Effects of acivicin and dichloroallyl lawsone upon pyrimidine biosynthesis in mouse L1210 leukemia cells

Acivicin (NSC 163501) and dichloroallyl lawsone (NSC 126771) are potent inhibitors of nucleotide biosynthesis with consequent anti-cancer activity against certain experimental tumors. To determine in detail the metabolic events induced by each inhibitor, we have devised a new two-dimensional chromatographic procedure for measurement of the concentrations of all pyrimidine intermediates and some purine nucleotides from 100 microliter of an extract of cells grown in the presence of [14C]bicarbonate. Addition of acivicin (25 microM) to mouse L1210 leukemia cells causes severe depletion in the cellular levels of CTP and GTP, accumulation of uridine nucleotides, and abrupt but transient increases in the concentrations of the early intermediates of both the pyrimidine and purine pathways. Addition of dichloroallyl lawsone (25 microM) results in a rapid depletion of uridine and cytidine nucleotides; carbamyl aspartate and dihydroorotate accumulate to high levels in an equilibrium ratio of 20.5:1, and orotate, orotidine, and UMP increase transiently before decreasing to levels approaching their original steady states. The predominant inhibitory effects of acivicin are upon the reactions UTP----CTP and XMP----GMP, but there is also an initial transient activation of both the pyrimidine and purine pathways by acivicin. The data obtained with dichloroallyl lawsone are consistent with inhibition of the conversion of UMP----UDP initially followed by potent inhibition of dihydroorotate----orotate.     

Rhodamine 123 alters the mitochondrial ultrastructure of cultured L1210 cells

Exposure of exponentially growing L1210 cells in vitro to 5-10 micrograms/ml of rhodamine 123 (R123) for 16-48 hr inhibits cell proliferation and induces cell arrest in the G1A phase of the cell cycle. The cells remain viable during the arrest and resume growth after removal of R123; extended exposure to R123 is cytotoxic. Exposure to R123 results in morphological alterations in mitochondria of all cells observed; specifically, mitochondria of R123-treated cells are characterized by a distention of the intracristal spaces and a significant increase in the number of matrix granules. Gross morphological changes of mitochondria include formation of extended organelles and the appearance of doughnut-shaped structures.     

Alteration in de novo pyrimidine biosynthesis during uridine reversal of pyrazofurin-inhibited DNA synthesis

Pyrazofurin, a pyrimidine nucleoside analogue with antineoplastic activity, inhibits cell proliferation and DNA synthesis in cells by inhibiting uridine 5'-phosphate (UMP) synthase. It has been previously shown in concanavalin A (con A)-stimulated guinea pig lymphocytes (23) that pyrazofurin-inhibited DNA synthesis could be selectively reversed by exogenous uridine (Urd). In this report, we have examined possible mechanisms for the Urd reversal with experiments that determine the ability of exogenous Urd to (a) interfere with either the intracellular transport of pyrazofurin, or the conversion of pyrazofurin to its intracellularly active form, pyrazofurin-5'-phosphate; (b) reverse the pyrazofurin block of [14C]orotic acid incorporation into DNA; and (c) alter the pattern of exogenous [3H]Urd incorporation into DNA-thymine (DNA-Thy) and DNA-cytosine (DNA-Cyt) during pyrazofurin inhibition of pyrimidine de novo biosynthesis. The results of these experiments showed that Urd reversal does not occur through altered pyrazofurin transport or intracellular conversion to pyrazofurin-5'-phosphate, nor does it alter the distribution of [3H]Urd in DNA-Thy and DNA-Cyt. Instead, these findings indicate that the primary mechanism for exogenous Urd reversal of pyrazofurin inhibition of DNA synthesis involves the reversal of pyrazofurin inhibition of UMP synthase, thus restoring orotic acid incorporation into lymphocyte DNA through the pyrimidine de novo pathway.     

Effect of a 400-kilocalorie carbohydrate diet on human plasma uridine and hypoxanthine concentrations

Seven healthy female volunteers were fed a 400-kcal carbohydrate diet for 4 days after eating a standardized diet for 3 days. Plasma uridine and hypoxanthine concentrations were measured by high-performance liquid chromatography. After 4 days on the 400-kcal diet, the plasma uridine concentration decreased by 35% but the plasma hypoxanthine concentration remained stable.     

Pyrimidine nucleosides enhance the efficacy of inhibitors of pyrimidine biosynthesis in cultured hepatocellular carcinoma cells

Inhibition of colony formation in cultured hepatocellular carcinoma cells of the rat was used to test the efficacy of inhibitors of de novo pyrimidine biosynthesis as potential anticancer drugs. N-(phosphonacetyl)-L-aspartic acid (PALA) (10 and 100 micrograms/ml) and 5-aza-5,6-dihydroorotic acid (DHOX) (100 micrograms/ml) inhibited the formation of colonies and these inhibitions were completely reversed by inclusion of 0.1 mM uridine, the end product of de novo pyrimidine biosynthesis, in the culture medium. With some lots of fetal bovine serum where PALA and DHOX had little effect on inhibiting colony formation, addition of 0.1 mM cytidine restored the inhibitory characteristics of PALA and, to some extent, DHOX. The results demonstrate that cytidine levels modulate the inhibitions of hepatoma colony formation by both PALA and DHOX and that co-administration of these drugs together with cytidine provides a simple expedient to increase drug efficacy.     

Radioiodination of L 1210 cells.

The lymphoid leukaemia L 1210 cells of mice were labelled with 125I. The cell homogenates were fractionated and from the microsomal fraction 90 per cent of the radioactive material could be precipitated with perchloric acid, whereas only 4 per cent was precipitated from the soluble fraction. Papain bound with Enzacryl AH released 31 per cent of radioactivity. It was concluded therefrom that the surface proteins of the cells were labelled. Electrophoretic separation of these proteins in polyacrylamide gel with sodium dodecyl sulphate was performed and 6--8 radioactive fractions of surface peptides were found.     

Effect of 6-azauridine on de novo pyrimidine biosynthesis in cultured Ehrlich ascites cells. Orotate inhibition of dihydroorotase and dihydroorotate dehydrogenase.

The inhibition of dihydro-orotase (E 3.5.2.3) and dihydroorotate (DHO) dehydrogenase (dihydro-orotate oxidase, EC 1.3.3.1) by cellular orotate (OA) in Ehrlich ascites cells was studied by measuring the accumulation of the intermediates of de novo pyrimidine biosynthesis at various times after the addition of 6-azauridine to the culture medium. The addition of 6-azauridine resulted in the accumulation of orotidine, OA, DHO, and carbamyl aspartate (CAA). The use of the observed ratios of [CCA]/[OA] and [DHO]/[OA] and other known constants allowed us to calculate that the increased cellular OA concentration caused primarily an inhibition of DHO dehydrogenase rather than an inhibition of dihydroorotase. A constant ratio of [CAA]/[DHO] was observed which probably indicates that the interconversion of these two intermediates catalyzed by dihydroorotase is near equilibrium in these cells as has been observed in vitro (Christopherson, R.I., Matsuura, T., and Jones, M.E. (1978) Anal. Biochem. 89, 225-234). It is suggested that the probable intracellular accumulation of CAA in patients with oroticaciduria may have significant secondary effects.     

Ethylglyoxal bis(guanylhydrazone) as an inhibitor of polyamine biosynthesis in L1210 leukemia cells

Ethylglyoxal bis(guanylhydrazone), a close derivative of the known anti-cancer drug methylglyoxal bis(guanylhydrazone), is also a powerful inhibitor of S-adenosylmethionine decarboxylase (EC 4.1.1.50), the enzyme needed for the synthesis of spermidine and spermine. There were, however, marked differences between the ethyl and methyl derivatives of glyoxal bis(guanylhydrazone) when tested in cultured L1210 cells. The cellular accumulation of ethylglyoxal bis(guanylhydrazone) represented only a fraction (20-25%) of that of the methyl derivative. Moreover, polyamine depletion, which is known to strikingly stimulate the uptake of methylglyoxal bis(guanylhydrazone), decreased, if anything, the uptake of ethylglyoxal bis(guanylhydrazone) by L1210 cells. The compound produced spermidine and spermine depletion fully comparable to that achieved with methylglyoxal bis(guanylhydrazone) at micromolar concentrations. Ethylglyoxal bis(guanylhydrazone) was growth-inhibitory to L1210 cells and produced an additive antiproliferative action when used together with 2-difluoromethylornithine. Ethylglyoxal bis(guanylhydrazone) was distinctly less effective than methylglyoxal bis(guanylhydrazone) in releasing bound polyamines from isolated cell organelles in vitro. Ethylglyoxal bis(guanylhydrazone) was also devoid of the early and profound mitochondrial toxicity typical to methylglyoxal bis(guanylhydrazone). These findings may indicate that this compound is a more specific inhibitor of polyamine biosynthesis with less intracellular polyamine 'receptor-site' activity than methylglyoxal bis(guanylhydrazone).     

Galactosyltransferase activity and cell growth: uridine diphosphate (UDP)galactose inhibition of murine leukemic L1210 cells

UDPgalactose inhibits the growth of mouse leukemic L1210 cells. In calf serum supplemented Dulbecco's medium (CS-DMEM), 1.2 mM UDPgalactose (UDPgal) inhibited cell growth by 50% (IC50), and 5 mM UDPgalactose inhibited cell growth by 92%. Other nucleotide sugars as well as galactose, glucose, and galactose-1-phosphate had little or no effect on cell growth. Uridine nucleotides, which inhibit galactosyltransferase activity, protected L1210 cells from the growth inhibitory effect of UDPgalactose when both were added simultaneously to culture media. Unlike mouse 3T12 cells, in which no inhibition of cell growth was observed with heat-inactivated calf serum (HICS)-DMEM, 5 mM UDPgalactose inhibited L1210 cell growth in HICS-DMEM to the same degree as that observed in CS-DMEM. In contrast to 3T12 cells, L1210 cells secrete significant galactosyltransferase activity into the media. Complete inhibition of 3T12 cell growth by UDPgal was observed if HICS-DMEM medium was first conditioned by L1210 cells for 48 hours. No difference in cell growth or [3H]thymidine uptake was detected after 6 hours of exposure to UDPgalactose, but both were significantly decreased at 24 and 48 hours. Flow cytometric analysis of UDPgalactose effects on L1210 cells revealed no differences in the distribution of cells in G1, S, or G2-M of the cell cycle after 6 hours of incubation, but after 16 hours of UDPgalactose treatment, L1210 cells were arrested in early S phase. These cells were completely viable and morphologically similar to control L1210 cells. Normal growth was resumed when UDPgal was removed. The data suggest that UDPgalactose inhibition of cell growth requires extracellular galactosyltransferase activity and that the effect is mediated via the cell membrane.     

Effect of plasma membrane contacts between cells on uridine incorporation and RNA polymerase activity

The effect of lateral plasma membrane contacts between neighbouring cells on uridine incorporation and endogenous RNA polymerase activity has been studied in kidney epithelial cells growing on a solid substrate in culture. A gradual increase of intercellular membrane contacts was parallelled by a gradual decrease in the rate of uridine incorporation. In contrast, the endogenous RNA polymerase activity remained unaltered when cells established membrane contacts between each other. The results are interpreted as reflecting a decreased uptake of uridine through the plasma membrane rather than a decline in RNA synthesis in cells with intercellular membrane contacts.     

关键基因的修饰对枯草芽孢杆菌尿苷合成的影响

【目的】探究磷酸核糖焦磷酸(PRPP)合成酶(prs)和氨甲酰磷酸合成酶(pyr AA/pyr AB)的点突变,以及异源5′-核苷酸酶(sdt1)的过表达,对枯草芽孢杆菌尿苷生物合成的影响。【方法】依据推断的变构位点,分别在prs基因和pyr AB基因编码序列中引入点突变;将点突变的prs基因在染色体xyl R位点整合表达,pyr AB基因则在染色体原位被修饰;sdt1基因在染色体sac B位点整合过表达。通过对重组菌摇瓶发酵液中尿苷、胞苷和尿嘧啶的分析,表征相关基因修饰对尿苷合成的影响。【结果】在PRPP合成酶中引入Asn120Ser、Leu135Ile和Glu52Gly或Val312Ala点突变,分别导致尿苷积累量提高67%和96%。进一步在氨甲酰磷酸合成酶中引入Ser948Phe、Thr977Ala和Lys993Ile点突变,导致尿苷积累量又增加了182%,达到6.97 g/L。在此基础上,过表达异源5′-核苷酸酶,导致尿苷产量增加17%,达到8.16 g/L。【结论】PRPP合成酶和氨甲酰磷酸合成酶的酶活或反馈抑制调节机制,是限制尿苷过量合成的重要因素。PRPP合成酶的Asn120Ser和Leu135Ile点突变,以及氨甲酰磷酸合成酶的Ser948Phe、Thr977Ala和Lys993Ile点突变,能够显著促进尿苷合成。PRPP合成酶附加的Glu52Gly或Val312Ala点突变,有利于尿苷合成。异源的嘧啶专一性5′-核苷酸酶的引入,也对尿苷的合成有明显的促进作用。