Abstract
HER-2/neu overexpression in tumor cells caused abnormalities of MHC class I surface expression due to impaired expression of components of the antigen-processing machinery (APM) including the low molecular weight proteins, the transporter associated with antigen processing (TAP), and the chaperone tapasin, whereas the expression of MHC class I heavy chain as well as β(2)-microglobulin was only marginally affected. This oncogene-mediated deficient APM component expression could be reverted by interferon-γ treatment, suggesting a deregulation rather than structural alterations as underlying molecular mechanisms. To determine the level of regulation, the transcriptional activity of APM components was analyzed in HER-2/neu(-) and HER-2/neu(+) cells. All major APM components were transcriptionally down-regulated in HER-2/neu(+) when compared with HER-2/neu(-) cells, which was accompanied by a reduced binding of RNA polymerase II to the APM promoters. Site-directed mutagenesis of the p300- and E2F-binding sites in the APM promoters did not reconstitute the oncogene-mediated decreased transcription rate with the exception of tapasin, which was restored in HER-2/neu(+) cells to levels of wild type tapasin promoter activity in HER-2/neu(-) fibroblasts. The E2F-directed control of tapasin expression was further confirmed by chromatin immunoprecipitation analyses showing that E2F1 and p300 bind to the tapasin and APM promoters in both cell lines. Moreover, siRNA-mediated silencing of E2F1 was associated with an increased tapasin expression, whereas transient overexpression of E2F1 launch a reduced tapasin transcription, suggesting that E2F1 is an essential transcription factor for tapasin.
Highlights
Grant SE-589/11-1, 11-2 and Grant SE-581/9-2. □S The on-line version of this article contains supplemental Table 1. 1 To whom correspondence should be addressed: Magdeburger Strasse 2, 06112
HER-2/neu overexpression caused a dramatic decrease of MHC class I surface expression, which was accompanied by an impaired transcription of tapasin, TAP1, TAP2, and LMP2 (Fig. 1B) and a reduced protein expression of major antigen-processing machinery (APM) components as representatively shown for tapasin (Fig. 1C)
When compared with HER-2/neuϪ cells, the different APM promoter activities were strongly reduced in HER-2/neuϩ transfectants, but to a different extent varying from 64% for TAP1, 84% for LMP2, 62% for TAP2, and 75% for tapasin (Fig. 2A)
Summary
Grant SE-589/11-1, 11-2 and Grant SE-581/9-2. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1. 1 To whom correspondence should be addressed: Magdeburger Strasse 2, 06112. Described in tumors of distinct origin that allow their escape from immune cell recognition (4 – 8). These defects often result in decreased immunogenicity of tumors, disease progression, and reduced survival of patients [9, 10]. The molecular mechanisms underlying altered MHC class I surface expression have been attributed to impaired expression of the low molecular weight proteins (LMP)-2, -7, and -10, the proteasome activator (PA), the transporter associated with antigen processing (TAP) 1 and 2, 2-microglobulin (2-m), and the MHC class I heavy chain (HC). Methylation and altered histone acetylation were found with a low frequency for 2-m, HC, and TAP1 in tumors and were associated with a decreased MHC class I surface expression (8, 28 –32). Tapasin expression might represent a key event in overcoming immune surveillance in these tumors [35]
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