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Dennis M. Defoe, David J. Harris, III, Preston D. Moore, Jarrod D. Sword, Theresa A. Harrison; Cell Cycle-specific Function of p27 in Individual Corneal Endothelial Cells In Vivo. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6435.
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© ARVO (1962-2015); The Authors (2016-present)
Previous mRNA and protein expression studies, as well as targeted gene deletion experiments, have indicated a role for the cyclin-dependent kinase (CDK) inhibitor p27 in arresting cell proliferation during corneal endothelium development and in adult monolayer cultures (Joyce et al. 1996; Yoshida et al., 2004). Recently, p27 has been shown to have cyclin-CDK-independent functions and, in certain tissues, to act as both a pro-proliferative and anti-proliferative factor (Besson et al., 2007). To specifically address its cell cycle-related function we have examined a mouse knock-in strain in which four amino acid substitutions in the cdkn1b gene product prevent its interaction with cyclins and CDKs (p27CK-), but leave its other functions intact. To exclude the possibility of non-cell autonomous gene function, we have also made use of mosaic analysis with double markers (MADM), a technique that allows gene knockout or mutation in single cells or cell clones (Muzumdar et al., 2007).
Wild-type and p27CK- corneas were labeled with Alexa Fluor 488-phalloidin, flat-mounted and visualized by confocal microscopy. For MADM, two mouse lines, each with reciprocally chimeric transgenes consisting of partial coding sequences for green and red fluorescent proteins (GFP and RFP), separated by an intron-embedded LoxP site, were interbred with a Cre recombinase-expressing strain. The p27CK- allele was introduced distal to the MADM transgene cassette via meiotic recombination. Following the limited occurrence of Cre-mediated interchromosomal recombination during mitosis, functional GFP and RFP were reconstituted and each expressed in one of the two daughter cells, with green cells being homozygous mutant and red cells homozygous wild-type.
Endothelial monolayers from p27CK- animals, similar to those of p27 knock-out mice, exhibit approximately a 50% increase in cell density relative to wild-type monolayers. In mosaic animals, the ratio of green to red cells was 2.5-3.0, indicating a dramatically greater expansion of mutant cells compared to wild-type cells.
In corneal endothelium, p27 influences cell number by acting cell autonomously solely through its interaction with cyclin-CDK. Furthermore, the difference between tissue-wide versus mosaic expression of the mutation indicates that there are global size mechanisms that ultimately limit the extent of proliferative change.
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