Mark Paulik

IS THE DRUG-RESPONSIVE NADH OXIDASE OF THE CANCER CELL PLASMA MEMBRANE A MOLECULAR TARGET FOR ADRIAMYCIN?

Enhanced growth inhibition and antitumor responses to adriamycin have been observed repeatedly from several laboratories using impermeant forms of adriamycin where entry into the cell was greatly reduced or prevented. Our laboratory has described an NADH oxidase activity at the external surface of plasma membrane vesicles from tumor cells where inhibition by an antitumor sulfonylurea, N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY181984), and by the vanilloid, capsaicin (8-methyl-N-vanillyl-6-noneamide) correlated with inhibition of growth. Here we report that the oxidation of NADH by isolated plasma membrane vesicles was inhibited, as well, by adriamycin. An external site of inhibition was indicated from studies where impermeant adriamycin conjugates were used. The EC50 for inhibition of the oxidase of rat hepatoma plasma membranes by adriamycin was several orders of magnitude less than that for rat liver. Adriamycin cross-linked to diferric transferrin and other impermeant supports also was effective in inhibition of NADH oxidation by isolated plasma membrane vesicles and in inhibition of growth of cultured cells. The findings suggest the NADH oxidase of the plasma membrane as a growth-related adriamycin target at the surface of cancer cells responsive to adriamycin. Whereas DNA intercalation remains clearly one of the principal bases for the cytotoxic action of free adriamycin, this second site, possibly related to a more specific antitumor action, may be helpful in understanding the enhanced efficacy reported previously for immobilized adriamycin forms compared to free adriamycin.

Transition vesicle formationin vitro

SummaryIsolated fractions enriched in transition elements derived from part rough—part smooth regions of endoplasmic reticulum of rat liver respondin vitro to ATP plus a concentrated fraction of cytoplasmic proteins by formation of ca. 60 nm vesicles with nap-like coats resembling those of transition vesicles of the intact cell. Similar vesicles are normally considered to function in the transfer of materials from endoplasmic reticulum to cis elements of the Golgi apparatus.

Identification of antitumor sulfonylurea binding proteins of HeLa plasma membranes.

Plasma membranes of cultured HeLa S cells bound the tritiated antitumor sulfonylurea [3H]LY181984 with high affinity (Kd of about 25 nM). The number of binding sites, estimated to represent 30 to 35 pmol/mg protein, would represent a low abundance protein of the total plasma membrane proteins. The binding proteins appeared to contain one or more thiols in the binding site as high affinity binding of [3H]LY181984 was reduced by treatment with the covalent thiol blocking reagent, N-ethylmaleimide (NEM), or by oxidation with dilute hydrogen peroxide but was protected by glutathione or dithiothreitol. Elimination of binding of [3H]LY181984 by NEM was prevented by excess unlabeled LY181984 (an active sulfonylurea) but less so by excess LY181985 (an inactive sulfonylurea). The binding proteins were specifically labeled with thiol reagents following reaction of unprotected thiols with unlabeled thiol reagents. Binding proteins at ca. 34 kDa were labeled. Plasma membrane proteins after solubilization with SDS under strongly reducing conditions still bound sulfonylurea. [3H]LY181984 binding to plasma membrane proteins resolved on SDS-PAGE correlated as well with proteins in the 30-40 kDa range.

Transitional endoplasmic reticulum membranes and vesicles isolated from animals and plants

SummaryThe process of formation from endoplasmic reticulum and transfer to Golgi apparatus of small 50–70 nm transition vesicles has been reconstituted in a cell-free system. Fractions enriched in transition elements derived from part-rough, part-smooth transitional regions of the endoplasmic reticulum were prepared from elongation zones of hypocotyls of etiolated seedlings of soybean and coleoptiles of maize and were compared with those from rat liver. When activated with nucleoside triphosphate, cytosol and an ATP regenerating system, time- and temperature-dependent transfer of membranes to Golgi apparatus acceptor was demonstrated. The fractions enriched in transition elements were radioiodinated with125I by the Bolton-Hunter procedure. Acceptor Golgi apparatus stacks were immobilized to nitrocellulose strips to facilitate analysis. In heterologous transfer experiments, the plant and animal acceptors and donors could be interchanged. The transfer was limited primarily by the donor (rat liver > soybean hypocotyl > maize coleoptiles) and determined secondarily by the source of the acceptor. The acceptor fractions were most efficacious when prepared from the same source as the donor. Thus, 50–70 nm vesicles bud from transitional endoplasmic reticulum elements of plants function in a manner similar to those of animal cells to transfer membrane materials to the Golgi apparatus. The recognition signals that determine vesicle fusion appear to be conserved both among species and between the plant and animal kingdoms to the extent that donor and acceptor sources may be interchanged with only small reductions in overall efficiency of transfer.

Retinoid modulation of cell-free membrane transfer between endoplasmic reticulum and Golgi apparatus

Cell-free transfer of radiolabeled membrane proteins from part-rough, part-smooth transitional elements of the endoplasmic reticulum to Golgi apparatus immobilized to nitrocellulose in the presence of nucleoside triphosphate, an ATP-regenerating system and a cytosol fraction was promoted by retinol. At an optimum concentration of 1 microgram/ml, the rate and amount of transfer was approximately doubled over 1 to 2 h of incubation in the cell-free system. The transition vesicles induced to form in the cell-free system were concentrated by preparative free-flow electrophoresis in order to study separately the steps of vesicle formation from transitional endoplasmic reticulum and the steps of vesicle fusion with Golgi apparatus. The retinol effect was on vesicle formation as evidenced by an approx. 2-fold increase in transition vesicle numbers, as determined by electron microscope morphometry, and amount from protein determinations on the isolated fractions enriched in transition vesicles. The retinol response in the complete transfer could be eliminated by addition of concentrated cytosol, including cytosol depleted of retinol. An interaction of retinol with some component of the vesicle formation process, possibly involving guanine nucleotides, is indicated.

Identification of the 16°C compartment of the endoplasmic reticulum in rat liver and cultured hamster kidney cells

Summary— In many systems transfer between the endoplasmic reticulum and the Golgi apparatus is blocked at temperatures below 16°C. In virus‐infected cells in culture, a special membrane compartment is seen to accumulate. Our studies with rat liver show a similar response to temperature both in situ with slices and in vitro with isolated transitional endoplasmic relticulum fractions. With isolated transitional endoplasmic reticulum fractions, when incubated in the presence of nucleoside triphosphate and a cytosol fraction, temperature dependent formation of vesicles occurred with a Q10 of −2 but was apparent only at temperatures greater than 12°C. A similar response was seen in situ at 12°C and 16°C where fusion of transition vesicles with cis Golgi apparatus, but not their formation, was blocked and transition vesicles accumulated in large numbers. At 18°C and below and especially at 8°C and 12°C, the cells responded by accumulating smooth tubular transitional membranes near the cis Golgi apparatus face. With cells and tissue slices at 20°C neither transition vesicles nor the smooth tubular elements accumulated. Those transition vesicles which formed at 37°C were of a greater diameter than those formed at 4°C both in situ and in vitro. The findings show parallel responses between the temperature dependency of transition vesicle formation in vitro and in situ and suggest that a subpopulation of the transitional endoplasmic reticulum may be morphologically and functionally homologous to the 16°C compartment observed in virally‐infected cell lines grown at low temperatures.

NADH-activated cell-free transfer between Golgi apparatus and plasma membranes of rat liver

This report concerns development of a cell-free system from rat liver to study transport of membrane constituents from the Golgi apparatus to the plasma membrane. Highly purified Golgi apparatus as donor and a mixture of sheets and vesicles as plasma membrane acceptor fractions were combined to analyze requirements for lipid and protein transport. In the reconstituted system, the Golgi apparatus donor was in suspension. To measure transfer, membrane constituents of the donor membranes were radiolabeled with [3H]acetate (lipids) or [3H]leucine (proteins). The plasma membrane vesicles were used as the acceptor and were unlabeled and immobilized on nitrocellulose for ease of recovery and analysis. The reconstituted cell-free transfer was dependent on temperature, but even at 37 degrees C, the amount of transfer did not increase with added ATP, was not specific for any particular membrane fraction or subfraction nor was it facilitated by cytosol. ATP was without effect both in the presence or absence of a cytosolic fraction capable of the support of cell-free transfer in other systems. In contrast to results with ATP, NADH added to the reconstituted system resulted in an increased amount of transfer. A further increase in transfer was obtained with NADH plus a mixture of ascorbate and dehydroascorbate to generate ascorbate free radical. The transfer of labeled membrane constituents from the Golgi apparatus to the plasma membrane supported by NADH plus ascorbate radical was stimulated by a cytosol fraction enriched in less than 10 kDa components. This was without effect in the absence of NADH/ascorbate radical or with ATP as the energy source. Specific transfer was inhibited by both N-ethylmaleimide and GTP gamma S. The findings point to the possibility of redox activities associated with the trans region of the Golgi apparatus as potentially involved in the transport of membrane vesicles from the Golgi apparatus to the cytoplasmic surface of the plasma membrane.

Comparison of [35S]GTPγS binding to plasma membranes and endomembranes prepared from soybean and rat

SummaryPlasma membranes were prepared from soybean hypocotyls and roots by aqueous two-phase partitioning and subsequent free-flow electrophoresis. The highly purified plasma membranes bound [35S]GTPγS with a relatively high affinity (Kd≈10nM). The binding was saturable and specific as it was indicated by the displacement of bound [35S]GTPγS by unlabeled GTPγS and GTP, but not by ATPγS, ATP, UTP or CTP. ITP was intermediate in its ability to displace [35S]GTPγS. When soybean plasma membrane proteins were separated by SDS-PAGE and displayed by autoradiography, two major [35S]GTPγS binding proteins were revealed with apparent molecular weights of 24 and 28 kDa. Results with plasma membranes from soybean hypocotyls and roots were similar but differed from those with plasma membranes prepared from rat liver and adipocytes where only a single major [35S]GTPγS binding activity with a molecular weight of 28 kDa was observed.

Retinoic acid and calcitriol inhibition of growth and NADH oxidase of normal and immortalized human keratinocytes

Plasma membranes were isolated by aqueous two-phase partition from normal human keratinocytes (HKc) and from human keratinocytes immortalized with human papillomavirus type 16 DNA (HKc/HPV16). The NADH oxidase of plasma membrane vesicles of normal HKc was stimulated by epidermal growth factor whereas that of HKc/HPV16 was not. The NADH oxidase of the plasma membranes from both normal HKc and HKc/HPV16 was inhibited by calcitriol (1 alpha-1,25-dihydroxy vitamin D-3) and retinoic acid. However, with plasma membranes from HKc/HPV16 the NADH oxidase was more susceptible to inhibition by retinoic acid than were membranes from normal HKc. Similarly, clonal growth of HKc/HPV16 was inhibited by retinoic acid at lower concentrations than normal HKc whereas inhibition of clonal growth of normal HKc and HKc/HPV16 by calcitriol showed similar dose-dependencies. Comparing normal HKc and HKc/HPV16, the results demonstrate parallel inhibition of clonal growth and NADH oxidase by both retinoic acid and calcitriol of HKc/HPV16 but not of normal HKc. These results suggest that an increased sensitivity of the plasma membrane NADH oxidase of HKc/HPV16 to retinoic acid may be related to the increased sensitivity of these cells to growth control by retinoic acid. In addition, since plasma membrane NADH oxidase of HKc/HPV16 shows altered responsiveness to growth modulators such as EGF, retinoic acid and calcitriol, it appears that HKc/HPV16 express an NADH oxidase with different characteristics than those of normal HKc.

INHIBITION BY BREFELDIN A OF NADH OXIDATION ACTIVITY OF RAT LIVER GOLGI APPARATUS ACCELERATED BY GDP

Reduced pyridine nucleotide has been reported to enhance cell‐free transfer of membrane material from a radiolabeled Golgi apparatus donor fraction from rat liver to an acceptor fraction consisting of inside‐out plasma vesicles immobilized on nitrocellulose [(1992) Biochim. Biophys. Acta 1107, 131]. As part of a continuing effort to identify NADH‐requiring enzymes in the Golgi apparatus which may be important to membrane trafficking, highly purified fractions of Golgi apparatus from rat liver were tested for their ability to oxidize NADH and the inhibition of the oxidation of NADH by brefeldin A. The isolated Golgi apparatus fractions were found to oxidize NADH with a specific activity comparable to that of the plasma membrane of rat liver. The activity was inhibited by brefeldin A and this inhibition was augmented by GDP. At near optimal concentrations of 7μM brefeldin A and 1 μM GDP, the activity was > 90% inhibited. Brefeldin A inhibition of NADH oxidation by the Golgi apparatus was time‐dependent and GDP appeared to accelerate the inhibition by brefeldin A.