Differential effects of monoclonal antibodies on activating transcription factor-1 and cAMP response element binding protein interactions with DNA.

Activating transcription factor-1 (ATF1) and cAMP response element binding protein (CREB) have been implicated in cAMP-, calcium-, and virus-induced transcriptional alterations. Although CREB and ATF1 share extensive homology, they appear to mediate distinct cellular functions. We investigated the effect on DNA binding and in vitro transcription of four monoclonal antibodies (mAb) that bound to domains in either the regulatory region (mAb 1 and 3) or unique regions near the DNA-binding domains (mAb 4 and 5) of ATF1.mAb 1 and 3 supershifted both ATF1 and CREB in a DNA binding assay but did not affect in vitro transcription. mAb 4 prevented ATF1-DNA binding while supershifting CREB.DNA complexes and inhibited in vitro transcription by 95% from the CRE-containing murine proliferating cell nuclear antigen promoter. mAb 5 reacted specifically with ATF1 and did not prevent DNA binding or affect in vitro transcription. The mAb 4 epitope was located within ATF1 amino acid residues 205-219, including the first 3 basic residues in the putative DNA-binding domain. Secondary structural analysis predicted that this region comprises a transition site from alpha-helix to a turn-like conformation in ATF1. The transition to turn-like motifs is predicted to occur in CREB after 5 additional residues, with a correspondingly longer alpha-helical domain. Although regulatory domains distinct from DNA binding regions are thought to account for most of the differences in activity of members of the CREB subfamily, our results suggest that small structural variations adjacent to DNA binding regions may also contribute to the distinct functional activities of ATF1 and CREB.

Expression of MUC1, MUC2, MUC3 and MUC4 mucin mRNAs in human pancreatic and intestinal tumor cell lines.

We examined the steady-state expression levels of mRNA for the MUC1, MUC2, MUC3 and MUC4 gene products in 12 pancreatic tumor cell lines, 6 colon tumor cell lines, and one ileocecal tumor cell line. The results showed that 10 of 12 pancreatic tumor cell lines expressed MUC1 mRNA and that 7 of these 12 lines also expressed relatively high levels of MUC4 mRNA. In contrast, MUC2 mRNA was expressed at only low levels and MUC3 was not detected in the pancreatic tumor cell lines. All 7 intestinal tumor cell lines examined expressed MUC2, and 5 of 7 expressed MUC3; however only one expressed significant levels of MUC1 and 2 expressed low levels of MUC4 mRNA. This report of high levels of MUC4 mRNA expression by pancreatic tumor cells raises the possibility that mucin carbohydrate epitopes defined by antibodies such as DuPan 2 may be expressed on a second mucin core protein produced by pancreatic tumor cells.

Phorbol esters modulate the phosphorylation of human T-cell leukemia virus type I Tax.

The Tax protein from human T-cell leukemia virus type I (HTLV-I) is a 40-kDa phosphoprotein capable of activating transcription from its own long terminal repeat (LTR), as well as increasing the transcription of cellular genes. Transcriptional activation of the HTLV-I LTR has been demonstrated via a cyclic-AMP-responsive element within the 21-bp Tax-responsive elements of the LTR. Phorbol esters also upregulate expression via the LTR. Since phosphorylation of Tax may play a role in these processes, we investigated the relative effects of kinase-stimulating agents on 32P incorporation into Tax. Our studies demonstrated that the phorbol ester 4 beta-phorbol-12 beta-myristate-13 alpha-acetate greatly stimulated Tax phosphorylation in a time- and dose-dependent manner. In contrast, 8-bromoadenosine 3'-5'-cyclic monophosphate induced little stimulation of Tax phosphorylation. Tax phosphorylation occurred only on serine residues and was mapped to a single tryptic fragment in both Tax-producing human lymphocytes and mouse fibroblast cells.

Inhibition of mouse skin tumorigenesis by dexamethasone occurs through a Ha-ras-independent mechanism.

The Ha-ras oncogene has been shown to be point-mutated and overexpressed in papillomas induced by the two-stage skin tumorigenesis regimen of 7,12-dimethylbenz[a]anthracene (DMBA) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Glucocorticoids inhibit mouse skin tumorigenesis when applied with the initiation agent or with the promoting agent. We have extended these studies to evaluate whether dexamethasone (Dex) treatment could inhibit development of already established tumors. An additional objective of this study was to investigate whether glucocorticoids directly inhibit Ha-ras gene expression at the level of transcription during skin tumorigenesis. In this report we demonstrate that topical Dex treatments significantly suppressed the formation of additional tumors relative to the acetone control group. However, Northern blot analysis of total RNA isolated from representative tumors during a series of sequential weeks of promotion indicated that Dex did not have a direct effect on Ha-ras steady-state mRNA levels despite the decrease in additional tumor numbers in the Dex-treated groups. We also investigated short-term effects of Dex on endogenous Ha-ras expression in normal mouse epidermis. Topical Dex administration had no effect on endogenous Ha-ras steady-state mRNA levels in normal skin after 2 or 24 h. To ensure that endogenous corticosterone levels in the SENCAR mouse were not influencing our results, Ha-ras mRNA levels in epidermis from SENCAR mice adrenalectomized 48 h prior to being killed were compared to Ha-ras levels in normal epidermis by Northern blot analysis. The data from this analysis revealed that bilateral adrenalectomy had no effect on Ha-ras steady-state mRNA levels in epidermis compared to ras levels in normal mouse epidermis. In summary, our results demonstrate that although Dex can inhibit further tumor development in DMBA/TPA-treated mouse epidermis, it does not do so by directly effecting Ha-ras gene expression in mouse epidermis.

Binding of the 97 kDa glucocorticoid receptor to the 5' upstream flanking region of the mouse c-Ha-ras oncogene.

Glucocorticoids regulate transcription of specific genes through the interaction of glucocorticoid hormone receptor complexes with DNA binding sites called glucocorticoid response elements (GREs). The GRE consensus sequence has been defined to be the imperfect palindromic sequence 5'-GGTACANNNTGTTCT-3', the most highly conserved portion being the 5'-TGTTCT-3' hexamer. We have identified 5 potential GREs in the 5' upstream noncoding region of the mouse c-Ha-ras oncogene, two with the same hexanucleotide sequence and three with a similar sequence. When subcloned fragments of the mouse c-Ha-ras 5' upstream region (containing the 2 hexamer GREs of exact homology) were fused to the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into HeLa cells, CAT expression driven from the ras promoter was induced up to 3-fold in the presence of dexamethasone. To determine whether the 5' upstream region of the mouse Ha-ras gene was capable of specifically interacting with the glucocorticoid receptor complex, we performed Southwestern blot analysis showing that cloned DNA fragments from the 5' upstream region of the mouse c-Ha-ras gene were able to bind a 97 kDa protein in whole cell extracts from both primary SENCAR mouse epidermal cells and HeLa G cells. Immunodepletion of the epidermal cell extract with a monoclonal antibody to the glucocorticoid receptor verified that the 97 kDa protein bound by the Ha-ras 5' region was indeed the glucocorticoid receptor protein. Our results demonstrate that the upstream noncoding region of the mouse c-Ha-ras gene binds the glucocorticoid receptor. Furthermore, the presence of glucocorticoids enhances the transcription of the mouse Ha-ras promoter region in a transient gene expression assay.

Elevated expression of Ha-ras is an early event in two-stage skin carcinogenesis in SENCAR mice.

Alterations in the expression of the Ha-ras oncogene were investigated in SENCAR mice epidermis at various stages of initiation and promotion during two-stage skin carcinogenesis in SENCAR mice. Adult SENCAR mice were treated with 200 nmol of the (+) enantiomer of benzo[a]pyrene 7,8-diol 9,10-epoxide-anti (BPDE-anti), a potent initiating agent, followed by repetitive treatments with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Other mice were 'sham'-initiated with the (-) enantiomer of BPDE-anti, which is inactive as a tumor initiator. Polyadenylated RNA was isolated from pre-tumor epidermis and tumors at eight different stages of tumorigenesis and analyzed for changes in Ha-ras expression using Northern blot hybridization. Significantly enhanced levels of Ha-ras RNA were observed in TPA-promoted papillomas as early as 7 weeks after initiation. Only trace amounts of Ha-ras RNA were present in untreated epidermis or epidermis treated with the (+) or (-) enantiomer followed by 2-12 treatments with TPA (pre-papilloma stage). Southern blot hybridization of tumor DNA indicated that the increased expression of the Ha-ras oncogene was not due to gene amplification. We conclude that elevated levels of Ha-ras expression can occur at an early stage of tumor development in mouse epidermis in vivo and may play a role in tumorigenesis.