Early growth response-1 mediates downregulation of telomerase in cervical cancer

Abstract

Early growth response (Egr)-1 is a transcription factor that triggers transcription of downstream genes within 15-30 min of various stimulations. These genes are expressed rapidly through specific promoter activation and mediate cell growth and angiogenesis. Following the previous computational identification of a site that was thought to be an Egr-1 consensus binding site at -273 to -281 in the human telomerase reverse transcriptase (hTERT) promoter region, the present study was conducted to evaluate the role of Egr-1 in the regulation of hTERT and telomerase in uterine cervical cancer. First, the expression of Egr-1 and hTERT at the mRNA level was examined in cervical cancer tissues. Egr-1 and hTERT were expressed much higher in cervical cancer tissues than in the normal cervix. However, a negative correlation was noted in the expression between Egr-1 and hTERT. By luciferase assay using hTERT promoter constructs, hTERT transcriptional activation was shown to be inhibited when Egr-1 was overexpressed. Furthermore, Egr-1 overexpression decreased hTERT protein production as well as hTERT mRNA as observed by western blotting analysis and real-time reverse transcription-polymerase chain reaction, respectively. The present study suggests that Egr-1 plays an important regulatory role in the transcriptional activation of hTERT.

Figure 1

(a) Determination of human telomerase reverse transcriptase (hTERT) and early growth response (Egr)‐1 expression by real‐time reverse transcription–polymerase chain reaction. Cervivical cancers (n = 18), and normal cervical epithelia (n = 13). Bars are ±SD in triplicate assays. (b) Relationship between the expression of hTERT and Egr‐1 mRNA in the cervical cancers. X‐axis, relative Egr‐1 mRNA; y‐axis, relative hTERT mRNA. Egr‐1 and hTERT mRNA levels were negatively correlated (P < 0.01).

Figure 2

Early growth response (Egr)‐1 transactivates human telomerase reverse transcriptase (hTERT) promoter activity. CaSki and SiHa cells were transfected with pGL3‐1375‐luc and 20 ng, 50 ng, or 100 ng Egr‐1 expression construct or empty vector, pcDNA3.1. The empty vector pcDNA3.1 was used to keep total plasmid DNA constant. Luciferase assays were carried out after 24 h, and enzymatic activities reported in relative luminescence units. Bars, ±SD in triplicate assays.

Figure 3

(a) Human telomerase reverse transcriptase (hTERT) mRNA levels in CaSki and SiHa cells transfected with early growth response (Egr)‐1. CaSki and SiHa cells were transfected with plasmid constructs expressing Egr‐1 and pcDNA3.1 using Lipofectamine 2000. Total RNA isolated after transfection was subjected to real‐time reverse transcription–polymerase chain reaction. (b) Egr‐1 decreases endogenous hTERT protein. hTERT levels in CaSki and SiHa cells transfected with Egr‐1 are shown. CaSki and SiHa cells were transfected with plasmid constructs expressing Egr‐1 and pcDNA3.1 using Lipofectamine 2000. Cell lysates were prepared and subjected to western blot analysis, using antibodies to hTERT, Egr‐1, and β‐actin. Equal amounts of cellular protein (40 µg) were loaded. In addition, normal TIG3 cells were used as a hTERT negative control.

Figure 4

Human telomerase reverse transcriptase (hTERT) reporter constructs with and without the WT1‐binding site and early growth response (Egr)‐1 effect. (a) pGL3‐1375‐luc, pGL3‐1175‐luc, pGL3‐776‐luc, and pGL3‐378‐luc contained the WT1 site, pGL181‐luc did not contain the WT1 site. (b) Relative reporter luciferase activities in CaSki and SiHa cell lines cotransfected with hTERT reporter constructs and pcDNA3.1‐pcDNA3.1 plasmid.

Figure 5

Effect of early growth response (Egr)‐1 on pGL3‐1375‐luc reporter gene espression. (a) pGL3‐1375‐luc reporter construct and (b) pGL3‐1375 Egr‐1 mt‐luc with GGG replaced with AAA in the Egr‐1‐binding site are represented. CaSki and SiHa cells were transfected with pGL3‐1375‐luc and pGL3‐1375 Egr‐1 mt‐luc and 2 µg pcDNA3.1‐Egr‐1 or control pcDNA3.1. Luciferase assays were carried out after 24 h. Bars show ±SD in triplicate assays.

Figure 6

Analysis of early growth response (Egr)‐1 interaction with WT1 response element in the human telomerase reverse transcriptase (hTERT) promoter. (a) Egr‐1 consensus oligonucleotide was end‐labeled with ³²P and used in the binding reaction with Egr‐1 as above. For the supershift assay, anti‐Egr‐1 antibody was added to the binding reaction. For competitor assays, a 50‐fold and a 200‐fold molar excess of WT1 and hTERT consensus oligonucleotides were added to the binding reaction. (b) Oligonucleotides containing the WT1 binding site of the hTERT promoter were labeled with ³²P and used in binding reactions with Egr‐1 prepared in rabbit reticulocyte lysates. For competitor assays, a 50‐fold molar excess of Egr‐1 consensus oligonucleotide was used. Shifted and supershifted bands as well as the labeled probe are indicated.