Molecular analysis of HLA-DR gene expression induced by IFN-gamma in malignant melanoma cell lines

Abstract

Human leukocyte antigen (HLA) class II molecules are polymorphic cell surface glycoproteins that are crucial for the cellular interaction in immune response. The expression of class II molecules is regulated in a tissue-specific and cytokine-inducible manner, and is mainly restricted to the antigen presenting cells. However, some tumor cells also express class II molecules, and in some class-II-negative tumor cells, class II expression is inducible by interferon (IFN)-gamma. However, their expression varies, even though the tumor cells originate from the same histological origin; some tumor cells show strong expression, others show weak or no expression. To determine whether this differential expression of class II molecules on tumor cells is transcriptionally regulated, FACS analysis and Northern hybridization were performed using a panel of melanoma cell lines, IGR3, Malme-3M, SK-Mel-24, and SK-Mel-28 to analyze the cell surface expression and mRNA transcription rate of HLA-DR before and after treatment with IFN-gamma. FACS analysis showed that before IFN-gamma treatment, IGR3 and Malme-3M cells barely expressed HLA-DR. On the contrary, almost all of the SK-Mel-24 cells (> 90%) and a relatively high rate (> 50%) of SK-Mel-28 cells expressed HLA-DR. After IFN-gamma treatment, HLA-DR expression was induced in Malme-3M cells and SK-Mel-28 cells which displayed elevated levels of HLA-DR expression in a time-dependent manner. However, IGR3 cells never responded to IFN-gamma. Northern analysis showed that treatment with IFN-gamma led to the steady-state mRNA augmentation of the HLA-DR gene in Malme-3M and SK-Mel-28, whereas in IGR3, IFN-gamma did not augment the transcriptional rate of the HLA-DR gene. To further clarify this differential modulation, sequencing analysis of PCR product of the HLA-DR proximal promoter region was done, since the transcription rate of the class II gene is controlled by the well-conserved proximal promoter region. Six independent clones from PCR products of the HLA-DRA proximal promoter region and 16 clones from PCR products of the HLA-DRB proximal promoter region were isolated from the above cell lines and sequenced. Comparison of the nucleotide sequences of all 6 clones of DRA promoter showed that the sequences are extremely similar in both regulatory sequences and their intervening sequences. Sixteen clones of HLA-DRB promoter showed sequence variations such as substitution and insertion/deletion, and these 16 clones could be further grouped into 6 homologues with sequence homology. These data established that the melanoma cell lines studied here showed a differential susceptibility to IFN-gamma on the modulation of HLA-DR molecules, that this modulation is transcriptionally regulated, and that the difference in promoter activity by sequence variation might contribute to such a differential transcriptional regulation at the promoter level.