Downregulation of MHC class I expression due to interference with p105-NF kappa B1 processing by Ad12E1A.

We have previously demonstrated that expression of major histocompatibility complex (MHC) class I genes is repressed in baby rat kidney cells transformed by early region 1 of oncogenic adenovirus type 12 (Ad12E1). Reduced expression of MHC class I antigens contributes to the escape of Ad12-transformed cells from T-cell-mediated immune surveillance and to tumour induction. In this study, we show that repression of MHC class I expression by Ad12E1A is mediated via the H2TF1 element of the MHC class I promoter. This element binds NF kappa B and KBF1, two factors which play a major role in the regulation of MHC class I expression in vivo. In extracts from Ad12E1-transformed cells, binding of KBF1 and NF kappa B to the H2TF1 element is decreased. This is caused by reduced production of p50-NF kappa B1, the 50 kDa subunit shared by KBF1 and NF kappa B, due to interference with p105-NF kappa B1 processing by Ad12-13S-E1A protein. Overexpression of the p105-NF kappa B1 cDNA, or of a truncated p105-NF kappa B1 cDNA that codes for p50-NF kappa B1, restores MHC class I expression in Ad12E1-transformed cells. These data demonstrate that downregulation of MHC class I expression in Ad12E1-transformed cells is due to interference with processing of p105-NF kappa B1 by the Ad12-13S-E1A protein.     

Adenovirus E1A,not human papillomavirus E7, sensitizes tumor cells to lysis by macrophages through nitric oxide- and TNF-alpha-dependent mechanisms despite up-regulation of 70-kDa heat shock protein

Expression of adenovirus (Ad) serotype 2 or 5 (Ad2/5) E1A or human papillomavirus (HPV)16 E7 reportedly sensitizes cells to lysis by macrophages. Macrophages possess several mechanisms to kill tumor cells including TNF-alpha, NO, reactive oxygen intermediates (ROI), and Fas ligand (FasL). E1A sensitizes cells to apoptosis by TNF-alpha, and macrophages kill E1A-expressing cells, in part through the elaboration of TNF-alpha. However, E1A also up-regulates the expression of 70-kDa heat shock protein, a protein that inhibits killing by TNF-alpha and NO, thereby protecting cells from lysis by macrophages. Unlike E1A, E7 does not sensitize cells to killing by TNF-alpha, and the effector mechanism(s) used by macrophages to kill E7-expressing cells remain undefined. The purpose of this study was to further define the capacity of and the effector mechanisms used by macrophages to kill tumor cells that express Ad5 E1A or HPV16 E7. We found that Ad5 E1A, but not HPV16 E7, sensitized tumor cells to lysis by macrophages. Using macrophages derived from mice unable to make TNF-alpha, NO, ROI, or FasL, we determined that macrophages used NO, and to a lesser extent TNF-alpha, but not FasL or ROI, to kill E1A-expressing cells. Through the use of S-nitroso-N-acetylpenicillamine, which releases NO upon exposure to an aqueous environment, E1A was shown to directly sensitize tumor cells to NO-induced death. E1A sensitized tumor cells to lysis by macrophages despite up-regulating the expression of 70-kDa heat shock protein. In summary, E1A, but not E7, sensitized tumor cells to lysis by macrophages. Macrophages killed E1A-expressing cells through NO- and TNF-alpha-dependent mechanisms.     

Mechanism of action of aryl hydrocarbon receptor antagonists: inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced CYP1A1 gene expression.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 gene expression as determined by increased CYP1A1 mRNA levels and ethoxyresorufin O-deethylase (EROD) activity in mouse Hepa 1c1c7, rat hepatoma H-4II E and human Hep G2 cancer cell lines. In contrast, treatment of these cell lines with either alpha-naphthoflavone (alpha NF) or 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) at concentrations as high as 10(-6) M resulted in only minimal induction of CYP1A1 mRNA levels or EROD activity. Cotreatment of the cells with 10(-9) M TCDD plus different concentrations (10(-8)-10(-6) M) of MCDF or alpha NF resulted in a concentration-dependent decrease in TCDD-induced CYP1A1 mRNA levels and EROD activity in the three cell lines. Moreover, using 10(-9) M [3H]TCDD, it was shown that the alpha NF- and MCDF-mediated antagonism of TCDD-induced CYP1A1 gene expression was paralleled by a decrease in levels of the nuclear [3H]TCDD-Ah receptor complex as determined by velocity sedimentation analysis of the nuclear extracts. The binding of nuclear extracts from the treated cells to a synthetic consensus dioxin responsive element (DRE) (a 26-mer) was determined by gel retardation studies using 32P-DRE. In cells treated with 10(-9) M TCDD or TCDD plus 10(-8)-10(-6) M alpha NF, the concentration-dependent decrease in TCDD-induced CYP1A1 gene expression by alpha NF was also paralleled by decreased levels of a retarded band associated with the nuclear Ah receptor-DRE complex. In contrast, the results of the gel shift assay of nuclear extracts treated with 10(-9) M TCDD or TCDD plus 10(-8)-10(-6) M MCDF indicated that there were relatively high levels of nuclear MCDF-Ah receptor complex in the cells co-treated with TCDD plus the antagonist but this was not accompanied by induced CYP1A1 gene expression. The results suggest that alpha NF and possibly MCDF compete with TCDD for cytosolic Ah receptor binding sites; however, MCDF may also inhibit the induction response by competing for and/or partially inactivating genomic binding sites.