Standardized kinetic microassay to quantify differential chemosensitivity on the basis of proliferative activity

Abstract

Summary. Conventionally in vitro cytotoxicity assays are performed as single-end-point determinations. To compensate for the diversity of growth rates among different cell lines in this report we describe a computerized kinetic chemosensitivity assay based on quantification of biomass by staining cells with crystal violet. As a prerequisite four human breast cancer cell lines (MDA-MB-231, MCF-7, T-47-D and ZR-75-1) were characterized with regard to oestrogen and progesterone receptor content, modal chromosome number and proliferation kinetics depending on the number of passages in culture. With prolonged time in culture for ZR-75-1 exposed to various concentrations of cisplatinum a dose-related increase in drug effect was observed. Owing to a correction of the T/C values for the initial cell mass (at the time when drug is added) a sharp distinction between cytostatic and cytocidal drug effects becomes obvious in plots of corrected T/C values versus time of incubation. The influence of the untreated control on the corrected T/C values and possible time courses of theoretical inhibition profiles (reflecting cytostatic, transient cytotoxic or cytocidal drug effects as well as development of resistance) and their relationship to the corresponding growth curves of drugtreated cells are discussed. Chemosensitivity assays with diethylstilbestrol dipropionate, tamoxifen, melphalan, cisplatinum, vinblastine, Adriamycin and 5-fluorouracil prove the theoretical considerations to be true for MDA-MB-231, MCF-7, T-47-D and ZR-75-1 human breast cancer cell lines in practice.

Bombesin antagonists inhibit proangiogenic factors in human experimental breast cancers

“…After an incubation period of 1 h, the medium was removed and kept at À701C for RIAs. After an additional incubation in serum-free medium for 72 h, in vitro cell growth was estimated by the crystal violet assay (Bernhardt et al, 1992). The OD at 600 nm of each well was measured using a Beckman (Palo Alto, CA, USA) plate reader.…”

Chemosensitivity of human MCF-7 breast cancer cells to diastereoisomeric diaqua(1,2-diphenylethylenediamine) platinum(II) sulfates and specific platinum accumulation

“…We have recently reported the principle and the applications of the kinetic crystal violet assay [2,23]. In brief, cells were seeded (100 gl/well) in 96-well flat-bottomed microtitration plates (Falcon Plastics 3075).…”

“…The mean protein-mass concentration was determined in a separate experiment as described above. Cell-associated platinum expressed in moles per liter was calculated by the equation CPt,c = (mpt,csp,NAA/MPt) X ~prt, C/Wprt,csp X mcsp,NAA), (2) where Mvt represents the molar mass of platinum.…”

“…According to Eq. 1, any growth curve for a drug-treated cell population can be reconstructed from the T/Ccorr values and the growth-curve data of the corresponding control [2]. For compounds I and II and cisplatin, a clear-cut dosedependent inhibition of cell proliferation that resulted in final equiactivity at 5 gM was observed, whereas compound III was ineffective at 1 and 5 gM.…”

“…For this purpose, we compared the diastereoisomeric diaqua[ 1,2-bis(4-fluorophenyl)ethylenediamine]platinum(II) sulfates (compounds I and II) and meso-diaqua[1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine]platinum(II) sulfate (compound III) with cisplatin in terms of their stability in culture medium, their dose-dependent antitumor activity, the time required to obtain a cytocidal drug effect, the kinetics of platinum accumulation by the hormone-sensitive MCF-7 human breast cancer cell line [2,3] and the extent of DNA platination. …”