The inhibitory effect of GCV on MGPC proliferation prompted us to examine the expression of cell cycle–related genes after GCV injection. Quantitative PCR analysis showed that GCV significantly decreased the expression of cyclin A2, cyclin B1, and cyclin D1 but had no effect on cyclin E1 at 3 dpi (
Fig. 6A). Ganciclovir also significantly decreased the expression of Cdk1 and Cdk2 (
Fig. 6A). Strikingly, high induction of a Cdk-dependent inhibitor p21
cip1 (gene
cdkn1a,
Fig. 6B) was found after GCV treatment at 3 dpi. Ganciclovir had no effect on the mRNA level of p27
kip1 and p57
kip2 (
Supplementary Fig. S3A). Since it is well known that p21
cip1 can be directly regulated by the tumor suppressor p53,
30,31 we next examined the expression of p53 by qPCR and Western blot analysis. Indeed, GCV induced a small but significant increase of p53 at both the mRNA and protein level at 3 dpi (
Figs. 6C,
6D). We also examined the expression of cell cycle genes at 2 dpi, when GCV has not yet had any observable effect on MGPC proliferation. At this time point, we observed a significant induction of p21
cip1 and p53 (
Figs. 6B,
6C), whereas the expression of most other cell cycle–related genes was comparable to that of control except for cyclin A2 (
Supplementary Fig. S3B). Therefore, induction of p53 and p21
cip1 was an early event after GCV treatment. Since p21
cip1 is a well-known cell cycle inhibitor, our data are consistent with the idea that the induction of p53 and p21
cip1 by GCV caused a cell cycle arrest and thus inhibited MGPC proliferation.
It has been shown that the transcription factors Pax6a and Pax6b are required only for MGPC proliferation, and the impact of GCV on MGPC proliferation is similar to that of Pax6a/6b knockdown.
18 We therefore examined the expression level of Pax6a/6b mRNA in PBS- and GCV-treated retina at 2 and 3 dpi. Quantitative PCR analysis showed that the expression of Pax6a and Pax6b in GCV-treated retina was comparable to that of control at 2 dpi (
Supplementary Fig. S3C). A small but significant decrease of Pax6b expression in GCV-treated retina was found at 3 dpi, whereas no significant difference in Pax6a expression was observed at this time point (
Supplementary Fig. S3C). Since Pax6b was reported to specifically regulate the first cell division of MGPCs,
18 it is possible that in addition to the induction of P53 and P21
cip1, GCV may also downregulate Pax6b expression to inhibit MGPC proliferation.