Simple, semiautomatic assay of cytostatic and cytotoxic effects of antitumor drugs by laser scanning cytometry: effects of the bis-intercalator WP631 on growth and cell cycle of T-24 cells.
BACKGROUND
Common assays of drug-induced cytotoxicity on adherent cells rely on cell trypsinization followed by count of live and dead cells. To estimate the cell cycle effects, cellular DNA content is analyzed by flow cytometry. This procedure is laborious and time consuming. The alternative viability assays, e.g., based on reduction of 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide, although rapid and convenient, do not provide information about individual cells or cell cycle effects and may be biased by growth imbalance.


METHODS
The bladder carcinoma T-24 cells were seeded onto eight-chamber microscope slide-based tissue culture vessels. The novel antitumor drug, the bis-intercalating anthracycline WP631, was administered at various concentrations to different chamber cultures on the same slide; the control cultures were left untreated. After 24, 48, and 72 h, the cultures were fixed, and cellular DNA was stained with 4,6-diamidino-2-phenyl indole (DAPI). The slides were scanned by laser scanning cytometry (LSC) to obtain the number of attached cells per culture chamber and reveal their cell cycle distribution.


RESULTS
The cell growth and viability plots in the absence and presence of WP621 were constructed from the frequency of the attached cells per chamber. A 50% reduction in cell number was observed at the 75 nM concentration of WP321. Mitotic and postmitotic cells were identified based on high intensity of maximal pixel of DAPI fluorescence. An increase in proportion of cells in G2 was seen at 75-300 nM of WP631. Relatively few (<12%) apoptotic cells, identified by the presence of DNA strand breaks, remained attached in the WP631-treated cultures.


CONCLUSIONS
Because late apoptotic cells detach during culturing, the cells that remain attached in the multi-chamber cultures represent predominantly live cells; the deficit in their number compared with the untreated cultures, recorded by LSC during scanning, provides information about the degree of cytostatic and cytotoxic effects of the studied drug. The possibility to demonstrate the cell cycle distribution, including a distinction between G2 and M cells, provides an additional advantage of this assay. Other parameters that may be associated with the cell cycle perturbation or with induction of apoptosis also can be measured in the same cultures by using the multiparameter capabilities of LSC. Each measured cell can be relocated for imaging or measurement after subsequent staining with other probes.
Pozarowski
Piotr
P
Brander Cancer Research Institute, New York Medical College, Valhalla, New York, USA.
Huang
Xuan
X
Gong
Richard W
RW
Priebe
Waldemar
W
Darzynkiewicz
Zbigniew
Z
D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000633:toxicity; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division; D045744:Cell Line, Tumor; D003630:Daunorubicin / Q000031:analogs & derivatives* / Q000494:pharmacology* / Q000633:toxicity; D005434:Flow Cytometry / Q000379:methods; D006696:Holography; D006801:Humans; D019044:Image Cytometry / Q000379:methods*; D007364:Intercalating Agents / Q000494:pharmacology / Q000633:toxicity; D001749:Urinary Bladder Neoplasms / Q000378:metabolism
D000970:Antineoplastic Agents; D007364:Intercalating Agents; C104426:WP 631; D003630:Daunorubicin