p63 is useful in the diagnosis of mammary metaplastic carcinomas

AIMS: p63 has been recently reported to be expressed in sarcomatoid/metaplastic carcinoma of the breast, in addition to its role as a myoepithelial marker. A large series of 34 metaplastic carcinomas, including cases with pure epithelial component (squamous cell and adenosquamous carcinomas), biphasic tumours with carcinomatous and sarcomatoid components and monophasic tumours with only spindle cell component, were evaluated for p63 expression with respect to the different cellular components. METHODS: All of the metaplastic carcinomas were assessed for p63 and conventional epithelial and mesenchymal markers of AE1/3, CAM5.2 and vimentin by immunohistochemistry. RESULTS: All of the different categories of metaplastic carcinomas showed similar clinico-pathological features (patient age, tumour size, nuclear grade, mitotic activity, lymph node status and hormonal receptor status). For metaplastic carcinoma with epithelial component only, p63 was only expressed in the squamous cell component, but not the adenocarcinoma component. Eight of the 10 tumours were positive for p63. For the tumours with sarcomatoid component, either singly or together with carcinomatous component, p63 was positive in 14 of 24 cases. Pure sarcomas and carcinomas were all negative for p63 staining by immunohistochemistry, thus rendering p63 staining highly specific for diagnosing metaplastic carcinoma. CONCLUSIONS: Using p63 for diagnosis of metaplastic carcinoma gives a sensitivity of 65%, a specificity of 96%, a positive predictive value of 96%, and a negative predictive value of 66% and an accuracy of 78%. p63 may be used as an adjunct marker in the diagnosis of metaplastic carcinoma.     

p63 is a prostate basal cell marker and is required for prostate development

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.     

p63 immunohistochemical staining is limited in soft tissue tumors

p63 is a p53 homolog that is expressed in various normal epithelial tissues and epithelial malignancies. Its expression in mesenchymal lesions has not been examined in depth; therefore, we studied p63 expression by immunohistochemical analysis in 650 soft tissue tumors. We found that p63 expression is limited in soft tissue tumors. The majority of tumors studied were p63-, including all cases of angiosarcoma, lipomatous neoplasms, dermatofibrosarcoma protuberans, solitary fibrous tumor, schwannoma, neurofibroma, gastrointestinal stromal tumor, and leiomyosarcoma. Nuclear p63 reactivity was found in a subset of soft tissue myoepithelioma and myoepithelial carcinoma of soft tissue, cellular neurothekeoma, soft tissue perineurioma, Ewing sarcoma/peripheral neuroectodermal tumor, diffuse-type giant cell tumor, and giant cell tumor of soft parts. Infrequent, weak, or focal p63-staining patterns were observed in low-grade fibromyxoid sarcoma, malignant peripheral nerve sheath tumor, extraskeletal myxoid chondrosarcoma, myxofibrosarcoma, proximal-type epithelioid sarcoma, synovial sarcoma, embryonal rhabdomyosarcoma, desmoplastic small round cell tumor, atypical fibroxanthoma, and spindle cell melanoma. Absent p63 expression is typical for most soft tissue tumors, including most (but not all) that would be in the differential diagnosis of spindle cell squamous carcinoma.     

p63 Coordinates anogenital modeling and epithelial cell differentiation in the developing female urogenital tract

p63 is a p53 homologue required for cutaneous development that is expressed in immature squamous epithelium and reserve cells of the cervix. Humans with p63 mutations exhibit defects in limb, accessory organ (skin appendage, breast, prostate), and genitourinary development. Because p63 expression patterns imply a strong role of the gene in the female genital tract development, newborn female p63-/-, +/-, and +/+ mice were examined in situ, dissected, and compared. Nuclear p63 protein was localized to the skin, vagina, bladder, urethra, and basal columnar cells of the caudal uterus in p63+/+ and +/- animals. p63-/- mice exhibited abnormal genital morphogenesis with hypoplastic genitalia, a single cloacal opening, and persistence of columnar epithelium at lower genital tract sites that normally undergo squamous and urothelial differentiation. The defects observed support p63-dependent pathways of genital tract development that permit externally, ectodermal basal cell replenishment integral to reciprocal epithelial stromal signaling, urorectal septation, and modeling of the external genitalia; and internally, the emergence of basal epithelial cell populations capable of divergent epithelial cell differentiation in the vagina, cervix, and urinary tract. Defects in the first pathway explain imperforate anus, vaginal septum, genital hypoplasia, and micropenis reported in humans with p63 mutations. The second is necessary for the generation of multipotential reserve cells in the cervix and may be operative in other epithelial stromal interactions integral to the emergence of uterine basal cells later in life.     

Characterization of specific p63 and p63-N-terminal isoform antibodies and their application for immunohistochemistry

The TP63 gene gives rise to protein isoforms with different properties and functions due to the presence (TAp63) or absence (ΔNp63) of an N-terminal p53-like transactivation domain. Immunohistochemistry for p63 has clinical value for certain tumour types, but investigations have been hampered by a lack of well characterized antibodies and the inability to discriminate between these N-terminal isoforms with opposite functional properties. We have extensively characterized a series of monoclonal antibodies to recombinant human TAp63 and two commercial p63 monoclonals by Western blot, immunostaining and phage display epitope mapping. Twenty-eight of 29 (96.6 %) novel monoclonals that recognized all p63 isoforms showed substantial cross-reactivity with p73, as did the commercial antibody, 4A4. One novel clone, PANp63-6.1, showed slight cross-reaction with p73 by Western blotting but not immunohistochemistry and the SFI-6 monoclonal did not cross-react with p73 or p53. Phage display revealed that the PANp63-6.1 epitope has one amino acid difference between p63 and p73, the 4A4 epitope is identical in both, whereas the SFI-6 epitope is unique to p63, accounting for these findings. We also produced and characterized a TAp63-specific clone that does not recognize p53 or p73, and we prepared polyclonal sera specific for ΔNp63 isoforms. Immunohistochemistry demonstrated that TAp63 is expressed in a variety of epithelial and other cell types during development, often in a converse pattern to ΔNp63, but has a very limited expression in normal adult tissues and is independent of ΔNp63. TAp63 was expressed in 17.6 % of squamous cancers of cervix that expressed p63, unlike normal cervix where TAp63 was not expressed. TAp63 did not associate with proliferative index, but cervical carcinomas with TAp63 expression showed improved survival. These data highlight the need for rigorous antibody characterization and indicate that p63-isoform identification may improve the clinical value of p63 expression analyses.     

p63 - Key molecule in the early phase of epithelial abnormality in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is the most common lung disease predisposing lung cancer. To clarify the early phase of epithelial abnormalities in IPF, we used an in vitro squamous metaplasia model, transforming growth factor beta1 (TGF beta1)-treated airway epithelial cells (BEAS-2B). The model repeated the expression of squamous epithelial character, such as involucrin, and keratin 6 and 14. DNA microarray analysis disclosed a unique expression signature in TGF beta1-treated airway epithelial cells, 20 specifically up-regulated genes including p63, jagged 1 (jag1) and the genes of structure proteins. Western blotting and RT-PCR analysis revealed that DeltaNp63alpha was the dominant isoform of p63 in our experimental model. Immunohistochemical analysis demonstrated the expression of p63 and jag1 in lung tissues of IPF. Inhibition of p63 with siRNA caused the down-regulation of jag1 expression, but not of involucrin, or keratin 6 and 14. Interestingly, the up-regulation of p63 was totally suppressed by N-acetyl-l-cysteine (NAC), but not by dexamethasone or pirfenidone. Thus, the p63-jag1 pathway may be up-regulated at an early phase of epithelial abnormalities in IPF, which can be overcome by NAC even in the TGF beta1-rich milieu.     

Antibodies targeting p63 react specifically in the cytoplasm of breast epithelial cells exhibiting secretory differentiation

AIMS: The nuclear detection of p63 in myoepithelial cells of the breast has been useful in identifying possibly invasive carcinomas. While examining myoepithelial cells for p63 a very strong cytoplasmic reaction product was noted in secretory cells. The aim was to determine whether this reaction is specific for p63 and indicative of all breast secretory cells. METHODS: Thirty breast specimens were tested immunohistochemically for p63 protein. These included seven with benign secretory changes, 10 secretory carcinomas (nine invasive), one microglandular adenosis, three lobular neoplasias, four invasive ductal carcinomas, three clear cell carcinomas, one squamous cell carcinoma and one mucinous carcinoma. RESULTS: Only cells exhibiting secretory changes or secretory carcinoma were cytoplasmically reactive for p63. The positive reaction was also present as an intraluminal secretory product. This reaction was not seen in cells undergoing apocrine differentiation or in other cells containing secretory vacuoles. CONCLUSIONS: Cells with secretory changes contain p63 protein or an antigenic equivalent. The detection of p63 protein continues to have considerable value for the identification of myoepithelial cells and thus the determination of invasion, but will also have value in the determination of secretory carcinomas of the breast and in understanding their development.     

p63 deficiency activates a program of cellular senescence and leads to accelerated aging

The p53 tumor suppressor plays a key role in organismal aging. A cellular mechanism postulated to drive the aging process is cellular senescence, mediated in part by p53. Although senescent cells accumulate in elderly individuals, most studies have relied on correlating in vitro senescence assays with in vivo phenotypes of aging. Here, using two different mouse models in which the p53-related protein p63 is compromised, we demonstrate that cellular senescence and organismal aging are intimately linked and that these processes are mediated by p63 loss. We found that p63(+/-) mice have a shortened life span and display features of accelerated aging. Both germline and somatically induced p63 deficiency activates widespread cellular senescence with enhanced expression of senescent markers SA-beta-gal, PML, and p16(INK4a). Using an inducible tissue-specific p63 conditional model, we further show that p63 deficiency induces cellular senescence and causes accelerated aging phenotypes in the adult. Our results thus suggest a causative link between cellular senescence and aging in vivo, and demonstrate that p63 deficiency accelerates this process.     

Hic-5 regulates an epithelial program mediated by PPARgamma

PPARgamma is a dominant regulator of fat cell differentiation. However, this nuclear receptor also plays an important role in the differentiation of intestinal and other epithelial cell types. The mechanism by which PPARgamma can influence the differentiation of such diverse cell lineages is unknown. We show here that PPARgamma interacts with Hic-5, a coactivator protein expressed in gut epithelial cells. Hic-5 and PPARgamma colocalize to the villus epithelium of the small intestine, and their expression during embryonic gut development correlates with the transition from endoderm to a specialized epithelium; expression of both these factors is reduced in tumors. Forced expression of Hic-5 in colon cancer cells enhances the PPARgamma-mediated induction of several gut epithelial differentiation/maturation markers such as L-FABP, kruppel-like factor 4 (KLF4), and keratin 20. siRNA directed against Hic-5 specifically reduces PPARgamma-mediated induction of gut epithelial genes in colon cells and in an ex vivo model of embryonic gut differentiation. Finally, forced expression of Hic-5 during 3T3-L1 preadipocyte differentiation inhibits adipogenesis while inducing inappropriate expression of several mRNAs characteristic of gut epithelium in these mesenchymal cells. These results indicate that Hic5 is an important component in determining an epithelial differentiation program induced by PPARgamma.     

Hay-Wells syndrome is caused by heterozygous missense mutations in the SAM domain of p63

Hay-Wells syndrome, also known as ankyloblepharon-ectodermal dysplasia-clefting (AEC) syndrome (OMIM 106260), is a rare autosomal dominant disorder characterized by congenital ectodermal dysplasia, including alopecia, scalp infections, dystrophic nails, hypodontia, ankyloblepharon and cleft lip and/or cleft palate. This constellation of clinical signs is unique, but some overlap can be recognized with other ectodermal dysplasia syndromes, for example ectrodactyly--ectodermal dysplasia--cleft lip/palate (EEC; OMIM 604292), limb--mammary syndrome (LMS; OMIM 603543), acro-dermato-ungual-lacrimal-tooth syndrome (ADULT; OMIM 103285) and recessive cleft lip/palate--ectodermal dysplasia (CLPED1; OMIM 225060). We have recently demonstrated that heterozygous mutations in the p63 gene are the major cause of EEC syndrome. Linkage studies suggest that the related LMS and ADULT syndromes are also caused by mutations in the p63 gene. Thus, it appears that p63 gene mutations have highly pleiotropic effects. We have analysed p63 in AEC syndrome patients and identified missense mutations in eight families. All mutations give rise to amino acid substitutions in the sterile alpha motif (SAM) domain, and are predicted to affect protein--protein interactions. In contrast, the vast majority of the mutations found in EEC syndrome are amino acid substitutions in the DNA-binding domain. Thus, a clear genotype--phenotype correlation can be recognized for EEC and AEC syndromes.     

Evidence for a human IgG1 class switch program

In activated murine B lymphocytes, immunoglobulin class switch recombination occurs as a highly regulated process which is targeted to distinct switch regions. Here we present first evidence that in human B lymphocytes, switch recombination is targeted to distinct switch regions as well. In a panel of clonally unrelated IgG1-expressing human B cells, immortalized by Epstein-Barr virus (EBV) transformation, seven out of nine cells show switch recombination between Sμ and Sγ1 on both alleles, the active and inactive one. The remaining cells show no switch recombination on the inactive IgH locus. The very strong correlation of switch recombination on both alleles of IgG1-expressing cells proves that class switch recombination to IgG1 is not random but directed in human B lymphocytes.     

非小细胞肺癌p63基因的表达及其与3号染色体位点变化的关系

目的　探讨非小细胞肺癌 p6 3基因的蛋白表达水平及其与定位在 3号染色体 2 7～ 2 9区域改变的关系。 方法　应用比较基因组杂交 (CGH)技术对 70例原发性肺鳞状细胞癌和肺腺癌标本进行染色体不平衡性分析。采用组织芯片技术构建12 2例原发性非小细胞肺癌石蜡包埋标本组织芯片 ,并采用免疫组织化学方法检测p6 3蛋白表达情况。比较 p6 3蛋白表达及其与 3号染色体末端改变的关系。结果　CGH分析结果发现 ,30例鳞状细胞癌 2 4例出现 3q2 7～ 2 9区域DNA拷贝数目的增加 ,4 0例腺癌仅 8例发现 3q2 7～ 2 9区域DNA获得。p6 3免疫组化染色结果显示 :5 0例 (84 75 % )鳞状细胞癌免疫组化染色为阳性 ;3例大细胞肺癌中 2例 (6 6 6 6 % )为阳性反应 ;腺癌中仅有 1例 (1 6 7% )为阳性。p6 3蛋白的阳性表达率与患者的年龄、性别、肿瘤的分级、肿瘤的转移以及生存率无关 (P >0 0 5 )。p6 3免疫组化阳性率与 3q2 7～ 2 9区域的改变比较结果显示 :p6 3免疫组化阳性反应与 3号染色体长臂 2 7～ 2 9区域的DNA扩增呈正相关 (P <0 0 1)。结论　p6 3基因的扩增与肺鳞状细胞癌发生和发展有密切的关系