Cytotoxicity of Cinnamic Aldehyde on Leukemia L1210 Cells

ABSTRACT

Cytotoxic effect of cinnamic aldehyde (CA) on L1210 mouse leukemia cells was tested. Addition of CA in Fischer's medium at 4.8 μg/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 sulf-hydryl-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.     

六味地黄多糖体外抗肿瘤作用的初步研究

目的　六味地黄多糖 Ｃ Ａ４ ３ Ｂ 和 Ｐ ３ 是具有免疫调节作用的活性多糖,在此基础上进一步观察其体外对肿瘤细胞增殖的抑制作用。方法　用［３ Ｈ］ Ｔｄ Ｒ 参入法,琼脂克隆形成法及细胞形态学和 Ｎ Ｂ Ｔ 还原染色等方法观察其对小鼠淋巴细胞白血病细胞系 Ｌ１２１０ 、人早幼粒白血病细胞系 Ｈ Ｌ６０增殖反应、克隆存活率的影响以及对 Ｈ Ｌ６０ 细胞的分化诱导作用。结果　 Ｃ Ａ４ ３ Ｂ和 Ｐ ３ 对 Ｌ１２１０ 和 Ｈ Ｌ６０ 细胞的体外增殖反应和克隆形成率具有不同程度的抑制作用,对 Ｈ Ｌ６０细胞的分化形态及 Ｎ Ｂ Ｔ 阳性细胞率无明显影响,但可以使 Ｈ Ｌ６０ 细胞体积明显缩小。结论　六味地黄多糖 Ｃ Ａ４ ３ Ｂ 和 Ｐ ３ 对肿瘤细胞生长有抑制作用,可能是具有免疫调节和直接抑瘤双重作用的活性多糖;另外提示 Ｃ Ａ４ ３ Ｂ 和 Ｐ ３ 对 Ｈ Ｌ６０ 细胞分化有一定的诱导作用     

Reversal of the cytotoxicity of 3'-amino-3'-deoxythymidine by pyrimidine deoxyribonucleosides.

Mammalian cell replication is strongly inhibited by 3′-amino-3′deoxythymidine (3′-aminothymidine). This cytotoxieity can be specifically prevented or reversed by pyrimidine 2′-deoxyribonucleosides. The addition of 50 μM 2′-deoxycytidine to L1210 cells treated with 10 μM 3′ the population doubling time from about 38 hr to 17 hr. The control cells doubled every 13 hr. Another cytotoxic effect produced by 3′-aminothymidine is a dose- and time-dependent increase in cell volume. 2′-Deoxycytidine can effectively prevent and reverse this increase. 3′-Aminothymidme appears to be a potent selective inhibitor of DNA synthesis in L1210 cells. The incorporation of [³H]thymidine into DNA was inhibited by 50 per cent at 1 μM 3′-aminothymidine, a concentration which reduced L1210 replication by about 65 per cent. The rate of incorporation of [³H] adenine into DNA, another measure of DNA synthesis, was reduced similarly by 3′-aminothymidine. and 2′-deoxycytidine eliminated this inhibition as well. An effect on RNA or protein synthesis was not detected. The incorporation of [³H] uridine or [³H] adenine into RNA, or of tritiated amino acids into protein, was not reduced by 25 μM 3′-aminothymidine. These results suggest that selective disruption of DNA metabolism may account for the cytotoxicity of 3′-aminothymidine.     

Effect of the dialdehyde derivative of 5'-deoxyinosine on pyrimidine deoxyribonucleoside metabolism in L1210 cells

The effects of the dialdehyde derivatives of inosine (Inox) and 5'-deoxyinosine (5'-dInox) on L1210 cells were compared. The growth of L1210 cells was inhibited to a greater extent by 5'-dInox than by Inox. The increased inhibition of L1210 cell growth by 5'-dInox was also reflected by the increased inhibition of the incorporation of precursors into RNA, DNA and proteins. Even though 5'-dInox was a more potent inhibitor, Inox accumulated in the L1210 cells to levels 4- to 5-fold greater than 5'-dInox. The metabolism of [5-3H]deoxycytidine and [5-3H]deoxyuridine by L1210 cells in culture, in the presence of Inox or 5'-dInox, indicated that dCMP deaminase was an intracellular site of action for 5'-dInox. The dCMP deaminase activity in cell-free extracts prepared from 5'-dInox-treated cells was reduced markedly. This decrease in activity was not reversed by increased substrate concentrations nor was the activity subject to allosteric activation by dCTP. Deoxyuridine and deoxycytidine were able to reverse the effects of 5'-dInox on the inhibition of L1210 cell growth.     

Tumor cell adherence to cultured capillary endothelial cells is promoted by activators of protein kinase C

Stimulation of cells with protein kinase C (PKC)-specific activators such as phorbol esters increased in a reversible manner the rate of adherence of [3H]leucine-labelled L1210 cells to cultured bovine cerebral cortex capillary endothelial cells (CEC). This effect was not specific for L1210 cells since 12-O-tetradecanoyl phorbol 13-acetate (TPA) strongly increased the binding of various other tumor cell lines. Phorbol esters increased the rate of L1210 cell adhesion to CEC by enhancing their binding capacity without affecting the apparent affinity of L1210 cells for CEC. This stimulation was specific to the phorbol analogs which activate PKC since it was not effected by 4 alpha-phorbol didecanoate, known to be inactive for PKC. Down-regulation experiments showed that adhesion enhancement was entirely attributable to an effect on tumor cells without contribution of CEC intracellular PKC. PKC inhibitors like staurosporine, sphingosine and H-7 showed strong antagonistic activity towards TPA-induced L1210 cell adherence to CEC (IC50 = 0.5 nM, 160 nM and 10 microM, respectively). Adhesive proteins such as vitronectin, fibrinogen, fibronectin and the tetrapeptide RGDS, an active sequence from their cell-binding domains, exhibited potent, dose-dependent inhibition of PKC-induced tumor cell adhesion.     

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.     

Carrier-mediated transport of folate compounds in L1210 cells. Initial rate kinetics and extent of duality of entry routes for folic acid and diastereomers of 5-methyltetrahydrohomofolate in the presence of physiological anions

Comparison of the kinetic parameters for influx of highly purified [3H]folic acid versus [3H]methotrexate in L1210 cells under anionic buffer conditions showed a marked discordancy. In addition, the kinetics for influx of [3H]folic acid were unchanged in variant L1210 cells defective in [3H]methotrexate transport. In these variant cells, the Vmax for methotrexate was reduced 17-fold and the Km was increased 3-fold. The results show that [3H]folic acid influx is mediated by a system which has a low affinity, but a 20-fold higher capacity, for folate compounds than the classical high-affinity system mediating [3H]methotrexate influx. Since the latter system also exhibits very low affinity for [3H]folic acid, it would not be expected to contribute significantly to the total influx of [3H]folic acid. The high-capacity system for [3H]folic acid influx is different from that believed to mediate pterin influx in L1210 cells since it was not inhibited by adenine, a potent inhibitor of pterin influx. However, exposure of cells to [3H]folic acid in a nonanionic buffer resulted in marked stimulation of initial influx, and a fraction of influx under these conditions was inhibited by methotrexate. These results suggest that anions modulate the extent of multiplicity of [3H]folic acid influx by their known effects on the high-affinity, reduced folate/methotrexate system. The diastereomers, at carbon 6, of [14C]5-methyltetrahydrohomofolate shared both transport systems. The influx Km for the natural diastereomer was one-half that of the unnatural form for both transport systems. Both diastereomers showed a much greater differential in affinity between the two transport systems than did [3H]folic acid. Our results suggest that an analog which could be effectively transported by the low-affinity/high-capacity route may be useful in the treatment of tumors resistant to methotrexate due to a defective high-affinity/low capacity influx system. We also found that incubation of L1210 cells with [3H]folic acid or the natural diastereomer [14C]5-methyltetrahydrohomofolate for 10 min resulted in the formation of a nonexchangeable fraction of radioactivity amounting to 20-40% of the total accumulation. This non-exchangeable fraction may be explained by the accumulation of metabolites other than polyglutamates. Preloading of cells with methotrexate prior to incubation with [3H]folic acid prevented the accumulation of radioactivity as a nonexchangeable fraction.     

Alterations in [3H]thymidine incorporation into DNA and [3H]uridine incorporation into RNA induced by 5-azacytidine in vivo.

Administration in vivo of 5-azacytidine (5-aza-CR) caused suppression of [³H]thymidine ([³H]TdR) incorporation into DNA of bone marrow and gastrointestinal mucosa of mice and a more prolonged suppression of L1210 ascites tumor. Single doses of 5-aza-CR caused a modest and short-lived suppression of incorporation of [³H]uridine ([³H]UR) into nuclear RNA of L1210 ascites tumor cells. No suppression of [³H]UR incorporation into RNA of bone marrow or gastrointestinal mucosa was observed. L1210 tumor cells resistant to the other active cytidine analogue, cytosine arabinoside, demonstrated less disruption of [³H]TdR incorporation after exposure to 5-aza-CR, suggesting some cross resistance in the effects of these two drugs on DNA synthesis. Survival studies carried out in mice bearing both the sensitive and resistant L1210 tumor cell lines confirmed cross resistance of the anti-tumor effects of the two cytidine analogues. Second doses of 5-aza-CR, with the timing og administration based upon the differing patterns of recovery of [³H]TdR incorporation between normal tissues and tumor cells, led to a prolongation of survival in mice bearing the sensitive L1210 ascites tumor.     

Tumor cell permeability to peplomycin

The uptake of [3H]peplomycin-Cu(II) ([3H]PEP-Cu(II)) into various tumor cell lines was studied. The time course of [3H]PEP-Cu(II) uptake into AH66, AH66F, Ehrlich and P388 cells was biphasic. The first phase of uptake was completed within 5 minutes. The second, slower phase, of uptake into AH66, AH66F and Ehrlich cells increased linearly with incubation time, but that into P388 cells reached a plateau level. In L1210 cells, only the first rapid uptake was observed. The lower uptake into P388 and L1210 cells during the second phase may be related to their insensitivity to PEP. However, the uptake into AH66F cells was higher than that into AH66 cells, although AH66F cells were less sensitive to PEP than AH66 cells. Deamide PEP was detected in intact cells which had taken up [3H]PEP-Cu(II) during 4 hours. This confirmed that PEP-Cu(II) was transported into the cell, the copper removed and PEP metabolized to deamide PEP. [3H]PEP-Cu(II) uptake into AH66 and AH66F cells increased in proportion to the extracellular concentration of drug up to at least 200 micrograms/ml, suggesting that uptake was not mediated by a carrier system. Metabolic inhibitors such as NaN3 and 2,4-dinitrophenol enhanced [3H]PEP-Cu(II) uptake, but did not influence efflux. Uptake was also enhanced by membrane modifiers such as dibucaine and chlorpromazine which increase the fluidity of lipid membranes. The results suggest that PEP-Cu(II) was taken up into tumor cells by passive diffusion, controlled by an energy-dependent cell membrane barrier.     

Synthesis of 2,8-disubstituted imidazo[1,5-a]pyrimidines with potent antitumor activity

Seventeen 1,2,3,4-tetrahydroimidazo[1,5-a]pyrimidine derivatives bearing electron-withdrawing substituents were designed and synthesized by novel ring closure as potential antitumor agents. They were screened for their activities against mouse leukemia L1210 and human oral epidermoid carcinoma KB cell lines, and relationships of structure and antitumor activity in vitro are discussed. It was found that 8-thiocarbamoyl-1,2,3,4-tetrahydroimidazo[1, 5-a]pyrimidin-2(1H)-thione (8c) exhibited activity comparable to that of 5-fluorouracil against both L1210 and KB cells. The existence of both 2-thioxo and 8-substituent with a thioxo group in the molecule is crucial for the cytotoxicity against L1210 and KB cells. A novel procedure for introduction of a double bond between C-3 and C-4 in 8c was developed. Introduction of the 3,4-double bond increased the activity against L1210, but against KB cells the activity decreased by 4-fold. Cytotoxicity of compounds 8c and 8-thiocarbamoyl-1,2-dihydroimidazo[1,5-a]pyrimidin-2(1H)-thione (11c) against human solid tumor and leukemia cell lines was further evaluated. The saturation of the 3,4-double bond led to a significant increase in cytotoxicity against tumor cell lines tested.     

Growth inhibition by homofolate in tumor cells utilizing a high-affinity folate binding protein as a means for folate internalization

A subline (JT-1) of L1210 mouse leukemia cells that contains elevated levels of a high-affinity folate binding protein is sensitive to growth inhibition by homofolate. Inhibition was observed at nanomolar concentrations of folate or 5-formyltetrahydrofolate where the high-affinity binding protein is the predominant uptake route for folate compounds. At 1.0 nM folate, inhibition of growth by 50% occurred at 0.7 nM homofolate, and maximal inhibition exceeded 90% at homofolate concentrations above 10 nM. Homofolate also inhibited the uptake of 1.0 nM [3H]folate by L1210/JT-1 cells in 72-hr cultures, and the extent of uptake inhibition by 1.0 and 20 nM homofolate was comparable to the inhibition of cell growth by the same concentrations of homofolate. At a growth-limiting concentration of 5-formyltetrahydrofolate (0.5 nM), half-maximal inhibition of L1210/JT-1 cell growth occurred at 1.0 nM homofolate. When excess concentrations of folate (5 microM) or 5-formyltetrahydrofolate (0.5 microM) were added to the medium, no growth inhibition was observed for homofolate at concentrations up to 100 microM. Parental cells lacking the folate binding protein did not respond to homofolate either at growth-limiting (0.5 microM) or excess (5.0 microM) levels of folate. Binding measurements showed that homofolate has a high affinity for the folate-binding protein (Ki = 0.03 nM) but interacts poorly with the reduced-folate transport system (Ki = 203 microM). These results indicate that homofolate inhibits the growth of L1210 cells when intracellular folates are acquired via the high-affinity folate binding protein. The basis for this inhibition appears to be competition by homofolate for substrate binding and internalization.     

Cisplatin-resistant derivatives of murine L1210 leukemia cells are not susceptible to growth-inhibiting and apoptosis-inducing actions of transforming growth factor-beta1.

Murine L1210 leukemia cells possessing an increased resistance to cisplatin were found to be refractory to transforming growth factor (TGF)-beta1-induced growth inhibition, while the parental L1210 cells were strongly inhibited by this cytokine. Growth inhibition was estimated on the basis of [3H]thymidine incorporation, cell counting and colony-forming assay. Cisplatin-resistant L1210 cells were also shown to be much more resistant than the parental cells to both cisplatin- and TGF-beta1-induced apoptosis. These results suggest the existence of cross-resistance to cisplatin and TGF-beta1 in the studied leukemia cells.     

4-[4″-(2″,2″,6″,6″-四甲基哌啶氮氧自由基)氨基]-4′-去甲表鬼臼毒对L1210细胞系增殖、克隆形成和DNA合成的影响

新合成的鬼臼毒自旋标记衍生物4-(4″-(2″,2″,6″,6″-四甲基哌啶氮氧自由基)氨基)-4′-去甲表鬼臼毒(GP-7)显著抑制体外培养的L1210细胞生长。抑制作用和浓度及处理时间正相关,作用特点和鬼臼乙叉甙(VP-16)相似,24,48hIC_(50)分别为1.51和0.13μmol/L。GP-7和VP-16对L1210细胞软琼脂克隆形成均有抑制作用,且有浓度依赖性,IC_(50)分别为3.29和2.82μmol/L。GP-70.08～100μmol/L对L1210细胞DNA合成抑制率为18.4～80.7％。本文结果表明GP-7体外抗肿瘤作用与VP-16相似。     

海南哥纳香醇甲(GHM-10)对L1210细胞生物大分子合成的抑制作用

目的旨在研究GHM-10对L1210细胞生物大分子合成的影响。用[³H]标记前体参入试验。结果表明,GHM-104～12μg·ml^-1作用6h,可使L1210细胞的DNA,RNA及蛋白质的生物合成发生明显抑制,其中以抑制DNA合成的作用最强。用[³H]TdR参入曲线法、紫外吸收光谱移动法和荧光光谱移动法研究了GHM-10抑制DNA生物合成的机制。结果表明,GHM-10 6～8μg·ml^-1作用L1210细胞1h后,DNA合成的抑制已不可逆。提示GHM 10可能引起DNA分子结构的损伤,但GHM-10不能嵌入DNA分子或直接破坏DNA的结构。     

SB203580, a specific inhibitor of p38-MAPK pathway, is a new reversal agent of P-glycoprotein-mediated multidrug resistance.

P-glycoprotein (P-gp) is the plasma membrane transport pump responsible for efflux of chemotherapeutic agents from cells and is one of the systems that secures multidrug resistance (MDR) of neoplastic cells. In the present study, drug sensitive L1210 and multidrug resistant L1210/VCR (characterized by overexpression of P-gp) mouse leukemic cell lines were used as an experimental model. We have found that SB203580, a specific inhibitor of p38-MAPK pathway, significantly reduced the degree of the vincristine resistance in L1210/VCR cells. This phenomenon was accompanied by a decrease in the LC(50) value of vincristine from 3.203+/-0.521 to 0.557+/-0.082 microM. The LC(50) value of sensitive cells for vincristine was about 0.011 microM. The effect of SB203580 on L1210/VCR cells was associated with significantly increased intracellular accumulation of [3H]-vincristine in the concentration dependent manner. Prolonged exposure of resistant cells to 30 microM SB203580 did neither significantly influence the gene expression of P-gp, nor change the protein levels of p38-MAPK. Western blot analysis revealed that the MDR phenotype in L1210/VCR cells was associated with increased level and activity of cytosolic p38-MAPK. In resistant cells, the enhanced phosphorylation of both, p38-MAPK and ATF-2 (endogenous substrate for p38-MAPK) was found as well. In conclusion we could remark that SB203580, an inhibitor of p38 kinase pathway, reversed the MDR resistance of L1210/VCR cells. MDR phenotype of these cells is connected with increased levels and activities of p38-MAPK. These findings point to the possible involvement of the p38-MAPK pathway in the modulation of P-gp mediated multidrug resistance in the L1210/VCR mouse leukemic cell line. However, the mechanisms of SB203580 action should be further investigated.     

Substrate properties of analogs of myo-inositol

The hydrolysis of the minor cell membrane lipid phosphatidylinositol-4,5-bisphosphate mediates the action of many growth factors and hormones. As an approach to the development of specific inhibitors of this process, we have synthesized a series of analogs of myo-inositol and have evaluated their ability to serve as substrates for phosphatidylinositol synthetase. Modification at the 2-, 3-, or 4-positions produced compounds unable to serve as substrates, but several 5-modified analogs retained activity as substrates of phosphatidylinositol synthetase. The product formed from 5-deoxy-5-fluoro-myo-[3H]inositol by phatidylinositol synthetase was hydrolyzed by phospholipase D and gave 5-deoxy-5-fluoro-myo-inositol as the radiolabeled product. Two analogs, 5-deoxy-myo-inositol and 5-deoxy-5-fluoro-myo-inositol, were shown to permeate L1210 leukemia cells and be incorporated into cellular phospholipid. Analysis of the radiolabeled lipids formed on incubation of L1210 cells with 5-deoxy-5-fluoro-myo-[3H]inositol indicated that the fradulent lipid formed was further phosphorylated to the monophosphate but not to the diphosphate form.     

一些植物成分对实验肿瘤的作用

观察了18个植物成分对S180、Lewis肺癌、B16黑色素瘤、Ehrlich腹水癌、白血病P388、L1210和L615等小鼠肿瘤的疗效。以白血病P388和L1210最为敏感,L615最不敏感。此外,试验了高三尖杉酯碱、美登素、羟基喜树碱和长春新碱对Friend白血病的疗效,仅高三尖杉醋碱有明显疗效。部分样品还观察了对~3H标记的前体参入肿瘤细胞核酸和蛋白质的影响。     

The biochemical pharmacology of the thymidylate synthase inhibitor, 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583).

2-Desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) is a more water-soluble analogue of the quinazoline-based thymidylate synthase (TS) inhibitor, N10-propargyl-5,8-dideazafolic acid (CB3717). A 3-fold loss in TS inhibitory activity (murine and human TS, Ki = 10 nM) was accompanied by a 40-fold increase in growth inhibitory potency against L1210 and W1L2 cells in vitro (IC50 = 0.085 and 0.05 microM, respectively) when compared with CB3717. In L1210 cells a concentrative uptake mechanism was demonstrated for [3H]ICI 198583 (Kt = 2.9 microM). The L1210:1565 cell line, with an impaired ability to transport reduced folates or methotrexate (MTX), was resistant (100-fold relative to the wild-type L1210 line) to ICI 198583 (but not CB3717) and did not take up [3H]ICI 198583 significantly. The measurement of folylpolyglutamate synthetase (FPGS) substrate activity demonstrated a Km of 40 microM for ICI 198583 and a Vmax/Km (relative to folic acid) of 3.5. The formation of intracellular polyglutamate derivatives was demonstrated in both L1210 (mouse) and WIL2 (human) cells grown in vitro after exposure to 1 microM [3H]ICI 198583. In L1210 cells, by 4 hr, approximately 50% of the intracellular 3H(approximately 1 microM) was found as polyglutamate forms of ICI 198583, principally as tri- and tetraglutamates. After 24 hr the ICI 198583 polyglutamate pool had expanded, the tetraglutamate metabolite predominated and there was significant formation of the pentaglutamate. Upon resuspension of L1210 cells in drug free medium, ICI 198583 was largely lost from the cells but the polyglutamates were preferentially retained, after 24 hr approximately 70% remained. Synthetic ICI 198583 polyglutamates were shown to be up to 100-fold more potent as inhibitors of isolated TS than the parent compound. Following in vivo administration (500 mg/kg i.v.) ICI 198583 was cleared rapidly from the plasma of mice (T1/2 beta = 16 min, clearance = 42 mL/min/kg). Despite this clearance there was prolonged, dose-dependent inhibition of TS in L1210:NCI cells in vivo. Thus, following 500 mg/kg i.v. the flux through TS was inhibited by greater than 80% for at least 24 hr. Administration of five doses at 5 mg/kg daily of ICI 198583 to L1210:ICR tumour-bearing mice resulted in greater than 60% of the mice being cured, a 10-fold improvement in potency over CB3717. The maximum tolerated dose (MTD) for ICI 198583 using this schedule was greater than 500 mg/kg/day compared with 200 mg/kg/day of CB3717.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of a membrane sugar analogue, 6-deoxy-6-fluoro-d-galactose,on the L1210 leukemic cell ectosialyltransferase system

In L1210 leukemia cells, 6-deoxy-6-fluoro-d-galactose specifically inhibited the incorporation of [³H]-d-galactose, while that of other precursors of glycoconjugate biosynthesis, including mannose and glucosamine, was unaffected. The activation of [6-³H]-6-deoxy-6-fluoro-d-galactose to a nucleotide sugar was similar to that found for [³H]-d-galactose. The incorporation of either sugar after 1 hr was visualized by electron microscopic autoradiography to be in the Golgi region. Treatment of L1210 cells with 6-deoxy-6-fluoro-d-galactose in vitro or in vivo resulted in a specific, dose- and time-dependent decrease in the activity of cell surface sialyltransferase (ectosialyltransferase) but not of 5′-nucleotidase, a plasma membrane marker enzyme. The decrease in ectosialyltransferase activity appeared to be selective and is suggested to be due to structural modification of the cell surface galactoprotein acceptors for this enzyme. The data indicate that 6-deoxy-6-fluoro-d-galactose is an effective modifier of cellular glycoconjugate in that its incorporation into certain cell surface components results in a modification of plasma membrane structure and function.     

Decreased expression of the reduced folate carrier and folypolyglutamate synthetase is the basis for acquired resistance to the pemetrexed antifolate (LY231514) in an L1210 murine leukemia cell line

Pemetrexed (LY231514) is a new-generation antifolate that, in its polyglutamyl forms, is a potent inhibitor of thymidylate synthase and glycinamide ribonucleotide formyltransferase (GAR transformylase). This study explored the mechanisms of resistance to pemetrexed in L1210 murine leukemia cells using chemical mutagenesis with 5-formyltetrahydrofolate (5-formylTHF) as the growth substrate. A cell line, MTA-13, was identified that was 8.5-fold resistant to pemetrexed with comparable cross-resistance to ZD1694 (Tomudex) and lesser cross-resistance (5-fold) to ZD9331 [(2S)-2-(O-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydro-quinazolin-6-ylmethyl)-N-(prop-2-ynyl)amino]benzamido)-4-(tetrazol-5-yl)-butyric acid], DDATHF (dideazatetrahydrofolate) (3.5-fold), and methotrexate (MTX) (2.7-fold) but comparable sensitivity to trimetrexate. Influx of pemetrexed, MTX, and 5-formylTHF into MTA-13 cells was decreased by 56, 47, and 38% compared to wild-type cells. Folate receptor expression was negligible in both cell lines. Net drug uptake declined within 15min to a slower, constant rate over the next 45min, reflecting the rate of accumulation of pemetrexed polyglutamate derivatives. This rate in the MTA-13 line was half that of the wild-type cells. Accumulation of 50nM [3H]pemetrexed, 25nM [3H]5-formylTHF, or 50nM [3H]DDATHF after 3 days was decreased to 35, 46, and 56% the level of L1210 cells. The reduced folate carrier (RFC) message and protein were decreased by 50%, and folypolyglutamate synthetase (FPGS) message was decreased by 65% in MTA-13 cells. No mutations were detected in either protein by DNA sequence analysis. There was a slight decrease (approximately 25%) in thymidylate synthase mRNA, without mutations in the protein, and there was no change in GAR transformylase message. The data indicate that resistance to pemetrexed in the MTA-13 cell line was due to changes in both RFC and FPGS expression, two proteins that act in tandem to regulate polyglutamation of folates and antifolates in cells, resulting in cellular depletion of these active pemetrexed congeners.