Plasma membrane ectoglycosyltransferase activity of L1210 murine leukemic cells

L1210 murine leukemia cells after treatment with Cl. perfringens neuraminidase at pH 7.0 incorporated six times more N-acetylneuraminic acid-[C¹⁴] than control cells when incubated for 30 minutes with cytidine 5′-monophosphate N-acetylneuraminic-[C¹⁴] acid and three times more galactose-[C¹⁴] when incubated with uridine diphosphate galactose-[C¹⁴]. These sugars were incorporated in a 10% trichloracetic acid insoluble fraction and more than 75% of the incorporated N-acetylneuraminic acid- [C¹⁴] could be removed by further treatment of these cells with neuraminidase. The incorporation of N-acetylneuraminic acid- [C¹⁴] as a function of time was divided into two rates: a rapid one, active during the first 30 minutes followed by a slower one, similar to the rate observed with untreated cells. The addition of Ba⁺⁺ and Ca⁺⁺ ions at 8.3 mM increased the incorporation of N-acetylneuraminic acid- [C¹⁴] by 25% while 8.3 mM EDTA decreased activity by 58% . The addition of Zn⁺⁺ or Hg⁺⁺ at similar concentrations abolished the incorporation almost completely. The optimal pH for the incorporation of N-acetylneuraminic acid- [C¹⁴] by these neuraminidase treated cells was 6.5. These data suggest that ectoglycosyltransferases are present on the outer surface of the plasma membrane of L1210 cells and are able to catalyze the addition of radiolabeled nucleotide sugars onto macromolecular acceptors (cell surface glycoproteins and glycolipids) prepared by prior incubation of the cells with neuraminidase. Use of these procedures for labeling outer cell surfaces may also prove to be valuable for the study of plasma membrane glycoprotein and glycolipid structure, synthesis, and turnover.     

Competitive inhibition and substrate activity of uridine diphosphate 6-deoxygalactose for Escherichia coli uridine diphosphate galactose 4-epimerase (Short Communication)

UDP-6-deoxygalactose inhibits the UDP-galactose 4-epimerase (EC 5.1.3.2) from Escherichia coli in a competitive manner with respect to the substrate UDP-galactose, giving K(i) 1.3x10(-3)m. As a substrate for the enzyme, it is transformed into UDP-6-deoxyglucose, although the reaction stops before equilibrium is attained. Possible causes of this behaviour are discussed.     

Biochemical and ultrastructural studies of ectoglycosyltransferase systems of murine L1210 leukemic cells

Intact murine L1210 leukemic cells incorporated significant quantities of [³H]-N-acetylneuraminic acid directly from CMP-N-acetylneuraminic acid. When pretreated with Vibrio cholerae neuraminidase, incorporation increased sixfold to tenfold. Biochemical studies comparing incorporation of N-acetyl-neuraminic acid from the nucleotide sugar with that from free sugar demonstrated that the relatively high levels of incorporation from CMP-N-acetyl-neuraminic acid could not be due to the incorporation of free sugar generated by extracellular degradation of the nucleotide sugar. Very little N-acetylneuraminic acid was taken up or incorporated by L 1210 cells from free sugar and this incorporation was not increased by neuraminidase pretreatment. Moreover, extracellular breakdown of CMP-N-acetylneuraminic acid during incubations with L 1210 cells was rather insignificant. Electron microscope autoradiography of cells incubated with CMP-N-acetylneuraminic acid demonstrated that greater than 84% of the incorporated radioactivity was associated with the plasma membrane and less than 1% with the Golgi apparatus. These findings are consistent with the conclusion that incroporation of N-acetylneuraminic acid from CMP-N-acetylneuraminic acid is the consequence of a cell surface sialytransferase system. Pretreatment of cells with the nonpenetrating reagent, diazonium salt of sulfonilic acid, significantly inhibited this ectoenzyme system while only marginally affecting galactose uptake and incorporation at the Golgi apparatus. Interestingly, incorporation from CMP-N-acetylneuraminic acid declined as the viability of the cell population declined. When taken together, the above evidence develops a rigorous argument for the presence of a sialyltransferase enzyme system at the cell surface of L 1210 cells. Studies directed towards the detection of a similar ectogalactosyltransferase system were also undertaken. Cells incubated in the presence of UDP-[³H]-galactose incorporated radioactivity into a macromolecular fraction. The presence of excess unlabeled galactose in the incubation medium significantly reduced this incorporation. Electron microscope autoradiographs of cells incubated with UDP-[³H]-galactose, demonstrated that incorporation occurred primarily at the Golgi apparatus. The grain distribution in these autoradiographs was similar to that for free galactose. Thus, the incorporation observed for L-1210 cells incubated in UDP-[³H]-galactose was due primarily to the intracellular utilization of free galactose generated by extracellular degradation of the nucleotide sugar. Inability t o demonstrate an ectogalacto-syltransferase system on L1210 cells does not rule out the possibility that the enzyme is present but undetectable due t o the absence of appropriate cell surface acceptor molecules.     

Suppressive effect of adrenalectomy on growth of L1210 leukemic cells in ascites

This study was designed to evaluate the effect of adrenalectomy on growth of L1210 leukemic cells in ascites of BDF1 mice. Varying doses of 1.5 x 10(4), 5.0 x 10(5), and 1.5 x 10(6) viable tumour cells were inoculated intraperitoneally into groups of either adrenalectomized or sham-operated mice. At days 4 to 7 after the inoculation, adrenalectomized mice inoculated with 1.5 x 10(4) or 5.0 x 10(5) tumour cells had a smaller number of tumour cells in ascites than sham-operated controls. However, after inoculation of 1.5 x 10(6) cells, no significant differences were found at days 2 to 4 between adrenalectomized and sham-operated mice. The growth retardation by adrenalectomy was not observed in adrenalectomized mice supplemented with 4 or 6 micrograms dexamethasone per day per mouse. It suggested that the ablation of glucocorticoids was at least partially responsible for the growth retardation observed in adrenalectomized mice. Cell kinetic analysis revealed that the difference in a potential doubling time could not explain these results. Tumour retention in the peritoneal cavity was measured using [125I]-iododeoxyuridine-labelled tumour cells as a tracer. At days 4 to 6 after inoculation of 5.0 x 10(5) labelled cells, radioactivity in the peritoneal cavity in adrenalectomized mice was about 70 per cent of that in sham-operated mice. This ratio was almost equivalent to the ratio of the number of cells in ascites of adrenalectomized mice to that of sham-operated ones. Consequently, growth retardation observed in adrenalectomized mice resulted from an increase in tumour cell migration and/or in tumour cell death, but not from an increase in doubling time.     

Isolation of temperature-sensitive mutants from murine leukemic cells (L5178Y)

Two temperature-sensitive mutants (ts1 and ts3) have been isolated from murine leukemic cells, L5178Y, after mutagenesis and cytosine arabinoside selection. Both ts1 and ts3 grew normally at the permissive temperature (33 °C) but not at the non-permissive temperature (39 °C). Consistent results were obtained with the growth patterns in suspension culture as well as the plating efficiencies in soft agar. Temperature shift experiments showed that the mutant cells remained viable after extended exposure to the non-permissive temperature. Labeling studies with radioactive precursors indicated that the synthesis of DNA, but not of RNA or protein, was affected in these mutants at 39 °C. The defective function of ts3 cells was substantially corrected by supplementing alanine, hypoxanthine, and pyruvate.     

p-(Bromoacetamido)phenyl uridyl pyrophosphate: an active-site-directed irreversible inhibitor for uridine diphosphate galactose 4-epimerase.

The synthesis of p-(bromoacetamido)phenyl uridyl pyrophosphate (BUP) is described. This compound is an active-site-directed irreversible inhibitor of Escherichia coli UDP-galactose 4-epimerase. The inactivation follows pseudo-first-order kinetics at pH 8.5 in nonnucleophilic buffers, and a saturation effect is seen in the pseudo-first-order rate constant as the concentration of BUP is increased. The half-saturation parameter for BUP in the inactivation is 0.21 +/- 0.02 mM, which compares favorably with the inhibition constant of 0.3 +/- 0.05 mM for BUP acting as a competitive reversible inhibitor of the enzyme. The inactivation rate is slow, however, with a minimum half-time of 12 h at pH 8.5 and 27 degrees C. Both specific alkylation and nonspecific alkylation by BUP occur, but nonspecific alkylation is faster than the inactivation and the rate of inactivation correlates well with the rate of covalent incorporation of one molecule of [14C]BUP at the active site.     

CTP: CMP phosphotransferase activity in L1210 cells

Substrate converting enzymes interfering with the measurement of ribonucleotide reductase were assessed in cell-free extracts prepared from L1210 cells. Data show the presence of a myokinase-type enzyme activity (CTP:CMP phosphotransferase) which catalyzes the reaction: 2CDP in equilibrium CMP + CTP. This enzyme is not removed by passage of cell extracts over ATP-agarose columns. Monitoring of nucleoside diphosphate substrate level is, therefore, mandatory for obtaining accurate measurements of CDP reductase activity in crude cell extracts.     

Inhibitory potency of 5-benzyluracil, 5-phenylcytosine and 5-phenylpyrimidin-2-one nucleosides against uridine phosphorylase from mouse leukemic L1210 cells

The inhibitory activity of a series of novel sugar-modified nucleosides derived from 5-benzyluracil, 5-phenylcytosine and 5-phenylpyrimidin-2-one against uridine phosphorylase purified from mouse leukemic L-1210 cells was investigated. Significant activity was encountered with O2,2'-anhydro-5-benzylcytidine hydrochloride, 2',3'-dideoxy-5-benzyluridine, 2',3'-dideoxy-4-thiouridine and alpha- and beta-anomers of 5-benzyl-1-(2-deoxy-D-arabino-hexopyranosyl)uracil.     

Early explorations of the pathways of uridine diphosphate galactose in man and in microorganisms

Thirty years ago, a number of human inborn errors in carbohydrate metabolism were explored with specific enzymatic tests on blood samples (hemolysates). Hereditary galactosemia was the first example. When the inoperative step in galactose metabolism was specified, the basis for the diet therapy used on the galactosemic infants, namely galactose-free diet, could be shown to be securely founded. As far as galactose metabolism is concerned, the cells of the infant are faced with two problems: (i) the conversion of dietary lactose (galactosyl glucose) to glucose and its catabolites involved in energy metabolism, and (ii) the conversion of dietary glucose or lactose to galactosyl units of glycolipids and glycoprotein cell structures. Subsequent studies on microorganisms revealed several types of hereditary defect in galactose metabolism. One type which permits the bacteria to develop a normal carbohydrate pattern in their cell walls includes an enzyme defect, like that described in the cells of the galactosemic infant. Two other types, with the inability to synthesize UDPGlc or UDPGal from glucose, do not permit the bacteria to build the fabric of the normal bacterial cell wall. This is the subject for discussion.     

Selective inhibition by bis(2-chloroethyl)methylamine (nitrogen mustard) of the Na+/K+/Cl- cotransporter of murine L1210 leukemia cells

Incubation of L1210 murine leukemia cells in vitro with 10 microM of the bifunctional alkylating agent bis(2-chloroethyl)methylamine (nitrogen mustard, HN2) for 10 min brought about a fall of more than 99.9% in their ability to form colonies when the cells were suspended in 0.5% nutrient agar. Incubation with HN2 also inhibited the influx of the potassium congener 86Rb+ to exponentially proliferating L1210 cells in a concentration-dependent manner. This inhibition was specific and was accounted for by a reduction of a diuretic-sensitive component of 86Rb+ influx, identified in the preceding paper (Wilcock, C. and Hickman, J.A. (1988) Biochim. Biophys. Acta 946, 359-367) as being mediated by a Na+/K+/Cl- cotransporter. Inhibition by 10 microM HN2 was complete after a 3-h incubation. There was no inhibition at this time of the ouabain-sensitive component of 86Rb+ influx, mediated by Na+/K+-ATPase. After 3 h of incubation with 10 microM HN2 there was also no change in the membrane potential of the treated cells as measured by the distribution of the [3H]TPMP+, no decrease in cellular ATP concentration and no change in intracellular pH, and the ability of the cells to exclude the vital dye Trypan blue was not significantly different from control values. These effects of HN2, therefore, appeared to follow lethal damage, but precede cell death. In the stationary phase of L1210 cell growth, the component of HN2 and diuretic-sensitive K+ influx to L1210 cells was reduced, whilst the component constituting the HN2-insensitive ouabain-sensitive sodium pump was increased. The monofunctional alkylating agent MeHN1 (2-chloroethyldimethylamine) which cannot cross-link cellular targets and has no antitumor activity, did not inhibit 86Rb+ influx to L1210 cells when incubated at equimolar or equitoxic concentrations to HN2. Intracellular potassium concentration was maintained close to control values of 138 +/- 10 mM in HN2-treated cells because of an approx. 35% fall in cell volume. The results suggest that the Na+/K+/Cl- cotransporter is a selectively inhibitable target for HN2, and the lesion is discussed with reference to the cytotoxic effects of this agent.     

The biochemical and ultrastructural effects of tunicamycin and D-glucosamine in L1210 leukemic cells

Tunicamycin was found to specifically inhibit the incorporation of a number of sugars into L1210 leukemia cell glycoproteins. This inhibition of glyco-protein biosynthesis led to a cessation of cell growth which was reversible in a dose-dependent and time-dependent manner. After removal of the antibiotic from L1210 cell cultures resumption of sugar incorporation preceded that of thymidine incorporation and the recovery of cell growth. The treatment of cells with tunicamycin resulted in a significant increase in the intracellular pool of UDP-N-acetylglucosamine which occurred concurrently with alterations in cell ultrastructure including distentions of the endoplasmic reticulum and nuclear membranes. Similar ultrastructural changes and increases in the intracellular pools of UDP-sugars were observed in L1210 cells exposed to 5 mM D-glucosamine, which suggested that the antiproliferative effects of tunicamycin may be related to the accumulation in the endoplasmic reticulum of one or more nucleotide sugar precursors of asparagine-linked glycoprotein biosynthesis. However, the biological effects of tunicamycin could be distinguished from those caused by D-glucosamine. Exposure of L1210 cells to tunicamycin resulted in specific alterations in the biochemical composition of the plasma membrane and in the inhibition of cellular agglutination by wheat germ agglutinin which were not apparent following exposure to equitoxic concentrations of the aminosugar. These studies, together with those which demonstrated that recovery of the cellular capacity to synthesize glycoproteins was obligatory for the recovery of cellular proliferation in tunicamycin-treated cells, suggested that inhibition of the synthesis of glycoproteins was the major factor limiting L1210 leukemic cell proliferation.     

Selective inhibition by bis(2-chloroethyl)methylamine (nitrogen mustard) of the Na/K/Cl cotransporter of murine L1210 leukemia cells

Incubation of L1210 murine leukemia cells in vitro with 10 μM of the bifunctional alkylating agent bis(2-chloroethyl)methylamine (nitrogen mustard, HN2) for 10 min brought about a fall of more than 99.9% in their ability to form colonies when the cells were suspended in 0.5% nutrient agar. Incubation with HN2 also inhibited the influx of the potassium congener ⁸⁶Rb⁺ to exponentially proliferating L1210 cells in a concentration-dependent manner. This inhibition was specific and was accounted for by a reduction of a diuretic-sensitive component of ⁸⁶Rb⁺ influx, identified in the preceding paper (Wilcock, C. and Hickman, J.A. (1988) Biochim. Biophys. Acta 946, 359–367) as being mediated by a Na⁺/K⁺/Cl⁻ cotransporter. Inhibition by 10 μM HN2 was complete after a 3-h incubation. There was no inhibition at this time of the ouabain-sensitive component of ⁸⁶Rb⁺ influx, mediated by Na⁺/K⁺-ATPase. After 3 h of incubation with 10 μM HN2 there was also no change in the membrane potential of the treated cells as measured by the distribution of the [³H]TPMP⁺, no decrease in cellular ATP concentration and no change in intracellular pH, and the ability of the cells to exclude the vital dye Trypan blue was not significantly different from control values. These effects of HN2, therefore, appeared to follow lethal damage, but precede cell death. In the stationary phase of L1210 cell growth, the component of HN2 and diuretic-sensitive K⁺ influx to L1210 cells was reduced, whilst the component constituting the HN2-insensitive ouabain-sensitive sodium pump was increased. The monofunctional alkylating agent MeHN1 (2-chloroethyldimethylamine) which cannot cross-link cellular targets and has no antitumour activity, did not inhibit ⁸⁶Rb⁺ influx to L1210 cells when incubated at equimolar or equitoxic concentrations to HN2. Intracellular potassium concentration was maintained close to control values of 138 ± 10 mM in HN2-treated cells because of an approx. 35% fall in cell volume. The results suggest that the Na⁺/K⁺/Cl⁻ cotransporter is a selectively inhibitable target for HN2, and the lesion is discussed with reference to the cytotoxic effects of this agent.