Department of Molecular and Cellular Pathology, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK
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Abstract |
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Abbreviations: LMS, limb mammary syndrome; PBST, phosphate-buffered saline and 0.1% Tween 20.
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Introduction |
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The complexity of the regulation of the p53 pathway, and of its downstream consequences, is well recognized. Control of the pathway is mediated by regulation of protein stability, modulation of post-translational modifications such as phophorylation and acetylation, by a range of proteinprotein interactions and by manipulation of the subcellular localization of p53 protein (2,18,19). The cellular insults that can activate the p53 pathway are diverse (1,2). They include DNA damage, as a consequence of a wide range of physical and chemical agents, as well as alterations in pO2 tension, altered cellsubstrate interactions and changes in cell anchorage, changes in ribonucleotide pools, heat shock and the actions of growth factors and cytokines, as well as the effects of activated oncogenes via an ARF-dependent pathway (2,18). The downstream consequences of p53 activation remain incompletely understood, although most data are consistent with the view that p53 is a transcription factor capable of activating (and in some cases repressing) the activity of target genes via binding to canonical p53-responsive elements (20). The physiological activities so elicited include (but are certainly not restricted to) apoptosis and growth arrest. The p53 pathway is carefully regulated in a cell type- and tissue type-specific manner which remains poorly understood (21,22).
Recent studies have shown that there are, in addition to the p53 gene encoded by a locus on chromosome 17, at least two further genes with remarkable structural and sequence homology to p53 (2). Kaghad et al. (23) reported the existence of a locus on human chromosome 1p36 encoding a protein termed p73. This has considerable homology to mammalian p53, containing an N-terminal transactivation domain, a region with considerable identity to the DNA-binding domain of p53, including absolute conservation of the critical DNA contact residues and much identity and similarity in the oligomerization domain. p73 differs from p53 in having a long C-terminal extension which as a result of alternative splicing exists in two isoforms, a long form (p73) and a short form (p73ß). p73 has some properties in common with p53, being able to bind to the canonical p53 consensus DNA-binding motif and being able to induce apoptosis when overexpressed (24). More recently, Yang et al. (25) described a second locus (at human 3q2729) encoding a p53 homologue that they termed p63. This is the human homologue of a rat clone, KET, identified by Schmale and Bamberger (26) and independently identified by other groups and variously named p40 (27), p51 (28), p73L (29) and CUSP (GenBank accession no. AF091627). Like p73, p63 can undergo alternative splicing with three C-terminal variants (
, ß and
), two N-terminal variants (TA and
N) and an interstitial splice variant in which 12 bases (encoding four amino acids) may be deleted from exon 9 (25). p73 and p63 have considerable homology at the amino acid level as well as similar genomic architectures and, in the regions homologous to p53, this is similar to the p53 gene on chromosome 17. Interestingly, both p73 and p63 are remarkably similar to a cDNA clone found in the invertebrate cephalopod Loligo forbesi (the north European squid) (30).
The existence of a family of p53-like genes is perhaps not a surprise given that other critical cellular regulators exist in gene families (31). In particular, elements of a parallel tumour suppressor pathway, the retinoblastoma pathway, show components with multiple forms encoded by similar but distinct genes (e.g. Rb, p107, p130 and cyclins D1, D2 and D3), but many other molecular systems show this phenomenon, often with consequent functional redundancy. At present little is known about the biology and function of the newly reported p53 homologues. The close similarity in the overall structure of p73 and p63 suggests that both can act as transcription factors and may possibly act on a similar or even identical sets of target genes to that regulated by p53. An understanding of the novel p53 homologues will require well-characterized reagents coupled with analysis of the expression of these proteins in a variety of in vivo and in vitro systems. Here we describe the generation and characterization of antisera reacting with both p63 and
Np63
and we define the expression of these and other immunologically related isoforms in normal human tissues by both biochemical and immunohistochemical assays. Surprisingly, and in striking contrast to p53, p63
and
Np63
are widely expressed at high levels, particularly being found in the nuclei of proliferating cells. Furthermore, in stark contrast to p53 and p73, p63 expression is suppressed by DNA damage.
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Materials and methods |
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Immunoprecipitation
One hundred and fifty micrograms of all tissue and cellular lysates were added to antibody binding buffer (1% NP40, 50 mM HEPES, pH 7.5, 0.15 M NaCl, 1 mM phenylmethylsulphonyl fluoride, 10 mM NaF, 1 mM dithiothreitol) to make a final volume of 100 µl. An aliquot of 50 µl of a 10% slurry of protein A beads covalently linked to either SC1 or pre-immune serum was then added and incubated overnight on a spinning wheel at 4°C. The beads were then quickly spun down, the supernatant removed and the beads washed 10 times in 1 ml of buffer (1% NP40, 50 mM HEPES, pH 7.5, 0.15 M NaCl) with subsequent centrifugation and supernatant removal between each wash. Fifty microlitres of EDTA sample buffer was added and mixed with the beads (32). Samples were then analysed by SDSPAGE and western blotting.
In vitro transcriptiontranslation
Plasmids containing the full-length sequences for mouse p53, rat KET (26) and p73 (23) were in vitro translated using the TNT T7 coupled Wheat Germ Extract System (Promega). Antibody cross-reactions and co-immunoprecipitations were analysed by incubation with either anti-p53 (CM5) (21) or anti-p63
serum (SC1) and harvesting using 50 µl of a 10% slurry of protein A beads on an orbital shaker at 4°C overnight. Aliquots of 50 µl of Laemmli buffer were added and 10 µl analysed on 10% SDSPAGE gels which were subsequently fixed for a period of 15 min in fix solution (50% methanol, 10% acetic acid, 40% water). Protein signals were then amplified in a fluorographic reagent (Amplify; Amersham life Sciences) according to the manufacturer's instructions. The gels were dried under vacuum and then exposed to hypersensitive film (Amersham) for an appropriate length of time.
Tissues and immunohistochemistry
Tumour samples were collected from patients undergoing gastrointestinal surgery and immediately fixed in 10% buffered formalin for 24 h prior to processing to paraffin wax using standard procedures. Paraffin sections (4 µm) were cut from paraffin wax blocks and used for diagnosis or immunohistochemistry using SC1. Tissue sections were dewaxed and rehydrated using standard procedures. After blocking endogenous peroxidase activity, non-specific binding activity was reduced by incubation in a 20% solution of normal goat serum in phosphate-buffered saline for 20 min at room temperature. Primary antibody (SC1) or pre-immune serum from rabbit was diluted to 1:2000 with neat goat serum, added to the slides and incubated overnight at 4°C. The sections were then washed three times for 5 min to remove unbound antibody. The antibody signal was then amplified by a further incubation in a 1:100 solution of goat anti-rabbit horseradish peroxidase (Dakopatts) prepared in neat goat serum for 1 h at room temperature. Sections were washed three times for 5 min prior to amplification and visualization using the rabbit StriAvigen supersensitive immunohistochemistry kit (Biogenex) according to the manufacturer's instructions. After immunostaining, sections were washed in distilled water, lightly counterstained with Mayer's haematoxylin, dehydrated, cleared and mounted in DPX mounting medium (Histological Supplies Ltd) according to the manufacturer's instructions. Negative controls consisted of pre-immune serum used at double the concentration of the test SC1 antibody and using otherwise identical conditions.
Tissues and western blotting
Normal human tissue obtained at autopsy or at biopsy (adjacent to lesional tissue) was homogenized in lysis buffer (1% NP40, 25 mM HEPES, 0.4 mM KCl, 5 mM EDTA, 10 mM NaF, 5 mM phenylmethylsulphonyl fluoride) using a plastic pellet homogenizer (BDH) and left on ice for 15 min. The samples were spun in a centrifuge at 14 000 r.p.m. for 10 min. The supernatant was removed and aliquots taken for protein determination and for further analysis. Protein concentration was determined by Bradford assay and 30 µg of total protein was analysed on a 10% SDSpolyacrylamide gel, transferred to nitrocellulose (Hybond C; Amersham) and blocked (5% Marvel, 0.1% Tween 20 in phosphate-buffered saline) for 1 h at room temperature. The SC1 anti-p63 serum was added to the blot at 1:1000 concentration in blocking buffer and incubated for 1 h at room temperature. The nitrocellulose was washed four times for 5 min with PBST (phosphate-buffered saline and 0.1% Tween 20) and incubated for 1 h with the secondary peroxidase-conjugated antibody at a 1:1000 dilution. The nitrocellulose was washed three times for 5 min in PBST. The nitrocellulose was immersed in ECL reagent (Enhanced Chemiluminescence kit; Amersham) for 1 min, dried and then exposed to hypersensitive film (Amersham) for an appropriate length of time (32).
Cell culture
HaCat cells (33) (a kind gift of Prof. E.B.Lane) and other established cell lines were grown under standard conditions at 37°C in Dulbecco's modified Eagle's medium supplemented with 10% foetal calf serum for all experiments. Medium was removed from cells which were subsequently washed with phosphate-buffered saline prior to UV light treatment. Cells were treated with UV light using a Stratalinker (Stratagene); 25 J were administered to cells which were replenished with medium and harvested at a range of time points thereafter (0, 6, 12, 24 and 96 h). Control cells were treated in an identical fashion with the omission of UV radiation. In other experiments, HaCat cells were harvested at timed intervals after exposure to 25 J/m2 UV. Cells were harvested following removal of medium by scraping in cold phosphate-buffered saline. Cell pellets were immediately frozen in liquid nitrogen until required. Cell pellets were lysed in lysis buffer (above) for protein extraction and left on ice for 15 min. The samples were spun in a centrifuge at 14 000 r.p.m. for 10 min. The supernatant was removed and aliquots taken for protein determination and for further analysis.
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Results |
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Immunoblot analysis of p63 and
Np63
in normal and neoplastic tissues
The expression of p63 and its isoforms was examined in a range of normal human tissues obtained at autopsy or from surgical resections. As is the case with established cell lines, a range of isoforms could be immunologically detected (Figure 3
). The slowest mobility form in a range of human tissues (p63
) has a slightly higher mobility than the species identified in HaCat cells. The difference may be due to an as yet uncharacterized modification such as phosphorylation or acetylation. In some tissues (ovary, breast, colon, smooth muscle, skin and lung) this species predominates. In other tissues (oesophagus, liver and pancreas), higher mobility forms are seen, including a species with similar mobility to that seen in HaCat cells. In yet other tissues (nerve, peripheral blood lymphocytes, spleen, kidney, liver, pancreas and thyroid) an intermediate form not detected in HaCat cells, but present in mdm2 null and wild type mouse embryo fibroblasts, is seen. The biochemical nature of these species remains unclear but the existence of multiple splice variants as well as alternativee promoter usage (25) provides a probable molecular basis for their existence.
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While there is a strong association between SC1 immunoreactivity and the proliferative compartments in some tissues, this is by no means absolute. For example, in the thymus there was staining of cortical epithelial cells (predominantly but not exclusively cytoplasmic) but the highly proliferative cortical thymocytes (34) did not show detectable staining with SC1. Similarly, there were many tissues in which there is minimal proliferation but where considerable SC1 immunoreactivity was found. For example, in endocrine tissues such as adrenal, pancreatic islet, thyroid and parathyroid there was heterogeneous but prominent nuclear immunoreactivity with SC1. The proportion of cells labelled was between 10 and 25% of the endocrine cells present, many fold higher than the usual proliferative state of such tissues.
In ducted glands such as salivary gland, pancreas and breast there was considerable nuclear, and in some duct structures cytoplasmic, labelling with SC1. For example, in both pancreas and parotid a proportion of acinar cells showed strong nuclear labelling with SC1 while the ductal epithelium of both organs showed variable nuclear and prominent cytoplasmic staining. In the adult, non-pregnant breast lobular epithelium showed extensive nuclear staining although non-stained cells were easily identified. As with parotid and pancreas, the duct epithelium showed both nuclear and cytoplasmic staining. Skin adnexal structures such as eccrine and apocrine glands showed a mixture of nuclear and cytoplasmic staining. In apocrine cells in particular there was an accentuation of cytoplasmic staining in association with the lumenal aspect of the cell membranes. In all these tissues the proportion of cells showing SC1 staining was far in excess of the proportion of cells that a plethora of other studies have demonstrated as being in the cell cycle (35). Other tissues that showed this phenomenon were the liver, where many hepatocytes and biliary epithelial cells showed nuclear immunoreactivity, and the kidney, where the bulk of tubular epithelial cells had SC1 staining.
Non-epithelial structures also had extensive SC1 immunoreactivity. Most notably smooth muscle had strong nuclear immunoreactivity in the gastrointestinal tract and myometrium. Endothelium often showed nuclear and occasional cytoplasmic staining in most sites examined. Peripheral neural tissue also had extensive SC1 staining, with the ganglion cells of the Meissner's and Auerbach's plexus being stained. All peripheral nerve trunks showed intense staining in association with the neurilemma. As with the other staining patterns reported, this was not observed with pre-immune serum. As seen in Figure 3, two isoforms of p63
were seen in nerve, similar to the species identified in other tissues, including small intestinal mucosa. The higher mobility species similar to that seen in peripheral blood lymphocytes predominated.
The effect of UV-induced DNA damage on p63 expression
DNA damage can induce the expression of both p53 (1,2) and p73 (3638). To determine the effect of DNA damage on expression of p63 protein we treated HaCat cells with 25 J/m2 UVC and harvested cells at a range of time points thereafter. Western analysis (Figure 5
) showed that the p63
protein was constitutively expressed at a high level in untreated cells, as previously described. The expression of p63
, however, was down-regulated at 24 h post-treatment but the cells began to recover after this time period until the baseline level was achieved again at ~96 h. There is a clear dose dependency of this response, as shown by examining p63
expression 24 h after treatment with increasing doses of UVC. From this information it would appear that the p63
isoform is down-regulated in response to UV and had the ability to recover expression levels with advancing time after the initial treatment. Methyl methane sulphonate treatment had a similar effect on p63
in HaCat cells, although actinomycin D and ionizing radiation had no effect (not shown). Interestingly, there is no alteration in expression of the
Np63
species. In addition, in MCF7 cells, which only express the higher mobility
Np63
, there is no alteration in expression of the expressed isoforms although p53 is induced (not shown). In preliminary studies to determine the effect of UV radiation on human skin, we studied human skin at intervals after 1.5 minimal erythema dose of solar simulated light (39). In comparison with expression of p53, which was up-regulated in response to UV radiation (39), p63
expression in the skin mirrored the changes observed in the HaCat cell line. p63
immunoreactivity diminished from a high baseline in response to UV then recovered with advancing time after the initial dose.
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Discussion |
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Recent studies (51,52) using homologous recombination have resulted in p63 +/ and / mice being generated. These studies revealed that p63 is a critical component of biochemical networks involved in ectodermal differentiation during embryogenesis. Mice without functioning p63 lack all squamous epithelia and their derivatives, including salivary glands, breast epithelium and lachrymal glands. The biological role of the various p63 isoforms was not revealed by these studies as they resulted in the complete ablation of all p63 isoforms. It is of interest that in the current study and a related investigation of p63 expression in oral epithelia, p63 and
Np63
immunoreactivity is found in the nuclei of many cells, including the proliferative compartments of stratified squamous epithelium. However, the expression of p63
and
Np63
is by no means restricted to proliferating cells. A role of p63 in human development is strongly suggested by the recent recognition of a limb mammary syndrome (LMS) (53) which has similarities to the phenotype of the p63 null mice. In this syndrome there are severe hand, foot and limb defects and developmental defects in the breast (53) and the critical region for LMS has been localized to a 3 cM interval on chromosome 3q27 which includes the p63 locus. Other genes at this locus have been excluded as being responsible for LMS and p63 is now an attractive candidate.
The study of p63 and definition of its role in neoplasia and other physiological and pathological situations will require the detailed characterization of both the biochemical properties of p63 and also its in vivo expression. The data provided in this study represent a major step in our cataloguing of p63 isoform expression in man. We have shown that the serum SC1 specifically recognizes, by immunoprecipitation, native p63 protein generated in coupled in vitro transcriptiontranslation reactions, but that this serum does not recognize either p73
or murine p53 in similar assays. Furthermore, SC1 does not react with recombinant human p53 protein in western assays. Interestingly, serum CM5 (21) recognizes p63 in this highly stringent assay. This presumably reflects the similarity of the core DNA-binding domain between p53 (the immunogen for CM5) and p63. It is of course possible that CM5 also recognizes p73. While this observation raises issues concerning the specificity of CM5, CM5 immunoreactivity was not observed in immunohistological studies of p53 null tissues (22). Nevertheless, it will be important to revisit the expression patterns of p53 in murine tissues, in the light of our new knowledge of the existence of p53 homologues. Such studies of p63 expression in murine and other rodent tissues are in progress.
There is abundant p63 immunoreactivity in a range of murine and human cell lines (this work and K.Nylander, P.J.Coates and P.A.Hall, submitted for publication). Two species are recognized by SC1 in cell lines and are interpreted to represent p63 and
Np63
. Three observations provide evidence for this assertion. First, the species are similar to those reported by Yang et al. (25) as being p63
and
Np63
. Second, it has recently been shown that using monoclonal antibody 4A4 (a generous gift of Dr F. McKeon), which recognizes p63, identical mobility species are seen (K.Nylander, P.J.Coates and P.A.Hall, submitted for publication). Finally, RTPCR studies have shown that p63
and
Np63
mRNA expression reflects the immunochemical analysis of SC1 immunoreactivity (K.Nylander, P.J.Coates and P.A.Hall, submitted for publication). Interestingly,
Np63
is the predominant form in all cell lines studied and is also found in tissues (this work) including normal and neoplastic gastrointestinal epithelia (S.J.Campbell et al., unpublished observations). This form of p63 would appear to represent a dominant negative species capable of inhibiting transcriptional activation by p53 and other p53-like proteins, such as p73 and the full-length forms of p63. It may well be that this can act as an important check on the activity of p53-like proteins which are potentially dangerous molecules in that they can effect both growth arrest and apoptotic functions and potentially other critical cellular activities. In this context the novel observation of the down-regulation of p63 in HaCat cells by UV irradiation is striking and intriguing. Further studies of this phenomenon in order to determine its generality and mechanism are needed.
Examination of the western blot data from cells and tissues (Figures 2 and 3) reveals additional complexity. For example, the mobility of p63
in HaCat cells appears slower than in the tissues examined. The reason for this is unclear but may reflect some form of post-translational modification, polymorphism or mutation. Examining the tissue blot (Figure 3
) shows that four distinct species are identified in tissues. In oesophagus we find the slow mobility species (presumed to be p63
) and a form of similar mobility to the fastest mobility form in HaCat (and other cells), which is presumed to be
Np63
. An even higher mobility species of uncertain nature is found at low abundance in lung. Finally, a high abundance form is found in nerve, peripheral blood lymphocytes and other tissues. The nature and significance of this species is uncertain at present. None of these bands are identified by western blot studies using pre-immune serum at the same concentration as used for SC1. Furthermore, as stated above, the two forms interpreted to be p63
and
Np63
are also recognized by the 4A4 anti-p63 monoclonal antibody. It may be that over and above the isoforms already described (25), p63 shows similar complexity to that reported for p73, where at least four isoforms exist as a consequence of alternative splicing (23,40).
Despite having been only recently described it is clear that p63 is a complex gene capable of encoding a range of different species. The immunochemical data presented here provides further evidence of this complexity and the use of our SC1 serum in immunohistological assays is complicated by the multiple species recognized and we must therefore consider resultant immunostaining with caution. Nevertheless, it seems from our data that p63 immunoreactivity is widespread in tissues, with differential expression of various p63
isoforms. A fuller understanding of the biology of p63 and all its isoforms will only come with further studies and the availability of a panel of antibodies that can recognize and distinguish all the known isoforms. The work presented here is a step on that road.
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Acknowledgments |
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Notes |
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References |
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