Rafael E. Alcalde

The E-cadherin gene is silenced by CpG methylation in human oral squamous cell carcinomas

Reduction of E‐cadherin strongly relates to invasiveness and metastasis in vitro. To clarify CpG methylation around the promoter region of the E‐cadherin gene in oral squamous cell carcinoma (SCC), we examined the DNA samples of various human SCC cell lines and primary oral SCC tissues by methylation‐specific polymerase chain reaction (MSP). CpG methylation of the E‐cadherin gene markedly correlated to the reduction of E‐cadherin expression in human oral SCC cell lines. In primary oral SCC tissues, only 1 of 5 preserved E‐cadherin‐expressing tissues was methylated, whereas methylation was found in 17 (94.4%) of 18 E‐cadherin‐reduced tissues. Our results suggest that reduction of E‐cadherin expression is associated with CpG methylation of the E‐cadherin gene promoter. We recently established two cell lines with high and low metastatic potential, UM1 and UM2, from SCC primary tongue tissue of a patient. E‐cadherin expression of high‐metastatic UM1 was clearly lower than that of low‐metastatic UM2, and MSP results showed CpG methylation in the UM1 but not the UM2 cell line. To investigate whether demethylation of CpG methylation of the E‐cadherin gene could restore expression and function of E‐cadherin, we treated UM1 with the demethylating agent 5‐azacytidine (5‐aza) and found that E‐cadherin expression was indeed restored by demethylation. Moreover, in the demethylated UM1, invasion of the collagen gel was clearly suppressed compared with the untreated UM1. These results suggested that inactivation of E‐cadherin expression resulted from CpG methylation of the gene promoter; a correlation between CpG methylation of the E‐cadherin gene promoter and invasive potential was also suggested.

Cephalometric norms in Japanese adults

PURPOSE Knowledge of the normal dentofacial patterns of adults belonging to various ethnic and age groups is important for clinical and research purposes. Lateral cephalometric standards of normal Japanese adults were developed using the Burstone and Legan comprehensive cephalometric analyses that are specific for orthognathic surgery. PATIENTS AND METHODS Cephalometric radiographs of 217 Japanese adults were analyzed, and the mean values of their hard and soft tissue measurements were compared with those of white American adults. RESULTS Statistically significant differences were found in the Japanese sample, who had a shorter maxilla, larger upper anterior face height, and lower posterior dental height than Burstones white sample. A less prominent chin was observed in the Japanese male group. Soft tissue analysis of the Japanese subjects showed a retrognathic maxilla and mandible in relation to the soft tissue glabella and bilabial protrusion when compared with the white adult standards. CONCLUSION Lateral cephalometric norms are specific for the racial group, but these values should not be interpreted as treatment goals. Normative data represents an aid for the diagnosis and treatment planning of orthognathic surgery according to the needs and expectations of each individual patient.

Effect of a newly developed bisphosphonate, YH529, on osteolytic bone metastases in nude mice

YH529, [1‐hydroxy‐2‐(imidazo [1,2‐a] pyridin‐3‐yl) ethylidene]‐bisphosphonic acid monohydrate, is a newly developed third‐generation bisphosphonate with a potent inhibitory activity toward osteoclastic bone resorption. The primary cellular mechanism of osteolysis associated with metastatic cancer is osteoclast‐mediated. It is likely that bisphosphonates would be efficacious in this situation. In the present study, we examined the effect of YH529 in a nude mice bone metastasis model, in which the intracardiac injection of a human breast cancer cell line, MDA‐MB‐231(MDA‐231), leads to osteolytic bone metastases. To examine whether YH529 would prevent such bone metastasis, we administered YH529 s.c. to nude mice simultaneously with cancer cell inoculation through the entire experimental period (protocol 1) or performed short‐term prophylactic administration before inoculation of the MDA‐231 cells (protocol 2). In addition, to examine the possible therapeutic effects of the drug on established bone metastases, we injected YH529 after radiographically small but distinct osteolytic bone metastases had been detected (protocol 3). In all protocols, YH529 (2 μg/mouse/day) markedly inhibited bone metastases as well as the progression of established metastatic foci that were quantified on the radiographs. Histological examination and histomorphometrical analysis revealed that YH529 markedly reduced the number of osteoclasts and the size of the tumor at the metastatic bone sites. Our results suggest that YH529 may suppress metastasis formation and tumor growth in bone through inhibition of osteoclastic bone resorption. Int. J. Cancer 77:279–285, 1998.© 1998 Wiley‐Liss, Inc.

Prognostic significance of ERRB3 overexpression in oral squamous cell carcinoma

Immunohistochemical analysis of erbB3, as the third member of epidermal growth factor receptor gene family, was performed on 41 cases of oral squamous cell carcinoma, correlating the staining pattern with clinical outcome. High expression of erbB3 protein (ERBB3) was significantly associated with lymph node metastasis (P < 0.05), survival rate (P < 0.05) and mode of invasion (P < 0.01) in this series. These results demonstrated that ERBB3 expression may be helpful in identifying those oral squamous cell carcinomas with higher malignant potential.

Odontogenic ghost cell carcinoma: Report of a case and review of the literature

The calcifying odontogenic cyst (COC) was first recognized by Gorlin et al’ in 1962 and described by the World Health Organization’ in 1971 as a cystic lesion that shows an epithelial lining with a well-defined basal layer of columnar cells, an overlying layer that may resemble the stellate reticulum, and masses of ghost cells. A carcinoma forming in a COC was originally shown in the same publication on odontogenie tumors.’ The term odontogenic ghost cell carcinoma (OGCC)” was used later to defined this rare entity that has features of COC and an infiltrative pattern, epithelial cell atypia (pleomorphic epithelial cells with pleomorphic and vesicular nuclei), numerous mitoses, and necrotic foci. Only six cases of simultaneous occurrence of COC and its malignant transformation have been reported in the English language literature.2*4-7 The present article describes a case of OGCC of the maxilla in a 72-yearold woman and discusses the treatment and prognosis of the cases previously reported.

Establishment of High and Low Metastasis Cell Lines Derived from a Human Tongue Squamous Cell Carcinoma

Malignant tumors are composed of cells with different phenotypic properties and only certain cell subpopulations present metastatic potential. The establishment of cell lines with high or low metastatic potential is necessary to investigate the molecular mechanisms of the metastatic process. However, human oral squamous cell carcinoma (SCC) cell lines that are suitable for the above investigation are scarce. High and low metastatic cells were obtained from a primary lesion of a patient with tongue carcinoma who had not received any therapy. Two distinct cell lines were selected, UM1 with a scattered growth pattern and loose cell-cell adhesion, and UM2 with a colony-formed growth pattern and firm cell-cell adhesion. The expression of E-cadherin in UM1 was clearly lower than that in UM2. UM1 exhibited a higher motility, invasive and metastatic activity than UM2 in vivo and in vitro. A low invasive and a metastatic oral SCC cell line, useful to investigate invasion and metastasis mechanisms, have been established.

p53 and MDM2 Expression in Oral Squamous Cell Carcinoma

The p53 tumor suppressor gene is the most commonly mutated gene in human cancer and is a frequent abnormality in oral squamous cell carcinoma and its precancerous lesions. MDM2 (murine double minute-2), a new proto-oncogene, may be associated with p53 gene products and may negatively affect the transcriptional activating function of p53. The purpose of this study was to investigate the incidence of MDM2 and its relationship to the expression of p53 in oral squamous cell carcinoma and precancerous lesions. Overexpression of p53 and MDM2 proteins was detected in 52 and 40% of oral squamous cell carcinomas, respectively. p53 gene mutation, absent in normal oral epithelium was observed in 31% of the carcinoma cases. Our finding suggested that MDM2 protein may be an alternative mechanism causing p53 protein dysfunction in oral squamous cell carcinoma.

ANGIOGENESIS AND EXPRESSION OF PLATELET-DERIVED ENDOTHELIAL CELL GROWTH FACTOR IN ORAL SQUAMOUS CELL CARCINOMA

It has been demonstrated that angiogenesis is required in the process of tumor progression and metastasis. Microvessel density (MVD) estimates tumor angiogenesis and is an independent indicator for predicting tumor metastasis in a variety of carcinomas. Platelet-derived endothelial cell growth factor (PD-ECGF) is known to be an angiogenic factor in vitro and in vivo. Of 55 patients with oral squamous cell carcinoma (OSCC), regional metastasis was absent in 35 and present in 20. Cases with lymph node metastasis showed significantly higher MVD (mean 61.0 +/- 28.8) than those without metastasis (mean 29.3 +/- 15.1; p < 0.001). A total of 37 cases (67.3%) were PD-ECGF-positive with a high MVD (mean 47.8 +/- 27.9) and 18 (32.7%) showed a negative PD-ECGF expression with a low MVD (mean 26.6 +/- 13.2). PD-ECGF expression was significantly correlated with the increment of MVD (p < 0.01). We suggest that MVD can be used as an independent prognostic indicator for predicting metastasis and that PD-ECGF activity plays an important role in the neovascularization of OSCC.

Extracellular matrices expression in invasion area of adenoid cystic carcinoma of salivary glands

Adenoid cystic carcinoma (ACC) is a salivary malignant tumor with poor long-term prognosis, that is known to have predilection for invasion of the adjacent stroma and neural tissues. This carcinoma has shown a high incidence of recurrence and distal metastasis. Invasive carcinomas have been associated with the distributions of extracellular matrices (ECM). Cell proliferation as a marker of tumor growth has been related to poor prognosis in oral carcinomas. Immunohistochemical analysis of 15 cases of ACC was done using antibodies to laminin, type IV collagen, fibronectin, tenascin and anti-proliferating nuclear antigen (PCNA). Laminin and type IV collagen were totally or partially absent in the ACC invasive areas. Tenascin was expressed in the stroma and cytoplasm and was associated with tumor cell proliferation. It can be concluded that basement membrane represents a barrier that is lost during cell invasion and tenascin may be involved in the detachment of cancer cells, increasing the invasive potential of ACC.

Regulation of Apoptosis Reduction in the Cisplatin-Resistant A431 Cell Line by Bcl-2 and CPP32

Cisplatin (cis-diamminedichloroplatinum(II), CDDP) is one of the most important chemotherapeutic agents; however, the mechanisms of resistance to this drug are still unknown. Recent reports have demonstrated that chemotherapy can induce apoptosis in some cancer cells, indicating that apoptosis may play a very important role in cancer therapy. Therefore, we used a CDDP-resistant cell line from the human epidermoid carcinoma cell line A431 to investigate whether the modulation of apoptosis influences CDDP resistance. In the CDDP-resistant cell, the cell cycle was not perturbed after CDDP treatment. DNA gel electrophoresis and ELISA of the CDDP-resistant cell showed reduced apoptosis when compared with A431 cells treated with CDDP. We determined the p53, Bcl-2, Bax and CPP32 protein levels by Western blotting. This analysis demonstrated a marked increase in Bcl-2 protein levels and a reduction in CPP32 protein levels in CDDP-resistant cells. Our results indicate that the reduction of apoptosis was one of the CDDP-resistant mechanisms, and that reduced apoptosis in CDDP-resistant cells was influenced by Bcl-2 and CPP32 proteins.