Phase I pharmacokinetic study of the novel antitumor agent SR233377.

SR233377 is a novel thioxanthenone analogue that demonstrated solid tumor selectivity in vitro with activity confirmed in vivo against several murine tumors including those of colon, pancreas, and mammary origin. Its primary preclinical dose-limiting toxicities included myelosuppression and neurological toxicity. The neurological toxicity was acute and could be ameliorated in mice when the drug was administered as a 1-h infusion instead of rapid i.v. injection. As a result of its preclinical efficacy profile, SR233377 entered Phase I clinical investigation. The compound was administered i.v. over 2 h on day 1 repeated every 28 days. The starting dose was 33 mg/m2 (one-tenth the mouse LD10). Escalations continued to 445 mg/m2 (six escalations), where dose-limiting toxicity was observed. At this dose, acute ventricular arrhythmias, including one patient with torsades de pointes and transient cardiac arrest, occurred. Because this toxicity might have been related to the plasma peak, the protocol was amended to a 24-h infusion beginning at 225 mg/m2. With this dose, prolongation of the corrected QT interval (QTc) over the pretreatment levels resulted. Because prolonged QTc is a known forerunner to acute ventricular arrhythmias, clinical development of SR233377 was stopped. However, preclinical antitumor and toxicity studies with analogues are underway with hopes of identifying a new clinical candidate with similar antitumor effects that is devoid of cardiac toxic effects.

Is gamma-actin a regulator of amino acid transport?

A mutated yeast cell line incapable of growth in minimal medium with proline as the sole nitrogen source was restored to normal growth by transfection with a cDNA from mouse Ehrlich cells. The cloned cDNA (E51) was found to be 90% homologous to gamma-actin. Immediately after transfection with E51 cDNA, both alpha-aminoisobutyric acid (AIB) and proline uptake in the mutated yeast were increased, particularly at pH 5. The expression of the same E51 cDNA also enhanced amino acid uptake in Xenopus laevis oocytes after injection into the Xenopus nuclei. A mutated mammalian lymphocyte cell line (GF-17), deficient in system A transport, also showed increased Na(+)-dependent transport after transfection with E51 cDNA. Whereas the mock transfected GF-17 cells failed to grow in the selection medium, the transfectants with E51 cDNA grew better than the untransfected cells. The data are consistent with the conclusion that expression of E51 cDNA can modify inactive, endogenous amino acid transporters, permitting substantial amino acid uptake in cells deficient in amino acid transporter(s) and permitting rapid cell growth. The data suggest that the gamma-actin-like protein coded for by E51 cDNA may play a significant regulatory role in amino acid transport.

VLA-beta 1 integrin subunit-specific monoclonal antibodies MB1.1 and MB1.2: binding to epitopes not dependent on thymocyte development or regulated by phorbol ester and divalent cations.

We report here the isolation of two new monoclonal antibodies (MB1.1 and MB1.2) against mouse VLA-beta 1 integrin subunit. Characterization by flow cytometry demonstrated binding of MB1.1 and MB1.2 to freshly isolated thymocytes, primary bone marrow mast cell lines, as well as cell lines of distinct lineage each expressing different combination of VLA integrins. The specificity of MB1.1 and MB1.2 was determined by (1) their binding to antigen with M(r) about 120 kDa, and (2) the ability of antiserum against the carboxyl terminal of VLA-beta 1 subunit to deplete antigens for MB1.1 and MB1.2 in sequential immunoprecipitation experiments. The epitopes for MB1.1 and MB1.2 were in close proximity to each other since preincubation of cells with one MAb inhibited the binding of the other. However, MB1.1 and MB1.2 differed in their affinity for the beta 1 subunit. In addition, neither MAbs had any effect on cell adhesion to matrix proteins indicating that the epitopes involved are distant from VLA integrin ligand-binding sites. MB1.1 and MB1.2 appear to differ from the two MAbs so far reported against mouse VLA-beta 1 subunit, KMI6 and 9EG7. Thus, the epitopes for MB1.1 and MB1.2 were readily detectable on unfractionated thymocytes whereas KMI6 has been reported to bind only a fraction of CD4-8- and CD4-8+ thymocytes. Phorbol ester and Mn2+, which have been shown to regulate the binding of 9EG7, had no effect on MB1.1 and MB1.2 binding to VLA-beta 1 integrin subunit.

Identification of the integrin alpha 3 beta 1 as a component of a partially purified A-system amino acid transporter from Ehrlich cell plasma membranes.

We have previously reported [McCormick and Johnstone (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7877-7881] the partial purification of the Na(+)-dependent A-system amino acid transporter from Ehrlich cell plasma membranes and have suggested that a 120-130 kDa peptide, a major component of the purified fraction [octyl glucoside (OG) extract], is involved in Na(+)-dependent amino acid transport. In the present study, N-terminal sequence analysis of the 120-130 kDa peptide revealed a sequence similar to that of the alpha 3 subunit of the integrin alpha 3 beta 1. The presence of alpha 3 beta 1 was confirmed by Western blots of the OG extract probed with anti-alpha 3 or -beta 1 antibodies. Western blots also showed that an antibody originally raised against the 120-130 kDa peptide crossreacts with both the alpha 3 and beta 1 integrin subunits. Co-purification of alpha 3 beta 1 and Na(+)-dependent transport activity suggested that the two activities might be associated. Evidence that alpha 3 plays a role in transport is shown by the fact that an antibody against human alpha 3, but not beta 1, removed transport activity (approximately 25% loss) from cholate-solubilized Ehrlich membranes. Further purification of OG extracts using concanavalin A and wheat-germ lectin columns resulted in the separation of transport activity from the bulk (but not all) of alpha 3 beta 1 integrin without loss of the transport activity. These results indicate that the integrin itself is not essential for amino acid transport. Reconstitution of a purified alpha 3 beta 1-depleted protein fraction showed high levels of Na(+)-dependent, alpha-methylaminoisobutyric-acid-inhibitable amino acid transport in proteoliposomes, whereas reconstituted integrin alone showed little transport activity. However, in the integrin-depleted fractions, high amino acid uptake occurred in K+ which compromised the accurate measurement of the Na(+)-dependent component of uptake. The data suggest that alpha 3 may be associated with the A-system transporter and may modulate the activity of this carrier. Moreover, transfection of K562 and RD cells with human alpha 3 and alpha 2 cDNA showed that the former but not the latter increased A-system transport, thus providing more direct evidence that alpha 3 may modulate A-system transport activity.

Differentiation of two classes of "A" system amino acid transporters.

Injection of mRNA from GF-14 cells (a mouse lymphocyte cell line) into Xenopus oocytes led to the expression of a transport activity characteristic of amino acid transport system A, i.e., Na+ dependent and recognizing aminoisobutyric acid and its methyl derivative (AIB and meAIB). Sucrose density gradient fractionation of GF-14 mRNA showed that the maximum level of expression of activity was associated with a 2.2-kb RNA fraction. Pretreatment of GF-14 cells with insulin caused a twofold increase in system A transport activity in the cells themselves and the mRNA from the insulin-treated cells induced a comparable increase in A system transport over control mRNA when expressed in oocytes. mRNA from cells treated with insulin in presence of actinomycin D did not show a response to insulin, suggesting that GF-14 cells synthesized new transporter in response to insulin. Similar experiments with mRNA from the Ehrlich ascites cells, showed that expression of system A type transport was associated chiefly with a 4.2-kb mRNA fraction. Although insulin treatment of Ehrlich cells also caused an increase in A system transport activity in the cells themselves (nearly twofold), the response to insulin was not blocked by actinomycin D or cycloheximide. Moreover, mRNA from insulin-treated and untreated Ehrlich cells gave the same level of expression in oocytes of A type amino acid transport. In contrast to GF-14 cells, Western blots of plasma membranes showed that insulin treatment of Ehrlich cells increased the amount therein of a 120- to 130-kDa peptide, previously associated with amino acid transport (Proc. Natl. Acad. Sci. USA 85, 7877, 1988), suggesting that in the Ehrlich cells, but not in GF-14, cells this polypeptide was translocated from intracellular sites in response to insulin. The data associate two distinctly different responses to insulin with A-type amino acid transporters and are consistent with the existence of more than a single A type amino acid transporter in mouse cells.

Molecular size of a Na(+)-dependent amino acid transporter in Ehrlich ascites cell plasma membranes estimated by radiation inactivation.

Radiation inactivation was used to estimate the molecular size of a Na(+)-dependent amino acid transport system in Ehrlich ascites cell plasma membrane vesicles. Na(+)-dependent alpha-aminoisobutyric acid uptake was measured after membranes were irradiated at -78.5 degrees C in a cryoprotective medium. Twenty-five percent of the transport activity was lost at low radiation doses (less than 0.5 Mrad), suggesting the presence of a high molecular weight transport complex. The remaining activity (approximately 75% of total) decreased exponentially with increasing radiation dose, and a molecular size of 347 kDa was calculated for the latter carrier system. Vesicle permeability and intravesicular volume were measured to verify that losses in transport activity were due to a direct effect of radiation on the transporter and not through indirect effects on the structural integrity of membrane vesicles. Radiation doses 2-3-fold higher than those required to inactivate amino acid transport were needed to cause significant volume changes (greater than 15%). Vesicle permeability was unchanged by the irradiation. The structural integrity of plasma membrane vesicles was therefore maintained at radiation doses where there was a dramatic decrease in amino acid transport. The relationship between the fragmentation of a 120-130-kDa peptide, a putative component of the Na(+)-dependent amino acid carrier [McCormick, J. I., & Johnstone, R. M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7877-7881], and loss of transport activity in irradiated membranes was also examined. Peptide loss was quantitated by Western blot analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of specific acidic lipids on the reconstitution of Na(+)-dependent amino acid transport in proteoliposomes derived from Ehrlich cell plasma membranes.

The effect of acidic phospholipids on the activity of a Na(+)-dependent amino acid transporter (A system) from Ehrlich ascites cell plasma membranes was examined. Plasma membranes were solubilized in cholate/urea and reconstituted with Ba2(+)-precipitated asolectin (soybean phospholipid free of anionic phospholipids) replenished with different acidic phospholipids. In the absence of added acidic phospholipids, transport activity was very low. However, three acidic lipids [cardiolipin greater than phosphatidic acid (PA) greater than phosphatidylinositol] were capable of restoring transport activity (in the order given) to proteoliposomes made from Ba2(+)-precipitated asolectin, while other acidic phospholipids (phosphatidylserine and phosphatidylglycerol) were much less active in this respect. For restoration of optimal activity, PA containing at least one unsaturated fatty acyl moiety, particularly in the beta position, was required. PA containing only saturated fatty acids in the beta and gamma positions was largely inactive. No difference in restoration of function was observed on varying the saturated fatty acyl chain length in PA from 10 carbons to 18 carbons. The specific effects of PA on the A-system transporter were not shared by the Na(+)-independent amino acid exchange system (L system) or the glucose transport system. Treatment with poly(ethylene glycol) 8000 was shown to reduce the nonspecific permeability of the reconstituted proteoliposomes and to enhance Na(+)-dependent amino acid transport.

Simple and effective purification of a Na+-dependent amino acid transport system from Ehrlich ascites cell plasma membrane.

A reconstitution assay was used to measure transport activity during purification of a Na+-dependent amino acid transporter from Ehrlich cell plasma membrane. Cholate/urea-solubilized membranes were fractionated on a Sepharose 6B column and transport activity was recovered in the column void volume. Centrifugation of the void volume fraction at 105,000 X g and reextraction of the pellet with 1% octyl glucoside led to recovery of an extract whose specific transport activity was nearly 30-fold higher than that of the original solubilized extract with a recovery of 38% of the original activity. The properties of amino acid uptake in the purified reconstituted transporter were identical to those in native plasma membrane vesicles. The major component present in the purified fraction had a molecular mass of 120-130 kDa. Strong evidence that this 120- to 130-kDa peptide contains a component of the amino acid transporter was obtained by immunoprecipitation of transport activity from solubilized membranes with an antibody against the 120- to 130-kDa peptide. This study tentatively identifies a component of the Na+-dependent amino acid transporter as a peptide with an apparent molecular mass of 120-130 kDa.

Volume enlargement and recovery of Na+-dependent amino acid transport in proteoliposomes derived from Ehrlich ascites cell membranes.

Na+-dependent amino acid transport can be reconstituted from solubilized Ehrlich cell plasma membranes by addition of asolectin vesicles, gel filtration, and a freeze-thaw cycle. Removal of phosphatidic acid (approximately 10% of the total lipid) by Ba2+ precipitation reduces the efficiency of reconstitution of Na+-dependent amino acid transport by approximately 73% and decreases intravesicular volume of the proteoliposomes by approximately 43%. The loss of transport activity is not due to exclusion of specific proteins during reconstitution. The phosphatidic acid-free liposomes are less permeable and require more time to attain an equilibrium distribution of solute. Transport activity and intravesicular volume can be restored to Ba2+-precipitated asolectin proteoliposomes by addition of egg-phosphatidic acid during reconstitution. The extent of recovery of transport activity is proportional to the change in intravesicular volume and depends on the amount of phosphatidic acid present. Replacement of phosphatidic acid with 20% phosphatidylserine or phosphatidylglycerol leads to increases in intravesicular volume with little or no increase in amino acid transport. Generation of phosphatidic acid in situ by treatment of Ba2+-precipitated proteoliposomes with phospholipase D also restored transport. The observed increase in transport activity (9-fold) is accompanied by a 46% increase in intravesicular volume, presumably caused by vesicle fusion. Phosphatidic acid is also required for successful reconstitution of Na+-dependent amino acid transport from pure phosphatidylcholine:phosphatidylethanolamine (1:1) mixtures with only a small change (approximately 16%) in intravesicular volume. The results provide evidence for both indirect and direct effects of phosphatidic acid on reconstitution of Na+-dependent amino acid transport. The indirect effects occur through enlargement of intravesicular volume, large vesicles showing higher rates of transport. However, there is also evidence to indicate a specific effect of phosphatidic acid on the Na+-dependent amino acid transporter, since other acidic lipids may change intravesicular volume without a commensurate change in transport activity.

Asymmetric reconstitution of the glucose transporter from Ehrlich ascites cell plasma membrane: role of alkali cations.

Gel chromatography of solubilized Ehrlich cell plasma membranes and preformed asolectin vesicles coupled to a freeze-thaw cycle results in the reconstitution of 3-O-methyl-D-glucose transport. The transport activity of the liposomes formed is critically dependent on the cation present during reconstitution. Liposomes formed in K+ show high levels of carrier-mediated 3-O-methyl-D-glucose uptake (495 pmol/min/mg protein) while those formed in Na+ do not (33 pmol/min/mg protein). The inactivity in Na+ is not due to a diminished incorporation of glucose transporter nor is it due to carrier molecules reconstituted with a different orientation from those in K+ liposomes. Instead, the low glucose transport level in Na+ liposomes is related to the small size of vesicles formed with Na+. A second freeze-thaw cycle in K+ causes a two- to threefold increase in the available intravesicular volume of Na+ liposomes and results in an eightfold increase in carrier-mediated 3-O-methyl-D-glucose uptake. K+ liposomes, treated in an identical manner, show only a twofold increase in uptake. The glucose transporter was identified as a protein with a molecular mass range of 44.7 to 66.8 kDa, by the D-glucose-inhibitable photoincorporation of [3H]cytochalasin B. The carrier protein is inserted in reconstituted vesicles in a nonrandom manner with at least 80% of the molecules oriented with the cytoplasmic domain accessible to the external medium. In contrast, the neutral Na+-dependent amino acid transport system appears to be randomly reconstituted.

Effect of alkali cations on freeze-thaw-dependent reconstitution of amino acid transport from Ehrlich ascites cell plasma membrane.

Na+-dependent amino acid transport can be reconstituted by gel filtration of disaggregated plasma membrane and asolectin vesicles coupled to a freeze-thaw cycle. The resultant transport activity is markedly affected by the nature of the reconstitution medium. Reconstitution in K+ permits the formation of active liposomes, whereas reconstitution in Na+, Li+, or choline does not. Electron micrographs of K+ liposomes show a wide variation in liposome sizes. Ficoll density gradient fractionation of K+ liposomes shows that the largest vesicles are lipid rich, have the lowest density, and have the highest level of Na+-dependent amino acid transport. Liposomes formed in Na+ have a 34% smaller trapped volume than K+ liposomes and lack a population of large vesicles. A second freeze-thaw in K+ restores activity to Na+ liposomes which now contain large low density active vesicles. Fluorescence measurements of freeze-thaw-induced mixing of vesicle lipids indicates that the absence of large vesicles in Na+ liposomes is due to inhibition by Na+ of lipid vesicle fusion events during freezing and thawing. The large vesicle fraction is enriched in a 125-kDa peptide. It has not yet been established whether this peptide is part of the transport system for neutral amino acids.

A simple and efficient method for reconstitution of amino acid and glucose transport systems from Ehrlich ascites cells.

Solubilized Ehrlich cell plasma membrane proteins were incorporated into lipid vesicles in the presence of added phospholipid, using Sephadex G-50 chromatography combined with a freeze-thaw step. Liposomes formed in K+ exhibited high levels of Na+-dependent, alpha-aminoisobutyric acid uptake which was electrogenic and inhibited by other amino acids. The transport activity reconstituted was similar to that observed in native plasma membrane vesicles. In addition to transport by system A, leucine exchange activity (system L), Na+-dependent serine exchange activity (system ASC), and stereospecific glucose transport activity were also reconstituted. The latter was inhibited by D-glucose, D-galactose, cytochalasin B, and mercuric chloride. The medium used for reconstitution was critical for the recovery of Na+-dependent amino acid transport. The use of Na+ in the reconstitution procedure led to formation of liposomes which displayed little Na+-dependent and gradient-stimulated amino acid uptake. In contrast, all transport activities studied were efficiently reconstituted in K+ medium.