Mitomycin C induces multidrug resistance in glaucoma surgery.

BACKGROUND: Despite the adjuvant use of mitomycin C during trabeculectomy, failures still occur. We investigated whether cultured human Tenon fibroblasts exposed to low-dose mitomycin C developed a multidrug resistance phenotype in vitro, and whether mitomycin C treatment during previous filtration surgery induces P-glycoprotein expression in vivo. METHODS: Cultured human Tenon fibroblasts treated with low-dose 0.01 nM mitomycin C for 2 weeks were subsequently treated with 0.1 to 100 microM mitomycin C in the absence or presence of 4 microM verapamil, and allowed to recover for 24 hours. Low-dose mitomycin C-treated fibroblasts were analysed for P-glycoprotein expression using flow cytometry, immunoblotting, and RT-PCR for mdr-1 mRNA. In addition, fibroblasts were treated with low dose 0.1 nM 5-fluorouracil for 2 weeks and analysed for P-glycoprotein expression using flow cytometry. Expression of P-glycoprotein was analysed in surgically removed Tenon tissue (n = 30) using immunohistochemistry. Of the 30 patients, 20 had a previous trabeculectomy, of which nine had previous adjuvant therapy with mitomycin C during trabeculectomy. RESULTS: Partial resistance to mitomycin C after low-dose mitomycin C pre-treatment was significantly neutralised by the addition of verapamil. Low-dose mitomycin C up-regulated P-glycoprotein expression, but not mdr-1 mRNA expression. 5-Fluorouracil did not induce P-glycoprotein expression. P-glycoprotein expression was detected in all nine patients exposed to mitomycin C during previous trabeculectomies. Only six of 21 specimens from patients not previously exposed to mitomycin C showed faint P-glycoprotein expression. CONCLUSION: The induction of P-glycoprotein by mitomycin C could explain some failures that occur after repeated use of mitomycin C during trabeculectomy. The concomitant use of verapamil or the use of 5-fluorouracil alone could increase the success rate of repeat trabeculectomies.

Effects of daunorubicin and CD95L on retinal function in superfused vertebrate retina.

Alternative treatments of proliferative vitreoretinopathy (PVR) are needed. The intravitreal application of daunorubicin combined with CD95 ligand (CD95L) could provide a new therapeutic strategy. The effects of this application on bovine retinal function were investigated. Bovine retina preparations were perfused with a standard solution preequilibrated with oxygen. The b-wave and, after the addition of aspartate, the photoreceptor potential P III of the electroretinogram (ERG) were recorded using Ag/AgCl electrodes. Stable ERG amplitudes were recorded, then daunorubicin was added to the solution for 45 minutes, also with the addition of CD95L antibody. Subsequently, the preparation was reperfused with the standard solution for 100 minutes, to allow for recovery. The reduction in b-wave amplitude was reversible and not significantly changed by the addition of 0.25 microg/mL CD95L antibody to 13 microM of daunorubicin. The reduction of the b-wave amplitude was significantly changed and only partly reversible within the recovery time using 40 microM and 80 microM of daunorubicin. The photoreceptor potential P III amplitude was not significantly changed for up to 80 microM of daunorubicin. The ERG showed toxic effects of daunorubicin above a concentration of 13 microM used therapeutically in humans. The combination with CD95L did not increase retinal toxicity. It is, therefore, concluded that daunorubicin may be applied intraocularly, combined with CD95L, without interfering with retinal function.

Apoptosis-mediating receptor-ligand systems in human retinal pigment epithelial cells.

BACKGROUND: To establish new strategies for the treatment of proliferative vitreoretinopathy (PVR), we investigated new members of a recently discovered apoptosis-inducing receptor-ligand system in human retinal pigment epithelial (RPE) cells. TRAIL (Apo2-L) and Apo3-L are capable of inducing cell death via their receptors Trail-R1 to Trail-R4 and TRAMP. The goal of this study was to prove the existence of these new apoptosis-inducing receptors and ligands in RPE cells. METHODS: Human RPE cells, cultured or prepared directly from the eye, were examined by RT-PCR. Immunohistochemistry of epiretinal membranes of traumatic PVR was performed for the detection of TRAIL and Trail-R1. Protein expression of Trail-R1 was examined in cultured human RPE cells by western blot. Cell death after TRAIL treatment of human RPE cells was measured by crystal violet staining. RESULTS: For RPE cells derived directly from the eye, we detected mRNAs of Trail-R2, Trail-R3, TRAIL, and APO3-L, but not Trail-R1, Trail-R4, and TRAMP. All the examined transcripts were detected in human P0 RPE cells in vitro. Immunohistochemical studies on PVR membranes identified TRAIL and Trail-R1. Western blot confirmed the presence of Trail-R1 in cultured human RPE cells. TRAIL failed to kill RPE cells in vitro, but showed a strong synergistic killing effect when coincubated with protein (cycloheximide) or RNA (actinomycin D) synthesis inhibitor. CONCLUSIONS: We detected a novel apoptosis-inducing receptor-ligand system in RPE cells. An induction of apoptosis as a treatment of PVR seems to be possible. Further investigations are needed including an animal model of PVR.