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Immunolocalization of PTCH Protein in Odontogenic Cysts and Tumors
1 Departmento de Clínica, Patologia e Cirurgia, and Correspondence: 4 corresponding author, rsgomez{at}mail.odonto.ufmg.br
The human patched gene (PTCH) functions in both embryologic development and tumor suppression. PTCH mutations have been found in odontogenic keratocysts. However, the expression and localization of the protein product of the gene have not been determined in odontogenic tumors and cysts. We investigated 68 odontogenic lesions by immunohistochemistry, and compared their PTCH expression with that in basal cell carcinomas. All odontogenic lesions, including two keratocysts with truncating mutations, were positive for PTCH. Different types of lesions had different amounts of staining. Lack of staining was noted in the majority of basal cell carcinomas. Taken together, these data suggest that odontogenic keratocysts arise with heterozygous mutations of the PTCH gene.
Key Words: odontogenic keratocyst • PTCH
The human homologue of the Drosophila segment polarity gene Patched encodes the transmembrane protein PTCH, which is a receptor for the morphogen Sonic Hedgehog (Stone et al., 1996). Sonic Hedgehog/Patched signaling controls cell fates, patterning, and growth in numerous tissues, including teeth (Bitgood and McMahon, 1995). Sonic Hedgehog signaling regulates growth and determines the shape of the tooth, but it is not essential for differentiation of ameloblasts or odontoblasts (Dassule et al., 2000). Hardcastle et al. (1998) demonstrated that addition of exogenous Sonic Hedgehog protein directly to early tooth progenitors and also adjacent to tooth progenitors results in abnormal epithelial invagination. Analysis of these data reinforces the role of Sonic Hedgehog signaling in epithelial cell proliferation during tooth development. Mutations in the PTCH gene were identified as the underlying genetic event in nevoid basal cell carcinoma syndrome (Hahn et al., 1996). The demonstration of frequent loss of heterozygosity within the region containing the PTCH gene in sporadic and hereditary odontogenic keratocysts (Lench et al., 1996; Levanat et al., 1996), and the subsequent finding of PTCH mutations in sporadic keratocysts have sparked intense interest in the role of this gene in odontogenic disorders (Barreto et al., 2000). Mutational inactivation of PTCH leads to overexpression of the mutant transcript owing to failure of a negative feedback mechanism (Undén et al., 1997; Nagano et al., 1999). Expression studies with in situ hybridization and reverse-transcription/polymerase chain-reaction have shown PTCH overexpression in basal cell carcinomas compared with normal skin, a finding not seen in other types of skin cancer (Gailani et al., 1996; Undén et al., 1997; Nagano et al., 1999). The paucity of data concerning the localization of PTCH protein in many lesions, coupled with the evidence of PTCH gene mutations in odontogenic keratocysts and the importance of the Hedgehog signaling pathway during tooth formation, prompted us to investigate PTCH protein expression and localization in various odontogenic cysts and tumors.
Tissue Samples of Odontogenic Lesions Sixty-eight odontogenic lesions, all from unrelated patients, were retrieved from the files of the Oral Pathology Laboratory, School of Dentistry, Universidade Federal de Minas Gerais. This study followed an informed consent protocol that was approved by the University’s Ethics Committee. The odontogenic lesions consisted of 15 radicular cysts, 29 odontogenic keratocysts, 1 glandular odontogenic cyst, 6 dentigerous cysts, 3 odontogenic myxomas, 6 calcifying odontogenic cysts, and 8 ameloblastomas. In 7 out of 29 odontogenic keratocysts included in the present study, sequencing analysis of the PTCH gene was previously performed (Table  ) (Barreto et al., 2000). The single glandular odontogenic cyst included in this study did not present a PTCH gene mutation (Barreto et al., 2001).
The majority of previous PTCH expression studies have been done with RNA-based methods in basal cell carcinomas (Gailani et al., 1996; Undén et al., 1997; Nagano et al., 1999). To compare the immunolocalization of PTCH protein present in the odontogenic epithelium with that of other types of epithelial cells, we also studied 11 basal cell carcinomas and 10 samples of normal oral mucosa. All tissues had been fixed in formalin and embedded in paraffin.
Antibody Production
Immunohistochemical Method
All tissue examined showed positive intra-cytoplasmic staining of PTCH (Figs. 1, 2 ), notwithstanding variable intensity. Positive staining in the intermediate and superficial layers of the cystic epithelium was detected in 14/15 of radicular cysts (Fig. 1A), with the basal layer also staining in two of them. PTCH staining was found in the intermediate and superficial cells of the epithelium in all odontogenic keratocysts (Fig. 1B). In addition, staining of the basal cell layer with loss of the characteristic features of a keratocyst and a dense inflammatory infiltrate was detected in two keratocysts (#1 and #3, Table ).
Immunoreactivity was present in the superficial cells of the epithelium of an odontogenic glandular cyst (Fig. 1C ). Similarly, immunostaining was found in the intermediate and superficial epithelial cells of dentigerous cysts (Fig. 1D), two of them showing weak staining of basal cells. In addition, hyaline bodies present in two dentigerous cysts showed a strong positivity (Fig. 1D). Ghost and superficial epithelium cells of calcifying odontogenic cysts had positive immunolabeling, as well as the central polyhedral and loosely connected angular cells of ameloblastoma (Fig. 2A). Only one ameloblastoma showed no staining. Immuno-positivity for PTCH was also noticed in the mesenchymal cells of odontogenic myxoma (Fig. 2B).
Fragments of normal oral mucosa revealed weaker staining than epidermis (Fig. 2C
Patched is both a member of the hedgehog pathway and a target of the pathway. PTCH mRNA expression is a marker of activation of the hedgehog pathway. High levels of PTCH mRNA are seen in virtually all basal cell carcinomas, which have either inactivating mutations of PTCH (a negative regulator of the hedgehog pathway) or activating mutations of SMOH (a positive regulator of the hedgehog pathway) (Gailani et al., 1996; Undén et al., 1997; Nagano et al., 1999; Tojo et al., 1999). There are relatively few data on PTCH protein expression in tumors and other lesions (Zedan et al., 2001). The purpose of the current study was to examine expression of PTCH in odontogenic tumors and cysts, which also can arise with mutations in PTCH. We used immunohistochemical methods to assess PTCH expression at the protein level. As a means of validating this technique, we examined PTCH protein in basal cell carcinomas. As expected, there was virtually complete lack of immunostaining of PTCH in the peripheral and central cells of tumor sheets. Although the tumor cells overexpress PTCH mRNA, PTCH protein would not be expected to be present in basal cell carcinomas, because the vast majority of these tumors have truncating PTCH mutations 5' to the region encoding the peptide against which our antibody was made. Our findings of marked staining in normal epidermis are in contrast with those of previous studies showing very little PTCH mRNA in skin (Gailani et al., 1996; Undén et al., 1997). The presence of PTCH protein in skin may reflect higher sensitivity of immunostaining than the in situ hybridization methods used by those authors. PTCH mRNA is detected in normal epidermis by RT-PCR (Hahn et al., 1996). In addition, it is possible that PTCH protein accumulates in skin cell due to high stability compared with PTCH mRNA. Immunostaining of odontogenic lesions revealed the presence of PTCH protein in virtually all cysts and tumors. In epithelial lesions, PTCH was commonly observed in all superficial layers but not basal cells. Increased PTCH mRNA levels may reflect a clonal genetic change, resulting in loss of autoregulation, causing mRNA overexpression (Undén et al., 1997). However, loss of PTCH regulation could also result from activation of the hedgehog signaling pathway by mutations in other genes (Undén et al., 1997). The finding of PTCH staining in normal epidermis could reflect a differentiation process occurring in the epithelia of these odontogenic lesions. The dentigerous cyst, a developmental odontogenic cyst, can also be caused by the functional loss of PTCH (Levanat et al., 2000). We demonstrated positive staining in the epithelium and an intense immunolabeling in hyaline bodies in two dentigerous cysts. Hyaline bodies are globulous structures seen within or below the epithelial lining of odontogenic cysts (Yamaguchi, 1980), but their origin remains unknown (Philippou et al., 1990). The intense immunolocalization of the PTCH in these hyaline bodies suggests that their formation is associated with PTCH accumulation in epithelial cells of the lesion. However, the importance of PTCH in the pathogenesis of hyaline bodies remains to be established. The positive labeling for PTCH in epithelial lesions (radicular cyst, glandular odontogenic cyst, calcifying epithelial odontogenic cyst, and ameloblastoma) and in a mesenchymal tumor (myxoma) is in agreement with both cell types expressing this protein during early odontogenesis (Hardcastle et al., 1998). The staining in all lesions was more intense and evident than in the epithelium of normal oral mucosa, consistent with a model whereby the Hedgehog pathway is activated in these lesions. It is unlikely that PTCH itself is mutated in all of these lesions, because most inactivating PTCH mutations would be expected to result in low levels of PTCH protein (as in basal cell carcinomas). More studies are needed to determine how this pathway is switched on. Loss of heterozygosity in PTCH was previously reported in 7 sporadic odontogenic keratocysts (Lench et al., 1996; Levanat et al., 1996). Two of the keratocysts reported in this study have PTCH mutations predicted to result in a truncated protein. One would expect no immunostaining of the epithelial cells of these lesions. Surprisingly, an immunoreactivity was detected, indicating that the epithelial cells may be heterozygous for the PTCH mutation. Therefore, these results suggest that odontogenic keratocyst may arise with haplo-insufficiency of PTCH. Consistent with this model, retention of one normal copy of PTCH in a mouse medulloblastoma with a heterozygous PTCH mutation was demonstrated (Zurawel et al., 2000).
This investigation was supported in part by grants from FAPEMIG, PRONEX, and CNPq, (Brazil) and by NIH R01-CA57605 (AEB). Received for publication December 5, 2001. Revision received August 7, 2002. Accepted for publication September 5, 2002.
Journal of Dental Research, Vol. 81, No. 11,
757-760 (2002) This article has been cited by other articles:
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