L5178Y Lymphoblasts Research Articles

Detection of a Factor Released by L5178Y Lymphoblasts That Inhibits Mouse Macrophage-Activation Induced by Lipopolysaccharides

Background Several factors inhibit cellular immune response by deactivating macrophages, but very few, such as those described here, prevent macrophage activation. Methods Ascites liquid from 12-day-old BALB/c mice bearing 5178Y lymphoma tumors was collected, and cell-free ascites liquid (CFAL) was separated from lymphoblasts. The supernatant (S1) was obtained from the homogenized and centrifuged lymphoblasts Then, macrophage cultures containing 0.2 × 10 6 cells from lymphoma-bearing or healthy mice were added to 10 μL of CFAL or S1, plus 5 μg of lipopolysaccharides (LPS)/mL, 40 U interferon-γ or a blend of both. Macrophages were incubated with CFAL or S1 prior to or after adding the activators to investigate whether any of the previously mentioned lymphoma fractions inhibited macrophage activation or whether they deactivated them. The effect of CFAL or S1 was estimated as the diminution of the amount of nitric oxide released by the experimental macrophage cultures with respect to controls (activated macrophages treated with none of the lymphoma fractions). Results LPS, IFN-γ, and the LPS/γ blend activated macrophages from both lymphoma-bearing and healthy mice. None of the lymphoma fractions deactivated macrophages. CFAL, but not S1, inhibited the macrophage activation, i.e., the percentage of inhibition of nitric oxide releasing 76.7% and 78.1% in macrophages from healthy and lymphoma-bearing mice, respectively. In addition, CFAL was unable to inhibit the macrophage-activation effect of IFN-γ or the LPS/IFN-γ blend. Conclusions Mouse L5178Y lymphoma releases a factor that in vitro inhibits the macrophage activation induced by LPS, but not by IFN-γ controls.

Cytotoxic and Mutagenic Effects of Radon and Radon Daughters on Murine L5178Y Lines Differing in DNA Repair

The effects of 222Rn were measured in mouse L5178Y (LY) lymphoblasts that differ in repair capabilities. Line LY-S1 is deficient in the repair of X-radiation-induced DNA doublestrand breaks, while lines LY-R16 and LY-R83 are presumed to be deficient in the excision of UV-radiation-induced pyrimidine dimers. Line LY-R83 is hemizygous while the other two lines are heterozygous at the thymidine kinase (tk) locus. After exposure to radon the D0's were found to be very similar for the three lines (0.30-0.31 Gy), whereas for X radiation the D0 for line LY-S1 is lower (0.7 Gy) than that for the two LY-R lines (1.3 Gy). Mutant frequencies at the tk locus were higher per gray after treatment with radon than X radiation, but at equitoxic doses the mutant frequencies were similar for X and alpha-particle radiation. A low radon-induced mutant frequency was observed for the hemizygous line, in agreement with the hypothesis that multilocus lesions were induced by the alpha-particle radiation and that mutants bearing intergenic lesions were not recovered in the TK+/- line. The entire active tk allele was lost by 81% of the TK-/- mutants of line LY-R16. In lines LY-S1 and LY-R16, 39-43% of the TK-/- mutants exhibited loss of galactokinase activity, indicating that the mutational lesion inactivating the tk gene frequently extended to the neighboring galactokinase gene.

Modulation of melphalan uptake in murine L5178Y lymphoblasts in vitro by changes in ionic environment

The alkylating agent melphalan is actively transported in mammalian cells by two amino acid transport carriers: the sodium-dependent carrier with substrate preference for alanine-serine-cysteine (system ASC), and a sodium-independent carrier with preference for leucine (system L). The effect of altering the ionic environment of murine L5178Y lymphoblasts was investigated in order to determine not only the direct effects of hydrogen and calcium ions on these transport systems, but also the indirect effects of agents or modulators known to alter intracellular calcium. Melphalan transport followed a bell-shaped distribution curve over a pH range from 3 to 9 with a pH optimum of 4.3 and 4.6 for transport by systems ASC and L, respectively. Those agents that could cause a decrease in cytosolic calcium such as the calcium channel blockers verapamil, diltiazem and nitrendipine, the calcium chelator (ethyleneglycol-bis-(β-aminoethylether) N, N, N′, N′-tetraacetic acid (EGTA) and reduction of pH were found to augment melphalan uptake, whereas conditions that would elevate intracellular calcium such as the calcium ionophore A23187, the calcium channel agonist (−) Bay K 8644, elevation of extracellular calcium and the calcium pump inhibitor trifluoperazine were all found to decrease melphalan uptake. These findings suggest that modification of ionic environment directly or indirectly by agents known to alter intracellular calcium can modulate melphalan uptake

Induction of multilocus lesions by UVC-radiation in mouse L5178Y lymphoblasts

The survival, the mutant frequency and the nature of the DNA alteration responsible for the inactivation of the thymidine kinase ( tk) locus were investigated in 5 strains of mouse L5178Y lymphoblasts exposed to UVC radiation. The nature of the DNA alteration was investigated in independent TK −/− mutants using Southern blot analysis. The concomitant loss of galactokinase (GK) activity in homogenates of individual TK −/− mutants was taken as an indication that the lesion inactivating the tk allele extended to the neighboring galactokinase ( gk) allele. The survival of strains LY-R16 and LY-R83 was decreased to a greater extent than that of strains LY-S1, LY-SR1, and LY-3.7.2C, reflecting a deficiency in excision repair in strains derived from LY-R cells. The TK −/− mutant frequency of strain LY-R83, which is monosomic for chromosome 11 and thus hemizgyous for the tk and gk genes, was only 50% of the mutant frequency of strain LY-R16 which is heterozygous for the tk gene. Moreover, a greatly reduced percentage of individual spontaneous and UVC-induced TK −/− mutants of strain LY-R83 showed loss of GK activity in comparison to the other strains. This result indicates that UVC irradiation induces intergenic mutations and that such mutants are poorly recovered in the hemizygous strain. Strain LY-3.7.2C appears to have only one active galactokinase ( gk) allele, and very few TK −/− mutants of this strain showed loss of GK activity, possibly because this strain, although heterozyogous for the tk gene, is hemizygous in the region of the gk gene. Strains LY-R16 and LY-S1 are deficient in the repair of UVC- and X-radiation-induced damage, respectively, and the percentage of TK −/− mutants with intergenic mutations was higher for strain LY-R16 after UVC-radiation and for strain LY-S1 after X-radiation. These results indicate that unrepaired DNA lesions lead to an increase in intergenic mutations.

Uptake and Binding of 3′-(3-Cyano-4-morpholinyl)-3′-deaminoadriamycin and 3′-(4Morpholinyl)-3′-deaminoadriamycin in L5178Y Lymphoblasts In Vitro

3'-(3-Cyano-4-morpholinyl)-3'-deaminoadriamycin (CMA) and 3'-(4-morpholinyl)-3'-deaminoadriamycin (MA) are analogues of Adriamycin, with altered cytotoxic activity. CMA is 100- to 1500-fold more cytotoxic than Adriamycin and possesses unique DNA crosslinking activity. Intact MA does not crosslink DNA and has a cytotoxicity equivalent to Adriamycin, but it retains its activity in anthracycline-resistant cells. In this study, uptake and binding of [3H]CMA and [3H]MA in L5178Y lymphoblasts were examined. Both CMA and MA were rapidly taken up by cells at 37 degrees C and concentrated almost exclusively in the nucleus. All of the intracellular MA was TCA-soluble, but only 45% of this drug effluxed from the cells by 4 hr. More than 50% of CMA in the cells was TCA-insoluble, and approximately 40% effluxed from the cells by 4 hr through loss of the TCA-soluble fraction. CMA differed from other alkylating agents in that more than 97% of the bound drug was associated with DNA. The bound drug was partially lost from the DNA by a process that may have involved DNA repair.

Diacylglycerols Enhance the Anti‐tumor Effect of Glucocorticoid on L5178Y Lymphoblasts in vivo

1,2‐Dioleoyl‐rac‐glycerol, a potent activator of protein kinase C, was found to enhance the growth‐inhibitory effect of triamcinolone acetonide on L5178Y lymphoblasts in adrenalectomized, male DBA/2 mice. On the other hand, in mice without adrenalectomy, it markedly inhibited tumor growth without increasing the plasma level of corticosterone or adrenocorticotropic hormone or reducing the body weight. These results suggest that diacylglycerol enhances the action of endogenous glucocorticoid to a sufficient level to inhibit the growth of lymphoblasts. Of various diacylglycerols with different carbon chain lengths tested, 1,2‐dioleoyl‐rac‐glycerol was the most potent growth inhibitor and was maximally effective at a dose of above 30 μg/100 g body weight. This finding suggests that diacylglycerols may he useful for enhancing the antitumor effect of a low dose of glucocorticoid or endogenous glucocorticoid on lymphoblasts without any significant side effect.

Modulation of membrane transport of alkylating agents and amino acids by an analog of vasopressin in murine L5178Y lymphoblasts in vitro

The synthetic vasopressin analog 1-deamino-8- d-arginine vasopressin (dDAVP) has been shown to influence a wide range of cell-membrane-related events. Accordingly, the effect of dDAVP on membrane transport of various alkylating agents and amino acids was evaluated in L5178Y lymphoblasts in vitro. dDAVP stimulated melphalan uptake but conversely inhibited uptake of nitrogen mustard, choline (the natural transport substrate for the nitrogen mustard carrier), and leucine. No effect on the uptake of cyclophosphamide or glutamine was observed. Increased melphalan uptake was due to effects on both substrate influx and efflux. The effect of dDAVP on melphalan influx was particularly complex: dDAVP stimulated melphalan influx by amino acid transport system ASC but inhibited influx by system L, resulting in a net increase in unidirectional drug influx. Melphalan efflux was inhibited by dDAVP. Decreased uptake of nitrogen mustard, choline and leucine was due, at least in part, to decreased substrate influx. However, the mechanisms of inhibition were dissimilar: inhibition of substrate influx was non-competitive for choline but competitive for leucine. In conclusion, dDAVP induced diverse but apparently specific effects on membrane transport of several alkylating agents and amino acids. Since the accumulation of alkylating agents such as melphalan within tumor cells is a major determinant of cytotoxicity, dDAVP may have a role as a biological response modifier.

Studies on biomodulators of glucocorticoid actions; the nature and the modes of actions of glucocorticoid potency amplifiers

We have found many compounds that amplify the action of glucocorticoid without themselves having any glucocorticoid-like action and have proposed the concept of ‘Glucocorticoid Action Biomodulators’. These biomodulators consist of ‘Glucocorticoid Sensitivity Amplifiers’, which greatly amplify the action of glucocorticoid at doses of glucocorticoid that alone have minimal effects, and ‘Glucocorticoid Potency Amplifiers’, which markedly enhance the effect of glucocorticoid at doses that have maximal effects. Potent activators of protein kinase C, such as 1,2-racemic dioctanoylglycerol, 12- o-tetradecanoyl-phorbol-13-acetate, and epidermal growth factor (EGF), markedly enhanced the induction of tyrosine aminotransferase and ornithine decarboxylase by dexamethasone in adrenalectomized rats in vivo and in primary cultures of adult rat hepatocytes in vitro. They amplified enzyme induction by even a large amount of dexamethasone that had a maximal effect, but had no effect in the absence of glucocorticoid. These modes of amplification show that these compounds are ‘Glucocorticoid Potency Amplifiers’. They amplified not only enzyme induction in liver but also growth inhibition by glucocorticoid of solid tumor L5178Y lymphoblasts. They specifically amplified the actions of glucocorticoids and did not amplify the actions of other steroids, such as 17-β estradiol, glucagon and insulin. The induction of tyrosine aminotransferase by glucocorticoid and its amplification by EGF were both inhibited by 1-(5-iso-quinoline-sulfonyl)-2-methyl-piperazine, an inhibitor of protein kinase C, and not by N-[2-(methylamino)-ethyl]-5-isoquinoline-sulfonamide, an inhibitor of cyclic nucleotide dependent protein kinases, suggesting that the induction and the amplification are mediated by protein kinase C.

Studies on the mechanism of action of quinone antitumor agents

The presence of a quinone group in the structure of a compound has been shown to produce cell kill and DNA strand breaks by a mechanism involving free radicals and active oxygen species. The ability of the compound to bind to DNA appeared to increase the DNA damage induced and the cytotoxic activity. A new series of model compounds has been used to investigate further the role of the quinone group in the mechanism of action of quinone antitumor agents. Bis(dimethylamino)benzoquinone, which contains a quinone group, produced significant cell kill of L5178Y lymphoblasts and induced concentration-dependent single-strand and double-strand breaks in the DNA of these cells. Benzoquinone dimustard, which possesses a quinone moiety and active alkylating groups, was approximately 2500 times more cycotoxic to L5178Y cells than was bis(dimethylamino)benzoquinone and was approximately 200-fold more active in inducing DNA double-strand breaks than was the quinone agent. Benzoquinone dimustard induced no apparent DNA single-strand breaks, but produced significant DNA cross-linking, a process which interferes with the assay for single-strand breaks. The cell kill produced by both quinone agents was inhibited by catalase, but not by superoxide dismutase. The cytotoxic activity of bis(dimethylamino)benzoquinone and two other quinone model compounds, hydrolyzed benzoquinone mustard and benzoquinone mustard, appeared to correlate with the induction of DNA strand breaks, while there appeared to be no correlation between cell kill and DNA doublestrand breaks induced by benzoquinone dimustard. However, the cytotoxicity of benzoquinone dimustard appeared to be related to the cross-linking activity of this agent. These studies have provided additional evidence that the presence of a quinone group in the structure of a compound can result in significant cell kill by a mechanism that appears to involve active oxygen species. Quinone containing agents can induce DNA strand breaks, and this effect is enhanced when the agent is able to bind to DNA. The induction of DNA strand breaks appeared to correlate with cytotoxic activity for bis(dimethylamino)benzoquinone, hydrolyzed benzoquinone mustard and benzoquinone mustard, but not for benzoquinone dimustard, suggesting that the contribution of quinone-induced strand breaks to the overall cytotoxicity of an agent may vary considerably.

Induction of DNA single- and double-strand breaks by diethylstilbestrol in murine L5178Y lymphoblasts in vitro

The mechanism of action of the synthetic estrogen diethylstilbestrol (DES) was investigated in murine L5178Y lymphoblasts. The dose-survival curve of cells treated with DES in serum-free medium for 1 hr was characterized by a prominent shoulder followed by a simple exponential decline; the D 0, the dose of DES reducing cell survival to 1/ e, was 1.52 nmoles/ml. DNA single-strand breaks, as measured by the alkaline elution method, were observed in DES-treated cells, and these followed a dose-response relationship after an apparent threshold of 10 μM DES was exceeded. Protein-associated strand breaks, which represent the increment in single-strand breaks that occurs by exposing drug-treated cells to proteinase K, were also noted. DNA double-strand breaks as measured by filter elution technology at pH 9.6 were observed and increased markedly to reach a level of approximately 9000 rad equivalents at a DES concentration of 20 μM. The measured ratio (mean ± S.E.) of single- to double-strand breaks induced by DES in L5178Y limphoblasts was 0.09 ± 0.035. A comparison of the ratio of single- to double-strand breaks induced by DES to that observed following radiation suggested that all of the single-strand breaks produced by DES could be attributed to double-strand breaks. The close correspondence of the dose-response curve for cytocidal activity of DES with that obtained for induction of DNA double-strand breaks suggested that such breaks may play an important role in the mechanism of cell kill by DES.

Identification of differential drug responses and mechanism(s) of resistance in vincristine-resistant cell lines developed either by exposure to the drug or to fractionated radiation

Clinical resistance to antitumour drug therapy is known to occur not only following repeated courses of chemotherapy but also in patients who have received prior radiotherapy. For example: in head and neck cancer when chemotherapy is used as initial treatment response rates of approximately 70% are frequently encountered but the same drug protocols in previously irradiated patients result in only 40% to 50% of patients responding (15, 18); similarly, in testicular teratomas it is considered that radiotherapy may impair future chemotherapy ( 14). This reduced response generally is considered to be associated predominantly with impairment of the blood supply to the tumour resulting from radiation-induced vascular fibrosis. However, it should be noted that even in these patients with recurrent disease objective responses to chemotherapy frequently occur in slightly less than half of those treated but the duration of response is generally short. Hence, the addition of chemotherapy has not had a significant impact on overall survival figures. This initial, but unsustained response argues against markedly impaired drug delivery to previously irradiated tumours and implies involvement of cellular phenomena. For example, it is possible that radiatiqn treatment may have induced drug resistance and thus subsequent drug treatment could brovide a positive selection pressure for these resistant tumour cells, resulting in the out-gfowth of a drug-resistant population. We have, therefore, used continuous human cell lines in culture to determine (i) does X-ray treatment of tumour cells ib vitro lead to subsequent drug resistance? and if so, (ii) are these drug resistant cells qomparable to those in which resistance has been induced by repeated exposure to sbecific antitumour agents? This paper describes the derivation of these two types of drug resistant cell populations using murine L5178Y lymphoblasts, which exhibit resistance to vincristine, provides details of their patterns of

Uptake and decomposition of chlorambucil by L5178Y lymphoblasts in vitro

The uptake of [ 14C]chlorambucil by L5178Y lymphoblasts was studied using thin-layer chromatography to identify the various radioactive components that enter or leave cells. Theoretical calculations predicted that entry of chlorambucil into cells by simple diffusion would be rapid and essentially complete in 45 sec or less. Uptake of intact chlorambucil was rapid, reaching a cell/medium ratio of approximately 1.5 in less than 15 sec at both 37° and 4°, consistent with a simple diffusion mechanism. In cells treated with [ 14C]chlorambucil for 60 min, the intracellular level of intact drug decreased with time, and this decay was attributed to hydrolysis and alkylation. The level of intact drug in the medium decreased at a similar rate resulting in a nearly constant cell/medium distribution ratio. Intact chlorambucil in the cells was found to be entirely ethanol- and trichloroacetic acid-soluble. Efflux of intact chlorambucil was very rapid and temperature-insensitive. These findings suggest that chlorambucil efflux, as well as influx, is by a simple diffusion mechanism. A derivative of chlorambucil was found in ethanol solutions of the drug. This derivative, which may be the ethyl ester of the drug, is highly concentrated in cells and may interfere with pharmacological investigations of chlorambucil.

Heterogeneity of DNA methylation in murine L5178Y lymphoblasts.

Comparison of the extent of methylation in mouse DNA fragments rendered MgCl2 soluble after mild DNase II digestion of nuclei, with different reassociation rate and nucleoli-bound, revealed the existence of 3 regions of the genome particularly 5-methylcytosine-rich: the sequences considered to be related to the transcriptionally active chromatin with the highest content of this base and fast reassociating, as well as nucleolar DNA with somewhat lower proportion of the methylated cytosines.

Effects of asparagine synthetase inhibitors on asparaginase resistant tumors

Inhibitors of asparagine synthetase have been selected with an asparagine-independent sub-line of L5178Y lymphoblasts in culture. Five of nineteen asparagine analogs showed selective inhibition of asparagine synthetase. The order of inhibition in vitro of cell growth and asparagine synthetase is: l-β- aspartyl methylamide > l-β- aspartyl hydroxamate > l-β- aspartyl hydrazide > d,l- threo-β- met > d,l-α-N- methylasparagine . The most effective inhibitor of asparagine synthetase, β-aspartyl methylamide, was competitive with glutamine ( K i , = 2 × 10 −4M) and ammonia. The β-amide was also relatively resistant to hydrolysis by Escherichia coli and Erwinia carotovora asparaginases. The β-amide had a plasma half-life of 90 min in mice; was concentrated 5-fold in leukemic cells from ascites fluid; and was metabolized to an α- N-acetyl derivative which was excreted in the urine. The compound increased life span from 31 to 79 per cent when administered to mice bearing various asparaginase resistant tumors.

Studies on the transport and distribution of diaminopyrimidines in L5178Y lymphoblasts in cell culture

Studies on the transport and distribution of diaminopyrimidines in L5178Y lymphoblasts in cell culture

Levels of cyclic AMP in murine L5178Y lymphoblasts grown in different concentrations of serum

Murine L5178Y lymphoblasts were grown in different concentrations of horse and fetal calf serum. Intracellular levels of cyclic AMP, determined throughout the growth period of these cells, did not vary significantly. Furthermore, logarithmically growing cells that became stationary did not show the pronounced increase in levels of cyclic AMP that is characteristic of other cell types when they become confluent or quiescent. These results demonstrate that the unrestricted growth of lymphoblasts is independent of the serum concentration used and may be due to an aberration in the cyclic AMP system.

Comparison of the effects of methotrexate and pyrimethamine on L5178Y lymphoblasts in cell culture

The effects of methotrexate and pyrimethamine were compared against methotrexate sensitive and resistant sublines of L 5178Y lymphoblasts. Against the sensitive subline methotrexate on a molar basis was more effective in reducing cell viability. Against a subline that was resistant to methotrexate by virtue of impaired transport of the drug, pyrimethamine proved superior to methotrexate in reducing cell viability.

Incorporation of radioactivity into phospholipid fractions of L5178Y lymphoblasts after treatment with hydrolyzed nitrogen-14C mustard.

The distribution of radioactivity in the phospholipid fractions of L5178Y lymphoblasts following treatment with choline-14C and hydrolyzed nitrogen-14C mustard (HN2-OH-14C) was investigated. Incorporation of radioactivity into phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine, and neutral lipid was observed following treatment with HN2-OH-14C but radioactivity was found only in phosphatidyl choline with choline-14C serving as precursor. Enzymatic hydrolysis of phosphatidyl choline liberated most of the radioactivity into the aqueous fraction with choline-14C serving as precursor. However, most of the radioactivity remained in the non-aqueous phase after enzymatic or acid hydrolysis of lecithin with HN2-OH-14C acting as precursor and thin layer chromatography of the non-aqueous, acid hydrolysate revealed 25% of the activity in the lecithin fraction, 38% in the 1,2-diglyceride fraction, and 32% in the non-esterified fatty acid fraction, suggesting possible incorporation of drug into fatty acids. The degree of incorporation of HN2-OH into the lipid fraction compared quantitatively with the amount of active nitrogen mustard bound to DNA and RNA.

Schedule dependence of the lethal effects of 6-azauridine and cytosine arabinoside on murine leukemia cells (L5178Y) in culture

The antineoplastic drugs 6-azauridine and cytosine arabinoside exhibit a supra-additive lethal effect on murine L5178Y lymphoblasts if exposure to 6-azauridine precedes exposure to cytosine arabinoside; an additive effect is seen if cytosine arabinoside precedes 6-azauridine, while a sub-additive effect is obtained when the two drugs are present simultaneously. The potentiation of the effect of cytosine arabinoside by 6-azauridine increases for 2 1 2 hours following the removal of the 6-azauridine from the culture.

Repair replication in x-irradiated lymphoma cells in vitro.

SummaryRepair replication using isopycnic CsCl density—gradient techniques has been studied in P388 cells and in two lines of L5178Y lymphoblasts showing a large difference in radiosensitivity. The results indicate that in all three cell-lines only small numbers of bases < 10 are inserted/damaged site. The radiosensitive L5178Y cell line consistently showed greater base insertion/damaged site than the radioresistant line and P388 cells. DNA of the two L5178Y cell-lines was equally sensitive to breakage by x-irradiation, as shown by alkaline sucrose gradient sedimentation techniques. A component of DNA associated with a lipid containing material has been identified which contaminates native DNA after all extraction procedures used. Characterization of this material suggests that it represents DNA attached to a membrane fraction. Such material when present may be mistaken for ‘repair’ incorporation. The present data fit the generalization that only small numbers of bases are inserted/break and are in close ...