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Biomedical subjects

Y Shi

Publications and source records attributed to Y Shi.

At least 19 recordsLinked to original sources
PubMed1999

Cloning of an L-3-hydroxyacyl-CoA dehydrogenase that interacts with the GLUT4 C-terminus.

Evidence indicates that the carboxy-terminal cytoplasmic domain of glucose transporter 4 (GLUT4) is important for the regulation of GLUT4 in muscle and adipocytes. We cloned from a human skeletal muscle cDNA library a 34-kDa protein which interacts with GLUT4 C-terminal cytoplasmic domain in a two-hybrid system and also with GLUT4 C-terminus synthetic peptide in an in vitro binding assay. This protein, called YP10, showed a high degree (>90%) of sequence homology with l-3-hydroxyacyl-CoA dehydrogenase (HAD) and had a dehydrogenase activity similar to pig heart HAD, which was inhibited by GLUT4 C-terminus synthetic peptide. An antiserum raised against pig heart HAD also reacted with YP10. Western blot analysis using this antiserum revealed abundant immunoreactivity only in the mitochondria- and plasma membrane-enriched fractions of rat adipocytes. Northern blots revealed that YP10 mRNA is most abundant in skeletal and heart muscle. These findings suggest that YP10, a HAD isoform, interacts with GLUT4 at the plasma membrane and may play a role in cross-talk between glucose transport and fatty acid metabolism.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1999

Effect of mutations at the monomer-monomer interface of cAMP receptor protein on specific DNA binding.

To determine the thermodynamic role of binding of an operon to cAMP receptor protein (CRP) in the activation of transcription, isothermal titration calorimetry measurements were performed on the binding of three 40-base pair DNA sequences to the cyclic nucleoside complexes of CRP and its mutants at 296 K. The three 40-base pair sequences consisted of a consensus DNA (conDNA) duplex derived from the CRP-binding site sequences of the operons activated by CRP and two DNA sequences based on the CRP-binding site sequences of the lac operon (lacDNA) and of the gal operon (galDNA). The mutants of CRP consisted of a T127L mutant, a S128A mutant, and a mutant containing both mutations (CRP*) which not only alter the transcriptional activity of the CRP complexes but also are involved in the monomer-monomer interfacial interactions of the CRP dimer. The binding reactions of the DNA duplexes to the fully cNMP-ligated CRP-mutant complexes were endothermic with binding constants as high as 6.6 +/- 1.1 x 10(6) M-1 (conDNA.CRP(cAMP)2). ConDNA binding to the unligated T127L and CRP* mutants was observed as well as conDNA and lacDNA binding to CRP with cAMP bound to only one monomer. The reduction of the binding constants with increase in KCl concentration indicated the formation of two ion pairs for the cAMP-ligated CRP and S128A complexes and four ion pairs for the cAMP-ligated T127L and CRP* complexes. Reduction of the DNA binding constants upon substitution of D2O for H2O in the buffer, the large heat capacity changes, and the enthalpy-entropy compensation exhibited by the binding reactions indicate the importance of dehydration in the binding reaction. Small angle neutron scattering measurements on the lacDNA.CRP(cAMP)2 complex in D2O/H2O mixtures show that the DNA is bent around the cAMP-ligated protein in solution.

Base Sequence↗
PubMed1999

Molecular mimicry between a viral peptide and a myelin oligodendrocyte glycoprotein peptide induces autoimmune demyelinating disease in mice.

Semliki Forest Virus (SFV) induces an encephalomyelitis followed by demyelination in the brains of C57Bl6/J (B6) mice. To investigate the role of molecular mimicry in the pathogenesis of postviral demyelination, alignment algorithms were used and amino acid homologies between immunogenic epitopes of SFV and myelin autoantigens, myelin basic protein (MBP), myelin proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) were identified. Immunization of B6 mice with SFV proteins induced significant lymphocyte proliferation to SFV E2 peptides and to MOG peptide, 18-32 (which had molecular mimicry with E2 115-129), but not to MBP or PLP peptides. Both MOG 18-32 and E2 115-129, induced a later-onset chronic EAE-like disease that correlated with the presence of multifocal vacuolation in the CNS white matter. This histopathology was reminiscent of the secondary demyelination seen following SFV infection. Serum antibody responses to the peptides appeared late after immunizations and some samples cross-reacted with other myelin peptides, as well as with the mimicked MOG peptides. These findings suggest that following a CNS viral infection, antibody response to an epitope of virus that exhibits molecular mimicry with a peptide of MOG may contribute to autoimmune mediated injury to CNS myelin.

Acute Disease↗
PubMed1999

Characterization of a mutant pancreatic eIF-2alpha kinase, PEK, and co-localization with somatostatin in islet delta cells.

Phosphorylation of eukaryotic translation initiation factor-2alpha (eIF-2alpha) is one of the key steps where protein synthesis is regulated in response to changes in environmental conditions. The phosphorylation is carried out in part by three distinct eIF-2alpha kinases including mammalian double-stranded RNA-dependent eIF-2alpha kinase (PKR) and heme-regulated inhibitor kinase (HRI), and yeast GCN2. We report the identification and characterization of a related kinase, PEK, which shares common features with other eIF-2alpha kinases including phosphorylation of eIF-2alpha in vitro. We show that human PEK is regulated by different mechanisms than PKR or HRI. In contrast to PKR or HRI, which are dependent on autophosphorylation for their kinase activity, a point mutation that replaced the conserved Lys-614 with an alanine completely abolished the eIF-2alpha kinase activity, whereas the mutant PEK was still autophosphorylated when expressed in Sf-9 cells. Northern blot analysis indicates that PEK mRNA was predominantly expressed in pancreas, though low expression was also present in several tissues. Consistent with the high levels of mRNA in pancreas, the PEK protein was only detected in human pancreatic islets, and the kinase co-localized with somatostatin, a pancreatic delta cell-specific hormone. Thus PEK is believed to play an important role in regulating protein synthesis in the pancreatic islet, especially in islet delta cells.

Amino Acid Sequence↗
PubMed1999

[Pathogenic gene linkage analysis and hemopoietic characteristics in a kindred with sideroblastic anemia].

OBJECTIVE: Analysis of pathogenic gene linkage and hemopoietic characteristics in a kindred with sideroblastic anemia. METHODS: PCR amplification of the microsatellites DXS991,DXS1199 in chromosome Xp11.22 linked gene ALAS2 and of the microsatellite DXS1226 in Xp22. 13 linked another irrelevant gene and analysis of gene linkage in a kindred with 2 patients and 7 normal persons. The bone marrow hemopoietic cells from 2 patients were cultured in condition culture matrix with various cytokines added in and the CFU-E, CFU-GM and CFU-Meg formations were observed at different times. RESULTS: The kindred study revealed that pathogenic gene linked with DXS991 and DXS1199 but did not link with DXS1226.Hemopoietic cell culture showed that erythroid colonies of the two patients grew more vigorously than controls and they could grew in the absence of Epo except in common condition matrix. The erythroid colonies withered after a week and were smaller than the controls after 13 days. CONCLUSION: The kindred is subject to an X-linked sideroblastic anemia(XLSA) with the pathogenic gene ALAS2 involved. In XLSA,the function of stem cells is primarily normal before erythropoiesis, then the erythroid progenitors become dysplasia.

Adult↗
PubMed1999

Decreased expression of the pro-apoptotic protein Par-4 in renal cell carcinoma.

Par-4 is a widely expressed leucine zipper protein that confers sensitization to apoptosis induced by exogenous insults. Because the expression of genes that promote apoptosis may be down-regulated during tumorigenesis, we sought to examine the expression of Par-4 in human tumors. We present here evidence that Par-4 protein levels were severely decreased in human renal cell carcinoma specimens relative to normal tubular cells. Replenishment of Par-4 protein levels in renal cell carcinoma cell lines conferred sensitivity to apoptosis. Because apoptosis may serve as a defense mechanism against malignant transformation or progression, decreased expression of Par-4 may contribute to the pathophysiology of renal cell carcinoma.

Apoptosis↗
PubMed1999

Optimization of high-speed DNA sequencing on microfabricated capillary electrophoresis channels.

DNA sequencing separations have been performed in microfabricated electrophoresis channels with the goal of determining whether high-quality sequencing is feasible with these microdevices. The separation matrix, separation temperature, channel length and depth, injector size, and injection parameters were optimized. DNA fragment sizing separations demonstrated that 50-micron-deep channels provide the best sensitivity for our detection configuration. One-color sequencing separations of single-stranded M13mp18 DNA on 3% linear polyacrylamide (LPA) were used to optimize the twin-T injector size, injection conditions, and temperature. The best one-color separations were observed with a 250-micron twin-T injector, an injection time of 60 s, and a temperature of 35 degrees C. The first 500 bases appeared in 9.2 min with a resolution of > 0.5, and the separation extended to 700 bases. The best four-color sequencing separations were performed using 4% LPA, a temperature of 40 degrees C, and a 100-micron twin-T injector. These four-color runs were complete in only 20 min, could be automatically base-called using BaseFinder to over 600 bp after the primer, and were 99.4% accurate to 500 bp. These results significantly advance the quality of microchip-based electrophoretic sequencing and indicate the feasibility of performing high-speed genomic sequencing with microfabricated electrophoretic devices.

Base Sequence↗
PubMed1999

Role for p300 in stabilization of p53 in the response to DNA damage.

The nuclear p300/CBP proteins function as coactivators of gene transcription. Here, using cells deficient in p300 or CBP, we show that p300, and not CBP, is essential for ionizing radiation-induced accumulation of the p53 tumor suppressor and thereby p53-mediated growth arrest. The results demonstrate that deficiency of p300 results in increased degradation of p53. Our findings suggest that p300 contributes to the stabilization and transactivation function of p53 in the cellular response to DNA damage.

CREB-Binding Protein↗
PubMed1999

Role of ERAB/L-3-hydroxyacyl-coenzyme A dehydrogenase type II activity in Abeta-induced cytotoxicity.

Endoplasmic reticulum-associated amyloid beta-peptide (Abeta)-binding protein (ERAB)/L-3-hydroxyacyl-CoA dehydrogenase type II (HADH II) is expressed at high levels in Alzheimer's disease (AD)-affected brain, binds Abeta, and contributes to Abeta-induced cytotoxicity. Purified recombinant ERAB/HADH II catalyzed the NADH-dependent reduction of S-acetoacetyl-CoA with a Km of approximately 68 microM and a Vmax of approximately 430 micromol/min/mg. The contribution of ERAB/HADH II enzymatic activity to Abeta-mediated cellular dysfunction was studied by site-directed mutagenesis in the catalytic domain (Y168G/K172G). Although COS cells cotransfected to overexpress wild-type ERAB/HADH II and variant beta-amyloid precursor protein (betaAPP(V717G)) showed DNA fragmentation, cotransfection with Y168G/K172G-altered ERAB and betaAPP(V717G) was without effect. We thus asked whether the enzyme might recognize alcohol substrates of which the aldehyde products could be cytotoxic; ERAB/HADH II catalyzed oxidation of a variety of simple alcohols (C2-C10) to their respective aldehydes in the presence of NAD+ and NAD-dependent oxidation of 17beta-estradiol. Addition of micromolar levels of synthetic Abeta(1-40) to purified ERAB/HADH II inhibited, in parallel, reduction of S-acetoacetyl-CoA (Ki approximately 1.6 microM), as well as oxidation of 17beta-estradiol (Ki approximately 3.2 microM) and (-)-2-octanol (Ki approximately 2.6 microM). Because micromolar levels of Abeta were required to inhibit ERAB/HADH II activity, whereas Abeta binding to ERAB/HADH II occurred at much lower concentrations (Km approximately 40-70 nM), the latter more closely simulating Abeta levels within cells, Abeta perturbation of ERAB/HADH II was likely to result from mechanisms other than the direct modulation of enzymatic activity. Cells cotransfected to overexpress ERAB/HADH II and betaAPP(V717G) generated malondialdehyde-protein and 4-hydroxynonenal-protein epitopes, which were detectable only at the lowest levels in cells overexpressing either ERAB/HADH II or betaAPP(V717G) alone. Generation of such toxic aldehydes was not observed in cells contransfected to overexpress Y168G/K172G-altered ERAB and betaAPP(V717G). We conclude that the generalized alcohol dehydrogenase activity of ERAB/HADH II is central to the cytotoxicity observed in an Abeta-rich environment.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1999

Tissue inhibitor of metalloproteinases-1 (TIMP-1) mRNA is elevated in advanced stages of thyroid carcinoma.

Tumour cell invasion and metastasis is a multistep process that involves the degradation of extracellular matrix proteins by matrix metalloproteinases (MMPs). Tissue inhibitors of metalloproteinases (TIMPs) act as negative regulators of MMPs and thus prevent tumour cell invasion and metastasis by preserving extracellular matrix (ECM) integrity. In the present study we examined the expression of one member of TIMPs, TIMP-1, in 39 thyroid tumour specimens and two thyroid carcinoma cell lines (NPA and SW579). We also investigated the effect of high TIMP-1 expression on the invasive potential of NPA cells. Northern blot analysis showed that TIMP-1 mRNA levels correlated directly with tumour aggressiveness: the highest number of TIMP-1 transcripts was found in stages III and IV vs benign goitre (P < 0.0001). However, TIMP-1 expression was not increased in NPA and SW579 cells, both of which are derived from poorly differentiated thyroid tumours. Immunohistochemical study showed strong TIMP-1 staining in the stroma cells of advanced stages of carcinomas. Overexpression of TIMP-1 by gene transfer resulted in a significant suppression of the malignant phenotype of NPA cells as judged by an in vitro tumour invasion assay. These results suggest that high levels of TIMP-1 transcripts in advanced stages of thyroid carcinoma likely come from stroma rather than thyroid cancer cells, and TIMP-1 may function as a thyroid tumour invasion/metastasis suppressor.

Adult↗
PubMed1999

Furazolidone-containing short-term triple therapies are effective in the treatment of Helicobacter pylori infection.

BACKGROUND: A furazolidone-containing therapeutic regimen for Helicobacter pylori infection has attracted special interest in the face of a rising world-wide metronidazole resistant H. pylori, and the expense of currently used antimicrobial regimens. AIM: To evaluate the efficacy of furazolidone-containing regimens in eradicating H. pylori. METHODS: One-hundred and forty H. pylori positive patients with endoscopically confirmed duodenal ulcer or functional dyspepsia received one of four different regimens to eradicate H. pylori. In the first trial, the patients were randomly assigned to receive a 1-week course of furazolidone 100 mg b.d. and clarithromycin 250 mg b.d., with either tripotassium dicitrato bismuthate (TDB) 240 mg b.d. (FCB group) or lansoprazole 30 mg daily (FCL group). In the second trial, the patients were randomly assigned to receive a 1-week course of clarithromycin 250 mg b.d. and omeprazole 20 mg daily, with either furazolidone 100 mg b.d. (FCO group) or metronidazole 400 mg b.d. (MCO group). Endoscopy was repeated 4 weeks following completion of therapy with re-assessment of H. pylori status on gastric biopsies by histology and culture. RESULTS: Four patients (1 in FCB, 1 in FCO and 2 in MCO groups) dropped out because they refused a follow-up endoscopy. Eradication rates of H. pylori on an intention-to-treat basis in the FCB, FCL, FCO and MCO groups were 91% (32/35, 95% CI: 82-99%), 91% (32/35, CI: 82-99%), 86% (30/35, CI: 74-97%) and 74% (26/35, CI: 60-89%) (all P > 0.05), respectively. Mild side-effects occurred in 15% of the 140 patients. In MCO group, the eradication rate in the patients infected with metronidazole-sensitive isolates of H. pylori was 86%, but dropped to 67% in those with metronidazole-resistance strains (P = 0.198). CONCLUSION: One-week regimens containing furazolidone and clarithromycin in combination with TDB or a proton pump inhibitor fulfil the criteria for successful H. pylori therapy.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed1999

Factors influencing the levels of fatty acid synthase complex activity in fowl.

There is a notable discrepancy between the FAS (fatty acid synthase) activity of four types of fowl (egg chicken, meat chicken, egg duck, and meat duck) with distinctively different body fat levels. There is a 14.8 fold difference per unit body weight between the maximum and minimum FAS activities. The three major factors affecting this discrepancy are liver weight per unit body weight, which is 2.3 times greater in meat ducks than in egg chickens, the amount of FAS protein per gram of liver, which is 1.85 times greater in meat ducks than in egg chickens, and the FAS specific activity in meat ducks, which is 3.5 times greater in meat ducks than in egg chickens. Within the same species of egg chickens, the abdomen fat per kg of body weight at 470 days after egg production is 66 times greater than 90 days before egg production and the liver FAS activity is increased 9.6 fold. The 9.6 fold FAS activity increase resulted from an increase in the specific activity, since the liver weight per kilogram of body weight remained constant at approx. 20 grams and the FAS weight per gram of liver also remained constant at approx. 4.5 mg. This shows that the control of the basic FAS activity level which is closely related to the level of body fat does not mainly arise from genetic control. For the same kind of fowl, the control of the basic FAS activity level occurs after gene expression. It is suggested that control may be imposed in the folding phase when new peptides give rise to functional proteins.

Adipose Tissue↗
PubMed1999

Molecular analysis of microbial community structures in pristine and contaminated aquifers: field and laboratory microcosm experiments.

This study used phylogenetic probes in hybridization analysis to (i) determine in situ microbial community structures in regions of a shallow sand aquifer that were oxygen depleted and fuel contaminated (FC) or aerobic and noncontaminated (NC) and (ii) examine alterations in microbial community structures resulting from exposure to toluene and/or electron acceptor supplementation (nitrate). The latter objective was addressed by using the NC and FC aquifer materials for anaerobic microcosm studies in which phylogenetic probe analysis was complemented by microbial activity assays. Domain probe analysis of the aquifer samples showed that the communities were predominantly Bacteria; Eucarya and Archaea were not detectable. At the phylum and subclass levels, the FC and NC aquifer material had similar relative abundance distributions of 43 to 65% beta- and gamma-Proteobacteria (B+G), 31 to 35% alpha-Proteobacteria (ALF), 15 to 18% sulfate-reducing bacteria, and 5 to 10% high G+C gram positive bacteria. Compared to that of the NC region, the community structure of the FC material differed mainly in an increased abundance of B+G relative to that of ALF. The microcosm communities were like those of the field samples in that they were predominantly Bacteria (83 to 101%) and lacked detectable Archaea but differed in that a small fraction (2 to 8%) of Eucarya was detected regardless of the treatment applied. The latter result was hypothesized to reflect enrichment of anaerobic protozoa. Addition of nitrate and/or toluene stimulated microbial activity in the microcosms, but only supplementation of toluene alone significantly altered community structures. For the NC material, the dominant subclass shifted from B+G to ALF, while in the FC microcosms 55 to 65% of the Bacteria community was no longer identifiable by the phylum or subclass probes used. The latter result suggested that toluene exposure fostered the proliferation of phylotype(s) that were otherwise minor constituents of the FC aquifer community. These studies demonstrated that alterations in aquifer microbial communities resulting from specific anthropogenic perturbances can be inferred from microcosm studies integrating chemical and phylogenetic probe analysis and in the case of hydrocarbon contamination may facilitate the identification of organisms important for in situ biodegradation processes. Further work integrating and coordinating microcosm and field experiments is needed to explore how differences in scale, substrate complexity, and other hydrogeological conditions may affect patterns observed in these systems.

Bacteria, Anaerobic↗
PubMed1999

Calcium-induced modification of protein conformation demonstrated by immunohistochemistry: What is the signal?

A recent study by Morgan et al. on the mechanism of the heating antigen retrieval (AR) has raised an interesting issue concerning calcium-induced modification of protein conformation demonstrated by immunohistochemistry (IHC). The current study is based on calcium-induced modification of thrombospondin (TSP) and Ki-67, as demonstrated by IHC using seven monoclonal antibodies (MAbs) to TSP and an MAb MIB1. Experiments were carried out on frozen tissue sections of bladder carcinoma and lymph node. Frozen sections were incubated with solutions of 50 mM CaCl2 and/or 10 mM EDTA at 4C overnight before formalin or acetone fixation for TSP and Ki-67, respectively. Sections were then fixed in 10% neutral buffered formalin or acetone before immunostaining. Seven MAbs to TSP, named Ab1 to 7 representing clone numbers of A4.1, D4.6, C6.7, A6.1, B5.2, A2.5, and HB8432, respectively, and MIB1 were utilized as primary antibodies. ABC was used as the detection system and AEC as the chromogen for immunohistochemical staining. An extracellular immunostaining pattern represented a positive result for TSP, and nuclear staining for MIB1. Frozen sections preincubated in 50 mM CaCl2 overnight at 4C showed significant loss of staining and/or altered staining pattern for six of the seven antibodies to TSP and MIB1 compared to positive controls not exposed to CaCl2. Lack of immunostaining of TSP and MIB1 attributable to exposure to CaCl2 could be partially recovered by incubating the frozen sections in EDTA. Calcium-induced modification of protein structure was demonstrated more than 10 years ago on the basis of immunochemical techniques. In this study, similar calcium-induced modification of protein was detectable by IHC in frozen tissue sections, suggesting that calcium-induced modification of protein structure may occur independently of fixation-induced modification. The fact that calcium binding may affect IHC staining is not surprising in view of the fact that antibody/antigen interactions are protein structure-dependent. However, in this experiment the change occurred before and independent of formalin fixation and does not necessarily imply a role for calcium in AR. There may be a valuable role for the use of chemical modification in visualization of protein structure changes in tissue sections by IHC. (J Histochem Cytochem 47:463-469, 1999)

Calcium↗
PubMed1999

Molecular and cellular basis of tissue remodeling during amphibian metamorphosis.

Amphibian metamorphosis involves systematic transformations of various tadpole organs/tissues. Three major types of changes take place during this process. These are remodeling, resorption, and de novo development, all of which appear to involve both cell proliferation and apoptosis (programmed cell death). All metamorphic changes are controlled by thyroid hormone (T3) and are organ-autonomous. Recent studies using primary cell cultures and a stably transformed cell line from tadpole tissues have implicated that T3 induces apoptosis cell-autonomously. This T3-induced, metamorphosis-associated apoptosis is similar to cell death in other animal species and involves similar cell death executioners. Both the activation of these executioners and the pathways leading to cell proliferation and differentiation are believed to be through transcriptional regulation by T3 receptors (TRs). TRs can activate or repress target gene transcription depending upon the presence or absence of T3, respectively. Many direct T3-response genes have been isolated and found to encode a variety of proteins that can affect both intra- and extra-cellular events. The determinations of the identities of these response genes through sequence analyses and studies on their expression profiles during development have provided strong clues toward their roles in metamorphosis. However, future studies using organ and cell culture systems and/or transient or stable transgenic technologies are required to understand how these genes transduce the T3 signal to activate the downstream cell death and proliferation/differentiation pathways.

Amphibians↗
PubMed1998

Solution structure of a fragment of the dimerization domain of DP-1 determined by 1H-nuclear magnetic resonance and distance geometry.

The structure of a synthesized peptide with the sequence of NHILPNESAYDQKNIRRRVYDALNVLMAMNIISK that corresponds to residues 151-184 of transcription factor DP-1 (Girling et al., Nature 362 (1993) 83-87) was determined by 1H-nuclear magnetic resonance in water and 40% d3-trifluoroethanol/water, respectively. Nuclear Overhauser effect cross peaks, alphaH chemical shifts and J-coupling constants of alphaH-NH show that the peptide consists a helix from Ser-8 to Ser-33 in solution. Fifty structures were constructed with 288 upper distance limits and 21 angle constraints by DIANA (Guntert et al., J. Mol. Biol. 217 (1991) 517-530). Although the N-terminal of the peptide exhibits a random conformation, the 20 best structures show a root mean square deviation of 0.89+/-0.36 A for backbone atoms and 1.80+/-0.34 A for heavy atoms from residue Ser-8 to Ser-33. This result supports the proposal that DP-1 and E2F-1 may dimerize with a coiled-coil type interaction.

Amino Acid Sequence↗
PubMed1998

Methylation of the androgen receptor promoter CpG island is associated with loss of androgen receptor expression in prostate cancer cells.

Androgen-independent metastatic prostate cancer is characterized by a heterogeneous loss of androgen receptor (AR) expression among tumor cells. In this study, we evaluate DNA hypermethylation as a potential transcriptional regulatory mechanism in AR-negative prostate cancer cell lines. Nucleotide sequence analysis demonstrates an approximately 15-kb CpG island in the AR gene that encompasses the transcription start site and exon 1. Using Southern blotting with methylation-sensitive restriction enzymes and methylation-specific PCR, we find aberrant methylation in the AR expression-negative cell lines Du145, DuPro, TSU-PR1, and PPC1. Incomplete methylation in the AR CpG island is also seen in normal female breast and ovarian tissues consistent with the inactivation of one X chromosome by hypermethylation. In contrast, prostate cancer cell lines LNCaP and PC3 express AR and are unmethylated. Normal prostate epithelial cell strains demonstrate no methylation. Exposure of AR-negative prostate cancer cell lines to 5-aza-2' deoxycytidine, a demethylating agent, induces the reexpression of AR RNA in DuPro and TSU-PR1. This reexpression is associated with a demethylation of this region. Prostate-specific antigen, an androgen-responsive gene, is also specifically induced in these lines after AR reexpression. Therefore, in vitro DNA methylation of the 5' CpG AR island may be associated with the loss of AR expression. Furthermore, our results demonstrate that treatment with demethylating agents may engender the reexpression and function of the androgen receptor in AR-negative cell lines.

CpG Islands↗