Transcriptional control of p53 expression participates in the generation of appropriate levels of active p53 in response to mitogenic stimulation. This prompted us to study the role of a putative AP-1 and a NF-kB motif in the human p53 promoter for transcriptional regulation. We show that mutation of the AP-1 or the NF-kB motif abolishes transcription from the human p53 promoter in HeLa, HepG2 and adenovirus type 5 E1-transformed 293 cells. In comparison, mutation of the previously characterized Myc/Max/USF binding site in the human p53 promoter reduces the transcription rate ®vefold. The AP-1 motif in the human p53 promoter binds c-Fos and c-Jun and the NF-kB motif binds p50 NF-kB1 and p65 RelA . The cooperative nature of transcriptional activation by these factors was documented by repression of c-fos or NF-kB1 translation: Pretreatment of the cells with a cfos or p50 NF-kB1 antisense oligonucleotide suppresses transcription from the human p53 promoter completely. In addition, we show that (a) the level of endogenous p53 mRNA and (b) transcription from the strictly p53-dependent human mdm2 promoter are reduced in the presence of c-fos, c-jun, p50 NF-kB1 , p65 RelA or c-myc antisense oligonucleotides, underscoring the importance of these transcription factors for the expression of functional p53.

No abstract

Adenovirus (Ad) E1A proteins are transcriptional regulators with antioncogenic but also transforming properties. We have previously shown that transformation-defective Ad5 E1A-derivatives are excellent tumor suppressors. For tumor-specific expression of the E1A-derivatives we intend to use tumor specific human telomerase reverse transcriptase (hTERT) core promoters. Here, we show that Spm2 and other E1A proteins with an intact amino terminus activated all hTERT constructs 10 -20-fold in malignant tumor cells but not in primary fibroblasts, without affecting the activity of endogenous telomerase. The transcription rate in tumor cells was in the range of transcription from the SV40 promoter, which qualifies an E1A-hTERT system as a putative tumor targeting/expression system. The activation of the hTERT promoter by E1A was enhanced upon deletion of the Wilms' tumor 1 negative regulatory element and maintained high after deletion of the adjacent c-Myc-responsive E-box, demonstrating an important role of the remaining sequences that contain several Sp1-motifs. E1A-mediated hTERT activation was independent from the presence of the conserved region 3 (CR3) of E1A but dependent on E1A's binding to p300/CBP and recruitment of its histone acetyltransferase activity. Moreover, E1A-Spm2 and histone deacetylase-1 behaved as antagonists with respect to the regulation of transcription from the hTERT promoter. Overall, hTERT promoter/E1A-Spm2 systems may turn out to be excellent tools for transcriptionally targeted anticancer gene therapy.

No abstract

Adenoviral E1A proteins exhibit a strong tumor-suppressive activity in human tumor cells. However, E1A is capable of transforming rodent and human cells in cooperation with other oncoproteins, such as activated RAS. Thus, the therapeutic use of wild-type E1A harbors the principal risk of enhancing tumor malignancy. This prompted us to construct E1A 13S cDNA-derived mutants that were unable to transform baby mouse kidney cells in cooperation with E1B and to test their tumor-suppressive activity in BLM human melanoma cells. Anchorage-independent growth in soft agar was reduced for those cell lines expressing the E1A delCR2 mutant, which lacks the entire conserved region 2 (CR2) sequences, or for cells expressing the E1A CR3Ex2 mutant, which contains CR3 plus exon 2 sequences. In contrast, cell lines expressing the entire E1A wild-type (E1A WT ) or only the exon 2 sequences (E1A Ex2 ) grew like the parental BLM cells. Moreover, inoculation of nude mice with BLM cells or cells expressing E1A Ex2 revealed large tumors after 2 weeks. In contrast, tumors derived from E1A delCR2 -or E1A CR3Ex2 -expressing cells exhibited a substantial delay in tumor growth accompanied by a loss of E1A expression in the outgrown tumors. Cell lines expressing E1A WT showed an intermediate phenotype. Thus, expression of CR3 plus exon 2 sequences is sufficient to enhance both the antioncogenic properties and the therapeutic safety of E1A in our system.