Association of p63 with Proliferative Potential in Normal and Neoplastic Human Keratinocytes
1999; Elsevier BV; Volume: 113; Issue: 6 Linguagem: Inglês
10.1046/j.1523-1747.1999.00780.x
ISSN1523-1747
AutoresRamine Parsa, Annie Yang, Frank McKeon, Howard Green,
Tópico(s)Cancer and Skin Lesions
Resumop63, 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. 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. The p53 tumor-suppressor gene encodes a multifunctional DNA-binding protein important in cell cycle and cell death regulation, and is the most frequently altered gene in human cancers (Ko and Prives, 1996Ko L.J. Prives C. p53: puzzle and paradigm.Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2220) Google Scholar;Levine, 1997Levine A.J. p53, the cellular gatekeeper for growth and division.Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6522) Google Scholar). The p53 protein contains three major functional domains: an N-terminal transactivation domain, a central, DNA-binding domain, and a C-terminal oligomerization domain. Regulation of cell growth and apoptosis by p53 occurs through its transactivation capability; mutations of p53 yielding either dominant-negative or novel gain-of-functions are thought to contribute to tumorigenesis (Levine, 1997Levine A.J. p53, the cellular gatekeeper for growth and division.Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6522) Google Scholar). The identification of a new protein, termed p73, having structural and functional similarities to p53, suggested that a family of p53-like proteins exists (Kaghad et al., 1997Kaghad M. Bonnet H. Yang A. et al.Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers.Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1508) Google Scholar). Recently, a new gene termed variously KET, p40, p51, and p63, located on human chromosome 3q, has been shown to possess homology with p53 and p73 (Schmale and Bamberger, 1997Schmale H. Bamberger C. A novel protein with strong homology to the tumor suppressor p53.Oncogene. 1997; 15: 1363-1367Crossref PubMed Scopus (245) Google Scholar;Osada et al., 1998Osada M. Ohba M. Kawahara C. et al.Cloning and functional analysis of human p51, which structurally and functionally resembles p53.Nat Med. 1998; 4: 839-843Crossref PubMed Scopus (463) Google Scholar;Trink et al., 1998Trink B. Okami K. Wu L. Sriuranpong V. Jen J. Sidransky D. A new human p53 homologue.Nat Med. 1998; 4: 747-748Crossref PubMed Scopus (219) Google Scholar;Yang et al., 1998Yang A. Kaghad M. Wang Y. et al.p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.Mol Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1764) Google Scholar). This gene, which we will refer to as p63, was detected in a variety of human and murine tissues (Osada et al., 1998Osada M. Ohba M. Kawahara C. et al.Cloning and functional analysis of human p51, which structurally and functionally resembles p53.Nat Med. 1998; 4: 839-843Crossref PubMed Scopus (463) Google Scholar;Trink et al., 1998Trink B. Okami K. Wu L. Sriuranpong V. Jen J. Sidransky D. A new human p53 homologue.Nat Med. 1998; 4: 747-748Crossref PubMed Scopus (219) Google Scholar;Yang et al., 1998Yang A. Kaghad M. Wang Y. et al.p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.Mol Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1764) Google Scholar) and encodes two categories of transcripts under the control of two alternative promoters (Yang et al., 1998Yang A. Kaghad M. Wang Y. et al.p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.Mol Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1764) Google Scholar). The first encodes proteins with an acidic N-terminal transactivation domain (TAp63) that, like p53 and p73, can activate transcription and induce apoptosis. The second encodes proteins lacking the N-terminal transactivation domain (Np63), and potentially acts in a dominant-negative manner to suppress transactivation by p53 and TAp63 (Yang et al., 1998Yang A. Kaghad M. Wang Y. et al.p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.Mol Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1764) Google Scholar). Cultured human keratinocytes predominantly express truncated, dominant-negative p63 isotypes (▵Np63) (Yang et al., 1998Yang A. Kaghad M. Wang Y. et al.p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.Mol Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1764) Google Scholar). Investigation of 66 human primary tumors of various cell types and 102 tumor cell lines has revealed point mutations of p63 only in three tumors, all of epidermal origin (Osada et al., 1998Osada M. Ohba M. Kawahara C. et al.Cloning and functional analysis of human p51, which structurally and functionally resembles p53.Nat Med. 1998; 4: 839-843Crossref PubMed Scopus (463) Google Scholar). All three of these mutations were located in the DNA-binding domain (Osada et al., 1998Osada M. Ohba M. Kawahara C. et al.Cloning and functional analysis of human p51, which structurally and functionally resembles p53.Nat Med. 1998; 4: 839-843Crossref PubMed Scopus (463) Google Scholar). We have now studied the expression of p63 in normal and neoplastic keratinocytes. p63 appears to be a marker for keratinocytes with proliferative capacity and may be useful for the prognostic study of neoplastic keratinocytes. Human epidermal keratinocytes derived from foreskin of a normal newborn (strain YF29) were grown with supporting 3T3-J2 cells (Rheinwald and Green, 1975Rheinwald J.G. Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.Cell. 1975; 6: 331-334Abstract Full Text PDF PubMed Scopus (3723) Google Scholar),using additives to the culture medium (Allen-Hoffmann and Rheinwald, 1984Allen-Hoffmann B.L. Rheinwald J.G. Polycyclic aromatic hydrocarbon mutagenesis of human epidermal keratinocytes in culture.Proc Natl Acad Sci USA. 1984; 81: 7802-7806Crossref PubMed Scopus (154) Google Scholar;Simon and Green, 1985Simon M. Green H. Enzymatic cross-linking of involucrin and other proteins by keratinocyte particulates.In Vitro Cell. 1985; 40: 677-683Scopus (162) Google Scholar), including 10% fetal calf serum (Hyclone, Logan, UT). Megacolonies of keratinocytes were grown on 150 mm dishes by inoculating 10–20 cells per dish and cultivating for 16–18 d (Tseng and Green, 1994Tseng H. Green H. Association of basonuclin with ability of keratinocytes to multiply and with absence of terminal differentiation.J Cell Biol. 1994; 126: 495-506Crossref PubMed Scopus (81) Google Scholar). Well-isolated colonies were then chosen for experiments. Keratinocytes were grown to confluence in 150 mm dishes, trypsinized, and collected by centrifugation. Centrifugal elutriation was performed as described previously (Tseng and Green, 1994Tseng H. Green H. Association of basonuclin with ability of keratinocytes to multiply and with absence of terminal differentiation.J Cell Biol. 1994; 126: 495-506Crossref PubMed Scopus (81) Google Scholar). A total of seven fractions were collected as cell suspensions. A drop of each cell suspension was photographed in a hemacytometer for size measurement and cell counts. The remaining cells were lysed for RNA preparation. Total RNA was isolated from cells, using RNeasy Total RNA kit (Qiagen, Santa Clarita, CA). Ten micrograms of total RNA from each cell fraction was loaded in each lane, separated on a 1% formaldehyde-agarose gel and transferred to nylon membranes (Amersham, Bucks, U.K.). Blots were prehybridized and hybridized at 68°C in Quickhyb hybridization solution (Stratagene, La Jolla, CA). The probes were labeled with (32P) dCTP by using a multiprime labeling system kit (Amersham). The RNA was first probed with a 420 bp fragment corresponding to a common region between TAp63 and Np63 cDNA (from nucleotides 765 to 1185 of TAp63 cDNA and from nucleotides 600 to 1020 of ▵Np63 cDNA) (Yang et al., 1998Yang A. Kaghad M. Wang Y. et al.p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.Mol Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1764) Google Scholar). The RNA was then dehybridized and reprobed sequentially, with cDNA encoding the human basonuclin and involucrin (Eckert and Green, 1986Eckert R.L. Green H. Structure and evolution of the human involucrin gene.Cell. 1986; 46: 583-589Abstract Full Text PDF PubMed Scopus (307) Google Scholar;Tseng and Green, 1994Tseng H. Green H. Association of basonuclin with ability of keratinocytes to multiply and with absence of terminal differentiation.J Cell Biol. 1994; 126: 495-506Crossref PubMed Scopus (81) Google Scholar). 18S and 28S rRNA probed with specific oligonucleotides were used as controls for loading variation and RNA integrity. In situ hybridization with digoxygenin-labeled cRNA probes corresponding to the p63 cDNA fragment (described above) were performed on frozen sections as described (Schaeren-Wiemers and Gerfin-MoSeries, 1993Schaeren-Wiemers N. Gerfin-MoSeries A. A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labelled cRNA probes.Histochemistry. 1993; 100: 431-440Crossref PubMed Scopus (1077) Google Scholar). The nuclei were counterstained with 1% methylene green (Fluka Chemical, Milwaukee, WI) for 5 min. RT-PCR analyses were performed on RNA preparations, using primers specific for human p63 TA and human p63▵ N as described (Yang et al., 1998Yang A. Kaghad M. Wang Y. et al.p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.Mol Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1764) Google Scholar). Cultured cells or frozen tissue sections were fixed as described previously (Tseng and Green, 1994Tseng H. Green H. Association of basonuclin with ability of keratinocytes to multiply and with absence of terminal differentiation.J Cell Biol. 1994; 126: 495-506Crossref PubMed Scopus (81) Google Scholar). The mouse monoclonal antibody to p63 (Yang et al., 1998Yang A. Kaghad M. Wang Y. et al.p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.Mol Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1764) Google Scholar) and the rabbit antibody to basonuclin (Iuchi and Green, 1997Iuchi S. Green H. Nuclear localization of basonuclin in human keratinocytes and the role of phosphorylation.Proc Natl Acad Sci USA. 1997; 94: 7948-7953Crossref PubMed Scopus (24) Google Scholar) were detected with sheep antisera to mouse or rabbit IgG coupled to HRP (Amersham). The rabbit antibodies to human involucrin (Biomedical Technologies, Stoughton, MA) and to human Ki-67 (Dako, Carpinteria, CA) were detected with goat antiserum to rabbit IgG coupled to FITC (Boehringer-Mannheim Biochemicals, Indianapolis, IN). DNA was stained with Hoechst 33258 (Fluka Chemical) at 1 μg per ml for 2 min. In both the epidermis and the stratified epidermal cultures, the growth and differentiated properties of keratinocytes have been correlated with their size and location (Rowden, 1975Rowden G. Ultrastructural studies of keratinized epithelia of the mouse. III. Determination of the Volumes of nuclei and cytoplasm of cells in murine epidermis.J Invest Dermatol. 1975; 64: 1-3Crossref PubMed Scopus (34) Google Scholar;Sun and Green, 1976Sun T.T. Green H. Differentiation of the epidermal keratinocyte in cell culture: formation of the cornified envelope.Cell. 1976; 9: 511-521Abstract Full Text PDF PubMed Scopus (384) Google Scholar;Bergstresser et al., 1978Bergstresser P.R. Pariser R.J. Taylor J.R. Counting and sizing of epidermal cells in normal human skin.J Invest Dermatol. 1978; 70: 280-284Crossref PubMed Scopus (49) Google Scholar). Proliferation is mostly confined to small cells in the basal layer. Keratinocytes that leave the basal layer withdraw from the cell cycle, undergo terminal differentiation, and enlarge as they move through suprabasal layers. In stratified epidermal cultures, enlargement and terminal differentiation of keratinocytes are accompanied by loss of both the ability to replicate their DNA (Sun and Green, 1976Sun T.T. Green H. Differentiation of the epidermal keratinocyte in cell culture: formation of the cornified envelope.Cell. 1976; 9: 511-521Abstract Full Text PDF PubMed Scopus (384) Google Scholar) and the ability to form colonies (Barandon and Green, 1985Barandon Y. Green H. Cell size as a determinant of the clone-forming ability of human keratinocytes.Proc Natl Acad Sci USA. 1985; 82: 5390-5394Crossref PubMed Scopus (274) Google Scholar;Tseng and Green, 1994Tseng H. Green H. Association of basonuclin with ability of keratinocytes to multiply and with absence of terminal differentiation.J Cell Biol. 1994; 126: 495-506Crossref PubMed Scopus (81) Google Scholar). Differentiating cells also show a marked reduction or total loss of basonuclin expression (Tseng and Green, 1994Tseng H. Green H. Association of basonuclin with ability of keratinocytes to multiply and with absence of terminal differentiation.J Cell Biol. 1994; 126: 495-506Crossref PubMed Scopus (81) Google Scholar) and begin to synthesize involucrin (Banks-Schlegel and Green, 1981Banks-Schlegel S. Green H. Involucrin synthesis and tissue assembly by keratinocytes in natural and cultured human epithelia.J Cell Biol. 1981; 90: 732-737Crossref PubMed Scopus (272) Google Scholar;Watt and Green, 1981Watt F.M. Green H. Involucrin synthesis is correlated with cell size in human epidermal cultures.J Cell Biol. 1981; 90: 738-742Crossref PubMed Scopus (212) Google Scholar;Tseng and Green, 1994Tseng H. Green H. Association of basonuclin with ability of keratinocytes to multiply and with absence of terminal differentiation.J Cell Biol. 1994; 126: 495-506Crossref PubMed Scopus (81) Google Scholar) and many other protein markers of terminal differentiation (Fuchs and Byrne, 1994Fuchs E. Byrne C. The epidermis: rising to the surface.Curr Opin Genet Dev. 1994; 4: 725-736Crossref PubMed Scopus (218) Google Scholar). We have examined the expression of p63 as the cells enlarge in stratified cultures of keratinocytes. Cells from confluent cultures were separated according to size by centrifugal elutriation. Seven fractions were collected. In each fraction, the average cell size was determined and the presence of p63, basonuclin, and involucrin mRNA was monitored by Northern analysis, using the coding regions of these genes as probes (Eckert and Green, 1986Eckert R.L. Green H. Structure and evolution of the human involucrin gene.Cell. 1986; 46: 583-589Abstract Full Text PDF PubMed Scopus (307) Google Scholar;Tseng and Green, 1994Tseng H. Green H. Association of basonuclin with ability of keratinocytes to multiply and with absence of terminal differentiation.J Cell Biol. 1994; 126: 495-506Crossref PubMed Scopus (81) Google Scholar;Yang et al., 1998Yang A. Kaghad M. Wang Y. et al.p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.Mol Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1764) Google Scholar). As shown in Figure 1, the expression of p63 mRNA is confined to the first four fractions, which contain small cells with a mean diameter of 11–16.5 μm. This population also contains basonuclin mRNA. Cells of this size range are known to include nearly all the colony-forming cells of the culture (Barandon and Green, 1985Barandon Y. Green H. Cell size as a determinant of the clone-forming ability of human keratinocytes.Proc Natl Acad Sci USA. 1985; 82: 5390-5394Crossref PubMed Scopus (274) Google Scholar;De D’Anna et al., 1988De D’Anna F. Luca M. Cancedda R. Zicca A. Franzi A.T. Elutriation of human keratinocytes and melanocytes from in vitro cultured epithelium.Histochem J. 1988; 20: 674-678Crossref Scopus (8) Google Scholar;Tseng and Green, 1994Tseng H. Green H. Association of basonuclin with ability of keratinocytes to multiply and with absence of terminal differentiation.J Cell Biol. 1994; 126: 495-506Crossref PubMed Scopus (81) Google Scholar). In the last three fractions, as the mean cell size increases above 16.5 μm in diameter, the abundance of p63 mRNA drops sharply, as does that of basonuclin mRNA. These cells accumulate involucrin mRNA, a characteristic of terminal differentiation. Cells of this size range do not synthesize DNA (Sun and Green, 1976Sun T.T. Green H. Differentiation of the epidermal keratinocyte in cell culture: formation of the cornified envelope.Cell. 1976; 9: 511-521Abstract Full Text PDF PubMed Scopus (384) Google Scholar) and are unable to form colonies (Barandon and Green, 1985Barandon Y. Green H. Cell size as a determinant of the clone-forming ability of human keratinocytes.Proc Natl Acad Sci USA. 1985; 82: 5390-5394Crossref PubMed Scopus (274) Google Scholar;De D’Anna et al., 1988De D’Anna F. Luca M. Cancedda R. Zicca A. Franzi A.T. Elutriation of human keratinocytes and melanocytes from in vitro cultured epithelium.Histochem J. 1988; 20: 674-678Crossref Scopus (8) Google Scholar;Tseng and Green, 1994Tseng H. Green H. Association of basonuclin with ability of keratinocytes to multiply and with absence of terminal differentiation.J Cell Biol. 1994; 126: 495-506Crossref PubMed Scopus (81) Google Scholar). These data clearly demonstrate that the p63 gene is expressed in the small colony-forming cells, and is absent from the larger cells that are undergoing terminal differentiation. Previous studies have shown that human keratinocytes express mainly truncated dominant-negative p63 isotypes (▵Np63) rather than those with the N-terminal transactivation domain (TAp63) (Figure 2a,Yang et al., 1998Yang A. Kaghad M. Wang Y. et al.p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.Mol Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1764) Google Scholar). Semi-quantitative RT-PCR reactions designed to amplify ▵Np63 transcripts in RNA preparations from fractionated cells showed that small keratinocytes express high levels of ▵Np63 transcripts (Figure 2b). As the cell size increased, the abundance of ▵Np63 transcripts decreased. Comparison of Figure 1 with Figure 2(b) shows that the decrease in ▵Np63 transcripts detected by RT-PCR follows that of the p63 mRNA detected by Northern analysis. These data indicate that the differentiation of human keratinocytes is associated with the disappearance of transcripts encoding truncated dominant-negative p63 isotypes. To determine the location of p63 mRNA within the epidermis, we performed in situ hybridization on frozen sections of human foreskin. A digoxygenin-labeled antisense p63 cRNA corresponding to the cDNA probe used in our Northern analysis revealed a strong signal in keratinocytes of the basal and lower spinous layers of the epidermis (Figure 3a); however, the level of p63 mRNA in different cells of the basal layer appeared variable. The basal cells with the highest level of p63 mRNA were sometimes clustered in patches separated by basal cells containing little or no p63. No hybridization was detected when the sense cRNA was used as a probe (Figure 3b). Frozen tissue sections of normal human foreskin were analyzed for the presence of p63 by immunofluorescence, using the 4A4 monoclonal antibody (Yang et al., 1998Yang A. Kaghad M. Wang Y. et al.p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.Mol Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1764) Google Scholar). Strong staining was confined to the nuclei of basal or immediately suprabasal keratinocytes (Figure 4); however, as suggested by the mRNA localization, the intensity of p63 protein staining in different cells of the basal layer appeared variable. The basal cells most brightly stained for p63 were clustered in patches, separated by basal cells less brightly stained or unstained. The protein was absent from the spinous layer, where terminal differentiation begins. In contrast to basonuclin, which is found in both nuclear and cytoplasmic compartments of the basal layer of human epidermis (Iuchi et al., 1999Iuchi S. Easley K. Matsuzaki K. Weiner L. O’Connor N. Green H. Alternative subcellular locations of keratinocyte basonuclin.Exp Dermatol. 1999Google Scholar), p63 appeared strictly localized in the nucleus (Figure 4). Double staining for p63 and involucrin showed that involucrin appears in the spinous layer beginning several layers above the first layer lacking p63. Therefore, in normal human epidermis, all cells lose p63 considerably prior to the appearance of involucrin (Figure 4a). It has been proposed that hair follicles are an important reservoir of epidermal stem cells (Kobayashi et al., 1993Kobayashi K. Rochat A. Barrandon Y. Segregation of keratinocyte colony-forming cells in the bulge of the rat vibrissa.Proc Natl Acad Sci USA. 1993; 90: 7391-7395Crossref PubMed Scopus (189) Google Scholar;Lavker et al., 1993Lavker R.M. Miller S. Wilson C. Cotsarelis G. Wei Z.G. Yang J.S. Sun T.T. Hair follicle stem cells: their location, role in hair cycle, and involvement in skin tumor formation.J Invest Dermatol. 1993; 101: 16S-26SAbstract Full Text PDF PubMed Scopus (150) Google Scholar;Fuchs and Byrne, 1994Fuchs E. Byrne C. The epidermis: rising to the surface.Curr Opin Genet Dev. 1994; 4: 725-736Crossref PubMed Scopus (218) Google Scholar;Rochat et al., 1994Rochat A. Kobayashi K. Barrandon Y. Location of stem cells of human hair follicles by clonal analysis.Cell. 1994; 76: 1063-1073Abstract Full Text PDF PubMed Scopus (447) Google Scholar). We stained mature anagen hair follicles for p63 and involucrin. As in the case of basonuclin (Tseng and Green, 1994Tseng H. Green H. Association of basonuclin with ability of keratinocytes to multiply and with absence of terminal differentiation.J Cell Biol. 1994; 126: 495-506Crossref PubMed Scopus (81) Google Scholar), the p63-containing cells are distributed in the basal layer of the outer root sheath, the part of the follicle continuous with the basal layer of the epidermis (Figure 5). In the upper region of the follicle, cell clusters brightly stained for p63 could be seen (Figure 5). Cells of this region have been shown to contain very little basonuclin (Weiner and Green, 1998Weiner L. Green H. Basonuclin as a cell marker in the formation and cycling of the murine hair follicle.Differentiation. 1998; 63: 263-272Crossref PubMed Google Scholar;Iuchi et al., 1999Iuchi S. Easley K. Matsuzaki K. Weiner L. O’Connor N. Green H. Alternative subcellular locations of keratinocyte basonuclin.Exp Dermatol. 1999Google Scholar). In the lower half of the follicle, p63 seems to be present in all layers of the outer root sheath. In the region close to the bulb, the cells of the outer root sheath narrow into a single layer of cells retaining p63. In the bulb, p63-containing keratinocytes surround the surface of the follicular papilla (Figure 5). In contrast to basonuclin (Weiner and Green, 1998Weiner L. Green H. Basonuclin as a cell marker in the formation and cycling of the murine hair follicle.Differentiation. 1998; 63: 263-272Crossref PubMed Google Scholar;Iuchi et al., 1999Iuchi S. Easley K. Matsuzaki K. Weiner L. O’Connor N. Green H. Alternative subcellular locations of keratinocyte basonuclin.Exp Dermatol. 1999Google Scholar), no difference was observed in the distribution of p63 staining between newborn and adult epidermis, nor in staining intensity along the hair shaft (data not show). p63 is absent from the dermal cells within the follicular papilla or surrounding the follicle, and from the differentiated cells that form the medulla and inner root sheath, where involucrin is expressed. It has been shown that the growth of megacolonies depends on outward migration of the rapidly proliferating cells located in a rim close to the colony perimeter (Barrandon and Green, 1987Barrandon Y. Green H. Three clonal types of keratinocyte with different capacities for multiplication.Proc Natl Acad Sci USA. 1987; 84: 2302-2306Crossref PubMed Scopus (1040) Google Scholar). The multiplication rate internal to this rim is much lower. Although the small cells located in the center of the colonies are quiescent, they remain capable of growth (Barrandon and Green, 1987Barrandon Y. Green H. Three clonal types of keratinocyte with different capacities for multiplication.Proc Natl Acad Sci USA. 1987; 84: 2302-2306Crossref PubMed Scopus (1040) Google Scholar;Tseng and Green, 1994Tseng H. Green H. Association of basonuclin with ability of keratinocytes to multiply and with absence of terminal differentiation.J Cell Biol. 1994; 126: 495-506Crossref PubMed Scopus (81) Google Scholar). Ki-67 is a cell proliferation-associated human nuclear antigen found in all stages of the cell cycle (Gerdes et al., 1984Gerdes J. Lemke H. Baisch H. Wacker H.-H. Schwab U. Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67.J Immunol. 1984; 133: 1710-1715PubMed Google Scholar). When located in the nuclei, Ki-67 has been reported as a specific marker of multiplying keratinocytes in human epidermis (Ando et al., 1989Ando M. Kawashima T. Kobayashi H. Ohkawara A. Proliferating cells in the normal and psoriatic epidermis detected by Ki67 monoclonal antibody.J Invest Dermatol. 1989; 92: 395Google Scholar;Miyauchi et al., 1990Miyauchi S. Hashimoto K. Miki Y. The innermost cell layer of the outer root sheath is positive with Ki-67.J Invest Dermatol. 1990; 95: 393-396Crossref PubMed Scopus (25) Google Scholar;Hennen et al., 1998Hennen M. Thiriar S. Noel J.C. Galand P. Ki-67 Immunostaining of normal human epidermis–-comparison with 3H-thymidine labelling and PCNA immunostaining.Dermatology. 1998; 197: 123-126Crossref PubMed Scopus (40) Google Scholar). Double staining for p63 and Ki-67 revealed that the rapidly proliferating cells located close to the megacolony perimeter contained both p63 and Ki-67 (Figure 6a). Ki-67 was concentrated in the nucleoli, a site from which p63 seemed to be excluded. In the central part of the colonies, virtually all cells in the basal layer of the stratified colony retain p63 but few cells contain Ki-67 (Figure 6b). Similarly, in normal human epidermis, most cells in the basal layer of epidermis are not engaged in multiplication (Boezeman et al., 1987Boezeman J.B. Bauer F.W. de Grood R.M. Flow cytometric analysis of the recruitment of G0 cells in human epidermis in vivo following tape stripping.Cell Tissue Kinet. 1987; 20: 99-107PubMed Google Scholar;Potten and Morris, 1988Potten C.S. Morris R.J. Epithelial stem cells in vivo.J Cell Sci Supplement. 1988; 10: 45-62Crossref PubMed Google Scholar;Clausen and Potten, 1990Clausen O.P. Potten C.S. Heterogeneity of keratinocytes in the epidermal basal cell layer.J Cutan Pathol. 1990; 17: 129-143Crossref PubMed Scopus (22) Google Scholar). Double staining for p63 and Ki-67 revealed that only a small proportion of the cells containing p63 also contain Ki-67, but all the cells containing Ki-67 contained p63 (Figure 7). Thus, in stratified epidermal cultures and in normal human epidermis, p63 is expressed in the nuclei of cells that are either proliferating or possess the ability to proliferate. Having shown the association of p63 with the ability of basal keratinocytes to multiply, we examined the expression of p63 in a squamous carcinoma cell line (SCC13) (Rheinwald and Beckett, 1980Rheinwald J.G. Beckett M.A. Defective terminal differentiation in culture as a consistent and selectable character of malignant human keratinocytes.Cell. 1980; 22: 629-632Abstract Full Text PDF PubMed Scopus (271) Google Scholar). RT-PCR analyses were performed on RNA preparations from these cells, using primers specific for each of the two different p63 N-termini, TA and ▵N (Yang et al., 1998Yang A. Kaghad M. Wang Y. et al.p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.Mol Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1764) Google Scholar). An RT-PCR reaction designed to amplify transcripts encoding the N-terminal transactivation domain showed little or no product. In contrast, a strong signal was detected in the RT-PCR reaction designed to amplify the truncated ▵Np63 transcript (data not shown). These data suggest that these cells, like normal keratinocytes, predominantly express truncated dominant-negative p63 isotypes (▵Np63). We also analyzed the expression of the p63 gene in squamous cell carcinomas derived from oral epithelium overlying the mandibular alveolus. Frozen sections from extensively, moderately, and poorly differentiated tumors were doubly stained for p63 and involucrin. In well-differentiated tumors, p63 was readily detected in the nuclei of cells arranged in a circle consisting of one or two layers of well-organized, basal-like cells. As in the normal epidermis, p63-containing cells were located at some distance from the terminally differentiated cells stained brightly for involucrin, and were generally absent from the zone immediately surrounding the involucrin-containing cells (Figure 8a compared to Figure 4). Less differentiated tumors showed a large increase in the number of cells containing p63 and those cells were irregularly distributed with respect to centers of terminal differentiation (Figure 8b,c). Some of the cells containing p63 were located very close to involucrin-containing zones, instead of remaining at some distance, as in normal epidermis and in well-differentiated tumors. In poorly differentiated squamous tumors, there were very few cells containing involucrin; most of the cells stained very brightly for p63 (Figure 8c), and the distribution of these p63-containing cells was chaotic with respect to centers of terminal differentiation. In this report, we show that in cultured keratinocytes p63 mRNA is confined to the small cells known to be capable of DNA synthesis and colony formation, and absent from the larger cells that are undergoing terminal differentiation. Our data indicate that the irreversible growth arrest and differentiation of human keratinocytes are associated with the disappearance of transcripts encoding truncated dominant-negative p63 isotypes. In the epidermis, the mRNA for p63 is concentrated in the basal and lower spinous layers, in agreement with the previously reported result for the rat homologue KET mRNA (Schmale and Bamberger, 1997Schmale H. Bamberger C. A novel protein with strong homology to the tumor suppressor p53.Oncogene. 1997; 15: 1363-1367Crossref PubMed Scopus (245) Google Scholar); however, the amount of p63 mRNA and protein in different cells of the basal layer appeared variable. Basal cells with abundant p63 mRNA or protein were clustered in patches, separated by basal cells with little or no p63 mRNA or protein. Heterogeneity of cells within the basal layer with respect to proliferative capacity and expression of protein markers is well known (Clausen and Potten, 1990Clausen O.P. Potten C.S. Heterogeneity of keratinocytes in the epidermal basal cell layer.J Cutan Pathol. 1990; 17: 129-143Crossref PubMed Scopus (22) Google Scholar;Jones et al., 1995Jones P.H. Harper S. Watt F.M. Stem cell patterning and fate in human epidermis.Cell. 1995; 80: 83-93Abstract Full Text PDF PubMed Scopus (698) Google Scholar). Whether the heterogeneity in p63 staining of the basal layer means that p63 is more highly expressed in epithelial stem cells deserved investigation. Interestingly, another marker of epidermal basal cells, basonuclin, is more uniformly present in cells of the basal cell layer than in p63 (Tseng and Green, 1994Tseng H. Green H. Association of basonuclin with ability of keratinocytes to multiply and with absence of terminal differentiation.J Cell Biol. 1994; 126: 495-506Crossref PubMed Scopus (81) Google Scholar;Weiner and Green, 1998Weiner L. Green H. Basonuclin as a cell marker in the formation and cycling of the murine hair follicle.Differentiation. 1998; 63: 263-272Crossref PubMed Google Scholar), supporting the notion that p63 distribution is differentially expressed in basal cell populations. Whereas more directed studies will be required to determine whether p63 is a reliable marker for epidermal stem cells, a functional link between p63 expression and the maintenance of the epidermal stem cell population is evident from the p63–/– mouse (Mills et al., 1999Mills A.A. Zheng B. Wang X.J. Vogel H. Roop D.R. Bradley A. p63 is a p53 homologue required for limb and epidermal morphogenesis.Nature. 1999; 398: 708-713Crossref PubMed Scopus (1631) Google Scholar;Yang et al., 1999Yang A. Schweitzer R. Sun D. et al.p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development.Nature. 1999; 398: 714-718Crossref PubMed Scopus (1825) Google Scholar). In its distribution, p63 is in many respect similar to that of basonuclin (Tseng and Green, 1994Tseng H. Green H. Association of basonuclin with ability of keratinocytes to multiply and with absence of terminal differentiation.J Cell Biol. 1994; 126: 495-506Crossref PubMed Scopus (81) Google Scholar), a zinc finger protein (Tseng and Green, 1992Tseng H. Green H. Basonuclin: a keratinocyte protein with multiple paired zinc fingers.Proc Natl Acad Sci USA. 1992; 89: 10311-10315Crossref PubMed Scopus (72) Google Scholar); however, there are two important differences: (i) in the case of p63, strictly nuclear localization has been demonstrated in this report and inYang et al., 1998Yang A. Kaghad M. Wang Y. et al.p63, a p53 homolog at 3q27–29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities.Mol Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1764) Google Scholar. In contrast, basonuclin, although often nuclear in its location, is sometimes cytoplasmic (Yang et al., 1997Yang Z. Gallicano G.I. Yu Q.C. Fuchs E. An unexpected localization of basonuclin in the centrosome, mitochondria, and acrosome of developing spermatids.J Cell Biol. 1997; 137: 657-669Crossref PubMed Scopus (51) Google Scholar;Mahoney et al., 1998Mahoney M.G. Tang W. Ziang M.M. Moss S.B. Gerton G.L. Stanley J.R. Tseng H. Translocation of the zinc finger protein basonuclin from the mouse germ cell nucleus to the midpiece of the spermatozoon during spermiogenesis.Biol Reprod. 1998; 59: 388-394Crossref PubMed Scopus (34) Google Scholar;Weiner and Green, 1998Weiner L. Green H. Basonuclin as a cell marker in the formation and cycling of the murine hair follicle.Differentiation. 1998; 63: 263-272Crossref PubMed Google Scholar;Iuchi et al., 1999Iuchi S. Easley K. Matsuzaki K. Weiner L. O’Connor N. Green H. Alternative subcellular locations of keratinocyte basonuclin.Exp Dermatol. 1999Google Scholar). The basis for cytoplasmic localization of basonuclin in keratinocytes has been demonstrated to reside in phosphorylated serine residues located very close to the nuclear localization signal sequence, particularly serine 541 (Iuchi and Green, 1997Iuchi S. Green H. Nuclear localization of basonuclin in human keratinocytes and the role of phosphorylation.Proc Natl Acad Sci USA. 1997; 94: 7948-7953Crossref PubMed Scopus (24) Google Scholar). There is no evidence on whether p63 possesses such a mechanism affecting its localization. (ii) In the hair follicles, p63 is strongly stained in the nuclei of the outer root sheath along its entire length, whereas nuclear staining for basonuclin is confined to the region below the sebaceous gland (Tseng and Green, 1994Tseng H. Green H. Association of basonuclin with ability of keratinocytes to multiply and with absence of terminal differentiation.J Cell Biol. 1994; 126: 495-506Crossref PubMed Scopus (81) Google Scholar;Weiner and Green, 1998Weiner L. Green H. Basonuclin as a cell marker in the formation and cycling of the murine hair follicle.Differentiation. 1998; 63: 263-272Crossref PubMed Google Scholar). In squamous cell carcinomas, we found that the number of cells containing p63 and their distribution depends on the degree of anaplasia of the tumor. 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, the number of cells containing p63 increases and their distribution becomes chaotic with respect to centers of terminal differentiation; in poorly differentiated tumors cell cells express p63 and there is virtually no relation between the distribution of cells containing p63 and the small zones of terminal differentiation. Similar experiments were carried out for basonuclin, but variability in its staining made it impossible to correlate its expression with the degree of anaplasia of the tumor (our unpublished data). Whereas our analysis was limited to only two examples of each well differentiated, moderately differentiated, and poorly differentiated tumors, the consistency of the findings suggest that p63 may be a valuable diagnostic and prognostic marker of anaplasia of keratinocyte tumors. Investigations of a much larger panel of tumors will be necessary to ascertain the clinical importance of p63 as a marker in this disease. The presence of p63 in the basal cells of the epidermis, in the cells of the outer root sheath of the hair follicle, and in the small colony-forming cells of stratified epidermal cultures suggests that this protein may have a function in maintaining the proliferative potential of keratinocytes and prevention of terminal differentiation. This concept is further supported by the much greater abundance of p63-containing cells in poorly and moderately differentiated squamous cell carcinomas than in well-differentiated ones. Finally, the strongest evidence for a stem-cell function of p63 is provided by the recent demonstration that the disruption of the p63 gene in mice results in embryonic failure to sustain keratinocyte multiplication, thus culminating in the total absence of all stratified squamous epithelia and their derivatives (Mills et al., 1999Mills A.A. Zheng B. Wang X.J. Vogel H. Roop D.R. Bradley A. p63 is a p53 homologue required for limb and epidermal morphogenesis.Nature. 1999; 398: 708-713Crossref PubMed Scopus (1631) Google Scholar;Yang et al., 1999Yang A. Schweitzer R. Sun D. et al.p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development.Nature. 1999; 398: 714-718Crossref PubMed Scopus (1825) Google Scholar). This research was supported by a grant from the National Cancer Institute.
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