DNA hypermethylation is a mechanism for loss of expression of the HLA class I genes in human esophageal squamous cell carcinomas

The three human leukocyte antigen (HLA) class I antigens, HLA-A, HLA-B and HLA-C, play important roles in the elimination of transformed cells by cyto toxic T cells. Frequent loss of expression of these antigens at the cell su rface has been observed in many human cancers. Various mechanisms for post- transcriptional regulation have been proposed and tested but the molecular mechanisms for transcriptional regulation are not clear. We show by immunoh istochemistry that the HLA class I antigens are absent in 26 of 29 (89%) sa mples of human esophageal squamous cell carcinomas (ESCC). Eleven of the 26 ESCC samples lost mRNA expression for at least one of the HLA genes, as sh own by RT-PCR. DNA from the 29 pairs of ESCC and neighboring normal epithel ium were examined for CpG island hypermethylation, homozygous deletion, mic rosatellite instability (MSI) and loss of heterozygosity (LOH). DNA from no rmal epithelial tissues had no detectable methylation of the CpG islands of any of these gene loci. Thirteen of 29 ESCC samples (45%) exhibited methyl ation of one or more of the three HLA loci and six samples (21%) exhibited methylation of all three loci. The HLA-B gene locus was most frequently met hylated (38%). HLA-B mRNA expression in an ESCC cell line, where HLA-B was hypermethylated and did not express mRNA, was activated after treatment wit h 5-aza-2 ' -deoxycytidine. Homozygous deletion of these three gene loci wa s not observed. Relatively low rates of LOH and MSI were observed for the m icrosatellite markers D6S306, D6S258, D6S273 and D6S1666, close to the HLA- A, -B and -C loci, although a high ratio of LOH was observed at a nearby lo cus (represented by the markers D6S1051 and D6S1560), where the tumor suppr essor gene p21(Waf1) resides. A strong correlation between genetic alterati ons and mRNA inactivation was observed in the ESCC samples. Our results ind icate that HLA class I gene expression was frequently down-regulated in ESC C at both the protein and mRNA levels and that hypermethylation of the prom oter regions of the HLA-A, -B and -C genes is a major mechanism of transcri ptional inactivation.