Cyclin-dependent kinase inhibitor roscovitine induces cell cycle arrest and apoptosis in rabbit retinal pigment epithelial cells.
OBJECTIVE
Cyclin-dependent kinases (CDKs) play essential roles in the intracellular control of the cell cycle. It has been postulated that roscovitine, a potent CDK2, CDK5, and CDC2 inhibitor, might inhibit cellular proliferation by arresting the cell cycle. This in vitro study investigated the antiproliferative and apoptotic effects of roscovitine in cultured rabbit retinal pigment epithelial (RPE) cells.


METHODS
Experiments using rabbit RPE from young pigmented rabbits were carried out using roscovitine dissolved in dimethylsulfoxide at concentrations ranging from 1 to 100 micromol. Cell proliferation was measured by an MTT assay. The cell cycle response of RPE cells to roscovitine was analyzed by flow cytometry of propidium iodide-stained nuclei. Proteins related to DNA damage in the RPE cells were then assayed by Western blot.


RESULTS
Roscovitine inhibited proliferation of RPE cells in a dose-dependent manner. Cell cycle analysis after treatment demonstrated an accumulation of cells arrested in the S- and G2/M phases. Flow cytometry showed that 40 microM of roscovitine increased the cell population in the sub-G1 peak, which is considered a marker of cell death by apoptosis. Western blot analysis revealed Bcl-2 decreased and Bax increased after treatment of RPE cells with roscovitine.


CONCLUSIONS
This study of the response of RPE cells to roscovitine demonstrated a bidirectional relationship between cell cycle control and apoptosis.
Wu
Pei-Chang
PC
Department of Ophthalmology, Chang Gung Memorial Hospital, Kaohsiung Medical Center, Kaohsiung Hsien, Taiwan.
Tai
Ming-Hong
MH
Hu
Dan-Ning
DN
Lai
Chien-Hsiung
CH
Chen
Yi-Hao
YH
Wu
Yi-Chen
YC
Tsai
Chia-Ling
CL
Shin
Shyi-Jang
SJ
Kuo
Hsi-Kung
HK
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D004847:Epithelial Cells / Q000187:drug effects*; D005434:Flow Cytometry; D006131:Growth Inhibitors / Q000494:pharmacology*; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis; D011687:Purines / Q000494:pharmacology*; D011817:Rabbits; D000077546:Roscovitine; D051028:bcl-2-Associated X Protein / Q000096:biosynthesis
D006131:Growth Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D011687:Purines; D051028:bcl-2-Associated X Protein; D000077546:Roscovitine; D004247:DNA; D018844:Cyclin-Dependent Kinases