ROCK Inhibitor Y-27632 Suppresses Dissociation-Induced Apoptosis of Murine Prostate Stem/Progenitor Cells and Increases Their Cloning Efficiency

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+CD49fhigh (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

Activation of NF-kappa B signaling promotes growth of prostate cancer cells in bone

Patients with advanced prostate cancer almost invariably develop osseous metastasis. Although many studies indicate that the activation of NF-κB signaling appears to be correlated with advanced cancer and promotes tumor metastasis by influencing tumor cell migration and angiogenesis, the influence of altered NF-κB signaling in prostate cancer cells within boney metastatic lesions is not clearly understood. While C4-2B and PC3 prostate cancer cells grow well in the bone, LNCaP cells are difficult to grow in murine bone following intraskeletal injection. Our studies show that when compared to LNCaP, NF-κB activity is significantly higher in C4-2B and PC3, and that the activation of NF-κB signaling in prostate cancer cells resulted in the increased expression of the osteoclast inducing genes PTHrP and RANKL. Further, conditioned medium derived from NF-κB activated LNCaP cells induce osteoclast differentiation. In addition, inactivation of NF-κB signaling in prostate cancer cells inhibited tumor formation in the bone, both in the osteolytic PC3 and osteoblastic/osteoclastic mixed C4-2B cells; while the activation of NF-κB signaling in LNCaP cells promoted tumor establishment and proliferation in the bone. The activation of NF-κB in LNCaP cells resulted in the formation of an osteoblastic/osteoclastic mixed tumor with increased osteoclasts surrounding the new formed bone, similar to metastases commonly seen in patients with prostate cancer. These results indicate that osteoclastic reaction is required even in the osteoblastic cancer cells and the activation of NF-κB signaling in prostate cancer cells increases osteoclastogenesis by up-regulating osteoclastogenic genes, thereby contributing to bone metastatic formation

THE RAT PROBASIN GENE PROMOTER DIRECTS HORMONALLY AND DEVELOPMENTALLY-REGULATED EXPRESSION OF A HETEROLOGOUS GENE SPECIFICALLY TO THE PROSTATE IN TRANSGENIC MICE

An expression cassette carrying 426 basepairs of the rat probasin (PB) gene promoter and 28 basepairs of 5’-untranslated region is sufficient to target the expression of the bacterial chloramphenicol acetyltransferase (CAT) gene specifically to the prostate in transgenic mice. The PB-CAT transgene was expressed in three of five (60%) independent lines of mice, and this expression, as reported previously for the endogenous rat gene, was male specific, restricted primarily to the lateral, dorsal, and ventral lobes of the prostate, with only very low levels of CAT activity detected in the anterior prostate and seminal vesicles. The developmental and hormonal regulation of the transgene also paralleled that reported for the rat gene, with a 70-fold increase in CAT activity in the mouse prostate observed between 2-7 weeks of age, a time corresponding to sexual maturation. PB-CAT activity in the prostate declined after castration to 3.5% of the precastration level, and the CAT activity in castrated males approached precastration levels when mice were supplemented with testosterone. Transgene expression in castrated males was not induced by dexamethasone. Coinjection of PB-CAT with a chicken lysozyme gene matrix attachment region resulted in their cointegration and further restricted the pattern of PB-CAT to the dorsolateral prostate, with suppressed expression observed in the ventral prostate. These studies demonstrate that a minimal rat probasin promoter can target heterologous gene expression specifically to the prostate in a developmentally and hormonally regulated fashion