STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF CLASS I MAJOR HISTOCOMPATIBILITY COMPLEX GENES IN TUMOURS

Abstract

Expression of class I MHC antigens on tumour cells are believed to be crucial as they can regulate the sensitivity to antitumoural immunological mechanisms. Overall or selective qualitative or quantitative changes in MHC molecules may modify the recognition of tumour cells by components of the immune system. In many experimental models studied, variation in the expression of MHC class I antigens have been shown to exert a decisive influence on local tumour growth and metastasis. Due to the significance of class I MHC gene products in the host immune responses, I have: 1) isolated and characterised a 'novel' class I MHC gene. 2) investigated the regulation of class I MHC gene expression in human breast carcinoma cell lines. 1) ISOLATION AND CHARACTERIZATION OF THE H-2K1k GENE FROM THE C3Hf/HeN MOUSE AND ITS EVOLUTIONARY IMPLICATIONS In this study, by isolating overlapping cosmid clones and performing 'chromosome walking' from the H-2Kk gene, I have obtained cosmid clones encoding the H-2K1k gene from two separate cosmid libraries. The nucleotide sequence of one of the clones was determined. In vitro analysis revealed that the cloned H-2K1k gene could yield a normal H-2 class I mRNA transcript of 1.7 kb. However, the deletion of four nucleotides in exon 3 of the H-2K1k gene results in a pre-mature translation termination codon at the beginning of exon 4. Sequence analysis also shows that the H-2K1k gene had undergone many substitutions of methylated CpGs (mcCpGs) and suggests that this gene is inactive in C3Hf/HeN mouse. Consistent with the above observations, when expressed in human cells, the cloned H-2K1k gene gave an aberrant polypetide of Mr 36 000. Therefore, although the H-2K1k gene is homologous to other class I MHC gene in its molecular organization and nucleotide sequence, it is a pseudogene. 2) REGULATION OF CLASS I HLA GENE EXPRESSION IN HUMAN BREAST CARCINOMA CELL LINES Under-expression of class I HLA mRNA could be detected in some human breast carcinoma cell lines. Using gel mobility shift assays to analyse possible molecular mechanisms that may be involved in the down-regulation of class I HLA gene expression, a novel 9 bp cis-acting consensus element was identified in exon 1 of HLA-A, -B and -C genes. This sequence is able to exert a negative regulatory effect on the activity of a heterologous TK promoter. Furthermore, with hormone addition, factors binding to the 9 bp sequence could participate in a negative fashion with cellular trans-acting factors which bind to the reported GRE sequence and not to the ERE sequence.