Distinct p53 transcriptional programs dictate acute DNA-damage responses and tumor suppression.

The molecular basis for p53-mediated tumor suppression remains unclear. Here, to elucidate mechanisms of p53 tumor suppression, we use knockin mice expressing an allelic series of p53 transcriptional activation mutants. Microarray analysis reveals that one mutant, p53(25,26), is severely compromised for transactivation of most p53 target genes, and, moreover, p53(25,26) cannot induce G(1)-arrest or apoptosis in response to acute DNA damage. Surprisingly, p53(25,26) retains robust activity in senescence and tumor suppression, indicating that efficient transactivation of the majority of known p53 targets is dispensable for these pathways. In contrast, the transactivation-dead p53(25,26,53,54) mutant cannot induce senescence or inhibit tumorigenesis, like p53 nullizygosity. Thus, p53 transactivation is essential for tumor suppression but, intriguingly, in association with a small set of novel p53 target genes. Together, our studies distinguish the p53 transcriptional programs involved in acute DNA-damage responses and tumor suppression-a critical goal for designing therapeutics that block p53-dependent side effects of chemotherapy without compromising p53 tumor suppression.

Global genomic profiling reveals an extensive p53-regulated autophagy program contributing to key p53 responses.

The mechanisms by which the p53 tumor suppressor acts remain incompletely understood. To gain new insights into p53 biology, we used high-throughput sequencing to analyze global p53 transcriptional networks in primary mouse embryo fibroblasts in response to DNA damage. Chromatin immunoprecipitation sequencing reveals 4785 p53-bound sites in the genome located near 3193 genes involved in diverse biological processes. RNA sequencing analysis shows that only a subset of p53-bound genes is transcriptionally regulated, yielding a list of 432 p53-bound and regulated genes. Interestingly, we identify a host of autophagy genes as direct p53 target genes. While the autophagy program is regulated predominantly by p53, the p53 family members p63 and p73 contribute to activation of this autophagy gene network. Induction of autophagy genes in response to p53 activation is associated with enhanced autophagy in diverse settings and depends on p53 transcriptional activity. While p53-induced autophagy does not affect cell cycle arrest in response to DNA damage, it is important for both robust p53-dependent apoptosis triggered by DNA damage and transformation suppression by p53. Together, our data highlight an intimate connection between p53 and autophagy through a vast transcriptional network and indicate that autophagy contributes to p53-dependent apoptosis and cancer suppression.

Inappropriate p53 activation during development induces features of CHARGE syndrome.

CHARGE syndrome is a multiple anomaly disorder in which patients present with a variety of phenotypes, including ocular coloboma, heart defects, choanal atresia, retarded growth and development, genitourinary hypoplasia and ear abnormalities. Despite 70-90% of CHARGE syndrome cases resulting from mutations in the gene CHD7, which encodes an ATP-dependent chromatin remodeller, the pathways underlying the diverse phenotypes remain poorly understood. Surprisingly, our studies of a knock-in mutant mouse strain that expresses a stabilized and transcriptionally dead variant of the tumour-suppressor protein p53 (p53(25,26,53,54)), along with a wild-type allele of p53 (also known as Trp53), revealed late-gestational embryonic lethality associated with a host of phenotypes that are characteristic of CHARGE syndrome, including coloboma, inner and outer ear malformations, heart outflow tract defects and craniofacial defects. We found that the p53(25,26,53,54) mutant protein stabilized and hyperactivated wild-type p53, which then inappropriately induced its target genes and triggered cell-cycle arrest or apoptosis during development. Importantly, these phenotypes were only observed with a wild-type p53 allele, as p53(25,26,53,54)(/-) embryos were fully viable. Furthermore, we found that CHD7 can bind to the p53 promoter, thereby negatively regulating p53 expression, and that CHD7 loss in mouse neural crest cells or samples from patients with CHARGE syndrome results in p53 activation. Strikingly, we found that p53 heterozygosity partially rescued the phenotypes in Chd7-null mouse embryos, demonstrating that p53 contributes to the phenotypes that result from CHD7 loss. Thus, inappropriate p53 activation during development can promote CHARGE phenotypes, supporting the idea that p53 has a critical role in developmental syndromes and providing important insight into the mechanisms underlying CHARGE syndrome.

Full p53 transcriptional activation potential is dispensable for tumor suppression in diverse lineages.

Over half of all human cancers, of a wide variety of types, sustain mutations in the p53 tumor suppressor gene. Although p53 limits tumorigenesis through the induction of apoptosis or cell cycle arrest, its molecular mechanism of action in tumor suppression has been elusive. The best-characterized p53 activity in vitro is as a transcriptional activator, but the identification of numerous additional p53 biochemical activities in vitro has made it unclear which mechanism accounts for tumor suppression. Here, we assess the importance of transcriptional activation for p53 tumor suppression function in vivo in several tissues, using a knock-in mouse strain expressing a p53 mutant compromised for transcriptional activation, p53(25,26). p53(25,26) is severely impaired for the transactivation of numerous classical p53 target genes, including p21, Noxa, and Puma, but it retains the ability to activate a small subset of p53 target genes, including Bax. Surprisingly, p53(25,26) can nonetheless suppress tumor growth in cancers derived from the epithelial, mesenchymal, central nervous system, and lymphoid lineages. Therefore, full transactivation of most p53 target genes is dispensable for p53 tumor suppressor function in a range of tissue types. In contrast, a transcriptional activation mutant that is completely defective for transactivation, p53(25,26,53,54), fails to suppress tumor development. These findings demonstrate that transcriptional activation is indeed broadly critical for p53 tumor suppressor function, although this requirement reflects the limited transcriptional activity observed with p53(25,26) rather than robust transactivation of a full complement of p53 target genes.

Chemical Screening in Zebrafish.

Phenotypic small molecule screens in zebrafish have gained popularity as an unbiased approach to probe biological processes. In this chapter we outline basic methods for performing chemical screens with larval zebrafish including breeding large numbers of embryos, plating larval fish into multi-well dishes, and adding small molecules to these wells. We also highlight important considerations when designing and interpreting the results of a phenotypic screen and possible follow-up approaches, including popular methods used to identify the mechanism of action of a chemical compound.

Analysis of p53 transactivation domain mutants reveals Acad11 as a metabolic target important for p53 pro-survival function.

The p53 tumor suppressor plays a key role in maintaining cellular integrity. In response to diverse stress signals, p53 can trigger apoptosis to eliminate damaged cells or cell-cycle arrest to enable cells to cope with stress and survive. However, the transcriptional networks underlying p53 pro-survival function are incompletely understood. Here, we show that in oncogenic-Ras-expressing cells, p53 promotes oxidative phosphorylation (OXPHOS) and cell survival upon glucose starvation. Analysis of p53 transcriptional activation domain mutants reveals that these responses depend on p53 transactivation function. Using gene expression profiling and ChIP-seq analysis, we identify several p53-inducible fatty acid metabolism-related genes. One such gene, Acad11, encoding a protein involved in fatty acid oxidation, is required for efficient OXPHOS and cell survival upon glucose starvation. This study provides new mechanistic insight into the pro-survival function of p53 and suggests that targeting this pathway may provide a strategy for therapeutic intervention based on metabolic perturbation.

Risk factors, prognostic indicators, and outcome of pyothorax in cats: 80 cases (1986-1999).

OBJECTIVE: To identify risk factors associated with development of pyothorax in cats, assess survival rates for cats that are treated, determine prognostic indicators, and determine recurrence rates. DESIGN: Retrospective study. ANIMALS: 80 cats with pyothorax and 212 control cats. PROCEDURE: History; month of evaluation; physical examination findings; results of hematologic, serum biochemical, and retrovirus testing; radiographic findings; outcome; recurrence rate; and necropsy findings were recorded. For control cats, age, sex, breed, indoor versus outdoor status, vaccination history, and single- versus multi-cat household status were recorded. RESULTS: Cats from multi-cat households were 3.8 times as likely (95% confidence interval, 1.9 to 8.2) to develop pyothorax, compared with cats from single-cat households. Indoor or outdoor status was not a risk factor. Cats with pyothorax were significantly younger (mean, 3.83 +/- 3.43 years) than controls (mean, 5.62 +/- 5.27 years). Nonsurvivors had significantly lower heart rates than survivors. Hypersalivation was significantly more common in nonsurvivors (11/39; 26.8%) than survivors (1/39; 3%). Overall, 48.8% (39/80) of cats survived. When cats that were euthanatized without treatment were excluded from analyses, the survival rate was 66.1% (39/59). Pyothorax recurred in 1 of 17 cats for which follow-up information was obtained. CONCLUSIONS AND CLINICAL RELEVANCE: Cats with pyothorax that received treatment had a fair to good prognosis, with low recurrence rates in survivors. Hypersalivation and low heart rate were associated with worse clinical outcome. Cats with pyothorax were likely to come from multi-cat households.

Local and systemic complications in a cat after poorly performed dental extractions.

This case report describes a cat that was presented with infected oral tissues and signs of systemic inflammatory response. Incomplete dental extractions had been performed 10-days earlier During a second dental procedure, 22 tooth root remnants were removed. Diabetic ketoacidosis and possible thromboembolism complicated the cat's recovery period After 13-days of hospitalization, the patient was stable enough to be sent home. Postoperative examinations at 1, 3, 6, 12, 16, 24, and 52-weeks indicated continued improvement with moderate glycemic control and chronic but stable renal failure. The patient died from further complications of diabetic ketoacidosis 20-months following root remnant extractions.

Intravital correlated microscopy reveals differential macrophage and microglial dynamics during resolution of neuroinflammation.

Many brain diseases involve activation of resident and peripheral immune cells to clear damaged and dying neurons. Which immune cells respond in what way to cues related to brain disease, however, remains poorly understood. To elucidate these in vivo immunological events in response to brain cell death we used genetically targeted cell ablation in zebrafish. Using intravital microscopy and large-scale electron microscopy, we defined the kinetics and nature of immune responses immediately following injury. Initially, clearance of dead cells occurs by mononuclear phagocytes, including resident microglia and macrophages of peripheral origin, whereas amoeboid microglia are exclusively involved at a later stage. Granulocytes, on the other hand, do not migrate towards the injury. Remarkably, following clearance, phagocyte numbers decrease, partly by phagocyte cell death and subsequent engulfment of phagocyte corpses by microglia. Here, we identify differential temporal involvement of microglia and peripheral macrophages in clearance of dead cells in the brain, revealing the chronological sequence of events in neuroinflammatory resolution. Remarkably, recruited phagocytes undergo cell death and are engulfed by microglia. Because adult zebrafish treated at the larval stage lack signs of pathology, it is likely that this mode of resolving immune responses in brain contributes to full tissue recovery. Therefore, these findings suggest that control of such immune cell behavior could benefit recovery from neuronal damage.