ized in a human melanoma cell line (MZ2-MEL). 4 Expression The MAGE-1 gene, originally isolated from a human of the MAGE-1 gene on HLA-A1 positive tumor cells directs melanoma cell line, directs the expression of a potential the expression of a nonapeptide that allows specific recognitumor-rejection antigen, MZ2-E. This antigen is recogtion by CTL in vitro. The MAGE-1 gene is expressed in many nized by autologous cytotoxic T lymphocytes in associatumor cell lines as well as in fresh tumor cells, including 40% tion with a major histocompatibility complex class I molof melanomas, 5 20% of breast tumors, 6 and 35% of nonsmall ecule (HLA-A1), and has provided a basis for specific cell lung tumors 7 while it has not been shown in normal cells immunotherapy for melanoma patients. Here we show except in testis. 5 However, literature search reveals there has a high frequency of expression of the MAGE-1 gene in been no report on MAGE-1 gene expression in HCC. hepatocellular carcinoma (HCC). We examined the ex-
We used reverse-transcription polymerase chain reaction pression of the MAGE-1 gene in cell lines originated from (RT-PCR) and Southern blotting techniques to examine the hepatoma cells and in tumor and nontumor tissues of expression of the MAGE-1 gene in tumor and normal liver livers with HCC by reverse-transcription polymerase tissues from patients with HCC. We found a high frequency chain reaction (RT-PCR) and subsequent Southern blotof expression of the MAGE-1 gene in HCC. ting. MAGE-1 messenger RNA (mRNA) was expressed in three of four HCC cell lines (75%) and in 16 of 20 (80%) MATERIALS AND METHODS resected HCCs though none was detected in nontumor Cell Lines. Seven human cell lines were studied. K562 cell derived tissues. The high frequency expression of MAGE-1 gene from a chronic myeloid leukemia in blastic crisis (generously proin HCCs suggests the possibility as a target for tumorvided from American Type Culture Collection [ATCC], Rockville, specific immunotherapy for HCC patients. (HEPATOLOGY MD), was used as a positive control for MAGE-1 gene expression. 8 1996;24:1437-1440.)
Chang liver cell derived from normal human liver (generously provided from Japanese Cancer Research Resources Bank [JCRRB], Tokyo), 9 PLC/PRF/5 derived from HCC cells of a black male with Hepatocellular carcinoma (HCC) is one of the most common chronic hepatitis B virus infection (from JCRRB), Hep G2 derived tumors in Japan. 1 Several treatments, such as transcatheter from a hepatoblastoma of a 15-year-old black male (from ATCC), 10,11 arterial embolization, chemo-lipiodolization, percutaneous HLE and HLF derived from HCC cells of a 68-year-old Japanese ethanol injection therapy, and surgical resection, are effective male (from JCRRB), 12 HuH 6 clone 5 derived from hepatoblastoma and have improved the prognosis of early to moderately adof a 1-year-old Japanese male, and HuH 7 derived from the HCC vanced-stage HCC patients. However, there are still many cells of a 57-year-old Japanese male (from JCRRB) 13,14 were used. patients with advanced-stage HCC for whom these modal-Liver Tissues. Twenty patients with HCC underwent surgical reities are ineffective. Many trials of the systemic chemothersection at the Kyushu University Hospital from March 1993 to Octo- ber 1994. Two liver specimens from each patient, one from the tumor apy and immunotherapy have not yet shown efficacy as a lesion and another from nontumorous tissue, were snap frozen in treatment for advanced HCC. liquid nitrogen and kept at 080ΠC until use.
There is now a consensus that human tumor cells possess Preparation of mRNA. Messenger RNA (mRNA) was extracted antigens encoding a tumor-specific peptide recognized by T from approximately 10 7 cells of cell line and 50 mg to 100 mg of liver cells. 2 The induction of cytotoxic T lymphocytes (CTLs), which tissue with a Micro prep mRNA extraction kit (Pharmacia Biotech, recognize these tumor-associated antigens can be efficacious Uppsala, Sweden). Extracted mRNA was dissolved in 40 mL of dis- in the prevention and treatment of tumors. 3 Thus, the generatilled water. tion of CTLs directed against HCC-associated antigens has Complementary DNA Synthesis. Each mRNA sample was incupotential for the development of specific immunotherapy of bated with 1 mL (1 nmol) of random hexamers (Takara Biomedicals, Ohtsu, Japan), 4 mL of 5 *reverse-transcriptase buffer (Life Technolo-HCC.
gies, Gaithersburg, MD), 2 mL of 0.1 mol/L dithiothreitol (Life Tech-Recently, the MAGE-1 gene has been identified as a gene nologies), 1 mL of dNTP mixture (2.5 mmol/L of each deoxyribonucleofor the tumor-rejection antigen MZ2-E that was characterside triphosphate) (Takara Biomedicals), and 200 units of Molony murine leukemia virus RT (Life Technologies) (final volume of 20 mL). After incubation at 37ΠC for 60 minutes, the RT products were used for PCR amplification.