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J Liao

Publications and source records attributed to J Liao.

At least 19 recordsLinked to original sources
PubMed2000

Molecular alterations of p73 in human esophageal squamous cell carcinomas: loss of heterozygosity occurs frequently; loss of imprinting and elevation of p73 expression may be related to defective p53.

p73 is structurally and functionally related to p53 and is possibly a tumor suppressor gene. Using 15 surgically resected frozen esophageal specimens containing both squamous cell carcinomas (ESCC) and neighboring normal epithelia, we studied p73 gene alterations and mRNA expression. Loss of heterozygosity of the p73 loci was found in nine of 14 informative cases (64%). A polymorphism at codon 173 (Thr) of p73 was identified (eight samples had ACC and seven had ACT), but mutation was not detected in tumor samples. Nine of the 15 ESCC samples (60%) displayed significantly elevated expression of p73 over the neighboring normal epithelium; of these nine samples, four displayed loss of imprinting (LOI) and one switched the expressed allele. Hypermethylation of exon 1 of the p73 gene was not detected, using the bisulfite modification method, in normal or tumor samples. Twelve of the 15 (80%) ESCC samples contained p53 defects, including missense mutation, non-frameshift small deletion or insertion, non-detectable transcripts and protein accumulation. The ESCC samples with p53 defects were significantly correlated with those which had elevated expression of p73 (Fisher's exact test, P < 0.05). The results suggest that increased expression of p73, including that by LOI, could be a partial compensatory mechanism for defective p53.

Carcinoma, Squamous Cell↗
PubMed2000

Transcriptional repression by blimp-1 (PRDI-BF1) involves recruitment of histone deacetylase.

B-lymphocyte-induced maturation protein (Blimp-1) is a transcriptional repressor that is considered to be a master regulator of terminal B-cell development because it is sufficient to trigger differentiation in the BCL(1)-cell model. Transcription of the c-myc gene is repressed by Blimp-1 during B-cell differentiation. In this study, we have explored the mechanism by which Blimp-1 represses transcription by using Gal4-fusion protein assays and assays in which Blimp-1 represses the natural c-myc promoter. The results show that Blimp-1 represses the c-myc promoter by an active mechanism that is independent of the adjacently bound activator YY1. Blimp-1 contains two regions that independently associate with histone deacetylase (HDAC) and endogenous Blimp-1 in nuclear extracts binds in vitro to the c-myc Blimp-1 site in a complex containing HDAC. The functional importance of recruiting HDAC for Blimp-1-dependent repression of c-myc transcription is supported by two experiments. First, the HDAC inhibitor tricostatin A inhibits Blimp-1-dependent repression in cotransfection assays. Second, a chromatin immunoprecipitation assay shows that expression of Blimp-1 causes deacetylation of histone H3 associated with the c-myc promoter, and this deacetylation depends on the Blimp-1 binding site in the c-myc promoter.

Acetylation↗
PubMed2000

Protein inhibitor of activated STAT-1 (signal transducer and activator of transcription-1) is a nuclear receptor coregulator expressed in human testis.

An androgen receptor (AR) interacting protein was isolated from a HeLa cell cDNA library by two-hybrid screening in yeast using the AR DNA+ligand binding domains as bait. The protein has sequence identity with human protein inhibitor of activated signal transducer and activator of transcription (PIAS1) and human Gu RNA helicase II binding protein (GBP). Binding of PIAS1 to human AR DNA+ligand binding domains was androgen dependent in the yeast liquid beta-galactosidase assay. Activation of binding by dihydrotestosterone was greater than testosterone > estradiol > progesterone. PIAS1 binding to full-length human AR in a reversed yeast two hybrid system was also androgen dependent. [35S] PIAS1 bound a glutathione S-transferase-AR-DNA binding domain (amino acids 544-634) fusion protein in affinity matrix assays. In transient cotransfection assays using CV1 cells with full-length human AR and a mouse mammary tumor virus luciferase reporter vector, there was an androgen-dependent 3- to 5-fold greater increase in luciferase activity with PIAS1 over that obtained with an equal amount of control antisense cDNA or mutant PIAS1. Constitutive transcriptional activity of the AR N-terminal+DNA binding domain was increased 6-fold by PIAS1. PIAS1 also enhanced glucocorticoid receptor transactivation in response to dexamethasone but inhibited progesterone-induced progesterone receptor transactivation in the same assay system. mRNA for PIAS1 was highly expressed in testis of human, monkey, rat, and mouse. In rat testis the onset of PIAS1 mRNA expression coincided with the initiation of spermatogenesis between 25-30 days of age. Immunostaining of human and mouse testis with PIAS1-specific antiserum demonstrated coexpression of PIAS1 with AR in Sertoli cells and Leydig cells. In addition, PIAS1 was expressed in spermatogenic cells. The results suggest that PIAS1 functions in testis as a nuclear receptor transcriptional coregulator and may have a role in AR initiation and maintenance of spermatogenesis.

Aged↗
PubMed1999

Treatment of multidrug resistant (MDR1) murine leukemia with P-glycoprotein substrates accelerates the course of the disease.

The prognosis of patients with tumors expressing P-glycoprotein (P-gp), the MDR1 gene product, is generally poor. It is assumed that this is due to decreased tumor responsiveness that results from decreased drug accumulation. We observed that treatment of animals bearing MDR1-transfected leukemic cells with P-gp substrates (i.e., drugs that are transported by P-gp) significantly worsened host survival compared to treatment with vehicle or non-P-gp substrates. This effect was seen with cancer chemotherapeutic agents (paclitaxel and vincristine) and with the MDR modulator, trans-flupenthixol. To determine the mechanism(s) underlying this observation, we studied alterations in pharmacokinetics, pharmacodynamics, and metastasis. We found that the drug-induced acceleration of disease was associated with increased metastases. P-gp(+) cells treated with P-gp substrates demonstrated several pro-metastatic features, including membrane ruffling and invasion through a hepatocyte monolayer. These results suggest that the treatment of MDR tumors with P-gp substrates may produce changes in malignant behavior that could adversely affect therapeutic outcomes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

CCAAT/enhancer-binding protein beta (C/EBPbeta) and C/EBPdelta contribute to growth hormone-regulated transcription of c-fos.

Using the c-fos enhancer as a model to analyze growth hormone (GH)-promoted gene expression, the roles of CCAAT/enhancer-binding proteins (C/EBPs) in GH-regulated transcription were investigated. In 3T3-F442A fibroblasts stably expressing the c-fos promoter mutated at the C/EBP binding site upstream of luciferase, c-fos promoter activity is stimulated by GH 6-7-fold; wild type c-fos promoter shows only a 2-fold induction by GH. This suggests that C/EBP restrains GH-stimulated expression of c-fos. Electrophoretic mobility shift assays with nuclear extracts from 3T3-F442A cells indicate that GH rapidly (2-5 min) increases binding of C/EBPbeta and C/EBPdelta, to the c-fos C/EBP binding site. Both liver activating protein (LAP) and liver inhibitory protein (LIP), forms of C/EBPbeta, are detected in 3T3-F442A cells by immunoblotting. GH increases the binding of LAP/LAP and LAP/LIP dimers. Overexpression of LIP interferes with GH-promoted reporter expression in CHO cells expressing GH receptors, consistent with the possibility that LIP restrains GH-stimulated c-fos expression. Overexpression of LAP elevates basal luciferase activity but does not influence promoter activation by GH, while overexpressed C/EBPdelta elevates basal promoter activity and enhances the stimulation by GH. GH stimulates the expression of mRNA for C/EBPbeta and -delta and increases levels of C/EBPdelta. Although C/EBPbeta is not detectably altered, GH induces a shift to more rapidly migrating forms of LIP and LAP upon immunoblotting. Treatment of extracts from GH-treated cells with alkaline phosphatase causes a shift of the slower migrating form to the rapidly migrating form, consistent with GH promoting dephosphorylation of LIP and LAP. These studies implicate C/EBPbeta and -delta in GH-regulated gene expression. They also indicate that GH stimulates the binding of C/EBPbeta and -delta to the c-fos promoter and promotes the dephosphorylation of LIP and LAP. These events may contribute to the ability of C/EBPbeta and -delta to regulate GH-stimulated expression of c-fos.

3T3 Cells↗
PubMed1999

Human keratinocytes and tumor-derived cell lines express alternatively spliced forms of transforming growth factor-alpha mRNA, encoding precursors lacking carboxyl-terminal valine residues.

The human transforming growth factor-alpha (TGF-alpha) gene is thought to contain five introns and six exons, encoding a transmembrane precursor (proTGF-alpha) from which the mature polypeptide is released by proteolytic cleavage. We identified a novel 32-nucleotide exon (exon alpha) within intron 5 and an alternative splice acceptor site in exon 6, splitting exon 6 into two segments: 6A and 6B. Therefore, in addition to wild type (wt) proTGF-alpha mRNA, which skips exon alpha, two novel proTGF-alpha variants are produced: Variant I (VaI), skipping exons alpha and 6A, and Variant II (VaII) which includes exon alpha and skips exon 6A. The only significant difference between variant and wt proTGF-alpha proteins is that the two wt carboxyl-terminal valines are replaced in the variants by five or four other amino acids, respectively. Both variant TGF-alpha mRNAs were readily detected in human keratinocytes and tumor-derived cell lines. Their protein products were cleaved as efficiently as wt TGF-alpha in response to the calcium ionophore A23187. However, both variants (but not wt) reduced serum requirements for proliferation in CHO cells. In addition, VaII-expressing CHO cells (not VaI or wt) formed foci in monolayer cultures. These results suggest that variant TGF-alpha precursors induce autonomous growth.

Amino Acid Substitution↗
PubMed1999

Regulation of GLUT1 gene transcription by the serine/threonine kinase Akt1.

We used mouse hepatoma (Hepa1c1c7) cells to study the role of the serine/threonine kinase Akt in the induction of GLUT1 gene expression. In order to selectively turn on the Akt kinase cascade, we expressed a hydroxytamoxifen-regulatable form of Akt (myristoylated Akt1 estrogen receptor chimera (MER-Akt1)) in the Hepa1c1c7 cells; we verified that hydroxytamoxifen stimulates MER-Akt1 activity to a similar extent as the activation of endogenous Akt by insulin. Our studies reveal that stimulation of MER-Akt1 by hydroxytamoxifen induces GLUT1 mRNA and protein accumulation to levels comparable to that induced by insulin; therefore, activation of the Akt cascade suffices to induce GLUT1 gene expression in this cell system. Furthermore, expression of a kinase-inactive Akt mutant partially inhibits the response of the GLUT1 gene to insulin. Additional studies reveal that the induction of GLUT1 mRNA by Akt and by insulin reflects increased mRNA synthesis and not decreased mRNA degradation. Our findings imply that the GLUT1 gene responds to insulin at the transcriptional level and that Akt mediates a step in the activation of GLUT1 gene expression in this system.

Animals↗
PubMed1999

Characterization of the major physiologic phosphorylation site of human keratin 19 and its role in filament organization.

Keratin polypeptide 19 (K19) is a type I intermediate filament protein that is expressed in stratified and simple-type epithelia. Little is known regarding K19 regulation or function, and the only other type I keratin that has been studied in terms of regulation is keratin 18 (K18). We characterized K19 phosphorylation as a handle to study its function. In vivo, serine is the major phosphorylated residue, and phosphopeptide mapping of 32PO4-labeled K19 generates one major phosphopeptide. Edman degradation suggested that the radiolabeled phosphopeptide represents K19 Ser-10 and/or Ser-35 phosphorylation. Mutation of Ser-10 or Ser-35 followed by transfection confirmed that Ser-35 is the major K19 phosphorylation site. Transfection of Ser-35 --> Ala K19 showed a filament assembly defect as compared with normal or with Ser-10 --> Ala K19. Comparison of K18 and K19 phosphorylation features in interphase cells showed that both are phosphorylated primarily at a single site, preferentially in the soluble versus the insoluble keratin fractions. K19 has higher basal phosphorylation, whereas K18 phosphorylation is far more sensitive to phosphatase type I and IIA inhibition. Our results demonstrate that Ser-35 is the major K19 interphase phosphorylation site and that it plays a role in keratin filament assembly. K19 and K18 phosphorylations share some features but also have distinct properties that suggest different regulation of type I keratins within the same cells.

Animals↗
PubMed1999

Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters.

Although the zebrafish has become a popular model organism for vertebrate developmental and genetic analyses, its use in transgenic studies still suffers from the scarcity of homologous gene promoters. In the present study, three different zebrafish cDNA clones were isolated and sequenced completely, and their expression patterns were characterized by whole-mount in situ hybridization as well as by Northern blot hybridization. The first clone encodes a type II cytokeratin (CK), which is specifically expressed in skin epithelia in early embryos and prominently expressed in the adult skin tissue. The second clone is muscle specific and encodes a muscle creatine kinase (MCK). The third clone, expressed ubiquitously in all tissues, is derived from an acidic ribosomal phosphoprotein P0 (arp) gene. In order to test the fidelity of zebrafish embryos in transgenic expression, the promoters of the three genes were isolated using a rapid linker-mediated PCR approach and subsequently ligated to a modified green fluorescent protein (gfp) reporter gene. When the three hybrid GFP constructs were introduced into zebrafish embryos by microinjection, the three promoters were activated faithfully in developing zebrafish embryos. The 2.2-kb ck promoter was sufficient to direct GFP expression in skin epithelia, although a weak expression in muscle was also observed in a few embryos. This pattern of transgenic expression is consistent with the expression pattern of the endogenous cytokeratin gene. The 1.5-kb mck promoter/gfp was expressed exclusively in skeletal muscles and not elsewhere. By contrast, the 0.8-kb ubiquitous promoter plus the first intron of the arp gene were capable of expressing GFP in a variety of tissues, including the skin, muscle, lens, neurons, notochord, and circulating blood cells. Our experiments, therefore, further demonstrated that zebrafish embryos can faithfully express exogenously introduced genes under the control of zebrafish promoters.

Animals↗
PubMed1999

The inhibition of endothelial activation by unsaturated fatty acids.

Dietary long-chain fatty acids (FA) may influence pathological processes involving endothelial activation and leukocyte-endothelial interactions, such as inflammation and atherosclerosis. We previously showed that the n-3 FA docosahexaenoate (22:6n-3, DHA) inhibits cytokine-stimulated expression of endothelial-leukocyte adhesion molecules and soluble cytokines in the range of nutritionally achievable plasma concentrations. More recently we assessed structural determinants of VCAM-1 inhibition by FA. Cultured endothelial cells were incubated first with various saturated, monounsaturated, n-6 or n-3 polyunsaturated FA alone and then together with interleukin-1 or tumor necrosis factor. Saturated FA did not inhibit cytokine-induced endothelial activation, while a progressive increase in inhibitory activity was observed, for the same chain length, with the increase in double bonds accompanying the transition from monounsaturates to n-6 and, further, to n-3 FA. Comparison of various FA indicated no role of the double-bond position or configuration; the greater number of double bonds could explain the greater inhibitory activity of n-3 vs. n-6 FA. In order to ascertain mechanisms for these effects, we demonstrated inhibition of nuclear factor-kappaB (NF-kappaB) activation by DHA in parallel with a reduction in hydrogen peroxide (a critical mediator of NF-kappaB activation) released by endothelial cells either extracellularly or intracellularly. This suggests that a property related to fatty acid peroxidability (the presence of multiple double bonds) is related to inhibitory properties of hydrogen peroxide release and, consequently, of endothelial activation.

Animals↗
PubMed1999

Principles for designing randomized preventive trials in mental health: an emerging developmental epidemiology paradigm.

An emerging population-based paradigm is now being used to guide the design of preventive trials used to test developmental models. We discuss elements of the designs of several ongoing randomized preventive trials involving reduction of risk for children of divorce, for children who exhibit behavioral or learning problems, and for children whose parents are being treated for depression. To test developmental models using this paradigm, we introduce three classes of design issues: design for prerandomization, design for intervention, and design for postintervention. For each of these areas, we present quantitative results from power calculations. Both scientific and cost implications of these power calculations are discussed in terms of variation among subjects on preintervention measures, unit of intervention, assignment, balancing, number of pretest and posttest measures, and the examination of moderation effects.

Bias↗
PubMed1999

A class of neuroD-related basic helix-loop-helix transcription factors expressed in developing central nervous system in zebrafish.

Neuronal basic helix-loop-helix (bHLH) transcription factors such as neuroD and neurogenin have been shown to play important roles in neuronal development. In the present study, several distinct bHLH DNA fragments were isolated from the zebrafish genomic DNA by a pair of degenerate polymerase chain reaction (PCR) primers deduced from the conserved bHLH domains of neuroD and neurogenins. Based on the bHLH fragments, three complete neuroD-related cDNA clones, including complete coding regions, ndr1a, ndr1b, and ndr2 (ndr for neuroD related), were isolated and assembled by 5' and 3' rapid amplification of cDNA ends (RACE). A phylogenetic analysis indicated the presence of four groups of neuroD-related genes in the neuroD subfamily in vertebrates: neuroD, ndr1a/ndr1b/MATH-2, ndr2/NDRF, and neuroM/MATH3. Expression of the newly isolated neuroD-related genes was examined by reverse transcriptase (RT)-PCR and whole-mount in situ hybridization. Unlike neuroD, which was expressed broadly in primary neurons during early zebrafish development starting from 10 h postfertilization (hpf), expression of ndr1a and ndr1b started relatively late (around 22 hpf) and was restricted to the olfactory system: olfactory bulbs in the telecephalon (ndr1a and ndr1b) and olfactory organs (ndr1b) starting around 22 hpf. Although a faint ndr2 mRNA signal was detected by RT-PCR in early embryos, no ndr2 mRNA was detected by whole-mount in situ hybridization in embryos up to 72 hpf, suggesting that it is expressed rather late. Our observations suggest that the two novel neuroD-related genes, ndr1a and ndr1b, are involved in the development of the olfactory system and perhaps contribute to its functional complexity.

Amino Acid Sequence↗
PubMed1999

Fast skeletal muscle-specific expression of a zebrafish myosin light chain 2 gene and characterization of its promoter by direct injection into skeletal muscle.

A zebrafish myosin light chain 2 cDNA clone was isolated and characterized. Sequence analysis of the clone revealed a high homology with the mammalian and avian genes encoding the fast skeletal muscle isoform, MLC2f. In situ hybridization and Northern blot hybridization analyses indicated that the zebrafish MLC2f mRNA is expressed exclusively in the fast skeletal muscle. Ontogenetically, the MLC2f mRNA appears around 16 hours postfertilization (hpf) in the first few well-formed anterior somites. At later stages, the MLC2f mRNA can also be detected in fin buds, eye muscles, and jaw muscles. To develop a useful model system for analyzing muscle gene regulation, the promoter of the zebrafish MLC2f gene was isolated and linked to the chloramphenicol acetyltransferase (CAT) reporter gene. The MLC2f/CAT chimeric constructs were analyzed by direct injection into the zebrafish skeletal muscle, and significant CAT activity was observed; in contrast, little or no CAT activity was generated from a similarly injected prolactin gene promoter/CAT gene construct. Within the 1 kb of the MLC2f promoter region, several MEF2-binding sites and E-boxes were identified, suggesting that MLC2f can be regulated by muscle transcription factors MEF2 and myogenic bHLH proteins. A 5' deletion analysis indicated that the proximal 79 nucleotides from the transcription start site, which contains a single MEF2-binding site, is sufficient to drive a high level of CAT activity in injected muscle. Internal deletion of the MEF2 element in the -79-bp construct caused an 80% decrease in CAT activity, whereas internal deletion of the same MEF2 element in a -1044-bp construct had no effect on induced CAT activity. These observations suggest that an MEF2 element is important to activate the MLC2f gene in muscle cells, and the effect of loss of the proximal MEF2 element can be compensated for by the presence of the upstream MEF2 elements. This study also demonstrated that direct injection of DNA into skeletal muscle is a valid and valuable approach to analyze muscle gene promoters in the zebrafish.

Amino Acid Sequence↗
PubMed1999

Sarcomatoid renal cell carcinoma: biologic behavior, prognosis, and response to combined surgical resection and immunotherapy.

PURPOSE: Sarcomatoid variants of renal cell carcinoma (RCC) are aggressive tumors that respond poorly to immunotherapy. We report the outcomes of 31 patients with sarcomatoid RCC treated with a combination of surgical resection and immunotherapy. PATIENTS AND METHODS: Patients were identified from the database of the University of California Los Angeles Kidney Cancer Program. We retrospectively reviewed the cases of 31 consecutive patients in whom sarcomatoid RCC was diagnosed between 1990 and 1997. Clinical stage, sites of metastasis, pathologic stage, and type of immunotherapy were abstracted from the medical records. The primary end point analyzed was overall survival, and a multivariate analysis was performed to distinguish any factors conferring an improved survivorship. RESULTS: Twenty-six percent of patients were male and 74% were female, and the median age was 59 years (range, 34 to 73 years). Length of follow-up ranged from 2 to 77 months (mean, 21.4 months). Twenty-eight patients (84%) had known metastases at the time of radical nephrectomy (67% had lung metastases and 40% had bone, 21% had liver, 33% had lymphatic, and 15% had brain metastases). Twenty-five patients (81%) received immunotherapy, including low-dose interleukin (IL)-2-based therapy (five patients), tumor-infiltrating lymphocyte-based therapy plus IL-2 (nine patients), high-dose IL-2-based therapy (nine patients), dendritic cell vaccine-based therapy (one patient), and interferon alpha-based therapy alone (one patient). Two patients (6%) achieved complete responses (median duration, 46+ months) and five patients (15%) achieved partial responses (median duration, 36 months). One- and 2-year overall survival rates were 48% and 37%, respectively. Using a multivariate analysis, age, sex, and percentage of sarcomatoid tumor (< or >50%) did not significantly correlate with survival. Improved survival was found in patients receiving high-dose IL-2 therapy compared with patients treated with surgery alone or any other form of immunotherapy (P = .025). Adjusting for age, sex, and percentage of sarcomatoid tumor, the relative risk of death was 10.4 times higher in patients not receiving high-dose IL-2 therapy. Final pathologic T stage did not correlate significantly with outcome, but node-positive patients had a higher death rate per year of follow-up than did the rest of the population (1.26 v 0.76, Cox regression analysis). CONCLUSION: Surgical resection and high-dose IL-2-based immunotherapy may play a role in the treatment of sarcomatoid RCCs in select patients.

Adult↗
PubMed1999

Pervanadate-mediated tyrosine phosphorylation of keratins 8 and 19 via a p38 mitogen-activated protein kinase-dependent pathway.

Glandular epithelia express the keratin intermediate filament (IF) polypeptides 8, 18 and 19 (K8/18/19). These proteins undergo significant serine phosphorylation upon stimulation with growth factors and during mitosis, with subsequent modulation of their organization and interaction with associated proteins. Here we demonstrate reversible and dynamic tyrosine phosphorylation of K8 and K19, but not K18, upon exposure of intact mouse colon or cultured human cells to pervanadate. K8/19 tyrosine phosphorylation was confirmed by metabolic 32PO4-labeling followed by phosphoamino acid analysis, and by immunoblotting with anti-phosphotyrosine antibodies. Pervanadate treatment increases keratin solubility and also indirectly increases K8/18 serine phosphorylation at several known sites, some of which were previously shown to be associated with EGF stimulation, extracellular signal-regulated kinase (ERK), or p38 kinase activation. However, K8/19 tyrosine phosphorylation is independent of EGF signaling or ERK activation while inhibition of p38 kinase activity blocks pervanadate-induced K8/19 tyrosine phosphorylation. Our results demonstrate tyrosine phosphatase inhibitor-mediated in vivo tyrosine phosphorylation of K8/19, but not K18, and suggest that tyrosine phosphorylation may be a general modification of other IF proteins. K8/19 tyrosine phosphorylation involves a pathway that utilizes the p38 mitogen-activated protein kinase, but appears independent of EGF signaling or ERK kinase activation.

Animals↗