Antisense epidermal growth factor receptor RNA transfection in human glioblastoma cells down-regulates telomerase activity and telomere length

Abstract

Epidermal growth factor receptor is overexpressed and/or amplified in up to 50% of glioblastomas, suggesting an important role of this gene in glial tumorigenesis and progression. In the present study we demonstrated that epidermal growth factor receptor is involved in regulation of telomerase activity in glioblastoma. Antisense-epidermal growth factor receptor approach was used to inhibit epidermal growth factor receptor expression of glioblastoma U87MG cells. Telomerase activity in antisense-epidermal growth factor receptor cells decreased by up to 54 folds compared with control cells. Moreover, the telomere lengths of antisense-epidermal growth factor receptor cells were shortened. In addition, the tumorigenicity of antisense-epidermal growth factor receptor cells was significantly inhibited. Taken together, there were strong correlations between tumorigenicity and epidermal growth factor receptor expression levels, and between tumorigenicity and telomerase activity. These results provide evidence that epidermal growth factor receptor plays an important role in the regulation of telomerase activity of glioma cells. Our findings provide new insights into both the biological functions of epidermal growth factor receptor and the regulation of telomerase activity. The inhibition of telomerase activity triggered by antisense-epidermal growth factor receptor treatment may reflect yet another mechanism of antisense-epidermal growth factor receptor approach in tumour suppression.

Figure 1

Telomerase activity in U87MG cells, empty vector-transfected cells (U87MG/pBabe), antisense-EGFR transfected cells AS-1 and AS-3. There was no significant difference in the telomerase activity between parental and empty vector-transfected cells. However, the telomerase activity reduced 54 folds and 2.5 folds in AS-1 (P<0.001) and AS-3 (P<0.05), respectively, when compared to that of the control cells. The negative control was lysis buffer and the positive control was provided by the TRAP-eze telomerase detection kit. TSR8 (control template) was used as a quantification control. TSK1 was the internal standard of PCR.

Figure 2

TRF length analysis by Southern blot of HaeIII-digested DNA with the telomere-specific probe (TTAGGG)_4. The densitometric peak in smear signal was taken as the peak TRF length. The peak TRF lengths were 7.6, 7.2, 3.8 and 4.1 kb in parental U87MG cell, empty vector-transfected cells (U87MG/pBabe), antisense-EGFR transfected clones AS-1 and AS-3, respectively. The peak TRF lengths of control cells and antisense-EGFR transfected cells correlated well with the telomerase activity.