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Adenoviral p53 gene transfer inhibits human Tenon's capsule fibroblast proliferation
K T M Johnson1, F Rödicker, K Heise
1Zentrum für Augenheilkunde, Abteilung für Erkrankungen des vorderen Augenabschnitts, Hufelandstrasse 55, D-45147 Essen, Germany.
Background/Aim:
Although antiproliferative drugs have been used successfully to prevent scarring after filtration surgery in patients with glaucoma, complications associated with their use (such as hypotony or endophthalmitis) energise the search for an alternative treatment. Single application of beta radiation leads to long term growth arrest and expression of p53 in human Tenon's capsule fibroblasts (hTf). The authors assume that the activation of p53 is one of the cellular triggers. Their aim was to analyse the effect of p53 overexpression on hTf and to determine which pathways are involved.
Methods:
A recombinant adenoviral vector (rAd.p53) containing transgenes encoding for human p53 and green fluorescent protein (GFP) was used to induce overexpression of p53 in hTF and a control vector (rAd.GFP). Transgene expression was detected by western blot (p53 and p21WAF-1/Cip1). Cell proliferation and viability were investigated using cell counts, 5'-bromodeoxyuridine incorporation (BrdU assay) and tetrazolium reduction (MTT assay).
Results:
Infection of hTf with rAd.p53 resulted in significant inhibition of cell proliferation, DNA synthesis, and metabolic activity in vitro. Western blot showed increased levels of p53 and p21WAF-1/Cip1 in rAd.p53 infected cells, but not in rAd.GFP and uninfected cells. Apoptosis was excluded with flow cytometry.
Conclusions:
Adenoviral p53 gene transfer leads to significant growth inhibition in hTf. P53 induces p21(WAF-1/Cip1) expression and does not cause apoptosis in hTf in vitro. p53 as an antiproliferative drug has the potential to replace mitomycin C and 5-fluorouracil in glaucoma surgery.
Insights
Adenoviral p53 gene transfer significantly inhibits human Tenon
Area of Science:
- Ophthalmology
- Cell Biology
- Molecular Biology
Background:
- Glaucoma surgery often uses antiproliferative drugs to prevent scarring.
- Complications like hypotony and endophthalmitis necessitate alternative treatments.
- Beta radiation induces growth arrest and p53 expression in human Tenon's capsule fibroblasts (hTf).
Purpose of the Study:
- To analyze the effect of p53 overexpression on hTf.
- To determine the cellular pathways involved in p53-mediated growth inhibition.
Main Methods:
- Recombinant adenoviral vector (rAd.p53) used for p53 overexpression in hTf.
- Western blot to detect p53 and p21WAF-1/Cip1 expression.
- Cell proliferation, DNA synthesis, and metabolic activity assessed via cell counts, BrdU, and MTT assays.
Main Results:
- rAd.p53 infection significantly inhibited hTf proliferation, DNA synthesis, and metabolic activity.
- Increased p53 and p21WAF-1/Cip1 levels observed in infected cells.
- Apoptosis was ruled out as a mechanism of growth inhibition.
Conclusions:
- Adenoviral p53 gene transfer effectively inhibits hTf growth.
- p53 induces p21WAF-1/Cip1 expression without causing apoptosis in hTf.
- p53 shows potential as an alternative antiproliferative drug for glaucoma surgery.
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