Berna S. Sayan

p63 is upstream of IKKα in epidermal development

The epidermis, the outer layer of the skin composed of keratinocytes, develops following the action of the transcription factor p63. The mouse Trp63 gene contains two promoters, driving the production of distinct proteins, one with an N-terminal trans-activation domain (TAp63) and one without (ΔNp63), although their relative contribution to epidermal development is not clearly established. To identify the relative role of p63 isoforms in relation to IKKα, also known to be essential for epithelial development, we performed both molecular and in vivo analyses using genetic complementation in mice. We found that the action of TAp63 is mediated at the molecular level by direct and indirect transactivation of IKKα and Ets-1, respectively. We also found that ΔNp63 upregulates IKKα indirectly, through GATA-3. Our data are consistent with a role for p63 directly upstream of IKKα in epithelial development.

p53 Is Cleaved by Caspases Generating Fragments Localizing to Mitochondria

The p53 tumor suppressor protein exerts most of its anti-tumorigenic activity by transcriptionally activating several pro-apoptotic genes. Accumulating evidence also suggests a transcription-independent function of p53 during apoptosis. It has recently been shown that, when activated, a fraction of p53 translocates to mitochondria, causing cytochrome c release. We now demonstrate a caspase-dependent cleavage of p53 resulting in the generation of four fragments, two of which lack a nuclear localization signal and consequently localize to cytosol. Moreover, these two fragments translocate to mitochondria and induce mitochondrial membrane depolarization in the absence of transcriptional activity. This novel feature of p53 supports the model whereby cytosolic p53 exerts major functions in apoptosis and also suggests the presence of a positive feedback loop in which activated caspases cleave p53 to augment mitochondrial membrane depolarization.

Acquired expression of transcriptionally active p73 in hepatocellular carcinoma cells.

p53 and p73 proteins activate similar target genes and induce apoptosis and cell cycle arrest. However, p53, but not p73 is considered a tumour-suppressor gene. Unlike p53, p73 deficiency in mice does not lead to a cancer-prone phenotype, and p73 gene is not mutated in human cancers, including hepatocellular carcinoma. Here we report that normal liver cells express only ΔN-p73 transcript forms giving rise to the synthesis of N-terminally truncated, transcriptionally inactive and dominant negative p73 proteins. In contrast, most hepatocellular carcinoma cells express TA-p73 transcript forms encoding full-length and transcriptionally active p73 proteins, in addition to ΔN-p73. We also show that together with the acquired expression of TA-p73, the ‘retinoblastoma pathway’ is inactivated, and E2F1-target genes including cyclin E and p14ARF are activated in hepatocellular carcinoma. However, there was no full correlation between ‘retinoblastoma pathway’ inactivation and TA-p73 expression. Most TA-p73-expressing hepatocellular carcinoma cells have also lost p53 function either by lack of expression or missense mutations. The p73 gene, encoding only ΔN-p73 protein, may function as a tumour promoter rather than a tumour suppressor in liver tissue. This may be one reason why p73 is not a mutation target in hepatocellular carcinoma.

P73 and caspase-cleaved p73 fragments localize to mitochondria and augment TRAIL-induced apoptosis

The p73 protein, a member of the p53 family, has both developmental and tumorigenic functions. Here we show that p73 is cleaved by caspase-3 and -8 both in vitro and in vivo during apoptosis elicited by DNA-damaging drugs and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor ligation. TAp73 and some of its cleavage products are localized to mitochondria. siRNA-mediated downregulation of p73 expression induced a small but significant change in the susceptibility of HCT116 cells to TRAIL-induced apoptosis. A transcription-deficient mutant of TAp73 enhanced TRAIL-induced apoptosis suggesting that p73 protein has transcription-independent functions during death receptor-mediated apoptosis. Additionally, recombinant p73 protein induced cytochrome c release from isolated mitochondria providing evidence that nonnuclear p73 may have additional functions in the progression of apoptosis.

Cleavage of the transactivation-inhibitory domain of p63 by caspases enhances apoptosis

p63 is a p53-related transcription factor. Utilization of two different promoters and alternative splicing at the C terminus lead to generation of six isoforms. The α isoforms of TAp63 and ΔNp63 contain a transactivation-inhibitory (TI) domain at the C termini, which can bind to the transactivation (TA) domain and inhibit its transcriptional activity. Consequently, TAp63α can directly inhibit its activity through an intramolecular interaction; similarly, ΔNp63α can inhibit the activity of the active TAp63 isoforms through an intermolecular interaction. In this work, we demonstrate that after induction of apoptosis, the TI domain of the p63α isoforms is cleaved by activated caspases. Cleavage of ΔNp63α relieves its inhibitory effect on the transcriptionally active p63 proteins, and the cleavage of TAp63α results in production of a TAp63 protein with enhanced transcriptional activity. In agreement with these data, generation of the N-terminal TAp63 fragment has a role in apoptosis because stable cell lines expressing wild-type TAp63 are more sensitive to apoptosis compared with cells expressing the noncleavable mutant. We also used a model system in which TAp63 expression was induced by trichostatin-A treatment in HCT116 cells. Trichostatin-A sensitized these cells to apoptosis, and this sensitization was associated with cleavage of up-regulated p63.

NAPO as a novel marker for apoptosis

Apoptosis or programmed cell death plays a pivotal role in embryonic development and maintenance of homeostasis. It is also involved in the etiology of pathophysiological conditions such as cancer, neurodegenerative, autoimmune, infectious, and heart diseases. Consequently, the study of apoptosis is now at center of both basic and clinical research applications. Therefore, sensitive and simple apoptosis detection techniques are required. Here we describe a monoclonal antibody–defined novel antigen, namely NAPO (negative in apoptosis), which is specifically lost during apoptosis. The anti-NAPO antibody recognizes two nuclear polypeptides of 60 and 70 kD. The antigen is maintained in quiescent and senescent cells, as well as in different phases of the cell cycle, including mitosis. Thus, immunodetection of NAPO antigen provides a specific, sensitive, and easy method for differential identification of apoptotic and nonapoptotic cells.

Generation of ΔTAp73 Proteins by Translation from a Putative Internal Ribosome Entry Site

Abstract:u2002 p73 belongs to a family of transcription factors, including p53 and p63, that mediate response to DNA damage and cellular stress by inducing DNA repair, cell cycle arrest, and apoptosis. TP73 gene contains two promotors and several splice variants resulting in up to 24 possible permutations of p73 proteins which underlies the complexity of the family and its regulatory mechanisms. p73 variants lacking the N‐terminal, denoted as ΔTAp73, are not transcriptionally competent and they act in a dominant negative fashion over TAp73. ΔTAp73 isoforms can be generated by alternative promotor usage, giving rise to ΔNp73, or alternative splicing of exons 2, 3 or 2, and 3 together. Such transcript isoforms potentially produce oncogenic proteins and they were shown to be present in primary tumors and tumor‐derived cell lines. We investigated the possibility of additional mechanisms by which p73 protein could be regulated and discovered a putative internal ribosome entry site (IRES) in exon 2. Translation initiation of TAp73 mRNA results in a ΔNp73‐like peptide, thus demonstrating an additional mechanism whereby a ΔTA p73 protein is produced from a transcript originally generated from the P1 promotor of the p73 gene.

Induction of TAp63 by histone deacetylase inhibitors

TAp63 belongs to the p53-tumour suppressor family and is capable of transactivating a set of target genes to induce cell cycle arrest and apoptosis. We showed that treatment of cancer cells with chemo-therapeutic drugs or the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) results in induction of TAp63 expression, which is in turn related with chemosensitivity. Indeed, induction of TAp63 by TSA affects sensitivity to chemo-therapeutic drugs via the cleavage of the trans-inhibitory domain of TAp63 by active caspases, resulting in generation of a transcriptionally hyper-active TAp63 fragment. Therefore therapeutic approaches that enhance TAp63 expression may offer an improvement in the management of chemoresistant tumours. In this study we tested the abilities of different HDAC inhibitors to induce TAp63 expression. We discovered that two HDAC inhibitors belonging to the hydroxamate group, namely TSA and LBH589, are the most efficient inducers of TAp63 expression. Finally, we found that induction of TAp63 expression in HCT116 cells depends on p53, as p53-negative HCT116 cells failed to induce significant TAp63 expression following treatment with different HDAC inhibitors.

A monoclonal antibody against DNA binding helix of p53 protein

Three monoclonal antibodies (Mabs) were generated against p53 DNA-binding core domain. When tested by immunoprecipitation, Western blot and immunofluorescence techniques, Mab 9E4, as well as 7D3 and 6B10 reacted with both wild-type and various mutant p53 proteins. The epitopes recognized by Mabs 7D3, 9E4 and 6B10 were located respectively within the amino acid residues 211-220, 281-290 and 291-300 of human p53 protein. The epitope recognized by 9E4 Mab coincides with helix 2, also called p53 DNA binding helix, which allows the direct contact of the protein with its target DNA sequences. This antibody may be useful to study transcription-dependent and transcription-independent activities of wild-type and mutant p53 proteins.

Nuclear Exclusion of p33ING1b Tumor Suppressor Protein: Explored in HCC Cells Using a New Highly Specific Antibody

Mouse monoclonal antibodies (MAb) were generated against p33ING1b tumor suppressor protein. 15B9 MAb was highly specific in recognizing a single protein band of approximately 33 kDa endogenous p33ING1b protein from HCC cell lines and normal liver tissue by Western blot analysis and by immunoprecipitation. Although p33ING1b mutations are rarely observed in cancer, differential subcellular distribution and nuclear exclusion of p33ING1b were reported in different cancer types. Therefore we analyzed the expression and subcellular localization of p33ING1b in HCC cell lines using 15B9 MAb. So far, p33ING1b mutations or differential subcellular localization are not reported in HCC. In this study, by indirect immunofluorescence using MAb 15B9, we demonstrate that nuclear localization of p33ING1b was highly correlated with well-differentiated HCC cell lines whereas poorly differentiated HCC cells have nuclear exclusion of the protein. Moreover no association was observed between differential subcellular localization of p33ING1b and p53 mutation status of HCC cell lines. Hence our newly produced MAb 15B9 can be used for studying cellular activities of p33ING1b under normal and cancerous conditions.