Identification of P2Y11 Receptor Expressed in Human Retinoblastoma Cells

PURPOSE: The present study aimed to identify the characteristics and physiological function of the P2Y11 receptor, a receptor likely expressed in human retinoblastoma cells. METHODS: We measured possible P2Y11 signaling in WERI-Rb-1 cells using a Ca2+ imaging technique and RT-PCR. RESULTS: 1) 10 micro M ATP elicited a strong but transient increase in Ca2+ in the WERI-Rb-1 cells, and this Ca2+ rise was well maintained after external Ca2+-depletion. 2) ATP-induced Ca2+ response arose entirely through Ca2+ mobilization. 3) P2Y11 agonist (BzATP, 100 micro M) increased Ca2+ by 31.2+/-3.7 % of ATP effect. 4) mRNA for P2Y11 subtype was identified using RT-PCR. CONCLUSIONS: P2Y11 purinergic activation can increase the intracellular calcium level through calcium mobilization in undifferentiated retinoblastoma cells, which may play an important role in cell proliferation, differentiation, and even pathologic processes.

Characteristics of Nicotinic Receptor Expressed in Human Retinoblastoma

PURPOSE: To identify the characteristics and physiological function of the nicotinic receptor expressed in human retinoblastoma cells. METHODS: We measured possible nicotinic signaling in WERI-Rb-1 cells using the Ca2+ imaging technique and the patch clamp method. RESULTS: 1) Nicotine-induced [Ca2+]i rise arose entirely through Ca2+ influx, which was completely abolished by hexamethonium (100 micro M). 2) Nicotine also induced remarkable depolarization from -56.6 +/- 3.7 mV to -29.6 +/- 3.6 mV (n=4) under current clamp mode, but it failed to directly activate the T-type Ca2+ channel expressed in retinoblastoma cells. CONCLUSIONS: Nicotinic activation can increase the intracellular calcium level through calcium influx in the undifferentiated retinoblastoma cells, which may play important roles in cell proliferation, differentiation, and cell death.

Chemosensitization to adriamycin by cyclosporin A and verapamil in human retinoblastoma cell lines

The chemosensitizing effects of cyclosporin A and verapamil on the cytotoxicity of adriamycin were investigated using MTT assay against two human retinoblastoma cell lines, Y79 and WERI-Rb-1. Y79 and WERI-Rb-1 were totally resistant to doses up to 5.0 micrograms/ml of verapamil. Cyclosporin A inhibited the survival of Y79 and WERI-Rb-1 dose-dependently, however, the maximum inhibition at the highest concentration tested (5.0 micrograms/ml) was less than 50% (% survival at 5.0 micrograms/ml of cyclosporin A: 65.6% and 66.9% in Y79 and WERI-Rb-1, respectively). Combination of cyclosporin A and verapamil did not further inhibit the survival of Y79 and WERI-Rb-1 compared with cyclosporin A alone. Adramycin inhibited the survival of Y79 and WERI-Rb-1 dose-dependently. The chemosensitizing effects of cyclosporin A and verapamil on the cytotoxicity of adriamycin were evaluated in terms of sensitizing index (SI: the ratio of IC50 to adriamycin alone to IC50 to adriamycin in the presence of cyclosporin A and/or verapamil). Cyclosporin A significantly enhanced SI and the addition of verapamil enhanced SI further: SI values at 5.0 micrograms/ml of cyclosporin A, 5.0 micrograms/ml of cyclosporin A plus 1.5 micrograms/ml of cyclosporin A plus 1.5 micrograms/ml of verapamil, 5.0 micrograms/ml of cyclosporin A plus 3.0 micrograms/ml of verapamil were 2.0, 2.6 and 2.8 in Y79 and 2.6, 5.8 and 9.7 in WERI-Rb-1, respectively. These results suggest that cyclosporin A and verapamil are promising chemosensitizers to adriamycin in the treatment of retinoblastoma.