Because of its amplification and/or overexpression in many human tumors, the HER-2/neu proto-oncogene represents an attractive target for T-cell-mediated vaccination strategies. However, overexpression of oncogenes is often associated with defective expression of components of the MHC class I antigen-processing machinery (APM), thereby resulting in an immune escape phenotype of oncogene-transformed cells. To determine whether HER-2/neu influences the MHC class I antigen-processing pathway, the expression pattern of different APM components was examined in murine in vitro models of constitutive and tetracycline-controlled HER-2/neu expression. In comparison with HER-2/neu ؊ control cells, HER-2/ neu ؉ fibroblasts exhibit reduced levels of MHC class I surface antigens that were associated with impaired expression and/or function of the peptide transporter associated with antigen processing, the proteasome subunits low molecular weight protein 2 and low molecular weight protein 10, the proteasome activators PA28␣ and PA28␤, and tapasin. These APM abnormalities resulted in reduced sensitivity to lysis by CTLs.

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V-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2; synonyms HER2, NEU) encodes a transmembrane glycoprotein with tyrosine kinase-specific activity that acts as a major switch in different signal-transduction processes. ERBB2 amplification and overexpression have been found in a number of human cancers, including breast, ovary and kidney carcinoma. Our aim was to detect ERBB2-

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 ␤ 2 -microglobulin was only marginally affected. This oncogene-mediated deficient APM component expression could be reverted by interferon-␥ 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.The expression of multiple components of the antigen-processing machinery (APM) 2 is a prerequisite for constitutive MHC class I surface expression and necessary for recognition of non-self antigens by CD8 ϩ cytotoxic T lymphocytes (1-3). Abnormalities in the MHC class I surface expression have been 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). Recently, 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)28, the transporter associated with antigen processing (TAP) 1 and 2, ␤ 2 -microglobulin (␤ 2 -m), and the MHC class I heavy chain (HC).Despite the identification of structural alterations in ␤ 2 -m, HC, and TAP, which are caused by deletions, mutations, loss of heterozygosity, and/or recombinations, their occurrence is rare (11)(12)(13)(14). Because in most cases MHC class I APM defects could be restored by treatment with proinflammatory and inflammatory cytokines such as tumor necrosis factor (TNF)-␣ and type I and type II interferons (IFN), their impaired expression appears to be mainly due to deregulation rat...

The products of the collagen-al (I) and -a2(I) genes form the triple helical molecule collagen type I, which constitutes the major ECM protein in tissue fibrosis. The collagen-al (I) gene is mainly transcriptionally regulated, and its promoter activity depends on the interaction of the transcription factors NF-I and Spi with a tandem repeat of evolutionary conserved NF-I/Spl switch elements. An increased affinity of Spl to these elements has been observed in experimental liver fibrosis. Here, we demonstrate that the DNA binding drug mithramycin displays a high affinity binding to the GC-rich elements in the collagenal (I) promoter as measured by DNAse I protection and gel retardation assays.Mithramycin interferes with Spl but not with NF-I binding to these sites. At a concentration of 100 nM, mithramycin efficiently reduces basal and TGF--stimulated al (I) gene expression in human primary fibroblasts. The transcriptional activity and mRNA steady state levels of other genes, including the collagenase gene, as well as the growth rate of fibroblasts remained unchanged on exposure to this drug. Taken together, our results indicate that the transcriptional activity of the type I collagen gene highly depends on its GC-rich regulatory elements, and further, that these elements can be differentially blocked, thereby changing the balance between ECM structural and degrading gene activities in human fibroblasts. (J.