Drug sensitivity assays were performed using a variation of a colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] assay on V79, CHO-AuxB1, CHRC5, NCI-H460, and NCI-H249 cell lines following optimization of experimental conditions for each cell line. Results from this assay were compared with data assimilated simultaneously by clonogenic assay and by dye exclusion assay. Good correlation was observed using the CHO-AuxB1 cell line and the pleiotropic drug-resistant mutant CHRC5, with similar degrees of relative resistance observed with both the MTT and clonogenic assays. Good correlation was observed between the clonogenic and MTT assays for 1-h drug exposures, although the MTT assay was more sensitive to vinblastine. In general, the clonogenic assay was more sensitive when continuous drug exposures were utilized, although this was primarily related to the increased drug exposure time. While the use of the MTT assay in drug sensitivity testing of primary tumor samples is limited, since contaminating normal cells may also reduce the tetrazolium, the MTT assay can be semiautomated, and therefore it offers a valid, simple method of assessing chemosensitivity in established cell lines.

https://cancerres.aacrjournals.org/content/canres/47/4/936.full.pdf

Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing.

For the past 30 years strategies for the preclinical discovery and development of potential anticancer agents have been based largely upon the testing of agents in mice bearing transplantable leukemias and solid tumors derived from a limited number of murine as well as human sources. The feasibility of implementing an alternate approach, namely combined in vitro/in vivo screening for selective cytotoxicity among panels of human tumor cell lines derived from a broad spectrum of human solid tumors is under investigation. A group of 30 cell lines acquired from a variety of sources and representing 8 lung cancer pathologies as well as 76 cell lines representing 10 other categories of human cancer (carcinomas of colon, breast, kidney, prostate, ovary, head and neck; glioma; leukemia; melanoma; and sarcoma) have exhibited acceptable growth characteristics and suitable colorimetric profiles in a single, standard culture medium. Measurements of in vitro growth in microculture wells by cell-mediated reduction of tetrazolium showed excellent correlation (0.89 less than r2 less than 0.98) with measurements of cellular protein in adherent cell line cultures as well as viable cell count in suspension cell line cultures (0.94 less than r2 less than 0.99). Since the microculture tetrazolium assay provides sensitive and reproducible indices of growth as well as drug sensitivity in individual cell lines over the course of multiple passages and several months' cultivation, it appears suitable for initial-stage in vitro drug screening.

https://cancerres.aacrjournals.org/content/canres/48/3/589.full.pdf

Feasibility of Drug Screening with Panels of Human Tumor Cell Lines Using a Microculture Tetrazolium Assay

Publisher Summary Lectins play an important role in the development of immunology. Lectins also find application in serological laboratories for typing blood and determining secretor status, separating leucocytes from erythrocytes, and agglutinating cells from blood in the preparation of plasma. They serve as reagents for the detection, isolation, and characterization of carbohydrate-containing macromolecules, including blood-group antigens. In their interaction with saccharides, lectins serve as models for carbohydrate-specific antibodies, with the important advantage to purify lectins in gram quantities. Lectins are classified according to their carbohydrate-binding specificity that includes D-mannose(D-glucose)-binding lectins and 2-acetamido-2-deoxy-D-glucose-binding lectins. The chapter considers only those lectins that have been purified to homogeneity, and studied with regard to their biophysical, biochemical, and carbohydrate-binding specificity. The chapter also describes the cell-binding and biological properties of lectins. The chapter concludes with the description of several glycopeptide structures showing the carbohydrate-binding loci with which various lectins interact.

The lectins : carbohydrate-binding proteins of plants and animals

Radiation survival curves were generated for V79 Chinese hamster and two human lung cancer cell lines (NCI-H460 and NCI-H249) with doubling times of 10, 20, and 85 h, respectively, using a standard clonogenic assay, a dye exclusion assay, and a semiautomated colorimetric assay utilizing a tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylformazan bromide. Comparable results for D0 and extrapolation number (n) were observed for all assays in the lines with doubling times of 10 and 20 h. In these instances the tumor cell lines had undergone seven or more doublings after radiation. For the tumor line (H249) with an 80-h doubling time the D0S were comparable between the assays while the extrapolation number was increased in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylformazan bromide assay, a result probably related to the lower number of doublings (less than 4) after radiation. We then tested the ability of the assays to detect radiation protection and sensitization using known agents. We found that cysteamine treatment resulted in radioprotection (by a factor of 8 at 8 Gy) while 5-bromo-2-deoxyuridine incorporation caused enhancement of radiation sensitivity in all three assays. We conclude that, while optimal conditions for each cell line (cell number plated and doubling time) must be established, using characterized tumor cell lines, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylformazan bromide assay could be automated and thus be of great value in screening large numbers of potential radiosensitizers or protectors.

https://cancerres.aacrjournals.org/content/canres/47/4/943.full.pdf

Evaluation of a Tetrazolium-based Semiautomated Colorimetric Assay: Assessment of Radiosensitivity

2 – Isolation, Physicochemical Characterization, and Carbohydrate-Binding Specificity of Lectins

Dissociated cancer cells are exposed to antineoplastic drugs (5 × 10 4 viable cells/drug for 1 hr or continuously) and cultured for 4 to 6 days in liquid medium. Cells are then stained with Fast green dye, sedimented onto slides with a Cytospin centrifuge, and counterstained with a modified hematoxylin and eosin technique. Dead cells stain with Fast green, and living cells stain with hematoxylin and eosin. Cell kill is calculated as percentage of control based on the relative numbers of living tumor cells, living non-tumor cells, and dead cells. Drug sensitivity could be assayed in 125 of 162 specimens of human neoplasms obtained from malignant effusions (16 of 18), excisional biopsies (31 of 44), needle biopsies (34 of 47), endoscopic biopsies (18 of 23), peripheral blood samples (19 of 20), and bone marrow aspirates (five of seven). The assays were successful (median of ten drugs tested) in: 46 of 64 adenocarcinomas; four of 11 squamous cell carcinomas; five of seven lymphomas; six of seven melanomas; two of four sarcomas; 18 of 20 transitional-cell carcinomas; 14 of 15 small-cell carcinomas; seven of eight myelomas; 12 of 12 chronic lymphocytic leukemias; seven of nine acute leukemias, and four of five "undifferentiated" carcinomas. The assay results demonstrated a strong correlation between the in vitro chemosensitivity of different types of tumors and the known clinical response patterns of these tumors. This assay can be used to determine which specific cells are killed in a heterogeneous cell population. Further work is needed to determine if this assay may be useful in blind screening trials for antineoplastic agents or if it may be of clinical use in predicting response to agents which are not cycle specific.

https://cancerres.aacrjournals.org/content/canres/43/2/749.full.pdf

A Novel Dye Exclusion Method for Testing in Vitro Chemosensitivity of Human Tumors

The following factors must be considered when dye exclusion assays are interpreted. (a) It may require several days for lethally damaged cells to lose their membrane integrity following a cytotoxic insult. (b) During this time, the "surviving" cells may continue to proliferate. (c) Also during this time, some lethally damaged cells may undergo an early disintegration, so that they are not present to be stained with dye at the end of the culture period. Factors b and c may cause an underestimate of cell kill when the results of the assay are based upon the traditional "percent viability" expression. In order to overcome these problems, an internal standard was developed and tested. This was based upon the addition of a constant number of permanently fixed duck erythrocytes to the cultures of cells from two different established tumor cell lines. Results were based upon comparisons of the ratios of "viable" tumor cells to duck erythrocytes on permanent cytocentrifuge slides prepared from the cultures. This novel "ratio" method was found to be a more sensitive index of drug-induced cell kill than the traditional percent viability method. A standard agar cloning assay gave somewhat higher estimates of cell kill than the ratio method, although both assays were in qualitative agreement for the drugs tested. All three assays demonstrated a clear dose-effect relationship for most of the drugs tested. Dye exclusion assays may have a useful role in chemosensitivity testing in vitro.

https://cancerres.aacrjournals.org/content/canres/43/1/258.full.pdf

Comparison of Dye Exclusion Assays with a Clonogenic Assay in the Determination of Drug-induced Cytotoxicity

Two hemagglutinating components purified from the lima bean and composed of identical 62,000-molecular-weight subunits have mitogenic activity in normal and leukemic human lymphocyte cultures. Component II (247,000 molecular weight), a tetramer with four saccharide binding sites, is severalfold more active than component III (124,000 molecular weight), a dimer with two binding sites per molecule, as a mitogen for normal lymphocytes. Component II also stimulates mitogenesis in populations of cultured leukemic lymphocytes and appears to have greater activity than phytohemagglutinin-P in leukemic lymphocyte cultures obtained from some patients. Lima bean component III has minimal mitogenic activity toward leukemic lymphocytes. Components II and III appear to compete for the same cell-surface binding sites, but component II has a much greater capacity than component III to trigger DNA synthesis at low concentrations (2.5-25 μg/ml) in normal lymphocyte cultures. These results suggest that the mitogenic activity of lima bean lectin components is directly related to their valence.

Stimulation of Mitogenesis in Normal and Leukemic Human Lymphocytes by Divalent and Tetravalent Lima Bean Lectins

Drug-resistant strains of Mycobacterium tuberculosis have increasingly begun to emerge (1). Many authorities agree that this situation has the potential to become a problem of serious proportions. One hopes that scientists in both public and private sectors are hard at work studying these resistant strains in the laboratory to discover new drugs and methods to circumvent the resistance to the old drugs. What if it were impossible to study these resistant strains in the laboratory? What if the only models for Mycobacterium tuberculosis were species of Escherichia colil What if we didn't have a clue whether or not a series of new drugs had any chance of working until we had tested them in thousands of dying human beings with advanced tuberculosis? Finally, how would we feel if most of the drugs which emerged from the Escherichia coli screen as potential antituberculosis candidates routinely inflicted life-threatening toxic effects in these dying human beings? One would think that the first order of business would be to make a high priority of developing better model systems. More precisely, one would look to develop laboratory methods to study directly the drug-resistant mycobacteria which are causing all of the trouble.

/pdf/antineoplastic-drug-screening-belongs-in-the-laboratory-not-49rts830px.pdf

Antineoplastic drug screening belongs in the laboratory, not in the clinic.

Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of nuclear non-histone proteins extracted with 0.15 and 0.30 m NaCl from human normal lymphocytes or leukemic leukocytes indicated a lack of bands in the highermolecular-weight regions of the gels. However, the corresponding fractions from phytohemagglutinin-stimulated lymphocytes and Burkitt lymphoma cells did contain high-molecular-weight protein bands. Moreover, high-molecular-weight bands were present in the non-histone protein fraction extracted with 1% SDS:4 m urea (“tightly bound” non-histone protein) from all types of cells studied, but proliferating cells were found to contain significantly greater amounts of protein in the 1% SDS: 4 m urea fraction than were present in largely nonproliferating cells.

Nonstimulated normal lymphocytes and leukemic leukocytes were found to have a nuclear-associated protease activity that was capable of markedly degrading proteins in Burkitt lymphoma cell nuclei within 2 hr at 0–4° (pH 7.2). The susceptibility of the Burkitt lymphoma cell nuclear proteins to protease degradation decreased in the following order: 0.15 m NaCl-extractable proteins ≥ 0.30 m NaCl-extractable proteins > F1 histone > other histones > 1% SDS: 4 m urea-extractable proteins.

Assays of protease and phosphatase activity in isolated nuclei demonstrated the following: ( a ) 10% of the nuclear protein in Burkitt lymphoma cells was rendered acid soluble during a 2-hr incubation at 37° (pH 7.5); ( b ) 20 to 90% of the nuclear protein in normal lymphocytes and leukemic leukocytes was rendered acid soluble under the same conditions; ( c ) phytohemagglutinin (1000 µg/ml) substantially reduced protein hydrolysis in this system; ( d ) 3H was rendered acid soluble at a rate comparable to that of 32P in nuclei from normal lymphocytes that had been prelabeled with ATP-γ-32P and 3H-labeled amino acids. However, 3H was rendered acid soluble at a much slower rate than 32P in nuclei isolated from Burkitt lymphoma cells.

We conclude that proteases may have an important role in regulating the content and function of nuclear proteins.

https://cancerres.aacrjournals.org/content/canres/33/11/2923.full.pdf

Larry M. Weisenthal

Papers

A Novel Dye Exclusion Method for Testing in Vitro Chemosensitivity of Human Tumors

Comparison of Dye Exclusion Assays with a Clonogenic Assay in the Determination of Drug-induced Cytotoxicity

Stimulation of Mitogenesis in Normal and Leukemic Human Lymphocytes by Divalent and Tetravalent Lima Bean Lectins

Antineoplastic drug screening belongs in the laboratory, not in the clinic.

Catabolism of nuclear proteins in control and phytohemagglutinin-stimulated human lymphocytes, leukemic leukocytes, and Burkitt lymphoma cells.