Mutant prion proteins in Gerstmann-Sträussler-Scheinker disease with neurofibrillary tangles.

Two families with Gerstmann-Sträussler-Scheinker disease (GSS) are atypical in possessing neocortical neurofibrillary tangles (NFTs), which are few or absent in other kindreds with GSS, in addition to amyloid plaques that react with prion protein (PrP) antibodies and protease-resistant PrP accumulation in the brain. A leucine substitution at PrP codon 102 has been genetically linked to GSS in some families. We examined the PrP gene in these families. A serine for phenylalanine substitution was found at codon 198 in the Indiana patients; arginine for glutamine substitution at codon 217 in the Swedish patients. These mutations in PrP are the first to be associated with the appearance of both PrP amyloid plaques and neocortical NFTs in GSS patients.

Cloning of a human nucleoside transporter implicated in the cellular uptake of adenosine and chemotherapeutic drugs.

In most mammalian cells nucleoside uptake occurs primarily via broad-specificity, es (e, equilibrative; 5, sensitive to NBMPR inhibition) transporters that are potently inhibited by nitrobenzylthioinosine (NBMPR). These transporters are essential for nucleotide synthesis by salvage pathways in hemopoietic and other cells that lack de novo pathways and are the route of cellular uptake for many cytotoxic nucleosides used in cancer and viral chemotherapy. They play an important role in adenosine-mediated regulation of many physiological processes, including neurotransmission and platelet aggregation, and are a target for coronary vasodilator drugs. We have previously reported the purification of the prototypic es transporter from human erythrocytes and have shown that this glycoprotein of apparent M, 55,000 is immunologically related to nucleoside transporters from several other species and tissues, including human placenta. Here we report the isolation of a human placental cDNA encoding a 456-residue glycoprotein with functional characteristics typical of an es-type transporter. It is predicted to possess 11 membrane-spanning regions and is homologous to several proteins of unknown function in yeast, nematodes, plants and mammals. Because of its central role in the uptake both of adenosine and of chemotherapeutic nucleosides, study of this protein should not only provide insights into the physiological roles of nucleoside transport but also open the way to improved therapies.

Human equilibrative nucleoside transporter 1 (hENT1) protein is associated with short survival in resected ampullary cancer.

BACKGROUND: Gemcitabine is an acceptable alternative to best supportive care in the treatment of advanced biliary tract cancers. The human equilibrative nucleoside transporter 1 (hENT1) is a ubiquitous protein and is the major means by which gemcitabine enters human cells. Moreover, recent reports indicate a significant correlation between immunohistochemical variations of hENT1 in tumor samples and survival after gemcitabine therapy in patients with solid tumors. MATERIALS AND METHODS: We used immunohistochemistry to assess the abundance and distribution of hENT1 in tumor samples from radically resected cancer of the ampulla, and sought correlations between immunohistochemical results and clinical parameters including disease outcomes. RESULTS: In the 41 individual tumors studied, 12 (29.3%) had uniformly high hENT1 immunostaining. Statistical analysis showed a significant correlation between hENT1 and Ki-67 (P = 0.04). No statistical significant differences were found between immunohistochemical findings and patient characteristics (sex, age, and tumor-node-metastasis). On univariate analysis, hENT1 and Ki-67 expression were associated with overall survival (OS). Specifically, those patients with overexpression of hENT1 showed a shorter OS (P = 0.022) and those with high Ki-67 staining showed a shorter survival (P = 0.05). CONCLUSIONS: hENT1 expression is a molecular prognostic marker for patients with resected ampullary cancer and holds promise as a predictive factor to assist in chemotherapy decisions.

Human equilibrative nucleoside transporter (ENT) family of nucleoside and nucleobase transporter proteins.

1. The human (h) SLC29 family of integral membrane proteins is represented by four members, designated equilibrative nucleoside transporters (ENTs) because of the properties of the first-characterized family member, hENT1. They belong to the widely distributed eukaryotic ENT family of equilibrative and concentrative nucleoside/nucleobase transporter proteins. 2. A predicted topology of eleven transmembrane helices has been experimentally confirmed for hENT1. The best-characterized members of the family, hENT1 and hENT2, possess similar broad permeant selectivities for purine and pyrimidine nucleosides, but hENT2 also efficiently transports nucleobases. hENT3 has a similar broad permeant selectivity for nucleosides and nucleobases and appears to function in intracellular membranes, including lysosomes. 3. hENT4 is uniquely selective for adenosine, and also transports a variety of organic cations. hENT3 and hENT4 are pH sensitive, and optimally active under acidic conditions. ENTs, including those in parasitic protozoa, function in nucleoside and nucleobase uptake for salvage pathways of nucleotide synthesis and, in humans, are also responsible for the cellular uptake of nucleoside analogues used in the treatment of cancers and viral diseases. 4. By regulating the concentration of adenosine available to cell surface receptors, mammalian ENTs additionally influence physiological processes ranging from cardiovascular activity to neurotransmission.

Gemcitabine transport in xenopus oocytes expressing recombinant plasma membrane mammalian nucleoside transporters.

BACKGROUND: Gemcitabine, a pyrimidine analogue of deoxycytidine, is an anticancer nucleoside drug that requires functional plasma membrane nucleoside transporter proteins to reach its intracellular targets and cause cytotoxicity. Because of technical difficulties inherent in studying nucleoside transport in human cells, we rigorously defined gemcitabine membrane transportability by producing each of the available human (h) and rat (r) recombinant nucleoside transporters (NTs) individually in Xenopus laevis oocytes. METHODS: Oocytes were microinjected with in vitro-transcribed RNAs derived from complementary DNAs encoding (C = concentrative) rCNT1, rCNT2, hCNT1, hCNT2, (E = equilibrative) rENT1, rENT2, hENT1, and hENT2. Uptake of [(3)H]gemcitabine and [(14)C] uridine was measured 3 days after microinjection to determine kinetic constants. We also used the two-electrode, voltage-clamp technique to investigate the electrophysiology of hCNT1-mediated gemcitabine transport. RESULTS: Gemcitabine was transported by most of the tested proteins (the exceptions being the purine-selective rCNT2 and hCNT2), with the greatest uptake occurring in oocytes producing recombinant rCNT1 and hCNT1. Influxes of gemcitabine mediated by hCNT1, hENT1, and hENT2 were saturable and conformed to Michaelis-Menten kinetics with apparent K(m) values of 24, 160, and 740 microM, respectively. Gemcitabine had a limited ability to cross the lipid bilayer of oocyte membranes by simple diffusion. External application of gemcitabine to oocytes producing recombinant hCNT1 induced an inward current, which demonstrated that hCNT1 functions as a Na(+)/nucleoside co-transport protein and confirmed the transporter's ability to transport gemcitabine. CONCLUSIONS: Mammalian nucleoside transporters vary widely in their affinity and capacity to transport gemcitabine. Variation in the tumor and tissue distribution of plasma membrane nucleoside transporter proteins may contribute to the solid tumor activities and schedule-dependent toxic effects of gemcitabine.

Inhibition of methotrexate-induced differentiation of cultured human choriocarcinoma (BeWo) cells by thymidine.

During exposure to methotrexate, cultured human choriocarcinoma (BeWo) cells stop proliferating, enlarge, and undergo a complex differentiative response that resembles in utero development of quiescent syncytiotrophoblasts. In the present work, complete inhibition of proliferation and maximal cell enlargement required exposure to 1 microM methotrexate, whereas colony-forming ability, determined after transfer of cells to drug-free medium, was unaffected over a wide range of concentrations (10(-12)-10(-5) M). BeWo cells were sensitive to the antifolate effects of methotrexate since thymidylate synthase activity and incorporation of [14C]formate into DNA, RNA, and protein were reduced by greater than 90% after short drug exposures, and progression of cells through S phase of the cell cycle was blocked by prolonged drug exposures. When methotrexate was coadministered with hypoxanthine and thymidine or leucovorin, its antiproliferative and differentiative effects were blocked. When methotrexate was coadministered with either hypoxanthine or thymidine, its antiproliferative activity was unaffected, whereas expression of syncytiotrophoblastic markers was blocked in the presence of thymidine but not in the presence of hypoxanthine. Exposure of BeWo cells to fluorodeoxyuridine also stimulated cell enlargement and expression of syncytiotrophoblastic markers, and these effects were blocked by coadministration of thymidine. Thus BeWo cells, which were sensitive to the antifolate effects of methotrexate, were not killed during cytostasis but instead entered a reversible differentiated state, apparently resulting from thymidylate starvation and consequent inhibition of DNA synthesis.

Differential cellular retention of vincristine and vinblastine by cultured human promyelocytic leukemia HL-60/Cl cells: the basis of differential toxicity.

Differential toxicity of vincristine and vinblastine against cells of a cloned subline of human promyelocytic leukemia (HL-60/Cl) was dependent on exposure conditions. During continuous exposures of 48 h, vincristine and vinblastine were equitoxic with drug concentrations that inhibited proliferation rates by 50% of 7.6 and 8.1 nM, respectively. When cells were subjected to 4-h exposures and transferred to drug-free medium, the drug concentration of vinblastine that inhibited proliferation rates by 50% (1.1 microM) was significantly greater than that of vincristine (41 nM). Analysis by flow cytometry of the effects of equitoxic drug exposures on cell-cycle progression suggested that vincristine and vinblastine acted by the same mechanism (G2-M phase inhibition). [3H]Vincristine and [3H]vinblastine were bound to serum proteins in growth medium to the same extent (25%) over a wide range of concentrations, and the amounts of "free" extracellular drug did not decrease during prolonged exposures. Analysis by high-performance liquid chromatography of extracts of cultures incubated with growth-inhibitory concentrations of [3H]vincristine or [3H]vinblastine indicated little, if any, metabolism of either drug by cells or culture fluids; after 24 h, 85-95% of radioactivity was recovered from cells or growth medium as unchanged vincristine or vinblastine. At concentrations from 6 nM to 6 microM, vinblastine entered cells rapidly, reaching maximum levels within 0.5-2 h, and the relationship between maximal cell-associated drug and extracellular free vinblastine was linear. Although uptake of vincristine was slower than that of vinblastine, the cellular content of vincristine reached that of vinblastine during prolonged (12-24 h) exposures, and the amounts of cell-associated drug, relative to extracellular drug concentrations, indicated considerable "concentrative" accumulation (intra: extracellular ratios, greater than 100). When drug exposures were ended by transfer of cells to drug-free medium, vinblastine was released from cells more rapidly and to a greater extent than vincristine, independent of whether exposures were 4 or 24 h. Rates of uptake and release of vinblastine (50 nM) were unaffected by depletion of cellular adenosine triphosphate, suggesting that rapid release was not mediated by an energy-dependent efflux system.

Resistance to 9-beta-D-arabinofuranosyladenine in cultured leukemia L 1210 cells.

A cultured line of L1210 leukemia cells, designated L1210/ara-A, was selected for resistance to 9-beta-D-arabinofuranosyladenine (ara-A) by a series of 72-hr exposures to increasing concentrations of ara-A in the presence of 1 microM deoxycoformycin. Cells of the resistant line were about one-tenth as sensitive as were cells of the parent line to the effects of ara-A on proliferation, viability, and tumorigenicity. Cross-resistance, as determined by comparison of drug effects on rates of proliferation of L1210/C2 and L1210/ara-A cells, was seen with adenosine, deoxyadenosine, methylmercaptopurine ribonucleoside, tubercidin, and cordycepin but not with 1-beta-D-arabinofuranosylcytosine or with 9-beta-D-arabinofuranosyl-2-fluoroadenine. The levels of resistance to methylmercaptopurine ribonucleoside, cordycepin, and tubercidin were considerably greater than that seen with ara-A itself. L1210/C2 and L1210/ara-A cells were compared with respect to the effects of ara-A on cell size distributions, DNA distributions, labeling indices, and apparent rates of DNA synthesis, and the differences seen were consistent with inhibition of DNA synthesis and unbalanced growth as the major mechanism of ara-A cytotoxicity. The decreased sensitivity of DNA synthesis in L1210/ara-A cells treated with ara-A, relative to L1210/C2 cells, was due to reduced intracellular accumulation of ara-A phosphates in the resistant line. Phosphorylation of ara-A, adenosine, and tubercidin, but not deoxyadenosine or deoxycytidine, was greatly reduced in intact L1210/ara-A cells, relative to L1210/C2 cells, and adenosine kinase activity in extracts of L1210/ara-A cells was negligible. Resistance to ara-A, and cross-resistance to tubercidin, methylmercaptopurine ribonucleoside, and cordycepin is attributed to loss of adenosine kinase activity.

Antiproliferative effects of 9-beta-D-arabinofuranosyladenine 5'-monophosphate and related compounds in combination with adenosine deaminase inhibitors against mouse leukemia L1210/C2 cells in culture.

The antiproliferative activity of 9-beta-D-arabinofuranosyladenine 5'-monophosphate against a cultured line of mouse leukemia cells (L1210/C2) was enhanced by addition of either 2'-deoxycoformycin or erythro-9-(2-hydroxy-3-nonyl)adenine. The activity of 9-beta-D-arabinofuranosyladenine 5'-monophosphate, alone or in combination with either of the two inhibitors of adenosine deaminase, was comparable to that of 9-beta-D-arabinofuranosyladenine (ara-A), apparently reflecting the rapid conversion of 9-beta-D-arabinofuranosyladenine 5'-monophosphate to ara-A by L1210/C2 cells. Several ara-A analogs were assayed for antiproliferative activity against L1210/C2 cells; of these, only 9-beta-D-arabinofuranosyladenine 5'-O-methylphosphate and 2'-deoxy-2'-amino-9-beta-D-arabinofuraosyladenine were active. Addition of 2'-deoxycoformycin to cell culture fluids enhanced the activity of 9-beta-D-arabinofuranosyladenine 5'-O-methylphosphate suggesting conversion to an adenosine deaminase-sensitive intermediate, presumably ara-A.

Combination therapy of mouse leukemia L1210 by 1-beta-D-arabinofuranosylcytosine and 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine.

Nitrobenzylthioinosine (NBMPR), an inhibitor of nucleoside transport, was tested in combination with 1-beta-D-arabinofuranosylcytosine (ara-C) for therapeutic activity against mouse leukemia L1210. NBMPR alone had no activity, whereas therapy with NBMPR and ara-C in combination was significantly better than with ara-C alone. The therapeutic potentiation resulting from the combination of NBMPR and ara-C appeared to be host mediated since NBMPR alone was not toxic to cultured L1210 cells. NBMPR treatment of normal mice increased the plasma half-time of ara-C and decreased rates of urinary excretion of ara-C and 2'-deoxycytidine; however, these effects were not large enough to explain the therapeutic potentiation. Because the drug combination appeared to be no more effective than ara-C alone in therapy of mouse leukemia L1210/TG (a thiopurine-resistant L1210 subline lacking hyposanthine-guanine phosphoribosyltransferase), the host-mediated therapeutic potentiation was attributed in in vivo breakdown of NBMPR to 6-mercaptopurine.

Initial rate kinetics of the transport of adenosine and 4-amino-7-(beta-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (tubercidin) in cultured cells.

A procedure is described for determining early time courses of nucleoside uptake by cultured cells in suspension. Replicate samples of cell suspensions were exposed to medium containing 3H-nucleosides for brief intervals (sec) ended by addition of nitrobenzylthioinosine, a potent inhibitor of nucleoside transport that terminated nucleoside uptake virtually instantaneously. Time courses of nucleoside uptake were constructed from the cellular content of nucleoside acquired by the replicate samples during graded intervals of exposure to the labeled permeant. Such time courses were definitive of cellular uptake of nucleosides during the first few sec of exposure to permeant and yielded initial rates of uptake of adenosine and 4-amino-7-(beta-d-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (tubercidin). Defining initial rates of nucleoside uptake as rates of inward transport, relationships between transport rates and extracellular concentrations of these permeants were evaluated in HeLa cells and in two cultured lines of mouse lymphoma L5178Y cells that differ in their abilities to phosphorylate adenosine and tubercidin. Transport rates for these permeants were similar in the two L5178Y cell types and were saturable in the 3 cell lines with Km values between 14 and 38 microM. Adenosine and tubercidin were mutually competitive permeants in L5178Y cells, indicating that they are substrates for the same transport mechanism.

Enhancement of 9-beta-d-arabinofuranosyladenine cytotoxicity to mouse leukemia L1210 in vitro by 2'-deoxycoformycin.

2'-Deoxycoformycin (2'-dCF), a potent inhibitor of adenosine deaminase, was tested in combination with 9-beta-D-arabinofuranosyladenine (ara-A) and 9-beta-D-arabinofuranosyladenine 5'-formate for cytotoxic activity against mouse leukemia L1210 in culture. 2'-dCF, which alone had no activity, significantly enhanced cytostatic and cytotoxic activities of ara-A and its more soluble derivative, 9-beta-D-arabinofuranosyladenine 5'-formate; the latter 2 agents, when tested at equimolar concentrations, were equivalent in their effects on proliferation and viability. The therapeutic response of mice bearing the in vitro line of L1210 cells (L1210/C2) to combination therapy with 2'-dCF and 9-beta-D-arabinofuranosyladenine 5'-phosphate was comparable to that reported elsewhere for therapy of mice bearing the parent in vivo line. Continuous exposure of cultured L1210 cells to ara-A and 2'-dCF induced a prolonged period of unbalanced growth, characterized by inhibition of proliferation and DNA synthesis while RNA and protein synthesis continued; exposure periods in excess of a single population doubling were required to achieve significant cell kill. Potentiation of ara-A activity against the relatively insensitive mouse leukemia L1210 was attributed to increased stability of ara-A resulting from 2'-dCF inhibition of adenosine deaminase.

A comparison of the abilities of nitrobenzylthioinosine, dilazep, and dipyridamole to protect human hematopoietic cells from 7-deazaadenosine (tubercidin).

Nitrobenzylthioinosine, dilazep, and dipyridamole are potent inhibitors of equilibrative transport of nucleosides that may have pharmacological applications in modulating the therapeutic index of nucleoside antimetabolites used in cancer chemotherapy. We have compared the relative abilities of these inhibitors to reduce the toxicity of in vitro exposures to tubercidin against clonogenic progenitor cells of normal human bone marrow (CFU-GEMM, BFU-E, CFU-GM) and of two leukemic human cell lines (HL-60/C1, CCRF-CEM) that differ in their expression of transporter subtypes. Short (1-h) exposures to 1 microM tubercidin alone inhibited colony formation (a) of normal human hematopoietic progenitors (CFU-GEMM, BFU-E, CFU-GM) by 100%, and (b) of HL-60/C1 and CCRF-CEM cells by > 90%. Pretreatment (30 min) with nitrobenzylthioinosine, dilazep, or dipyridamole followed by simultaneous treatment (1 h) with these transport inhibitors during tubercidin exposures reduced toxicity against hematopoietic progenitors and cell lines. Greater reductions of toxicity were consistently seen with bone marrow progenitors and CCRF-CEM cells than with HL-60/C1 cells. For CFU-GEMM, BFU-E, and CFU-GM cells, reductions in tubercidin toxicity of 50-100% were achieved at these concentrations: > or = 0.1 microM (nitrobenzylthioinosine); > or = 0.1 microM (dilazep); and > or = 3.0 microM (dipyridamole). Pretreatment (30 min) followed by simultaneous treatment (1 h) with any of the transport inhibitors (> or = 0.1 microM) and 0.1 microM [3H]-tubercidin blocked the uptake of radioactivity completely in CCRF-CEM cells and only partially in HL-60/C1 cells. These effects, which were consistent with the nucleoside transport phenotypes of CCRF-CEM cells (inhibitor-sensitive) and HL-60/C1 cells (inhibitor-sensitive and inhibitor-resistant), suggested that protection was due to the inhibition of tubercidin uptake via equilibrative nucleoside transport system(s). Light-density mononuclear cells from human bone marrow, of which the clonogenic progenitors represented only a minor (< 0.01%) subpopulation, possessed far fewer nitrobenzylthioinosine-binding sites (2 x 10(4) sites/cell, Kd = 0.7 nM) than either HL-60/C1 cells (1.7 x 10(5) sites/cell, Kd = 0.9 nM) or CCRF-CEM cells (3.3 x 10(5) sites/cell, Kd = 0.5 nM). Initial rates of uptake of 1 microM [3H]adenosine (0-6 s, 20 degrees C) by human bone marrow mononuclear cells were reduced partially by 0.1 microM inhibitor (nitrobenzylthioinosine > dipyridamole > dilazep) and completely by 10 microM inhibitor.(ABSTRACT TRUNCATED AT 400 WORDS)

Transport of uridine and 3-deazauridine in cultured human lymphoblastoid cells.

The transport of uridine and 3-deazauridine was compared in two lines of cultured human lymphoblastoid cells that differ in their sensitivity to 3-deazauridine apparently because of reduced uridine-cytidine kinase activity in the resistant line. The kinetic parameters (+/- S.E.) of uridine transport were similar in the two cell lines: Km, 0.23 +/- 0.02 and 0.25 +/- 0.07 mM; and Vmax, 35 +/- 2 and 57 +/- 10 pmol/microliter of cell water per sec, respectively, for 6410/MP (parental) and 6410/MP/DU (resistant) cells. 3-Deazauridine, while transported with similar kinetic characteristics in both cell lines, was not as good a substrate for the nucleoside transporter as was uridine, and its transport was dependent on pH. Kinetic parameters, determined using calculated concentrations of the undissociated form of 3-deazauridine (pKa, 6.5), were: Km, 0.52 +/- 0.01 and 0.51 +/- 0.03 mM; and Vmax, 28 +/- 0.5 and 24 +/- 0.9 pmol/microliter of cell water per sec, respectively, for 6410/MP and 6410/MP/DU cells. At pH 8, a condition in which 97% of 3-deazauridine molecules are ionized, rates of transport were almost zero. It is concluded that the undissociated form of 3-deazauridine is the substrate for the nucleoside transporter.

Biological effects of inhibition of guanine nucleotide synthesis by mycophenolic acid in cultured neuroblastoma cells.

Mycophenolic acid, an inhibitor of inosinate dehydrogenase, had cytostatic and cytotoxic effects on cultured neuroblastoma cells. Proliferation was inhibited by 50% when cells were incubated with 0.07 micrometerM mycophenolic acid, and cell viability was reduced by 83% when cells were treated with 10 micrometerM mycophenolic acid for 24 hr. Treatment of monolayer cultures with mycophenolic acid reduced intracellular concentrations of guanosine triphosphate by 70% within 3 hr, whereas cytidine triphosphate and uridine triphosphate concentrations were significantly elevated, and adenosine triphosphate concentrations were increased only slightly. Reduction of cellular guanine nucleotides had differential effects on rates of macromolecular synthesis: incorporation of radioactive thymidine into acid-insoluble material was inhibited by mycophenolic acid to a much greater extent than was that of adenosine and leucine. Although proliferation of neuroblastoma cells was inhibited, differentiation, as judged by formation of neuronlike processes in serum-free medium, was unaffected by decreased intracellular concentrations of guanosine triphosphate.

Differential activity of vincristine and vinblastine against cultured cells.

Vincristine and vinblastine exhibit differential activity against tumors and normal tissues. In this work, a number of cultured cell lines were assayed for their sensitivity to the antiproliferative and cytotoxic effects of the two drugs following short-term (4 hr) or during continuous exposures. Differential activity was not seen when cells were subjected to continuous exposures. The concentrations of vincristine and vinblastine, respectively, that inhibited growth rates by 50% were: mouse leukemia L1210 cells, 4.4 and 4.0 nM; mouse lymphoma S49 cells, 5 and 3.5 nM; mouse neuroblastoma cells, 33 and 15 nM; HeLa cells, 1.4 and 2.6 nM; and human leukemia HL-60 cells, 4.1 and 5.3 nM. In contrast, differential toxicity was seen when cells were subjected to 4-hr exposures and transferred to drug-free medium: the 50% growth-inhibitory concentrations for vincristine and vinblastine, respectively, for inhibition (a) of proliferation of L1210 cells were 100 and 380 nM and of HL-60 cells were 23 and 900 nM and (b) of colony formation of L1210 cells were 6 and greater than 600 nM and of HeLa cells were 33 and 62 nM. Uptake and release of [3H]-vincristine and [3H]vinblastine were examined in L1210 cells under the conditions of growth experiments. Uptake of both drugs was dependent on the pH of culture media, and significantly greater amounts of [3H]vinblastine than of [3H]vincristine were associated with cells after 4-hr exposures to equal concentrations of either drug. When cells were transferred to drug-free medium after 4-hr exposures, vinblastine was released much more rapidly from cells than was vincristine, and by 0.5 hr after resuspension of cells, the amount of vincristine associated with the cells was greater than the amount of vinblastine and remained so for up to at least 6 hr.

Mouse transporter protein, a membrane protein that regulates cellular multidrug resistance, is localized to lysosomes.

Mouse transporter protein (MTP), a small, highly conserved mammalian intracellular membrane protein with four putative transmembrane domains, has been implicated in the transport of nucleosides and/or related molecules across intracellular membranes. The production of recombinant MTP in Saccharomyces cerevisiae alters sensitivity of yeast cells to a heterogeneous group of compounds (e.g., antimetabolites, antibiotics, anthracyclines, ionophores, and steroid hormones) by changing the subcellular compartmentalization of these drugs, suggesting that MTP functions similarly in higher organisms. The present study was undertaken to define the intracellular location of MTP in mammalian cells. Native MTP was not detected by indirect immunofluorescence in cell types that expressed MTP mRNA; therefore, a hemagglutinin (HA) epitope-tagged version of MTP was produced in cultured BHK21 cells by transient transfection, and its distribution within cells was determined by confocal microscopy using antibodies directed against the HA epitope and various organellar proteins. Antibodies directed against HA-MTP colocalized with antibodies against late endosomal and lysosomal proteins but not with antibodies against either Golgi or early endosomal proteins. Analysis of subcellular fractions from rat liver by immunoblotting with antibodies directed against MTP demonstrated the presence of a MTP-like protein in Golgi- and lysosome-enriched membranes but not in mitochondria. These results indicate that MTP resides in late endosomes and lysosomes, a finding that is consistent with the proposed role for MTP in the movement of a variety of small molecules across endosomal and lysosomal membranes. MTP shares a number of characteristics with other lysosome-associated proteins. We, therefore, propose that it be redesignated murine lysosome-associated protein transmembrane 4.

Fluctuations in nucleoside uptake and binding of the inhibitor of nucleoside transport, nitrobenzylthioinosine, during the replication cycle of HeLa cells.

Binding of the potent nucleoside transport inhibitor 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine (NBMPR) and rates of uptake of several nucleosides were examined at 4-hr intervals during the replication cycle of HeLa S3 cells. Monolayer cultures of synchronous cells, obtained by mitotic detachment, were assayed for high-affinity binding of NBMPR and for rates of uptake of thymidine, uridine, cytidine, adenosine, inosine, and guanosine. The number of NBMPR binding sites per cell doubled between 4 and 16 hr after detachment (late G1 and S phase); during this interval, V max 'S for uptake of cytidine and adenosine doubled, and for uridine and thymidine uptake increased about 4- and 8-fold, respectively. Rates of inosine and guanosine uptake at extracellular concentrations below saturation increased 2-fold between G1 and S phase of the cell cycle. Km 'S for cellular uptake of thymidine, uridine, cytidine, and adenosine did not change with progress through the cycle. The results presented suggest that changes in nucleoside uptake during the HeLa cell cycle were due, in part, to changes in the activity of NBMPR-sensitive transport elements in the membrane.

Effect of lithium on the myelosuppressive and chemotherapeutic activities of vinblastine.

Serum concentrations of lithium after i.p. administration to normal mice declined with biphasic kinetics. Treatment of mice bearing ascitic L1210 leukemia with lithium resulted in a modest protection against the myelosuppressive effects of vinblastine. Lithium as a single agent was without effect on the survival times of mice bearing either the L1210 or P388 leukemia. Similarly, there was no evidence of significant antiproliferative or cytotoxic activity when cultured L1210 leukemia and murine neuroblastoma cells were exposed to lithium at levels comparable to those observed in the serum of lithium-treated mice. The therapeutic activity of vinblastine against the L1210 and P388 leukemias was not significantly altered by either simultaneous or subsequent administration of lithium. Lithium did not antagonize the antiproliferative or cytotoxic action of vinblastine against L1210 leukemia or murine neuroblastoma cells and was without effect in experiments with neuroblastoma cells that assessed vinblastine inhibition of a biological function dependent on formation of cytoplasmic microtubules (neurite formation induced by serum deprivation). The results obtained suggest that administration of lithium to reduce myelosuppression is not likely to counteract the tumoricidal activity of vinblastine.

Effect of duration of exposure to verapamil on vincristine activity against multidrug-resistant human leukemic cell lines.

Verapamil sensitizes multidrug-resistant cell lines to various heterocyclic anticancer drugs by inhibition of energy-dependent release of drug, presumably by interaction with membrane glycoproteins involved in drug efflux. This work assessed verapamil sensitization of human multidrug-resistant lymphocytic and myeloid leukemic cell lines (CEM/VLB100, HL-60/AR) to vincristine during exposures of short duration (4 h). When cells were transferred to drug-free medium immediately after simultaneous 4-h exposures to vincristine and verapamil, the antiproliferative activity of vincristine was not altered in CEM/VLB100 cells and was only moderately increased in HL-60/AR cells. In contrast, when cells were transferred to verapamil-containing medium, vincristine activity was greatly increased against both CEM/VLB100 and HL-60/AR cells. Verapamil enhanced accumulation and inhibited release of [3H]vincristine by CEM/VLB100 and HL-60/AR cells, indicating that the sensitization was due to an increase in cell-associated vincristine after transfer of cells to vincristine-free medium. Slot blot analysis of cellular RNA with the pMDR1 probe revealed high levels of expression of the mdr1 gene in CEM/VLB100 cells but no detectable expression in HL-60/AR cells. Consistent with this finding, polypeptides (Mr 170,000 to 180,000) that were recognized by a monoclonal antibody (C219) against P-glycoprotein were greatly overexpressed in CEM/VLB100 cells, but were expressed at low levels, if at all, in HL-60/AR cells. These results demonstrate the importance of duration of exposure to verapamil in reversing multidrug resistance, not only in cells that overexpress P-glycoprotein but also in cells, such as HL-60/AR, that express little, if any, P-glycoprotein.