title: ROCK inhibitor Y-27632 suppresses dissociation-induced apoptosis of murine prostate stem/progenitor cells and increases their cloning efficiency.
abstract: Activation of the RhoA/ROCK signaling pathway has been shown to contribute to dissociation-induced apoptosis of embryonic and neural stem cells. We previously demonstrated that approximately 1 out of 40 Lin(-)Sca-1(+)CD49f(high) (LSC) prostate basal epithelial cells possess the capacities of stem cells for self-renewal and multi-lineage differentiation. We show here that treating LSC cells with the ROCK kinase inhibitor Y-27632 increases their cloning efficiency by 8 fold in an in vitro prostate colony assay. Y-27632 treatment allows prostate colony cells to replate efficiently, which does not occur otherwise. Y-27632 also increases the cloning efficiency of prostate stem cells in a prostate sphere assay and a dissociated prostate cell regeneration assay. The increased cloning efficiency is due to the suppression of the dissociation-induced, RhoA/ROCK activation-mediated apoptosis of prostate stem cells. Dissociation of prostate epithelial cells from extracellular matrix increases PTEN activity and attenuates AKT activity. Y-27632 treatment alone is sufficient to suppress cell dissociation-induced activation of PTEN activity. However, this does not contribute to the increased cloning efficiency, because Y-27632 treatment increases the sphere-forming unit of wild type and Pten null prostate cells to a similar extent. Finally, knocking down expression of both ROCK kinases slightly increases the replating efficiency of prostate colony cells, corroborating that they play a major role in the Y-27632 mediated increase in cloning efficiency. Our study implies that the numbers of prostate cells with stem/progenitor activity may be underestimated based on currently employed assays, supports that dissociation-induced apoptosis is a common feature of embryonic and somatic stem cells with an epithelial phenotype, and highlights the significance of environmental cues for the maintenance of stem cells.
authors.affiliation: Department of Molecular and Cell Biology, Baylor College of Medicine, Houston, Texas, United States of America.
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Valdez
authors.forename: Boyu
authors.initials: B
authors.lastname: Zhang
authors.forename: Lei
authors.initials: L
authors.lastname: Wei
authors.forename: Jiang
authors.initials: J
authors.lastname: Chang
authors.forename: Li
authors.initials: L
authors.lastname: Xin
mesh_terms: D000577:Amides / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001681:Biological Assay; D015415:Biomarkers / Q000378:metabolism; D002999:Clone Cells; D003114:Colony-Forming Units Assay; D015536:Down-Regulation / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D008297:Male; D051379:Mice; D051059:PTEN Phosphohydrolase / Q000378:metabolism; D011467:Prostate / Q000166:cytology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology*; D018874:Spheroids, Cellular / Q000166:cytology / Q000187:drug effects; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D000577:Amides; D015415:Biomarkers; D047428:Protein Kinase Inhibitors; D011725:Pyridines; C108830:Y 27632; D051057:Proto-Oncogene Proteins c-akt; D054460:rho-Associated Kinases; D051059:PTEN Phosphohydrolase