DNA microarrays identification of primary and secondary target genes regulated by p53.
TL;DR: The results suggest that p53 activates concerted opposing signals and exerts its effect through a diverse network of transcriptional changes that collectively alter the cell phenotype in response to stress.
Abstract: The transcriptional program regulated by the tumor suppressor p53 was analysed using oligonucleotide microarrays. A human lung cancer cell line that expresses the temperature sensitive murine p53 was utilized to quantitate mRNA levels of various genes at diAerent time points after shifting the temperature to 328C. Inhibition of protein synthesis by cycloheximide (CHX) was used to distinguish between primary and secondary target genes regulated by p53. In the absence of CHX, 259 and 125 genes were up or down-regulated respectively; only 38 and 24 of these genes were up and down-regulated by p53 also in the presence of CHX and are considered primary targets in this cell line. Cluster analysis of these data using the super paramagnetic clustering (SPC) algorithm demonstrate that the primary genes can be distinguished as a single cluster among a large pool of p53 regulated genes. This procedure identified additional genes that co-cluster with the primary targets and can also be classified as such genes. In addition to cell cycle (e.g. p21, TGF-b, Cyclin E) and apoptosis (e.g. Fas, Bak, IAP) related genes, the primary targets of p53 include genes involved in many aspects of cell function, including cell adhesion (e.g. Thymosin, Smoothelin), signaling (e.g. H-Ras, Diacylglycerol kinase), transcription (e.g. ATF3, LISCH7), neuronal growth (e.g. Ninjurin, NSCL2) and DNA repair (e.g. BTG2, DDB2). The results suggest that p53 activates concerted opposing signals and exerts its eAect through a diverse network of transcriptional changes that collectively alter the cell phenotype in response to stress. Oncogene (2001) 20, 2225‐2234.
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Daniel C. Koboldt1, Robert S. Fulton1, Michael D. McLellan1, Heather Schmidt1 +352 more•Institutions (35)
TL;DR: The ability to integrate information across platforms provided key insights into previously defined gene expression subtypes and demonstrated the existence of four main breast cancer classes when combining data from five platforms, each of which shows significant molecular heterogeneity.
Abstract: We analysed primary breast cancers by genomic DNA copy number arrays, DNA methylation, exome sequencing, messenger RNA arrays, microRNA sequencing and reverse-phase protein arrays. Our ability to integrate information across platforms provided key insights into previously defined gene expression subtypes and demonstrated the existence of four main breast cancer classes when combining data from five platforms, each of which shows significant molecular heterogeneity. Somatic mutations in only three genes (TP53, PIK3CA and GATA3) occurred at >10% incidence across all breast cancers; however, there were numerous subtype-associated and novel gene mutations including the enrichment of specific mutations in GATA3, PIK3CA and MAP3K1 with the luminal A subtype. We identified two novel protein-expression-defined subgroups, possibly produced by stromal/microenvironmental elements, and integrated analyses identified specific signalling pathways dominant in each molecular subtype including a HER2/phosphorylated HER2/EGFR/phosphorylated EGFR signature within the HER2-enriched expression subtype. Comparison of basal-like breast tumours with high-grade serous ovarian tumours showed many molecular commonalities, indicating a related aetiology and similar therapeutic opportunities. The biological finding of the four main breast cancer subtypes caused by different subsets of genetic and epigenetic abnormalities raises the hypothesis that much of the clinically observable plasticity and heterogeneity occurs within, and not across, these major biological subtypes of breast cancer.
9,355 citations
TL;DR: Understanding the complex mechanisms that regulate whether or not a cell dies in response to p53 will ultimately contribute to the development of therapeutic strategies to repair the apoptotic p53 response in cancers.
Abstract: Compared with many normal tissues, cancer cells are highly sensitized to apoptotic signals, and survive only because they have acquired lesions — such as loss of p53 — that prevent or impede cell death. We are now beginning to understand the complex mechanisms that regulate whether or not a cell dies in response to p53 — insights that will ultimately contribute to the development of therapeutic strategies to repair the apoptotic p53 response in cancers.
3,242 citations
TL;DR: The various signaling pathways known to be activated in response to oxidative stress in mammalian cells, the mechanisms leading to their activation, and their roles in influencing cell survival are discussed.
Abstract: Reactive oxygen species (ROS), whether produced endogenously as a consequence of normal cell functions or derived from external sources, pose a constant threat to cells living in an aerobic environment as they can result in severe damage to DNA, protein, and lipids. The importance of oxidative damage to the pathogenesis of many diseases as well as to degenerative processes of aging has becoming increasingly apparent over the past few years. Cells contain a number of antioxidant defenses to minimize fluctuations in ROS, but ROS generation often exceeds the cell's antioxidant capacity, resulting in a condition termed oxidative stress. Host survival depends upon the ability of cells and tissues to adapt to or resist the stress, and repair or remove damaged molecules or cells. Numerous stress response mechanisms have evolved for these purposes, and they are rapidly activated in response to oxidative insults. Some of the pathways are preferentially linked to enhanced survival, while others are more frequently associated with cell death. Still others have been implicated in both extremes depending on the particular circumstances. In this review, we discuss the various signaling pathways known to be activated in response to oxidative stress in mammalian cells, the mechanisms leading to their activation, and their roles in influencing cell survival. These pathways constitute important avenues for therapeutic interventions aimed at limiting oxidative damage or attenuating its sequelae.
2,222 citations
TL;DR: A robust approach is described that couples chromatin immunoprecipitation (ChIP) with the paired-end ditag (PET) sequencing strategy for unbiased and precise global localization of transcription-factor binding sites (TFBS).
1,180 citations
Cites background from "DNA microarrays identification of p..."
...In the past decade, numerous efforts have been made to identify p53-targeted genes through various gene-expression techniques, including microarray and SAGE combined with bioinformatics tools (Yu et al., 1999; Zhao et al., 2000; Kannan et al., 2001; Kho et al., 2004)....
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TL;DR: This review covers applications of microarray devices and systems for pharmacogenomic research and drug discovery, infectious and genetic disease and cancer diagnostics, and forensic and genetic identification purposes.
Abstract: ▪ Abstract In this review, recent advances in DNA microarray technology and their applications are examined. The many varieties of DNA microarray or DNA chip devices and systems are described along with their methods for fabrication and their use. This includes both high-density microarrays for high-throughput screening applications and lower-density microarrays for various diagnostic applications. The methods for microarray fabrication that are reviewed include various inkjet and microjet deposition or spotting technologies and processes, in situ or on-chip photolithographic oligonucleotide synthesis processes, and electronic DNA probe addressing processes. The DNA microarray hybridization applications reviewed include the important areas of gene expression analysis and genotyping for point mutations, single nucleotide polymorphisms (SNPs), and short tandem repeats (STRs). In addition to the many molecular biological and genomic research uses, this review covers applications of microarray devices and sys...
1,122 citations
Cites background from "DNA microarrays identification of p..."
...The following is a compilation of microarray publications on some of the broad areas of application: general applications of microarrays for gene expression analysis (179–181); gene expression analysis on high-density microarrays (182, 183); gene expression analysis for cancer (184–191); gene expression analysis for drug discovery, metabolism, and toxicity (192, 193); gene expression analysis for neuroscience applications (194–196); gene expression analysis for microbiological and infectious disease (197–200); use of microarrays for genotyping (201–204); general articles on use of microarrays for cancer (205, 206); infectious disease diagnostics (207–209); use of microarrays for disease diagnostics (210–212); and applications of microarrays for plant biology (213–215)....
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TL;DR: The author regrets the lack of citations for many important observations mentioned in the text, but their omission is made necessary by restrictions in the preparation of review manuscripts.
7,653 citations
"DNA microarrays identification of p..." refers background in this paper
...This may be the basis for p53 protection of cells against DNA damage and various stress conditions that lead usually to growth arrest or apoptosis (Levine, 1997)....
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TL;DR: It is proposed that the Mdm2-promoted degradation of p53 provides a new mechanism to ensure effective termination of the p53 signal.
Abstract: The p53 tumour-suppressor protein exerts antiproliferative effects, including growth arrest and apoptosis, in response to various types of stress. The activity of p53 is abrogated by mutations that occur frequently in tumours, as well as by several viral and cellular proteins. The Mdm2 oncoprotein is a potent inhibitor of p53. Mdm2 binds the transcriptional activation domain of p53 and blocks its ability to regulate target genes and to exert antiproliferative effects. On the other hand, p53 activates the expression of the mdm2 gene in an autoregulatory feedback loop. The interval between p53 activation and consequent Mdm2 accumulation defines a time window during which p53 exerts its effects. We now report that Mdm2 also promotes the rapid degradation of p53 under conditions in which p53 is otherwise stabilized. This effect of Mdm2 requires binding of p53; moreover, a small domain of p53, encompassing the Mdm2-binding site, confers Mdm2-dependent detstabilization upon heterologous proteins. Raised amounts of Mdm2 strongly repress mutant p53 accumulation in tumour-derived cells. During recovery from DNA damage, maximal Mdm2 induction coincides with rapid p53 loss. We propose that the Mdm2-promoted degradation of p53 provides a new mechanism to ensure effective termination of the p53 signal.
4,311 citations
"DNA microarrays identification of p..." refers background in this paper
...It was shown previously that at 328C p53Val135 is relocalized to the nucleus (Ginsberg et al., 1991) and that it may therefore be protected from degradation in the presence of CHX due to a lack of nuclear exclusion and the absence of hDM2 synthesis (Haupt et al., 1997; Kubbutat et al., 1997)....
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TL;DR: It is shown that interaction with Mdm2 can also result in a large reduction in p53 protein levels through enhanced proteasome-dependent degradation, which may contribute to the maintenance of low p53 concentrations in normal cells.
Abstract: The tumour-suppressor p53 is a short-lived protein that is maintained at low, often undetectable, levels in normal cells. Stabilization of the protein in response to an activating signal, such as DNA damage, results in a rapid rise in p53 levels and subsequent inhibition of cell growth. Tight regulation of p53 function is critical for normal cell growth and development, and one mechanism by which p53 function is controlled is through interaction with the Mdm2 protein. Mdm2 inhibits p53 cell-cycle arrest and apoptic functions and we show here that interaction with Mdm2 can also result in a large reduction in p53 protein levels through enhanced proteasome-dependent degradation. Endogenous levels of Mdm2 are sufficient to regulate p53 stability, and overexpression of Mdm2 can reduce the amount of endogenous p53. Because mdm2 is transcriptionally activated by p53, this degradative pathway may contribute to the maintenance of low p53 concentrations in normal cells. Furthermore, mechanisms regulating the Mdm2-induced degradation of p53 may play a role in controlling the extent and duration of the p53 response.
3,298 citations
"DNA microarrays identification of p..." refers background in this paper
...It was shown previously that at 328C p53Val135 is relocalized to the nucleus (Ginsberg et al., 1991) and that it may therefore be protected from degradation in the presence of CHX due to a lack of nuclear exclusion and the absence of hDM2 synthesis (Haupt et al., 1997; Kubbutat et al., 1997)....
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TL;DR: The author would like to thank S. H. Roan for all her help and members of the Massague laboratory for insightful discussions.
2,303 citations
"DNA microarrays identification of p..." refers background in this paper
...Other genes in our list of primary genes like endoglin (a coreceptor of TGF-b) and versican may also be part of the TGF-b activating system (Massague et al., 2000)....
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...The p15 is an inhibitor of cdk4 and cdk6 and its overexpression may lead to G1 arrest (Massague et al., 2000), thus TGF-b may behave as a tumor suppressor (Tang et al., 1998)....
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TL;DR: In this paper, a consensus binding site with a striking internal symmetry was identified, consisting of two copies of the 10 base pair motif 5′-PuPuC(A/T)(T/A)GPyPyPy-3′ separated by 0-13 base pairs.
Abstract: Recent experiments have suggested that p53 action may be mediated through its inter action with DNA. We have now identified 18 human genomic clones that bind to p53 in vitro. Precise mapping of the binding sequences within these clones revealed a consensus binding site with a striking internal symmetry, consisting of two copies of the 10 base pair motif 5′-PuPuPuC(A/T)(T/A)GPyPyPy-3′ separated by 0-13 base pairs. One copy of the motif was insufficient for binding, and subtle alterations of the motif, even when present in multiple copies, resulted in loss of affinity for p53. Mutants of p53, representing each of the four “hot spots” frequently altered in human cancers, failed to bind to the consensus dimer. These results define the DNA sequence elements with which p53 interacts in vitro and which may be important for p53 action in vivo.
1,996 citations