Active oxidative decarboxylation of malate by mitochondria isolated from L-1210 ascites tumor cells

Mitochondria from L-1210 mouse ascites tumor show a very high rate of oxidation of L-malate in comparison with mitochondria from normal tissues. They were found to contain large amounts of malic enzyme (E.C.1.1.1.39) catalyzing oxidative decarboxylation of L-malate to pyruvate. Malic enzyme in extracts of tumor mitochondria requires Mn²⁺ or Mg²⁺, utilizes either NAD⁺ or NADP⁺ as electron acceptor, and shows positive cooperativity in binding of L-malate. These observations suggest that L-1210 tumor mitochondria actively convert excess tricarboxylate cycle intermediates and their precursors into pyruvate for further oxidation.     

Regulation of spermidine transport in l1210 cells

• 1.1. 1 mM 2-amino isobutyric add (AIB), glutamine or asparagine when preincubated for 3 hr with L1210 cells promoted a marked increase in the rate of spermidine uptake.
• 2.2. Cycloheximide also increased the transport rate and completely prevented the increase due to AIB.
• 3.3. Trifluoperazine and iso-H7 inhibited the uptake of spermidine, much less the uptake of AIB.
• 4.4. Adenosine promoted an increase in the uptake of AIB, a decrease in that of spermidine.
• 5.5. Hypotonic stress also increased the rate of spermidine transport. This modification was only partially prevented by cycloheximide.
• 6.6. Okadaic arid had no effect on this increase, whereas it prevented the increase of ODC activity.

     

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.     

Induction of gamma-glutamylcysteine synthetase by prostaglandin A2 in L-1210 cells

Effects of prostaglandin A2 (PGA2) on glutathione (GSH) status in L-1210 cells were examined. When the cells were cultured in the presence of PGA2, a persistent rise of cellular GSH concentration was observed 6 h after the addition of PGA2. This stimulatory effect of PGA2 was abolished if the cells were pretreated with an enzyme inhibitor of GSH synthesis, buthionine sulfoximine. Subsequent study with cell free extract of cultured L-1210 has revealed that PGA2 stimulated the biosynthesis of gamma-glutamylcysteine synthetase (EC 6.3.2.2). Actinomycin D inhibited this stimulatory effect of PGA2 on cultured cells. The optimal pH, Km value for glutamic acid and sensitivity to inhibitors of gamma-glutamylcysteine synthetase from PGA2 treated and nontreated cells were virtually the same. Thus, our findings suggest that PGA2 induced gamma-glutamylcysteine synthetase in cultured L-1210 cells which is responsible for the elevated level of GSH in these cells.     

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.     

Sodium-dependent nucleoside transport in mouse leukemia L1210 cells

Nucleoside permeation in L1210/AM cells is mediated by (a) equilibrative (facilitated diffusion) transporters of two types and by (b) a concentrative Na(+)-dependent transport system of low sensitivity to nitrobenzylthioinosine and dipyridamole, classical inhibitors of equilibrative nucleoside transport. In medium containing 10 microM dipyridamole and 20 microM adenosine, the equilibrative nucleoside transport systems of L1210/AM cells were substantially inhibited and the unimpaired activity of the Na(+)-dependent nucleoside transport system resulted in the cellular accumulation of free adenosine to 86 microM in 5 min, a concentration three times greater than the steady-state levels of adenosine achieved without dipyridamole. Uphill adenosine transport was not observed when extracellular Na+ was replaced by Li+, K+, Cs+, or N-methyl-D-glucammonium ions, or after treatment of the cells with nystatin, a Na+ ionophore. These findings show that concentrative nucleoside transport activity in L1210/AM cells required an inward transmembrane Na+ gradient. Treatment of cells in sodium medium with 2 mM furosemide in the absence or presence of 2 mM ouabain inhibited Na(+)-dependent adenosine transport by 50 and 75%, respectively. However, because treatment of cells with either agent in Na(+)-free medium decreased adenosine transport by only 25%, part of this inhibition may be secondary to the effects of furosemide and ouabain on the ionic content of the cells. Substitution of extracellular Cl- by SO4(-2) or SCN- had no effect on the concentrative influx of adenosine.     

Pentoxifylline influences drug transport activity of P-glycoprotein and decreases mdrl gene expression in multidrug resistant mouse leukemic L1210/VCR cells

The effects of pentoxifylline (PTX) on intracellular accumulation of doxorobicin (DOX), DOX cytotoxicity and expression of Pgp in multidrug resistant L1210/VCR cell line were investigated. PTX (100 mg/l) was able to enhance the DOX accumulation in resistant cells. The maximum intracellular levels of DOX were reached after treatment with PTX for 24 hours (total duration of PTX-treatment was 72 hours). The levels of mdrl mRNA (measured by RT-PCR method) were decreased 2-fold in the presence of 100 mg/l PTX (minimum reached within 48 hours) in comparison to control cells.     

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.     

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.     

Rejection of reovirus-treated L1210 leukemia cells by mice

Summary L1210 leukemia cells were treated in vitro with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and reovirus to determine their interactive effects on rejection of these tumor cells by mice. The cells were treated with BCNU at concentrations of 0, 3, or 10 M, incubated for 48 h, then treated with reovirus at a multiplicity of infection of 0, 10, 30, or 100 for 2, 6, or 12 h. The survival of mice injected with cells treated with any amount of reovirus, regardless of BCNU treatment, was greater than that of mice injected with untreated cells. Exposure of the cells to reovirus for 6 or 12 h increased the survival of mice injected with these cells as compared with that of mice injected with cells exposed to reovirus for 2 h. Of the survivors, 76% were resistant to subsequent challenge with untreated L1210 cells. These results suggest that activities associated with reovirus replication may cause modifications of L1210 cells that enable them to induce an immune response, thus facilitating their rejection. A lack of correlation between differences in DNA synthesis (measured by ³H-thymidine uptake) by treated cells and the ability of those cells to kill recipient mice indicates that rejection of cells treated with reovirus or BCNU is not due to a decrease in their ability to proliferate or, presumably, to generate lethal tumors. The survival of mice injected with treated L1210 cell preparations containing as few as 2.9% reovirus-infected cells was enhanced to the same degree as that of mice injected with those containing as many as 14.6% infected cells, indicating that modification of only a minor component of the tumor cell population is sufficient to alter the ability of the cells to generate a lethal tumor.This work was supported by a research grant from the Miami University Faculty Research Committee and a Sigma Xi Grant-in-Aid of Research     

Isolation and charaterization of surface glycoproteins from L-1210, P-388 and HeLa cells

A method is described that permits the rapid extration of the cell surface glycoproteins of two murine leukemic cells, the P-388 and the L-1210 cells as well as those of the human adenocarcinoma cells, the HeLa cells.Proof of the surface location of these glycoproteins is provided by labeling the intact cells; (a) with ¹²⁵I by the lactoperoxidase iodination technique; (b) with ³H by the galactose oxidase-reductive tritiation method. Most of these glycoproteins were also shown to incorporate radioactive glucosamine and fucose. By these criteria as well as by the distribution of molecular weights, the surface glycoproteins of the two murine cells are indistinguishable; however, they differ markedly from the surface glycoproteins of HeLa cells. The extracts of the murine cells wee shown to contain lectin receptor activity as determined by their ability to inhibit the lectin-induced agglutination of the intact cells.     

Early transport changes during erythroid differentiation of Friend leukemic cells

Early transport changes occurring during Friend erythroleukemic cell differentiation are reported. A decrease in the rate of 86Rb transport was observed beginning approximately five hours after stimulation with 1.5% dimethylsulfoxide (DMSO), a potent inducer of Friend cell differentiation. By 12 to 14 hours after DMSO addition, the transport rate had stabilized at close to 60% of control level. This decrease in the rate of 86Rb transport preceded a previously reported decrease in cell volume. Other chemical inducers of Friend cells, such as hypoxanthine and ouabain, also caused early decreases in 86Rb influx. In contrast, xanthine, which does not induce Friend cell differentiation, also did not affect 86Rb influx. The transport of two amino acid analogues, alpha-aminoisobutyric acid and 2-aminobicyclo [2,2,1]-heptane-2-carboxylic acid, which differ in their mode of uptake, was also measured following induction by DMSO. The transport rates of both compounds decreased after a 12-hour exposure to DMSO. In contrast, the uptake of 3H-colchicine, a drug which diffuses passively across the cell membrane, was not significantly affected. Studies with several variant cell lines which do not synthesize hemoglobin in response to DMSO indicate that these non-inducible cells can be divided into two classes--those that demonstrate early changes in transport very similar to the changes observed in inducible cell lines and those which exhibit only small changes in transport. Results obtained using a revertant clone have helped to distinguish between those transport changes which are associated with the induction of hemoglobin synthesis and those which are not. In addition, these early transport changes may be useful in defining the stage in the differentiation process at which a particular variant line is blocked.     

Nucleoside transport in L1210 murine leukemia cells. Evidence for three transporters

L1210 murine leukemia cells have two nucleoside transport activities that differ in their sensitivity to nitrobenzylmercaptopurine riboside (NBMPR). This study re-examines NBMPR-insensitive nucleoside transport in these cells and finds that it is mediated by two components, one Na(+)-dependent and the other Na(+)-independent. A mutant selected previously for loss of NBMPR-insensitive transport lacks only the Na(+)-independent activity. When NBMPR is used to block efflux via the NBMPR-sensitive transporter, uptake of formycin B (a nonmetabolized analog of inosine) is concentrative in both the parental and mutant cells, but the intracellular concentration of the nucleoside is 5-fold lower in the parental cells. Decreased accumulation of formycin B in the parental cells is due to efflux of the nucleoside via the NBMPR-insensitive, Na(+)-independent transporter that the mutant lacks. The Na(+)-dependent transporter appears to accept most purine, but not pyrimidine, nucleosides as substrates. Two exceptions are uridine, a good substrate, and 7-deazaadenosine, a poor substrate. In contrast, all of the nucleosides tested are substrates for the Na(+)-independent transporter. We conclude that L1210 cells have three distinct nucleoside transporters and that the specificity of the Na(+)-dependent transporter is similar to that of one of the two Na(+)-dependent nucleoside transporters seen in mouse intestinal epithelial cells.