Soft agar

Altered cell morphology Increased growth rate Increased saturation density Formation of multilayers Reduced adhesion to substratum Colony formation in soft agar Reduced serum requirement Altered growth factor requirement Tumor formation in athymic nude mice... 

The soft agar is allowed to set at room temperature and the plates are inverted and incubated (within 1 h of pouring) at 37°C in the dark. Incubation is continued for 2-3 days. 

The soft agar overlays are prepared and maintained at 45°C prior to use. 

To 2.0 ml aliquots of soft agar overlay medium (0.6% and 0.5% sodium chloride in distilled water) containing a trace of histidine and excess biotin and maintained at 45° C in a dry block, add 100 pi at a solution of the test article. Use only one plate per dilution. 

Repeat step (3), using 0.5 ml of 8 percent S9 mix per 2.0 ml aliquot of soft agar in addition to the test compound and tester strain. The S9 mix is kept on ice during the experiment. 

Two main protocols have been devised for carrying out mutation assays with mouse lymphoma L5178Y cells, plating the cells in soft agar or a fluctuation test approach. The latter is described in the following section, based on Cole et al. (1986). The reader is referred to Clive et al. (1987) for a full description of the soft-agar method. 

Oberly, T.J., Bewsey, B.J. and Probst, G.S. (1987). A procedure for the CHO/HGPRT mutation assay involving treatment of cells in suspension culture and selection of mutants in soft agar. Mutation Res. 182 99-111. 

Moved] Cranberry fruit of Early Black cultivar was fractionated chromatographically and fractions were analyzed for flavonoid content. The effects of the flavonoid fractions and ursolic acid, an abundant triterpenoid in cranberry peel, were assessed in two models of colon cancer and one model of breast cancer. Clonogenic soft agar assays were used to determine the effect of these compounds on tumor colony formation in HCT-116, HT-29 and MCF-7 cells. MTT and trypan blue assays were performed to assess their ability to inhibit tumor cell proliferation. TUNEL assays were performed to assess apop-totic response to the cranberry compounds. The proanthocyanidins inhibited tumor colony formation in HCT-116 and HT-29 cells in a dose-dependent manner, with greater effect on the HCT-116 cell line. Ursolic acid strongly inhibited tumor colony formation in both colon cell lines. These compounds also decreased proliferation in all three tumor cell lines with the HCT-116 cell line most strongly affected. (150 words)... 

In addition to UCN-01 and other staurosporine or maleimide derivatives (e.g., compounds 17 and 18 Fig. 4) [102,103,105-107], PDKl kinase activity is inhibited by aminopyrimidines. A representative example of this compound class is BX-320 (compound 19, Fig. 4), a PDKl inhibitor (IC50 = 30 nM) that displays good selectivity over protein kinase A (PKA, 35-fold) [108]. BX-320 blocks the growth in soft agar of a wide range of tumor cell lines (IC50 = 0.093 to 1.32 xM), and shows efficacy in a metastasis mouse model (200 mg/kg bid). 

Horowitz, D. and King, A.G. (2000) Colorimetric determination of inhibition of hematopoietic progenitor cells in soft agar. Journal of Immunological Methods, 244, 49-58. 

Gel diffusion assays (biological target is mixed in soft agar and spread as a thin film the compound library is spread on the surface of the film after allowing for compound... 

It is assumed that CVS produces some factors) accelerating hematopoiesis. A significant level of CSF activity assessed by in vitro soft-agar colony formation in bone marrow cells was detected in the sera of a few hours after the final injection of 50 mg/kg CVS. 

No morphologically transformed colonies were seen in control cultures. Cells transformed in this way also exhibited the ability to grow in soft agar and formed anaplastic fibrosarcomas when transplanted into the cheek pouches of four-week old male hamsters (J5 0. Tsuda et l. (33) also demonstrated morphological transformation in primary cultures of Syrian hamster embryo cells by crude tryptophan pyrolysates. In this experiment, a concentration of 50 pg/ml of pyrolysate induced 1.8% transformed colonies. The control transformation frequency was 0.028%. 

In addition to Ras, a number of other proteins are known to be substrates for FTase (many of unknown identity or function, determined by 2-D gel shift experiments in the presence and absence of FTI) these may also play a part in determining relative susceptibility to FTase inhibition.46 In particular, RhoB, a farnesylated protein involved with the formation of actin stress fibers and cytoskeletal organization, has been implicated in the growth-inhibitory consequences of FTase inhibition.47,48 Another study using the FTI 19 found that growth inhibition in soft agar of a panel of human tumor cell lines correlated with the mutational status of the H-and N-Ras proteins, but not with K-Ras mutations.49 In this study also, cells with genetic defects in addition to ras were sensitive to FTase inhibition. 

Compound Z n FTase 1C50 (nM) GGTase IC q (nM) MTL ( iM) Soft Agar EC5g (Ufh-ras... 

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