Antisense epidermal growth factor receptor RNA transfection in human malignant glioma cells leads to inhibition of proliferation and induction of differentiation

Abstract

The epidermal growth factor receptor (EGFR) is a protooncogene that is frequently observed with alterations in late stage gliomas, suggesting an important role of this gene in glial tumorigenesis and progression. In this study we evaluated an antisense EGFR approach as an alternative therapeutic modality for glioblastomas. We transfected U-87MG cells with an antisense EGFR construct and obtained several clones stably expressing lower or undetectable levels of EGFR protein. These clones were found to have impaired proliferation as well as a reduced transforming potential to grow in soft agarose. The number of cells positive for the cell cycle-specific nuclear antigen Ki-67 was also significantly decreased (P < 0.05) in antisense EGFR-transfected clones compared with parental or empty vector-transfected cells. Flow cytometric analysis revealed that the proportion of cells in G0/G1 phases of the cell cycle in the antisense clones increased by up to 31% compared with control cells, whereas the proportion of cells in S phase decreased by up to 58%. In addition, the antisense EGFR-transfected cells showed higher expression of glial fibrillary acidic protein and a more differentiated form, with smaller cell bodies possessing fine tapering cell processes. These results suggest that EGFR plays a major role in modulating cell growth and differentiation in glioblastoma cells. Our experimental model of antisense EGFR provides a basis for future development of antisense EGFR oligodeoxynucleotides in treatment of glioblastomas.

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

“…The stable transfectants expressed low or undetectable levels of EGFR protein (Tian et al, 1998). These transfected cells were found to be weakly-or non-responsive to epidermal growth factor induced cell growth (Tian et al, 1999).…”

“…The construction of the antisense-EGFR construct and the cloning of the transfected cells have been described previously (Tian et al, 1998). The EGFR cDNA fragment (BamHI-BamHI) derived from PE7 was inserted in reverse orientation at the BamHI site of the pBabe-puro vector (Morgenstern and Land, 1990).…”

“…Cells were transfected with the antisense-EGFR constructs using the Transfectam reagent (Promega Corp., Madison, WI, USA). Clones stably expressing undetectable or low levels of EGFR protein (AS-1, AS-3), were selected in 1 mg ml 71 puromycin (Sigma Chemical Co., St. Louis, MO, USA) as described previously (Tian et al, 1998). U87MG cells were also transfected with pBabe-puro vector to serve as a control.…”

“…The protein level of EGFR was reduced about five-fold in antisense-EGFR clone AS-3, and was undetectable in antisense-EGFR clone AS-1. These successfully transfected antisense-EGFR clones were found to have impaired proliferation, reduced transforming potential as well as cellular differentiation (Tian et al, 1998). In this study, antisense-EGFR clones AS-1 (the most inhibited clone) and AS-3 (the intermediately inhibited clone) were further analysed in subsequent experiments.…”

“…In glioblastoma, EGFR is found to be overexpressed and /or amplified in up to 50% of all cases, suggesting an important role of this gene in glial tumorigenesis and progression . The use of antisense-EGFR RNA to block the function of enhanced EGFR in human glioblastoma cells has been reported to inhibit cellular proliferation and induce differentiation (Tian et al, 1998). Antisense-EGFR transfection in rat C6 glioma cells was also found to inhibit cellular proliferation and induce apoptosis (Pu et al, 2000).…”