Biomedical subjects

Dik van Leenen

Publications and source records attributed to Dik van Leenen.

7 recordsLinked to original sources

PubMed2006

Dissection of a metastatic gene expression signature into distinct components.

BACKGROUND: Metastasis, the process whereby cancer cells spread, is in part caused by an incompletely understood interplay between cancer cells and the surrounding stroma. Gene expression studies typically analyze samples containing tumor cells and stroma. Samples with less than 50% tumor cells are generally excluded, thereby reducing the number of patients that can benefit from clinically relevant signatures. RESULTS: For a head-neck squamous cell carcinoma (HNSCC) primary tumor expression signature that predicts the presence of lymph node metastasis, we first show that reduced proportions of tumor cells results in decreased predictive accuracy. To determine the influence of stroma on the predictive signature and to investigate the interaction between tumor cells and the surrounding microenvironment, we used laser capture microdissection to divide the metastatic signature into six distinct components based on tumor versus stroma expression and on association with the metastatic phenotype. A strikingly skewed distribution of metastasis associated genes is revealed. CONCLUSION: Dissection of predictive signatures into different components has implications for design of expression signatures and for our understanding of the metastatic process. Compared to primary tumors that have not formed metastases, primary HNSCC tumors that have metastasized are characterized by predominant down-regulation of tumor cell specific genes and exclusive up-regulation of stromal cell specific genes. The skewed distribution agrees with poor signature performance on samples that contain less than 50% tumor cells. Methods for reducing tumor composition bias that lead to greater predictive accuracy and an increase in the types of samples that can be included are presented.

Carcinoma, Squamous Cell↗

PubMed2005

Mediator expression profiling epistasis reveals a signal transduction pathway with antagonistic submodules and highly specific downstream targets.

Mediator is an evolutionarily conserved coregulator of RNA polymerase II transcription. Microarray structure-function analysis of S. cerevisiae Mediator reveals functional antagonism between the cyclin-dependent kinase (Cdk) submodule and components from the Tail (Med15, Med2, Med3), Head (Med20, Med18), and Middle (Med31). Certain genes exhibit increased or decreased expression, depending on which subunit is deleted. Epistasis analysis with expression-profile phenotypes shows that MED2 and MED18 are downstream of CDK8. Strikingly, Cdk8-mediated modification of a single amino acid within Mediator represses the regulon of a single transcription factor, Rcs1/Aft1. Highly specific gene regulation is thought to be determined by activators and combinatorial use of cofactors. Here, subtle modification of the general transcription machinery through one of its own components is shown to determine highly specific expression patterns. Expression profiling can therefore precisely map regulatory cascades, and our findings support a role for Mediator as a direct processor of signaling pathways for determining specificity.

Chromosomal Proteins, Non-Histone↗

PubMed2005

Genome-wide analyses reveal RNA polymerase II located upstream of genes poised for rapid response upon S. cerevisiae stationary phase exit.

The resting state of eukaryotic cells (G0) is relatively uncharacterized. We have applied DNA microarray expression profiling of S. cerevisiae to reveal multiple transitions during a complete 9-day growth cycle between stationary phase (SP) exit and entry. The findings include distinct waves of transcription after the diauxic shift (DS), identification of genes active in SP, and upregulation of over 2500 genes during the first minutes of lag phase. This provides a framework for analyzing large-scale reprogramming of gene expression. Despite global repression, the general transcription machinery is found to be present in quiescent cells but is largely inactive. Genome-wide location analysis by chromatin immunoprecipitation (ChIP on chip) reveals that RNA polymerase II is more predominantly bound at intergenic regions in SP, upstream of hundreds of genes immediately induced upon exit. In contrast to current models of activation-coupled recruitment, the results show that RNA polymerase II is located and maintained upstream of many inactive genes in quiescence.

Chromatin Immunoprecipitation↗

PubMed2004

Broadly altered gene expression in blood leukocytes in essential hypertension is absent during treatment.

We assessed whether large-scale expression profiling of leukocytes of patients with essential hypertension reflects characteristics of systemic disease and whether such changes are responsive to antihypertensive therapy. Total RNA from leukocytes were obtained from untreated (n=6) and treated (n=6) hypertensive patients without apparent end-organ damage and from normotensive controls (n=9). RNA was reverse-transcribed and labeled and gene expression analyzed using a 19-K oligonucleotide microarray using dye swaps. Samples of untreated and of treated patients were pooled for each sex and compared with age- and sex-matched controls. In untreated patients, 680 genes were differentially regulated (314 up and 366 down). In the treated patients, these changes were virtually absent (4 genes up, 3 genes down). A myriad of changes was observed in pathways involved in inflammation. Inflammation-dampening interleukin receptors were decreased in expression. Intriguingly, inhibitors of cytokine signaling (the PIAS family of proteins) were differentially expressed. The expression of several genes that are involved in regulation of blood pressure were also differentially expressed: angiotensin II type 1 receptor, ANP-A receptor, endothelin-2, and 3 of the serotonin receptors were increased, whereas endothelin-converting enzyme-1 was decreased. Strikingly, virtually no changes in gene expression could be detected in hypertensive patients who had become normotensive with treatment. This observation substantiates the long-standing idea that hypertension is associated with a complex systemic response involving inflammation-related genes. Furthermore, leukocytes display differential gene expression that is of importance in blood pressure control. Importantly, treatment of blood pressure to normal values can virtually correct such disturbances.

Adult↗

PubMed2004

Twenty-two novel mutations in the lysosomal alpha-glucosidase gene (GAA) underscore the genotype-phenotype correlation in glycogen storage disease type II.

Patients with glycogen storage disease type II (GSDII, Pompe disease) suffer from progressive muscle weakness due to acid alpha-glucosidase deficiency. The disease is inherited as an autosomal recessive trait with a spectrum of clinical phenotypes. We have investigated 29 cases of GSDII and thereby identified 55 pathogenic mutations of the acid alpha-glucosidase gene (GAA) encoding acid maltase. There were 34 different mutations identified, 22 of which were novel. All of the missense mutations and two other mutations with an unpredictable effect on acid alpha-glucosidase synthesis and function were transiently expressed in COS cells. The effect of a novel splice-site mutation was investigated by real-time PCR analysis. The outcome of our analysis underscores the notion that the clinical phenotype of GSDII is largely dictated by the nature of the mutations in the GAA alleles. This genotype-phenotype correlation makes DNA analysis a valuable tool to help predict the clinical course of the disease.

Adolescent↗

PubMed2003

Monitoring global messenger RNA changes in externally controlled microarray experiments.

Expression profiling is a universal tool, with a range of applications that benefit from the accurate determination of differential gene expression. To allow normalization using endogenous transcript levels, current microarray analyses assume that relatively few transcripts vary, or that any changes that occur are balanced. When normalization using endogenous genes is carried out, changes in expression levels are calculated relative to the behaviour of most of the transcripts. This does not reflect absolute changes if global shifts in messenger RNA populations occur. Using external RNA controls, we have set up microarray experiments to monitor global changes. The levels of most mRNAs were found to change during yeast stationary phase and human heat shock when external controls were included. Even small global changes had a significant effect on the number of genes reported as being differentially expressed. This suggests that global mRNA changes occur more frequently than is assumed at present, and shows that monitoring such effects may be important for the accurate determination of changes in gene expression.

Bacillus subtilis↗

PubMed2003

Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector.

We have designed a doxycycline-regulated form of the H1 promoter of RNA polymerase III that allows the inducible knockdown of gene expression by small interfering RNAs (siRNAs). As a proof-of-principle, we have targeted beta-catenin in colorectal cancer (CRC) cells. T-cell factor (TCF) target-gene expression is induced by accumulated beta-catenin, and is the main transforming event in these cells. We have shown previously that the disruption of beta-catenin/TCF4 activity in CRC cells by the overexpression of dominant-negative TCF induces rapid G1 arrest and differentiation. Stable integration of our inducible siRNA vector allowed the rapid production of siRNAs on doxycycline induction, followed by specific downregulation of beta-catenin. In these CRC cells, TCF reporter-gene activity was inhibited, and G1 arrest and differentiation occurred. The inhibition of two other genes using this vector system shows that it should be useful for the inducible knockdown of gene expression.

Base Sequence↗