A sensitive assay for the evaluation of cytotoxicity and its pharmacologic modulation in human solid tumor-derived cell lines exposed to cancer-therapeutic agents.
OBJECTIVE
Reliable in vitro cytotoxicity assays are essential for determining the responses of human normal and cancer-derived cells to therapeutic agents and also for the identification and pre-clinical evaluation of new drugs capable of selectively augmenting the susceptibility of cancer cells to conventional therapies. The clonogenic survival assay is considered as the "gold standard" in this regard because it measures the sum of all modes of cell death, encompassing both early and late events such as delayed growth arrest. In this assay, however, the impact of cell-to-cell communication is disregarded because the cells are plated out at very low densities. In addition, here we provide evidence that human breast cancer cell lines cannot be reliably evaluated by clonogenic assays. We developed a novel long-term, High Density Survival (HDS), assay that circumvents the various intrinsic shortcomings of the conventional cytotoxicity assays.


METHODS
In the HDS assay, the cells are maintained at a high density for 24 h prior to, and for 24 h after, exposure to a DNA-damaging agent to facilitate intercellular communication. After a carefully scheduled subculturing for approximately 7 days, cultures are assessed for the extent of growth.


RESULTS
The degree of radiosensitivity and cisplatin sensitivity evaluated by the HDS assay in human cancer cells was comparable to that measured by the clonogenic assay. Pharmacological inhibitors of CaMKII and/or PI3K signaling elicited a greater degree of radiosensitization when determined by the HDS assay than the clonogenic assay. In all these experiments, there was no relationship between the degree of cytotoxicity measured by the clonogenic survival and viability assays. In the HDS assay, all seven human breast cancer cell lines that we tested exhibited a high degree of radioresistance.


CONCLUSIONS
The novel HDS assay appears to be a powerful tool for evaluating cancer cell responses to therapeutic agents under conditions which incorporate some aspects of intercellular communication.
Mirzayans
Razmik
R
Department of Oncology, University of Alberta, Cross Cancer Institute, Edmonton, Alberta T6G 1Z2, Canada. razmikm@cancerboard.ab.ca
Andrais
Bonnie
B
Scott
April
A
Tessier
Amy
A
Murray
David
D
D000970:Antineoplastic Agents / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000188:drug therapy*; D002450:Cell Communication; D002452:Cell Count; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D005260:Female; D006801:Humans; D009369:Neoplasms / Q000188:drug therapy*; D011836:Radiation Tolerance; D015398:Signal Transduction / Q000187:drug effects
D000970:Antineoplastic Agents; D002945:Cisplatin