The more effective chemosensitivity tests should be developed as useful tools in cancer biology research, anticancer drug screening, individualized chemotherapy, and development of other cancer treatment modalities. For practical use of chemosensitivity tests enormous efforts are necessary to improve previous tests or to develop new ideal tests. In this study, the experiments to achieve more efficient experimental conditions such as proper plating cell numbers and reasonable anticancer drug exposure (concentration and timel in the clonogenic assay using 5 human cancer cell lines were performed, and the inhibitory effects of anticancer agents on in vitro tumor marker levels of 2 cancer cell lines were evaluated, and then the value of simple dye exclusion assay was reassessed. The human cancer cell lines used in this study were SC-I of colon cancer, PLC/PRF/5 of hepatoma. ZR-75-1 of breast cancer, G 361 and Bowes melanoma of malignant melanoma. The anticancer drugs used were 5 FU, methotrexate, mitomycin C, adriamycin, cisplatin and BCUN. The clonogenic assay of SC-1 was done at various plating cell numbers, and clonogenic assay as chemosensitivity test was performed at various drug concentrations with continuous or 1-hour exposures. Clonogenic assays of 5 human cancer cell lines were carried out at the drug concentrations of 1/10 peak plasma concentra- tion (PPC) and 1/100 PYC of continuous exposure, or 1/1(l PPC of I-hour exposure. The tumnor marker inhibition of SC-I and PLC/RRF/5 by anticancer drug was measured by radioimmunoassay on the third and the fifth incubation day, and the cell survival fraction by dye exclusion assay with trypan blue was calculated. In clonogenic assay of SC-I; the number of colrmies was increased with the increase of plating cell number, and the piating of: 5 x 10(4) cells/mi formed the sufficient number of colonies (over 500 per well) with reasonable coefficiency of variance. Other cell lines also formed sufficient number of colonies with the plating of 5 x 10(4) cells/mi. In clonogenic assay of SCI as chemosensitivity test; continuous exposures of 1/10 PPC and 1/100 PPC of anticancer drug and 1-hour exposure of 1/10 PPC showed valuable results which were able to differentiate more sensitive drugs from less sensitive drugs. In clonogenic assay of 5 cancer cell lines: continuous exposures of 1/10 and 1/100 PPC identified sensitive drugs more clearly than 1-hour exposure of 1/10 PPC. Most anticancer drugs of long in vitro half life revealed more sensitive activity than those of short half life in the clonogenic assay of continuos exposure. Among three kinds of in vitro chemosensitivity tests of continuous drug exposure, the clonogenic assay showed more sensitive data in anticancer activity than the tumor marker inhibition test and the dye exclusion assay. And the results of 3 tests were closely interrelated in many series of the experiment. Anticancer drugs of longer in vitro half life usually revealed more sensitive activity also in the tumor marker inhibition test and the dye exclusion assay of continuous exposure. In conclusion: in the experiments of in vitro chemasensitivity tests using human cancer cell lines, the proper plating numbers of the clonogenic assay were approximately 5x10(4) cells/ml, and the continuous drug exposures (1/10 PPC and 1/100 PPC) identified sensitive drugs more clearly than the 1-hour exposure (1/10 PPC), and the effects of the exposure time and especially the in vitro drug half life in media as well as the in vitro anticancer drug concentration were significant on the sensitivity data in the clonogenic assay and also in the tumor marker inhibition test and the dye exclusion assay, and the results of three tests were closely interrelated in many series of the experiment.
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