Gerald J. Goldenberg

Transport of Nitrogen Mustard on the Transport-Carrier for Choline in L5178Y Lymphoblasts

The uptake of choline by L5178Y lymphoblasts occurs by a carrier mechanism and is an active process. Transport of nitrogen mustard and its hydrolyzed derivative is mediated by this same carrier. This finding is an example of drug transport by a carrier whose primary function is transport of a naturally occurring substrate.

Anatagonism of the cytocidal activity and uptake of melphalan by tamoxifen in human breast cancer cells in vitro

The effect of the antiestrogen tamoxifen on the cytocidal activity and uptake of melphalan in human breast cancer cells was investigated. A clonogenic assay was used to obtain dose-survival curves of estrogen receptor-positive MCF-7 cells and of estrogen receptor-negative Evsa T cells following treatment with melphalan and/or tamoxifen. Isobolograms derived from these dose-survival curves were concave downward, suggesting that the drug interaction was antagonistic. The effect of tamoxifen on melphalan uptake by breast cancer cells was evaluated at steady-state conditions. Thin-layer chromatography revealed that the intracellular level of free intact melphalan (mean ± S.E.) in control cells was 6.47 ± 1.21 fmoles/cell and that in cells treated with tamoxifen was 3.60 ± 0.35 fmoles/cell; this 44% reduction in cellular melphalan was statistically significant (P = 0.006). Thus, the antagonistic cytocidal effect of melphalan and tamoxifen against breast cancer cells appeared to be due to inhibition of melphalan uptake at the steady state by the antiestrogen. Further investigation revealed that tamoxifen inhibited unidirectional melphalan influx in human breast cancer cells both by the sodium-independent system L and by the sodium-dependent system ASC. Tamoxifen also appeared to stimulate melphalan efflux from human breast cancer cells. The first-order rate constant K for melphalan efflux from control cells was 0.085 ± 0.008 and that from cells treated with tamoxifen was 0.129 ± 0.005; the difference was highly significant (P < 0.001). Therefore, the antagonistic effect of tamoxifen on the uptake and cytocidal activity of melphalan in breast cancer cells appeared to be due to inhibition of melphalan influx and stimulation of drug efflux.

Membrane transport, sulfhydryl levels and DNA cross-linking in Chinese hamster ovary cell mutants sensitive and resistant to melphalan☆

The mechanism of resistance to the alkylating agent melphalan was investigated in drug-sensitive and -resistant mutants of Chinese hamster ovary cells. Melphalan-resistant cells (MelR6), selected by a single exposure to melphalan, were 4.5-fold more resistant to drug than sensitive AUXB1 parental cells. Colchicine-resistant cells (CHRC5), which are cross-resistant to melphalan, were 15-fold more resistant than wild type cells. The kinetic parameters for drug influx were not significantly different in sensitive and resistant cells. The steady-state drug level in both MelR6 and CHRC5 cells was approximately 25 and 35% lower respectively than that in sensitive cells and this difference was accounted for by a more rapid rate of drug efflux from the resistant mutants. However, the level of drug resistance could not be explained entirely by this difference in drug transport. Sulfyddryl group levels were elevated in both MelR6 and CHRC5 cells relative to sensitive cells and these differences were statistically significant (P < 0.001). Furthermore, DNA interstrand cross-link formation was significantly lower in resistant cells than in sensitive cells. A similar rate of repair of DNA interstrand cross-links was observed in sensitive and resistant cells with the possible exception of a slower rate of repair in MelR6 cells. A higher level of DNA-protein cross-link activity, which may represent a mechanism for drug inactivation was observed in MelR6 cells. These studies suggest that resistance to melphalan in MelR6 and CHRC5 Chinese hamster ovary cell mutants is multifactorial involving lowered steady-state drug levels, enhanced drug efflux, elevated levels of sulfhydryl groups and decreased DNA interstrand cross-linking.

The effect of trypsin and neuraminidase on the circulation and organ distribution of tumor cells

The effect of trypsin and neuraminidase on the circulation and organ distribution of 51Cr‐labeled normal lymphocytes, Murphy‐Sturm lymphosarcoma, and Walker 256 carcinosarcoma cells was investigated in Wistar male rats. Radioactivity in various organs and blood was measured up to 72 hours after intravenous injection of the three cell lines. Trypsin treatment was followed by increased localization of both tumor cell lines to the spleen and peripheral blood and decreased retention in the lung at early time intervals; trypsin treatment also reduced hepatic uptake of lymphosarcoma but not that of carcinoma cells. Neuraminidase treatment was associated with increased localization of normal lymphocytes and both tumor cell lines to the liver, decreased retention of carcinoma cells in the lung, and fewer lymphosarcoma cells in the blood. The findings suggest that the organ distribution of tumor cells and normal lymphocytes may be related to cell surface constituents which are susceptible to modification by enzymatic treatment.

PROPERTIES OF L5178Y LYMPHOBLASTS HIGHLY RESISTANT TO NITROGEN MUSTARD

The distinct possibility exists that there is no single mode of action for alkylating agents because of their capacity to react with several nucleophilic groups in the A technique that has proven useful in investigating mechanisms of action of other chemotherapeutic agents, particularly the antimetabolites, has been the comparison of physical and chemical properties of two closely related cell lines differing only with respect to sensitivity to a particular Therefore, the object of this study was to obtain a cell line markedly resistant to nitrogen mustard that might serve as a model for the investigation of the mechanism of action of that drug by comparing the properties of sensitive and resistant cells.

Uptake and decomposition of chlorambucil by L5178Y lymphoblasts in vitro

The uptake of [14C]chlorambucil by L5178Y lymphoblasts was studied using thin-layer chromatography to identify the various radioactive components that enter or leave cells. Theoretical calculations predicted that entry of chlorambucil into cells by simple diffusion would be rapid and essentially complete in 45 sec or less. Uptake of intact chlorambucil was rapid, reaching a cell/medium ratio of approximately 1.5 in less than 15 sec at both 37 degrees and 4 degrees, consistent with a simple diffusion mechanism. In cells treated with [14C]chlorambucil for 60 min, the intracellular level of intact drug decreased with time, and this decay was attributed to hydrolysis and alkylation. The level of intact drug in the medium decreased at a similar rate resulting in a nearly constant cell/medium distribution ratio. Intact chlorambucil in the cells was found to be entirely ethanol- and trichloroacetic acid-soluble. Efflux of intact chlorambucil was very rapid and temperature-insensitive. These findings suggest that chlorambucil efflux, as well as influx, is by a simple diffusion mechanism. A derivative of chlorambucil was found in ethanol solutions of the drug. This derivative, which may be the ethyl ester of the drug, is highly concentrated in cells and may interfere with pharmacological investigations of chlorambucil.

A comparison of melphalan transport in human breast cancer cells and lymphocytes in vitro

Evidence is presented that melphalan uptake by MCF-7 human breast cancer cells and peripheral blood lymphocytes from normal human subjects is an active process involving 2 amino acid carriers. Quantitative differences were observed in drug transport between the 2 cell types. Cell/medium distribution ratios of melphalan at equilibrium conditions were approx. 4-fold higher in breast cancer cells than in lymphocytes. The transport capacity, Vmax, for both carrier systems was at least 50-fold greater in MCF-7 cells than in lymphocytes, whereas the Michaelis constants were similar. These findings, in part, provide a rational basis for the use of melphalan in the chemotherapy of breast cancer.

Mechanisms of resistance to chlorambucil in chronic lymphocytic leukemia

The postulated biochemical mechanisms responsible for clinical resistance to chlorambucil (CLB) in chronic lymphocytic leukemia (CLL) have been examined. The total sulfhydryl, non-protein-bound sulfhydryl, protein-bound sulfhydryl (PSH) and glutathione (GSH) levels, in addition to glutathione S-transferase (GST) activities, were measured in the leukemic cells of 18 CLL patients. In addition, the formation and repair of DNA cross-links were measured following incubation of the cells with 100 microM chlorambucil in vitro. These parameters were then correlated with the subsequent clinical responses of the patients, as measured by the percent fall in lymphocyte count 3 weeks following 0.9 mg/kg chlorambucil. No correlations were observed between any of the individual parameters and clinical response, although a slight positive correlation was observed between the PSH:GSH ratio and clinical response. These findings suggest that multiple mechanisms may contribute to CLB-resistance in CLL.

Induction of DNA single- and double-strand breaks by diethylstilbestrol in murine L5178Y lymphoblasts in vitro☆

The mechanism of action of the synthetic estrogen diethylstilbestrol (DES) was investigated in murine L5178Y lymphoblasts. The dose-survival curve of cells treated with DES in serum-free medium for 1 hr was characterized by a prominent shoulder followed by a simple exponential decline; the Do, the dose of DES reducing cell survival to 1/e, was 1.52 nmoles/ml. DNA single-strand breaks, as measured by the alkaline elution method, were observed in DES-treated cells, and these followed a dose-response relationship after an apparent threshold of 10 microM DES was exceeded. Protein-associated strand breaks, which represent the increment in single-strand breaks that occurs by exposing drug-treated cells to proteinase K, were also noted. DNA double-strand breaks as measured by filter elution technology at pH 9.6 were observed and increased markedly to reach a level of approximately 9000 rad equivalents at a DES concentration of 20 microM. The measured ratio (mean +/- S.E.) of single- to double-strand breaks induced by DES in L5178Y limphoblasts was 0.09 +/- 0.035. A comparison of the ratio of single- to double-strand breaks induced by DES to that observed following radiation suggested that all of the single-strand breaks produced by DES could be attributed to double-strand breaks. The close correspondence of the dose-response curve for cytocidal activity of DES with that obtained for induction of DNA double-strand breaks suggested that such breaks may play an important role in the mechanism of cell kill by DES.

Modulation of membrane transport of alkylating agents and amino acids by an analog of vasopressin in murine L5178Y lymphoblasts in vitro

The synthetic vasopressin analog 1-deamino-8-D-arginine vasopressin (dDAVP) has been shown to influence a wide range of cell-membrane-related events. Accordingly, the effect of dDAVP on membrane transport of various alkylating agents and amino acids was evaluated in L5178Y lymphoblasts in vitro. dDAVP stimulated melphalan uptake but conversely inhibited uptake of nitrogen mustard, choline (the natural transport substrate for the nitrogen mustard carrier), and leucine. No effect on the uptake of cyclophosphamide or glutamine was observed. Increased melphalan uptake was due to effects on both substrate influx and efflux. The effect of dDAVP on melphalan influx was particularly complex: dDAVP stimulated melphalan influx by amino acid transport system ASC but inhibited influx by system L, resulting in a net increase in unidirectional drug influx. Melphalan efflux was inhibited by dDAVP. Decreased uptake of nitrogen mustard, choline and leucine was due, at least in part, to decreased substrate influx. However, the mechanisms of inhibition were dissimilar: inhibition of substrate influx was non-competitive for choline but competitive for leucine. In conclusion, dDAVP induced diverse but apparently specific effects on membrane transport of several alkylating agents and amino acids. Since the accumulation of alkylating agents such as melphalan within tumor cells is a major determinant of cytotoxicity, dDAVP may have a role as a biological response modifier.