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RESEARCH REPORT |
1 Departmento de Clínica, Patologia e Cirurgia, and
2 Pharmacology, Faculdade de Odontologia, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte-MG, Brazil CEP 31270-901;
3 Department of Genetics, Yale University School of Medicine, New Haven, CT 06520-8005, USA;
4 corresponding author, rsgomez{at}mail.odonto.ufmg.br
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: odontogenic keratocyst • PTCH
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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) (Barreto et al., 2000). The single glandular odontogenic cyst included in this study did not present a PTCH gene mutation (Barreto et al., 2001).
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Antibody Production
Polyclonal antibody was raised in rabbit against a human patched peptide from the carboxy-terminal region of the protein (RLPTPSPEPPPSVVRFAMP). Validation of this antibody was previously described (Karpen et al., 2001).
Immunohistochemical Method
PTCH protein staining was performed by the streptavidin-biotin method. Briefly, 3-µm sections were de-waxed in xylene and hydrated with graded ethanol. Removal of formolic pigment was performed. Endogenous peroxidase was blocked by the incubation of sections in 6% (v/v) H2O2/methanol. Slides were subjected to microwave pre-treatment (Shi et al., 1997) and incubated with the primary antibody (anti-PTCH) for 18 hrs at 4°C. After being washed in 20 mmol/L Tris-HCl buffer (pH 7.4) containing 0.9% NaCl, sections were incubated for 30 min at room temperature with biotinylated multi-link swine anti-goat, mouse, and rabbit immunoglobulin (Dako, Carpinteria, CA, USA). Sections were washed and incubated for 30 min at room temperature with 1:100 horseradish-peroxidase-conjugated streptavidin. The peroxidase activity was visualized by the application of 0.01% diaminobenzidine tetrahydrochloride and 0.03% H2O2. Sections were counterstained with Meyer’s hematoxylin and mounted in Permount®. Negative controls consisted of omission of the primary or the secondary antibody or primary incubation in the presence of non-immunized rabbit serum instead of the primary antibody. Immunoreactions were independently analyzed by two investigators unaware of the clinical data. Staining was qualitatively analyzed as negative or positive and graded semi-quantitatively.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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), 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
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). 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). In the epithelium of mucosa, staining was weak but present in all layers, with the superficial layer occasionally showing a more evident staining. Mesenchymal cells of the connective tissue did not stain. In fragments of basal cell carcinomas, the epidermis showed strong staining, while the vast majority of the neoplastic cells were negative. Only focal cells at the central portion of the tumor sheets, non-epithelial dendritic cells, and scattered inflammatory cells of the stroma were immuno-positive (Fig. 2D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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).
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ACKNOWLEDGMENTS |
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Received December 5, 2001; Last revision August 7, 2002; Accepted September 5, 2002
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Undén AB, Zaphiropoulos PG, Bruce K, Toftgard R, Stahle-Backdahl M (1997). Human patched (PTCH) mRNA is overexpressed consistently in tumor cells of both familial and sporadic basal cell carcinoma. Cancer Res 57:2336–2340.
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