Abstract 1921: The effect of Hoechst 33342 and Hoechst 33258 on side population (SP) cells or stem cell-like population

Abstract Hoechst 33342 side population (SP) analysis is widely used for identifying and sorting side population (SP) or stem cell-like population from a variety of tissues and species including cancer cell lines. However, Hoechst 33342, but not its derivative Hoechst 33258, is an apoptotic inducer. Our previous results showed that Hoechst 33342 induced apoptosis in seven mesothelioma cell lines through cytochrome C release, caspase activation and degradation of Poly (ADP-ribose) polymerase. We hypothesize Hoechst 33342 may cause SP cell death during Hoechst 33342 staining and Hoechst 33258 may be a safe indicator for identification and isolation of SP cells. The aims of this study are 1) to detect the effect of H 33342 on SP cells, and 2) to identify if H33258 can be an indicator for stem cell isolation. Procedure: The effect of Hoechst dyes on the tumorigenicity of H2373 mesothelioma cells was analyzed by soft agar clonogenic colony formation. MTT assay have been employed to estimate the effect of Hoechst dyes on cell growth in H2373 mesothelioma cells, and Hoechst 33342 and Hoechst 33258 SP analysis was performed on a FACSDiVa cell sorter. Results: Our result demonstrated that Hoechst 33342 staining can causes SP cell damage and also decreases cell colony formation and cell proliferation after using the routine staining doses and intervals for SP cell analysis. In contrast, Hoechst 33258 has little effect on the cell colony formation and cell proliferation. We have found that verapamil, ABC transporter inhibitor, significantly inhibits the efflux of intracellular Hoechst 33258, which means Hoechst 33258 may be used as a marker in isolating ABC transporter positive cells, as Hoechst 33342 does. Conclusion: 1) Hoechst 33342 can induce apoptosis of SP cells of mesothelioma cancer cells 2) Hoechst 33258 may be a safe indicator and replace Hoechst 33342 for identification and isolation of side population. Future Studies: 1) More in vitro methods such as clonogenic assay, drug resistant assay, and invasion assay are required for further determination of stem cell-like properties of side population isolated by Hoechst 33258 when compared to that isolated by Hoechst 33342. 2) Xenograft (or animal) study is ultimately required to determine the capacity of in vivo tumor formation of the side population cells isolated by Hoechst 33258 after the completion of in vitro study Note: This abstract was not presented at the meeting. Citation Format: Naimei Tang, Crystal Zhang, Cyndi Noraian, Anil Wali, Harvey Pass, Michael Harbut, Xinbo Zhang. The effect of Hoechst 33342 and Hoechst 33258 on side population (SP) cells or stem cell-like population. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1921. doi:10.1158/1538-7445.AM2014-1921