V De Laurenzi

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

Human ΔNp73 regulates a dominant negative feedback loop for TAp73 and p53

Inactivation of the tumour suppressor p53 is the most common defect in cancer cells. p53 is a sequence specific transcription factor that is activated in response to various forms of genotoxic stress to induce cell cycle arrest and apoptosis. Induction of p53 is subjected to complex and strict control through several pathways, as it will often determine cellular fate. The p73 protein shares strong structural and functional similarities with p53 such as the potential to activate p53 responsive genes and the ability to induce apoptosis. In addition to alternative splicing at the carboxyl terminus which yields several p73 isoforms, a p73 variant lacking the N-terminal transactivation domain (ΔNp73) was described in mice. In this study, we report the cloning and characterisation of the human ΔNp73 isoforms, their regulation by p53 and their possible role in carcinogenesis. As in mice, human ΔNp73 lacks the transactivation domain and starts with an alternative exon (exon 3′). Its expression is driven by a second promoter located in a genomic region upstream of this exon, supporting the idea of two independently regulated proteins, derived from the same gene. As anticipated, ΔNp73 is capable of regulating TAp73 and p53 function since it is able to block their transactivation activity and their ability to induce apoptosis. Interestingly, expression of the ΔNp73 is strongly up-regulated by the TA isoforms and by p53, thus creating a feedback loop that tightly regulates the function of TAp73 and more importantly of p53. The regulation of ΔNp73 is exerted through a p53 responsive element located on the ΔN promoter. Expression of ΔNp73 not only regulates the function of p53 and TAp73 but also shuts off its own expression, once again finely regulating the whole system. Our data also suggest that increased expression of ΔNp73, functionally inactivating p53, could be involved in tumorogenesis. An extensive analysis of the expression pattern of ΔNp73 in primary tumours would clarify this issue.

'Tissue' transglutaminase ablation reduces neuronal death and prolongs survival in a mouse model of Huntington's disease.

By crossing Huntingtons disease (HD) R6/1 transgenic mice with ‘tissue’ transglutaminase (TG2) knock-out mice, we have demonstrated that this multifunctional enzyme plays an important role in the neuronal death characterising this disorder in vivo. In fact, a large reduction in cell death is observed in R6/1, TG2−/− compared with R6/1 transgenic mice. In addition, we have shown that the formation of neuronal intranuclear inclusions (NII) is potentiated in absence of the ‘tissue’ transglutaminase. These phenomena are paralleled by a significant improvement both in motor performances and survival of R6/1, TG2−/− versus R6/1 mice. Taken together these findings suggest an important role for tissue transglutaminase in the regulation of neuronal cell death occurring in Huntingtons disease.

Differential roles of p63 isoforms in epidermal development : selective genetic complementation in p63 null mice

Epidermal development requires the transcription factor p63, as p63−/− mice are born dead, without skin. The gene expresses two proteins, one with an amino-terminal transactivation domain (TAp63) and one without (ΔNp63), although their relative contribution to epidermal development is unknown. To address this issue, we reintroduced TAp63α and/or ΔNp63α under the K5 promoter into p63−/− mice by in vivo genetic complementation. Whereas p63−/− and p63−/−;TA mice showed extremely rare patches of poorly differentiated keratinocytes, p63−/−;ΔN mice showed significant epidermal basal layer formation. Double TAp63α/ΔNp63α complementation showed greater patches of differentiated skin; at the ultrastructural level, there was clear reformation of a distinct basal membrane and hemidesmosomes. At the molecular level, ΔNp63 regulated expression of genes characteristic of the basal layer (K14), interacting (by Chip, luc assay) with the third p53 consensus site. Conversely, TAp63 transcribed the upper layers genes (Ets-1, K1, transglutaminases, involucrin). Therefore, the two p63 isoforms appear to play distinct cooperative roles in epidermal formation.

BAG3: a multifaceted protein that regulates major cell pathways

Bcl2-associated athanogene 3 (BAG3) protein is a member of BAG family of co-chaperones that interacts with the ATPase domain of the heat shock protein (Hsp) 70 through BAG domain (110–124 amino acids). BAG3 is the only member of the family to be induced by stressful stimuli, mainly through the activity of heat shock factor 1 on bag3 gene promoter. In addition to the BAG domain, BAG3 contains also a WW domain and a proline-rich (PXXP) repeat, that mediate binding to partners different from Hsp70. These multifaceted interactions underlie BAG3 ability to modulate major biological processes, that is, apoptosis, development, cytoskeleton organization and autophagy, thereby mediating cell adaptive responses to stressful stimuli. In normal cells, BAG3 is constitutively present in a very few cell types, including cardiomyocytes and skeletal muscle cells, in which the protein appears to contribute to cell resistance to mechanical stress. A growing body of evidence indicate that BAG3 is instead expressed in several tumor types. In different tumor contexts, BAG3 protein was reported to sustain cell survival, resistance to therapy, and/or motility and metastatization. In some tumor types, down-modulation of BAG3 levels was shown, as a proof-of-principle, to inhibit neoplastic cell growth in animal models. This review attempts to outline the emerging mechanisms that can underlie some of the biological activities of the protein, focusing on implications in tumor progression.

Additional complexity in p73: Induction by mitogens in lymphoid cells and identification of two new splicing variants ε and ζ [2]

Additional complexity in p73: induction by mitogens in lymphoid cells and identification of two new splicing variants e and ζ

Structure, function and regulation of p63 and p73.

The p53 tumor suppressor gene is one of the most frequently mutated genes in human cancers.* p53 is a sequence-specific transcription factor and plays a critical role in activating the expression of genes involved in cell cycle arrest or apoptosis under conditions of genotoxic stress., For over two decades, p53 was thought to be the only gene of its kind in the vertebrate genomes. This strong conviction, which was widely accepted in the p53 field, has now been proven to be incorrect. Two genes, referred to as p63 and p73, have been found to encode proteins that share a significant amino-acid identity in the transactivation domain, the DNA binding domain, and the oligomerization domain with p53. In the short period since their cloning, a number of investigators have reported on the structure, the function and the regulation of p63 and p73. This review summarizes the current information on the p63 and the p73 genes, with a focus on the differences between the three members in this newly defined p53-gene family.

FLASH is required for histone transcription and S-phase progression

Cajal bodies are nuclear subdomains that are involved in maturation of small ribonucleoproteins and frequently associate with small nuclear RNA and histone gene clusters in interphase cells. We have recently identified FADD-like IL-1β-converting enzyme (FLICE) associated huge protein (FLASH) as an essential component of Cajal bodies. Here we show that FLASH associates with nuclear protein, ataxia-telangiectasia, a component of the cell-cycle-dependent histone gene transcription machinery. Reduction of FLASH expression by RNA interference results in disruption of the normal Cajal body architecture and relocalization of nuclear protein, ataxia-telangiectasia. Furthermore, FLASH down-regulation results in a clear reduction of histone transcription and a dramatic S-phase arrest of the cell cycle. Chromatin immunoprecipitation reveals that FLASH interacts with histone gene promoter sequences. These results identify FLASH as an important component of the machinery required for histone precursor mRNA expression and cell-cycle progression.

p53: 25 years of research and more questions to answer

1 Department of Surgery and Molecular Oncology, Ninewells Hospital and Medical School, Cancer Research UK, Cell Transformation Research Group, University of Dundee, Dundee DD1 9SY, UK 2 Biochemistry Laboratory, IDI-IRCCS, c/o Department of Experimental Medicine and Biochemical Sciences, University of Rome ‘Tor Vergata’, 00133 Rome, Italy 3 MRC Toxicology Unit, Hodgkin Building, University of Leicester, PO Box 138, Lancaster Road, Leicester LE1 9HN, UK * Corresponding author: D. Lane. E-mail: j.bourdon@dundee.ac.uk

FLASH is an essential component of Cajal bodies

Cajal bodies are small nuclear organelles with a number of nuclear functions. Here we show that FLICE-associated huge protein (FLASH), originally described as a component of the apoptosis signaling pathway, is mainly localized in Cajal bodies and is essential for their structure. Reduction in FLASH expression by short hairpin RNA results in disruption of the normal architecture of the Cajal body and relocalization of its components. Because the function of FLASH in the apoptosis receptor signaling pathway has been strongly questioned, we have now identified a clear function for this protein.