Expressions of MAGE-3, PCNA, p21, and p53 Proteins in the Hypopharyngeal Squamous Cell Carcinoma Cell Line (PNUH-12) Analysed by Bivariate Flow Cytometry

ABSTRACT

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.