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Biomedical subjects

Christoph Plass

Publications and source records attributed to Christoph Plass.

At least 19 recordsLinked to original sources
PubMed2006

A novel mutation in the tyrosine kinase domain of ERBB2 in hepatocellular carcinoma.

BACKGROUND: Several studies showed that gain-of-function somatic mutations affecting the catalytic domain of EGFR in non-small cell lung carcinomas were associated with response to gefitinib and erlotinib, both EGFR-tyrosine kinase inhibitors. In addition, 4% of non-small cell lung carcinomas were shown to have ERBB2 mutations in the kinase domain. In our study, we sought to determine if similar respective gain-of-function EGFR and ERBB2 mutations were present in hepatoma and/or biliary cancers. METHODS: We extracted genomic DNA from 40 hepatoma (18) and biliary cancers (22) samples, and 44 adenocarcinomas of the lung, this latter as a positive control for mutation detection. We subjected those samples to PCR-based semi-automated double stranded nucleotide sequencing targeting exons 18-21 of EGFR and ERBB2. All samples were tested against matched normal DNA. RESULTS: We found 11% of hepatoma, but no biliary cancers, harbored a novel ERBB2 H878Y mutation in the activating domain. CONCLUSION: These newly described mutations may play a role in predicting response to EGFR-targeted therapy in hepatoma and their role should be explored in prospective studies.

Carcinoma, Hepatocellular↗
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

Epigenetic modulation of tumor suppressor CCAAT/enhancer binding protein alpha activity in lung cancer.

BACKGROUND: Loss of tumor suppressor CCAAT/enhancer-binding protein-alpha (C/EBPalpha) expression is seen in several human malignancies, including acute myelogenous leukemia and lung cancer. We hypothesized that DNA methylation and histone acetylation of the C/EBPalpha promoter may modulate C/EBPalpha expression in lung cancer. METHODS: We analyzed C/EBPalpha expression in 15 human lung cancer cell lines and in 122 human lung primary tumors by northern blotting, immunoblotting, and immunohistochemistry. C/EBPalpha promoter methylation was assessed using bisulfite sequencing, combined bisulfite restriction analysis, methylation-specific polymerase chain reaction, and Southern blotting. We examined the acetylation status of histones H3 and H4 at the C/EBPalpha promoter by chromatin immunoprecipitation. Binding of methyl-CpG-binding proteins MeCP2 and MBD2 and upstream stimulatory factor (USF) to the C/EBPalpha promoter was assayed in cell lines that were untreated or treated with histone deacetylase inhibitor trichostatin A and demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) by chromatin immunoprecipitation and by electrophoretic mobility shift assays. RESULTS: DNA methylation and histone acetylation in the upstream region (-1422 to -896) of the C/EBPalpha promoter were associated with low or absent C/EBPalpha expression in 12 of 15 lung cancer cell lines and in 81 of 120 primary lung tumors. MeCP2 and MBD binding to the upstream C/EBPalpha promoter was detected in C/EBPalpha-nonexpressing cell lines; USF binding was detected in C/EBPalpha-expressing cell lines; however, in C/EBPalpha-nonexpressing cell lines USF binding was detected only after trichostatin A and 5-aza-dC treatment. CONCLUSIONS: DNA hypermethylation of the upstream C/EBPalpha promoter region, not the core promoter region as previously reported, is critical in the regulation of C/EBPalpha expression in human lung cancer.

Acetylation↗
PubMed2006

Contour area filtering of two-dimensional electrophoresis images.

We describe an algorithm, Contour Area Filtering, for separating background from foreground in gray scale images. The algorithm is based on the area contained within gray scale contour lines. It can be viewed as a form of local thresholding, or as a seed growing algorithm, or as a type of watershed segmentation. The most important feature of the algorithm is that it uses object area to determine the segmentation. Thus, it is relatively impervious to brightness and contrast variations across an image or between different images. Contour Area Filtering was designed specifically for image analysis of 2D electrophoresis gels, although it can be applied to other gray scale images. A typical gel image is an electrophoretogram or a phosphor image of 1000-2500 spots representing protein or DNA restriction fragments. The images are quantitative with spot intensities reflective of the number of proteins or the DNA fragment copy number. The background intensity can vary widely across the image caused both by variation in spot density and by the physical laboratory process of creating a gel. Analyzing and comparing gel images entails extracting and segmenting spots, registering images and matching spots, and measuring differences between spots. We present experimental results which show that Contour Area Filtering is a quick, efficient method for separating electrophoresis gel background from foreground with extremely high accuracy.

Algorithms↗
PubMed2006

DNA motifs associated with aberrant CpG island methylation.

Epigenetic silencing involving the aberrant methylation of promoter region CpG islands is widely recognized as a tumor suppressor silencing mechanism in cancer. However, the molecular pathways underlying aberrant DNA methylation remain elusive. Recently we showed that, on a genome-wide level, CpG island loci differ in their intrinsic susceptibility to aberrant methylation and that this susceptibility can be predicted based on underlying sequence context. These data suggest that there are sequence/structural features that contribute to the protection from or susceptibility to aberrant methylation. Here we use motif elicitation coupled with classification techniques to identify DNA sequence motifs that selectively define methylation-prone or methylation-resistant CpG islands. Motifs common to 28 methylation-prone or 47 methylation-resistant CpG island-containing genomic fragments were determined using the MEME and MAST algorithms (). The five most discriminatory motifs derived from methylation-prone sequences were found to be associated with CpG islands in general and were nonrandomly distributed throughout the genome. In contrast, the eight most discriminatory motifs derived from the methylation-resistant CpG islands were randomly distributed throughout the genome. Interestingly, this latter group tended to associate with Alu and other repetitive sequences. Used together, the frequency of occurrence of these motifs successfully discriminated methylation-prone and methylation-resistant CpG island groups with an accuracy of 87% after 10-fold cross-validation. The motifs identified here are candidate methylation-targeting or methylation-protection DNA sequences.

Algorithms↗
PubMed2006

Accurate quantification of DNA methylation using combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform.

DNA methylation is the best-studied epigenetic modification and describes the conversion of cytosine to 5-methylcytosine. The importance of this phenomenon is that aberrant promoter hypermethylation is a common occurrence in cancer and is frequently associated with gene silencing. Various techniques are currently available for the analysis of DNA methylation. However, accurate and reproducible quantification of DNA methylation remains challenging. In this report, we describe Bio-COBRA (combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform), as a novel approach to quantitative DNA methylation analysis. The combination of a well-established method, COBRA, which interrogates DNA methylation via the restriction enzyme analysis of PCR-amplified bisulfite treated DNAs, with the Bioanalyzer platform allows for the rapid and quantitative assessment of DNA methylation patterns in large sample sets. The sensitivity and reproducibility of Bio-COBRA make it a valuable tool for the analysis of DNA methylation in clinical samples, which could aid in the development of diagnostic and prognostic parameters with respect to disease detection and management.

Azacitidine↗
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

Quantitative assessment of DNA methylation: Potential applications for disease diagnosis, classification, and prognosis in clinical settings.

Deregulation of the epigenome is now recognized as a major mechanism involved in the development and progression of human diseases such as cancer. As opposed to the irreversible nature of genetic events, which introduce changes in the primary DNA sequence, epigenetic modifications are reversible and leave the original DNA sequence intact. There is now evidence that the epigenetic landscape in humans undergoes modifications as the result of normal aging, with older individuals exhibiting higher levels of promoter hypermethylation compared to younger ones. Thus, it has been proposed that the higher incidence of certain disease in older individuals might be, in part, a consequence of an inherent change in the control and regulation of the epigenome. These observations are of remarkable clinical significance since the aberrant epigenetic changes characteristic of disease provide a unique platform for the development of new therapeutic approaches. In this review, we address the significance of DNA methylation changes that result or lead to disease, occur with aging, or may be the result of environmental exposure. We provide a detailed description of quantitative techniques currently available for the detection and analysis of DNA methylation and provide a comprehensive framework that may allow for the incorporation of protocols which include DNA methylation as a tool for disease diagnosis and classification, which could lead to the tailoring of therapeutic approaches designed to individual patient needs.

Chromatography, High Pressure Liquid↗
PubMed2006

CpG island methylation and expression of the secreted frizzled-related protein gene family in chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (CLL) is characterized by a clonal accumulation of mature neoplastic B cells indicating disruption of apoptosis. Restriction Landmark Genome Scanning was done to identify novel target genes silenced by CpG island methylation in CLL. Secreted frizzled-related protein 4 (SFRP4), a negative regulator of the Wnt signaling pathway, was found to be frequently methylated in CLL samples. Wnt signaling has been shown to control normal apoptotic behavior and is required for normal B-cell development whereas aberrant activation of this pathway has been observed in CLL. We show aberrant DNA methylation and silencing of SFRP4, as well as of additional SFRP family members, in primary CLL samples. Induction of their expression in a dose-dependent manner following treatment with a demethylating agent, 5-aza-2'-deoxycytidine, was shown. Of the five SFRP family members studied in detail, SFRP1 was hypermethylated and down-regulated in all CLL patient samples studied, suggesting that this epigenetic event is a critical step during leukemogenesis. Our results suggest that silencing of SFRPs by CpG island methylation is one possible mechanism contributing to aberrant activation of Wnt signaling pathway in CLL.

Azacitidine↗
PubMed2006

Epigenetic regulation of the tumor suppressor gene TCF21 on 6q23-q24 in lung and head and neck cancer.

The identification of tumor suppressor genes has classically depended on their localization within recurrent regions of loss of heterozygosity. According to Knudson's two-hit hypothesis, the remaining allele is lost, either genetically or, more recently identified, through epigenetic events. To date, retrospective analyses have determined promoter methylation as a common alternative alteration in cancer cells to silence cancer-related genes. Here we report an application of restriction landmark genomic scanning that allows for DNA methylation profiling along a region of recurrent loss of heterozygosity at chromosome 6q23-q24. This approach resulted in the identification of a tumor suppressor gene, TCF21, which is frequently lost in human malignancies. We demonstrate that TCF21 is expressed in normal lung airway epithelial cells and aberrantly methylated and silenced in the majority of head and neck squamous cell carcinomas and non-small-cell lung cancers analyzed. TCF21 is known to regulate mesenchymal cell transition into epithelial cells, a property that has been shown to be deficient in carcinomas. We further demonstrate that exogenous expression of TCF21 in cells that have silenced the endogenous TCF21 locus resulted in a reduction of tumor properties in vitro and in vivo.

Animals↗
PubMed2006

20q11.1 amplification in giant-cell tumor of bone: Array CGH, FISH, and association with outcome.

The goal of this study was to identify recurrent regions of genomic gain or loss in giant-cell tumor of bone (GCTb). Array comparative genomic hybridization (aCGH) was performed for 20 frozen tumor samples of GCTb. A separate subset of 59 GCTb with outcome data was used for validation. The most frequent region of change identified by aCGH was gain of a 1-Mbp region at 20q11.1. In the validation arm of 59 cases the minimal common region of copy number gain at 20q11.1, seen in 54% of the samples, was BAC clone RP11-4O9, which contained the genes TPX2 and BCL2L1. For most cases, amplification was restricted to the mononuclear component and was not present in the multinucleated giant cells. Southern blot for TPX2 and BCL2L1 identified the former as the gene with the highest level of amplification for these two proposed candidate genes of importance. Immunohistochemistry for TPX2 expression correlated with amplification, while BCL2L1 expression was not identified. Kaplan-Meier curves for progression-free survival showed a statistically significant difference for cases with 20q11.1 amplification (P = 0.0001). Univariate analysis involving Cox proportional hazards models did not show a significant difference for initial treatment type (curettage versus resection) (P = 0.575), age (</=50 vs. >50) (P = 0.543), or sex (P = 0.268), but did correlate with 20q11.1 amplification (P = 0.001). By multivariate analysis, it was found that 20q11.1 amplification (P = 0.001) was the only factor to reach statistical significance. 20q11.1 amplification can be used as a marker of prognostic importance in GCTb. We propose TPX2 as a candidate oncogene in the core-amplified region at 20q11.1.

Bone Neoplasms↗
PubMed2006

Epigenetics in chronic lymphocytic leukemia.

Enormous evidence has accumulated in the past decades that establishes the importance of epigenetic modifications in cancer and has resulted in shifting the focus from entirely genetic-based studies to integrated studies involving both genetic and epigenetic alterations. Chronic lymphocytic leukemia (CLL) is one such example where studies involving epigenetic aberrations have accelerated the search for affected genes, which was initially restricted to commonly deleted chromosomal regions. Many novel genes that are epigenetically silenced in CLL have been identified. Advances in the understanding of post-translational histone modifications and DNA methylation in normal and in CLL cells have proven to be extremely beneficial in finding powerful diagnostic markers, as well as in exploring novel therapies. At present, the field of epigenetics is at an evolving stage, but there is no doubt that further unraveling of its cause and effects in transformed cells will bring a new revolution in cancer therapeutics.

Animals↗
PubMed2005

Lymphoid cell growth and transformation are suppressed by a key regulatory element of the gene encoding PU.1.

Tight regulation of transcription factors, such as PU.1, is crucial for generation of all hematopoietic lineages. We previously reported that mice with a deletion of an upstream regulatory element (URE) of the gene encoding PU.1 (Sfpi1) developed acute myeloid leukemia. Here we show that the URE has an essential role in orchestrating the dynamic PU.1 expression pattern required for lymphoid development and tumor suppression. URE deletion ablated B2 cells but stimulated growth of B1 cells in mice. The URE was a PU.1 enhancer in B cells but a repressor in T cell precursors. TCF transcription factors coordinated this repressor function and linked PU.1 to Wnt signaling. Failure of appropriate PU.1 repression in T cell progenitors with URE deletion disrupted differentiation and induced thymic transformation. Genome-wide DNA methylation assessment showed that epigenetic silencing of selective tumor suppressor genes completed PU.1-initiated transformation of lymphoid progenitors with URE deletion. These results elucidate how a single transcription factor, PU.1, through the cell context-specific activity of a key cis-regulatory element, affects the development of multiple cell lineages and can induce cancer.

Animals↗
PubMed2005

Epigenetic differences arise during the lifetime of monozygotic twins.

Monozygous twins share a common genotype. However, most monozygotic twin pairs are not identical; several types of phenotypic discordance may be observed, such as differences in susceptibilities to disease and a wide range of anthropomorphic features. There are several possible explanations for these observations, but one is the existence of epigenetic differences. To address this issue, we examined the global and locus-specific differences in DNA methylation and histone acetylation of a large cohort of monozygotic twins. We found that, although twins are epigenetically indistinguishable during the early years of life, older monozygous twins exhibited remarkable differences in their overall content and genomic distribution of 5-methylcytosine DNA and histone acetylation, affecting their gene-expression portrait. These findings indicate how an appreciation of epigenetics is missing from our understanding of how different phenotypes can be originated from the same genotype.

5-Methylcytosine↗
PubMed2005

TWIST2 demonstrates differential methylation in immunoglobulin variable heavy chain mutated and unmutated chronic lymphocytic leukemia.

PURPOSE: Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease for which natural history can be predicted based on the presence or absence of immunoglobulin (Ig) variable heavy chain (V(H)) gene mutations. Herein we report selective epigenetic silencing of the transcription factor TWIST2 (DERMO1) in Ig V(H) mutated CLL and describe a semiquantitative assay to study promoter methylation of this gene in primary tumor cells. MATERIALS AND METHODS: TWIST2 promoter methylation was identified by restriction landmark genome scanning. Southern blot (SB), bisulfite sequencing, and combined bisulfite restriction analysis (COBRA), and quantitative SB-COBRA was performed to study methylation of the TWIST2 promoter. Reverse transcription polymerase chain reaction assays were used to study TWIST2 expression in CLL cells. RESULTS: Following identification and confirmation of TWIST2 methylation in CLL patients, we demonstrated that expression of this transcription factor is related to the degree of promoter methylation. Expression of TWIST2 in a CLL cell line in which the promoter is methylated was increased following decitabine treatment. We next studied 53 patients by COBRA and demonstrated that 72% of patient samples with mutated Ig V(H) show TWIST2 methylation, while only 16% of patient samples with unmutated Ig V(H) were methylated (P < .001). In a subset of patients, methylation of TWIST2 correlated with mRNA expression. CONCLUSION: TWIST2 is differentially methylated in CLL cells relative to Ig V(H) mutational status and can be quantitatively monitored by SB-COBRA. Based on the known role of TWIST2 in silencing p53 function in other malignancies, future studies should focus on the role of TWIST2 in CLL and related lymphoproliferative diseases.

Antimetabolites, Antineoplastic↗
PubMed2005

The MLL partial tandem duplication: evidence for recessive gain-of-function in acute myeloid leukemia identifies a novel patient subgroup for molecular-targeted therapy.

MLL (ALL-1) chimeric fusions and MLL partial tandem duplications (PTD) may have mechanistically distinct contributions to leukemogenesis. Acute myeloid leukemia (AML) blasts with the t(9;11)(p22; q23) express MLL-AF9 and MLL wild-type (WT) transcripts, while normal karyotype AML blasts with the MLL(PTD/WT) genotype express MLL PTD but not the MLL WT. Silencing of MLL WT in MLL(PTD/WT) blasts was reversed by DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, and MLL WT induction was associated with selective sensitivity to cell death. Reduction of MLL PTD expression induced MLL WT and reduced blast colony-forming units, supporting opposing functions for MLL PTD and MLL WT whereby the MLL PTD contributes to the leukemic phenotype via a recessive gain-of-function. The coincident suppression of the MLL WT allele with the expression of the MLL PTD allele, along with the functional data presented here, supports the hypothesis that loss of WT MLL function via monoallelic repression contributes to the leukemic phenotype by the remaining mutant allele. These data from primary AML and the pharmacologic reversal of MLL WT silencing associated with a favorable alteration in the threshold for apoptosis suggest that these patients with poor prognosis may benefit from demethylating or histone deacetylase inhibitor therapy, or both.

Acute Disease↗