Quantitative evaluation of the expression of MAGE genes in tumors by limiting dilution of cDNA libraries

The MACE-A genes are expressed in tumor cells but not in healthy tissues, except in male germ line cells and in placenta. They encode tumor-specific antigens recognized by autologous cytolytic T lymphocytes (CTLs), On the basis of semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) assays, 6 of the 12 members of the MACE-A family, including MAGE-A1, were previously reported to have a high level of expression in tumors, whereas 5 other members, including MACE-A In, were expressed at a much lower level, deemed to be insufficient for CTL recognition. However, analysis with antibodies has shown that some melanoma cell lines contain equivalent amounts of MAGE-A10 and MRGE-A10 proteins, This discrepancy appeared to be due to the low efficacy of the primers that had been used for the previous MAGE-A10 RT-PCR assays. This led us to develop a method that is independent of the efficacy of the PCR primers to evaluate MACE-A gene expression. cDNA libraries from tumor cell lines were introduced into bacteria, of which 200 pools of about 500 bacteria were maintained in microcultures. The frequencies of the MACE-A cDNA clones in each library were evaluated by performing PCR assays on each of these pools. The abundance of MACE-A10 cDNAs was found to be similar to that of MAGE-A1 in 3 of the libraries that were analyzed, including 2 with high expression (1/6,400), confirming that MAGE-A10 is expressed at a high level, MACE-AZ, A3, A4, A6 and A12 cDNAs were also confirmed often to be present at a frequency of more than 1/10,000, a level of expression that should suffice for recognition of antigenic peptides encoded by these genes by cytolytic T cells. The remaining MAGE genes ave either not expressed in tumors or are expressed at a very low level, with the exception of MAGE-A8 and I I, which show high expression in a very small number of tumors. This method also allowed us to isolate 5 MACE-A cDNAs that we had not obtained previously, enabling us to delineate the exons in the sequences of genes MACE-A5, A8, A9, A10 and A11. (C) 1999 Wiley-Liss, Inc