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Antisense epidermal growth factor receptor transfection impairs the proliferative ability of human rhabdomyosarcoma
C De Giovanni1, L Landuzzi, F Frabetti
1Cancer Research Institute, University of Bologna, Italy.
Abstract:
Human rhabdomyosarcoma cells express membrane epidermal growth factor receptor (ECF-R), which could confer responsiveness to EGF and transforming growth factor-alpha (TGF-alpha) of autocrine or paracrine origin. To study the role played by this growth factor circuit in the proliferation and differentiation of myogenic neoplastic cells, human rhabdomyosarcoma EGF-R-expressing cells (RD/18 clone) have been transfected with a plasmid containing a fragment of the EGF-R cDNA in the antisense orientation. In vitro growth and differentiative ability were studied on six antisense-transfected clones (AS) in comparison to parental RD/18 cells and to cells transfected with the plasmid containing only the neomycin resistance gene (NEO). A reduced EGF-R membrane expression was found in AS clones by decreased immunofluorescence with an anti-EGF-R monoclonal antibody. All AS transfectants had a greatly impaired proliferative ability, even when cultured in fetal bovine serum-containing medium. Proliferation of AS clones was completely blocked in medium supplemented with 2% horse serum. The differentiation ability of AS clones was heterogeneous, ranging from clones with a percentage of myosin-positive cells higher than controls to clones with a negligible myosin expression. Therefore, the growth impairment determined by the loop interruption is not sufficient to switch on the differentiation program. The role played by EGF-R in the proliferation of human rhabdomyosarcoma cells suggests that this receptor could constitute a target for a therapeutic approach.
Insights
Human rhabdomyosarcoma cells
Area of Science:
- Oncology
- Cell Biology
- Molecular Biology
Background:
- Human rhabdomyosarcoma cells express epidermal growth factor receptor (EGF-R).
- EGF-R may mediate responsiveness to autocrine or paracrine growth factors like EGF and TGF-α.
- The role of this growth factor circuit in myogenic neoplastic cell proliferation and differentiation is unclear.
Purpose of the Study:
- To investigate the role of the epidermal growth factor receptor (EGF-R) signaling pathway in the proliferation and differentiation of human rhabdomyosarcoma cells.
- To determine if inhibiting EGF-R expression impacts the growth and differentiation capabilities of these cancer cells.
Main Methods:
- Human rhabdomyosarcoma cells (RD/18 clone) were transfected with an antisense EGF-R cDNA plasmid.
- Six antisense-transfected clones (AS) were generated and compared to parental cells and neomycin-transfected controls (NEO).
- EGF-R membrane expression was assessed via immunofluorescence; proliferation and differentiation (myosin expression) were evaluated in vitro.
Main Results:
- Antisense transfection significantly reduced membrane-bound EGF-R expression in AS clones.
- All AS transfectants exhibited markedly impaired proliferative ability, even in serum-rich media.
- Proliferation was completely inhibited in AS clones cultured in low-serum conditions.
- Differentiation ability varied among AS clones, with some showing increased and others negligible myosin expression.
Conclusions:
- Inhibition of EGF-R expression severely impairs the proliferation of human rhabdomyosarcoma cells.
- Disrupting the EGF-R signaling loop is insufficient to induce a differentiation program in these cells.
- EGF-R represents a potential therapeutic target for human rhabdomyosarcoma treatment.