In melanoma cell lines, two different patterns of MHC class II expression have been described, either an IFN γ-inducible expression of HLA-DR and HLA-DP, with a faint or null expression of HLA-DQ, resembling that described for melanocytes, or a constitutive expression, i.e., IFN-γ independent, of all three HLA-D isotypes. As this latter phenotype has been associated with a more rapid progression of melanoma tumors, we have analyzed in different melanoma cell lines the molecular mechanisms leading to this abnormal pattern of MHC class II expression. In agreement with the evidence of a coordinate transcription of the HLA-D genes in these cell lines, we have shown the constitutive expression of CIITA (class II transactivator) transcripts, CIITA being known as the master switch of MHC class II expression. Unexpectedly, these transcripts initiate from promoter III of the CIITA gene, a promoter that is mainly used constitutively in B lymphocytes. This expression was further shown to occur through factor(s) acting on the enhancer located upstream of CIITA promoter III, which was previously described in epithelioid cells as an IFN-γ-response sequence. The hypothesis of a general abnormality of the IFN-γ transduction pathway was dismissed. Constitutive transcription of CIITA from promoter III having been observed in unrelated melanoma cell lines, we propose the hypothesis that this phenomenon might not be a random event, but could be linked to the neoplasic state of the melanoma cells.

The class II transactivator (CIITA) controls both the constitutive and IFN-gamma inducible expression of HLA-D genes. In addition, through the squelching of another transactivator CREB-binding protein, CIITA was more recently shown to have a wider cellular function, including cell cycle control or cellular response to IFN-gamma and IL-4. However, due to its low expression level, its analysis mainly relies on the study of recombinant overexpressed forms of the protein. We report here the analysis of native CIITA in various cell types. We first show the precise timing of CIITA protein expression in a fibroblast cell line in response to IFN-gamma. This expression is observed 2 h after the cytokine addition with a peak of expression ranging from 16 to 24 h. We next show the existence of two major isoforms of the CIITA protein differentially expressed in fibroblast, B lymphocyte or melanoma cell lines. We present the first demonstration that these isoforms originate from alternative translation initiation codons. We finally show that CIITA isoforms translocate to the nucleus with an apparently similar efficiency. Our data therefore demonstrate the existence of CIITA isoforms whose respective ratio depends on the cell type examined. However, we present evidence for a modulation of this ratio in a melanoma cell line with an abnormal constitutive expression of MHC class II molecules.

MHC class II expression defects have been evidenced in several human tumor cell lines originating from lung cancers or retinoblastoma. Accordingly, the mouse adenocarcinoma and fibrosarcoma cell lines, RAG and L(tk-), do not express I-A and I-E molecules even when treated with IFN-gamma. Here we show that fusion of both cell lines restores the inducible expression of MHC class II, thereby demonstrating that they present different and recessive alterations outside the MHC class II locus. CIITA, the MHC class II transactivator, controls the tissue-specific expression of MHC class II genes and creates the architecture of the transcriptional complex that binds to the MHC class II gene promoters. In L(tk-) cells, C2ta transcripts, expressed from the gene encoding CIITA, were indeed detected in severely limited amounts, with a defect in C2ta transcription initiation. In agreement we show here that the L(tk-) cell line does not express the CIITA protein. In contrast, in the RAG cell line, C2ta transcripts were expressed at normal levels, from the proper initiation site. The nucleotide sequencing of the CIITA cDNA from RAG did not reveal any mutation. However, the CIITA protein was not detected. These data evidence a new type of defect in a MHC class II-defective tumor cell line, as we show here that the alteration in the RAG cells occurs downstream of C2ta transcription. The RAG mutation might therefore reside in the C2ta transcript nuclear export or translation, or in the stability of the CIITA protein.