[show abstract][hide abstract] ABSTRACT: p73 and p63, the two ancestral members of the p53 family, are involved in neurogenesis, epithelial stem cell maintenance and quality control of female germ cells. The highly conserved oligomerization domain (OD) of tumor suppressor p53 is essential for its biological functions, and its structure was believed to be the prototype for all three proteins. However, we report that the ODs of p73 and p63 differ from the OD of p53 by containing an additional alpha-helix that is not present in the structure of the p53 OD. Deletion of this helix causes a dissociation of the OD into dimers; it also causes conformational instability and reduces the transcriptional activity of p73. Moreover, we show that ODs of p73 and p63 strongly interact and that a large number of different heterotetramers are supported by the additional helix. Detailed analysis shows that the heterotetramer consisting of two homodimers is thermodynamically more stable than the two homotetramers. No heterooligomerization between p53 and the p73/p63 subfamily was observed, supporting the notion of functional orthogonality within the p53 family.
Cell death and differentiation 09/2009; 16(12):1582-9. · 8.24 Impact Factor
[show abstract][hide abstract] ABSTRACT: p63 is a p53-related DNA-binding protein that helps regulate differentiation and proliferation in epithelial progenitor cells. Its expression has never been evaluated in the human gastrointestinal tract. The aim of this study was to evaluate the expression of p63 in the esophagus and related metaplastic and neoplastic disorders to gain insight into the pathogenesis of these processes. Of particular interest was the expression of p63 in Barrett esophagus (BE) and in BE-associated multilayered epithelium. Multilayered epithelium has been postulated to represent an early precursor to the development of BE primarily because it shares morphologic and immunophenotypic features of both squamous and columnar epithelium, and has been shown prospectively to be highly associated with BE. Routinely processed mucosal biopsy or resection specimens that contained normal esophageal squamous epithelium (n = 20), squamous dysplasia (n = 4), squamous cell carcinoma (n = 7), BE (n = 10), BE-associated multilayered epithelium (n = 13), esophageal mucosal gland ducts (n = 10), BE-associated dysplasia (n = 12), and BE-associated adenocarcinoma (n = 7) were immunostained for p63 to determine the extent and location of staining. p63 staining was compared with the staining patterns observed for p53, Ki 67 (proliferation marker), and cytokeratins (CKs) 13 (squamous marker), 14 (basal squamous marker), 8/18 (columnar marker), and 19 (basal/columnar marker). Expression of p63 messenger RNA (mRNA) isoforms was also analyzed by reverse-transcription polymerase chain reaction of freshly isolated tissues. In the normal esophagus, p63 was expressed in the basal and suprabasal layers of the squamous epithelium and in basal cells that line the mucosal gland ducts but was negative in all other epithelia of the gastrointestinal tract, including the stomach, small intestine, and colon. Similarly, p63 was not expressed in BE, but it, was present in the basal layer of multilayered epithelium in 9 of 13 cases (69%). p63-positive cells in multilayered epithelium and in the mucosal gland duct epithelium were positive for CK8/18 (100%) and CK13 (67% and 30%, respectively) and negative for CK14 (0%), in contrast to p63-positive cells in squamous epithelium, which were positive for CK14 and CK13 (100%) but negative for CK8/18. In neoplastic tissues, p63 was diffusely expressed in all cases of esophageal squamous cell dysplasia and carcinoma but was negative in all cases of esophageal and colorectal adenocarcinoma. The DeltaN isoform of p63 mRNA predominated in all benign and neoplastic squamous tissues examined. p63 may represent a marker of 2 distinct epithelial progenitor cells (basal squamous epithelium and gland duct epithelium) in the esophagus. P63 is upregulated in squamous neoplastic conditions and in this manner may play a role in squamous carcinogenesis. These data also indicate that multilayered epithelium is phenotypically similar to, and may share a lineage relationship with, mucosal gland duct epithelium.
Human Pathlogy 12/2001; 32(11):1157-65. · 2.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: The proliferative compartment of stratified squamous epithelia consists of stem and transient amplifying (TA) keratinocytes. Some polypeptides are more abundant in putative epidermal stem cells than in TA cells, but no polypeptide confined to the stem cells has yet been identified. Here we show that the p63 transcription factor, a p53 homologue essential for regenerative proliferation in epithelial development, distinguishes human keratinocyte stem cells from their TA progeny. Within the cornea, nuclear p63 is expressed by the basal cells of the limbal epithelium, but not by TA cells covering the corneal surface. Human keratinocyte stem and TA cells when isolated in culture give rise to holoclones and paraclones, respectively. We show by clonal analysis that p63 is abundantly expressed by epidermal and limbal holoclones, but is undetectable in paraclones. TA keratinocytes, immediately after their withdrawal from the stem cell compartment (meroclones), have greatly reduced p63, even though they possess very appreciable proliferative capacity. Clonal evolution (i.e., generation of TA cells from precursor stem cells) is promoted by the sigma isoform of the 14-3-3 family of proteins. Keratinocytes whose 14-3-3final sigma has been down-regulated remain in the stem cell compartment and maintain p63 during serial cultivation. The identification of p63 as a keratinocyte stem cell marker will be of practical importance for the clinical application of epithelial cultures in cell therapy as well as for studies on epithelial tumorigenesis.
Proceedings of the National Academy of Sciences 04/2001; 98(6):3156-61. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human p73, a novel homolog of p53, has recently been cloned and mapped at chromosome 1p36.3, the locus for putative tumor suppressor gene(s) of neuroblastoma (NBL) and other cancers. p73, like p53, inhibits growth and induces apoptosis in neuroblastoma and osteosarcoma cell lines.
To test the hypothesis that p73 is a NBL suppressor gene, we examined expression, allelo-typing, and mutation of the p73 gene in primary human neuroblastomas. Loss of heterozygosity (LOH) for p73 was performed in 272 primary NBLs using a CT repeat polymorphic marker, which we found in intron 9 of the p73 gene.
p73 LOH was observed in 28 out of 151 (19%) informative cases. The high frequency of p73 LOH was significantly associated with sporadic neuroblastomas (P< 0.001), MYCN amplification (P< 0.001), and advanced stages (P< 0.05). Mutational analyses by PCR-SSCP (single strand conformation polymorphism) revealed two mis-sense mutations in 140 NBLs, one somatic and one germline.
Thus, the present results have shown that mutation of p73 is infrequent in NBLs, although the p73 locus is frequently lost in advanced stage tumors. These suggest that p73 may not be a tumor suppressor in the classic Knudson manner.
Medical and Pediatric Oncology 02/2001; 36(1):42-4.
[show abstract][hide abstract] ABSTRACT: The p53 homologue p63 encodes for different isotypes able to either transactivate p53 reporter genes (TAp63) or act as p53-dominant-negatives (DeltaNp63). p63 is expressed in the basal cells of many epithelial organs and its germline inactivation in the mouse results in agenesis of organs such as skin appendages and the breast. Here, we show that prostate basal cells, but not secretory or neuroendocrine cells, express p63. In addition, prostate basal cells in culture predominantly express the DeltaNp63alpha isotype. In contrast, p63 protein is not detected in human prostate adenocarcinomas. Finally, and most importantly, p63(-/-) mice do not develop the prostate. These results indicate that p63 is required for prostate development and support the hypothesis that basal cells represent and/or include prostate stem cells. Furthermore, our results show that p63 immunohistochemistry may be a valuable tool in the differential diagnosis of benign versus malignant prostatic lesions.
American Journal Of Pathology 01/2001; 157(6):1769-75. · 4.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Inactivation of the tumour suppressor p53 is the most common defect in cancer cells. The discovery of its two close relatives, p63 and p73, was therefore both provocative and confounding. Were these new genes tumour suppressors, p53 regulators, or evolutionary spin-offs? Both oncogenic and tumour-suppressor properties have now been attributed to the p53 homologues, perhaps reflecting the complex, often contradictory, protein products encoded by these genes. p63 and p73 are further implicated in many p53-independent pathways, including stem-cell regeneration, neurogenesis and sensory processes.
[show abstract][hide abstract] ABSTRACT: Squamous (CIN) and glandular (ACIS) intraepithelial lesions often coexist in the same cervical specimen. However, a less common and little studied variant consists of a stratified epithelium resembling CIN in which conspicuous mucin production is present (Stratified Mucin-producing Intraepithelial LEsions (SMILE). This report describes the phenotypic characteristics of the SMILE, its associated lesions, and its immunophenotype.
Eighteen SMILEs were identified by the presence of conspicuous cytoplasmic clearing or vacuoles in lesions otherwise resembling CIN. The morphologic spectrum of SMILEs was detailed; including associated intraepithelial and invasive cervical neoplasms. In addition, selected cases were stained for mucicarmine, markers of squamous cell/reserve cell differentiation (keratin-14 and p63), and proliferative activity (Mib-1).
Stratified neoplastic epithelial cells with a high Mib-1 index and a rounded or lobular contour at the epithelialstromal interface characterized SMILEs. In contrast to CIN, in which mucin droplets are confined to surface cells, mucin was present throughout the epithelium, varying from indistinct cytoplasmic clearing to discrete vacuoles. SMILEs were distinguished from benign metaplasia by nuclear hyperchromasia and a high Mib-1 index. All but three coexisted with either a squamous (CIN) or glandular (ACIS) precursor lesion. Nine of nine coexisting invasive carcinomas contained glandular, adenosquamous differentiation, or both. SMILEs stained negative for keratin-14 and variably for p63. When present, staining with p63 was confined to basal areas of SMILEs and was absent in areas of columnar differentiation.
SMILEs are unusual cervical intraepithelial lesions best classified as variants of endocervical columnar cell neoplasia based on immunophenotype. The distribution and immunophenotype of SMILEs are consistent with a neoplasm arising in reserve cells in the transformation zone. The coexistence of a wide spectrum of intraepithelial and invasive cell phenotypes suggests that SMILEs are a marker for phenotypic instability, emphasizing the importance of identifying SMILEs and ensuring a complete examination of specimens containing this unusual precursor lesion.
American Journal of Surgical Pathology 11/2000; 24(10):1414-9. · 4.87 Impact Factor
[show abstract][hide abstract] ABSTRACT: In the epidermis, p53 plays an important role in UV-B protection that led us to examine the role, if any, that p63, a p53 homologue highly expressed in the basal layer of the epidermis, might play in the epidermal UV-B response. One p63 isoform, deltaNp63alpha, decreased dramatically in normal keratinocytes or newborn epidermis at both the protein and RNA levels after UV-B irradiation. In an attempt to further investigate the significance of the UV-B-induced decrease of this p63 isoform as well as further delineate the function of p63 in the epidermis, we generated transgenic mice that constitutively express deltaNp63alpha in the mouse epidermis using the loricrin promoter (ML.deltaNp63alpha). The ML.deltaNp63alpha mouse epidermis developed normally, with no overt phenotype and an unaltered proliferation rate. When challenged by UV-B exposure, the ML.deltaNp63alpha mice exhibited a 40-45% decrease in the number of apoptotic cells in the epidermis as compared with nontransgenic littermates. These results suggest that aberrant expression of deltaNp63alpha altered the UV-B-induced apoptotic pathway in the transgenic epidermis, proving that down-regulation of deltaNp63alpha in response to UV-B is important to epidermal apoptosis. The forced overexpression of deltaNp63alpha may act via a dominant negative effect on the endogenous p53 transcriptional activity required for UV-B-induced apoptosis.
Cancer Research 09/2000; 60(15):4016-20. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: p53 plays an essential pro-apoptotic role, a function thought to be shared with its family members p73 and p63. Here, we show that p73 is primarily present in developing neurons as a truncated isoform whose levels are dramatically decreased when sympathetic neurons apoptose after nerve growth factor (NGF) withdrawal. Increased expression of truncated p73 rescues these neurons from apoptosis induced by NGF withdrawal or p53 overexpression. In p73-/- mice, all isoforms of p73 are deleted and the apoptosis of developing sympathetic neurons is greatly enhanced. Thus, truncated p73 is an essential anti-apoptotic protein in neurons, serving to counteract the pro-apoptotic function of p53.
[show abstract][hide abstract] ABSTRACT: The nuclear factors of activated T cells (NF-ATs) constitute a family of transcription factors that transduce calcium signals in the immune, cardiac, muscular and nervous systems. Like their distant relatives of the Rel family, including NF-kappaB, NF-ATs are cytoplasmic in resting cells and activated by means of induced nuclear import. Unlike NF-kappaB, however, NF-ATs show highly dynamic nuclear shuttling properties that have important implications for graded signaling by these molecules. This review focuses on recent advances in deciphering mechanisms by which calcium signaling regulates the nucleo-cytoplasmic shuttling,and therefore transactivation functions of the NF-ATs. These discoveries highlight the interplay between nuclear import and export signals on NF-ATs, and the roles of the calcium-activated phosphatase calcineurin and NF-AT kinases in controlling the activity of these signals. They also reveal that NF-ATs, as well as other transcription factors controlled at the level of nuclear import, face the very real prospect of futile cycling across the nuclear envelope as a consequence of conflicting nuclear import and export signals. We discuss the molecular mechanisms by which calcineurin suppresses futile cycling, as well as the major challenges to our understanding of NF-AT signaling in diverse biological systems.
Cellular and Molecular Life Sciences CMLS 04/2000; 57(3):411-20. · 5.62 Impact Factor
[show abstract][hide abstract] ABSTRACT: p63, a recently identified member of the p53 gene family, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. We show that in normal human epidermis, in hair follicles, and in stratified epidermal cultures, p63 protein is principally restricted to cells with high proliferative potential and is absent from the cells that are undergoing terminal differentiation. In normal human epidermis and in hair follicles, basal cells with abundant p63 are interspersed with cells with little or no p63. Whenever p63 mRNA is present, it encodes mainly truncated, potentially dominant-negative isotypes. In squamous cell carcinomas, the number of cells containing p63 and their distribution depends on the degree of anaplasia. In highly differentiated tumors, p63 is confined to a ring of basal-like cells surrounding, but at a distance from, centers of terminal differentiation. In less differentiated tumors, most cells contain p63 and their distribution is chaotic with respect to centers of terminal differentiation. p63 appears to be a valuable diagnostic marker for anaplastic keratinocytes.
Journal of Investigative Dermatology 01/2000; 113(6):1099-105. · 6.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: In fission yeast, overexpression of the replication initiator protein Cdc18p induces re-replication, a phenotype characterized by continuous DNA synthesis in the absence of cell division. In contrast, overexpression of Cdc6p, the budding yeast homolog of Cdc18p, does not cause re-replication in S. cerevisiae. However, we have found that Cdc6p has the ability to induce rereplication in fission yeast. Cdc6p cannot functionally replace Cdc18p, but instead interferes with the proteolysis of both Cdc18p and Rum1p, the inhibitor of the protein kinase Cdc2p. This activity of Cdc6p is entirely contained within a short N-terminal peptide, which forms a tight complex with Cdc2p and the F-box/WD-repeat protein Sud1p/Pop2p, a component of the SCF(Pop) ubiquitin ligase in fission yeast. These interactions are mediated by two distinct regions within the N-terminal region of Cdc6p and depend on the integrity of its Cdc2p phosphorylation sites. The data suggest that disruption of re-replication control by overexpression of Cdc6p in fission yeast is a consequence of sequestration of Cdc2p and Pop2p, two factors involved in the negative regulation of Rum1p, Cdc18p and potentially other replication proteins.
MGG - Molecular and General Genetics 11/1999; 262(3):473-80.
[show abstract][hide abstract] ABSTRACT: EEC syndrome is an autosomal dominant disorder characterized by ectrodactyly, ectodermal dysplasia, and facial clefts. We have mapped the genetic defect in several EEC syndrome families to a region of chromosome 3q27 previously implicated in the EEC-like disorder, limb mammary syndrome (LMS). Analysis of the p63 gene, a homolog of p53 located in the critical LMS/EEC interval, revealed heterozygous mutations in nine unrelated EEC families. Eight mutations result in amino acid substitutions that are predicted to abolish the DNA binding capacity of p63. The ninth is a frameshift mutation that affects the p63alpha, but not p63beta and p63gamma isotypes. Transactivation studies with these mutant p63 isotypes provide a molecular explanation for the dominant character of p63 mutations in EEC syndrome.
[show abstract][hide abstract] ABSTRACT: The nuclear envelope (NE) is a distinct subdomain of the ER, but few membrane components have been described that are specific to it. We performed a visual screen in tissue culture cells to identify proteins targeted to the NE. This approach does not require assumptions about the nature of the association with the NE or the physical separation of NE and ER. We confirmed that screening a library of fusions to the green fluorescent protein can be used to identify proteins targeted to various subcompartments of mammalian cells, including the NE. With this approach, we identified a new NE membrane protein, named nurim. Nurim is a multispanning membrane protein without large hydrophilic domains that is very tightly associated with the nucleus. Unlike the known NE membrane proteins, it is neither associated with nuclear pores, nor targeted like lamin-associated membrane proteins. Thus, nurim is a new type of NE membrane protein that is localized to the NE by a distinct mechanism.
The Journal of Cell Biology 08/1999; 146(1):29-44. · 10.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Ca2+-activated protein phosphatase calcineurin induces apoptosis, but the mechanism is unknown. Calcineurin was found to dephosphorylate BAD, a pro-apoptotic member of the Bcl-2 family, thus enhancing BAD heterodimerization with Bcl-xL and promoting apoptosis. The Ca2+-induced dephosphorylation of BAD correlated with its dissociation from 14-3-3 in the cytosol and translocation to mitochondria where Bcl-xL resides. In hippocampal neurons, L-glutamate, an inducer of Ca2+ influx and calcineurin activation, triggered mitochondrial targeting of BAD and apoptosis, which were both suppressible by coexpression of a dominant-inhibitory mutant of calcineurin or pharmacological inhibitors of this phosphatase. Thus, a Ca2+-inducible mechanism for apoptosis induction operates by regulating BAD phosphorylation and localization in cells.
[show abstract][hide abstract] ABSTRACT: The p63 gene, a homologue of the tumour-suppressor p53, is highly expressed in the basal or progenitor layers of many epithelial tissues. Here we report that mice homozygous for a disrupted p63 gene have major defects in their limb, craniofacial and epithelial development. p63 is expressed in the ectodermal surfaces of the limb buds, branchial arches and epidermal appendages, which are all sites of reciprocal signalling that direct morphogenetic patterning of the underlying mesoderm. The limb truncations are due to a failure to maintain the apical ectodermal ridge, a stratified epithelium, essential for limb development. The embryonic epidermis of p63-/- mice undergoes an unusual process of non-regenerative differentiation, culminating in a striking absence of all squamous epithelia and their derivatives, including mammary, lacrymal and salivary glands. Taken together, our results indicate that p63 is critical for maintaining the progenitor-cell populations that are necessary to sustain epithelial development and morphogenesis.
[show abstract][hide abstract] ABSTRACT: Ubiquitin-dependent proteolysis plays an important role in cell-cycle control  . In budding yeast, the protein Skp1p, the cullin-family member Cdc53p, and the F-box/WD-repeat protein Cdc4p form the SCFCdc4p ubiquitin ligase complex, which targets the cyclin-dependent kinase (Cdk) inhibitor Sic1p for proteolysis      . Sic1p is recruited to the SCFCdc4p complex by binding to the WD-repeat region of Cdc4p  , while Skp1p binds to the F-box of Cdc4p . In fission yeast, two distinct Cdc4p-related proteins, Pop1p/Ste16p   and the recently identified Sud1p/Pop2p , regulate the stability of the replication initiator Cdc18p and the Cdk inhibitor Rum1p. We show here that, despite their structural and functional similarities, the pop1 and pop2 genes fail to complement each other's deletion phenotypes, indicating that they perform non-redundant, but potentially interdependent, functions in proteolysis. Consistent with this hypothesis, Pop1p and Pop2p formed heterooligomeric complexes when overexpressed, and binding of Cdc18p to Pop2p was dependent on Pop1p. The Pop1p-Pop2p interaction was mediated by the amino-terminal domain of Pop2p which, when fused to full-length Pop1p, rescued the phenotype of a Deltapop1Deltapop2 double mutant. Thus, close physical proximity of two distinct F-box/WD-repeat proteins directs proteolysis mediated by the SCFPop ubiquitin ligase complex.
Current Biology 05/1999; 9(7):373-6. · 9.49 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nuclear import of the NF-AT transcription factors during T-cell activation requires the calcium-activated phosphatase calcineurin, which unmasks nuclear-location signals on NF-AT. We show here that the nuclear import of NF-ATs is not sufficient to activate NF-AT target genes, as NF-ATs are subject to a futile cycling across the nuclear envelope owing to engagement with the exportin protein Crm1. Calcineurin suppresses this futile cycling by a non-catalytic mechanism involving the masking of nuclear export signals on NF-AT targeted by Crm1. This clustering of binding sites for calcineurin and Crml on NF-AT establishes an inherent competition between these molecules that imparts exquisite calcium sensitivity to the shuttling dynamics of the NF-AT transcription factors. Such a balance between nuclear import and export may regulate the action of other transcription factors.
[show abstract][hide abstract] ABSTRACT: Recent findings from mouse models of BRCA2 genetic lesions have provided intriguing insights and important questions concerning modes of tumor development in familial breast and ovarian cancers. Fibroblasts from mice homozygous for the BRCA2Tr allele grow poorly and display an array of chromosomal abnormalities that are consistent with a role for BRCA2 in DNA repair. This growth defect can be overcome and cellular transformation promoted by the expression of defective, dominant negative alleles of p53 and of the mitotic checkpoint gene Bub1, both of which are known to induce chromosome instability. These findings are mirrored in the genetic lesions sustained in tumors found in the rare BRCA2Tr/Trmice that survive to adulthood, which include defects in p53 as well as the mitotic checkpoint proteins Bub1 and Mad3L. Together, these data hint that tumors in these mice evolve from an unusually intense selective pressure to remove DNA damage checkpoints, which in turn might be facilitated by chromosomal abolition of mitotic checkpoints and the consequent increase in shuffling of genetic information. How these genetic lesions co-operate to yield transformed cells and how these data relate to BRCA1 and BRCA2 defects in the human population are important questions raised by this work.
Breast Cancer Research 02/1999; 1(1):8-10. · 5.33 Impact Factor