ATP controls cell cycle and induces proliferation in the mouse developing retina.

Previous data suggest that nucleotides are important mitogens in the developing chick retina. Here, we extended the study on the mitogenic effect of ATP to newborn mouse retinal explants. Our results showed that P2Y(1) receptors were widely distributed in C57bl/6 mice retina and that the majority of PCNA positive cells co-localized with P2Y(1) receptor. To evaluate proliferation, retinal explants obtained from newborn mice were incubated with 0.5 microCi [(3)H]-thymidine or 3 microM BrDU 1h before the end of culture. Our data showed that ATP induced a dose-dependent increase in [(3)H]-thymidine incorporation, an effect that was mimicked by ADP but not by UTP and was blocked by the P2 antagonist PPADS in a dose-dependent manner. The increase in [(3)H]-thymidine incorporation induced by ATP was only observed in explants cultured for 3 days or less and was mimicked by the ectoapyrase inhibitor ARL 67156. It corresponded to an increase in the number of BrdU(+) cells in the neuroblastic layer (NL) of the tissue, suggesting that ATP, through activation of P2Y(1) receptors, induced proliferation of late developing progenitors in retinal explants of newborn mice. The increase in the number of BrdU(+) cells was observed across the whole NL when explants were incubated with ATP for 24h and no increase in the number of p-histone H3 labeled cells could be noticed at this time point. In longer incubations of 48h with ATP or 24h with ATP followed by a period of 24h in fresh medium, an increase in the number of BrdU(+) cells promoted by ATP was observed only in the middle and outer, but not in the inner NL. In these conditions, an increase in the number of p-histone H3 labeled cells was detected in the outer NL, suggesting that ATP induced cells to enter S and progress to G2 phase of the cell cycle in the first 24h period of incubation. ATP also induced an increase and a decrease in the expression of cyclin D1 and p27(kip1), respectively, in retinal progenitors of the NL. While the increase in the expression of cyclin D1 was observed when retinal explants were incubated for 3h or longer periods of time, the decrease in the expression of p27(kip1) was noticed only after 6h incubation with ATP. Both effects were blocked by the P2 receptor antagonist PPADS. These data suggest that ATP induces cell proliferation in retinal explants by inducing late developing progenitors to progress from G1 to S phase of cell cycle.