Biomedical subjects

Peter Lichter

Publications and source records attributed to Peter Lichter.

At least 55 records · Page 3Linked to original sources

PubMed2005

Distinct gene expression patterns associated with FLT3- and NRAS-activating mutations in acute myeloid leukemia with normal karyotype.

In acute myeloid leukemia (AML), constitutive activation of the FLT3 receptor tyrosine kinase, either by internal tandem duplications (FLT3-ITD) of the juxtamembrane region or by point mutations in the second tyrosine kinase domain (FLT3-TKD), as well as point mutations of the NRAS gene (NRAS-PM) are among the most frequent somatic gene mutations. To elucidate whether these mutations cause aberrant signal transduction in AML, we used gene expression profiling in a series of 110 newly diagnosed AML patients with normal karyotype. The different algorithms used for data analysis revealed highly concordant sets of genes, indicating that the identified gene signatures are specific for each analysed subgroup. Whereas samples with FLT3-ITD and FLT3-TKD could be separated with up to 100% accuracy, this did not apply for NRAS-PM and wild-type samples, suggesting that only FLT3-ITD and FLT3-TKD are associated with an apparent signature in AML. The set of discriminating genes included several known genes, which are involved in cell cycle control (CDC14A, WEE1), gene transcription (HOXB5, FOXA1), and signal transduction (SMG1). In conclusion, we showed that unique gene expression patterns can be correlated with FLT3-ITD and FLT3-TKD. This might lead to the identification of further pathogenetic relevant candidate genes particularly in AML with normal karyotype.

Gene Expression Regulation, Neoplastic↗

PubMed2005

Effective transcriptome amplification for expression profiling on sense-oriented oligonucleotide microarrays.

Gene expression analysis using microarrays of synthetic long oligonucleotides is limited in that it requires substantial amounts of RNA. To obtain these quantities from minute amounts of starting material, protocols were developed that linearly amplify mRNA by cDNA synthesis and in vitro transcription. Since orientation of the product is antisense (aRNA), it is inapplicable for dye-labelling by reverse transcription and hybridization to sense-oriented oligonucleotide arrays. Here, we introduce a novel protocol in which aRNA labelling is achieved by a combination of two reverse and one forward transcription reactions followed by dye-incorporation using Klenow fragment, generating fluorescent antisense cDNA. We demonstrate high fidelity in arrays using up to 10(5)-fold amplification, starting from 2 ng total RNA. The generated data are highly reproducible and maintain relative gene expression levels between samples. These results demonstrate that our protocol describes an efficient and reliable technique to expand the applicability of oligonucleotide arrays to studies where RNA is the limited source material.

DNA, Antisense↗

PubMed2005

Characterization of a nuclear compartment shared by nuclear bodies applying ectopic protein expression and correlative light and electron microscopy.

To investigate the accessibility of interphase nuclei for nuclear body-sized particles, we analyzed in cultured cells from human origin by correlative fluorescence and electron microscopy (EM) the bundle-formation of Xenopus-vimentin targeted to the nucleus via a nuclear localization signal (NLS). Moreover, we investigated the spatial relationship of speckles, Cajal bodies, and crystalline particles formed by Mx1 fused to yellow fluorescent protein (YFP), with respect to these bundle arrays. At 37 degrees C, the nucleus-targeted, temperature-sensitive Xenopus vimentin was deposited in focal accumulations. Upon shift to 28 degrees C, polymerization was induced and filament arrays became visible. Within 2 h after temperature shift, arrays were found to be composed of filaments loosely embedded in the nucleoplasm. The filaments were restricted to limited areas of the nucleus between focal accumulations. Upon incubation at 28 degrees C for several hours, NLS vimentin filaments formed bundles looping throughout the nuclei. Speckles and Cajal bodies frequently localized in direct neighborhood to vimentin bundles. Similarly, small crystalline particles formed by YFP-tagged Mx1 also located next to vimentin bundles. Taking into account that nuclear targeted vimentin locates in the interchromosomal domain (ICD), we conclude that nuclear body-sized particles share a common nuclear space which is controlled by higher order chromatin organization.

Animals↗

PubMed2005

Nuclear RNAs confined to a reticular compartment between chromosome territories.

RNA polymerase II transcripts are confined to nuclear compartments. A detailed analysis of the nuclear topology of RNA from individual genes was performed for transcripts from the marker gene coding for chloramphenicol acetyltransferase, expressed at a high level from the HTLV-1 LTR promoter. The construct was transfected into A293 cells where the RNA was organized as an extensive reticular network. We also studied the RNA distribution from combinations of neighboring HIV and bacterial resistance genes that co-integrated within the genome of COS-7 cells-revealing spherical or track-like accumulations of RNA that were extensively branched. There were many nuclei with distinct but overlapping RNA accumulations. Since the coding genes localized at the overlapping points, the RNAs are synthesized at a common region and diverge. The correlation between the frequency of the separation of the transcripts and the physical distance of the respective genes suggests a subcompartmentalization in the microenvironment of genes on the basis of geometric parameters. Thus, the more distant the genes are on the same chromosome, the more likely they are confined to separated subcompartments of an extensive reticular system. Co-delineation of the RNA transcripts with Cajal bodies and chromosome territories indicated the organization of nuclear RNA transcripts in a reticular interchromosome domain compartment.

Animals↗

PubMed2005

Candidate genes in breast cancer revealed by microarray-based comparative genomic hybridization of archived tissue.

Genomic imbalances in 31 formalin-fixed and paraffin-embedded primary tumors of advanced breast cancer were analyzed by microarray-based comparative genomic hybridization (matrix-CGH). A DNA chip was designed comprising 422 mapped genomic sequences including 47 proto-oncogenes, 15 tumor suppressor genes, as well as frequently imbalanced chromosomal regions. Analysis of the data was challenging due to the impaired quality of DNA prepared from paraffin-embedded samples. Nevertheless, using a method for the statistical evaluation of the balanced state for each individual experiment, we were able to reveal imbalances with high significance, which were in good concordance with previous data collected by chromosomal CGH from the same patients. Owing to the improved resolution of matrix-CGH, genomic imbalances could be narrowed down to the level of individual bacterial artificial chromosome and P1-derived artificial chromosome clones. On average 37 gains and 13 losses per tumor cell genome were scored. Gains in more than 30% of the cases were found on 1p, 1q, 6p, 7p, 8q, 9q, 11q, 12q, 17p, 17q, 20q, and 22q, and losses on 6q, 9p, 11q, and 17p. Of the 51 chromosomal regions found amplified by matrix-CGH, only 12 had been identified by chromosomal CGH. Within these 51 amplicons, genome database information defined 112 candidate genes, 44 of which were validated by either PCR amplification of sequence tag sites or DNA sequence analysis.

Breast Neoplasms↗

PubMed2005

Translocation t(X;11)(q13;q23) in B-cell chronic lymphocytic leukemia disrupts two novel genes.

Deletion of chromosome region 11q22-q23 defines a subgroup of patients with B-cell chronic lymphocytic leukemia (B-CLL) characterized by poor survival. Although the tumor-suppressor gene ATM in the consensus deletion region was found to be biallelically inactivated in about one third of B-CLL cases, in the majority of those who have this deletion, inactivation of the remaining ATM allele was not observed. To identify a second disease-associated gene, we investigated two B-CLL cases with translocation breakpoints in the critical 11q23 deletion region. In one case, a t(X;11)(q13;q23) was cloned and two novel genes were isolated. The breakpoint on 11q23 affected the ARHGAP20 gene, which encodes a protein predicted to be involved in the regulation of Rho family GTPases. The breakpoint on Xq13 occurred in BRWD3, which codes for a putative novel transcription factor. The rearrangement of ARHGAP20 and BRWD3 did not result in fusion transcripts, but it disrupted both genes. Mutation analysis of 28 B-CLL samples with monoallelic deletions and two B-CLL samples with 11q23 translocations detected no deleterious mutation in the remaining copy of ARHGAP20. Quantitative expression analysis in 22 B-CLLs revealed significant up-regulation of ARHGAP20 in CLL B cells, whereas BRWD3 was slightly down-regulated. Thus, deregulation of ARHGAP20 by altered gene expression or by gene disruption (but not point mutation) might be a general molecular mechanism of B-CLL leukemogenesis.

Chromosome Banding↗

PubMed2005

Detection of chromosomal imbalances in retinoblastoma by matrix-based comparative genomic hybridization.

The genetic hallmark of retinoblastoma is mutation or deletion of the RB1 gene, whereas other genetic alterations that are also required are largely unknown. To screen for genomic imbalances on a genomewide level, we studied a series of 17 primary retinoblastomas by matrix-based comparative genomic hybridization (matrix-CGH). The matrix-CGH chip contained 6,000 immobilized genomic DNA fragments covering the human genome, with an average resolution of about 500 kb. The most frequent imbalances detected were gains on chromosome arms 1q (12 of 17), 6p (10 of 17), 2p (5 of 17), and 19q (4 of 17) and loss on 16q (7 of 17). Candidate regions could be narrowed to small intervals by the identified minimally overlapping regions on 1q22, 1q32.1q32.2, 2p24.1, and 6p21.33-p21.31. Furthermore, two as-yet-unknown high-level amplifications were detected, each in a single patient, on chromosome bands 1p34.2 and 1p33. Thus, this study identified new chromosomal regions and therefore potential candidate genes that may play a role in retinoblastoma.

Allelic Imbalance↗

PubMed2005

Copy number gains on 22q13 in adenoid cystic carcinoma of the salivary gland revealed by comparative genomic hybridization and tissue microarray analysis.

Adenoid cystic carcinoma (ACC) of the salivary gland is a neoplasm characterized by slow but inevitable local progression and terminal hematogenous metastasis. To detect novel imbalanced chromosomal regions associated with tumorigenesis, we used chromosomal comparative genomic hybridization to screen 27 ACC. The most common aberration was copy number gain of 22q13 (nine cases) followed by gains of 16p (seven cases) and 17q (four cases) and copy number losses on 6q (six cases). To further delineate the prevalence of 22q13 copy number gains in ACC, fluorescence in situ hybridization was performed for five bacterial/phage artificial chromosome (BAC/PAC) probes from the 22q13 consensus region with 57 ACC on a tissue microarray. The overall prevalence of copy number gains on 22q13 was 30% of the tumors in the fluorescence in situ hybridization analysis, irrespective of histologic differentiation (cribriform/tubular vs. solid) or tumor event (primary vs. recurrent). We therefore assume that copy number gain of 22q13 is a novel frequent finding in ACC that may be involved in the initial pathogenesis of this neoplasm by proto-oncogene activation.

Adult↗

PubMed2005

Differential KIT expression in histological subtypes of adenoid cystic carcinoma (ACC) of the salivary gland.

Adenoid cystic carcinoma (ACC) of the salivary gland is characterized by a prolonged but inevitably unfavorable clinical course. Recent studies suggested the transmembrane tyrosine kinase KIT to be involved in ACC pathogenesis. To investigate KIT expression in histologically defined subgroups of ACC and to clarify whether KIT gene copy number gain contributes to KIT overexpression, tumor tissue microarray sections including 55 ACC tumors were analyzed by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). The prevalence of positive KIT immunostaining was 89% (49/55). Strong immunostaining of KIT was only found in cribriform and tubular but never in solid subtypes (p=0.02). Average KIT staining intensity was higher in cribriform and tubular (n=37) compared to solid (n=18) ACC subtypes (p=0.005). FISH analysis revealed copy number gains of the KIT gene in 6.1% (3/49) of tumors analyzed. Our results implicate that specific KIT tyrosine kinase inhibitors such as imatinib, might be used in future therapeutic approaches against subgroups of ACC.

Carcinoma, Adenoid Cystic↗

PubMed2005

Patient-based cross-platform comparison of oligonucleotide microarray expression profiles.

The comparison of gene expression measurements obtained with different technical approaches is of substantial interest in order to clarify whether inter-platform differences may conceal biologically significant information. To address this concern, we analyzed gene expression in a set of head and neck squamous cell carcinoma patients, using both spotted oligonucleotide microarrays made from a large collection of 70-mer probes and commercial arrays produced by in situ synthesis of sets of multiple 25-mer oligonucleotides per gene. Expression measurements were compared for 4425 genes represented on both platforms, which revealed strong correlations between the corresponding data sets. Of note, a global tendency towards smaller absolute ratios was observed when using the 70-mer probes. Real-time quantitative reverse transcription PCR measurements were conducted to verify expression ratios for a subset of genes and achieved good agreement regarding both array platforms. In conclusion, similar profiles of relative gene expression were obtained using arrays of either single 70-mer or multiple short 25-mer oligonucleotide probes per gene. Although qualitative assessments of the expression of individual genes have to be made with caution, our results indicate that the comparison of gene expression profiles generated on these platforms will help to discover disease-related gene signatures in general.

Base Sequence↗

PubMed2005

Report: workshop on mediastinal grey zone lymphoma.

There are several indications that classical Hodgkin lymphoma (cHL) and at least a proportion of cases of Primary Mediastinal B cell Lymphoma (PMBL) are derived from B cells at similar stages of differentiation and share common pathogenic mechanisms. The first indication was the existence of mediastinal grey zone lymphomas as identified in the 4th International Symposium on HL, with clinical, histological and immunohistochemical features intermediate between cHL and PMBL. Second, both tumor types resemble a cell that is developmentally situated in-between the germinal center reaction and a plasma cell. Third, cHL and PMBL were found to have similar gene expression profiles, including the lack of immunoglobulin expression and low levels of B cell receptor signalling molecules, and the secretion of molecules like the chemokine TARC and the prominent expression of IL-13 receptors. Fourth, both entities were found to have common genomic aberrancies, notably in 2p15 and 9p24, the sites of the REL oncogene and the tyrosine kinase gene JAK2, respectively. Further comparison of both lymphoma types may provide further insight in the pathogenic mechanisms and allow the design of diagnostic algorithms to sort out the small number of so-called mediastinal grey zone lymphomas, that appear to be intermediate between PMBL and cHL.

B-Lymphocytes↗

PubMed2005

hMOF histone acetyltransferase is required for histone H4 lysine 16 acetylation in mammalian cells.

Reversible histone acetylation plays an important role in regulation of chromatin structure and function. Here, we report that the human orthologue of Drosophila melanogaster MOF, hMOF, is a histone H4 lysine K16-specific acetyltransferase. hMOF is also required for this modification in mammalian cells. Knockdown of hMOF in HeLa and HepG2 cells causes a dramatic reduction of histone H4K16 acetylation as detected by Western blot analysis and mass spectrometric analysis of endogenous histones. We also provide evidence that, similar to the Drosophila dosage compensation system, hMOF and hMSL3 form a complex in mammalian cells. hMOF and hMSL3 small interfering RNA-treated cells also show dramatic nuclear morphological deformations, depicted by a polylobulated nuclear phenotype. Reduction of hMOF protein levels by RNA interference in HeLa cells also leads to accumulation of cells in the G(2) and M phases of the cell cycle. Treatment with specific inhibitors of the DNA damage response pathway reverts the cell cycle arrest caused by a reduction in hMOF protein levels. Furthermore, hMOF-depleted cells show an increased number of phospho-ATM and gammaH2AX foci and have an impaired repair response to ionizing radiation. Taken together, our data show that hMOF is required for histone H4 lysine 16 acetylation in mammalian cells and suggest that hMOF has a role in DNA damage response during cell cycle progression.

Acetylation↗

PubMed2004

Topology of genes and nontranscribed sequences in human interphase nuclei.

Knowledge about the functional impact of the topological organization of DNA sequences within interphase chromosome territories is still sparse. Of the few analyzed single copy genomic DNA sequences, the majority had been found to localize preferentially at the chromosome periphery or to loop out from chromosome territories. By means of dual-color fluorescence in situ hybridization (FISH), immunolabeling, confocal microscopy, and three-dimensional (3D) image analysis, we analyzed the intraterritorial and nuclear localization of 10 genomic fragments of different sequence classes in four different human cell types. The localization of three muscle-specific genes FLNA, NEB, and TTN, the oncogene BCL2, the tumor suppressor gene MADH4, and five putatively nontranscribed genomic sequences was predominantly in the periphery of the respective chromosome territories, independent from transcriptional status and from GC content. In interphase nuclei, the noncoding sequences were only rarely found associated with heterochromatic sites marked by the satellite III DNA D1Z1 or clusters of mammalian heterochromatin proteins (HP1alpha, HP1beta, HP1gamma). However, the nontranscribed sequences were found predominantly at the nuclear periphery or at the nucleoli, whereas genes tended to localize on chromosome surfaces exposed to the nuclear interior.

Base Sequence↗

PubMed2004

Microarray gene expression profiling of B-cell chronic lymphocytic leukemia subgroups defined by genomic aberrations and VH mutation status.

PURPOSE: Genomic aberrations and mutational status of the immunoglobulin variable heavy chain (VH) gene have been shown to be among the most important predictors for outcome in patients with B-cell chronic lymphocytic leukemia (B-CLL). In this study, we report on differential gene expression patterns that are characteristic for genetically defined B-CLL subtypes. MATERIALS AND METHODS: One hundred genetically well-characterized B-CLL samples, together with 11 healthy control samples, were analyzed using oligonucleotide arrays, which test for the expression of some 12,000 human genes. RESULTS: Aiming at microarray-based subclassification, class predictors were constructed using sets of differentially expressed genes, which yielded in zero or low misclassification rates. Furthermore, a significant number of the differentially expressed genes clustered in chromosomal regions affected by the respective genomic losses/gains. Deletions affecting chromosome bands 11q22-q23 and 17p13 led to a reduced expression of the corresponding genes, such as ATM and p53, while trisomy 12 resulted in the upregulation of genes mapping to chromosome arm 12q. Using an unsupervised analysis algorithm, expression profiling allowed partitioning into predominantly VH-mutated versus unmutated patient groups; however, association of the expression profile with the VH mutational status could only be detected in male patients. CONCLUSION: The finding that the most significantly differentially expressed genes are located in the corresponding aberrant chromosomal regions indicates that a gene dosage effect may exert a pathogenic role in B-CLL. The significant difference in the partitioning of male and female B-CLL samples suggests that the genomic signature for the VH mutational status might be sex-related.

Adult↗

PubMed2004

Identification of novel AP-1 target genes in fibroblasts regulated during cutaneous wound healing.

Mesenchymal-epithelial interactions are increasingly considered to be of vital importance for epithelial homeostasis and regeneration. In skin, the transcription factor AP-1 was shown to be critically involved in the communication between keratinocytes and dermal fibroblasts. After skin injury, the release of IL-1 from keratinocytes induces the activity of the AP-1 subunits c-Jun and JunB in fibroblasts leading to a global change in gene expression. To identify AP-1 target genes in fibroblasts, which are involved in the process of cutaneous repair, we performed gene expression profiling of wild-type, c-jun- and junB-deficient fibroblasts in response to IL-1, mimicking the initial phase of wound healing. Using a 15K cDNA collection, over 1000 genes were found to be Jun-dependent and additional 300 clones showed IL-1 responsiveness. Combinatorial evaluation allowed for the dissection of the specific contribution of either AP-1 subunit to gene regulation. Besides previously identified genes that are involved in cutaneous repair, we have identified novel genes regulated during wound healing in vivo and showed their expression by fibroblasts on wound sections. The identification of novel Jun target genes should provide a basis for understanding the molecular mechanisms underlying mesenchymal-epithelial interactions and the critical contribution of AP-1 to tissue homeostasis and repair.

Animals↗

PubMed2004

Nuclear body movement is determined by chromatin accessibility and dynamics.

Promyelocytic leukemia (PML) and Cajal bodies are mobile subnuclear organelles, which are involved in activities like RNA processing, transcriptional regulation, and antiviral defense. A key parameter in understanding their biological functions is their mobility. The diffusion properties of PML and Cajal bodies were compared with a biochemically inactive body formed by aggregates of murine Mx1 by using single-particle tracking methods. The artificial Mx1-yellow fluorescent protein body showed a very similar mobility compared with PML and Cajal bodies. The data are described quantitatively by a mechanism of nuclear body movement consisting of two components: diffusion of the body within a chromatin corral and its translocation resulting from chromatin diffusion. This finding suggests that the body mobility reflects the dynamics and accessibility of the chromatin environment, which might target bodies to specific nuclear subcompartments where they exert their biological function.

Adenosine Triphosphate↗

PubMed2004

Genomic DNA-chip hybridization reveals a higher incidence of genomic amplifications in pancreatic cancer than conventional comparative genomic hybridization and leads to the identification of novel candidate genes.

Genomic analyses aimed at the detection of high-level DNA amplifications were performed on 13 widely used pancreatic cancer cell lines and 6 pancreatic tumor specimens. For these analyses, array-based comparative genomic hybridization (Matrix-CGH) onto dedicated microarrays was used. In comparison with chromosomal CGH (eight amplifications), a >3-fold number of DNA amplifications was detected (n = 29). The most frequent amplifications mapped to 7p12.3 (three pancreatic cancer cell lines and three pancreatic tumor specimens), 8q24 (four pancreatic cancer cell lines and one pancreatic tumor specimen), 11q13 (three pancreatic cancer cell lines and three pancreatic tumor specimens), and 20q13 (four pancreatic cancer cell lines and three pancreatic tumor specimens). Genes contained in the consensus regions were MYC (8q24), EGFR (7p12.3), and FGF3 (11q13). In six of seven pancreatic cancer cell lines and pancreatic tumor specimens with 20q13 amplifications, the novel candidate gene NFAT C2, which plays a role in the activation of cytokines, was amplified. Other amplifications also affected genes for which a pathogenetic role in pancreatic carcinoma has not been described, such as BCL10 and BCL6, two members of the BCL family. A subset of amplified genes was checked for overexpression by means of real-time PCR, revealing the highest expression levels for BCL6 and BCL10. Thus, Matrix-CGH allows the detection of a high number of amplifications, resulting in the identification of novel candidate genes in pancreatic cancer.

Aged↗

PubMed2004

Microarray-based screening for molecular markers in medulloblastoma revealed STK15 as independent predictor for survival.

Medulloblastoma, a primitive neuroectodermal tumor of the cerebellum, is one of the most common central nervous system malignancies of childhood. Despite aggressive multimodal therapy, including surgery, irradiation, and chemotherapy, 5-year survival rates have only approached 50-60%. To identify potential candidate genes that predict for overall survival (OS), we performed a gene expression profiling analysis in 35 newly diagnosed medulloblastoma neoplasms. Subsequently, the nine most promising candidate genes were analyzed by immunohistochemistry and fluorescence in situ hybridization on tumor tissue microarrays representing a series of 180 tumors. We found 54 genes in which expression levels predicted for unfavorable survival in medulloblastoma. In line with the gene expression profiling analysis, a positive staining for STK15 (P = 0.0006), stathmin 1 (P = 0.001), and cyclin D1 (P = 0.03) was associated with an unfavorable OS, whereas cyclin B1, DAXX, Ki-67, MYC, NRAS, and p53 showed no statistical significant effect. In comparison to clinically defined parameters such as gender, age, metastatic stage, extent of tumor resection, application of chemotherapy, and tumor grade, positive staining for STK15 was identified as an independent prognostic factor for OS (P = 0.026). Moreover, additional gene copy numbers of MYC (P = 0.003) and STK15 (P = 0.05) predicted for poor survival. The combination of gene expression profiling with tissue microarray experiments allowed the identification of a series of candidate genes that predicts for survival in medulloblastoma. Of the results highlighted by the various data analysis procedures, genes associated with cell proliferation (cyclin D1), transcription (MYC), and especially mitosis (stathmin 1, STK15) appear particularly intriguing with respect to medulloblastoma pathomechanism.

Adolescent↗