin, kepone, resveratrol, and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE). Cells were transfected with a construct (pERE(3)) containing three tandem estrogen responsive elements (EREs) and either wild-type estrogen receptor alpha (ER-wt) or variants expressing activation function-1 (ER-AF1) or AF-2 (ER-AF2). The ER agonist activities of the synthetic mono and dihydroxy aromatic compounds are comparable in all three-cell lines, whereas the activities of naringenin, kepone and resveratrol are dependent on cell context and expression of wild-type or variant forms of ER alpha. In contrast, the ER antagonist activities for these compounds were highly complex and, with the exception of 3Cl-PCB-OH, all compounds inhibited E2-induced wild-type or variant ER action. Results of this in vitro study suggest that the estrogenic and antiestrogenic activity of structurally diverse synthetic and natural estrogenic compounds is complex, and this is consistent with published data that often give contradictory results for these compounds.

Benzhydryl Compounds↗
PubMed2001

Targeted gene correction by small single-stranded oligonucleotides in mammalian cells.

We demonstrate that relatively short single-stranded oligodeoxynucleotides, 25-61 bases homologous to the target sequence except for a single mismatch to the targeted base, are capable of correcting a single point mutation (G to A) in the mutant beta-galactosidase gene, in nuclear extracts, episome, and chromosome of mammalian cells, with correction rates of approximately 0.05%, 1% and 0.1%, respectively. Surprisingly, these short single-stranded oligonucleotides (ODN) showed a similar gene correction frequency to chimeric RNA-DNA oligonucleotide, measured using the same system. The in vitro gene correction induced by ODN in nuclear extracts was not dependent on the length or polarity of the oligonucleotide. In contrast, the episomal and chromosomal gene corrections were highly dependent on the ODN length and polarity. ODN with a homology of 45 nucleotides showed the highest frequency and ODN with antisense orientation showed a 1000-fold higher frequency than sense orientation, indicating a possible influence of transcription on gene correction. Deoxyoligonucleotides showed a higher frequency of gene correction than ribo-oligonucleotides of the identical sequence. These results show that a relatively short ODN can make a sequence-specific change in the target sequence in mammalian cells, at a similar frequency as the chimeric RNA-DNA oligonucleotide.

Animals↗
PubMed2001

Differential expression of tissue-specific promoters by gene gun.

BACKGROUND: Tissue-specific promoters are becoming increasingly important in light of their effects on gene expression in gene therapy experiments. The regulation of gene expression may be as important as the delivery of the gene itself. OBJECTIVES: To determine the effects of the involucrin (INV), keratin 14 (K14) and cytomegalovirus (CMV) promoters on the expression of the reporter gene beta-galactosidase. METHODS: In vivo, plasmid DNA was introduced to BALB/c mice by gene gun. Skin biopsies were taken after 24 h for histology and beta-galactosidase staining. In tissue culture cells, plasmid DNA was introduced by transient transfection to cell lines 293 (transformed primary human embryonal kidney cells), NIH 3T3 (immortalized mouse fibroblasts) and human keratinocytes. Reporter gene expression was assayed by histochemical staining and chemiluminescence. RESULTS: The K14 and INV promoter constructs showed beta-galactosidase gene expression only in the epidermis, while the CMV promoter showed gene expression in both the dermis and epidermis. In cell culture, the INV and K14 promoter constructs demonstrated significant beta-galactosidase expression in human keratinocytes, but minimal expression in 293 and NIH 3T3 cell types. CMV promoter constructs demonstrated significant expression in all cell types. CONCLUSIONS: Gene expression can be regulated by different promoters both in vivo and in cell culture. Based on the physiological expression of the different promoters, gene expression can be restricted to certain cell types, tissues and skin layers.

Animals↗
PubMed2001

Toxicology of environmental estrogens.

It has been hypothesized that environmental contaminants that modulate endocrine signaling pathways may be causally linked to adverse health effects in humans. There has been particular concern regarding synthetic estrogens and their role in disrupting normal development of the male reproductive tract. Most estrogenic industrial compounds, such as bisphenol A (BPA) and nonylphenol, typically bind estrogen receptors alpha (ERalpha) and beta (ERbeta) and induce transactivation of estrogen-responsive genes/reporter genes, but their potencies are usually > or = 1,000-fold lower than observed for 17beta-estradiol (E2). Selective estrogen receptor modulators (SERMs) represent another class of synthetic estrogens that are being developed for treatment of hormone-dependent problems. The SERMs differentially activate wild-type ERalpha and variant forms expressing activation function 1 (ER-AF1) and AF2 (ER-AF2) in human HepG2 hepatoma cells transfected with a pC3-luciferase construct, and these in vitro differences reflect their unique in vivo biologies. The HepG2 cell assay has also been used in our laboratories to investigate the estrogenic activities of the following structurally diverse synthetic and phytoestrogens: 4'-hydroxytamoxifen; BPA; 2',4',6'-trichloro-4-biphenylol; 2',3',4',5'-tetrachloro-4-biphenylol; p-t-octylphenol; p-nonylphenol; naringenin; kepone; resveratrol; and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE). The results show that synthetic and phytoestrogens induce distinct patterns of gene activation in HepG2 and U2 osteogenic sarcoma cells, suggesting that these compounds will induce tissue-specific in vivo ER agonist or antagonist activities. The predicted differences between these compounds, based on results of the in vitro bioassay, have been confirmed. For example, BPA inhibits E2-induced responses in the rodent uterus, and HPTE and structurally related compounds are ERalpha agonists and ERbeta antagonists in assays carried out in HepG2 and other cancer cell lines.

Animals↗
PubMed2001

Specific double-stranded RNA interference in undifferentiated mouse embryonic stem cells.

Specific mRNA degradation mediated by double-stranded RNA (dsRNA) interference (RNAi) is a powerful way of suppressing gene expression in plants, nematodes, and fungal, insect, and protozoan systems. However, only a few cases of RNAi have been reported in mammalian systems. Here, we investigated the feasibility of the RNAi strategy in several mammalian cells by using the enhanced green fluorescent protein gene as a target, either by in situ production of dsRNA from transient transfection of a plasmid harboring a 547-bp inverted repeat or by direct transfection of dsRNA made by in vitro transcription. Several mammalian cells including differentiated embryonic stem (ES) cells did not exhibit specific RNAi in transient transfection. This long dsRNA, however, was capable of inducing a sequence-specific RNAi for the episomal and chromosomal target gene in undifferentiated ES cells. dsRNA at 8.3 nM decreased the cognate gene expression up to 70%. However, RNAi activity was not permanent because it was more pronounced in early time points and diminished 5 days after transfection. Thus, undifferentiated ES cells may lack the interferon response, similar to mouse embryos and oocytes. Regardless of their apparent RNAi activity, however, cytoplasmic extracts from mammalian cells produced a small RNA of 21 to 22 nucleotides from the long dsRNA. Our results suggest that mammalian cells may possess RNAi activity but nonspecific activation of the interferon response by longer dsRNA may mask the specific RNAi. The findings offer an opportunity to use dsRNA for inhibition of gene expression in ES cells to study differentiation.

Animals↗
PubMed2000

Targeted single-base correction by RNA-DNA oligonucleotides.

An oligonucleotide composed of a contiguous stretch of RNA and DNA residues has been developed to facilitate correction of single-base mutations of episomal and chromosomal targets in mammalian cells. We demonstrated that an RNA-DNA oligonucleotide (RDO) induced heritable correction of a point mutation in the tyrosinase gene at the level of genomic sequence, protein, and phenotype of albino mouse melanocytes and albino mouse skin. Such RDOs might hold promise as a therapeutic method for the treatment of skin diseases. However, the general application of RDO technology has been hampered by the absence of a standardized system to measure the gene conversion in a particular cell type in a rapid and reproducible manner. For this purpose, we established an in vitro system in which nuclear extracts from mammalian cells showed RDO-mediated gene correction of a shuttle vector containing a point mutation in the E. coli beta-galactosidase gene. This sensitive and convenient assay has been utilized to optimize the design of RDOs and to compare frequencies of gene conversion among different cell types. The general application of the RDO for site-specific gene correction or mutation would benefit from such mechanistic studies.

Animals↗