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Anobile, Cristina J.
Languages: English
Types: Doctoral thesis
Subjects: RC0254
Class n major histocompatibility complex antigens are cell surface ap-heterodimeric glycoproteins which function as important immune recognition molecules for the stimulation of CD4+-T lymphocyte-mediated immune responses. Their expression by tumour cells, and the possible implications, has been the subject of much debate. In this investigation aspects of the cytokine-mediated regulation of the transcription of the gene of the human class nMHC molecule lll..A-DRa were investigated in human cell lines derived from colorectal and neural tumours.\ud \ud The transcriptional regulation of HLA-DRa has been shown to be controlled through discrete promoter elements termed the W, X and Y boxes; the W and X elements were investigated here. Nuclear proteins were purified from inducible (colo 201, colo 205 and U373MG) and non-inducible (caco-2 and LS180) cell lines and were shown to associate with oligonucleotide probes corresponding to the W and X regulatory elements of the lll..A-DRa promoter using bandshift (gel retardation) experiments. Differences were observed in the populations ofW and X box-binding proteins between the cell lines. The IFN-y treatment of class n inducible colorectal cell lines resulted in the binding of different populations of transcription factors to these probes: novel factors were bound as well as proposed repressor proteins dissociating from these probes.\ud \ud When a promoter probe corresponding to the 470 base pairs upstream of the cap site was employed, differences in the binding of factors between inducible and non-inducible cell lines were again observed. Treatment of the colorectal tumour cell line colo 205 with IFN-y resulted in the appearance of a novel binding factor after 6hr treatment. Longer treatment resulted in the net loss of binding factors from the W-X box region in addition to the loss of a postulated "maintenance repressor factor" which associated less specifically with the W-X-Y box region.\ud \ud The use of reporter gene assays in the glioblastoma cell line U138MG showed that IFN-ap and TGF-p are both capable of suppression of IFN-y-induced CAT expression through a 680bp and a 320bp HLA-DRa promoter fragment. The levels of IFN-y-induced CAT expression were also greater for the 320bp promoter fragment expression vector. The suppression observed was greater with the larger promoter fragment. Concurrent IFN-y (IU/ml; 48hr) and IFN-ap (100U/ml; 48hr) reduced DRa680CAT expression to 47% and DRa320CAT expression to 84% of that observed with IFN-y alone; simultaneous treatment (with lU/ml IFN-y and 1000U/ml IFN-ap) of cells transiently transfected with pDRa320CAT resulted in CAT expression being 26% of that observed with IFN-y alone. For TGF-p (lOOU/ml) cotreatment with lU/ml IFN-y, the expression of CAT was 27% and 55% of that observed for IFN-y-treatment alone with pDRa680CAT and pDRa320CATtransfected U138MG, respectively. It was concluded that the 680bp fragment contained a repressor element absent form the truncated form. The 680bp fragment contained a putative IFN-ap response element which was removed by its truncation to yield the 320bp promoter region. The colorectal tumour cell lines were unable to express CAT driven by either HLA-DRa promoter fragment. It was hypothesised that a tissue-specific enhancer element was absent from both of these promoter fragments.\ud \ud This study demonstrated a variety of aspects of the cytokine-mediated regulation of HLA-DRa gene expression. Tissue-specific differences in the regulation of expression were shown between cells of neural and colorectal origin when they were interrogated with regards to their abilities for IFN-y-induced HLA-DRa promoter-driven gene expression. The binding of nuclear proteins to important regulatory elements of the HLA-DRa promoter also confirmed that tissue- and cell-line-specific differences occurred with this respect.

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