We have developed a rapid, sensitive, and inexpensive method for measuring the cellular protein content of adherent and suspension cultures in 96-well microtiter plates. The method is suitable for ordinary laboratory purposes and for very large-scale applications, such as the National Cancer Institute's disease-oriented in vitro anticancer-drug discovery screen, which requires the use of several million culture wells per year. Cultures fixed with trichloroacetic acid were stained for 30 minutes with 0.4% (wt/vol) sulforhodamine B (SRB) dissolved in 1% acetic acid. Unbound dye was removed by four washes with 1% acetic acid, and protein-bound dye was extracted with 10 mM unbuffered Tris base [tris (hydroxymethyl)aminomethane] for determination of optical density in a computer-interfaced, 96-well microtiter plate reader. The SRB assay results were linear with the number of cells and with values for cellular protein measured by both the Lowry and Bradford assays at densities ranging from sparse subconfluence to multilayered supraconfluence. The signal-to-noise ratio at 564 nm was approximately 1.5 with 1,000 cells per well. The sensitivity of the SRB assay compared favorably with sensitivities of several fluorescence assays and was superior to those of both the Lowry and Bradford assays and to those of 20 other visible dyes. The SRB assay provides a colorimetric end point that is nondestructive, indefinitely stable, and visible to the naked eye. It provides a sensitive measure of drug-induced cytotoxicity, is useful in quantitating clonogenicity, and is well suited to high-volume, automated drug screening. SRB fluoresces strongly with laser excitation at 488 nm and can be measured quantitatively at the single-cell level by static fluorescence cytometry.

We describe here the development and implementation of a pilot-scale, in vitro, anticancer drug screen utilizing a panel of 60 human tumor cell lines organized into subpanels representing leukemia, melanoma, and cancers of the lung, colon, kidney, ovary, and central nervous system. The ultimate goal of this disease-oriented screen is to facilitate the discovery of new compounds with potential cell line-specific and/or subpanel-specific antitumor activity. In the current screening protocol, each cell line is inoculated onto microtiter plates, then preincubated for 24-28 hours. Subsequently, test agents are added in five 10-fold dilutions and the culture is incubated for an additional 48 hours. For each test agent, a dose-response profile is generated. End-point determinations of the cell viability or cell growth are performed by in situ fixation of cells, followed by staining with a protein-binding dye, sulforhodamine B (SRB). The SRB binds to the basic amino acids of cellular macromolecules; the solubilized stain is measured spectrophotometrically to determine relative cell growth or viability in treated and untreated cells. Following the pilot screening studies, a screening rate of 400 compounds per week has been consistently achieved.

The National Cancer Institute (NCI) is implementing a large-scale in vitro drug-screening program that requires a very efficient automated assay of drug effects on tumor cell viability or growth. Many laboratories worldwide have adopted a microculture assay based on metabolic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). However, because of certain technical advantages to use of the protein-binding dye sulforhodamine B (SRB) in a large-scale screening application, a detailed comparison of data generated by each type of assay was undertaken. The MTT and SRB assays were each used to test 197 compounds, on simultaneous days, against up to 38 human tumor cell lines representing seven major tumor categories. On subsequent days, 38 compounds were retested with the SRB assay and 25 compounds were retested with the MTT assay. For each of these three comparisons, we tabulated the differences between the two assays in the ratios of test group values to control values (T/C) for cell survival; calculated correlation coefficients for various T/C ratios; and estimated the bivariate distribution of the values for IC50 (concentration of drug resulting in T/C values of 50%, or 50% growth inhibition) for the two assays. The results indicate that under the experimental conditions used and within the limits of the data analyses, the assays perform similarly. Because the SRB assay has practical advantages for large-scale screening, however, it has been adopted for routine use in the NCI in vitro antitumor screen.

A eukaryotic growth medium (Program Development Research Group Basal Growth Medium) was developed for CO2-independent maintenance and propagation of human and nonhuman tumor cell lines representing diverse histologies (e.g., cancers of the brain, colon, lung, ovary, and kidney, as well as leukemia and melanoma). It was also shown to be suitable for the maintenance and propagation of nontumor cells of human and nonhuman derivation. The medium derives its buffering capacity primarily from beta-glycerophosphate, exhibits a stable physiologic pH of 7.3-7.4, and is optimized to facilitate growth in atmospheric CO2. It is also useful in cellular growth and cytotoxicity assays based on either the metabolic reduction of tetrazolium reagents or protein staining. The 50% inhibitory concentration values obtained with carmustine, doxorubicin, and tamoxifen in cell lines maintained in the new medium under atmospheric CO2 were closely comparable to those obtained with these drugs against cells maintained in RPMI-1640 under a 5% CO2 environment.

D-Glucosamine is toxic to several malignant cell lines and in vivo tumors at concentrations that have little effect upon normal host tissues. Evidence is presented to support the hypothesis that cellular membranes may be the primary targets of glucosamine's tumoricidal activity. Treatment of rat C6 glioma cells with a cytotoxic concentration of glucosamine (20 mM) caused fragmentation of rough endoplasmic reticulum, proliferation of Golgi complexes, evagination of outer nuclear and mitochondrial membranes, and the accumulation of membranous vacuoles and lipid droplets in the cytoplasm. These changes were detected within the first 3 hr after treatment of cultures with glucosamine and became increasingly severe until cell lysis occurred between 24 and 48 hr of treatment. The cytotoxicity of glucosamine was potentiated by the local anesthetic lidocaine, and by other membrane-active drugs, at concentrations that were growth inhibitory but nonlytic. Most of these drugs possessed local anesthetic activity and inhibited glioma sterol synthesis. Within the same period of time required or ultrastructural changes in cellular membranes, glucosamine inhibited the incorporation of [2-14Clacetate into sterols and into an unidentified 400-dalton lipid that migrated close to sterols on thin-layer chromatograms. This inhibition was potentiated by lidocaine and increased over the same range of D-glucosamine concentrations that led to increased cell toxicity after a 48-hr treatment. These findings suggest that the effects of glucosamine upon cellular membranes may be central to its tumoricidal activity and that glucosamine, in combination with membrane-active drugs, may be useful in the treatment of certain types of tumors, particularly those of the central nervous system.