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GR Welsandt, A Hueber, N Kociok, P Esser, GK Krieglstein, H Mietz; Effect of Combined Cytotoxic Therapy on Cell Proliferation, RNA-Synthesis and Apoptosis . Invest. Ophthalmol. Vis. Sci. 2002;43(13):869.
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Purpose: Enhancement of glaucomatous filtering procedures by the use of antimetabolites is associated with toxic side-effects which are related to the amount and concentration of the specific drug used. Currently, only single drugs, mostly 5-Fluorouracil and Mitomycin, are used clinically. We performed human Tenon´s fibroblast cell culture studies to assess how the combination of two synergistic acting cytotoxic drugs possibly influence cell migration, RNA-synthesis and apoptosis. The aim was to reduce the toxic side effects of these drugs by achieving a similar strong antiproliferative effect as compared to the use of mitomycin or 5-fluorouracil. Methods: Cell proliferation was assessed by use of the scratch assay (Grisanti et al, 1998). With this technique, it was determined how much cells can proliferate two-dimensionally under standard conditions within a specific time period. RNA synthesis was measured with the (5,6-3H) uridine incorporation assay. Cells were pulse-labeled for 1h with 0.5µCi/ml (5,6-3H) uridine, and cell lysates were counted in a liquid scintillation counter. Experiments were performed in triplicate. Apoptosis was determined with the TUNEL assay using the cultured fibroblasts. Results: Proliferation was significantly inhibited by all combinations used in our studies. The strongest effect was observed with the combination of lomustin and fas-ligand (1.1±16.0% of proliferation), while the other combinations such as staurosporin and mitomycin (12.8±5.3%), staurosporin and doxorubicin (11.5±11.1%) or staurosporin and lomustin (19.2±4.8%) had less strong effects. RNA-synthesis was significantly inhibited by all combinations tested. Inhibition found included lomustin and fas-ligand (10.4%), staurosporin and mitomycin (81.7%), staurosporin and doxorubicin (28.7%) or staurosporin and lomustin (84.6%). The strongest inhibition was detected with the combination of staurosporin and vincristin (86.4%). TUNEL positive cells were observed with all combinations used here to a variable extent exceeding a proportion of 50% in most combinations tested. Conclusion: These results suggest that the use of single substances to inhibit postoperative scarring following glaucomatous filtering procedures can be substituted by combinations of cytotoxic drugs. Interestingly, it appears that specific combinations have variable strong effects on cell proliferation and RNA-synthesis, so that it remains to be determined which of these effects has the largest impact clinically.
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