MAGE (melanoma antigen gene) is a tumor specific shared antigen, presented by HLA class I molecules, which is recognized by cytotoxic T lymphocytes. MAGE proteins are expressed in malignant tumor cells, in contrast to no expression in normal or benign tissues except for testis and placenta. MAGE might be a potential target for immunotherapy of malignant tumors. However, its biological aspects associated with cell cycle are not yet described. The flow cytometry is a useful tool for objective and quantitative analyses of heterogenous tumor cell population. To understand the status of MAGE related to cell cycle and its relationship with p53 as the G1 checkpoint regulator, p21, and PCNA as a proliferative index, we investigated expression of MAGE-3 protein, mutant p53, p21, and PCNA by flow cytometry and immunohistochemical stain. In addition, double stains for MAGE-3/p53, p53/PCNA, and p53/p21 were analysed with bivariate flow cytometry. DNA histograms using MAGE-3/PI (DNA) and p53/PI (DNA) were also analysed. The cell line (PNUH- 12) used for this study originated from a hypopharyngeal squamous cell carcinoma, which has point mutation (exon 7, C-->G) of p53. The expression rate of MAGE-3 was 83%, PCNA 85%, and p53 81%. No expression for p21 was identified. MAGE-3 was expressed in cytoplasm, while both PCNA and p53 were expressed in nuclei of tumor cells. With bivariate analyses, coexpression rates of MAGE-3/p53 and p53/PCNA were 0.96 and 0.97, respectively. Both MAGE-3 and p53 showed constantly high level throughout the cell cycle. These results suggest that expression of MAGE-3 and mutant p53 is not dependent on the cell cycle. p21 seems to be inactivated.
Carcinoma, Squamous Cell* ; Cell Cycle ; Cell Line* ; Coloring Agents ; Cytoplasm ; DNA ; Flow Cytometry* ; Immunotherapy ; Mutant Proteins ; Placenta ; Point Mutation ; Proliferating Cell Nuclear Antigen* ; T-Lymphocytes, Cytotoxic ; Testis

PURPOSE: MAGE (melanoma antigen gene) is a tumor associated antigen, presented by HLA class I molecules, which is recognized by cytotoxic T lymphocytes. The expression of MAGE proteins are confined to malignant tumor tissues, except for the normal testis and placental tissues. Therefore, MAGE may be a potential target for immunotherapy of malignant tumors. However, biological aspects associated with the cell cycle are not yet described. MATERIALS AND METHODS: The material used for this study was a novel human squamous cell carcinoma cell line (PNUH-12) from the hypopharynx, which had one point mutation of 78th base, C to G, in exon 7 of p53 gene. To understand the role of MAGE in relation to cell cycle and its relationship with p53 as the Gl checkpoint regulator, the expressions of MAGE-3 protein and mvtant p53 (Mtp53) were accessed by flow cytometry and immunohistochemistry. Double stains for MAGE-3/Mtp53 was analyzed with bivariate flow cytometry. DNA histograms using MAGE-3/PI (DNA) and Mtp53/PI (DNA) were also analyzed. RESULTS: The expression rate of MAGE-3 and Mtp53 were 83% and 85%, respectively. MAGE-3 was expressed in cytoplasm, while M:p53 were expressed in the nuclei of the tumor cells on the immunohistochemical sections. With bivariate analyses, coexpression rate of MAGE-3/Mtp53 was 0.96, and MAGE-3 and Mtp53 constantly showed high levels throughout the cell cycle except Go. CONCLUSIONS: These results mean that (I) MAGE-3 might have yet unknown relationship with mutant p53, (2) expressions of MAGE-3 and Mtp53 are not dependent on the cell cycle in PNUH-12 hypopharyngeal squamous carcinoma cell line, and suggest that MAGE-3 might have a role as important as p53 during the development of malignant tumors.
Carcinoma, Squamous Cell ; Cell Cycle* ; Cell Line ; Coloring Agents ; Cytoplasm ; DNA ; Exons ; Flow Cytometry* ; Genes, p53 ; Humans ; Hypopharynx ; Immunohistochemistry ; Immunotherapy ; Point Mutation ; T-Lymphocytes, Cytotoxic ; Testis