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Microplate Screening for Apoptosis with Antibody to Single-Stranded DNA Distinguishes Anticancer Drugs from Toxic Chemicals

Radiation Oncology Department (R-71), University of Miami Medical School, 1550 NW 10th Ave., PAP Bldg. Room 128, Miami, FL 33136. apostain@bellsouth.net

Awtar Krishan

The effect of anticancer drugs and toxic compounds on cultures of human leukemic cells was evaluated by an enzyme-linked immunosorbent assay (Apoptosis ELISA) that uses a monoclonal antibody against single-stranded DNA to quantitate the apoptotic cells. The concentrations of 13 anticancer drugs, which increased Apoptosis ELISA absorbance, were close to the cytotoxic concentrations determined by the long-term cell survival assay. Short-term tetrazolium-based microculture tetrazolium (MTT) assay was significantly less sensitive than the Apoptosis ELISA and the cell survival assay for all anticancer drugs. For 6 drugs, cytotoxic concentrations measured by the MTT assay were at least 1 log higher than the concentrations inducing apoptosis. Importantly, in contrast to the anticancer drugs, 14 toxic chemicals did not increase the Apoptosis ELISA absorbance at cytotoxic concentrations. The difference in apoptosis induction by the anticancer drugs and the toxic chemicals was especially large in cultures treated with drug concentrations 2-fold higher than the IC₅₀ dose. Although all of the anticancer drugs tested induced intense ELISA reaction (mean absorbance 2.0), all toxic chemicals tested did not induce apoptosis. The Apoptosis ELISA assay could have useful applications in drug development as it can distinguish between clinically useful anticancer drugs and toxic compounds, has sensitivity similar to that of the long-term cell survival assay, and provides insight into the mechanism of drug cytotoxicity by differentiating between compounds killing cells by apoptosis and necrosis. (Journal of Biomolecular Screening 2003:185-190)

Key Words: apoptosis • microplates • drug screening • single-stranded DNA

Journal of Biomolecular Screening, Vol. 8, No. 2, 185-190 (2003)
DOI: 10.1177/1087057103253326


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