Monitoring the accumulation of fluorescently labeled phospholipids in cell cultures provides an accurate screen for drugs that induce phospholipidosis.
A large number of cationic amphiphilic drugs (CADs) are known to cause phospholipidosis (PLD) in vivo. In the present study, we have built upon our previous findings to further qualify the use of a fluorescently labeled phospholipid-based cell-culture assay to detect PLD-inducing drugs. In this paper, we demonstrate that 12 PLD-negative compounds and 11 drugs known to cause PLD in vivo are all correctly identified by using this assay. Interestingly, we found that in cells treated with certain CADs, the fluorescent phospholipid was sequestered in a very specific punctate pattern, which overlapped strongly with the staining pattern seen with a lysosomal marker protein. Our data also show that false positives can be generated with the fluorescence assay when compounds are used at concentrations that cause a >30% decrease in cell number in this assay. Confocal microscopy demonstrated that the staining pattern of fluorescent phospholipids in these cases may be differentiated from those of true positives by the fact that diffuse, rather than punctuate, fluorescence is observed. These studies confirm and expand our previous results showing that the fluorescent phospholipid assay is a highly sensitive, specific tool for detecting PLD-inducing drugs, if care is taken to rule out cytotoxicity-related artifact.
Nioi
Paul
P
The Schering-Plough Research Institute, Summit, New Jersey 07901, USA. paul.nioi@spcorp.com
Pardo
Ingrid D R
ID
Snyder
Ronald D
RD
D016477:Artifacts; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D008064:Lipidoses / Q000139:chemically induced* / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D018613:Microscopy, Confocal; D015336:Molecular Probe Techniques; D010743:Phospholipids / Q000378:metabolism*; D015203:Reproducibility of Results; D013997:Time Factors
D005456:Fluorescent Dyes; D010743:Phospholipids