PubMed Health⌕ Search

Biomedical subjects

Tim H-M Huang

Publications and source records attributed to Tim H-M Huang.

At least 19 recordsLinked to original sources
PubMed2006

Diverse gene expression and DNA methylation profiles correlate with differential adaptation of breast cancer cells to the antiestrogens tamoxifen and fulvestrant.

The development of targeted therapies for antiestrogen-resistant breast cancer requires a detailed understanding of its molecular characteristics. To further elucidate the molecular events underlying acquired resistance to the antiestrogens tamoxifen and fulvestrant, we established drug-resistant sublines from a single colony of hormone-dependent breast cancer MCF7 cells. These model systems allowed us to examine the cellular and molecular changes induced by antiestrogens in the context of a uniform clonal background. Global changes in both basal and estrogen-induced gene expression profiles were determined in hormone-sensitive and hormonal-resistant sublines using Affymetrix Human Genome U133 Plus 2.0 Arrays. Changes in DNA methylation were assessed by differential methylation hybridization, a high-throughput promoter CpG island microarray analysis. By comparative studies, we found distinct gene expression and promoter DNA methylation profiles associated with acquired resistance to fulvestrant versus tamoxifen. Fulvestrant resistance was characterized by pronounced up-regulation of multiple growth-stimulatory pathways, resulting in estrogen receptor alpha (ERalpha)-independent, autocrine-regulated proliferation. Conversely, acquired resistance to tamoxifen correlated with maintenance of the ERalpha-positive phenotype, although receptor-mediated gene regulation was altered. Activation of growth-promoting genes, due to promoter hypomethylation, was more frequently observed in antiestrogen-resistant cells compared with gene inactivation by promoter hypermethylation, revealing an unexpected insight into the molecular changes associated with endocrine resistance. In summary, this study provides an in-depth understanding of the molecular changes specific to acquired resistance to clinically important antiestrogens. Such knowledge of resistance-associated mechanisms could allow for identification of therapy targets and strategies for resensitization to these well-established antihormonal agents.

Breast Neoplasms↗
PubMed2006

Mapping geographic zones of cancer risk with epigenetic biomarkers in normal breast tissue.

PURPOSE: Genetic alterations were previously identified in normal epithelia adjacent to invasive cancers. The aim of this study was to determine DNA methylation in histologically normal tissues from multiple geographic zones adjacent to primary breast tumors. EXPERIMENTAL DESIGN: First, methylation status of a 4-kb region of RASSF1A promoter was interrogated using oligonucleotide-based microarray in 144 samples (primary tumors, 47; adjacent normals, 69; reduction mammoplasty tissues, 28). Second, allelic imbalance (AI)/loss of heterozygosity (LOH) surrounding RASSF1A promoter were analyzed in 30 samples (tumors, 8; adjacent normals, 22). Third, global methylation screening of 49 samples (tumors, 12; adjacent normals, 25; reduction mammoplasty, 12) was done by differential methylation hybridization. Real-time quantitative methylation-specific PCR was used to validate the microarray findings. RESULTS: DNA methylation in the core RASSF1A promoter was low in reduction mammoplasty tissues (P=0.0001) when compared with primary tumors. The adjacent normals had an intermediate level of methylation. The regions surrounding the core were highly methylated in all sample types. Microsatellite markers showed AI/LOH in tumors and some of the adjacent normals. Concurrent AI/LOH and DNA methylation in RASSF1A promoter occurred in two of six tumors. Global methylation screening uncovered genes more methylated in adjacent normals than in reduction mammoplasty tissues. The methylation status of four genes was confirmed by quantitative methylation-specific PCR. CONCLUSIONS: Our findings suggest a field of methylation changes extending as far as 4 cm from primary tumors. These frequent alterations may explain why normal tissues are at risk for local recurrence and are useful in disease prognostication.

Allelic Imbalance↗
PubMed2006

ChIP-chip comes of age for genome-wide functional analysis.

In the post-genome era, attention has focused on the functions of genome sequences and how they are regulated. The emerging epigenomic changes and the interactions between cis-acting elements and protein factors may play a central role in gene regulation. To understand the crosstalk between DNA and protein on a genome-wide scale, one emerging technique, called ChIP-chip, takes the strategy of combining chromatin immunoprecipitation with microarray. This new high-throughput strategy helps screen the targets of critical transcription factors and profile the genome-wide distribution of histone modifications, which will enable the feasibility of conducting a large-scale study, such as the Human Epigenome Project.

Animals↗
PubMed2006

A mixture model-based discriminate analysis for identifying ordered transcription factor binding site pairs in gene promoters directly regulated by estrogen receptor-alpha.

MOTIVATION: To detect and select patterns of transcription factor binding sites (TFBSs) which distinguish genes directly regulated by estrogen receptor-alpha (ERalpha), we developed an innovative mixture model-based discriminate analysis for identifying ordered TFBS pairs. RESULTS: Biologically, our proposed new algorithm clearly suggests that TFBSs are not randomly distributed within ERalpha target promoters (P-value < 0.001). The up-regulated targets significantly (P-value < 0.01) possess TFBS pairs, (DBP, MYC), (DBP, MYC/MAX heterodimer), (DBP, USF2) and (DBP, MYOGENIN); and down-regulated ERalpha target genes significantly (P-value < 0.01) possess TFBS pairs, such as (DBP, c-ETS1-68), (DBP, USF2) and (DBP, MYOGENIN). Statistically, our proposed mixture model-based discriminate analysis can simultaneously perform TFBS pattern recognition, TFBS pattern selection, and target class prediction; such integrative power cannot be achieved by current methods. AVAILABILITY: The software is available on request from the authors. CONTACT: lali@iupui.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Algorithms↗
PubMed2006

Prognostic DNA methylation biomarkers in ovarian cancer.

PURPOSE: Aberrant DNA methylation, now recognized as a contributing factor to neoplasia, often shows definitive gene/sequence preferences unique to specific cancer types. Correspondingly, distinct combinations of methylated loci can function as biomarkers for numerous clinical correlates of ovarian and other cancers. EXPERIMENTAL DESIGN: We used a microarray approach to identify methylated loci prognostic for reduced progression-free survival (PFS) in advanced ovarian cancer patients. Two data set classification algorithms, Significance Analysis of Microarray and Prediction Analysis of Microarray, successfully identified 220 candidate PFS-discriminatory methylated loci. Of those, 112 were found capable of predicting PFS with 95% accuracy, by Prediction Analysis of Microarray, using an independent set of 40 advanced ovarian tumors (from 20 short-PFS and 20 long-PFS patients, respectively). Additionally, we showed the use of these predictive loci using two bioinformatics machine-learning algorithms, Support Vector Machine and Multilayer Perceptron. CONCLUSION: In this report, we show that highly prognostic DNA methylation biomarkers can be successfully identified and characterized, using previously unused, rigorous classifying algorithms. Such ovarian cancer biomarkers represent a promising approach for the assessment and management of this devastating disease.

Adenocarcinoma↗
PubMed2006

T-bet binding to newly identified target gene promoters is cell type-independent but results in variable context-dependent functional effects.

Recently developed target gene identification strategies based upon the chromatin immunoprecipitation assay provide a powerful method to determine the localization of transcription factor binding within mammalian genomes. However, in many cases, it is unclear if the binding capacity of a transcription factor correlates with an obligate role in gene regulation in diverse contexts. It is therefore important to carefully examine the relationship between transcription factor binding and its ability to functionally regulate gene expression. T-bet is a T-box transcription factor expressed in several hematopoietic cell types. By utilizing a chromatin immunoprecipitation assay coupled to genomic microarray technology approach, we identified numerous promoters, including CXCR3, IL2Rbeta, and CCL3, that are bound by T-bet in B cells. Most surprisingly, the ability of T-bet to associate with the target promoters is not dependent upon the cell type background. Several of the promoters appear to be functionally regulated by T-bet. However, we could not detect a functional consequence for T-bet association with many of the identified promoters in overexpression studies or an examination of wild type and T-bet-/- primary B, CD4+, and CD8+ T cells. Thus, there is a high variability in the functional consequences, if any, that result from the association of T-bet with individual target promoters.

Animals↗
PubMed2006

Combinatorial analysis of transcription factor partners reveals recruitment of c-MYC to estrogen receptor-alpha responsive promoters.

In breast cancer and normal estrogen target tissues, estrogen receptor-alpha (ERalpha) signaling results in the establishment of spatiotemporal patterns of gene expression. Whereas primary target gene regulation by ERalpha involves recruitment of coregulatory proteins, coactivators, or corepressors, activation of these downstream promoters by receptor signaling may also involve partnership of ERalpha with other transcription factors. By using an integrated, genome-wide approach that involves ChIP-chip and computational modeling, we uncovered 13 ERalpha-responsive promoters containing both ERalpha and c-MYC binding elements located within close proximity (13-214 bp) to each other. Estrogen stimulation enhanced the c-MYC-ERalpha interaction and facilitated the association of ERalpha, c-MYC, and the coactivator TRRAP with these estrogen-responsive promoters, resulting in chromatin remodeling and increased transcription. These results suggest that ERalpha and c-MYC physically interact to stabilize the ERalpha-coactivator complex, thereby permitting other signal transduction pathways to fine-tune estrogen-mediated signaling networks.

Animals↗
PubMed2006

MPromDb: an integrated resource for annotation and visualization of mammalian gene promoters and ChIP-chip experimental data.

We have developed Mammalian Promoter Database (MPromDb), a novel database that integrates gene promoters with experimentally supported annotation of transcription start sites, cis-regulatory elements, CpG islands and chromatin immunoprecipitation microarray (ChIP-chip) experimental results with intuitively designed presentation. Release 1.0 of MPromDb currently contains 36,407 promoters and first exons (19,170 from human, 15,953 from mouse and 1284 from rat), 3739 transcription factor (TF)-binding sites (2027 from human, 1181 mouse and 531 rat) and 224 TFs with links to PubMed and GenBank references. Target promoters of TFs that have been identified by ChIP-chip assay are integrated into the database. MPromDb serves as a portal for genome-wide promoter analysis of data generated by ChIP-chip experimental studies. MPromDb can be accessed from http://bioinformatics.med.ohio-state.edu/MPromDb.

Animals↗
PubMed2005

Hypermethylation of 18S and 28S ribosomal DNAs predicts progression-free survival in patients with ovarian cancer.

PURPOSE: Repetitive ribosomal DNA (rDNA) genes are GC-rich clusters in the human genome. The aim of the study was to determine the methylation status of two rDNA subunits, the 18S and 28S genes, in ovarian tumors and to correlate methylation levels with clinicopathologic features in a cohort of ovarian cancer patients. EXPERIMENTAL DESIGN: 18S and 28S rDNA methylation was examined by quantitative methylation-specific PCR in 74 late-stage ovarian cancers, 9 histologically uninvolved, and 11 normal ovarian surface epithelial samples. In addition, methylation and gene expression levels of 18S and 28S rDNAs in two ovarian cancer cell lines were examined by reverse transcription-PCR before and after treatment with the demethylating drug 5'-aza-2'-deoxycytidine. RESULTS: The methylation level (amount of methylated rDNA/beta-actin) of 18S and 28S rDNAs was significantly higher (P < 0.05) in tumors than in normal ovarian surface epithelial samples. Methylation of 18S and 28S rDNA was highly correlated (R2= 0.842). Multivariate analysis by Cox regression found that rDNA hypermethylation [hazard ratio (HR), 0.25; P < 0.01], but not age (HR, 1.29; P = 0.291) and stage (HR, 1.09; P = 0.709), was independently associated with longer progression-free survival. In ovarian cancer cell lines, methylation levels of rDNA correlated with gene down-regulation and 5'-aza-2'-deoxycytidine treatment resulted in a moderate increase in 18S and 28S rDNA gene expressions. CONCLUSION: This is the first report of rDNA hypermethylation in ovarian tumors. Furthermore, rDNA methylation levels were higher in patients with long progression-free survival versus patients with short survival. Thus, rDNA methylation as a prognostic marker in ovarian cancer warrants further investigation.

Adult↗
PubMed2005

CpG Island microarray probe sequences derived from a physical library are representative of CpG Islands annotated on the human genome.

An effective tool for the global analysis of both DNA methylation status and protein-chromatin interactions is a microarray constructed with sequences containing regulatory elements. One type of array suited for this purpose takes advantage of the strong association between CpG Islands (CGIs) and gene regulatory regions. We have obtained 20,736 clones from a CGI Library and used these to construct CGI arrays. The utility of this library requires proper annotation and assessment of the clones, including CpG content, genomic origin and proximity to neighboring genes. Alignment of clone sequences to the human genome (UCSC hg17) identified 9595 distinct genomic loci; 64% were defined by a single clone while the remaining 36% were represented by multiple, redundant clones. Approximately 68% of the loci were located near a transcription start site. The distribution of these loci covered all 23 chromosomes, with 63% overlapping a bioinformatically identified CGI. The high representation of genomic CGI in this rich collection of clones supports the utilization of microarrays produced with this library for the study of global epigenetic mechanisms and protein-chromatin interactions. A browsable database is available on-line to facilitate exploration of the CGIs in this library and their association with annotated genes or promoter elements.

Base Sequence↗
PubMed2005

New anti-cancer strategies: epigenetic therapies and biomarkers.

Epigenetics is the study of chromatin modifications that affect gene expression without altering DNA nucleotide sequences. This review highlights a prominent role for epigenetic therapies, particularly those that reverse aberrant DNA methylation and histone acetylation, in the potential treatment of cancer. Administration of such therapies to reverse epigenetic "silencing" of tumor suppressors, including genes involved in chemotherapy responses, could prove useful in the management of cancer patients. In this review, we summarize recent advances in the use of methyltransferase and histone deacetylase inhibitors and possible synergistic combinations of these to achieve maximal tumor suppressor gene re-expression. Moreover, when used in combination with conventional chemotherapeutic agents, epigenetic-based therapies may provide a means to resensitize drug-resistant tumors to established treatments. As specific, aberrant epigenetic modifications are frequently associated with distinct cancer types, and likely occur early in tumorigenesis, these have potential utility as biomarkers. Finally, future directions are addressed, including alternative epigenetic targets, gene-specific modifications, and the use of bioinformatics.

Acetylation↗
PubMed2005

Chromatin immunoprecipitation (ChIP) on chip experiments uncover a widespread distribution of NF-Y binding CCAAT sites outside of core promoters.

The CCAAT box is a prototypical promoter element, almost invariably found between -60 and -100 upstream of the major transcription start site. It is bound and activated by the histone fold trimer NF-Y. We performed chromatin immunoprecipitation (ChIP) on chip experiments on two different CpG islands arrays using chromatin from hepatic HepG2 and pre-B cell leukemia NALM-6 cell lines, with different protocols of probe preparation and labeling. We analyzed and classified 239 known or predicted targets; we validated several by conventional ChIPs with anti-YB and anti-YC antibodies, in vitro EMSAs, and ChIP scanning. The importance of NF-Y binding for gene expression was verified by the use of a dominant negative NF-YA mutant. All but four genes are new NF-Y targets, falling into different functional categories. This analysis reinforces the notion that NF-Y is an important regulator of cell growth, and novel unexpected findings emerged from this unbiased approach. (i) A remarkable proportion of NF-Y targets, 40%, are complex transcriptional units composed of divergent, convergent, and tandem promoters. (ii) 40-50% of NF-Y sites are not in core promoters but are in introns or at distant 3' or 5' locations. The abundance of "unorthodox" CCAAT positions highlights an unexpected complexity of the NF-Y-mediated transcriptional network.

Amino Acid Sequence↗
PubMed2005

Tilling the chromatin landscape: emerging methods for the discovery and profiling of protein-DNA interactions.

Interactions between protein and DNA are essential for cellular function. The incremental process of developing global approaches to study chromatin began with the in vitro characterization of chromatin structural components and modifications of the versatile chromatin immunoprecipitation (ChIP) assay, capable of analyzing protein-DNA interactions in vivo. Among the emerging global approaches are ChIP cloning, ChIP display, differential chromatin scanning, ChIP-chip, DamID chromatin profiling, and chromatin array. These methods have been used to assess transcription-factor binding and (or) histone modification. This review describes these global methods and illustrates their potential in answering biological questions.

Acetylation↗
PubMed2005

Antimitogenic and chemosensitizing effects of the methylation inhibitor zebularine in ovarian cancer.

Deoxycytosine methylation within CpG islands of tumor suppressor genes plays a prominent role in the development and progression of drug-resistant ovarian cancer. Consequently, epigenetic therapies directed toward tumor suppressor demethylation/reexpression could potentially reverse malignant phenotypes and chemosensitize recalcitrant tumors. In this report, we examined the demethylating agent zebularine [1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one], in comparison with the well-known methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC), for its ability to inhibit ovarian cancer cell proliferation and to demethylate and induce tumor suppressor genes. Zebularine exerted significant (>5-aza-dC) antiproliferative effects against the ovarian cancer cell lines Hey, A2780, and the cisplatin-resistant A2780/CP in a dose-dependent manner (65% versus 35% inhibition at 48 hours, zebularine versus 5-aza-dC). Moreover, 48-hour treatment with 0.2 mmol/L zebularine significantly induced demethylation of the tumor suppressors ras-associated domain family 1A and human MutL homologue-1. RASSF1A gene reexpression was also observed, as was reexpression of two other tumor suppressors, ARHI and BLU, although levels differed from those induced by 5-aza-dC. Global analyses of DNA methylation revealed similar overall demethylation (2.5- to 3-fold) by 5-aza-dC and zebularine as determined by methyl acceptance assay. However, differences in demethylation of individual loci were observed as determined by differential methylation hybridization. Finally, we found that zebularine could resensitize the drug-resistant cell line A2780/CP to cisplatin, with a 16-fold reduction in the IC50 of that conventional agent. In summary, zebularine seems to be a promising clinical candidate, singly or combined with conventional regimens, for the therapy of drug-resistant ovarian cancer.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2005

Differential DNA methylation of gene promoters in small B-cell lymphomas.

Improved care of patients with small B-cell lymphomas (SBCLs) is likely to result from the ongoing discovery of molecular markers that better define these malignant neoplasms. We identified multiple gene loci whose DNA methylation patterns differed between 3 types of SBCL: B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma, mantle cell lymphoma, and grades I and II follicular lymphoma. This analysis was performed using an oligonucleotide microarray that allowed determination of the DNA methylation status of 156 loci in 38 genes. Combined bisulfite restriction analysis and methylation-specific polymerase chain reaction were used to validate the differential methylation of 6 of these genes. By using non-Hodgkin lymphoma cell lines as models, these genes were examined further for methylation and gene expression relationships. This study illustrates nonrandom epigenetic alterations in SBCLs that seem to preferentially involve lymphomas of germinal center derivation.

DNA Methylation↗
PubMed2004

Identifying estrogen receptor alpha target genes using integrated computational genomics and chromatin immunoprecipitation microarray.

The estrogen receptor alpha (ERalpha) regulates gene expression by either direct binding to estrogen response elements or indirect tethering to other transcription factors on promoter targets. To identify these promoter sequences, we conducted a genome-wide screening with a novel microarray technique called ChIP-on-chip. A set of 70 candidate ERalpha loci were identified and the corresponding promoter sequences were analyzed by statistical pattern recognition and comparative genomics approaches. We found mouse counterparts for 63 of these loci and classified 42 (67%) as direct ERalpha targets using classification and regression tree (CART) statistical model, which involves position weight matrix and human-mouse sequence similarity scores as model parameters. The remaining genes were considered to be indirect targets. To validate this computational prediction, we conducted an additional ChIP-on-chip assay that identified acetylated chromatin components in active ERalpha promoters. Of the 27 loci upregulated in an ERalpha-positive breast cancer cell line, 20 having mouse counterparts were correctly predicted by CART. This integrated approach, therefore, sets a paradigm in which the iterative process of model refinement and experimental verification will continue until an accurate prediction of promoter target sequences is derived.

Animals↗
PubMed2004

Loss of estrogen receptor signaling triggers epigenetic silencing of downstream targets in breast cancer.

Alterations in histones, chromatin-related proteins, and DNA methylation contribute to transcriptional silencing in cancer, but the sequence of these molecular events is not well understood. Here we demonstrate that on disruption of estrogen receptor (ER) alpha signaling by small interfering RNA, polycomb repressors and histone deacetylases are recruited to initiate stable repression of the progesterone receptor (PR) gene, a known ERalpha target, in breast cancer cells. The event is accompanied by acquired DNA methylation of the PR promoter, leaving a stable mark that can be inherited by cancer cell progeny. Reestablishing ERalpha signaling alone was not sufficient to reactivate the PR gene; reactivation of the PR gene also requires DNA demethylation. Methylation microarray analysis further showed that progressive DNA methylation occurs in multiple ERalpha targets in breast cancer genomes. The results imply, for the first time, the significance of epigenetic regulation on ERalpha target genes, providing new direction for research in this classical signaling pathway.

Base Sequence↗