Transfer of E2F-1 to human glioma cells results in transcriptional up-regulation of Bcl-2.

Strong evidence exists to support the tenet that activation of E2F transcription factors, via alterations in the p16-cyclin D-Rb pathway, is a key event in the malignant progression of most human malignant gliomas. The oncogenic ability of E2F has been related to the E2F-mediated up-regulation of several proteins that positively regulate cell proliferation. However, E2F may indirectly enhance proliferation by activating antiapoptotic molecules. In this work, we sought to ascertain whether E2F-1-mediated events involve the up-regulation of the antiapoptotic molecule Bcl-2. Western blot analyses showed up-regulation of Bcl-2 but not of Bcl-x(L) by 24 h after the transfer of E2F-1. Northern blot studies showed that transfer of E2F-1 also up-regulated Bcl-2 RNA. In support of these findings and the concept that E2F-1 has a direct effect in the induction of Bcl-2, we found a putative E2F binding site within the Bcl-2 sequence. Subsequent gel-mobility shift and supershift experiments involving the CTCCGCGC site in the bcl-2 promoter showed that E2F-1 bound Bcl-2. Transactivation experiments consistently showed that ectopic E2F-1 activated responsive elements located in the -1448/-1441 region in the P1 promoter region of the bcl-2 gene. As expected, other members of the E2F family of transcription factors such as E2F-2 and E2F-4 also transactivated the bcl-2 promoter. Our results demonstrate that E2F-1 modulates the expression of the antiapoptotic molecule Bcl-2 and suggest that up-regulation of Bcl-2 may favor the oncogenic role of E2F-1 and other members of the E2F family of transcription factors.

Inhibition of cell growth in human glioblastoma cell lines by farnesyltransferase inhibitor SCH66336.

Ras activation occurs through stimulation of an upstream growth factor receptor such as epidermal growth factor receptor (EGFR). The ultimate effect of Ras is to induce nuclear transcription via a signaling pathway sequentially involving Raf, MAP kinase kinase (MEK), and mitogen-activated protein kinase (MAPK). To transform cells, Ras oncoproteins must be posttranslationally modified with a farnesyl group in a reaction catalyzed by farnesyl protein transferase. Farnesyltransferase inhibitors, therefore, have been proposed as potent anticancer agents. This study demonstrates the growth-inhibitory effects of farnesyltransferase inhibitor SCH66336 on human glioblastoma cell lines U-251 MG, U-251/E4 MG (a stably transfected cell line with elevated EGFR expression), and U-87 MG. As determined by (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) (MTS) and viability assays, the concentration required to achieve 50% inhibition (IC50) ranged from 30 microM (single 24-h treatment) to 10 microM (5-day treatment). U-251/E4 MG with overexpression of EGFR were more sensitive than U-251 MG parental cells. These observations were also supported by soft agar analysis. Cells treated with SCH66336 underwent G2 arrest. Western blot analysis revealed a decrease in phospho-MAPK levels upon treatment with 10 microM SCH66336, whereas MAPK levels were unaffected by the drug. Interestingly, increased expression of EGFR was observed in U-251 MG and U-251/E4 MG but not in U-87 MG in the presence of the inhibitor. These results demonstrate that SCH66336 inhibits viability and anchorage-independent growth in a time- and dose-dependent manner in glioblastoma cell lines U-251 MG, U-251/E4 MG, and U-87 MG via a signal transduction pathway involving the down-regulation of phospho-MAPK. Overexpression of EGFR appears to alter cellular sensitivity to farnesyltransferase inhibitors. This may have a particularly important implication in glioblastoma, where over 50% of tumors have amplification and overexpression of EGFR.

Phosphorylation of glucose by a guanosine-5'-triphosphate (GTP)-dependent glucokinase in Fibrobacter succinogenes subsp. succinogenes S85.

Cell extracts of Fibrobacter succinogenes subsp. succinogenes S85 phosphorylated glucose with a GTP-dependent glucokinase. The enzyme showed little activity with ATP (12% of that with GTP). Of other phosphate donors tested, only dGTP and ITP gave high glucokinase activities. Dialyzed extracts required Mg+2 and K+ for maximal activity. In potassium phosphate buffer, glucokinase showed maximum activity at pH 7.5 with glucose-6-phosphate dehydrogenase as the coupling enzyme. In this assay, glucokinase was active with glucose (100%), 2-deoxy-D-glucose (40%), and mannose (20%). Partially purified glucokinase had a molecular weight of 82,000 and a pI of 4.82. Double-reciprocal plots of substrate concentration versus velocity were linear and the enzyme had apparent Km values of 55 microM for glucose and 72 microM for GTP. Dialyzed cell extracts of Fibrobacter intestinalis C1A also contained a GTP-dependent glucokinase that showed little activity with ATP. Potassium also stimulated the activity of this enzyme. These results suggest that this unusual glucokinase may be characteristic of the genus Fibrobacter.

Properties of a 4-ene-3-ketosteroid-5 alpha-reductase in cell extracts of the intestinal anaerobe, Eubacterium sp. strain 144.

When Eubacterium sp. 144 was grown in the presence of progesterone, extracts of these cells contained a 4-ene-3-ketosteroid-5 alpha-reductase (5 alpha-reductase). No evidence for the presence of a 5 beta-steroid-reductase or a 5 alpha to 5 beta-steroid-isomerase was found. 5 alpha-Reductase activity was dependent on reduced methyl viologen as the electron donor and this could be generated biologically by adding pyruvate or H2 to cell extracts or chemically by adding sodium dithionite. NADH or NADPH with or without flavin nucleotides were not electron donors for 5 alpha-reductase. Most of the 5 alpha-reductase activity (60-65%) of crude extracts was located in the membrane fraction and the enzyme was solubilized by treatment with 1% Triton X-100. Optimum 5 alpha-reductase activity occurred at pH 7.0-7.5 in potassium phosphate buffer but was stimulated by Tris-HCl buffer (pH 8.0-9.0). 5 alpha-Reductase activity was highest at 10% (v/v) methanol and was progressively inhibited by higher methanol concentrations. Sulfhydryl reagents strongly inhibited 5 alpha-reductase but the enzyme was not affected by other metabolic inhibitors. Extracts prepared from cells induced with 16-dehydroprogesterone and grown without hemin contained 5 alpha-reductase and 16-dehydroprogesterone-reductase activities equivalent to those found in extracts of induced cells grown with hemin. This indicates that hemin is not required for the synthesis of active steroid double bond-reductases in strain 144.

Characteristics of 16-dehydroprogesterone reductase in cell extracts of the intestinal anaerobe, Eubacterium sp. strain 144.

16-Dehydroprogesterone reductase (16-DHPR) activity was present in cell extracts of Eubacterium sp. strain 144 only when the organism was grown in the presence of steroids containing a delta 16-17 double bond and C-20-ketone. Cells grown with 16-dehydropregnenolone contained 16-DHPR activity but lacked delta 4-5-3-keto steroid reductase activity. Pyruvate or sodium dithionite served as electron donors for 16-DHPR and both reactions required methyl viologen as an electron carrier. Neither NADH nor NADPH, with or without flavin nucleotides, were used by 16-DHPR. Enzyme activity was detected in the cytoplasmic fraction (40%) and membrane fraction (20%) of crude cell extracts, but 40% of the activity was unaccounted for following ultracentrifugation. 16-DHPR activity was unaffected by pH in potassium phosphate buffer over the range 5.0 to 8.5, but was inhibited by Tris-HCl above pH 7.0. 16-DHPR activity was inhibited by sulfhydryl reagents, but inhibitors of electron transport reactions or metal chelators did not affect the enzyme.

Cellobiose uptake by the cellulolytic ruminal anaerobe Fibrobacter (Bacteroides) succinogenes.

Cellobiose transport by the cellulolytic ruminal anaerobe Fibrobacter (Bacteroides) succinogenes was measured using randomly tritiated cellobiose. When assayed at the same concentration (1 mM), total cellobiose uptake was one-fourth to one-third that of total glucose uptake. The abilities of F. succinogenes to transport cellobiose or glucose were not affected by the sugar on which the cells were grown. Aspects of the simultaneous transport of [14C(U)]glucose and [3H(G)]cellobiose, the failure of high concentrations of cold glucose to compete with hypothetical [3H(G)]glucose (derived externally from [3H(G)]cellobiose), and differential metal-ion stimulation of cellobiose transport indicate a cellobiose permease, rather than cellobiase plus glucose permease, was responsible for cellobiose transport. Glucose (10-fold molar excess) partially inhibited cellobiose transport. This was enhanced by prior incubation of the cells with glucose, suggesting subsequent metabolism of the glucose was responsible for the inhibition. Compounds interfering with electron transport or maintenance of transmembrane ion gradients inhibited cellobiose uptake, indicating that active transport rather than a phosphoenolpyruvate:phosphotransferase system catalyzed cellobiose transport. Na+, but not Li+, stimulated cellobiose transport.

Glucose uptake by the cellulolytic ruminal anaerobe Bacteroides succinogenes.

Glucose uptake by Bacteroides succinogenes S85 was measured under conditions that maintained anaerobiosis and osmotic stability. Uptake was inhibited by compounds which interfere with electron transport systems, maintenance of proton or metal ion gradients, or ATP synthesis. The most potent inhibitors were proton and metal ionophores. Oxygen strongly inhibited glucose uptake. Na+ and Li+, but not K+, stimulated glucose uptake. A variety of sugars, including alpha-methylglucoside, did not inhibit glucose uptake. Only cellobiose and 2-deoxy-D-glucose were inhibitory, but neither behaved as a competitive inhibitor. Metabolism of both sugars appeared to be responsible for the inhibition. Cells grown in cellobiose medium transported glucose at one-half the rate of glucose-grown cells. Spheroplasts transported glucose as well as whole cells, indicating glucose uptake is not dependent on a periplasmic glucose-binding protein. Differences in glucose uptake patterns were detected in cells harvested during the transition from the lag to the log phase of growth compared with cells obtained during the log phase. These differences were not due to different mechanisms for glucose uptake in the cell types. Based on the results of this study, B. succinogenes contains a highly specific, active transport system for glucose. Evidence of a phosphoenolpyruvate-glucose phosphotransferase system was not found.

Purification and properties of 16 alpha-hydroxyprogesterone dehydroxylase from Eubacterium sp. strain 144.

Eubacterium sp. strain 144 converts 16 alpha-hydroxyprogesterone to 17-isoprogesterone. The first step of this reaction is catalyzed by 16 alpha-hydroxyprogesterone dehydroxylase (16 alpha-dehydroxylase). This enzyme was purified 40-70-fold and characterized. 16 alpha-Dehydroxylase was found to be active in two molecular weight forms of Mr 181 000 and 326 000. A subunit relative molecular weight of 42 400 was determined by sodium dodecyl sulfate gel electrophoresis of the purified enzyme. Although active with both 16 alpha-hydroxyprogesterone and 16 alpha-hydroxypregnenolone, the affinity of 16 alpha-dehydroxylase for the latter steroid was twice that of the former based on the apparent Km values. Evidence of possible substrate inhibition at high concentrations was seen with 16 alpha-hydroxypregnenolone. 16-Ketoprogesterone was found to be a competitive inhibitor of 16 alpha-dehydroxylase with respect to both steroid substrates. Although generally unaffected by low concentrations of non-ionic detergents, 16 alpha-dehydroxylase activity was stimulated 3-7-fold by sodium dodecyl sulfate and inhibited strongly by cetyltrimethylammonium bromide.

Stimulation of 16-dehydroprogesterone and progesterone reductases of Eubacterium sp. strain 144 by hemin and hydrogen or pyruvate.

Suspensions of Eubacterium sp. strain 144, prepared from cells grown with 16-dehydroprogesterone, catalyzed the reduction of this steroid to 17-isoprogesterone at a very low rate. Modifications of the assay to optimize the pH (5.5) and increase the steroid solubility (10% [vol/vol] methanol) did not significantly enhance the reaction. However, growth of strain 144 in the presence of hemin was found to stimulate 16-dehydroprogesterone reductase during the initial 30 min of incubation, giving a biphasic time course. These biphasic kinetics could be eliminated by providing the cells with an exogenous electron donor. Strain 144 used either H2 or pyruvate for this purpose, and 17-isoprogesterone formation was nearly complete after 20 to 30 min of incubation. However, under these conditions, strain 144 further converted 17-isoprogesterone to products which lacked UV absorbance (254 nm). When progesterone was used as a substrate, it was found that strain 144 could reduce the C4-C5 double bond of this steroid by a progesterone reductase to give mostly 5 beta-pregnadione and some 5 alpha-pregnadione. Furthermore, the 3-keto group of 5 beta-pregnadione steroid was also reduced to a hydroxy function. The maximum activities of both 16-dehydroprogesterone and progesterone reductases in cell suspensions required the growth of strain 144 with hemin and 16-dehydroprogesterone and the presence of H2 or pyruvate.

Biotransformation of 16-dehydroprogesterone by the intestinal anaerobic bacterium, Eubacterium sp. 144.

Eubacterium sp. 144 biotransformed 16-dehydroprogesterone by initially hydrating approx 50% to 16 alpha-hydroxyprogesterone. The detection of this reaction was dependent, in part, on the solubility state of 16-dehydroprogesterone and was less extensive when the concentration of methanol was insufficient to solubilize the steroid. Cultures containing a mixture of 16-dehydroprogesterone and 16 alpha-hydroxyprogesterone formed isoprogesterone as a final steroid end product. However, the extent of the reductive reaction was influenced by culture age at the time of 16-dehydroprogesterone addition and decreased in older cultures. Moreover, both mid- and late-log phase cells also formed progesterone as a reduced steroid end product. The enzyme(s) responsible for isoprogesterone formation (16-dehydroprogesterone reductase) appeared to be inducible because activity was not evident until 3-6 h after the addition of 16-dehydroprogesterone to early log-phase cultures. Growth inhibitory concentrations of chloramphenicol or rifampin prevented isoprogesterone formation, but not the production of progesterone. At lower concentrations, chloramphenicol delayed both growth and isoprogesterone formation by strain 144. Interestingly, rifampin partially inhibited the 16 alpha-hydroxyprogesterone dehydratase (hydration reaction) in cultures of strain 144, but did not affect the enzyme's activity in cell extracts.

Biotransformation of 16 alpha-hydroxyprogesterone by Eubacterium sp. 144: non-enzymatic addition of L-cysteine to delta 16-progesterone.

Eubacterium sp. 144 dehydroxylated 16 alpha-hydroxy-progesterone; however, the expected intermediate, delta 16-progesterone, did not accumulate to significant concentrations in the culture medium. Moreover, the final end product of this biotransformation, 17 alpha-progesterone, was produced at a very slow rate. It was discovered that, under our culture conditions, delta 16-progesterone reacted chemically with L-cysteine to form a highly water-soluble derivative. The ability of delta 16-progesterone to react with L-cysteine in culture media was considerably reduced when L-cysteine was autoclaved in the presence of complex medium components. delta 16-Progesterone also reacted chemically with D-cysteine, L-homocysteine, glutathione, and 2-mercaptoethylamine. The reaction was favored by alkaline pH (greater than or equal to pH 8.0) and required both an unhindered thiol group and a proximal amino group on the mercapto compound. Chromatography of the putative delta 16-progesterone L-[U-14C]-cysteine reaction product by HPLC showed a single UV-absorbing, radioactive peak (RT 4.31 min).

16 alpha-dehydration of corticoids by bacteria isolated from rat fecal flora.

Two strains (No. 144 and No. 146) of rat intestinal anaerobic bacteria, phenotypically similar to Eubacterium lentum, were isolated and found capable of 16 alpha-dehydrating corticoids. The initial step in the 16 alpha-dehydration of 16 alpha-hydroxyprogesterone was dehydration at the C-16 and C-17 position with the accumulation of 16-dehydroprogesterone. This step required the side chain at C-17. In bacterial cultures the 16-dehydroprogesterone was then slowly reduced to iso-progesterone. 16 alpha-Hydroxypregnanolone was also converted to iso-pregnanolone by these bacteria. 16 alpha-Dehydratase was easily demonstrated in cell fractions of strain No. 144 incubated either aerobically or anaerobically. The same extracts did not convert 16-dehydroprogesterone to iso-progesterone under similar assay conditions. 16 alpha-Dehydration occurred at all substrate concentrations tested (20 to 200 micrograms/ml) provided the pH of the growth medium was between 6 abd 8 and the Eh below -130 mV. Strain No. 146 had both 16 alpha-dehydration and 21-dehydroxylation activities. The two enzymes functioned independently. A role for intestinal bacteria in the biotransformation of biliary 16 alpha-hydroxylated steroids and subsequent excretion in the urine is proposed.

Characterization of a pyridine nucleotide-nonspecific glutamate dehydrogenase from Bacteroides thetaiotaomicron.

An oxidized nicotinamide adenine dinucleotide phosphate/oxidized nicotinamide adenine dinucleotide (NADP+/NAD+) nonspecific L-glutamate dehydrogenase from Bacteroides thetaiotaomicron was purified 40-fold (NADP+ or NAD+ activity) over crude cell extract by heat treatment, (NH4)2SO2 fractionation, diethylaminoethyl-cellulose, Bio-Gel A 1.5m, and hydroxylapatite chromatography. Both NADP+- and NAD+-dependent activities coeluted from all chromatographic treatments. Moreover, a constant ratio of NADP+/NAD+ specific activities was demonstrated at each purification step. Both activities also comigrated in 6% nondenaturing polyacrylamide gels. Affinity chromatography of the 40-fold-purified enzyme using Procion RED HE-3B gave a preparation containing both NADP+- and NAD+-linked activities which showed a single protein band of 48,5000 molecular weight after sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The dual pyridine nucleotide nature of the enzyme was most readily apparent in the oxidative direction. Reductively, the enzyme was 30-fold more active with reduced NADP than with reduced NAD. Nonlinear concave 1/V versus 1/S plots were observed for reduced NADP and NH4Cl. Salts (0.1 M) stimulated the NADP+-linked reaction, inhibited the NAD+-linked reaction, and had little effect on the reduced NADP-dependent reaction. The stimulatory effect of salts (NADP+) was nonspecific, regardless of the anion or cation, whereas the degree of NAD+-linked inhibition decreased in the order to I- greater than Br- greater than Cl- greater than F-. Both NADP+ and NAD+ glutamate dehydrogenase activities were also detected in cell extracts from representative strains of other bacteroides deoxyribonucleic acid homology groups.

Transformation of 4-androsten-3,17-dione by growing cultures and cell extracts of Clostridium paraputrificum.

Growing cultures of Clostridium paraputrificum transformed 4-androsten-3,17-dione to 3 alpha-hydroxy-5 beta-androstan-17-one in a sequential manner with 5 beta-androstan-3,17-dione as an intermediate. The addition of 1.5 mM menadione to log-phase cultures which had formed 5 beta-androstan-3,17-dione resulted in a partial reoxidation of this steroid to 4-androsten-3,17-dione. However, this treatment also resulted in transient inhibition of culture growth. Resumption of growth was accompanied by complete reduction of 4-androsten-3,17-dione to 5 beta-androstan-3,17-dione. Cell extracts of C. paraputrificum were capable of carrying out these reductive transformations in the absence of added cofactors. However, Sephadex G-25 treated extracts required NADH or NADPH for these reactions. A flavin nucleotide, either FAD (plus NADH or NADPH) or FMN (plus NADH) was highly stimulatory for 4-androsten-3,17-dione reduction to 5 beta-androstan-3,17-dione. NADH was the preferred reduced pyridine nucleotide for reduction of the C4-C5 double bond, while time-course measurements suggested that NADPH was the preferred donor for reduction of the 3-keto group.

Synthesis of guanosine tetra- and pentaphosphates by the obligately anaerobic bacterium Bacteroides thetaiotaomicron in response to molecular oxygen.

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) were detected in formic acid extracts of air-exposed culutres of Bacteroides thetaiotaomicron. The identification of ppGpp and pppGpp in B. thetaiotaomicron was based on the following results: (i) cochromatography of 32P-labeled hyperphosphorylated nucleotides in two different two-dimensional solvent systems with authentic ppGpp and pppGpp; (ii) incorporation of [3H]guanosine into the putative ppGpp and pppGpp; (iii) alkaline lability; and (iv) resistance, to periodate oxidation. There was a marked increase in the concentration of ppGpp and pppGpp after shift from anaerobic to aerobic conditions, and accumulation of both ppGpp and pppGpp was blocked under these conditions by pretreatment of the culture with rifampin or tetracycline. Growth and incorporation of [3H]guanosine, [3H]tymidine, [14C]succinate, and L-[35S]methionine into macromolecules were inhibited immediately upon exposure to air. The accumulation of ppGpp and pppGpp in B. thetaiotaomicron upon exposure to air may represent a novel signal for synthesis of these compounds.

Partial purification of ferredoxin from Ruminococcus albus and its role in pyruvate metabolism and reduction of nicotinamide adenine dinucleotide by H2.

Extracts of Ruminococcus albus were not able to convert pyruvate to acetyl phosphate, CO2, and H2 after passage through a diethylaminoethyl (DEAE)-cellulose column. Activity was restored by a brown protein fraction eluted from the column with 0.4 M Cl-. The protein was partially purified and shown to have the spectral and biological characteristics of ferredoxin. R. albus ferredoxin, Clostridium pasteurianum ferredoxin, and methyl viologen restored activity for pyruvate decomposition by DEAE-cellulose-treated R. albus extracts. R. albus or C. pasteurianum ferredoxin restored the ability of DEAE-cellulose-treated C. pasteurianum extracts to form H2 and acetyl phosphate from pyruvate. Ferredoxin-free extracts of R. albus reduced nicotinamide adenine dinucleotide (NAD) when supplemented with R. albus or C. pasteurianum ferredoxin or with methyl viologen. These extracts reduced NADP with H2 poorly unless both ferredoxin and NAD were added, which indicates the presence of an NADH:NADP transhydrogenase. Flavin mononucleotide and flavin adenine dinucleotide were rapidly reduced by H2 by ferredoxin-free extracts in the absence of ferredoxin.