Energy-dependent reduced drug binding as a mechanism of Vinca alkaloid resistance in human leukemic lymphoblasts.

We studied the accumulation of [3H]vinblastine (VLB) by lines of CCRF-CEM cultured human leukemic lymphoblasts that were either sensitive or resistant to the drug. Neither cell line metabolized VLB, nor selectively retained any radioactive impurities. There was an apparent "instantaneous" accumulation of VLB by cells of both lines, resulting in cell to medium ratios greater than 1.0 within 1 sec after drug addition. Experiments between 0 and 60 sec revealed that the presumed undirectional initial rate of VLB accumulation by resistant cells, termed CEM/VLB100, was about one-half that of sensitive CEM cells. In experiments carried out over 60 min, the VLB-resistant cells accumulated considerably less [3H]VLB than did the sensitive cells. Drug accumulation by both cell lines was temperature-sensitive, since incubation of cells at 4 degrees resulted in only minimal uptake beyond that observed at zero time. CEM/VLB100 cells retained less drug than did CEM cells, apparently because of a larger fraction of readily releasable VLB compared with CEM cells. The accumulation of VLB by either cell line was related in part to cellular levels of ATP. Although depletion of ATP was associated with decreased accumulation of VLB by CEM cells, it was related to enhanced drug accumulation by CEM/VLB100 cells. Restoration of ATP levels to near control values by addition of glucose also had opposite effects on the two cell lines, causing further accumulation of VLB by the sensitive line but leading to apparent drug efflux from the resistant line. Potentially competing substrates (VM-26, colchicine, daunorubicin, and doxorubicin) failed to block this glucose-mediated release of VLB from the CEM/VLB100 cells. In experiments with energy-depleted CEM/VLB100 cells preloaded with VLB and then incubated in drug-free medium, initial drug loss was shown to be independent of cellular metabolism, being roughly the same for both metabolically intact and metabolically depleted cells. Glucose (energy) was required only for subsequent release of what appeared to be a more tightly bound cell-associated fraction of VLB. Results of zero-time binding studies tended to confirm that VLB binding by resistant cells has two components, one requiring and the other not requiring metabolic energy. Differences in the proportions of these two components between the sensitive and resistant cells suggest a mechanism for resistance to VLB and similar compounds.

The modulation of lymphocyte functions by molecules secreted by macrophages. I. Description and partial biochemical analysis.

Culture fluids of peritoneal exudate cells rich in macrophages stimulated DNA synthesis of thymocytes and, to lesser extent, of spleen cells. We also investigated the effects of culture fluids from macrophages on the in vitro response to a hapten-carrier protein (fluorescein-menocyanin) using spleen cells from immune mice. Macrophage culture fluids contained an activity that increased the plaque-forming cell response of both IgG and IgM class. This increase was observed in the absence of any added hapten protein to the culture. The helper function of T lymphocytes (as evidenced by challenging with the hapten on the homologous carrier) was also increased by the macrophage culture fluid. However, this enhancement was best observed in conditions of relatively low T-cell activity. Also, the macrophage fluid allowed spleen cells of nude athymic mice to make a plaque-forming cell response to sheep red blood cells of both the IgM and IgG class. The macrophage was the cell source of the stimulatory molecule since it was generated only in cultures of macrophages devoid of significant number of lymphocytes. Stimulatory activity was not found in cultures of lymphocytes, mouse embryo cells, or 3T3 cells. The thymocyte stimulatory molecule did not contain H-2 antigens, was resistant to diisopropylfluorophosphate treatment, eluted from Sephadex with a size ranging from 15,000 to 21,000 daltons, and was sensitive to chymotrypsin and pepsin.