Biomedical subjects

Peter J Munson

Publications and source records attributed to Peter J Munson.

25 records · Page 2Linked to original sources

PubMed2003

Diagnostic markers that distinguish colon and ovarian adenocarcinomas: identification by genomic, proteomic, and tissue array profiling.

Colon and ovarian cancers can be difficult to distinguish in the abdomen, and the distinction is important because it determines which drugs will be used for therapy. To identify molecular markers for that differential diagnosis, we developed a multistep protocol starting with the 60 human cancer cell lines used by the National Cancer Institute to screen for new anticancer agents. The steps included: (a) identification of candidate markers using cDNA microarrays; (b) verification of clone identities by resequencing; (c) corroboration of transcript levels using Affymetrix oligonucleotide chips; (d) quantitation of protein expression by "reverse-phase" protein microarray; and (e) prospective validation of candidate markers on clinical tumor sections in tissue microarrays. The two best candidates identified were villin for colon cancer cells and moesin for ovarian cancer cells. Because moesin stained stromal elements in both types of cancer, it would probably not have been identified as a marker if we had started with mRNA or protein profiling of bulk tumors. Villin appears at least as useful as the currently used colon cancer marker cytokeratin 20, and moesin also appears to have utility. The multistep process introduced here has the potential to produce additional markers for cancer diagnosis, prognosis, and therapy.

Adenocarcinoma↗

PubMed2003

Impact of p53 knockout and topotecan treatment on gene expression profiles in human colon carcinoma cells: a pharmacogenomic study.

To uncover transcriptional stress responses related to p53, we used cDNA microarrays (National Cancer Institute Oncochips comprising 6500 different genes) to characterize the gene expression profiles of wild-type p53 HCT-116 cells and an isogenic p53 knockout counterpart after treatment with topotecan, a specific topoisomerase I inhibitor. The use of the p53 knockout cells had the advantage over p53-overexpressing systems in that p53 activation is mediated physiologically. RNA was extracted after low (0.1 microM)- and high (1 microM)-dose topotecan at multiple time points within the first 6 h of treatment. To facilitate simultaneous study of the p53 status and pharmacological effects on gene expression, we developed a novel "cross-referenced network" experimental design and used multiple linear least squares fitting to optimize estimates of relative transcript levels in the network of experimental conditions. Approximately 10% of the transcripts were up- or down-regulated in response to topotecan in the p53+/+ cells, whereas only 1% of the transcripts changed in the p53-/- cells, indicating that p53 has a broad effect on the transcriptional response to this stress. Individual transcripts and their relationships were analyzed using clustered image maps and by a novel two-dimensional analysis/visualization, gene expression map, in which each gene expression level is represented as a function of both the genotypic/phenotypic difference (i.e., p53 status) and the treatment effect (i.e., of topotecan dose and time of exposure). Overall, drug-induced p53 activation was associated with a coherent genetic program leading to cell cycle arrest and apoptosis. We identified novel p53-induced and DNA damage-induced genes (the proapoptotic SIVA gene and a set of transforming growth factor beta-related genes). Genes induced independently of p53 included the antiapoptotic cFLIP gene and known stress genes related to the mitogen-activated protein kinase pathway and the Fos/Jun pathway. Genes that were negatively regulated by p53 included members of the antiapoptotic protein chaperone heat shock protein 70 family. Finally, among the p53-dependent genes whose expression was independent of drug treatment was S100A4, a small Ca(2+)-binding protein that has recently been implicated in p53 binding and regulation. The new experimental design and gene expression map analysis introduced here are applicable to a wide range of studies that encompass both treatment effects and genotypic or phenotypic differences.

Antineoplastic Agents↗

PubMed2003

Protein microarrays: meeting analytical challenges for clinical applications.

Protein microarrays, one emerging class of proteomic technologies, have broad applications for discovery and quantitative analysis. A rapidly expanding use of this technology is the acquisition of information about the posttranslational modifications of proteins reflecting the activity state of signal pathways and networks, and is now employed for the analysis of biopsy samples in clinical trial research.

Animals↗

PubMed2003

Spatial proximity of translocation-prone gene loci in human lymphomas.

Cancer cells frequently have disease-specific chromosome rearrangements. It is poorly understood why translocations between chromosomes recur at specific breakpoints in the genome. Here we provide evidence that higher-order spatial genome organization is a contributing factor in the formation of recurrent translocations. We show that MYC, BCL and immunoglobulin loci, which are recurrently translocated in various B-cell lymphomas, are preferentially positioned in close spatial proximity relative to each other in normal B cells. Loci in spatial proximity are non-randomly positioned towards the nuclear interior in normal B cells. This locus proximity is the consequence of higher-order genome structure rather than a property of individual genes. Our results suggest that the formation of specific translocations in human lymphomas, and perhaps other tissues, is determined in part by higher-order spatial organization of the genome.

DNA-Binding Proteins↗

PubMed2002

Conservation of relative chromosome positioning in normal and cancer cells.

Chromosomes exist in the interphase nucleus as individual chromosome territories. It is unclear to what extent chromosome territories occupy particular positions with respect to each other and how structural rearrangements, such as translocations, affect chromosome organization within the cell nucleus. Here we analyze the relative interphase positioning of chromosomes in mouse lymphoma cells compared to normal splenocytes. We show that in a lymphoma cell line derived from an ATM(-/-) mouse, two translocated chromosomes are preferentially positioned in close proximity to each other. The relative position of the chromosomes involved in these translocations is conserved in normal splenocytes. Relative positioning of chromosomes in normal splenocytes is not due to their random distribution in the interphase nucleus and persists during mitosis. These observations demonstrate that the relative arrangement of chromosomes in the interphase nucleus can be conserved between normal and cancer cells and our data support the notion that physical proximity facilitates rearrangements between chromosomes.

Animals↗

PubMed2002

Metallothionein promotes laminin-1-induced acinar differentiation in vitro and reduces tumor growth in vivo.

Laminin-1 was found previously to promote the morphological differentiation of a salivary gland cell line (HSG) into acinar-like structures with polarized nuclei (1). Here, microarray analysis showed that laminin-1 induced mainly one gene family of proteins, the metal binding metallothioneins (MTs), out of more than 10,500 cDNAs screened. Northern and protein analyses demonstrated that MT was increased some 5-10-fold by laminin-1 as early as 6 h after incubation. Cells transfected with this gene formed 3-5-fold larger acinar-like structures on exposure to laminin-1 in vitro and smaller, more differentiated tumors in vivo. We conclude that MTs are important in acinar cell morphological differentiation and may have novel functions other than metal binding.

Animals↗

PubMed2002

Both protein activation and gene expression are involved in early vascular tube formation in vitro.

PURPOSE: Gene expression and protein translation regulate and direct endothelial cell proliferation and differentiation. We initiated an unbiased global search for transcriptional changes occurring during endothelial cell vascular differentiation in vitro, focusing on genes not previously implicated in vascularization and angiogenesis. EXPERIMENTAL DESIGN: cDNA and protein from human umbilical vein endothelial cells forming vascular tubes on the basement membrane surrogate, Matrigel, were collected and subjected to a global unbiased search for alterations in expression of genes not previously linked to angiogenesis. RESULTS: Transcriptional inhibitors blocked vascular tube formation only when present within the first hour of incubation (P < 0.05). cDNA array analysis yielded 31 differentially regulated transcripts (of 5100 queried; false positive rate, 0.4%) from gene classes representing transcription, translational regulation, cell structure, and cell adhesion. mRNA levels of caldesmon, a cytoskeleton-associated protein not previously linked to angiogenesis, were markedly reduced during early tube formation. Caldesmon protein quantity was also markedly decreased as demonstrated by laser capture microdissection of tubule cells followed by immunoblotting. Strikingly, no significant changes in transcription of genes previously demonstrated to contribute to angiogenesis, invasion, or signal transduction contained on the array were observed. To investigate the possibility that posttranslational rather than transcriptional changes were involved in facilitating tube formation, we evaluated the activation status of two dominant signal pathways, RAS/mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT. A net 3-fold reduction in phospho-AKT and a 4-fold reduction in phospho-extracellular signal-regulated kinase-1/2 occurred in a transcription-independent fashion. CONCLUSIONS: These data suggest that both changes in gene expression and transcription-independent activation of signal transduction pathways may be involved in vascular tube formation. A combination of transcriptional and proteomic analysis has the potential to identify novel transcription-dependent and -independent molecular targets of angiogenesis.

Calmodulin-Binding Proteins↗