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.

BackgroundAlveolar soft-part sarcoma (ASPS) is an extremely rare, highly vascular soft tissue sarcoma affecting predominantly adolescents and young adults. In an attempt to gain insight into the pathobiology of this enigmatic tumor, we performed the first genome-wide gene expression profiling study.MethodsFor seven patients with confirmed primary or metastatic ASPS, RNA samples were isolated immediately following surgery, reverse transcribed to cDNA and each sample hybridized to duplicate high-density human U133 plus 2.0 microarrays. Array data was then analyzed relative to arrays hybridized to universal RNA to generate an unbiased transcriptome. Subsequent gene ontology analysis was used to identify transcripts with therapeutic or diagnostic potential. A subset of the most interesting genes was then validated using quantitative RT-PCR and immunohistochemistry.ResultsAnalysis of patient array data versus universal RNA identified elevated expression of transcripts related to angiogenesis (ANGPTL2, HIF-1 alpha, MDK, c-MET, VEGF, TIMP-2), cell proliferation (PRL, IGFBP1, NTSR2, PCSK1), metastasis (ADAM9, ECM1, POSTN) and steroid biosynthesis (CYP17A1 and STS). A number of muscle-restricted transcripts (ITGB1BP3/MIBP, MYF5, MYF6 and TRIM63) were also identified, strengthening the case for a muscle cell progenitor as the origin of disease. Transcript differentials were validated using real-time PCR and subsequent immunohistochemical analysis confirmed protein expression for several of the most interesting changes (MDK, c-MET, VEGF, POSTN, CYP17A1, ITGB1BP3/MIBP and TRIM63).ConclusionResults from this first comprehensive study of ASPS gene expression identifies several targets involved in angiogenesis, metastasis and myogenic differentiation. These efforts represent the first step towards defining the cellular origin, pathogenesis and effective treatment strategies for this atypical malignancy.

Novel structure-activity relationships (SAR) distinct from known SAR for ellipticines have been revealed for certain ellipticinium salts. In particular, ellipticiniums such as the prototypical 9-methoxy-2-methylellipticinium (I- or OAc-) were found to be preferentially cytotoxic to the brain tumor cell line subpanel of the NCI 60 cell-line screening panel. Similar specificity also was apparent with 9-unsubstituted ellipticiniums, or others bearing 9-methyl or 9-chloro substituents. In contrast, 9-hydroxy-substituted ellipticiniums, as well as all nonquaternized ellipticines tested, were devoid of brain tumor specificity. Therefore, it did not appear that this unusual preference was correlated with the relative availability of redox cycling mechanisms, since redox cycling presumably is blocked in 9-methyl- and 9-chloroellipticiniums. Indeed, related investigations have indicated that the brain tumor specificity is mediated by preferential uptake and intracellular accumulation of the specific ellipticiniums. The present study further supports that the NCI in vitro "disease-oriented" primary screen can facilitate the discovery of novel, selectively cytotoxic leads for in vivo and mechanistic investigations.

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.

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AF has noteworthy antitumor activity against certain human tumor renal cell lines in vitro and in vivo, which correlates with drug metabolism to covalently binding metabolites after CYP1A1 and CYP1B1 gene expression. We hypothesize that it leads to apoptosis induction. AF sensitive renal cell strains are predominantly of the papillary histological type. These results are limited by the small numbers of cell lines and cell strains but they are suggestive of the need for further testing in larger collections of cell strains.

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