Biomedical subjects

Dawei Xu

Publications and source records attributed to Dawei Xu.

At least 19 recordsLinked to original sources

PubMed2006

Human normal T lymphocytes and lymphoid cell lines do express alternative splicing variants of human telomerase reverse transcriptase (hTERT) mRNA.

Alternative splicing of telomerase reverse transcriptase (hTERT) mRNA is known to contribute to regulation of telomerase activity in normal and cancerous cells, however, previous studies indicated that normal human T and B cells exhibited constitutive expression of full-length hTERT mRNA without splicing variants and that activation of telomerase upon stimulation of the cells was due to the shuttling of hTERT protein from cytoplasm to nucleus [Proc. Natl. Acad. Sci. USA 96 (1999) 5147; J. Immunol. 166 (2001) 4826]. We found that typical variants of hTERT mRNA were widespread in human lymphocyte-derived cell lines and normal stimulated T cells. In activated T cells, induction of the full-length hTERT mRNA was coupled with increased hTERT protein expression and telomerase activity. Collectively, human normal and malignant lymphocytes, like other human cells, express splicing variants of hTERT mRNA and require transcriptional activation of the hTERT gene to acquire telomerase activity.

Alternative Splicing↗

PubMed2006

Switch to full-length of XAF1 mRNA expression in prostate cancer cells by the DNA methylation inhibitor.

X-linked inhibitor of apoptosis protein (XIAP) suppresses apoptotic cell death by binding to caspases and inhibiting their functions, while the XIAP-associated factor1 (XAF1), a zinc finger protein, antagonizes XIAP activities, thereby promoting apoptosis. The aberrant silence of the XAF1 gene has recently been found in various types of cancer cells, which is suggested to be one of the potential mechanisms underlying survival advantages of malignant cells. In the present study, we investigated the XAF1 expression in prostate cancer cells. Compared with normal tissues where a full-length of XAF1 mRNA is predominant, LNCaP and DU145 prostate cancer cell lines only expressed a short form of XAF1 transcripts, whereas PC3 cells exhibited a complete silence of the XAF1 gene. Inhibition of DNA methylation led to a switch to the full length of XAF1 mRNA expression in LNCaP and DU145 cells. The down-regulation of XAF1 expression was also observed in 6/8 tumor samples derived from patients with prostate cancer. Our findings suggest that splicing alterations or downregulation of the XAF1 transcript may occur during the development of prostate cancers due to the aberrant DNA methylation. The alternative splicing of XAF1 mRNA leads to formation of a truncated XAF1 protein with 19 amino acid deletion in its zinc finger domain, which likely affects its functional interaction with XIAP, and consequently, contributes to the pathogenesis of prostate cancers by disrupting balance of the apoptosis machinery.

Adaptor Proteins, Signal Transducing↗

PubMed2006

An impedance biosensor array for label-free detection of multiple antigen-antibody reactions.

An electrochemical impedance biosensor array with protein-modified electrodes was designed and fabricated in this report. To demonstrate its feasibility of the detection of multiple antigen-antibody binding reactions based on a label-free approach, human IgG (hIgG), rat IgG (rIgG), human globin and bovine serum albumin were immobilized, respectively, on the gold electrodes and then the resultant array was incubated with goat anti-hIgG, goat anti-rIgG, anti-human globin antibody and the mixture of three antibodies, respectively. The results indicated that the electron transfer resistance of the electrodes was significantly changed due to formation of the antigen-antibody conjugated layer. In addition, experimental conditions such as the protein concentration for the immobilization and screen were studied and optimized. Furthermore, the surface of various protein-modified electrodes was imaged with atomic force microscopy and the height distribution of protein particles was obtained with the Particle Analysis Software. The relative results were fully in accordance with the ones from the electrochemical impedance spectroscopy.

Animals↗

PubMed2006

Mitogen-activated protein kinase cascade-mediated histone H3 phosphorylation is critical for telomerase reverse transcriptase expression/telomerase activation induced by proliferation.

Telomerase activity and telomerase reverse transcriptase (hTERT), the key component of the telomerase complex, are tightly proliferation regulated in normal and malignant cells both in vitro and in vivo; however, underlying mechanisms are unclear. In the present study, we identified mitogen-activated protein kinase (MAPK) cascade-mediated histone H3 ser10 phosphorylation to be a molecular link between proliferation and induction of hTERT/telomerase activity. In normal human T lymphocytes and fibroblasts, growth or stress stimuli known to drive H3 phosphorylation through the MAPK signaling induce hTERT expression and/or telomerase activity that was preceded by phosphorylated histone H3 (ser10) at the hTERT promoter. Blockade of the MAPK-triggered H3 phosphorylation significantly abrogates hTERT induction and ser10 phosphorylation at this promoter. However, H3 ser10 phosphorylation alone resulted in low, transient hTERT induction, as seen in fibroblasts, whereas H3 phosphorylation followed by its acetylation at lys14 robustly trans-activated the hTERT gene accompanying constitutive telomerase activity in normal and malignant T cells. H3 acetylation without phosphorylation similarly exerted weak effects on hTERT expression. These results define H3 phosphorylation as a key to hTERT transactivation induced by proliferation and reveal a fundamental mechanism for telomerase regulation in both normal human cells and transformed T cells.

Acetylation↗

PubMed2005

hTERT gene copy number is not associated with hTERT RNA expression or telomerase activity in colorectal cancer.

In a majority of malignant human tumors telomerase activity can be detected, suggesting an immortal phenotype. Expression of the reverse transcriptase subunit, hTERT, in the human telomerase complex is required for telomerase activity. The regulation of hTERT, from gene level to a fully functional protein, is still a poorly understood process. Increased copy number of the hTERT gene has been demonstrated in a significant portion of established cell lines and tumors of different origin but its relevance for telomerase activity levels is unclear. In the present study, we examined the hTERT gene copy number using fluorescence in situ hybridization (FISH) in samples from 64 colorectal carcinomas and an increased copy number (> or = 3 hTERT gene copies/nucleus) was observed in 31 cases (48%). No statistical association existed between hTERT gene copy number and hTERT RNA expression or telomerase activity. However, a significant relationship was found between an increase in hTERT gene copy number and p53 protein accumulation (p = 0.002) and aneuploidy (p = 0.036). Only 4 tumors showed microsatellite instability, 3 of which had a normal hTERT gene copy number. The data indicated that the increased copy number of the hTERT gene in colorectal carcinoma was a result of genomic instability with no obvious consequence for telomerase activity levels.

Aneuploidy↗

PubMed2005

Label-free electrochemical detection for aptamer-based array electrodes.

An electrochemical impedance spectroscopy method of detection for aptamer-based array electrodes is reported in which the binding of aptamers immobilized on gold electrodes leads to impedance changes associated with target protein binding events. Human IgE was used as a model target protein and incubated with the aptamer-based array consisting of single-stranded DNA containing a hairpin loop. To increase the binding efficiency for proteins, a hybrid modified layer containing aptamers and cysteamine was fabricated on the photolithographic gold surface through molecular self-assembly. Atomic force microscopy analysis demonstrated that human IgE could be specifically captured by the aptamer and stand well above the self-assembled monolayer (SAM) surface. Compared to immunosensing methods using anti-human IgE antibody as the recognition element, impedance spectroscopy detection could provide higher sensitivity and better selectivity for aptamer-modified electrodes. The results of this method show good correlation for human IgE in the range of 2.5-100 nM. A detection limit of 0.1 nM (5 fmol in a 50-microL sample) was obtained, and an average of the relative standard deviation was <10%. The method herein describes the first label-free detection for arrayed electrodes utilizing electrochemical impedance spectroscopy.

Aptamers, Nucleotide↗

PubMed2005

Differential expression of full-length telomerase reverse transcriptase mRNA and telomerase activity between normal and malignant renal tissues.

Activation of telomerase, a key event during immortalization and malignant transformation, requires expression of the telomerase reverse transcriptase (hTERT). Consistently, lack of telomerase activity and hTERT expression occurs in most normal human somatic cells. However, it has been observed that both normal and cancerous renal tissues express hTERT whereas only the latter exhibits telomerase activity. The mechanism underlying the dissociation between hTERT expression and telomerase activity is unclear. In the present study, we examined telomerase activity and alternative splicing of hTERT transcripts in renal cell carcinoma (RCC) specimens and adjacent normal tissues from 33 patients with RCC. Telomerase activity was detectable in 27 of 33 (82%) RCC samples but none in their normal counterparts. Thirty-two of 33 tumors expressed overall hTERT mRNA and 27 of them contained full-length hTERT transcripts, all with telomerase activity. Although 42% (14 of 33) of normal renal samples expressed hTERT mRNA, none of them had full-length hTERT transcripts, coinciding with lack of telomerase activity. The presence of full-length hTERT mRNA and telomerase activity was significantly associated with c-MYC induction. In tumors, absence of full-length hTERT mRNA or telomerase activity defines a subgroup of nonmetastatic, early-stage RCCs. Taken together, telomerase repression in normal renal tissues is attributed to the absence of full-length hTERT transcripts, whereas telomerase activation is achieved via induction of or switch to expression of full-length hTERT mRNA during the oncogenic process of kidneys, and associated with aggressive RCCs.

Adult↗

PubMed2005

Epstein-Barr virus latent membrane 2A (LMP2A) down-regulates telomerase reverse transcriptase (hTERT) in epithelial cell lines.

The latent membrane protein (LMP) 2A, one of the membrane-spanning polypeptides encoded by the Epstein-Barr virus (EBV), has been implicated in the maintenance of viral latency and appears to function in part by inhibiting B-cell receptor (BCR) signaling through its ITAM motifs. It has also been suggested that LMP2A is involved in tumorigenesis mediated by EBV. In our study, we determined regulatory effects of LMP2A on the telomerase reverse transcriptase (hTERT) expression. We observed a significant and constant reduction of hTERT mRNA accompanied by decreased telomerase activity in epithelial cells expressing LMP2A. It was further shown that LMP2A inhibited the hTERT promoter activity in transient transfections of both B cells and epithelial cells, and that the ITAM motif was required for this inhibition. Thus LMP2A expression leads to the transcriptional repression of the hTERT gene through specific pathways, which may thereby contribute to the control of EBV-latency.

Carcinoma↗

PubMed2004

Telomere attrition predominantly occurs in precursor lesions during in vivo carcinogenic process of the uterine cervix.

Although human papillomavirus (HPV) has been defined as the pathogen for cervical carcinomas, molecular events underlying the oncogenic process are unclear. As telomere dysfunction-mediated chromosomal instability and telomerase activation have been suggested as key events in carcinogenesis, we dissected the dynamic changes in telomere length, checkpoint response, and temporal profile of telomerase expression during the evolution from precursor lesions (cervical intraepithelial neoplasia, CINs) to invasive cancers of the uterine cervix in sequential samples from 16 patients. Telomeres were significantly shortened in all CIN samples and no further substantial attritions occurred in most cases with the acquisition of malignant phenotype. Very short telomeres were coupled with constitutive activation of the DNA damage response pathway (Chk2 phosphorylation) and increased cellular proliferation in those cervical specimens. Telomerase reverse transcriptase (hTERT) expression was preferably induced at advanced CINs or invasive cancers. The present finding demonstrates that excessive telomere shortening predominantly occurs in the early carcinogenesis of the uterine cervix largely prior to telomerase activation. Widespread over-erosion of telomeres or telomere dysfunction in very early stages of cervical tumorigenesis might fuel transformation processes by driving chromosomal instability.

DNA Damage↗

PubMed2004

Transcriptional regulation of 15-lipoxygenase expression by promoter methylation.

15-Lipoxygenase type 1 (15-LO), a lipid-peroxidating enzyme implicated in physiological membrane remodeling and the pathogenesis of atherosclerosis, inflammation, and carcinogenesis, is highly regulated and expressed in a tissue- and cell-type-specific fashion. It is known that interleukins (IL) 4 and 13 play important roles in transactivating the 15-LO gene. However, the fact that they only exert such effects on a few types of cells suggests additional mechanism(s) for the profile control of 15-LO expression. In the present study, we demonstrate that hyper- and hypomethylation of CpG islands in the 15-LO promoter region is intimately associated with the transcriptional repression and activation of the 15-LO gene, respectively. The 15-LO promoter was exclusively methylated in all examined cells incapable of expressing 15-LO (certain solid tumor and human lymphoma cell lines and human T lymphocytes) while unmethylated in 15-LO-competent cells (the human airway epithelial cell line A549 and human monocytes) where 15-LO expression is IL4-inducible. Inhibition of DNA methylation in L428 lymphoma cells restores IL4 inducibility to 15-LO expression. Consistent with this, the unmethylated 15-LO promoter reporter construct exhibited threefold higher activity in A549 cells compared to its methylated counterpart. Taken together, demethylation of the 15-LO promoter is a prerequisite for the gene transactivation, which contributes to tissue- and cell-type-specific regulation of 15-LO expression.

Acetylation↗

PubMed2004

Inhibition of histone deacetylases.

Reversible histone acetylation, governed dynamically by histone acetyltransferases (HATs) and histone deacetylases (HDACs), plays a pivotal role in regulation of gene expression through remodeling chromatin structure. Manipulation of the equilibrium between acetylation and deacetylation of histones by specific HDAC inhibitors is thus a useful tool to study functional role(s) for histone hyper-/hypoacetylation in controlling gene transcription and many other cellular activities. By using the trans-activating effect of trichostatin A (TSA), a widely used HDAC inhibitor, on the telomerase reverse transcriptase (hTERT) gene as an example, we summarize various aspects of HDAC inhibitors and provide a general strategy for their in vitro application in studies of gene regulation.

Acetylation↗

PubMed2004

Enhanced sensitivity of prostate cancer DU145 cells to cisplatinum by 5-aza-2'-deoxycytidine.

During the oncopathogenic process aberrant DNA methylation frequently occurs, leading to silencing of sets of genes involved in cell cycle and apoptosis control pathways and other important biological functions. Targeting such a change has been suggested as a novel strategy for cancer prevention and therapy. In the present study, we examined whether suppression of DNA methylation was capable of enhancing sensitivity of prostate cancer DU145 cells to cisplatinum. 5-aza-2'-deoxycytidine (5-aza), a specific DNA methylation inhibitor, when added into DU145 cell culture alone, did not induce significant apoptosis. However, a combination of 5-aza with the chemotherapeutic agent cisplatinum showed great synergy in triggering apoptotic death of DU145 cells. The present finding provides a rationale to evaluate therapeutic effects of the DNA methylation inhibition and chemotherapy in patients with prostate cancer.

Antimetabolites, Antineoplastic↗

PubMed2003

Molecular characterization of human telomerase reverse transcriptase-immortalized human fibroblasts by gene expression profiling: activation of the epiregulin gene.

Reconstitution of telomerase activity by ectopic expression of telomerase reverse transcriptase (hTERT) results in an immortal phenotype in various types of normal human cells, including fibroblasts. Despite lack of transformation characteristics, it is unclear whether hTERT-immortalized cells are physiologically and biochemically the same as their normal counterparts. Here, we compared the gene expression profiles of normal and hTERT-immortalized fibroblasts by using a cDNA microarray containing 20,736 cDNA clones and identified 172 dysregulated genes or expressed sequence tags (ESTs). One of the highly expressed genes in the hTERT-immortalized fibroblasts (hTERT-BJ cells) encodes epiregulin, a potent growth factor. Blockade of epiregulin reduced the growth of hTERT-BJ cells and colony formation of hTERT-transformed fibroblasts. Moreover, inhibition of epiregulin function in immortal hTERT-BJ cells triggered a senescence program. Our results suggest that both activation of telomerase and subsequent induction of epiregulin are required for sustained cell proliferation. Given the significant difference in gene expression profiles between normal and hTERT-immortalized fibroblasts and the close relationship between epiregulin and tumorigenesis, we conclude that hTERT-immortalized cells may not replace their normal counterparts for studies of normal cell biology and that the use of hTERT for expansion of normal human cells for therapeutic purposes must be approached with caution.

Cell Division↗

PubMed2003

Deletion of the telomerase reverse transcriptase gene and haploinsufficiency of telomere maintenance in Cri du chat syndrome.

Cri du chat syndrome (CdCS) results from loss of the distal portion of chromosome 5p, where the telomerase reverse transcriptase (hTERT) gene is localized (5p15.33). hTERT is the rate-limiting component for telomerase activity that is essential for telomere-length maintenance and sustained cell proliferation. Here, we show that a concomitant deletion of the hTERT allele occurs in all 10 patients with CdCS whom we examined. Induction of hTERT mRNA in proliferating lymphocytes derived from five of seven patients was lower than that in unaffected control individuals (P<.05). The patient lymphocytes exhibited shorter telomeres than age-matched unaffected individuals (P<.0001). A reduction in replicative life span and a high rate of chromosome fusions were observed in cultured patient fibroblasts. Reconstitution of telomerase activity by ectopic expression of hTERT extended the telomere length, increased the population doublings, and prevented the end-to-end fusion of chromosomes. We conclude that hTERT is limiting and haploinsufficient for telomere maintenance in humans in vivo. Accordingly, the hTERT deletion may be one genetic element contributing to the phenotypic changes in CdCS.

Adolescent↗

PubMed2003

Different effects of metabolic inhibitors and cyclosporin A on daunorubicin transport in leukemia cells from patients with AML.

The objective of this study was to determine the role of transport proteins in daunorubicin (Dnr) accumulation and efflux in leukemia cells from 36 patients with acute myeloid leukemia (AML). Mononuclear cells were isolated and incubated with 1 microM Dnr with/without addition of 3 microM cyclosporin A (CyA) or metabolic inhibitors (MI). Cellular Dnr concentration in leukemia blast cells was measured with flow cytometry. After washing and reincubation of the cells in drug-free medium, Dnr efflux was followed with/without addition of CyA or MI. Levels of mRNA expression for mdr1, multidrug resistance associated protein (mrp) and lung resistance protein (lrp) were determined with reverse transcriptase-polymerase chain reaction (RT-PCR). MI enhanced cellular Dnr accumulation to a higher extent than CyA whereas CyA reduced Dnr efflux more efficiently than MI (P<0.001). There was a significant difference in Dnr accumulation between samples with low and high mdr1 mRNA levels but only in the presence of MI or CyA. Our results imply that other factors than P-glycoprotein (Pgp) are of major importance for in vitro Dnr accumulation in AML blasts and that the role of Pgp as a drug efflux pump is not conclusive.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2002

A new procedure for preparation of carboxylic acid hydrazides.

The standard method for preparing carboxylic acid hydrazides is hydrazinolysis of esters in alcoholic solutions. However, when applied to alpha,beta-unsaturated esters, the main product typically is the pyrazolidinone resulting from an undesired Michael-type cyclization. Other alternative methodologies reported for direct preparation of hydrazides from acids are inefficient. We developed an efficient and general process, involving preforming activated esters and/or amides followed by reaction with hydrazine, for the preparation of hydrazides including those of alpha,beta-unsaturated acids. This process gives the desired hydrazides in excellent yield and purity under mild conditions.

Journal Article↗

PubMed2002

The histone deacetylase inhibitor trichostatin A derepresses the telomerase reverse transcriptase (hTERT) gene in human cells.

Activation of telomerase, essential for cellular immortalization and transformation, requires the induction of its catalytic component, telomerase reverse transcriptase (hTERT). However, biochemical and genetic mechanisms for the control of hTERT expression remain undefined. In the present study, we demonstrate that the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) induces hyperacetylation of histones at the hTERT proximal promoter, directly transactivates the hTERT gene in normal human telomerase-negative cells, and upregulates hTERT expression in telomerase-positive tumor cells. Overexpression of HDAC1 leads to repression of the hTERT promoter activity. TSA-mediated activation of the hTERT promoter is abolished by the mutation of Sp1 sites at the proximal promoter, suggesting that the effect of TSA is regulated through Sp1 motifs. We also show a physical interaction of Sp1 with HDAC1 and the presence of HDAC1 at the hTERT promoter region. Moreover, hyperacetylation of histones at the hTERT promoter is associated with the natural up-regulation of hTERT expression that occurs in activated T lymphocytes. Taken together, histone acetylation/deacetylation may be a common underlying feature to hTERT transactivation/repression in human normal and malignant cells.

Acetylation↗

PubMed2002

Amplification of the telomerase reverse transcriptase (hTERT) gene in cervical carcinomas.

The expression of telomerase reverse transcriptase (hTERT), the catalytic component of the telomerase complex, is required for activation of telomerase during immortalization and transformation of human cells. However, the biochemical and genetic mechanisms governing hTERT expression remain to be elucidated. In the present study, we examined hTERT amplification as a potential genetic event contributing to telomerase activation in cervical carcinomas. An amplification of the hTERT gene was found in 1/4 cervical cancer cell lines and 21/88 primary tumor samples derived from the patients with cervical carcinomas. An increase in the hTERT copy number was significantly correlated with higher levels of hTERT protein expression. Moreover, the hTERT alterations with the enhanced hTERT expression were exclusively observed in those tumors with high-risk human papillomavirus infection. Taken together, the hTERT gene amplification, directly or indirectly targeted by human papillomavirus, may be one of the driving forces responsible for upregulation of hTERT expression and activation of telomerase in cervical cancers.

Carcinoma↗