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Biomedical subjects

John N Weinstein

Publications and source records attributed to John N Weinstein.

8 recordsLinked to original sources
PubMed2003

Human apurinic/apyrimidinic endonuclease (Ape1) and its N-terminal truncated form (AN34) are involved in DNA fragmentation during apoptosis.

We previously isolated a 34-kDa nuclease (AN34) from apoptotic human leukemia cells. Here, we identify AN34 as an N-terminally truncated form of human AP endonuclease (Ape1) lacking residues 1-35 (delta35-Ape1). Although Ape1 has hitherto been considered specific for damaged DNA (specific to AP site), recombinant AN34 (delta35-Ape1) possesses significant endonuclease activity on undamaged (normal) DNA and in chromatin. AN34 also displays enhanced 3'-5' exonuclease activity. Caspase-3 activates AN34 in a cell-free system, although caspase-3 cannot cleave Ape1 directly in vitro. We also found that Ape1 itself preferentially cleaves damaged chromatin DNA isolated from cells treated with apoptotic stimuli and that silencing of Ape1 expression decreases apoptotic DNA fragmentation in DFF40/CAD-deficient cells. Thus, we propose that AN34 and Ape1 participate in the process of chromatin fragmentation during apoptosis.

Amino Acid Sequence↗
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

MatchMiner: a tool for batch navigation among gene and gene product identifiers.

MatchMiner is a freely available program package for batch navigation among gene and gene product identifier types commonly encountered in microarray studies and other forms of 'omic' research. The user inputs a list of gene identifiers and then uses the Merge function to find the overlap with a second list of identifiers of either the same or a different type or uses the LookUp function to find corresponding identifiers.

Algorithms↗
PubMed2003

GoMiner: a resource for biological interpretation of genomic and proteomic data.

We have developed GoMiner, a program package that organizes lists of 'interesting' genes (for example, under- and overexpressed genes from a microarray experiment) for biological interpretation in the context of the Gene Ontology. GoMiner provides quantitative and statistical output files and two useful visualizations. The first is a tree-like structure analogous to that in the AmiGO browser and the second is a compact, dynamically interactive 'directed acyclic graph'. Genes displayed in GoMiner are linked to major public bioinformatics resources.

Computer Graphics↗
PubMed2003

Apoptotic susceptibility of cancer cells selected for camptothecin resistance: gene expression profiling, functional analysis, and molecular interaction mapping.

To study the molecular mechanisms by which drug resistance develops, we compared DU145 humanprostate cancer cells with a subline selected for resistance to camptothecin. Differences in gene expression level were assessed by hybridizing the two cell types against each other using quadruplicate "Oncochip" cDNA microarrays that included 1648 cancer-related genes. Expression levels differing by a factor of >1.5 were detected for 181 of the genes. These differences were judged statistically reliable on the basis of a stratum-adjusted Kruskal-Wallis test, after taking into account a dye-dependent variable. The 181 expression-altered genes included a larger than expected number of the "apoptosis-related" genes (P = 0.04). To assess whether this observation reflected a generalized resistance of RCO.1 to apoptosis, we exposed the cells to a range of stresses (cisplatin, staurosporine, UV, ionizing radiation, and serum starvation) and found greatly reduced apoptotic responses for RC0.1 (relative to DU145) using flow cytometric Annexin V and terminal deoxynucleotidyl transferase-mediated nick end labeling assays. We next examined the apoptosis-related genes in the context of a molecular interaction map and found expression differences in the direction "expected" on the basis of the apoptosis-resistance of RC0.1 for BAD, caspase-6, and genes that signal via the Akt pathway. Exposure of the cells to wortmannin, an inhibitor of the Akt effector phosphatidylinositol 3-kinase, provided functional support for involvement of the Akt pathway. However, closer examination of the molecular interaction map revealed a paradox: many of the expression differences observed for apoptosis-related genes were in the direction "contrary" to that expected given the resistance of RC0.1. The map indicated that most of these unexpected expression differences were associated with genes involved in the nuclear factor kappa B and transforming growth factor beta pathways. Overall, the patterns that emerged suggested a two-step model for the selection process that led to resistance in RC0.1 cells. The first hypothesized step would involve a decrease in apoptotic susceptibility through changes in the apoptosis-control machinery associated with the Bcl-2 and caspase gene families, and also in antiapoptotic pathways operating through Akt/PKB. The second step would involve changes in multifunctional upstream genes (including some genes in the nuclear factor kappa B and transforming growth factor beta pathways) that can facilitate apoptosis but that would also tend to contribute to cell proliferation in the presence of drug. Thus, we propose that a downstream blockade of apoptosis was "permissive" for the selection of upstream pathway changes that would otherwise have induced apoptosis. This model is analogous to one suggested previously for the relationship between oncogene function and apoptosis in carcinogenesis.

Androstadienes↗
PubMed2002

Transcriptional regulation of mitotic genes by camptothecin-induced DNA damage: microarray analysis of dose- and time-dependent effects.

cDNA microarray technology can be used to establish associations between characteristic gene expression patterns and molecular responses to drug therapy. In this study, we used cDNA microarrays of 1694 cancer-related genes to monitor the gene expression consequences of the treatment of HCT116 colon cancer cells with the topoisomerase I inhibitor camptothecin (CPT). To obtain a more homogeneous cellular response, we synchronized the cells in S-phase using aphidicolin (APH) before CPT treatment. Brief incubation with 20 and 1000 nM CPT caused reversible and irreversible G(2) arrest, respectively, and the patterns of gene expression change (with reference to untreated controls) were strikingly different at the two concentrations. Thirty-three genes, mainly divided into three groups, showed characteristic changes in the first 20 h as a consequence of treatment. Northern blots performed for five of these genes (each under eight experimental conditions) were quite consistent with the microarray results (average correlation coefficient, 0.86). Several p53-activated stress response genes were up-regulated after treatment with 1000 nM CPT or prolonged exposure to APH, but it seemed that the up-regulation did not directly cause cell cycle arrest because the up-regulation induced by prolonged treatment with APH did not prevent cell cycle progression after removal of APH. In contrast, cell cycle-dependent up-regulation of a group of mitosis-related genes was delayed or blocked after CPT treatments. The interrupted up-regulation of this group of genes was directly associated with G(2) arrest. In addition, we observed down-regulation of gene expression in cells that were recovering from cell cycle delay. The observations reported here suggest a fundamental difference at the gene expression level between the molecular mechanism of reversible G(2) delay that follows mild DNA damage and the mechanism of permanent G(2) arrest that follows more extensive DNA damage.

Aphidicolin↗
PubMed2002

'Omic' and hypothesis-driven research in the molecular pharmacology of cancer.

Given the layered, evolutionary complexity of biological systems, it will not be possible to understand them comprehensively on the basis of hypothesis-driven research alone. Likewise, it will not be possible to do so solely through 'omic' studies of genes, proteins and other molecules in aggregate. The two modes of research are complementary and synergistic. The twin complexities of cancer biology and drug discovery demand such synergy as a basis for the next generation of oncopharmacomic research.

Animals↗