Abstract
The DNA intercalating dye Hoechst 33342 or its close analog DCV are actively removed from cells by the multidrug resistance transporter ABCG2, a protein overexpressed in metastatic cells and somatic stem cells. In bivariate blue-red flow cytometry fluorescent plots active Hoechst or DCV efflux combined with a concentration dependent bathochromic shifts of these nuclear dyes leads to the segregation of the transporter-rich cells into a distinct cell cohort tilted towards the shorter wavelength axis of the plot, the cohort is generically known as the side population (SP). This feature has facilitated the surface marker-independent isolation of live stem cells. A drawback, though, is the known toxicity of Hoechst dyes. In this study we show that JC1, a bathochromic mitochondrial membrane potential-sensitive dye applied at proper concentration, can yield flow cytometry fluorescent emission bivariate plots containing a low JC1 accumulation (JC1low) cohort. Using a combination of multiple cell lines, ABC-transporter inhibitors and viral vector-driven insertion of the ABCG2 gene or ABCG2 and ABCB1 shRNAs we demonstrate that JC1low can be generated by either of the two aforementioned multidrug resistance transporters. Complete wash out of mitochondrial bound JC1 required more than 24 h. In spite of this tight binding, the dye did not affect either the mitochondrial membrane potentials or the proliferation rate. In contrast, contemporaneous with its nuclear accumulation, Hoechst 33342 or DVC, caused changes in the fluorescent emission of mitochondrial membrane potential sensitive dyes resembling the effects caused by the mitochondrial uncoupler FCCP. In a number of cell lines exposure to Hoechst resulted in marked slow-down of proliferation and abolition of ABCG2 transport activity during the subsequent 2 days but in K562 cells the exposure induced cell extended death. Overall, its lack of toxicity vis. a vis. the toxicity and genotoxicity of the DNA intercalating dyes makes JC1 an ideal tool for isolating live cells expressing high multidrug resistance transport activity.
Highlights
The incubation of live cells with the DNA binding supravital dye Hoechst 33342 (Hoechst) frequently results in flow cytometry bivariate 450/670 nm emission plots incorporating small cell cohorts displaying, a) lower absolute fluorescence, and b) a higher 450/670 nm ratio than the main cell population
A) JC1 is transported with comparable efficiency by the two main MDR transporter proteins, ABCG2 /BCRP and ABCB1/p-glycoprotein; b) JC1 washout is remarkably slow with a half time of dissociation exceeding 24 h; c) in spite of this prolonged residence in the mitochondria the JC1 did not have any measurable late effect on either membrane potential (MMPT), gross physical cell parameters or cell proliferation of transformed and tissue derived cell lines; and d) unlike JC1, incubation with Hoechst 3342 or DCV at the concentrations and periods used for the identification of side population (SP) markedly affected the MMPT during or after the dye incubation period and resulted in either delay in reduced marked proliferation rate proliferation in most cell line studied and cell death in K562 cells
These results indicate that development of a side population pattern with the mitochondrial dye results from the fact that, while the MMPT drives the intra-mitochondrial JC1 aggregation associated with the bathochromic shift, it can elicit the latter effect only if the dye concentration reaches a certain minimal threshold
Summary
The incubation of live cells with the DNA binding supravital dye Hoechst 33342 (Hoechst) frequently results in flow cytometry bivariate 450/670 nm emission plots incorporating small cell cohorts displaying, a) lower absolute fluorescence, and b) a higher 450/670 nm ratio than the main cell population. We have shown that incubation of ocular surface epithelial cells with the mitochondrial membrane potential (MMPT) sensitive dye JC1 [8, 9] results in bivariate 525/585 nm green/orange emission plots (JC1 image) containing a low dye accumulation cohort (JC1low) situated to the green-side of the main cell cohort [6,10] which constitute a JC1-SP This cell subset was efficiently abolished by the ABCG2 specific inhibitors fumitremorgen C (FTC) and Ko143 [11, 12] and contained the great majority of clonogenic cells within the outgrowths that develop from limbal biopsies explants [10] we and others [13] have used this JC1 greenside population as an alternative means to identify and/or quantitate ABCG2 transport activity in the ocular surface epithelial cells. A) JC1 is transported with comparable efficiency by the two main MDR transporter proteins, ABCG2 /BCRP and ABCB1/p-glycoprotein; b) JC1 washout is remarkably slow with a half time of dissociation exceeding 24 h; c) in spite of this prolonged residence in the mitochondria the JC1 did not have any measurable late effect on either MMPT, gross physical cell parameters or cell proliferation of transformed and tissue derived cell lines; and d) unlike JC1, incubation with Hoechst 3342 or DCV at the concentrations and periods used for the identification of SPs markedly affected the MMPT during or after the dye incubation period and resulted in either delay in reduced marked proliferation rate proliferation in most cell line studied and cell death in K562 cells
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
