A method is described by which the growth inhibitory effects of cytotoxic compounds and fermentation broth cultures on adherent tumor cell lines can be quantitated.Cells are seeded into 96-well microtiter plates and 16 hours later the test compounds or broths are added to the wells. Cell growth is measured after three days (B16 mouse melanoma cells) or six days (HT-29, human colon carcinoma cells) by first fixing adherent cells, staining with Giemsa stain, washing away excess stain, then solubilizing stained cells with HCl. Absorbance is determined using a microELISA spectrophotometer and the data are transferred to and analyzed by a computer. The assay is rapid and reproducible and can be used to identify fermentation broths with cytotoxic components.Addition of DNA into the assay mixture (cells plus compound) inhibits the cytotoxic activities of certain DNA-reactive agents. The results of this study demonstrate the application of this assay system for primary and secondary evaluation of fermentation broths for in vitro antitumor activity.

U937 human monoblast cells incubated with leukotriene D4 (LTD4) rapidly released arachidonic acid metabolites into the culture medium. Release was suppressed by the high-affinity LTD4 receptor antagonist SK&F 104353. Arachidonic acid release induced by LTD4 has been linked to a rapid induction of gene expression, and the propagation of the receptor binding signal is probably associated with enzymes that regulate gene expression. We have studied the participation of DNA topoisomerase I in LTD4 signal transduction. LTD4-specific release of arachidonic acid metabolites was inhibited (60-80%) by the topoisomerase I inhibitor camptothecin. LTD4 increased protein-linked DNA strand breakage induced by camptothecin in U937 cells; this enhancement was prevented by coincubation of the cells with LTD4 plus the receptor antagonist SK&F 104353. In addition, LTD4 produced a rapid transient increase in extractable topoisomerase I activity, which was maximum within the first 10 min after addition of LTD4 to the culture medium. Incubation of cultures for greater than 10 min with LTD4 before the addition of camptothecin resulted in no enhancement of camptothecin-induced DNA strand breakage, consistent with a reversal of topoisomerase I activation. Staurosporine, an inhibitor of protein kinase C, blocked LTD4-induced arachidonic acid release and attenuated the effect of LTD4 on camptothecin-induced DNA strand breakage. These results are consistent with the view that the regulation of topoisomerase I activity is involved in the propagation of LTD4-mediated signals in U937 cells.

Incubation of cultured rat aortic smooth muscle cells (A-10, ATCC CRL 1476) with [8-arginine]vasopressin (AVP) or thrombin increased the amount of DNA strand breakage induced by camptothecin, an inhibitor of topoisomerase I (DNA topoisomerase; EC 5.99.1.2) and transiently stimulated the extractable activity of this enzyme. Both topoisomerase-related responses were prevented by treatment of the cells with AVP or thrombin plus the appropriate receptor antagonist. The increase in strand breakage mediated by AVP and thrombin depended on the concentration of hormone. Neither AVP nor thrombin had any effect on strand breaks obtained with the epipodophyllotoxin VM-26, an inhibitor of topoisomerase II [DNA topoisomerase (ATP-hydrolysing); EC 5.99.1.3]. Pretreatment of the cells with pertussis toxin partially inhibited thrombin-mediated increases in camptothecin-induced strand breakage whereas AVP-mediated increases were unaffected. These results are consistent with the notion that AVP and thrombin induce a transient increase in intracellular topoisomerase I activity via interactions with their respective cell surface receptors and that the effects of the activation of these receptors are mediated by different G-proteins.