In RASSF1A-ectopically expressing cells, in that regulation of the cyclin
In RASSF1A-ectopically expressing cells, in that regulation of the cyclin D1 promoter does not appear to be inhibited upon RASSF1A expression [11]. However, expression of BLU in CNE-2 cells significantly inhibited the activity of the cyclin D1 promoter, as manifested by the different levels of the luciferase gene co-expressed with pCD316-BLU (Figure 3B). Transcription of cyclin D1 has been suggested to be regulated by the c-Jun transcription factor, because of the presence of a c-Jun activation site on its promoter [11]. Modulation of JNK activity by BLU was tested by cotransfection of the AP1 reporter, in PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/29045898 which luciferase expression is driven by activated JNK. We found that BLU dramatically blocks the reporter (Figure 3A) and leads to the inhibition of c-Jun phosphorylation at a dose of 100 PFU per cell (Figure 3C and D).Conclusions In recent years, human adenovirus (Ad) has been widely used as a vector for gene transfer to mammalian cells, owing to its ability to effectively infect a wide variety of cells [20]. Replication deficient viruses proliferate for weeks in hosts with cancer or other diseases, enabling the restored expression of a defected gene to achieve therapeutic goals. In the present study, a putative tumor suppressor BLU was efficiently transferred to an NPCderived line CNE-2. CNE-2, together with cell lines of identical histological origin, i.e. undifferentiated tumors arising in the nasopharynx from the endemic region, exhibit downregulated BLU expression as a result of promoter methylation [8]. A dose-dependent infection efficiency was observed. The genesis of cancer is a multi-step event, and aberrations involving genes on the 3p21 region, including homozygous deletions and promoter hypermethylation affecting cluster of TSGs, are early PX-478 supplier molecular changes in various human tumors [2]. Re-expression of the lost TSGs complementary to other interventions, for example, enhancement of host immune surveillance,Zhang et al. BMC Cancer 2012, 12:267 http://www.biomedcentral.com/1471-2407/12/Page 6 ofFigure 3 BLU expression downregulates cyclin D1 promoter and JNK activity, and inhibits phosphorylation of c-Jun. (A) CNE-2 cells were transfected with pCD316 vector or pCD316-BLU, and cell lysates were immunoblotted and probed with goat anti-human BLU polyclonal antibody. The membranes were stripped and re-probed with anti-actin mAb clone C4. (B) Expression of BLU inhibits cyclin D1 promoter activity. pCD316 and pCD316p-BLU plasmids were co-transfected with cyclin D1 promoter reporter at concentration ratios of 1:1.5 and 1:4, respectively. The luciferase activity was measured for the two conditions, and the reporter activity was presented as the ratio of the two. The data are presented as the mean ?SD, and are derived from at least three independent tests. *Indicates t < 0.05 when compared with the measured values from the two groups. (C) pCD316-BLU and empty vector were co-transfected with JNK reporter at concentration ratios of 1:1.5 and 1:4, and the reporter activity was calculated. The data re presented as mean ?SD, and were derived from at least three independent tests. *Indicates P < 0.05 when comparing the calculated values for the relative light units between the two groups. (D) CNE-2 cells were infected with 0, 10, 50 and 100 PFU Ad BLU (lanes 1, 2, 3, 4), and ectopic expression was demonstrated by immunoblotting and probing with anti BLU goat polyclonal, anti actin mAb, anti phospho-c-Jun (P-c-Jun) and anti-c-Jun rabbit p.
