Family friction as ΔNp73 antagonises p73 and p53

p53 and its homolog p73 are responsible for guarding the genome and regulating cellular responses to genotoxic damage. However, life is never simple and in fact multiple isoforms of each gene exist which may have opposing functions. ΔNp73 is a truncated isoform of p73 which lacks the N-terminal transactivation domain and is up-regulated in a number of diverse primary tumour types. Although its exact cellular function is unclear, upregulation of ΔNp73 has been linked to various pro-tumour activities. Here we review the current literature surrounding this mysterious protein and reveal its potentially important functions in tumourigenesis and treatment resistance.     

Cell density-dependent acetylation of ΔNp63α is associated with p53-dependent cell cycle arrest

ΔNp63α is a p63 isoform that is predominantly expressed in the epidermal stem cells and in cancer. To find the regulatory pathways of ΔNp63α, we assessed whether ΔNp63α is acetylated and determined the functional implications of acetylation. First, the hinge region of p63 was shown to be acetylated by PCAF, similarly to other p53 family members. Second, acetylation synergistically induced cytoplasmic localization of ΔNp63α. Finally, acetyl-ΔNp63α was induced during high-density culture, suggesting that acetylation of ΔNp63α may reinforce cell cycle arrest upon cell contact. Altogether, these findings suggest that acetylation of ΔNp63α contributes to the epidermal homeostasis.     

DNA damage down-regulates ΔNp63α and induces apoptosis independent of wild type p53

The tumor suppressor p53 is pivotal in cell growth arrest and apoptosis upon cellular stresses including DNA damage. Mounting evidence indicates that p63 proteins, which are homologs of p53, are also involved in apoptosis under certain circumstances. In this study, we found that treatment with DNA damage agents leads to down-regulation of ΔNp63α and induces apoptosis in FaDu and HaCat cells carrying mutant p53. Further study shows that DNA damage reduces steady-state mRNA level of ΔNp63α, but has little effect on its protein stability. In addition, knockdown of endogenous ΔNp63α directly induces apoptosis and sensitizes cells to DNA damage, while exogenous expression of ΔNp63α partially confers cellular resistance to DNA damage. Together, these data suggest that DNA damage down-regulates ΔNp63α, which may directly contribute to DNA damage-induced apoptosis.     

Protein Interaction Between p53 and △113p53 Is Required for the Anti-Apoptotic Function of △113p53

Zebrafish △113p53, an N-terminal truncated p53 isoform, is a p53-target gene that antagonises p53-mediated apoptotic activity.Interestingly, △113p53 does not act on p53 in a dominant-negative manner, but rather interferes with the p53 function by differentially modulating p53-target gene expression to protect cells from apoptosis. Previous studies showed that over-expressed △113p53 and p53proteins formed a complex. However, it is not known whether endogenous p53 and △113p53 proteins also interact with each other, and if this interaction is required for △113p53 to inhibit the apoptotic activity of full-length p53. In this study, we used two available zebrafish p53 antibodies to address these questions. One, Zfp53-N, only recognises full-length p53, whereas the other, Zfp53-A7C10, detects both full-length p53 and △113p53. Using Zfp53-N for immunoprecipitation and Zfp53-A7C10 for detection, we demonstrated that endogenous △113p53 and full-length p53 induced by a DNA-damaging drug formed a complex in vivo. Furthermore, of the six △113p53 mutants we generated with different point mutations in the oligomerisation domain, two failed to interact with p53 and lost the ability to modulate p53-target gene expression and inhibit p53-induced cell apoptosis. However, those △113p53 mutants that could interact with p53 retained the ability to antagonise the apoptotic activity of p53. Therefore, our data demonstrated that proteineprotein interaction between △113p53and p53 is essential for the anti-apoptotic function of △113p53. In addition, the two △113p53 mutants that failed to interact with p53 are also useful for the study of the mechanisms of other functions of △113p53.     

Protein Interaction Between p53 and △113p53 Is Required for the Anti-Apoptotic Function of △113p53

Zebrafish △113p53, an N-terminal truncated p53 isoform, is a p53-target gene that antagonises p53-mediated apoptotic activity. Interestingly, △113p53 does not act on p53 in a dominant-negative manner, but rather interferes with the p53 function by differentially modulating p53-target gene expression to protect cells from apoptosis. Previous studies showed that over-expressed △113p53 and p53 proteins formed a complex. However, it is not known whether endogenous p53 and △113p53 proteins also interact with each other, and if this interaction is required for △113p53 to inhibit the apoptotic activity of full-length p53. In this study, we used two available zebrafish p53 antibodies to address these questions. One, Zfp53-N, only recognises full-length p53, whereas the other, Zfp53-A7C10, detects both full-length p53 and △113p53. Using Zfp53-N for immunoprecipitation and Zfp53-A7C 10 for detection, we demonstrated that endogenous △113p53 and full-length p53 induced by a DNA-damaging drug formed a complex in vivo. Furthermore, of the six △113p53 mutants we generated with different point mutations in the oligomerisation domain, two failed to interact with p53 and lost the ability to modulate p53-target gene expression and inhibit p53-induced cell apoptosis. However, those △113p53 mutants that could interact with p53 retained the ability to antagonise the apoptotic activity of p53. Therefore, our data demonstrated that protein--protein interaction between △113p53 and p53 is essential for the anti-apoptotic function of △113p53. In addition, the two △113p53 mutants that failed to interact with p53 are also useful for the study of the mechanisms of other functions of △113p53.     

Differential regulation of p53 function by the N-terminal ΔNp53 and Δ113p53 isoforms in zebrafish embryos

Background  

The p53 protein family coordinates stress responses of cells and organisms. Alternative promoter usage and/or splicing of p53 mRNA gives rise to at least nine mammalian p53 proteins with distinct N- and C-termini which are differentially expressed in normal and malignant cells. The human N-terminal p53 variants contain either the full-length (FL), or a truncated (ΔN/Δ40) or no transactivation domain (Δ133) altogether. The functional consequences of coexpression of the different p53 isoforms are poorly defined. Here we investigated functional aspects of the zebrafish ΔNp53 ortholog in the context of FLp53 and the zebrafish Δ133p53 ortholog (Δ113p53) coexpressed in the developing embryo.     

Differential regulation of cutaneous oncoprotein HPVE6 by wtp53, mutant p53R248W and ΔNp63α is HPV type dependent

UV exposure and p53 mutations are major factors in non-melanoma skin cancer, whereas a role for HPV infections has not been defined. Previous data demonstrated the wtp53-mediated degradation of cutaneous HPV20E6 by caspase-3. ΔNp63α and hot-spot mutant p53R248W conveyed a protective effect on HPV20E6 under these conditions. We demonstrate a differential regulation by wtp53 of the E6 genes of cutaneous types HPV4, HPV5, HPV7, HPV27, HPV38, HPV48, HPV60 and HPV77. Caspase- or proteasome-mediated down-regulation was HPV type dependent. Mutant p53R248W up-regulated expression of all these E6 proteins as did ΔNp63α except for HPV38E6 which was down-regulated by the latter. None of these cellular proteins affected HPV41E6 expression. Ectopic expression of both mutp53R248W and ΔNp63α in the normal NIKS keratinocyte cell line harbouring endogenous p53 and p63however led to a down-regulation of HPV20E6. We demonstrate that HPV20E6 expression in these cells is modulated by additional, yet unidentified, cellular protein(s), which are not necessarily involved in apoptosis or autophagy. We further demonstrate proliferation of HPV20E6-expressing keratinocytes. Levels of proteins involved in cell cycle control, cyclin-D1, cdk6 and p16(INK4a), phosphorylated pRB, as well as c-Jun and p-c-Jun, were all increased in these cells. HPV20E6 did not compete for the interaction between p16(INK4a) with cyclin-D1 or cdk6. Phosphorylation of pRB in the HPV20E6 expressing cells seems to be sufficient to override the cytokenetic block induced by the p16(INK4a)/pRB pathway. The present study demonstrates the diverse influence of p53 family members on individual cutaneous HPVE6 proteins. HPV20E6 expression also resulted in varying protein levels of factors involved in proliferation and differentiation.     

Loss of ΔNp63α promotes mitotic exit in epithelial cells

Protein p63 is a key regulator in cell proliferation and cell differentiation in stratified squamous epithelium. ΔNp63α is the most commonly expressed p63 isoform, which is often overexpressed in human tumor. In the present work we report the potential involvement of ΔNp63α in cell cycle regulation. ΔNp63α accumulated in mitotic cells but its expression decreased during mitotic exit. Moreover, ΔNp63α knockdown promoted mitotic exit. ΔNp63α shares a conserved destruction box (D-box) motif with other potential targets of the Anaphase-Promoting Complex/Cyclosome (APC/C). Overexpression of APC/C coactivator Cdh1 destabilized ΔNp63α. Our results suggest that ΔNp63α level is cell cycle-regulated and may play a role in the regulation of mitotic exit.     

Over-expression of ΔNp63α facilitates rat corneal wound healing in vivo

To investigate the roles of ΔNp63α during corneal wound healing and the genes regulated by ΔNp63α in limbal epithelial cells. Adenovirus or shRNA targeting ΔNp63α were pre-injected into the anterior chamber of rat eyeballs and the central corneal epithelium was then wounded with NaOH. The effects of ΔNp63α expression during wound healing were observed by propidium iodide staining. In addition, limbal epithelial cells were cultured and ectopically expressed ΔNp63α by transfecting Ad-ΔNp63α. Total RNA was extracted from transfected epithelial cells and subjected to a gene expression microarray assay. The results showed that over-expression of ΔNp63α accelerated the process of corneal wound healing while knockdown of ΔNp63α impaired the process. ΔNp63α positively up-regulated several cell growth promoter genes and could be referred as a positive regulator of limbal epithelial cell proliferation. It might also inhibit cell differentiation and cell death by differential target gene regulation.     

WITHDRAWN: Expression of p53, p63, and p73 isoforms in squamous cell carcinoma and adenocarcinoma of esophagus☆,☆☆

Diabetic microangiopathy is often observed in diabetic patients, but there is little evidence regarding the relationship between post-prandial glycemia or insulinemia and the incidence of diabetic microangiopathy. In this study, to elucidate the relationship between post-prandial glycemia (or insulinemia) and diabetic microangiopathy, we performed a cross-sectional study of 232 subjects with type 2 diabetes mellitus who were not being treated with insulin injections. A multiple regression analysis showed that post-prandial hyperglycemia independently correlated with the incidence of diabetic retinopathy and neuropathy. Post-prandial hyperglycemia also correlated, although not independently, with the incidence of diabetic nephropathy. In addition, interestingly, post-prandial hypoinsulinemia independently correlated with the incidence of diabetic retinopathy, although not correlated with diabetic neuropathy or nephropathy. In conclusion, post-prandial hyperglycemia, rather than fasting glycemia or hemoglobin A1c levels, is an important predictor of the incidence of diabetic microangiopathy in Japanese type 2 diabetic patients.     

Analysis of the intracellular localization of p73 N-terminal protein isoforms TAp73 and ∆Np73 in medulloblastoma cell lines

The protein homologous to the tumor suppressor p53, p73, has essential roles in development and tumorigenesis. This protein exists in a wide range of isoforms with different, even antagonistic, functions. However, there are virtually no detailed morphological studies analyzing the endogenous expression of p73 isoforms at the cellular level in cancer cells. In this study, we investigated the expression and subcellular distribution of two N-terminal isoforms, TAp73 and ΔNp73, in medulloblastoma cells using immunofluorescence microscopy. Both proteins were observed in all cell lines examined, but differences were noted in their intracellular localization between the reference Daoy cell line and four newly established medulloblastoma cell lines (MBL-03, MBL-06, MBL-07 and MBL-10). In the new cell lines, TAp73 and ΔNp73 were located predominantly in cell nuclei. However, there was heterogeneity in TAp73 distribution in the cells of all MBL cell lines, with the protein located in the nucleus and also in a limited non-random area in the cytoplasm. In a small percentage of cells, we detected cytoplasmic localization of TAp73 only, i.e., nuclear exclusion was observed. Our results provide a basis for future studies on the causes and function of distinct intracellular localization of p73 protein isoforms with respect to different protein–protein interactions in medulloblastoma cells.     

p53 Mutants: the Achilles’ Heel of Human Cancers?

Recent scientific discoveries have thrust mutants of the tumor suppressor protein p53 into the forefront of the war on cancer, and hold out eventual hope for a small molecule drug that will be useful in treating human cancers with mutant p53 protein.     

Δ122p53, a mouse model of Δ133p53α, enhances the tumor-suppressor activities of an attenuated p53 mutant

Growing evidence suggests the Δ133p53α isoform may function as an oncogene. It is overexpressed in many tumors, stimulates pathways involved in tumor progression, and inhibits some activities of wild-type p53, including transactivation and apoptosis. We hypothesized that Δ133p53α would have an even more profound effect on p53 variants with weaker tumor-suppressor capability. We tested this using a mouse model heterozygous for a Δ133p53α-like isoform (Δ122p53) and a p53 mutant with weak tumor-suppressor function (mΔpro). The Δ122p53/mΔpro mice showed a unique survival curve with a wide range of survival times (92–495 days) which was much greater than mΔpro/- mice (range 120–250 days) and mice heterozygous for the Δ122p53 and p53 null alleles (Δ122p53/-, range 78–150 days), suggesting Δ122p53 increased the tumor-suppressor activity of mΔpro. Moreover, some of the mice that survived longest only developed benign tumors. In vitro analyses to investigate why some Δ122p53/mΔpro mice were protected from aggressive tumors revealed that Δ122p53 stabilized mΔpro and prolonged the response to DNA damage. Similar effects of Δ122p53 and Δ133p53α were observed on wild-type of full-length p53, but these did not result in improved biological responses. The data suggest that Δ122p53 (and Δ133p53α) could offer some protection against tumors by enhancing the p53 response to stress.The p53 tumor suppressor is most important for preventing cancers. p53 controls cell fate in response to stress by inducing apoptosis, cell cycle arrest/senescence, DNA repair (reviewed in Braithwaite et al.,^{1, 2} Oren,³ and Speidel⁴) or possibly restricting supply of basic substrates for metabolism.^{5, 6, 7} The regulation of p53 function has recently become more complex with the discovery of 13 isoforms, which may interfere with the normal functioning of full-length (FL) p53.^{8, 9, 10, 11, 12, 13, 14} An alternative promoter in intron 4 generates the Δ133p53 isoforms (Δ133p53α, and with additional alternative splicing in intron 9, Δ133p53β, and Δ133p53γ¹¹).The Δ133p53α isoform is expressed in many tissues, but elevated levels have been found in several cancers.^{11, 15, 16} Although the function(s) of Δ133p53α are not fully understood, growing evidence suggests it may have tumor-promoting capacities. Reducing Δ133p53α levels in the U87MG glioblastoma cell line reduced its ability to migrate and stimulate angiogenesis.¹⁷ Δ133p53α may also interfere with the tumor-suppressor functions of FLp53. The zebrafish ortholog of Δ133p53α, Δ113p53, inhibited p53-mediated apoptosis,¹⁸ and overexpression of Δ133p53α inhibited p53-directed G₁ cell cycle arrest.¹⁶Previously, we reported the construction and characterization of a mouse expressing an N-terminal truncation mutant of p53 (designated Δ122p53) that is very similar to Δ133p53α, providing the first mouse model of the Δ133p53α isoform.^{19, 20} Δ122p53 was found to increase cell proliferation and in p53 null cells transduced with a Δ122p53 expressing retrovirus, inhibited the transactivation of CDKN1a (encoding) p21^CIP1 and MDM2 by FLp53.^{19, 20} As well as elevating cell proliferation, homozygote Δ122p53 mice exhibited a profound pro-inflammatory phenotype, including increased serum interleukin-6 (IL-6) and γ-interferon (γ-IFN), and features of autoimmune disease.^{19, 20} The mice were tumor-prone displaying a complex tumor spectrum, but predominantly B-cell lymphomas and osteosarcomas. Thus, most evidence supports a role for the Δ133p53α isoform as a dominant oncogene that may interfere with normal FLp53 tumor-suppressor functions, but also has additional ''gain-of-function'' properties to promote tumor progression, probably through inflammatory mechanisms.²¹Given the above data, we reasoned that in an environment where p53 tumor-suppression capacity is compromised, such as in the context of the R72P allele^{22, 23, 24} or where p53 levels are reduced,^{25, 26, 27} the influence of Δ133p53α isoform on FLp53 function would be greater, leading to rapid tumor formation with a phenotype that would resemble that of the isoform alone. To test this, we generated mice heterozygous for Δ122p53 and a p53 mutant (mΔpro) that we previously described, that has attenuated tumor-suppressor activity.^{28, 29} The mΔpro mouse model is missing part of the p53 proline rich domain (PRD, amino acids 58–88). These mice are defective for DNA damage-induced apoptosis, and show a delayed and impaired cell cycle arrest response. Homozygous mΔpro mice develop late onset follicular B-cell tumors, while mΔpro heterozygotes developed few tumors in the presence of a wild-type p53 allele, or an early onset T-cell lymphoma in a p53-null background. In the latter case, the onset and tumor spectrum are indistinguishable from p53-null mice.²⁸In the current study, we found that, in contrast to our hypothesis, many Δ122p53/mΔpro mice showed extended survival compared with Δ122p53 homozygotes. In vitro analyses to explain this phenomenon suggested that Δ122p53 allele can enhance mΔpro tumor-suppressor functions, in particular cell cycle arrest.     

Truncation Mutants Highlight a Critical Role for the N- and C-termini of the Spirulina Δ6 Desaturase in Determining Regioselectivity

The results of our previous study on heterologous expression in Escherichia coli of the gene desD, which encodes Spirulina Δ⁶ desaturase, showed that co-expression with an immediate electron donor—either cytochrome b ₅ or ferredoxin—was required for the production of GLA (γ-linolenic acid), the product of the reaction catalyzed by Δ⁶ desaturase. Since a system for stable transformation of Spirulina is not available, studies concerning Spirulina-enzyme characterization have been carried out in heterologous hosts. In this present study, the focus is on the role of the enzyme’s N- and C-termini, which are possibly located in the cytoplasmic phase. Truncated enzymes were expressed in E. coli by employing the pTrcHisA expression system. The truncation of the N- and C-terminus by 10 (N10 and C10) and 30 (N30 and C30) amino acids, respectively, altered the enzyme’s regioselective mode from one that measures from a preexisting double bond to that measuring from the methyl end of the substrate.