The hypothesis that activation of apoptosis and DNA fragmentation is involved in TNF-mediated cytolysis of U937 tumor cells was investigated. Morphological, biochemical, and kinetic criteria established that TNF activates apoptosis as opposed to necrosis. Within 2-3 h of exposure to TNF, U937 underwent the morphological alterations characteristic of apoptosis. This was accompanied by cleavage of DNA into multiples of nucleosome size fragments. Both of these events occurred 1-2 h prior to cell death as defined by trypan blue exclusion or 51Cr release. DNA fragmentation was not a non-specific result of cell death since U937 cells lysed under hypotonic conditions did not release DNA fragments. The percentage of cells undergoing apoptosis depended on the concentration of TNF and was augmented by the addition of cycloheximide. A TNF-resistant variant derived from U937 did not undergo apoptosis in response to TNF, even in the presence of cycloheximide. Furthermore, TNF could still activate NFkB in this variant, suggesting that this pathway is not involved in TNF-mediated cytotoxicity. Two agents known to inhibit TNF-mediated cytotoxicity, ZnSO4 and 3-aminobenzamide, were shown to inhibit TNF-induced apoptosis. Taken altogether, these data support the hypothesis that activation of apoptosis is at least one essential step in the TNF lytic pathway in the U937 model system.

This study has addressed the question of whether there may be some common mechanism underlying the induction or expression of acquired cytokine and drug resistance in a tumor cell line. This study employed the tumor-necrosis-factor(TNF)-sensitive U937 tumor cell line as a model system to determine if selection of a tumor cell variant for cytokine resistance would also result in drug resistance and vice versa. Variants were selected by culturing in the presence of purified recombinant TNF or a mixed-lymphokine-containing supernatant derived from concanavalin-A-stimulated peripheral blood lymphocytes. The resulting variants were resistant not only to TNF, but also to certain chemotherapeutic drugs. The variants were most resistant to colchicine and the Vinca alkaloids, requiring drug concentrations 50- to 5000-fold higher to mediate levels of cytotoxicity comparable to that seen with the parental U937. The variants were moderately resistant to cycloheximide, actinomycin D, and mitomycin C. In contrast, these lines were relatively sensitive to doxorubicin or daunomycin. This phenomenon was not unique to U937 cells since we obtained a similar pattern of drug resistance by selecting TNF-resistant variants of the WEHI-164 tumor cell line. The cytokine-selected U937 variants were still lysed by NK cells, although they were somewhat less sensitive than the parental U937. Both variants were relatively resistant to lysis by activated macrophages, probably because of their TNF resistance. In an alternative selection procedure, U937 variants were derived by culturing in the presence of increasing concentrations of colchicine. The resulting variants were relatively resistant to TNF, providing further support for the existence of some common mechanism operating in induction or expression of acquired cytokine and drug resistance. The resistance mechanism apparently does not involve the P glycoprotein since the cytokine-selected U937 variants do not overexpress the mdr gene. This study has demonstrated that selection of TNF-resistant variants results in coexpression of a unique form of drug resistance that is characterized by resistance to microtubule-active drugs but not to the anthracycline antibiotics and is not associated with overexpression of the mdr gene.