title: The slow cell death response when screening chemotherapeutic agents.
abstract: OBJECTIVE
  To examine the correlation between cell death and a common surrogate of death used in screening assays, we compared cell death responses to those obtained with the sulforhodamine B (SRB) cell protein-based "cytotoxicity" assay.
  
  
  METHODS
  With the SRB assay, the Hill equation was used to obtain an IC50 and final cell mass, or cell mass present at infinite agent concentrations, with eight adherent cell lines and four agents (32 agent/cell combinations). Cells were treated with high agent concentrations (well above the SRB IC50) and the death response determined as the time-dependent decrease in cells failing to bind both annexin V and vital fluorochromes by flow cytometry.
  
  
  RESULTS
  Death kinetics were categorized as fast (5/32) (similar to the reference nonadherent Jurkat line), slow (17/32), or none (10/32), despite positive responses in the SRB assay in all cases. With slow cell death, a single exposure to a chemotherapeutic agent caused a slow, progressive increase in dead (necrotic) and dying (apoptotic) cells for at least 72 h.
  
  
  CONCLUSIONS
  Cell death (defined by annexin and/or fluorochrome binding) did not correlate with the standard SRB "cytotoxicity" assay. With the slow cell death response, a single exposure to an agent caused a slow conversion from vital to apoptotic and necrotic cells over at least 72 h (the longest time point examined). Here, increasing the time of exposure to agent concentrations modestly above the SRB IC50 provides a method of maximizing cell kill. If tumors respond similarly, sustained low doses of chemotherapeutic agents, rather than a log-kill, maximum tolerated dose strategy may be an optimal strategy of maximizing tumor cell death.
authors.affiliation: The Center for Translational Nuclear Medicine and Molecular Imaging, Massachusetts General Hospital, Charlestown, MA 02129, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Blois
authors.forename: Adam
authors.initials: A
authors.lastname: Smith
authors.forename: Lee
authors.initials: L
authors.lastname: Josephson
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002166:Camptothecin / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects*; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm; D004354:Drug Screening Assays, Antitumor; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D005472:Fluorouracil / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D017239:Paclitaxel / Q000494:pharmacology; D012235:Rhodamines; D013997:Time Factors; D014747:Vinblastine / Q000494:pharmacology
chemical_list: D017304:Annexin A5; D000970:Antineoplastic Agents; D018906:Antineoplastic Agents, Alkylating; D000972:Antineoplastic Agents, Phytogenic; D005456:Fluorescent Dyes; D012235:Rhodamines; C022027:lissamine rhodamine B; D014747:Vinblastine; D017239:Paclitaxel; D005472:Fluorouracil; D002166:Camptothecin