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.     

Changes in NF-kappa B and ISGF3 DNA binding activities are responsible for differences in MHC and beta-IFN gene expression in Ad5- versus Ad12-transformed cells.

Changes in MHC class I expression are frequently observed in tumors, which represents at least one mechanism by which tumor cells escape immune surveillance. MHC class I expression is often suppressed in type 12 adenovirus (Ad12)-transformed rodent cells, but is highly induced in Ad5-transformed cells. This difference helps to explain why Ad12 but not Ad5 can induce tumors in immunocompetent syngeneic rats. In this report we demonstrate that only Ad5- but not Ad12-transformed rodent fibroblasts constitutively express beta-IFN which results in ISGF3 factor induction, and stimulation of MHC class I expression. Furthermore, we demonstrate that in contrast to Ad12-transformed cells, Ad5-transformed cells show constitutive levels of nuclear NF-kappa B-like DNA binding activity. This is of particular interest since both the beta-IFN and the MHC class I promoters contain an NF-kappa B DNA binding site. Thus, high levels of MHC class I expression in Ad5-transformed cells are due to a combinatorial stimulation of two cis-regulatory sequences of the MHC class I promoter: the NF-kappa B binding site and the interferon stimulated response element (ISRE), which binds the ISGF3 factor complex. The failure of Ad12-transformed cells to activate this pathway explains their low levels of MHC class I expression and their greater oncogenicity.     

Adenovirus E1A-mediated regulation of class I MHC expression.

Expression of class I MHC transplantation antigens has been shown to be reduced in baby rat kidney (BRK) cells transformed by highly oncogenic adenovirus type 12 (Ad12), as compared with untransformed cells and cells transformed by non-oncogenic Ad5. Here we show that this reduction of class I expression also occurs in a variety of other primary cell cultures transformed by Ad12, and that reduction of class I gene expression occurs for all class I loci. Transfection of Ad5E1 into class I-negative Ad12-transformed BRK cells leads to complete restoration of class I expression. Introduction of Ad12E1 into most class I-positive established cell lines does not result in suppression of class I expression. However, transfection of the Ad12E1A region into a class I-positive cell line which was immortalized by a mutant Ad12E1A region resulted in suppression of class I gene expression, implying that the suppression of class I activity in Ad12-transformed cells is due to an active switching-off process.     

The SV40 TC-II(kappa B) enhanson binds ubiquitous and cell type specifically inducible nuclear proteins from lymphoid and non-lymphoid cell lines.

We have characterized the complexes resulting from the specific binding in vitro of proteins present in nuclear extracts of several lymphoid and non-lymphoid cell lines to the TC-I and TC-II sequences of the simian virus 40 (SV40) enhancer. No proteins could be detected, binding selectively to the TC-I sequence, but two proteins TC-IIA and TC-IIB were identified interacting specifically with both the TC-II/kappa B enhanson, 5'-GGAAAGTCCCC-3' (important for the activity of the SV40 enhancer in vivo), and with the related H-2Kb enhanson, 5'-TGGGGATTCCCCA-3'. The binding of these two proteins to mutated TC-II enhansons correlates with the effect of these mutations in vivo, suggesting that both proteins may be important for SV40 enhancer activity. The TC-IIA binding activity was present in nuclear extracts of mature lymphoid B cells and was increased in pre-B cell nuclear extracts by lipopolysaccharide (LPS) and cycloheximide treatment. Furthermore, complex formation between the TC-IIA protein and the TC-II enhanson was efficiently competed by the kappa B motif from the kappa chain enhancer, indicating that TC-IIA is the NF-kappa B factor or a closely related protein. However, in contrast to previous reports, a TC-IIA/NF-kappa B-like protein whose properties could not be distinguished from those of the TC-IIA protein present in lymphoid B cells, was found in nuclear extracts of several untreated non-lymphoid cell lines, notably of HeLa cells, but not of undifferentiated F9 embryonal carcinoma (EC) cells [F9(ND)]. The TC-IIA binding activity which was moderately increased in HeLa cell nuclear extracts by 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or cycloheximide treatment could be induced in nuclear extracts of F9(ND) cells by cycloheximide, but not by TPA. Moreover, the TC-IIA binding activity could be induced in cytosolic fractions from F9(ND) cells by treatment with deoxycholate, indicating that these cells contain an inhibitor protein similar to the previously described NF-kappa B inhibitor, I kappa B. The second TC-II enhanson binding protein, TC-IIB, which could be clearly distinguished from the TC-IIA/NF-kappa B-like protein, by a number of differential properties, resembles the previously described KBF1/H2TF1 protein as it binds with a higher affinity to the H-2Kb enhanson than to the TC-II/kappa B enhanson, and its pattern of methylation interference on the H-2Kb and TC-II/kappa B enhansons is identical to that reported for the KBF1/H2TF1 protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of adenovirus type 12 early region 1 in KB cells transformed by recombinants containing the gene.

The adenovirus type 12 (Ad12) early region 1 (E1) gene was introduced into KB cells by using a dominant selection vector, pSV2-gpt, and over 80 Gpt+ KB cell clones were established. Three types of recombinant DNAs (gAE1A, gARC, and gABA) were constructed. They contained the AccI-H, EcoRI-C, and BamHI-A fragments, respectively, of Ad12 DNA in pSV2-gpt. Five of 50 (10%) gABA-transformed cell clones, 12 of 18 (67%) gAE1A-transformed cell clones, and 10 of 18 (56%) gARC-transformed cell clones complemented the growth of Ad5 dl312 (deletion in E1A) and were designated as Gpt+ Ad+ cell clones. In these cell clones at their early passages, recombinant genome sequences were detected in cellular DNA and were expressed. T antigen g (the E1A gene product) was detected by immunofluorescence. The Gpt+ Ad+ cell clones supported the growth of Ad5 deletion mutants in parallel with the expression of Ad12 E1A or E1A plus E1B genes. After infection of Gpt+ Ad+ cell clones with Ad5 dl312, the early genes of dl312 were efficiently transcribed, indicating the expression of the pre-early function of the Ad12 E1A gene. Two clones each from gAE1A-,gARC-, and gABA-transformed cells were subcultured for a long period to determine the stability of the transfecting DNAs. Subculture in a nonselective medium resulted in cells which lost the transfecting DNAs. Subculture in a selective medium resulted in the selection of cells which maintained the gpt gene expression but lost the Ad12 gene expression. These results indicate that the transfecting DNA is present in an unstable state in KB cells.     

Expression of pancreatic islet MHC class I, insulin, and ICA 512 tyrosine phosphatase in low-dose streptozotocin-induced diabetes in mice.

Activated immune cells contribute to the development of diabetes mellitus in multiple low-dose streptozotocin-treated mice. However, a role in the process for MHC Class I restricted T-cells remains a matter of debate. In this study, we examined by confocal microscopy the pancreatic expression of MHC Class I protein, insulin, and ICA 512 protein tyrosine phosphatase in C57BL/Ks mice given 40 mg/kg bw streptozotocin IP on 5 consecutive days. All animals were hyperglycemic from Day 7 and onwards. A loss of ICA 512 from the central portions of the islets was noted on Day 3. On Day 7, an increase in MHC Class I expression, confined primarily to immune cells in the exocrine pancreas and the periinsular areas, was detected. Later, several MHC class I/glucagon and some MHC class I/insulin double-positive cells were found. The insulitis was maximal on Day 14 and declined thereafter. The induction of MHC Class I expression in endocrine cells, occuring only after the cellular infiltration and when the animals were diabetic, indicates that the immune component of the disease does not depend on MHC Class I-restricted cytotoxic T-cells but rather comprises a non-antigen-specific process. (J Histochem Cytochem 48:761-767, 2000)     

Two purified factors bind to the same sequence in the enhancer of mouse MHC class I genes: one of them is a positive regulator induced upon differentiation of teratocarcinoma cells

The MHC class I murine and beta-2-microglobulin genes are silent in embryonal carcinoma (EC) cells but are induced upon differentiation of these cells. We have previously shown that enhancer-like sequences located in the promoter of the H-2Kb gene are non-functional in F9 and PCC3 cells. We have previously purified a 48 kd protein (KBF1) from a mouse T cell line which binds to a palindromic sequence located in this enhancer and to a similar sequence in the promoter of the beta-2-microglobulin gene. We describe here the purification of a second protein (KBF2, 58 kd) which also binds to this sequence. While both activities are present in differentiated cells, KBF1 binding activity is absent in undifferentiated EC cells, where the palindromic sequence shows no enhancer activity. Upon differentiation, KBF1 binding activity is induced and the palindromic sequence becomes active as an enhancer. Thus, the absence of KBF1 activity in undifferentiated EC cells is at least in part responsible for the lack of expression of H-2 class I and beta-2-microglobulin genes in these cells and suggests that KBF1 activity is regulated during differentiation.