In vivo growth of mouse leukemia L1210 cells with metabolic alterations in the subunits of ribonucleotide reductase

Mouse leukemia L1210 cells selected for resistance to drugs targeted specifically at each of the protein subunits of ribonucleotide reductase were studied for their ability to grow in vivo. The life-span of the mice injected with hydroxyurea-resistant L1210 cells, which have elevated levels of the mRNA and protein for the non-heme iron (NHI, R2) subunit of ribonucleotide reductase, was approximately twice that of the mice injected with equal numbers of the parental wild-type L1210 leukemia cells. The life-span of mice injected with the L1210 cells that had alterations in the effector-binding subunit (EB, R1) was considerably shorter than the mice injected with the parental wild-type L1210 cells. These results provide direct evidence that tumor cells with alterations in the properties of ribonucleotide reductase grow differently in vivo, with defined effects on the host mouse that cause either an increased survival time or a decreased survival time compared to the effects of wild-type L1210 leukemia cells on tumor-bearing mice.     

N-Methylformycins. Reactivity with adenosine deaminase, incorporation into intracellular nucleotides of human erythrocytes and L 1210 cells and cytotoxicity to L 1210 cells.

The N-methyl derivatives of the C-nucleoside, formycin (7-amino-3(β-d-ribofurano-syl)pyrazolo[4, 3-d]pyrirnidine) were compared to formycin and adenosine with regard to their substrate activity with human erythrocytie adenosine deaminase (ADA), their ability to form intracellular nucleotides and their cytotoxicity to L1210 cells. Only 2-methylformycin (K_m = 6.1 mM, relative V_max = 396) and N^? -methylformycin (K_m = 0.1 mM, relative V_max = 3) showed substrate activity with ADA (corresponding kinetic parameters for adenosine were: K_m = 0.025 mM, relative V_max = 100). In contrast to previous hypotheses, these results suggest that the conformation (either syn or anti) of an adenosine analog is not a major factor in determining substrate activity with ADA. Neither 4-methylformycin nor 6-methylformycin formed their corresponding nucleotides when incubated with human erythrocytes, whereas both 1-methylfor-mycin and 2-methylformycin formed large amounts of their corresponding mono-, di- and triphosphate nucleotides. Inhibition of ADA by pretreatment of the erythrocytes with the potent ADA inhibitor, 2'-deoxycoformycin, had no effect on the incorporation of 1-methylformycin into erythrocytic nucleotides but greatly increased the incorporation of 2-methylformycin and N⁷-methylformycin. The conversion of both 1-methylformycin and 2-methylformycin into nucleotides was almost complete after 18 hr of incubation (in the presence of 2'-deoxycoformycin in the case of 2 methylformycin), whereas that of N⁷-methylformycin was only partially complete in the presence of 2'-deoxycoformycin. With both 1-methylformycin and N⁷-methylformycin, transient accumulation of the corresponding nucleoside 5'-monophosphate derivative was observed prior to the accumulation of the triphosphate nucleotide. Results, qualitatively similar to those found with erythrocytes, were obtained when the effects of 2'-deoxycoformycin on the incorporation of 1-methyl- and 2-methylformycins into the nucleotide pools of L 1210 cells in vitro were examined. Compounds capable of forming analog nucleotides in human erythrocytes or L1210 cells if deamination is prevented either by the molecular structure of the analog or by pretreatment of the cells with 2'-deoxycoformycin, also showed marked cytotoxicity to L1210 cells in culture, i.e. 1-methyl-, 2-methyl- and N⁷-methylformycin exhibited id₅₀ values of 0.5 to 2 μM, whereas 4-methyl- and 6-methylformyein were not significantly growth inhibitory. The potential usefulness of the various N-methyl derivatives of formycin (alone or in combination with an ADA inhibitor) as cytotoxic or antiviral agents is discussed.     

Formation of a prostaglandin A2-glutathione conjugate in L1210 mouse leukemia cells.

Prostaglandins containing a cyclopentenone moiety are potent antiviral and antigrowth compounds. Some evidence indicates that these prostaglandins are conjugated to glutathione by cells. However, the metabolism of one group, the prostaglandins of the A type, is unclear due to conflicting reports. We studied the uptake and metabolism of prostaglandin A2 (PGA2) in mouse L1210 leukemia and L929 fibroblast cell lines in which this prostaglandin has antiviral and antigrowth effects. Both cell types took up the PGA2 and then metabolized it to a more polar compound. Inside L1210 cells, PGA2 was initially conjugated to glutathione and then reduced at the 9-keto position to form 9-OH-PGA2-GSH. The 9-OH-PGA2-GSH was then secreted from the cells and apparently degraded to form the CysGly and Cys derivatives. Intracellular glutathione was decreased markedly by the addition of the PGA2 in L1210 and L929 cells. This result confirms that conjugation of PGA2 to glutathione occurs in both cell types. Formation of the 9-OH-PGA2-GSH and other glutathione-related conjugates was prevented when glutathione was depleted by growth in buthionine sulfoximine. The glutathione-depleted cells were insensitive to the cytotoxicity of the PGA2, suggesting that one of the glutathione-related conjugates may be involved in the cytotoxicity of PGA2. These results end the controversy over the metabolism of PGA2 and suggest mechanisms for its antiviral and antigrowth actions.     

Potentiation by 2'-deoxycoformycin of the inhibitory effect of xylosyladenine on nuclear RNA synthesis in L1210 cells in vitro

The effect of the adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF), on the inhibitory effect of 9-β-d-xylofuranosyladenine (XA) on nuclear RNA synthesis was examined in L1210 cells in vitro. Pretreatment of cells for 15 min with a 100 per cent inhibitory dose (1 × 10^?6 M) of dCF resulted in approximately a 3- to 8-fold reduction in the 50 per cent inhibitory dose (id₅₀) of XA for [³H]uridine and [³H]thymidine incorporation into RNA and DNA respectively. The id₅₀ for XA for RNA synthesis vs DNA synthesis was 5-fold lower in the absence of dCF and 20-fold lower in the presence of dCF, indicating the greater sensitivity of RNA synthesis to this inhibitor. Fractionation of nuclear RNA into rRNA, non-poly(A) heterogeneous RNA and poly(A)heterogeneous RNA revealed the latter species of RNA to be less sensitive to XA in the absence of dCF; however, in the presence of dCF, all three species of nuclear RNA showed similar sensitivities. Nuclear polyadenylic acid synthesis was among the most sensitive RNA fractions to XA, and was also inhibited to a greater degree by pretreatment of cells with dCF. These results indicate that XA is potentiated markedly by inhibition of adenosine deaminase, and that deamination serves as a major catabolic route for this drug.     

Ergosterol-conferred sensitivity to amphotericin B in cultured L1210 mouse leukemia cells

The sensitivity of cellular membranes to disruption by amphotericin B (AMP-B) depends on the sterol content of such membranes. L1210 murine leukemia cells, grown in medium containing ergosterol and then briefly treated with AMP-B, were more sensitive to the antibiotic than control populations. Cell lysis and damage were assessed by estimation of cell number, trypan blue uptake and cell viability by cloning in soft nutrient-agar. Cells grown in ergosterol required less than half the AMP-B needed for equivalent damage to controls, and were reduced in number and viability by over 99.9 per cent at an AMP-B concentration which barely affected the reproductive capacity of control cell populations.     

Membrane transport of mitoxantrone by L1210 leukemia cells

Transport of radiolabeled mitoxantrone, a new antineoplastic agent, was studied using cultured mouse L1210 leukemia cells. The initial velocity of influx remained linear for about 90 sec and was 110 pmoles/10(6) cells measured at 60 sec. The steady-state accumulation of about 480 pmoles/10(6) cells was not reached until 30 min. The unidirectional drug influx was linear from 0 to 1000 microM extracellular drug concentration. The initial uptake was relatively temperature independent between 37 degrees and 27 degrees, but accumulation at steady state was 17% lower at 27 degrees. None of six metabolic inhibitors had an appreciable effect on initial uptake. Efflux was initially exponential with a half-life of 2.8 min; this efflux and the residual drug concentration plateau were not affected by KCN or verapamil. Under steady-state conditions, about 86% of the cell-associated label was contained in parent drug and the remainder in an unidentified metabolite. These studies indicate that the mechanism of mitoxantrone uptake is passive diffusion. The efflux is not energy requiring, but there is considerable tight binding of the drug to cellular structures.     

Studies on inhibition of adenosine deaminase by isocoformycin in vitro and in vivo.

Isocoformycin is a structural isomer of coformycin which has been demonstrated to be a potent inhibitor of adenosine deaminase. Isocoformycin showed a weaker inhibition of this enzyme than coformycin; the binding of coformycin to enzyme was irreversible, but isocoformycin inhibition was competitive with substrate. The Ki value of isocoformycin was 4.5 approximately 10 X 10(-8) M. Following intraperitoneal injection of isocoformycin in mice, the adenosine deaminase activity of homogenates of several organs was determined and the following ED50 values (50% inhibition doses) were observed: 29 mg/kg for thymus, 13 mg/kg for spleen, 80 mg/kg for liver and 20 mg/kg for kidney. The inhibition of adenosine deaminase in rabbit blood in vitro was also tested in comparison with coformycin.     

Protein-associated deoxyribonucleic acid strand breaks produced in mouse leukemia L1210 cells by ellipticine and 2-methyl-9-hydroxyellipticinium

DNA intercalating agents, including ellipticine, had been found previously to produce protein-associated DNA single-strand breaks and double-strand breaks in mammalian cells. The relationship between these effects on DNA and cytotoxicity could not be determined reliably for ellipticine, because of the poor solubility characteristics of this compound. Studies were therefore carried out using the cationic derivative, 2-methyl-9-hydroxyellipticinium (2-Me-9-OH-E⁺), which has adequate water solubility and retains antitumor activity. DNA single-strand breaks (SSB) and DNA-protein crosslinks (DPC) were measured using the alkaline elution (pH 12) technique, and double-strand breaks (DSB) were measured by the neutral elution (pH 10) method. The effects of ellipticine and 2-Me-9-OH-E⁺ were compared in mouse leukemia L1210 cells. Like ellipticine, moderate concentrations of 2-Me-9-OH-E⁺ produced protein-associated SSB, indicated by the appearance of SSB and DPC at approximately equal frequencies and localized with respect to each other. Below 20 μM (1-hr treatments), the effects of the two drugs were comparable in magnitude. At higher concentrations, ellipticine produced extensive DNA breakage not associated with protein; this is perhaps secondary to an action on membranes or other non-DNA targets. However, 2-Me-9-OH-E⁺ produced only protein-associated strand breaks, even at 4-fold higher concentrations. The two compounds produced similar and relatively large extents of double-strand scission. The measured DSB/SSB ratio was higher than that produced by X-ray or certain other intercalators that have been similarly studied. The DNA effects of 2-Me-9-OH-E⁺, unlike those of ellipticine, were readily reversible and, therefore, permitted a meaningful correspondence between the magnitudes of the DNA effects and the inhibition of colony-forming ability. Comparison with two other types of intercalating agents indicated that neither the SSB nor the DSB predicts the magnitude of cell killing.     

Antibiotic binding to human polymorphonuclear neutrophils, mouse leukemia L1210 cells and mouse thymocytes

This report describes a system in which antibiotics could be compared for binding to different mammalian cells. These included functional phagocytes (human polymorphonuclear neutrophils; PMNs), non-phagocytic lymphocytes (mouse thymocytes), and non-functional leukocytes (mouse leukemia L1210 cells). When antibiotics bound to PMNs, they bound about the same to L1210 cells but much less to thymocytes. Combining these data with previous data, the ranking of cells that bound the greatest amount of antibiotics was: PMNs = L1210 cells = blood mononuclear leukocytes greater than thymocytes greater than erythrocytes. Thus, antibiotics bind differentially and not indiscriminately to mammalian cells.     

Induction of antitumor resistance to mouse leukemia L1210 by spergualins

Spergualin and its analog, 15-deoxyspergualin showed a marked antitumor effect against L1210 by intraperitoneal and oral administrations. After treatment with these substances 40- or 60-day survivors (cured mice of L1210) were resistant to reinoculation of L1210 cells. They were resistant only to L1210. The antitumor effector cells in these mice were determined to be T cells. NK activity of spleen cells was also enhanced by spergualins. The antitumor activity of 15-deoxyspergualin was markedly reduced in immuno-deficient mice. IL (interleukin)-2 production, but not IL-1, was enhanced in supernatant of mixed lymphocyte cultures by treatment with 15-deoxyspergualin. The mechanism of action of 15-deoxyspergualin on the immune system was discussed.     

Survival of L1210 leukemia cells and normal hematopoietic stem cells following in vivo administration of cyclocytidine

Summary The survival of L1210 cells following increasing doses of cyclocytidine (cyclo-C) was determined by spleen colony assay. For single dose, cyclo-C was effective reaching a fractional survival of less than 10^–3 at 10 mg/mouse. This is significantly more effective than a similar dose of arabinosyl cytosine (ara-C) given by rapid infusion. The time-survival curve demonstrated extended cytotoxicity following a single 10 mg/mouse dose of cyclo-C as expected for a long-acting agent. When a 24-hour infusion was examined, however, ara-C was more effective than cyclo-C by a factor of about 10. The clinical implication of this is discussed.     

Cytotoxicity of bacteriohopane-32-ol against mouse leukemia L1210 and P388 cells in vitro

To investigate the biological activities of the hopane group of pentacyclic triterpenoids, we isolated one hopanoid, bacteriohopane-32-ol from Rhodopseudomonas palustris and tested its cytotoxicity against mouse leukemia cells in vitro. The IC50 of the hopanoid for L1210 and P388 was 22 and 19 microM respectively. This activity was slightly reduced by co-incubation with cholesterol. As the mechanism of cytotoxic action, disturbances of membrane function and metabolism are discussed.     

Rapid determination of thymidylate synthase activity and its inhibition in intact L1210 leukemia cells in vitro

A rapid and convenient tritium release assay for measuring thymidylate (dTMP) synthase activity and its inhibition within intact mammalian cells is described in detail. Short-term incubation of murine leukemia L1210 cells with an appropriately labeled substrate precursor, either deoxyuridine ([5-3H]dUrd) or deoxycytidine ([5-3H]dCyd), allowed for: (1) uptake and intracellular conversion to the substrate deoxyuridylate ([5-3H]dUMP); and (2) the obligatory displacement of tritium from [5-3H]-dUMP during the dTMP synthase catalyzed reaction. Tritium released into the aqueous environment was quantitated after a quick one-step separation of tritiated H2O from other radiolabeled materials and cell debris. The amount of tritium released was evaluated as a function of a number of variables, including the concentration of labeled substrate precursors, cell number, and incubation time. Tritium from [5-3H]dCyd was released significantly faster than from [5-3H]dUrd under a variety of conditions. Both 5-fluorodeoxyuridine (1 microM) and methotrexate (10 microM), which effectively block intracellular dTMP synthesis, completely inhibited the release of tritium from either [5-3H]dCyd or [5-3H]dUrd demonstrating that the release of tritium is mediated exclusively by the dTMP synthase catalyzed reaction. In addition, there was a good correlation between tritium release, cellular uptake, and incorporation of [2-14C]dUrd into DNA. The inhibitory effects of antifolates such as methotrexate were independent of the type of labeled precursor used. In contrast, preferential interference with the release of tritium from [5-3H]-dCyd by dCyd derivatives and from [5-3H]dUrd by dUrd derivatives was observed, suggesting that competition for uptake and/or phosphorylation may contribute to the overall effects of certain nucleoside analogues on cellular dTMP synthase activity measured using the tritium release assay.     

In vitro and in vivo antitumor activity of YoshixolTR against murine L1210 leukemic cells.

In this report, antiproliferative effects of YoshixolTR in vitro and in vivo were investigated in murine L1210 cells. A proliferation of L1210 cells in vitro was inhibited by YoshixolTR in a dose- and time-dependent manner. This inhibition showed an arrest at the G0/G1 stage of the cell cycle, followed by a flow cytometric measurement. YoshixolTR induced apoptosis-like cell death identified by histological observations (scanning electron and transmission electron microscopy), DNA fragmentation, and a smaller increase in lactate dehydrogenase (LDH). In the in vivo experiments, YoshixolTR (5 microl/kg of body weight, on days 1, 3, and 5) was injected intraperitoneally in mice inoculated with L1210 cells. No marked prolongation of survival occurred between the control group and treated group. However, a survival curve in the treated group showed a shift toward a possible longer survival time. Additionally, on the basis of apoptosis-like cell death due to YoshixolTR as indicated above, a possibility of immunotherapy as a tumor vaccine has been examined. A vaccination of rabbit anti-serum, which consisted of components from the L1210 cells killed by YoshixolTR, produced a dramatic improvement of viability in the leukemic mice. In conclusion, YoshixolTR has an anti-leukemic potency with a new biological mechanism and an inductive potency of super-antigens as immunotherapeutic agents against malignant tumors.     

Initiation of autophagy and apoptosis by sonodynamic therapy in murine leukemia L1210 cells

Sonodynamic therapy (SDT) has shown great potential in target cancer therapy, but it induced cell death modes has not been fully investigated. This study was to examine autophagic and apoptotic responses to protoporphyrin IX (PpIX) mediated SDT in murine leukemia L1210 cells. After SDT, the occurrence of autophagy was identified by morphological observation and biochemical analysis. Meanwhile, the mitochondria dependent apoptosis pathway was examined to participate in SDT induced cell death. The relationship between autophagy and apoptosis was further investigated by applying pharmacological inhibition studies, which indicated that impairment of autophagy enhanced the anti-tumor effect of SDT through induction of apoptosis and necrosis, while caspase inhibition did not affect autophagic vacuoles formation or protect SDT induced cytotoxicity. The findings supported that autophagic vacuoles formed upstream and independently from caspase-dependent cell death. Additionally, the possible mechanism of SDT-induced autophagy was evaluated by measurement of ROS (reactive oxygen species) formation. Result suggested ROS play important role in initiating autophagy, possibly through the sono-damaged mitochondria being enclosed by autophagic vacuoles. All together, these data indicate that autophagy may be cytoprotective in our experimental system, and point to an important insight into how autophagy inhibitors, in combination with SDT may contribute a regimen for cancer therapy.     

Inhibition of ribonucleotide reductase and growth of human colon carcinoma HT-29 cells and mouse leukemia L1210 cells by N-hydroxy-N'-aminoguanidine derivatives

A series of N-hydroxy-N'-aminoguanidine (HAG) derivatives were studied and compared for their effects on ribonucleotide reductase activity in cell-free extracts; on nucleic acid synthesis and the growth of human colon carcinoma HT-29 cells; and on mouse leukemia L1210 cells in culture. The HAG derivatives [RCH=NNHC(=NH)NHOH-tosylate] studied could be grouped as: (1) hydroxybenzylidines; (2) methoxybenzylidines; and (3) nitrobenzylidines substituted at the R position. 2'-Hydroxybenzylidine-HAG, the lead compound, was relatively active in both HT-29 cells and L1210 cells (20 +/- 5 and 13 +/- 4 microM for 50% inhibition of HT-29 and L1210 cell growth respectively). The monohydroxybenzylidene compounds were generally more active than the dihydroxy- and trihydroxybenzylidene-HAG derivatives. The methoxybenzylidene-HAGs were as active as the monohydroxybenzylidene-HAGs. 2'-Hydroxy-4'-methoxybenzylidene-HAG was much more active than 2',4'-dihydroxybenzylidene-HAG. The mononitrobenzylidene-HAGs were more active than the dinitrobenzylidene-HAG compound. In general, L1210 cells were more sensitive to the effects of the HAG compounds than were HT-29 cells. There was good agreement between the concentration of drug required to inhibit the growth of HT-29 cells and that required to inhibit the growth of L1210 cells. There was also good correlation between the ability of HAG derivatives to inhibit ribonucleotide reductase activity and to inhibit tumor cell growth. Some derivatives, such as 2',3',4'- and 3',4',5'-trihydroxybenzylidene-HAG inhibited L1210 cell growth by 50% at lower concentrations (7.8 and 11.9 microM respectively) than the concentrations needed for 50% inhibition of HT-29 cell growth (196 and 234 microM respectively) and ribonucleotide reductase activity (122 and 188 microM respectively). The studies of nucleic acid synthesis in L1210 cells using [3H]cytidine as a precursor showed that 2',3',4'-trihydroxybenzylidine-HAG inhibited DNA synthesis at a lower concentration (29 microM for 50% inhibition) than was needed for the inhibition of RNA synthesis and formation of [3H]deoxycytidine nucleotides in the acid-soluble fraction (320 and 820 microM for 50% inhibition respectively). These results indicate that 2',3',4'-trihydroxybenzylidine-HAG inhibits DNA synthesis in L1210 cells through other mechanisms rather than exclusively through the inhibition of ribonucleotide reductase activity.