Multiparametric evaluation of apoptosis: effects of standard cytotoxic agents and the cyanoguanidine CHS 828.
A multiparametric high-content screening assay for measurement of apoptosis was developed. HeLa cells and lymphoma U-937 cells were exposed to cytotoxic drugs in flat-bottomed optical microtiter plates. After incubation, the DNA-binding dye Hoechst 33342, fluorescein-tagged probes that covalently bind active caspases and chloromethyl-X-rosamine to detect mitochondrial membrane potential (MMP) were added. Image acquisition and quantitative measurement of fluorescence in a defined number of cells per well was performed using the automated image capture and analysis instrument ArrayScan. The usefulness of the assay was tested in cells exposed to standard cytotoxic drugs as well as in experimental cytotoxic cyanoguanidine CHS 828. A time- and dose-dependent activation of caspase-3, decrease in MMP, and increase in nuclear fragmentation and condensation were observed for the standard drugs, with the ability to correlate the parameters on a single cell basis. CHS 828 induced caspase-3 activation and reduction in MMP with modest changes in nuclear morphology. The method described was considered to be a rapid and information-rich apoptosis assay suitable both for correlating morphological and biochemical apoptotic events in single cells as well as for screening and evaluation of novel substances with apoptosis-inducing capabilities.
Lövborg
Henrik
H
Department of Medical Sciences, Division of Clinical Pharmacology, University Hospital, Uppsala University, Uppsala, Sweden. Henrik.Lovborg@medsci.uu.se
Nygren
Peter
P
Larsson
Rolf
R
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D003486:Cyanides / Q000494:pharmacology*; D003603:Cytotoxins / Q000494:pharmacology*; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D006146:Guanidines / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D020298:U937 Cells
D000970:Antineoplastic Agents; D061945:Caspase Inhibitors; D003486:Cyanides; D003603:Cytotoxins; D006146:Guanidines; C401312:N-(6-chlorophenoxyhexyl)-N''-cyano-N''-4-pyridylguanidine; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; C004711:dicyandiamido