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G. M. Seigel, S. D. Narasipura, M. R. King, A. Lis, A. S. Hackam; Stem Cell Signaling Pathways in Human Retinoblastoma Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):28.
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© ARVO (1962-2015); The Authors (2016-present)
We hypothesize that stem cell signaling pathways, such as Notch and Wnt may play a role in the initiation, progression, and chemoresistance of retinoblastoma (RB) tumors. ABCG2, a universal stem cell marker, appears to detect RB cells with stem cell-like properties, including expression of human embryonic stem cell markers and side populations (Seigel, et al., 2005, 2007). In this study, we sought to investigate the role of Wnt and Notch pathways in the context of RB stem cell-like subpopulations.
Human retinoblastoma cell lines Y79 and WERI-RB27 were sorted into ABCG2+ and ABCG2- populations by fluorescence-activated cell sorting under sterile conditions using FACSVantage. Cytospin preparations of ABCG2+, ABCG2-, as well as unsorted cells, were examined for immunoreactivity of the interactive stem cell signaling markers Musashi-1 and Notch-1. Additionally, unsorted retinoblastoma cells were treated for 48h with the Wnt pathway agonist LiCl (0, 20, 40 mM), cytospun and examined for immunoreactivity of ABCG2, Musashi-1, as well as Ki67 (a cell division marker) by immunocytochemistry.
Virtually all RB cells sorted into ABCG2+ populations were also Musashi-1+ and Notch-1+, whereas ABCG2- RB cells were uniformly immunonegative for both Musashi-1 and Notch-1. In comparison, unsorted RB cells used as controls exhibited low numbers of cells immunoreactive to ABCG2 (2.5%), Musashi-1(3.7%) and Notch-1(3%). Treatment with the Wnt pathway agonist LiCl caused a concentration-dependent increase in the number of ABCG2+ and Musashi-1+ cells in both RB cell lines, with an accompanying decrease in cell division as evidenced by fewer Ki67+ cells.
We have identified an ABCG2+/Musashi-1+/Notch-1+ subpopulation of potential stem-like cells in human RB. Additionally, we have shown that the Wnt agonist LiCl increases the number of cells immunoreactive for ABCG2 and Musashi-1 in RB, as evidence for the significance of the Wnt signaling pathway in RB stem cell biology. A better understanding of the activities of the Wnt and Notch pathways, and their immunodetection in RB tumors may translate into improved prognostic tests, treatments, and outcomes in retinoblastoma.
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