Enhanced activity of cloned hamster TERT gene promoter in transformed cells

In 7,12-dimethylbenz[a]anthracene-treated hamster pouch epithelial cells, telomerase activity increased within 1 week of treatment and reached a 6–7-fold increase within 3 weeks. To investigate this phenomenon, we have cloned and sequenced the hamster telomerase catalytic subunit (hamTERT) promoter. Transient transfection with different genomic segments upstream of the ATG translation initiation codon linked to the luciferase reporter gene mapped the core promoter within a 250 bp region. Three major transcription initiation sites and several minor sites were found between −42 and −140 bp relative to the ATG site. Like the human and murine TERT promoters, the hamTERT promoter lacks TATA and CAT boxes and all three promoters share similar regulatory factor binding sites. DNase I footprint analysis revealed six protected regions which contain sequences homologous with known transcription factor binding sites. Three protein binding regions (I, II, and III) were essential for the promoter activity. Regions I and III bound to Sp1 and Sp3 transcriptional factors, whereas region II bound to an unknown factor. Transient transfection of a promoter-luciferase plasmid into Drosophila SL2 cells showed that Sp1 and Sp3 regulated the hamster TERT promoter in a concentration-dependent and synergistic manner. Telomerase activity showed a 2–4-fold and 8–10-fold increase in immortalized cells and tumor cells, respectively, but hamTERT expression was only increased 1.7-fold and 2.4-fold, respectively, in the same cells

The E7 Oncoprotein of Human Papillomavirus Type 16 Interacts with F-Actin in Vitro and in Vivo

AbstractWe report here that E7 oncoprotein of human papillomavirus type 16 (HPV-16) forms a complex in vivo and in vitro with actin, one of the components of the cellular cytoskeleton. The in vivo interaction was detected by immunofluorescent staining and confocal microscopic examination of normal human oral keratinocytes (NHOK) and CV-1 cells after transient expression of E7 employing the vaccinia virus–T7 RNA polymerase system and by coimmunoprecipitation from an immortalized, nontumorigenic cell line obtained after transfecting NHOK with the cloned HPV-16 DNA genome. The in vitro interaction was detected by cosedimentation of bacterially expressed E7 phosphorylated with rabbit reticulocyte lysate or purified casein kinase II (CKII) prior to incubation with F-actin. This interaction was inhibited if E7 phosphorylation by the rabbit reticulocyte lysate was prevented with heparin, a CKII inhibitor, or if the amino acids Ser-31 and Ser-32 in E7, which are phosphorylated by CKII, were replaced with amino acids that cannot be phosphorylated. Interestingly, a decrease in the amount of polymerized actin occurred in cells expressing E7