Dialdehyde derivative of 5'-deoxyinosine as a more potent analog of the dialdehyde derivative of inosine (NSC 118994).

The dialdehyde derivative of 5′-deoxyinosine (5′-deoxyinox) was prepared from 5′-deoxyadenosine by HNO₂ deamination and periodate oxidation. 5′-Deoxyinox was shown to inhibit ribonucleotide reductase activity in cell-free extracts and to inhibit RNA and DNA syntheses in intact cells. Further, 5′-deoxyinox inhibited the conversion of cytidine nucleotides to deoxycytidine nucleotides in intact cells. In comparative studies with the dialdehyde derivative of inosine (Inox), 5′-deoxyinox was shown to be more active on a molar basis in inhibiting RNA or DNA synthesis in intact cells. In addition, 5′-deoxyinox was more inhibitory to the growth of Novikoff hepatoma cells in culture than was Inox. 5′-Deoxyinox, in addition to being more active than Inox, also differed from Inox in its biochemical properties. Inox did not inhibit RNA polymerase activity when added to isolated nuclei. On the other hand, 5′-deoxyinox showed a marked inhibition of the RNA polymerase activity when added to the isolated nuclei. Further, inhibition of the RNA polymerase activity in the nuclei from Inox-treated cells was reversed completely by the addition of exogenous polydeoxyadenylate-deoxythymidylate as template, whereas the inhibition caused by 5′-deoxyi nox was not reversed by this treatment. These studies show that, in addition to the variation in activity caused by altering the purine component, the nature of the dialdehyde moiety also plays a role in the mode of action of this class of compounds.     

Regulation of pyrimidine biosynthesis in cultured L1210 cells by 3-deazauridine

The effect of 3-deazauridine on the synthesis of uracil nucleotides by de novo and salvage pathways was investigated in intact cultured L1210 cells. De novo pyrimidine biosynthesis, as measured by sodium [14C]bicarbonate incorporation into uracil nucleotides, was inhibited 40-85% at intracellular 3-deazauracil nucleotide concentrations of 1-6 nmoles/10(6) cells. The inhibition was not due to an increase in the size of the uracil nucleotide pool since this pool was only 97-66% of control level at 3-deazauracil nucleotide concentrations of 1-6 nmoles/10(6) cells. Furthermore, intracellular 3-deazauracil nucleotide concentrations of 0.5 to 5.2 nmoles/10(6) cells inhibited the salvage of [14C]uridine by 25-75%. The data indicate that 3-deazauridine may potentiate its own inhibition of CTP synthetase by reducing the concentration of competing uracil nucleotides by inhibiting de novo pyrimidine biosynthesis and pyrimidine salvage. It is postulated that the biochemical mechanism by which 3-deazauridine inhibits uracil nucleotide synthesis is by acting as a fraudulent allosteric regulator of carbamyl phosphate synthetase II and uridine/cytidine kinase.     

Modulation of fluoropyrimidine metabolism in L1210 cells by L-alanosine

L-Alanosine, an analogue of aspartic acid which inhibits the conversion of inosine monophosphate to adenosine monophosphate (AMP), was evaluated in L1210 cells as a modulator of 5-fluorouracil (FUra) and 5-fluorouridine (FUrd) metabolism. L-Alanosine resulted in increased intra-cellular levels of 5-phosphoribosyl-1-pyrophosphate (PRPP), enhanced FUra metabolism to ribonucleotide derivatives, and resulted in more FUra residues incorporated into RNA. Sequential addition of L-alanosine and FUra also resulted in synergistic cytotoxicity as determined by soft agar cloning. Adenine antagonized these biochemical and biological effects of L-alanosine. L-Alanosine also augmented the rate at which FUrd was metabolized and was also associated with a greater incorporation of the FUra residues into RNA. Cytotoxicity after sequential L-alanosine and FUrd was also synergistic. The mechanism by which L-alanosine altered the metabolism of FUrd, however, was different from the way in which it enhanced FUra metabolism in that aspartic acid and not adenine was able to reverse the effects of L-alanosine on FUrd metabolism and cytotoxicity. L-Alanosine appeared to augment the RNA-directed activity of FUra and FUrd in that there was no correlation between the enhanced metabolism and cytotoxicity of these two fluoropyrimidines and either levels of fluorodeoxyuridylate (FdUMP) which inhibits thymidylate (TMP) synthetase or inhibition of the ability of cells to incorporate deoxyuridylate into acid-precipitable material.     

Ribo- and deoxyribonucleoside effect on 3'-amino-2',3'-dideoxycytidine-induced cytotoxicity in cultured L1210 cells

3'Amino-2',3'-dideoxycytidine (3'-NH2-dCyd) is a potent inhibitor of the replication of cultured L1210 cells, with an IC50 of 1 microM. When ribo- and deoxyribonucleosides were examined for their effects on 3'-NH2-dCyd-induced cytotoxicity, only dCyd could both prevent and reverse these effects. Furthermore, even when the maximum increase in modal cell volume was allowed to develop (24 hr) in the presence of 2.5 microM 3'-NH2-dCyd, the addition of 25 microM dCyd to the medium containing 3'-NH2-dCyd reduced the modal cell volume nearly to control levels within 24 hr. Examination of the viability of these cells by colony formation in soft agar, following as much as a 9.5-hr exposure of 1, 2.5 and 10 microM 3'-NH2-dCyd, revealed that the lethal effects of the 3'-NH2-dCyd treatment were not observed only when 25 microM dCyd was added to the medium during this time. However, the lethality of a 24-hr exposure of 2.5 and 10 microM 3'-NH2-dCyd could not be prevented either by removal of the drug from the medium or by a 24-hr exposure of the medium containing 3'-NH2-dCyd to 25 microM dCyd. When the effect of 3'-NH2-dCyd on DNA biosynthesis in L1210 cells was examined, it was found that radiolabeled dAdo incorporation decreased by approximately 60, 80 or 90% following a 2.5-hr exposure to 2.5, 10 or 20 microM 3'-NH2-dCyd respectively. The addition of 25 microM dCyd under the same conditions resulted in a greater amount of dAdo incorporation compared to the unrescued cultures. Deamination of 3'-NH2-dCyd by partially purified human cytidine-deoxycytidine deaminase was about 2.5% that of either Cyd or dCyd deamination. The deaminated derivative, 3'-amino-2',3'-dideoxyuridine, was significantly less cytotoxic even at 50 microM.     

Effect of N8-acetylspermidine deacetylase inhibition on the growth of L1210 cells

A selective inhibitor of N8-acetylspermidine deacetylase has been employed to study the role of N8-acetylspermidine deacetylation in the regulation of L1210 cell growth. This inhibitor, 7-[N-(3-aminopropyl) amino] heptan-2-one (APAH), was found to stimulate the growth of L1210 cells at concentrations between 10 microM and 0.5 mM. Maximum stimulation was seen at 100 microM, resulting in significantly increased rates of cell division and maximum cell density. N8-Acetylspermidine levels in L1210 cells were shown to increase significantly after the APAH treatment as would be expected for deacetylase inhibition. The effects of deacetylase inhibition were mimicked by addition of N8-acetylspermidine to the culture medium at concentrations greater than 1 mM as indicated by a subsequent increase in rate of cell growth and maximum cell density. The magnitudes of the increases in growth observed were not large, but this might be expected in cells that are already in a rapid growth phase. Other exogenously added polyamines including N1-acetylspermidine, spermidine, putrescine, and spermine did not stimulate cell growth. These data suggest that stimulation of cell growth occurs as a consequence of N8-acetylspermidine accumulation and N8-acetylspermidine deacetylase inhibition.     

Effect of membrane potential on the cellular uptake of 2-N-methyl-ellipticinium by L1210 cells

Some quaternary ammonium derivatives of ellipticine are active antitumor drugs on both experimental and human tumors. Because of their positive charge, the cellular uptake of these molecules is expected to be influenced by the electric membrane potential. Experimental variations of the potential were produced by changing the external potassium concentration and the potassium permeability by the addition of valinomycin. Using the fluorescent lipophilic cationic dye 3,3-dihexyloxacarbocyanine iodide, the L1210 cell membrane potential was estimated at -35 mV by flow cytometric analysis, and the same technique was then used to study the effects of the membrane potential variations on 2-N-methyl-ellipticinium (NME) cellular uptake. Our results show that indeed NME uptake depends on the cell membrane potential, which might then influence its pharmacological properties.     

Cooperative sequestration of m-AMSA in L1210 cells

The anticancer drug 4′-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA) is known to bind to DNA by intercalation and to produce protein-associated DNA strand breaks in cells. Previous work [Zwelling et al., Biochemistry20, 6553 (1981)] had shown that m-AMSA is in rapid equilibrium between extracellular and intracellular compartments, and that the DNA strand breaks exist in a steady state of rapid formation and resealing. The current work reports an unusual uptake phenomenon of m-AMSA by mouse leukemia L1210 cells that occurs at higher drug concentrations than previously studied. The new uptake phenomenon was characterized by cooperativity, hysteresis, irreversibility, saturability, slowness and temperature dependence. It is concluded that m-AMSA concentrations above a critical value can initiate the irreversible sequestration of m-AMSA into a new phase, probably in an extranuclear compartment of the cell, from which the drug has no access to the nuclear DNA and probably does not contribute to cytotoxicity.     

Cytocidal action of homoharringtonine on L1210 cells in vitro

The proliferation of L 1210 cells ceased rapidly after they were exposed to homoharringtonine (HH) 1 microgram/ml during exponential growth phase. However, 25.3% of the cells were still able to form colonies in soft agar if HH was removed after 24 h of incubation (the colony-forming efficiency for control cells was 62.5%). The clonogenic cells survived from the treatment were still sensitive to HH-continuous exposure. The IC50 of the treated and control cells were 15 and 20 ng/ml, respectively. Yet, the sensitivity of the treated cells to cytarabine decreased enormously. For instance, the survival rate of HH-treated cells remained at 100% level after they were exposed to cytarabine 4-8 micrograms/ml for 1 h, but only 40% control cells survived from the same treatment. When cells were continuously exposed to HH 0.4 micrograms/ml, the colony-forming efficiency decreased exponentially as a function of exposure time. The T1/2 of the clonogenic cells was about 18 h. The DNA contents in L 1210 cells was measured with a flow-cytometer. The results showed that the cell-cycle progress in all cells was interrupted by HH, regardless which phase they belonged to. So the cells seemed to be in a "frozen" state and the histogram unchanged.     

Cytotoxicity of cinnamic aldehyde on leukemia L1210 cells

Cytotoxic effect of cinnamic aldehyde (CA) on L1210 mouse leukemia cells was tested. Addition of CA in Fischer's medium at 4.8 micrograms/ml could inhibit the growth of L1210 by 50 per cent. The terminal aldehyde-group of CA molecule was found to be responsible to the inhibition. Experiments of incorporating [3H]-uridine, [3H]-thymidine, and [3H]-leucine by the cells revealed that the syntheses of protein, DNA, and RNA were suppressed by the presence of CA in the culture solution with potency appeared in that order. The inhibitory effect of CA on glycolysis was insignificant. Direct reaction between aldehyde-groups of CA molecules and sulfhydryl-groups of cell components was proved. The results suggested that CA inhibited L1210 cells by blocking protein synthesis through trapping sulfhydryl-containing amino acids in the cell.     

2-乙氧甲酰亚甲基-4-氰基-5,6-二苯哒嗪-3-酮对L1210细胞周期的影响

本文研究了哒嗪(DPP)衍生物PD032对L1210细胞周期移行的影响。结果表明,PD032(2～10μmol/L)作用24h,G_1期细胞明显减少,G_2+M期细胞增加5～10倍。同时观察到有丝分裂指数增加,细胞体积增大及多核现象。S期在高浓度(10μmol/L)PD032时有减少,这时[~3H]TdR参入DNA也有轻度抑制。洗去药物后24～48h,上述变化有不同程度的恢复。以上结果说明PD032是有丝分裂抑制剂。     

Two mechanisms of adriamycin-DNA interaction in L1210 cells

Among the effects exerted by adriamycin (ADR), interaction with DNA is closely related to cytotoxicity. The interaction results in the formation of protein-associated DNA single-strand breaks (PA-SSB) and, at drug levels ? 2.8 × 10^?6 M, also in “direct” (nonenzymatic) DNA single-strand breaks (D-SSB). To characterize the two types of DNA lesions, euoxic mouse leukemia L1210 cells were treated with various antioxidant agents in the presence of 2.8 × 10^?6, × 10^?5, or × 10^?4 M concentrations of ADR. The enzymes superoxide dismutase (200 μg/ml) or catalase (250 μg/ml), the OH scavengers dimethyl sulfoxide (70 mM) or ethanol (70 mM), and an inhibitor of superoxide production, 2-deoxy-glucose (1 and 10 mM), reduced the frequency of D-SSB to 18.3 to 68.2% of its level in ADR-treated controls, while the frequency of PA-SSB remained unchanged. These observations seem to indicate that ADR-mediated free radicals cause discernible DNA damage in euoxic cells only at very high drug concentrations, greater than the peak plasma level achievable clinically following i.v. bolus. At lower ADR levels, relevant to clinical use, another type of interaction between the drug and DNA prevails, which apparently does not involve a free-radical mechanism.     

Effect of berberine on proliferation,cell cycle and apoptosis in HeLa and L1210 cells

Previous studies on the anticancer activity of protoberberine alkaloids against a variety of cancer cell lines were extended to human tumour HeLa and murine leukemia L1210 cell lines. An attempt was also made to investigate the relationship between the cytotoxic activity of berberine and its molecular mechanism of action. Cytotoxicity was measured in-vitro using a primary biochemical screening according to Oyama and Eagle, and the growth inhibition assay. The in-vitro cytotoxic techniques were complemented by cell cycle analysis and determination of apoptotic DNA fragmentation in L1210 cells. Berberine acted cytotoxically on both tumour cell lines. The sensitivity of leukemia L1210 cells to the berberine was higher than that of HeLa cells. The IC(100) was below 100 microg mL(-1) for HeLa cells and approached a 10 microg mL(-1) limit for the leukemia L1210 cells. For both cell lines the IC(50) was found to be less than 4 microg mL(-1), a limit put forward by the National Cancer Institute (NCI) for classification of the compound as a potential anticancer drug. In L1210 cells treated with 10-50 microg mL(-1) berberine, G(0)/G(1) cell cycle arrest was observed. Furthermore, a concentration-dependent decrease of cells in S phase and increase in G(2)/M phase was detected. In addition, apoptosis detected as sub-G(0) cell population in cell cycle measurement was proved in 25-100 microg mL(-1) berberine-treated cells by monitoring the apoptotic DNA fragmentation (DNA ladder) using agarose gel electrophoresis.     

DADAG诱导白血病L1210细胞凋亡

目的 以小鼠白血病细胞系L1210为对象,探讨半胱天冬氨酸蛋白酶（Caspases）家族成员在二乙酰二脱水卫矛醇（diacetyldianhydrogalactitol,DADAG）诱导肿瘤细胞凋亡过程中的作用。方法 MTT法测定DADAG对L1210细胞的杀伤作用;透射电镜和流式细胞术检测细胞凋亡;Western blot和Caspase-3试剂盒测定Caspase-3活性。结果 与对照组相比,L1210细胞经不同浓度的DADAG（12．0、17．2、24．5、35．0或50．0mg／L）处理72h后,其存活率,分别降至72％、63％、46％、35％和20％。DADAG50．0mg／L处理组的凋亡率、Caspase-3活性和PARP裂解片段较对照组明显升高。Caspase-3抑制剂可部分阻断此过程,而Caspases抑制剂可完全阻断此过程。结论 Caspases家族成员在DADAG诱导L1210细胞凋亡中发挥重要的作用。     

Effect of forphenicinol on the production of Ia-positive macrophages in mice with or without L1210 leukemia and on the growth of L1210 in immunized mice

Oral administration of forphenicinol, S-2-(3-hydroxy-4-hydroxymethylphenyl)glycine, increased the production of Ia-positive peritoneal macrophages in healthy mice. Moreover, forphenicinol increased Ia-positive macrophages in L1210-bearing mice. Though forphenicinol was ineffective against mouse leukemia L1210 when administered alone, in combination with L1210 vaccine it prolonged the survival time of L1210-bearing mice and the increase in the number of Ia-positive macrophages in peritoneal cavity coincided with the prolongation of the survival time. These results suggest that the initial action of forphenicinol is the preferential induction of Ia-positive macrophages, which play a role in the subsequent activation of various immune responses including macrophage activation.     

二乙酰二脱水卫矛醇对白血病L1210细胞增殖的抑制作用

目的探讨二乙酰二脱水卫矛醇(diacetyldianhydrogalactitol,Dadag)对小鼠白血病细胞株L1210细胞的增殖抑制作用及其机制。方法采用MTT比色法、平板集落形成实验和Hoechst 33258荧光染色法,分析Dadag对L1210细胞的生长抑制和凋亡影响。结果Dadag对L1210细胞有浓度依赖性的细胞毒性作用,抑制肿瘤细胞集落形成,并可诱导L1210细胞发生凋亡。结论Dadag对白血病L1210细胞的增殖抑制作用与其诱导肿瘤细胞发生凋亡密切相关。     

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.