Reinitiation Of Proliferation Research Articles

MICRORNAS REGULATE CARDIOMYOCYTE PROLIFERATION IN THE EARLY POSTNATAL PERIOD

The regenerative capacity of the mammalian heart is lost in the first week after birth when cardiomyocytes (CM) stop proliferating, become binucleated, and permanently exit the cell cycle. However, the molecular signals that drive CM from a proliferative to quiescent state remain poorly defined. We have previously shown that CM only proliferate in the first 12 hours after birth. Our transcriptomic analyses revealed that a differential expression of cell cycle factors exists during the cessation of cell division, binucleation and cell cycle exit in the first week of birth. Cell cycle factors have been shown to be regulated by microRNAs (miRNAs) which act as post-transcriptional gene silencers. Therefore, we hypothesized that specific miRNAs regulate cell cycle progression and that manipulation of these miRNAs promotes proliferation of CM in the neonate murine heart. Profiling of miRNAs from wild type murine hearts at 12h after birth (n=4/group) was performed using nCounter® miRNA mouse assays (Nanostring) and screened for cell cycle targets by miRSystem software. Preliminary analysis has shown that 14 miRNAs are differentially expressed during the proliferative burst that occurs at 12 hours after birth (p≤0.01) (Figure 1). These results have been confirmed in murine hearts by RT-qPCR. We have identified a unique subset of miRNAs that are significantly differentially expressed when CM transition from a proliferative to non-proliferative state 12h post-birth. These findings are promising and will guide our future work that will assess their role in the re-initiation of proliferation in the mammalian heart.

Abstract 4451: Long-term resistance to PI3K/mTOR inhibition requires increased expression of c-myc and YAP.

Abstract We have shown that extracellular matrix (ECM) contact contributes to PI3K/mTOR inhibitor resistance, where drug treatment leads to cytostasis but not cell death in ECM-attached cells. These cells do undergo proliferation arrest and this may reflect the situation in vivo, where PI3K/mTOR inhibitor treatment initially leads to growth arrest of the tumor cells. However this is often followed by re-initiation of proliferation, leading to relapse. In this study we have investigated the underlying molecular mechanisms that mediate long-term resistance to PI3K/mTOR inhibitors by selecting for cancer cells that proliferate after chronic inhibitor exposure in 3D cell cultures. To assess the mechanism of resistance, we performed reverse phase protein and RNA-seq arrays to identify signaling pathways that were altered in proliferating drug resistant cells. The protein arrays revealed up-regulated levels of c-myc and the Hippo pathway effector, YAP, in the resistant cells, and introduction of exogenous c-myc and YAP into drug sensitive cells rendered them resistant. While expression of c-myc alone induced partial resistance, YAP was also required, as knock-down of endogenous YAP resulted in loss of resistance. These data suggest that c-myc alone is not sufficient for generating resistance to PI3K/mTOR inhibitors, but an additional driver is required. Since the Hippo pathway is another major growth-promoting pathway (in addition to mTOR), it is conceivable that YAP compensates for the loss of mTOR signaling, contributing to resistance. Our current efforts are aimed at elucidating the mechanism by which YAP and c-myc mediate drug resistance to PI3K/mTOR inhibitors. Preliminary data suggests that c-myc-dependent glutamine metabolism contributes to drug resistance, as inhibition of glutaminase prevents resistance. Citation Format: Taru E. Muranen, Jonathan Coloff, Laura Selfors, Amy Bui, Gordon B. Mills, Joan S. Brugge. Long-term resistance to PI3K/mTOR inhibition requires increased expression of c-myc and YAP. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4451. doi:10.1158/1538-7445.AM2013-4451

Inhibition of rabbit aortic smooth muscle cell proliferation by selective inhibitors of protein kinase C.

1. We studied the effect of two structurally-related, selective inhibitors of protein kinase C, Ro 31-8220 and Ro 31-7549, on the reinitiation of proliferation in quiescent first passage rabbit aortic smooth muscle cells in response to (a) the direct activator of protein kinase C, phorbol dibutyrate (PDBu), (b) platelet-derived growth factor (PDGF), (c) a combination of PDGF and 5-hydroxytryptamine (5-HT) or (d) serum. 2. Ro 31-8220 and Ro 31-7549 concentration-dependently inhibited proliferation in response to each mitogen. The inhibitory potency (IC50) of Ro 31-8220 and Ro 31-7549, respectively, was similar against proliferation induced by PDBu (0.55 and 1.1 microM), PDGF (0.6 and 0.9 microM), PDGF and 5-HT (0.68 and 1.1 microM), although slightly less against serum (1.7 and 5 microM). The effects of the protein kinase C inhibitors on proliferation could not be ascribed to cytotoxicity. Neither Ro 31-8220 nor Ro 31-7549 (0.3-3 microM) inhibited PDGF receptor tyrosine phosphorylation. 3. The results show that Ro 31-8220 and Ro 31-7549 are potent inhibitors of smooth muscle cell proliferation in response to a direct activator of protein kinase C, the defined growth factors, PDGF and 5-HT, and the complex mixture of mitogens in serum. Protein kinase C activation thus appears to be an important growth transducing mechanism for each of these agents.

Growth factor-initiated proliferation of mouse embryonic fibroblasts induces cytotoxicity by natural killer cells and by a non-cytolysin cytotoxin in natural killer granules.

NK cells preferentially kill normal embryonic fibroblasts. Because embryonic cells are growth factor responsive and maintain high proliferative rates, we examined the requirement for growth factor-initiated proliferation for NK susceptibility. Murine embryonic fibroblasts made quiescent in defined medium lacking growth factors were relatively resistant to NK cytolysis. However, reinitiation of proliferation with basic fibroblast growth factor (bFGF) or epidermal growth factor enhanced lysis in a dose-dependent fashion. TGF-beta, which blocked cell division, did not enhance cytotoxicity. Additionally, growth inhibition by prolonged incubation at confluence suppressed lysis. The enhanced NK cytotoxicity of bFGF-stimulated fibroblasts was caused by a post-binding event because no difference in cold target inhibition could be demonstrated with bFGF-treated cells. NK cytotoxicity has largely been attributed to the action of cytotoxins released from cytoplasmic granules. In a 51Cr release assay, bFGF-treated fibroblasts were insensitive to NK granules isolated from the RNK large granular lymphocyte leukemia. However, these same cells exhibited marked sensitivity to lysis in an 18-h adhesion assay normally utilized to detect TNF-alpha. With the use of this assay, a dose-dependent increase in sensitivity of bFGF-treated fibroblasts was observed, whereas quiescent fibroblasts were resistant to the action of isolated NK granules. Granule cytotoxicity was not caused by cytolysin/perforin because inactivation of granule hemolytic activity with CaCl2 did not affect fibroblast killing, and bFGF-treated cells were insensitive to purified cytolysin/perforin. This suggested that another granule associated cytotoxin was responsible for enhanced NK sensitivity of actively proliferating fibroblasts.