Effects of the lipophilic anticancer drug teniposide (VM-26) on membrane transport.

The epipodophyllotoxin glucopyranosides have previously been shown to interact with membrane lipids and to alter the activity of several lipid-embedded membrane proteins. To determine if these agents are acting as general membrane perturbants, we have further examined their effects on membrane processes in Ehrlich ascites tumor cells. [3H]VM-26 and [3H]VP-16 were taken up rapidly and concentrated within the cells in proportion to their lipophilicity. Neither agent was found to have any significant effect on the influx of L-[3H]leucine or alpha-[3H]aminoisobutyric acid. Likewise, these drugs had no significant effects on the hexose transporter. The nucleoside transporter, which is structurally and functionally similar to the hexose transporter, was dramatically affected, however. VM-26 was a non-competitive inhibitor of equilibrium-exchange influx of cytosine arabinoside in Ehrlich cells with a Ki of 15 microM. Equilibrium-exchange influx increased with temperature in control cells (Q10 = 2) but not in VM-26-treated cells; thus, VM-26 was a more potent inhibitor at higher temperatures. VM-26 also significantly reduced zero-trans influx in Ehrlich, P388, L5178Y, and ML-1 cells, and these effects were immediate in onset. VM-26 inhibited high-affinity binding of the nucleoside transport inhibitor nitrobenzylmercaptopurine riboside (NBMPR), but VM-26 enhanced non-specific NBMPR binding to Ehrlich cells. The apparent specificity of the epipodophyllotoxins for the nucleoside transporter is discussed.

Role of uridine triphosphate in the phosphorylation of 1-beta-D-arabinofuranosylcytosine by Ehrlich ascites tumor cells.

Pyrimidine nucleotide pools were investigated as determinants of the rate of phosphorylation of 1-beta-D-arabinofuranosylcytosine (ara-C) by Ehrlich ascites cells and cell extracts. Cells were preincubated for 2 h with 10 microM pyrazofurin, 10 mM glucosamine, 50 microM 3-deazauridine, or 1 mM uridine in order to alter the concentrations of pyrimidine nucleotides. Samples of the cell suspensions were taken for assay of adenosine 5'-triphosphate (ATP), uridine 5'-triphosphate (UTP), cytidine 5'-triphosphate, guanosine 5'-triphosphate, deoxycytidine 5'-triphosphate (dCTP), and deoxythymidine 5'-triphosphate; then 1 microM [3H]ara-C was added and its rate of intracellular uptake was measured for 30 min. 3-Deazauridine lowered dCTP and stimulated ara-C uptake; however, pyrazofurin and glucosamine were potent inhibitors of ara-C uptake although they also decreased dCTP levels. Uridine stimulated ara-C uptake despite an increase in dCTP. A crude cytoplasmic extract was prepared by a procedure which permitted results of ara-C kinase assays to be expressed as pmol per min per 10(6) cells as in the cellular uptake studies. When assayed in the presence of mixtures of ribo- and deoxyribonucleoside triphosphates at concentrations close to their cellular levels, ara-C kinase activity closely approximated the cellular uptake rate for the five incubation conditions. Deletion of cytidine 5'-, guanosine 5'-, or deoxythymidine 5'-triphosphate from the assay mixture had little effect, while deletion of dCTP increased kinase activity 9-fold. Elimination of ATP also did not alter kinase activity in the presence of the remaining five ribo- and deoxyribonucleoside triphosphates; however, deletion of UTP reduced activity to 22% of the rate with the control mixture. When ara-C kinase was assayed with only 3 mM ATP, dCTP was a very potent inhibitor (50% inhibition concentration = 0.4 microM). Inhibition was complete at 10 microM dCTP, a concentration below the intracellular dCTP level in control cells (25 microM). With 0.9 mM UTP, enzyme activity was 2-fold greater in the absence of dCTP and the dCTP was 15-fold less potent as an inhibitor (50% inhibition concentration = 6 microM). We conclude that the actual phosphate donor for the phosphorylation of 1 microM ara-C in Ehrlich cells is UTP and not ATP. These observations suggest that successful combination protocols aimed at stimulating ara-C uptake by means of a decrease in dCTP levels must simultaneously preserve or increase UTP pools.

Inhibition of 1-beta-D-arabinofuranosylcytosine transport and net accumulation by teniposide and etoposide in Ehrlich ascites cells and human leukemic blasts.

The interactions of the epipodophyllotoxins, teniposide (VM-26) and etoposide (VP-16), with the nucleoside carrier were examined with emphasis on their effects on 1-beta-D-arabinofuranosylcytosine (ara-C) transport and net accumulation. VM-26 inhibited ara-C transport by Ehrlich ascites cells within 1 min of exposure, and inhibition was only partially reversed after 45 min in VM-26-free medium. ara-C transport was slowed by 50% by 7 microM VM-26 or by 35 microM VP-16. Since epipodophyllotoxins were noncompetitive inhibitors, fractional inhibition was independent of the ara-C concentration. Analysis of ara-C transport kinetics revealed only a single saturable transport route, and there was no indication of VM-26-insensitive transport. VM-26, VP-16, and ara-C were competitive inhibitors of the specific binding of nitrobenzylthioinosine to the nucleoside carrier with Ki values of 7.4 microM, 23 microM, and 2.2 microM, respectively. The rate of dissociation of nitrobenzylthioinosine (t 1/2 = 20.6 min) was accelerated by 5 microM ara-C (t 1/2 = 18.5 min) but slowed by 100 microM VM-26 (t 1/2 = 34.6). By these criteria, the interaction of VM-26 with the nucleoside carrier was qualitatively similar to that of dipyridamole. Although VM-26 inhibited ara-C transport, it did not significantly slow the rate of net intracellular accumulation of ara-C by Ehrlich cells, presumably because transport capacity far exceeds the capacity for phosphorylation in these cells. In freshly isolated human leukemic blasts, which have far less nucleoside transport activity, inhibition of ara-C accumulation by VM-26 was dependent on the ara-C concentration. At 1 microM ara-C, a concentration where transport was rate limiting for net uptake, VM-26 inhibited accumulation of ara-C over a 60-min time course. At 50 microM ara-C, transport was in excess, and VM-26 did not slow ara-C metabolism.

Binding of radiolabeled N-(phosphonacetyl)-L-aspartate to aspartate transcarbamylase from Ehrlich ascites tumor cells.

Binding of N-(phosphonacetyl)-[3H]L-aspartate (PALA) to aspartate transcarbamylase (ATCase, EC 2.1.3.1) in crude extracts from Ehrlich ascites tumor cells was examined. At pH 7.4, the dissociation constant was 1.39 +/- 0.22 nM; the maximal binding capacity indicated an average intracellular ATCase concentration of 0.13 microM. The presence of phosphate, MgCl2, or CaCl2 increased the apparent dissociation constant for [3H]PALA without altering the maximal binding capacity. Phosphate, a product of the ATCase reaction, probably acts as a competitor for the PALA binding site; Mg2+ and Ca2+ may inhibit [3H]PALA binding by forming a chelate which reduces the effective concentration of the free [3H]PALA. Carbamyl phosphate was a relatively weak inhibitor of [3H]PALA binding in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer alone. Addition of NaF, an inhibitor of nonspecific phosphatases, decreased the carbamyl phosphate "Ki" as an inhibitor of [3H]PALA binding to 7 microM, a value close to the Km. NaF appears to act as an inhibitor of carbamyl phosphatase activity present in the cell extract. A first-order dissociation rate constant of 0.050 +/- 0.004 min-1 (T1/2 = 14 min) was determined by following displacement of [3H]PALA with excess unlabeled PALA. The dissociation rate was strongly temperature dependent. A second-order rate constant of 3.6 X 10(7) liters mol-1 min-1 was calculated from this rate constant and the dissociation constant. Using these kinetic constants, a simple computer model predicted that PALA binding to ATCase is 95% complete within 14 min under the conditions of the assay; at intracellular ATCase concentrations, binding is slightly faster. These results are discussed in the context of both the kinetics of inhibition and the reversal of inhibition of pyrimidine synthesis within the intact cell.

Role of endocytosis and lysosomal pH in uptake of N-(phosphonacetyl)-L-aspartate and its inhibition of pyrimidine synthesis.

The mechanism of uptake and retention of N-(phosphonacetyl)-L-aspartate (PALA) was examined. Uptake of [3H]PALA by Ehrlich ascites tumor cells appeared to be biphasic. A small, variable quantity of PALA associated with cells within 5 min; the significance of this rapid uptake component is unclear. Between 15 min and 5 h, uptake was linear and consistent from experiment to experiment. The properties of the slow phase of PALA uptake are consistent with fluid-phase endocytosis. The intracellular PALA concentration approached the extracellular level very slowly, at a rate of approximately 1%/hr. The velocity of PALA uptake in these cells was proportional to the concentration in the media from 10(-6) to 10(-2) M. Uptake of PALA was identical to that of the extracellular marker inulin. Uptake of both PALA and inulin was inhibited by colchicine and stimulated by phorbol myristate acetate. The microtubule antagonist and the phorbol ester are known to, respectively, inhibit and stimulate endocytosis in other cell types. Phorbol myristate acetate enhanced the ability of PALA to inhibit incorporation of [14C]bicarbonate into pyrimidine nucleotides, presumably through an increase in PALA uptake. This inhibitory action of PALA was almost completely blocked by two agents known to neutralize lysosomal pH, NH4Cl and methylamine. These results suggest that intracellular PALA is initially compartmentalized in a pinosomal vesicle which may later fuse with cellular lysosomes. Neutralization of lysosomal pH prevents the protonation of some or all of the four negatively charged groups found in the structure of PALA which may be necessary for its diffusion across the lysosomal membrane and eventual inhibition of aspartate transcarbamylase in the cytoplasm. Since partitioning of the fully charged molecule into the lipid phase of the plasma membrane for diffusion out of the cell should be minimal, the effects of PALA on cellular metabolism are expected to be prolonged.

10 years' experience treating pancreatic and periampullary cancer.

The records of 136 patients with periampullary and pancreatic carcinoma were reviewed and the information compared with other reported series. The clinical presentation with jaundice without other manifestations is associated with the greatest number of potentially curable tumors. The majority of patients were treated by palliative bypass or had exploration and biopsy only. A tissue diagnosis is not imperative before radical excision, providing a systematic preoperative and operative evaluation indicates tumor. Ligation of the pancreatic duct with external drainage results in low morbidity and mortality and good functional results. Radical pancreaticoduodenectomy done in 21 per cent of our patients offers the best palliation and the only hope for cure.