Biomedical subjects

Christine Steinhoff

Publications and source records attributed to Christine Steinhoff.

14 recordsLinked to original sources

PubMed2006

Normalization and quantification of differential expression in gene expression microarrays.

Array-based gene expression studies frequently serve to identify genes that are expressed differently under two or more conditions. The actual analysis of the data, however, may be hampered by a number of technical and statistical problems. Possible remedies on the level of computational analysis lie in appropriate preprocessing steps, proper normalization of the data and application of statistical testing procedures in the derivation of differentially expressed genes. This review summarizes methods that are available for these purposes and provides a brief overview of the available software tools.

Algorithms↗

PubMed2005

Up-regulation of glucocorticoid-regulated genes in a mouse model of Rett syndrome.

Rett syndrome (RTT) is a severe form of mental retardation, which is caused by spontaneous mutations in the X-linked gene MECP2. How the loss of MeCP2 function leads to RTT is currently unknown. Mice lacking the Mecp2 gene initially show normal postnatal development but later acquire neurological phenotypes, including heightened anxiety, that resemble RTT. The MECP2 gene encodes a methyl-CpG-binding protein that can act as a transcriptional repressor. Using cDNA microarrays, we found that Mecp2-null animals differentially express several genes that are induced during the stress response by glucocorticoids. Increased levels of mRNAs for serum glucocorticoid-inducible kinase 1 (Sgk) and FK506-binding protein 51 (Fkbp5) were observed before and after onset of neurological symptoms, but plasma glucocorticoid was not significantly elevated in Mecp2-null mice. MeCP2 is bound to the Fkbp5 and Sgk genes in brain and may function as a modulator of glucocorticoid-inducible gene expression. Given the known deleterious effect of glucocorticoid exposure on brain development, our data raise the possibility that disruption of MeCP2-dependent regulation of stress-responsive genes contributes to the symptoms of RTT.

Animals↗

PubMed2004

Robust inference of groups in gene expression time-courses using mixtures of HMMs.

MOTIVATION: Genetic regulation of cellular processes is frequently investigated using large-scale gene expression experiments to observe changes in expression over time. This temporal data poses a challenge to classical distance-based clustering methods due to its horizontal dependencies along the time-axis. We propose to use hidden Markov models (HMMs) to explicitly model these time-dependencies. The HMMs are used in a mixture approach that we show to be superior over clustering. Furthermore, mixtures are a more realistic model of the biological reality, as an unambiguous partitioning of genes into clusters of unique functional assignment is impossible. Use of the mixture increases robustness with respect to noise and allows an inference of groups at varying level of assignment ambiguity. A simple approach, partially supervised learning, allows to benefit from prior biological knowledge during the training. Our method allows simultaneous analysis of cyclic and non-cyclic genes and copes well with noise and missing values. RESULTS: We demonstrate biological relevance by detection of phase-specific groupings in HeLa time-course data. A benchmark using simulated data, derived using assumptions independent of those in our method, shows very favorable results compared to the baseline supplied by k-means and two prior approaches implementing model-based clustering. The results stress the benefits of incorporating prior knowledge, whenever available. AVAILABILITY: A software package implementing our method is freely available under the GNU general public license (GPL) at http://ghmm.org/gql

Algorithms↗

PubMed2004

Gene expression changes in the course of neural progenitor cell differentiation.

The molecular changes underlying neural progenitor differentiation are essentially unknown. We applied cDNA microarrays with 13,627 clones to measure dynamic gene expression changes during the in vitro differentiation of neural progenitor cells that were isolated from the subventricular zone of postnatal day 7 mice and grown in vitro as neurospheres. In two experimental series in which we withdrew epidermal growth factor and added the neurotrophins Neurotrophin-4 or BDNF, four time points were investigated: undifferentiated cells grown as neurospheres, and cells 24, 48, and 96 hr after differentiation. Expression changes of selected genes were confirmed by semiquantitative RT-PCR. Ten different groups of gene expression dynamics obtained by cluster analysis are described. To correlate selected gene expression changes to the localization of respective proteins, we performed immunostainings of cultured neurospheres and of brain sections from adult mice. Our results provide new insights into the genetic program of neural progenitor differentiation and give strong hints to as yet unknown cellular communications within the adult subventricular zone stem cell niche.

Animals↗

PubMed2004

Different molecular mechanisms underlie placental overgrowth phenotypes caused by interspecies hybridization, cloning, and Esx1 mutation.

To obtain a deeper insight into the genes and gene networks involved in the development of placentopathies, we have assessed global gene expression in three different models of placental hyperplasia caused by interspecies hybridization (IHPD), cloning by nuclear transfer, and mutation of the Esx1 gene, respectively. Comparison of gene expression profiles of approximately 13,000 expressed sequence tags (ESTs) identified specific subsets of genes with changed expression levels in IHPD, cloned, and Esx1 mutant placentas. Of interest, only one gene of known function and one EST of unknown function were found common to all three placentopathies; however, a significant number of ESTs were common to IHPD and cloned placentas. In contrast, only one gene was shared between IHPD and Esx1 mutant, and cloned and Esx1 mutant placentas, respectively. These genes common to different abnormal placental growth genotypes are likely to be important in the occurrence of placentopathy.

Animals↗

PubMed2004

Decreased Fas expression in advanced-stage bladder cancer is not related to p53 status.

OBJECTIVES: The Fas-Fas ligand system is an important regulator of apoptosis and is involved in tumor development. Invasive cancers downregulate Fas expression to evade antitumor immune responses. Fas is a transcriptional target of p53, which is often mutated in bladder cancers. Therefore, Fas expression and its relation to p53 mutation was investigated. METHODS: Expression of Fas protein and p53 status was studied by immunohistochemistry in 83 bladder cancer specimens. In addition, mRNA levels for soluble (decoy) and membrane-bound forms of Fas were compared between 10 bladder cancer cell lines and primary uroepithelial cells by quantitative TaqMan polymerase chain reaction. Mutational analysis of the death domain of the Fas gene was performed in all cell lines. RESULTS: Organ-confined tumors maintained specific Fas staining at the cell membrane and often also in the cytoplasm. In higher stage carcinomas, Fas expression became restricted to a smaller fraction of cells or was lacking entirely. The correlation of Fas staining with tumor stage was highly significant but no correlation to tumor grade or survival was found. Furthermore, no statistically significant relationship was observed with either the presence or lack of mutated p53 accumulation. Membrane-bound Fas mRNA was decreased in most, and soluble Fas was increased in all transitional cell carcinoma lines compared with primary uroepithelial cells. No mutations in the death domain were detected. CONCLUSIONS: Fas downregulation occurring in advanced bladder cancer is unrelated to p53 mutations. The results of immunohistochemistry and mRNA studies of soluble and membrane-bound Fas in transitional cell carcinoma lines support the hypothesis of immune evasion in advanced bladder cancer.

Carcinoma, Papillary↗

PubMed2003

Decrease of DNA methyltransferase 1 expression relative to cell proliferation in transitional cell carcinoma.

In many common cancers such as transitional cell carcinoma (TCC), specific genes are hypermethylated, whereas overall DNA methylation is diminished. Genome-wide DNA hypomethylation mostly affects repetitive sequences such as LINE-1 retrotransposons. Methylation of these sequences depends on adequate expression of DNA methyltransferase I (DNMT1) during DNA replication. Therefore, DNMT1 expression relative to proliferation was investigated in TCC cell lines and tissue as well as in renal carcinoma (RCC) cell lines, which also display hypomethylation, as indicated by decreased LINE-1 methylation. Cultured normal uroepithelial cells or normal bladder tissue served as controls. In all tumor cell lines, DNMT1 mRNA as well as protein was decreased relative to the DNA replication factor PCNA, and DNA hypomethylation was present. However, the extents of hypomethylation and DNMT1 downregulation did not correlate. Reporter gene assays showed that the differences in DNMT1 expression between normal and tumor cells were not established at the level of DNMT1 promoter regulation. Diminished DNMT1:PCNA mRNA ratios were also found in 28/45 TCC tissues but did not correlate with the extent of DNA hypomethylation. In addition, expression of the presumed de novo methyltransferases DNMT3A and DNMT3B mRNAs was investigated. DNMT3B overexpression was observed in about half of all high-stage TCC (DNMT3B vs. tumor stage, chi(2): p = 0.03), whereas overexpression of DNMT3A was rarer and less pronounced. Expression of DNMT3A and DNMT3B in most RCC lines was higher than in TCC lines. Our data indicate that DNMT1 expression does not increase adequately with cell proliferation in bladder cancer. This relative downregulation probably contributes to hypomethylation of repetitive DNA but does not determine its extent alone.

Aged↗

PubMed2003

Gene expression profile of mouse bone marrow stromal cells determined by cDNA microarray analysis.

Bone marrow stromal cells (BMSC) have gained increased attention because of their multipotency and adult stem cell character. They have been shown to differentiate into other cell types of the mesenchymal lineage and also into non-mesenchymal cells. The exact identity of the original cells, which are isolated from bone marrow by their selective adherence to plastic, remains unknown to date. We have established and characterized mouse BMSC cultures and analyzed three independent samples by cDNA microarrays. The expression profile was compared with two previous expression studies of human BMSC and revealed a high degree of concordance between different techniques and species. To gain clues about the positional context and biology of the isolated cells within the bone marrow stroma, we searched our data for genes that encode proteins of the extracellular matrix, cell adhesion proteins, cytoskeletal proteins and cytokines/cytokine receptors. This analysis revealed a close association of BMSC with vascular cells and indicated that BMSC resemble pericytes.

Animals↗

PubMed2003

Activities of MAP-kinase pathways in normal uroepithelial cells and urothelial carcinoma cell lines.

It is often assumed that MAPK pathways drive proliferation of normal uroepithelial (UEC) and urothelial carcinoma (TCC) cells. To check this assumption, activities and inducibilities of promoters containing serum-response elements (SRE) or AP-1 binding sites were investigated in cultured UEC and seven TCC lines. Reporter plasmids dependent on SRE or AP-1 sites were highly active in UEC, but significantly less so in TCC lines. Reporter activity in TCC lines could be induced by constitutively active MEKK4 or TPA. Accordingly, phosphorylation of the MAPK pathway components MEK, ERK, and ELK1 was most pronounced in UEC and lower in TCC lines. MAPK-dependent promoter activities and bromodeoxyuridine incorporation decreased in UEC upon withdrawal of growth factors, but less so in TCC lines, in which serum diminution increased apoptosis. Likewise, E2F-dependent promoters responded to growth factors in UEC, but were more serum-independent in the TCC lines, which lack either RB1 or p16(INK4A). MEK inhibitors inhibited BrdU incorporation in UEC more strongly than in TCC lines. Thus, proliferation of normal uroepithelial cells is indeed associated with activation of MAPK pathways. However, autonomous proliferation of TCC lines--unexpectedly--appears much less dependent on MAPK activation and may rather be promoted by defects in cell cycle regulation.

Binding Sites↗

PubMed2003

Using hidden Markov models to analyze gene expression time course data.

MOTIVATION: Cellular processes cause changes over time. Observing and measuring those changes over time allows insights into the how and why of regulation. The experimental platform for doing the appropriate large-scale experiments to obtain time-courses of expression levels is provided by microarray technology. However, the proper way of analyzing the resulting time course data is still very much an issue under investigation. The inherent time dependencies in the data suggest that clustering techniques which reflect those dependencies yield improved performance. RESULTS: We propose to use Hidden Markov Models (HMMs) to account for the horizontal dependencies along the time axis in time course data and to cope with the prevalent errors and missing values. The HMMs are used within a model-based clustering framework. We are given a number of clusters, each represented by one Hidden Markov Model from a finite collection encompassing typical qualitative behavior. Then, our method finds in an iterative procedure cluster models and an assignment of data points to these models that maximizes the joint likelihood of clustering and models. Partially supervised learning--adding groups of labeled data to the initial collection of clusters--is supported. A graphical user interface allows querying an expression profile dataset for time course similar to a prototype graphically defined as a sequence of levels and durations. We also propose a heuristic approach to automate determination of the number of clusters. We evaluate the method on published yeast cell cycle and fibroblasts serum response datasets, and compare them, with favorable results, to the autoregressive curves method.

Algorithms↗

PubMed2003

Effect of routine repeat transurethral resection for superficial bladder cancer: a long-term observational study.

PURPOSE: We determined the long-term outcome in patients with superficial bladder cancer (Ta and T1) undergoing routine second transurethral bladder tumor resection (ReTURB) in regard to recurrence and progression. MATERIALS AND METHODS: We performed an inception cohort study of 124 consecutive patients with superficial bladder cancer undergoing transurethral resection and routine ReTURB (83) between November 1993 and October 1995 at a German university hospital. Immediately after transurethral resection all lesions were documented on a designed bladder map. ReTURB of the scar from initial resection and other suspicious lesions was performed at a mean of 7 weeks. Patients were followed until recurrence or death, or a minimum of 5 years. RESULTS: Residual tumor was found in 33% of all ReTURB cases, including 27% of Ta and 53% of T1 disease, and in 81% at the initial resection site. Five of the 83 patients underwent radical cystectomy due to ReTURB findings. The estimated risk of recurrence after years 1 to 3 was 18%, 29% and 32%, respectively. After 5 years 63% of the patients undergoing ReTURB were still disease-free (mean recurrence-free survival 62 months, median 87). Progression to muscle invasive disease was observed in only 2 patients (3%) after a mean observation of 61 months. CONCLUSIONS: These data suggest a favorable outcome regarding recurrence and progression in patients with superficial bladder cancer who undergo ReTURB. ReTURB is suggested at least in those at high risk when bladder preservation is intended.

Adult↗

PubMed2002

Activity of E2F-dependent promoters in bladder carcinoma cells and their use for tumour-specific targeting of p53-induced apoptosis.

Inactivation of P53 and RB functions are crucial changes in bladder cancer (TCC). High-level re-expression of P53 elicits apoptosis in TCC cell lines, but also--as shown here--in normal uroepithelial cells. Compromised RB function is thought to cause increased activity of E2F-dependent promoters in carcinoma cells. Indeed, several, but not all E2F-dependent promoters were stronger in TCC lines than in normal cells, with the highest activities in cell lines lacking RB rather than p16INK4A. Re-expression of p53 from an E2F-dependent promoter suppressed clone formation and induced apoptosis in TCC lines as efficiently as expression from the stronger RSV-LTR or LINE-1 promoters. In normal cells, p53 expression from an E2F-dependent promoter was tolerated, whereas expression from both stronger promoters was lethal. Thus, specific E2F-dependent promoters allow adjustment of p53 expression to selectively induce apoptosis in TCC vs. normal uroepithelial cells. This approach could be useful in targeting apoptosis to TCC and other carcinomas lacking p53 and RB function.

Apoptosis↗

PubMedDate not supplied

Analyzing gene expression time-courses.

Measuring gene expression over time can provide important insights into basic cellular processes. Identifying groups of genes with similar expression time-courses is a crucial first step in the analysis. As biologically relevant groups frequently overlap, due to genes having several distinct roles in those cellular processes, this is a difficult problem for classical clustering methods. We use a mixture model to circumvent this principal problem, with hidden Markov models (HMMs) as effective and flexible components. We show that the ensuing estimation problem can be addressed with additional labeled data-partially supervised learning of mixtures-through a modification of the Expectation-Maximization (EM) algorithm. Good starting points for the mixture estimation are obtained through a modification to Bayesian model merging, which allows us to learn a collection of initial HMMs. We infer groups from mixtures with a simple information-theoretic decoding heuristic, which quantifies the level of ambiguity in group assignment. The effectiveness is shown with high-quality annotation data. As the HMMs we propose capture asynchronous behavior by design, the groups we find are also asynchronous. Synchronous subgroups are obtained from a novel algorithm based on Viterbi paths. We show the suitability of our HMM mixture approach on biological and simulated data and through the favorable comparison with previous approaches. A software implementing the method is freely available under the GPL from http://ghmm.org/gql.

Algorithms↗

PubMedDate not supplied

Long-term maintenance of hematopoietic stem cells does not require contact with embryo-derived stromal cells in cocultures.

We recently established that two midgestation-derived stromal clones--UG26-1B6, urogenital ridge-derived, and EL08-1D2, embryonic liver-derived--support the maintenance of murine adult bone marrow and human cord blood hematopoietic repopulating stem cells (HSCs). In this study, we investigate whether direct HSC-stroma contact is required for this stem cell maintenance. Adult bone marrow ckit+ Ly-6C- side population (K6-SP) cells and stromal cells were cocultured under contact or noncontact conditions. These experiments showed that HSCs were maintained for at least 4 weeks in culture and that direct contact between HSCs and stromal cells was not required. To find out which factors might be involved in HSC maintenance, we compared the gene expression profile of EL08-1D2 and UG26-1B6 with four HSC-nonsupportive clones. We found that EL08-1D2 and UG26-1B6 both expressed 21 genes at a higher level, including the putative secreted factors fibroblast growth factor-7, insulin-like growth factor-binding proteins 3 and 4, pleiotrophin, pentaxin-related, and thrombospondin 2, whereas 11 genes, including GPX-3 and HSP27, were expressed at a lower level. In summary, we show for the first time long-term maintenance of adult bone marrow HSCs in stroma noncontact cultures and identify some secreted molecules that may be involved in this support.

Animals↗