Quantitative evaluation of the expression of MAGE genes in tumors by limiting dilution of cDNA libraries.
The MAGE-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 semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assays, 6 of the 12 members of the MAGE-A family, including MAGE-A1, were previously reported to have a high level of expression in tumors, whereas 5 other members, including MAGE-A10, 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-A1 and MAGE-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 MAGE-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 MAGE-A cDNA clones in each library were evaluated by performing PCR assays on each of these pools. The abundance of MAGE-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. MAGE-A2, 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 are either not expressed in tumors or are expressed at a very low level, with the exception of MAGE-A8 and 11, which show high expression in a very small number of tumors. This method also allowed us to isolate 5 MAGE-A cDNAs that we had not obtained previously, enabling us to delineate the exons in the sequences of genes MAGE-A5, A8, A9, A10 and A11.
Serrano
A
A
Ludwig Institute for Cancer Research, Brussels Branch, Brussels, Belgium.
Lethé
B
B
Delroisse
J M
JM
Lurquin
C
C
De Plaen
E
E
Brasseur
F
F
Rimoldi
D
D
Boon
T
T
D000951:Antigens, Neoplasm / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002874:Chromosome Mapping; D015972:Gene Expression Regulation, Neoplastic; D015723:Gene Library; D006801:Humans; D008297:Male; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010920:Placenta / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013737:Testis / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; C119317:MAGE-A10 antigen; C072011:MAGEA1 protein, human; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins; D012333:RNA, Messenger