Differential affinities of pyrimidine nucleoside analogues for deoxythymidine and deoxycytidine kinase determine their incorporation into murine leukemia L1210 cells

A number of 5-substituted pyrimidine deoxyribonucleoside (dThd or dCyd) derivatives have been evaluated for their effects on the incorporation of dThd and dCyd into the nucleotide pool and nucleic acids of murine leukemia L1210 cells. Several observations indicate that the dThd kinase and dCyd kinase activity of the cells and the differential affinities of these enzymes for the pyrimidine deoxyribonucleosides determine the incorporation of dThd and dCyd into the cells: (i) dThd and dCyd were not incorporated into mutant L1210 cells deficient in either dThd kinase or dCyd kinase activity; (ii) for a series of 5-substituted dThd and dCyd analogues a strong correlation was found between their inhibitory effects on the incorporation of dThd or dCyd into cell material and their Ki/Km for dThd kinase and dCyd kinase (r = 0.92 and 0.97, respectively); (iii) inhibitors of DNA synthesis (i.e. araC) and RNA synthesis (i.e. actinomycin D) suppressed the incorporation of dThd, most likely due to an inhibitory activity at the dThd kinase level (through the allosteric action of dTTP or slow regeneration of dThd kinase).     

Disruption of DNA synthesis and structure of mouse L1210 leukemia cells, sensitive and resistant to 1-methyl-1 nitrosourea and 1,3-bis(2-chloroethyl)-1-nitrosourea in vivo

Leukemia L1210 cells with acquired resistance to 1-methyl-1-nitrosourea (MNU) (L1210/MNU) and 1.3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (L1210/BCNU) were developed from leukemia L1210 cells sensitive to these drugs (L1210/0). The modal chromosome number of leukemia L1210/MNU and L1210/BCNU cells increases from 40 (L1210/0) to 41. It was shown that in leukemia L1210/MNU cells the inhibition of DNA synthesis after MNU administration in a therapeutic dose (80 mg/kg) is lasted within 24 hours, while that in leukemia L1210/0 cell--within 96 hours. After administration of BCNU (20 mg/kg) inhibition of DNA synthesis in leukemia L1210/BCNU cells reached of 50% of control in comparison with practically complete inhibition of DNA synthesis in leukemia L1210/0 cells. Centrifugation on alkaline sucrose density gradients revealed no differences in the rate of sedimentation of leukemia L1210/0, L1210/MNU and L1210/BCNU cell lysates. After 1 hour treatment with MNU of mice bearing L1210/MNU and L1210/0 leukemia cells single-strand breaks in DNA were determined. After 4 hours these strand-breaks retained in leukemia L1210/0 cells, but were eliminated in leukemia L1210/MNU cells. Administration of BCNU to mice with leukemia L1210/0 and L1210/BCNU cells resulted in both cases in the production of DNA aggregates. There is no complete cross-resistance between MNU and BCNU which allows a substitution of these drugs providing for the increase in their therapeutic efficiency.     

Effect of methotrexate on the leukemic cell cycle in mouse ascitic leukemia L1210]

In cells of L1210 ascite leukemia cells, methotrexate inhibited H3-thymidine incorporation, blocked shortly (during 4 hours) the G1 leads to S transition, and did not affect cells in G2-phase or in the late S phase. Almost half a cell population was degenerated and cells in S- and G1-phases were affected in equal proportion. This may suggest that methotrexate is not S-phase specific for cells of leukemia L1210. A simultaneous administration of vinblastine increases the antitumour effect of methotrexate. Cells in G2-phase constitute, presumably, a significant proportion of cells recovered after methotrexate administration. A comparison of the data obtained with literature evidence shows that in the sensitive (leukemia L1210) and resistant (acute mieloid leukemia of man) forms of leukemia, methotrexate affects cells that are in S-phase, whereas cells being in G1-phase are affected only when the sensitive tumours are treated.     

Role of protein kinase C activators and inhibitors, calmodulin antagonists and membrane sialic acids in polyamine transport in murine leukemia cells

The effects of activators and inhibitors of protein kinase C (phorbol esters and H-7) and antagonist to calmodulin (TFP) on polyamine transport in murine leukemia (L1210) cells are investigated. Phorbol esters and H-7 are found to enhance and curtail the uptake of 14C-Spermidine (Spd) respectively in L1210 cells. TFP also inhibits the uptake process. After desialation of cells with neuraminidase, phorbol esters are found to further increase the uptake of 14C-Spd by 35% compared to untreated cells. The sialic acid contents of the cells are regenerated by incubation with 14C-glucosamine for 18 hours. The regenerated cells mimic like untreated cells for the uptake of 14C-Spd i.e. after regeneration of sialic acids, the Spd uptake is curtailed significantly in comparison with desialated cells. Phorbol esters are found to enhance the activity of transglutaminase present in L1210 cells while H-7 and TFP exhibit reverse effects. The possible role of phorbol esters, H-7 and TFP and their effects on transglutaminase activity in relation with Spd transport process are discussed.     

Strategies for the measurement of the inhibitory effects of thymidine analogs on the activity of thymidylate synthase in intact murine leukemia L1210 cells

Two strategies have been pursued to monitor the inhibition of thymidylate (dTMP) synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) by thymidine (dThd) analogs in intact murine leukemia L1210 cells. The first method was based on the determination of tritium release from 2'-deoxy[5-3H]uridine [( 5-3H]dUrd) or 2'-deoxy[5-3H]cytidine [( 5-3H]dCyd); the second method was based on an estimation of the amount of dCyd incorporated into DNA as dTMP. The validity of these procedures was assessed by evaluating the inhibition of thymidylate synthase in murine leukemia L1210 cells by a series of 18 dThd analogs. There was a strong correlation between the inhibitory effects of the dThd analogs on the proliferation of L1210 cells on the one hand, and (i) their inhibitory effects on tritium release from [5-3H]dCyd (r = 0.926) and (ii) their inhibitory effects on the incorporation of dCyd into DNA dTMP (r = 0.921), on the other hand. Evaluation of tritium release from [5-3H]dCyd proved to be the most convenient method that has been described so far to measure thymidylate synthase activity and to follow the inhibitory effects of thymidylate synthase inhibitors in intact L1210 cells, since this method is rapid and very sensitive, and since it proved superior to the evaluation of tritium release from [5-3H]dUrd because it circumvents possible interactions of the inhibitors with thymidine kinase activity.     

Reversal effect of specific inhibitors of extracellular-signal regulated protein kinase pathway on P-glycoprotein mediated vincristine resistance of L1210 cells

Effect of specific inhibitors of extracellular-signal regulated protein kinase (ERK) pathway, PD98059 and U0126, on P-glycoprotein (Pgp)-mediated vincristine resistance of L1210/VCR cells was investigated. Both test inhibitors significantly reduced the survival of L1210/VCR cells in the presence of vincristine and this was associated with a decrease of LC50 values to vincristine from 2.65+/-0.43 to 0.67+/-0.28 micromol/l and to 0.69+/-0.09 micromol/l after treatment with 50 micromol/l PD98059 and 25 micromol/l UO126, respectively. Moreover, the effects of PD98059 are connected also with an increased intracellular accumulation of radiolabeled vincristine in resistant L1210/VCR cells in concentration dependent manner. The results of this study demonstrate that inhibitors of ERK signaling pathway are reversal agents of vincristine resistance in L1210/VCR cells. The precise mechanism of PD98059 and U0126 action in modulation of MDR is not resolved yet, but the role of ERK-mediated phosphorylation cascade could be considered.     

Phorbol esters augment polyamine transport by influencing Na(+)-K+ pump in murine leukemia cells.

In this report, we elucidate the role of Na(+)-K+ pump in the regulation of polyamine spermidine (Spd) transport in murine leukemia (L 1210) cells in culture. Ouabain, known to bind extracellularly to the alpha-subunit of the Na(+)-K+ pump, inhibits the pump activity. The L 1210 cells were found to possess ouabain binding sites at 7.5 fmol/10(6) cells. Ouabain significantly inhibited the Spd uptake in a dose-dependent manner. The maximum inhibition of Spd uptake by ouabain was observed beyond 200 microM. Spd transport was inversely correlated with the [3H]ouabain binding to L 1210 cells: an increase in the saturation of ouabain binding to L 1210 cells resulted in a decrease of the Spd uptake process. Treatment of L 1210 cells with protein kinase C activator phorbol esters increased the Spd transport and, also, ouabain-sensitive 86Rb+ uptake, a measure of the activity of the Na(+)-K+ pump. H-7, a protein kinase C inhibitor, significantly inhibited the ouabain-sensitive 86Rb+ uptake by L 1210 cells. Phorbol esters stimulated the level, but not the rate, of 22Na+ influx. Addition of H-7 to L 1210 cells inhibited the 22Na+ influx process. A concomitant phorbol ester-induced increase in 22Na+ influx, [14C]Spd uptake, together with the functioning of Na(+)-K+ pump, indicates the role of the "Na+ cycle" in the regulation of the polyamine transport process.     

The role of N-nitrosourea-induced changes in the nucleoid structure and activity of repair enzymes in the development of drug resistance in mice with leukemia L1210

Using centrifugation of the nucleoid in a neutral sucrose gradient, the damages in the secondary structure of DNA and the activity of repair enzymes, such as DNA-polymerases alpha and beta and poly(ADP-riboso) polymerase, induced by 1-methyl-nitrosourea (MNU) and 1.3-bis (2-chloroethyl)-1-nitrosourea (BCNU) injected at maximal nonlethal single doses to mice bearing parent leukemia cells (L1210/0) and resistant to MNU and BCNU leukemia L1210 cells (L1210/MNU and L1210/BCNU), were studied. The MNU-induced production of single-strand breaks in L1210/0 and L1210/MNU cells was more conspicuous in newly replicated DNA than in those in preexisting DNA. A more fast repair of the damages in newly replicated DNA was detected in L1210/BCNU and especially in L1210/MNU leukemia cells as compared with L1210/0 cells. The data obtained suggest that there are prone errors in the repair of DNA template, since most of the single-strand breaks were revealed in the newly replicated DNA synthesized on the repaired DNA. The repair of DNA damages in L1210/BCNU and especially in L1210/MNU cells was accompanied by the activation of DNA-polymerases alpha and beta and poly(ADP-riboso)polymerase. Both DNA-polymerases--alpha and beta--were shown to be involved in repair of DNA damages induced by MNU and only DNA-polymerase beta was involved in the repair of damages induced by BCNU.     

Cathepsin D of mouse leukemia L1210 cells. Unusual intracellular localization and biochemical properties.

Mouse leukemia L1210 cells contain lysosomes, but cathepsin D, a typical lysosomal enzyme, has an unusual localization. After fractionation of homogenates of L1210 cells by isopycnic density gradient centrifugation, most of the activity for all of the acid hydrolases studied, except cathepsin D, is sedimentable and shows a similar density distribution around a peak having a modal density of 1.16. In contrast, much more of the total activity for cathepsin D is not sedimentable, while the sedimentable activity has a distribution around a peak at a higher density of 1.18. After chromatography on Sephadex G-100 of cell extracts, two molecular weight forms of cathepsin D are found. One has an apparent molecular weight of approx. 45,000, similar to rat liver cathepsin D, while the apparent molecular weight of the second form is approx. 95,000. Both forms are 4-5 times more active than rat liver cathepsin D. The high molecular weight L1210 cathepsin D converts to the low molecular weight form with no loss in activity after treatment with beta-mercaptoethanol. In all respects the unusual intracellular localization and molecular weight forms of cathepsin D in mouse leukemia L1210 cells are similar to the situation found for rat thoracic duct lymphocytes.     

A nonfunctional protein in human leukemia cells reacts with antiserum to dihydrofolate reductase from L1210 leukemia cells

Antiserum raised in chickens to dihydrofolate reductase purified from L1210 leukemia cells by affinity chromatography inhibited the catalytic activity and the binding of methotrexate by the enzyme. Lysates of human chronic myelogenous leukemia cells, which had neither catalytic activity for dihydrofolate reductase nor binding of methotrexate, blocked the inhibiting effect of the antiserum on the function of the enzyme in L1210 cell lysates. In double immunodiffusion, these human leukemia cell lysates formed a single precipitin line against the antiserum. These findings indicate that nonfunctional dihydrofolate reductase in human leukemia cells share an antigenic determinant(s) with a functional form of the enzyme from L1210 murine leukemia cells.     

N-myristoyl-transferase activity in cancer cells. Solubilization, specificity and enzymatic inhibition of a N-myristoyl transferase from L1210 microsomes

The activity catalyzed by N-myristoyl transferase (NMT) is described for the first time in microsome-rich fractions from the murine leukemia cell line L1210, rat brain and mouse liver as biological sources. The enzyme from each source can accommodate various types of proteins (protein kinase A, virus structural gag protein or pp60src) as modelized by the use of their N-terminal derived peptides (GNAAAARR, GQTVTTPL and GSSKSKPKDP, respectively). As for some other types of membrane-bound enzymes, NMT activity can be enhanced by pretreatment with various types of detergents, amongst which Triton 770 and deoxycholate were the most potent. Further experiments on the L1210 microsome-rich fractions demonstrate that these two detergents were able to solubilize the microsomal enzyme, without modifying its substrate specificity. Finally, three compounds described in the literature to be inhibitors of NMT activity from other sources were tested for L1210 microsome-associated activity. None of them show any significant potency in inhibiting this activity. A new compound, myristoylphenylalanine, shows a slightly better inhibitory effect on the L1210 microsomal activity than the reference compounds with a median inhibitory concentration (IC50) of 0.2 mM.     

The effects of inhibitors of DNA biosynthesis on the cytotoxicity of 6-thioguanine

The effects of several metabolic inhibitors of DNA synthesis on the antiproliferative activity of 6-thioguanine (6-TG) were examined using cultured L1210 leukemia cels. The presence of hydroxyurea (HU), 1-beta-D-arabinofuranosylcytosine (araC), or 5-fluorodeoxyuridine (FUdR) in cultures of L1210 leukemic cells during exposure of 6-TG did not increase the degree of inhibition of cellular replication produced by the 6-thiopurine, but instead partially protected cells against the delayed cytotoxicity of 6-TG, implying that DNA replication was essential for the expression of cytotoxicity by the purine antimetabolite. Consistent with these results was the finding that synchronized L1210 cells exposed to 6-TG were the most susceptible to the cytotoxic action of the 6-thiopurine during G1/S and S phase. However, G2 phase cells were also sensitive to 6-TG indicating that at least two metabolic lesions are responsible for the production of cytotoxicity. Alkaline sucrose gradient sedimentation of L1210 cells exposed to 6-TG revealed that the purine analog causes structural changes in DNA suggesting that these hitherto unreported lesions may be involved in the cytotoxicity caused by this agent.     

Early progression from dimethyl sulfoxide-induced G(0)/G(1) arrest in L(1210) cells

Recently, dimethyl sulfoxide (DMSO) has been used as a convenient cryoprotectant for stem cells in stem cell transplantation using allogenic peripheral blood or umbilical cord blood. As the stem cells have a multipotency, clarification of the extent of cell proliferation after transplantation is difficult. In the present study, DMSO gradually induced G(0)/G(1) arrest in mouse leukemia L(1210) cells with good cell viability. After removal of DMSO, the cells proliferated appropriately, resulting in expression of the DNA-synthesizing enzymes thymidylate synthase and thymidine kinase within 6h, and the cells entering into S phase within 12h. The sequence was followed by the marked activation of both enzymes within 24h and the increase of bromodeoxyuridine (BrdU) immunoreactive (S phase) cells with rapid cell proliferation within 36 h. In conclusion, mouse leukemia L(1210) cells, which were treated with 1.5% DMSO for 96 h, tolerated the treatment and reversed the cell cycle arrest within 36 h.     

The 6S- and 6R-diastereomers of 5, 10-dideaza-5, 6, 7, 8-tetrahydrofolate are equiactive inhibitors of de novo purine synthesis

The diasteromers of 5,10-dideaza-5,6,7,8-tetrahydrofolate (DDATHF) differing in chirality about carbon 6 were resolved and studied as inhibitors of folate-dependent processes in mouse leukemia cells. Both diastereomers of DDATHF were found to be potent inhibitors of leukemia cell growth due to effects on de novo purine synthesis. Cell growth inhibition by these compounds was prevented by 5-formyltetrahydrofolate in a dose-dependent manner. This indicated that the effects of the DDATHF diastereomers were due to inhibition of folate-dependent processes. Metabolite reversal experiments indicated that 5'-phosphoribosylglycinamide formyltransferase was the major site of action of these compounds in mouse cells. Another site in de novo purine synthesis was affected at higher concentrations of diastereomer B in L1210 cells. Low concentrations of both diastereomers were found to inhibit pure L1210 5'-phosphoribosylglycinamide formyltransferase competitively with the folate substrate. The two diastereomers were also efficient substrates for mouse liver folylpolyglutamate synthetase. We conclude that the 6R- and 6S-diastereomers of DDATHF are remarkably similar and equiactive antimetabolites inhibitory to de novo purine synthesis and that the biochemical processes involved in their cytotoxicity display little stereochemical specificity.     

Induction of immune resistance against L1210 lymphatic leukemia in mice after lethal irradiation and reconstruction with fetal liver cells.

The immunohematopoietic potential of syngeneic fetal liver cells (SFLC) was examined and compared with syngeneic bone marrow cells (SBMC). SFLC generated about 3 times less 12th-day spleen colonies (CFU-S) than adult SBMC did. To test the SFLC ability for reconstitution of the immune system, mice were lethally total body irradiated (TBI) and transplanted i.v. with 3 x 10(7) SFLC or 1 x 10(7) SBMC. Thus, injected hematopoietic cells contained the same number of CFU-S. On days 28, 35, 42, and 49 after transplantation the mice were injected i.p. with 10(6) immunogenic L1210-Maf cells (L1210 leukemia cells treated in vitro with mafosfamide for inhibition of their growth in vivo) to test the ability for generation of immune response against L1210 leukemia. On day 56 the animals were challenged with 10(3) L1210 leukemia cells. Strong resistance against the leukemia was induced in TBI + SFLC and TBI + SBMC mice, suggesting that the SFLC similarly as SBMC are able to reconstitute immune system of the TBI host.     

The ultrastructural morphology of leukemia L 1210 ascites tumor cells transplanted with different frequency

The mouse lymphatic leukemia L 1210 ascites tumor cells were transplanted every 3rd (L1210/3) or 6th day (L 1210/6) and then examined by an electron microscope. In cytoplasm of L 1210 cells thick bundles of 8 nm filaments were observed. Lysosomes were more numerous in L 1210/6 cells. Results obtained from morphometric datas shown that the relative cell volume of L 1210/6 population was 1.59 time larger as compared with L 1210/3. The index of nuclear volume to cell volume was very similar in both populations. The relative volume of endoplasmic reticulum was more than twice larger in the L 1210/3 population. The cell surface area was larger in the L 1210/6 cells as compared with this in L 1210/3 cells, but the increase in the surface was not proportional to the increase of the cell volume. The relative surface area of endoplasmic reticulum cisternae was smaller in L 1210/6 cells than in L 1210/3 population.     

Generation of EPR-detectable nitrosyl-iron complexes in tumor target cells cocultured with activated macrophages.

After immunostimulation, murine macrophages oxidize L-arginine into nitric oxide (NO) which acts as an effector molecule. In this study, we attempted to establish whether activated macrophage-derived NO forms paramagnetic complexes in tumor target cells which do not express by themselves the L-arginine:NO pathway. Accordingly, murine L1210 leukemia cells were cocultivated with activated peritoneal macrophages from Bacillus-Calmette-Guérin-infected mice, or activated in vitro with interferon-gamma. In control experiments, macrophages were prevented from producing nitrogen oxides by incubation with NG-monomethyl-L-arginine, a specific inhibitor of the L-arginine:NO pathway. After coculture, L1210 cells were removed from adherent macrophage monolayers and analyzed by electron paramagnetic resonance at 77 K. In the L1210 cells cultured with activated macrophages, we detected a signal typical of nitrosyl-iron-sulfur complexes, with g values of 2.041 and 2.015. This signal was not present when L1210 cells were either cultured alone or cocultured with activated macrophages in the presence of NG-monomethyl-L-arginine. Mitochondria from activated macrophage-injured L1210 cells also exhibited the signal with g values of 2.041 and 2.015. These results show that when tumor target cells undergo cell-to-cell contact with activated macrophages during culture, the macrophages promote target cell nitrosylation in compartments like mitochondria.     

Synthesis and preliminary antitumor activity evaluation of a DHA and doxorubicin conjugate

A conjugate of DHA and doxorubicin (DHA-Dox) was synthesized, and its antitumor activity was evaluated in vitro against L1210 leukemia cells and in experimental animal tumor models including L1210 leukemia and B16 melanoma. DHA-Dox showed a greatly improved antitumor efficacy compared to free doxorubicin.     

Effect of S-adenosyl-1,12-diamino-3-thio-9-azadodecane, a multisubstrate adduct inhibitor of spermine synthase, on polyamine metabolism in mammalian cells

The effects of the potent spermine synthase inhibitor S-adenosyl-1,12-diamino-3-thio-9-azadodecane (AdoDatad) on polyamine biosynthesis have been studied in transformed mouse fibroblasts (SV 3T3 cells) and in mouse leukemia cells (L1210). A dose-dependent decrease in intracellular spermine concentration was observed in both cell lines when grown in the presence of the inhibitor. A major difference in the effects seen in these two cell lines was the cytotoxicity observed in L1210 cells exposed to the inhibitor, which contrasted with little or no effects on growth of SV 3T3 cells treated similarly. Oxidative metabolism of the drug in L1210 cells was suggested by the fact that addition of aminoguanidine, an amine oxidase inhibitor, to the cell cultures ablated the cytotoxic effects of the inhibitor. Complete analysis of intracellular polyamines was carried out, together with analysis of S-adenosylmethionine, decarboxylated S-adenosylmethionine, and the inhibitor. These analyses revealed that, although the inhibitor had a dramatic effect on spermine biosynthesis in the cells studied, a compensatory increase in spermidine biosynthesis was observed. This resulted in no change in total polyamine concentrations in cells treated with inhibitors of either spermine synthase or spermidine synthase (Pegg et al., 1982) alone or in combination. In all cases, the concentration of the aminopropyl donor decarboxylated S-adenosylmethionine increased dramatically, thus allowing for the observed maintenance of total polyamine levels even in the presence of either one or both potent inhibitors of the aminopropyltransferases. Oxidative metabolism of the inhibitor complicates the interpretation of experiments carried out in the absence of amine oxidase inhibitors such as aminoguanidine.(ABSTRACT TRUNCATED AT 250 WORDS)