Pyrimidine nucleoside analogues as inducers of pyrimidine nucleoside catabolizing enzymes in salmonella typhimurium

Various structural analogues of cytosine and uracil nucleosides were tested as potential inducers of the nucleoside catabolizing (cyt) enzymes in Salmonella typhimurium. Some analogues, e.g. 5′-O-alkyl cytidines and uridines, resistant to catabolic enzymes, were as effective as the natural inducers cytidine and uridine; but etherification of one of the cis 2′ or 3′hydroxyls fully abolished activity, pointing to a requirement of an intact ribose cis-glycol system for activity. A uridine analogue in the syn conformation, 6-methyluridine, a good substrate for uridine phosphorylase, was inactive as an inducer. The behaviour of various other analogues, in relation to their structure, conformation and substrate properties, indicated the absence of any correlation between inducing activity and substrate susceptibility. The overall findings are consistent with conclusions derived from genetic experiments. The active analogues apparently act via similar pathways, and probably affect the same regulatory mechanism(s) as the natural inducers.     

A -glucuronidase deficiency mucopolysaccharidosis: studies in cultured fibroblasts

Cytochrome P-450 cannot be detected spectrophotometrically in testis mitochondria of untreated rats because of the high cytochrome a₃ to Cytochrome P-450 ratio. Injection of human chorionic gonadotrophin (HCG) causes a large increase in mitochondrial cytochrome P-450. After 14 days injection, mitochondrial cytochrome P-450 levels are increased 15- to 30-fold (from 0.007 to 0.134 nmoles/mg protein) over control levels. Levels of cytochrome a + a₃ are not altered by this treatment. Mitochondrial cytochrome P-450 can also be demonstrated by injection of HCG into rats which were hypophysectomized 24 days previously. During hypophysectorny the mitochondrial cytochromes c + c_i, a + a₃ and mitochondrial protein decay with halflives of 14, 16, and 15.5 days, respectively. HCG treatment for 8 days increases mitochondrial cytochrome P-450 (from < 0.003 to 0.24 nmoles/mg protein) without altering the levels of the other mitochondrial cytochromes. The control of cytochrome P-450 levels in the mitochondria by HCG suggests that the level of this key component of cholesterol side-chain cleavage enzyme may be of importance in the regulation of steroidogenesis in the testis.     

Pyrimidine nucleoside, pseudouridine, and modified nucleoside excretion by growing and resting fibroblasts.

We are examining the relationship of RNA metabolism and de novo pyrimidine synthesis as parameters of malignant transformation. These initial experiments on normal hamster embryo fibroblasts have shown that excreted nucleosides are markers for intracellular RNA metabolism. We employed affinity chromatography to concentrate the nucleosides in the medium and sensitive column chromatographic procedures to quantitatively measure them. The excretion of pyrimidine nucleoside from hamster embryo fibroblasts in sulture was found to be dependent on the growth state of the cells, with the greatest accumulation occurring cell quiescence. The major nucleoside excretion products, uridine and cytidine, were both normal end products of RNA metabolism and the major nucleoside excretion products from cultured cells. The modified nucleosides N-1-methylguanosine, N-2-methylguanosine, N-2-dimethylguanosine, N-4-acetylcytidine, N-1-methylinosine, pseudouridine, N-1-methyladenosine, N-3-methylcytidine, and 5-methyleycytidine were found, as were several unidentified nucleosides.     

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.     

Cell surface-associated lysosomal enzymes in cultured human skin fibroblasts

Trypsin released from the surface of intact human skin fibroblasts β-N-acetylglucosaminidase. The amount of trypsin removable β-N-acetylglucosaminidase in 4 control and 14 mucopolysaccharidosis cell lines was equivalent to 1.5% (range 0.5–4.3%) of the intracellular activity. Cell surface-associated β-N-acetylglucosaminidase was absent in mucolipidosis II and III fibroblasts that form lysosomal enzymes defective in binding to the cell surface receptors of fibroblasts and in β-N-acetylglucosaminidase deficient fibroblasts (Sandhoff's disease). Indirect immunofiuorescence with monospecific antisera allowed the demonstration of β-N-acetylglucosaminidase, α-N-acetylglucosaminidase, α-mannosidase and β-glucuronidase on the cell surface of fibroblasts, whereas these enzymes were absent on the cell surface of mucolipidosis II and III fibroblasts. Simultaneous staining for β-glucuronidase and β-N-acetylglucosaminidase showed presence of both enzymes in almost identical areas of the same cell. Cross-reacting material was present on the cell surface of fibroblasts with a deficiency of β-N-acetylglycosaminidase, α-N-acetylglucosaminidase (mucopolysaccharidosis III B), α-mannosidase (mannosidosis) and β-glucuronidase (mucopolysaccharidosis VII). The demonstration of lysosomal enzymes on the cell surface is in agreement with the hypothesis that in fibroblasts transport of lysosomal enzymes to the lysosomal apparatus involves cycling of lysosomal enzymes via the cell surface.     

Surface enzymes in cultured fibroblasts from cystic fibrosis patients.

Membrane function was examined in cultured cells from cystic fibrosis patients by assaying several enzymes on intact skin fibroblasts attached to culture dishes. This technique required few cells and minimized disruption of cellular organization. Comparison of enzyme activities of intact and broken cells showed that 12% of total glucose-6-phosphate dehydrogenase, a cytoplasmic enzyme, was measurable using intact cells, while all adenosine monophosphatase was measurable using intact cells. Alkaline paranitrophenylphosphatase activity was divided between the cell surface and interior. Substrate competition experiments indicated that substrate specificities for adenosine monophosphatase and paranitrophenylphosphatase activities were different. Adenosine monophosphatase activities of 2 control and 2 cystic fibrosis strains fluctuated similarly during the cell culture cycle. The apparent Km values relative to adenosine monophosphate were similar in all strains. A chromatographic fraction of serum from a cystic fibrosis patient that was inhibitory to oyster ciliary activity had no effect on adenosine monophosphatase activity of normal fibroblasts. Furthermore, fractions of media from cystic fibrosis homozygote and heterozygote fibroblast cultures were not inhibitory to adenosine monophosphatase activities of intact normal fibroblasts or of part iculate fractions prepared from them. In light of previous studies that showed that factors from cystic fibrosis serum of culture medium disrupted specific membrane activities, it is proposed that the cystic fibrosis factor interacts with the plasma membrane, interfering most conspicuously with the protein functions that are sensitive to changes in their membrane environment.     

Argininosuccinase deficiency in fibroblasts cultured from patients with argininosuccinic aciduria

Fibroblasts cultured from the skin of four mentally retarded patients with argininosuccinic aciduria were markedly deficient in argininosuccinase activity.This work was supported by U.S. Public Health Service grants NB-05096 and CA-04670.     

Alpha-5-phosphoribosyl-1-pyrophosphate-independent salvage of purines in cultured Chinese hamster lung fibroblasts

A variant clone of cultured chinese hamster lung fibroblasts (V79), selected for resistance to 8-azaguanine (V79 azagrst), although lacking hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8), is able to convert hypoxanthine into IMP via purine-nucleoside phosphorylase (EC 2.4.2.1) and nucleoside kinase. In addition to the phosphoribosylation pathway, we also present evidence for the occurrence of a kinase-mediated pathway of recovery of hypoxanthine in the wild-type cells. The lower rate of formation of IMP in the V79 azagrst cells, apparently correlated with the phosphorylation of the nucleoside, suggests possible differences in the catalytic and/or regulatory properties of nucleoside kinase in the two cell lines. This fact might be of particular relevance in evaluating the mechanisms of resistance to purine analogs displayed by several cell types.     

Genetic complementation of propionyl-CoA carboxylase deficiency in cultured human fibroblasts.

Propionyl-CoA carboxylase (PCC) deficiency is an inherited metabolic disorder showing considerable variability of expression. We have investigated the possibility that there is a genetic basis for the clinical heterogeneity in this disorder by examining complementation in Sendai virus mediated heterokaryons of mutant fibroblast strains. Restoration of PCC activity was monitored in individual multinucleate cells in situ using a radioautographic procedure which detects the incorporation of 14C-propionate into trichloracetic acid precipitable material. Each mutant strain incorporated negligible amounts of radioactivity compared to control strains. Activity was not restored when different mutants were mixed without virus or when homokaryons were produced by self-fusion. Seven mutant strains were fused in all pairwise combinations and examined for increased 14C-propionate incorporation in heterokaryons. Two main complementation groups were revealed. One group was composed of three mutants. The other was a complex group composed of four mutants in which intragroup complementation was demonstrated. Two mutants showing excellent complementation by radioautography were examined for complementation by the direct assay of PCC ACTIVITY. The enzyme activity of virus-treated preparations with 23% multinucleate cells was 183 U (pmol/min/mg protein) compared to 16 U for the untreated mixture (normal range 450-850 u). We conclude that PCC deficiency resulted from mutations of heterogeneous origin, although the classification of mutants into complementation groups did not correlate with patterns of clinical heterogeneity.     

Effect of lectins on endocytosis and secretion of lysosomal enzymes by cultured fibroblasts.

1. Pretreatment of cultured human skin fibroblasts with convanavalin A and wheat germ agglutinin inhibited endocytosis of alpha-N-acetylglucosaminidase and increased extracellular accumulation of beta-N-acetylglucosaminidase. 2. These effects were dose-dependent, reversible and could be prevented by haptenic carbohydrates, such as methyl alpha-D-mannoside or N-acetylglucosamine. 3. Pretreatment of fibroblasts with di- and monovalent succinylated concanavalin A inhibited alpha-N-acetylglucosaminidase endocytosis, but had no effect on extracellular beta-N-acetylglucosaminidase accumulation. 4. Concanavalin A-alpha-N-acetylglucosaminidase complexes become internalized via the recognition of the lectin. Complex formation prevents recognition of the phosphorylated carbohydrate on lysosomal enzymes that interacts with cell surface receptors specific for lysosomal enzymes. The inhibitory effect of all lectins tested on lysosomal enzyme endocytosis suggests that the cell surface receptors for lysosomal enzymes interact either directly with lectins or are closely linked to lectin receptors. The effect of polyvalent lectins on extracellular lysosomal enzyme accumulation is ascribed to their alteration of membrane fluidity.     

Effect of 2-deoxyglucose on lysosomal enzymes in cultured human skin fibroblasts

Addition of 2-deoxyglucose, an inhibitor of glycosylation of proteins, to the medium of confluent cultures of human skin fibroblasts prevents the increase in specific activity of lysosomal enzymes that normally occurs after confluence. Maximal inhibition is obtained at a concentration of about 1 mM 2-deoxyglucose. The inhibition by 2-deoxyglucose is reversible. The K_m, pH dependence and electrophoretic mobility of the acid hydrolases tested was the same in cells cultured with or without 2-deoxyglucose. In homogenates of cultured human skin fibroblasts, about 95% of the β-hexosaminidase and α-galactosidase activity and about 65 % of the acid phosphatase activity with β-glycerolphosphate as substrate binds to concanavalin A (ConA); 2-deoxyglucose affects only the activity able to bind to ConA. In cells cultured in the presence of 2-deoxyglucose, the specific activity of alkaline phosphodiesterase I, a plasma membrane glycoprotein is lowered. 2-Deoxyglucose has no effect on the specific activity of succinate dehydrogenase, lactate dehydrogenase or total cellular protein.     

The effect of proteins released by cultured fibroblasts on density-dependent agglutination

An important criterion for the designation of non-transformed cell lines is density-dependent regulation of growth. The mechanism for control of such growth is believed to be lodged in the cell membrane. To elucidate this mechanism, the density dependence of surface-related processes has been examined. This report on ConA-mediated agglutination extends our earlier findings on another surface phenomenon, cell to substratum adhesion. Both these processes are density dependent and both are stimulated by cell proteins released into serum-free media by cultured human fibroblasts. It is suggested that these released proteins are transferable between cells and that they play a critical role in the mediation of density dependent surface phenomenon and in the density dependent regulation of cell growth.     

Characterization of lysosomes and lysosomal enzymes from Chediak-Higashi-syndrome cultured fibroblasts.

Chediak-Higashi-syndrome cultured skin fibroblasts were used to study the possible involvement of lysosomal enzymes and lysosomal dysfunction in this disorder. Our evidence indicated that Chediak-Higashi fibroblasts displayed a significant decrease in the specific activity of the acidic alpha-D-mannosidase (pH 4.2) compared with normal controls. Additional studies revealed a small, but significant, decrease in the rate of degradation of 125I-labelled beta-D-glucosidase that had been endocytosed into Chediak-Higashi cells.     

Uptake of retinol by cultured fibroblasts

Cultured fibroblasts of adult rats were used to determine whether they could take in retinol administered to the culture medium at physiological concentration. After the administration of retinol, cells were observed with a phase-contrast fluorescence light microscope (LM) and a transmission electron microscope (TEM). Retinol and retinyl fatty acyl esters (RFAE) stored in the cells were analyzed with high-performance liquid chromatography (HPLC). It was revealed that these fibroblasts could take in retinol in the medium at a concentration of 1 x 10(-7) M and store it in lipid droplets in the cytoplasm as retinyl palmitate and other RFAE.     

Transport enzymes in the cell membranes of cultured fibroblasts; alterations in dystrophic cells

In support of the widely held belief that membrane defects are present in the muscular dystrophies, alterations have been found in some transport-related enzymes of cells from affected donors. Cell membranes were isolated from cultured dermal fibroblasts of victims of myotonic muscular dystrophy, and of Duchenne's muscular dystrophy, and from cells of normal age- and sex-matched donors. Myotonic cells had an elevated Na+, K+ ATPase. gamma-Glutamyl transpeptidase was elevated in Duchenne cells. Among all cells' 5'nucleotide phosphatase exhibited a remarkably constant specific activity.     

Effects of dimethylsulfoxide on sphingomyelinase in cultured human fibroblasts and correction of sphingomyelinase deficiency in fibroblasts from Niemann-Pick patients

The effects of dimethylsulfoxide (DMSO) on sphingomyelinase activity were studied using human skin fibroblasts from normal individuals and Niemann-Pick patients. Sphingomyelinase activity in normal fibroblasts increased up to 230% of controls by 2% DMSO while the cell growth was inhibited. Other lysosomal hydrolases showed a rather smaller extent of increment in activity. There was no direct effect of DMSO in cell-free system and no evidence of any activating factor of sphingomyelinase in homogenates. Sphingomyelinase deficiency in fibroblasts from a Niemann-Pick patient (type C) was corrected by 2% DMSO with regaining its enzyme activity.     

Cytosolic thymidine kinase activity in cultured human fibroblasts from individuals with galactokinase deficiency

The structural genes for human galactokinase (GALK) and the human cytosolic form of thymidine kinase (TK1) are located on 17q21–q22. These two loci are tightly linked, and studies on Chinese hamster cell lines have shown that the expression of TK1 and GALK genes may alter simultaneously. We investigated the possibility of a dependent mutation of TK1 and GALK genes in cultured fibroblasts obtained from two patients homozygous for the GALK^G-deficient gene. Since we showed that the TK1 level varies as a function of the passage and the growth rate of a given strain, our experiments were performed on nonstored skin fibroblasts, between the third and the fifth passage for both controls and patients. We found that TK1 levels in GALK-deficient cells were almost 75% of those observed in control strains with a similar growth rate. Previous results in the literature have shown a pronounced decrease in TK1 activity in three GALK-deficient fibroblastic strains. We suggest that these disparities of TK1 levels in GALK-deficient fibroblasts may be related either to genetic heterogeneity of GALK deficiency or to differences in culture conditions. This work was supported in part by grants from La CNAMTS and l’Université de Paris-Sud (AI 86 10).