Stochastic modeling of apoptosis tolerance distributions measured by multivariate flow analysis of human leukemia cells.
BACKGROUND
Cytofluorometric analysis allows single-cell resolution of all-or-none programmed cell death (apoptosis) responses and permits direct measurement of cumulative frequency distributions (CFDs) of apoptosis sensitivity from which the median apoptosis tolerance can be estimated. Robust estimation of susceptibility to apoptosis within neoplastic cell populations provides a means of either accurately determining pharmacologically induced changes in apoptosis sensitivity or comparing cell population responses to different apoptosis inducers.


METHODS
Experimentally determined CFDs for VP-16 (etoposide)-induced apoptosis were measured by phosphotidylserine surface expression and mitochondrial membrane potential dissipation (DeltaPsi(m)) in BV173 leukemia cells. CFDs were modelled by a modified Hill equation using a four-parameter nonlinear regression from which median apoptosis tolerance (K) was estimated.


RESULTS
Median apoptosis tolerance (K) was estimated from nonlinear regression analysis of CFDs for DeltaPsi(m) collapse and loss of membrane asymmetry. The error distribution of K determined from nonlinear regression analysis of 100 simulated CFDs was shown to exhibit an asymmetrical distribution. The asymmetrical likelihood intervals for K were computed iteratively, thereby providing a measure of experimental error.


CONCLUSIONS
A distribution-based approach to apoptosis assay using multivariate flow analysis offers a powerful, quantitative technique for investigating the phenotypical basis of neoplastic cell responsiveness to apoptosis therapy, permitting separation of cell populations on the basis of apoptosis susceptibility.
Fennell
D A
DA
Department of Experimental Haematology, St Bartholomew's and The Royal London School of Medicine, London, United Kingdom.
Cotter
F E
FE
D017209:Apoptosis / Q000187:drug effects; D005047:Etoposide / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects; D007938:Leukemia / Q000378:metabolism / Q000473:pathology*; D008027:Light; D016013:Likelihood Functions; D016014:Linear Models; D008563:Membrane Lipids / Q000096:biosynthesis; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects; D015999:Multivariate Analysis; D010718:Phosphatidylserines / Q000096:biosynthesis; D012542:Scattering, Radiation; D012680:Sensitivity and Specificity; D013269:Stochastic Processes; D014407:Tumor Cells, Cultured
D008563:Membrane Lipids; D010718:Phosphatidylserines; D005047:Etoposide