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p27^kip1 Antisense-Induced Proliferative Activity of Rat Corneal Endothelial Cells

¹From the Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts; and the ²Department of Ophthalmology, Dokkyo University School of Medicine, Tochigi, Japan.

PURPOSE. To determine whether antisense downregulation of p27^kip1 will overcome G₁-phase arrest and promote cell cycle progression in rat corneal endothelial cells (CECs).

METHODS. Confluent cultures of rat CECs were incubated for 24 hours in the presence of p27^kip1 antisense (AS) oligonucleotides (oligoS) using nonliposomal lipid transfection. Control cultures were incubated under one of the following conditions: no oligos or lipid-containing buffer, lipid-containing buffer alone, or lipid-containing buffer plus missense (MS) p27^kip1 oligo. Viability was tested by a cell-viability assay after 0, 24, 48, and 72 hours. After postincubation for 0, 24, 48, or 72 hours, cultures were fixed and immunostained for p27^kip1, to test for downregulation, or for Ki67 or BrdU, to detect actively cycling cells. Western blot and immunocytochemistry (ICC) studies were conducted to determine the effect of p27^kip1 antisense treatment on the relative protein level and subcellular localization of several cell cycle proteins, including cyclin-D1, -E, -A, and -B1; CDK2 and -4; p21^cip1; and p15^INK4b. Proliferation was determined by direct counting of propidium iodide (PI) or 4',6'-diamino-2-phenylindole (DAPI)-stained cells.

RESULTS. Viability was not significantly affected by lipid-based oligo transfection for up to 48 hours, after which a decline was noted. The protein level of p27^kip1 was reduced after AS transfection in a time-dependent manner. Nuclear staining for p27^kip1 was greatly reduced in CECs incubated with AS oligo. No change in p27^kip1 levels was observed in controls at any time point tested. p27^kip1 AS oligo transfection increased cyclin-D1, -E, -A, and -B1 protein levels, and all cyclins were localized to the nucleus. No changes in protein level were observed for CDK2, CDK4, p21^cip1, or p15^INK4B. A time-dependent increase in the relative number of Ki67- and BrdU-positive cells was noted in CECs incubated with AS oligo. In contrast, no to few Ki67- or BrdU-positive cells were observed in CECs incubated with MS oligo or the buffer-treated control cells. The percentage increase in the number of cells transfected with AS oligo increased with time, compared with that of cells transfected with MS oligo.

CONCLUSIONS. Treatment with p27^kip1 antisense oligonucleotides followed by postincubation in 10% FBS lowers endogenous p27^kip1 protein levels and promotes proliferation in confluent cultures of rat CECs.

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