AIM:To explore different effects of 12 beticolins, Cercospora beticola toxins, on ras-transformed adrenocortical cell growth inhibition and their functional mechanism.METHODS:Beticolin-induced inhibition was measured with survival cell number determined by an automated photocolorimetric method. The penetration of beticolin was examined by confocal microscopy. Ras protein determined by Lowry method were separated by 14 % SDS-PAGE and electroblotted to Immobilon-P transfer membrane and detected with pan-Ras (Ab-3) monoclonal antibody. The Ca2+ chelation by beticolin was investigated using a calcium ionophore.RESULTS:Cell growth inhibition was found dose- and time-dependently at submicromolar level for beticolin-1, -2, and -13 (IC50 </= 250 nmol/L) and for beticolin-0, 6, and -11 (400 nmol/L < IC50 </= 500 nmol/L). The inhibition by beticolin-1 was immediate, independent of cell culture step and not reversible for 3-day treatment. Beticolin-3 and -4 were slightly active (1 micromol/L < IC50 </= 2 micromol/L) and beticolin-7, -9, -12, and -5 were inactive at micromolar level. The beticolin-induced cell growth inhibition was correlated with the hydrophobicity of these compounds. Beticolin-1 fluorescence in RTAC cells was detected by confocal microscopy whereas beticolin-3 and -12 were not even after a 24 h incubation period. Beticolin-1-induced cell growth inhibition was partially reverted by calcium ionophore suggesting a role of intracellular Ca2+ chelation by beticolin-1 on cell growth inhibition. Furthermore, beticolin-1 blocked up Ras p21 translocation to membrane and induced accumulation of Ras in the cytosol as an inactive form by different ways.CONCLUSION:Beticolins with high hydrophobicity inhibit tumorigenic cell proliferation by different ways.

Lipid transfer proteins (LTPs) and elicitins are both able to load and transfer lipidic molecules and share some structural and functional properties. While elicitins are known as elicitors of plant defence mechanisms, the biological function of LTP is still an enigma. We show that a wheat LTP1 binds with high affinity sites. Binding and in vivo competition experiments point out that these binding sites are common to LTP1 and elicitins and confirm that they are the biological receptors of elicitins. A mathematical analysis suggests that these receptors could be represented by an allosteric model corresponding to an oligomeric structure with four identical subunits.

Cryptogein is a proteinaceous elicitor of plant defense reactions which also exhibits sterol carrier properties. In this study, we report that this protein binds fatty acids. The stoichiometry of the fatty acid–cryptogein complex is 1:1. Linoleic acid and dehydroergosterol compete for the same site, but elicitin affinity is 27 times lower for fatty acid than for sterol. We show that C7 to C12 saturated and C16 to C22 unsaturated fatty acids are the best ligands. The presence of double bonds markedly increases the affinity of cryptogein for fatty acids. A comparison between elicitins and known lipid transfer proteins is discussed.

To test the Kirsten‐Ras (Ki‐Ras) alternative prenylation hypothesis in malignant transformation, we used a novel farnesyltransferase inhibitor competitive to farnesyl‐pyrophosphate, RPR130401, and a CaaX peptidomimetic geranylgeranyltransferase‐1 inhibitor GGTI‐298. In Ki‐Ras‐overexpressing transformed adrenocortical cells, RPR130401 at 1–10 μM inhibited very efficiently the [ 3 H]farnesyl but not [ 3 H]geranylgeranyl transfer to Ras. However, proliferation of these cells was only slightly sensitive to RPR130401 (IC 50 =30 μM). GGTI‐298 inhibited the growth of these cells with an IC 50 of 11 μM but cell lysis was observed at 15 μM. The combination of 10 μM RPR130401 and 10 μM GGTI‐298 inhibited efficiently (80%) cell proliferation. These combined inhibitors but not each inhibitor alone blocked the cell cycle in G 0 /G 1 and disrupted MAP kinase activation. Thus, combination of two inhibitors, at non‐cytotoxic concentrations, acting on the farnesyl‐pyrophosphate binding site of the farnesyltransferase and the CaaX binding site of the geranylgeranyltransferase‐1 respectively is an efficient strategy for disrupting Ki‐Ras tumorigenic cell proliferation.

Yeast autolysis affects membrane stability and induces a release of vacuolar enzymes into the cell cytoplasm. Consecutively, it was important to study the evolution of sterol content in Saccharomycescerevisiae for a fourteen day period of accelerated autolysis. Unesterified and esterified sterols were analyzed both in the biomass and in the autolysis medium. Ten sterols were identified by gas chromatography/mass spectrometry. A second group of six sterols was separated and partially characterized. Among the first group of 10 sterols, a dehydroergosterol was identified as ergosta-5, 7,9(11),22-tetraen-3beta-ol, not yet charaterized in S. cerevisiae. Yeast autolysis induced a decrease of esterified sterol content, especially first intermediates in the sequence of the ergosterol biosynthesis, as zymosterol. In contrast, the yeast autolysis resulted in the release of a low quantity of sterols into the medium. At the end of the fourteenth day of autolysis, 0.015% of the total sterol content of the initial biomass was found in the medium.

Theras-transformed newborn rat adrenocortical (RTAC) cells were obtained by transfection with the mutated c-Ha-rasEJoncogene. They are proliferative and tumorigenic cells characterized by expression of the c-Ha-rasEJoncogene and overexpression of a wild-typerasoncogene. The overproduced Ras p21 was identified here as Ki-Ras p21 by western blotting using a specific anti-Ki-Ras monoclonal antibody. Radioactivity derived from [14C]mevalonolactone was strongly incorporated into Ras p21 overproduced in RTAC cells. RTAC cells pretreated with lovastatin and labeled with either [3H]geranylgeranyl-pyrophosphate or [3H]farnesyl-pyrophosphate incorporated also radioactivity into Ras p21. These results showed that overproduced Ras proteins were geranylgeranylated as well as farnesylated in RTAC cells. These findings suggest that the strategy for inhibiting proliferation of Ki-ras-dependent tumorigenic cells should be directed against not only farnesylation but also geranylgeranylation of Ras p21.

Suspension-cultured cells of Nicotiana tabacum generated active oxygen species (AOS) when they were treated with the proteinaceous elicitor, cryptogein. This response was blocked by diphenylene iodonium, an inhibitor of the neutrophil NADPH oxidase. When microsomal extracts of tobacco cells were probed with an antibody directed against the human small G protein Rac2, two immunoreactive proteins were detected at 18.5 and 20.5 kDa. The same experiment performed with cytosolic extracts of tobacco cells led to the observation of a strong immunoreactive protein at 21.5 kDa only in the cryptogein-treated cells. The appearance of this cytosolic protein was related to the production of AOS by the elicited cells. These results provide evidence for the possible involvement of small G proteins, homologous to the neutrophil Rac2 protein, in the regulation of the elicitor-induced oxidative burst in plant.

The proliferation of GM16 and 4CDTras‐transformed newborn rat adrenocortical (RTAC) cells and Y1 mouse adrenal tumor cells was inhibited by beticolins, the fungal toxins extracted fromCercospora beticola, at submicromolar concentrations in a dose‐dependent manner. Inhibitory concentrations for half the maximum inhibition were 150, 75 and 25 nmfor beticolin‐1 and 230, 150 and 50 nmfor beticolin‐2 in GM16, 4CDT and Y1 cells respectively. Beticolins strongly inhibited the production of 11β‐hydroxysteroids on the second and third days of treatment in a dose‐dependent manner between 0.1 and 1 μm. Beticolins were shown by confocal microscopy to be localized in cytoplasmic organelles about 30–40 min after treatment. This finding favors a direct action of beticolins on mitochondrial steroid 11β‐hydroxylase albeit another less direct mechanism involving a cytoplasmic signaling pathway cannot be excluded.

A rapid high-performance liquid chromatographic (HPLC) method for the separation of phospholipids was developed for minute samples of total lipids (ca. 200 μg). The method was applied to the study of the phospholipid metabolism in adrenocortical cell cultures. A complete separation of the different cellular phospholipid classes was achieved in 40 min. Good resolution of the phospholipid peaks was obtained, which allowed the collection of each individual class of phospholipids for further analysis of radioactivity and fatty acid composition by gas chromatography. When cells were incubated with [U-14C]glycerol or [U-14C]palmitate the bulk of the radioactivity was found in cellular phosphatidylcholines. Exogenous phospholipids were incorporated into cellular lipids to a large extent, however without an increase in the cellular phospholipid content. 12-O-Tetradecanoyl-phorbol-13-acetate induced a 20% increase in the polyunsaturated fatty acid content of the cellular phosphatidylethanolamines, but no change was detected in the cellular phosphatidylcholines. The developed method is well-suited to the study of the phospholipid metabolism in adrenocortical cells where the phospholipid metabolism is closely linked to the specialized functions of the cells.

Adrenocortical cells were obtained by fractionated trypsination of newborn rat adrenal glands and transfected with a plasmid containing the EJT24-Ha-ras oncogene. Isolation of adhesive cells led to a proliferative cell line with an overexpression of 21 kDa ras protein. These cells incubated with corticosterone or deoxycorticosterone as the precursor produced a high level of 18-hydroxycorticosterone and aldosterone as identified by gas chromatography- mass spectrometry. ACTH and angiotensin II increased the basal production of aldosterone nineteen-fold and six-fold respectively. Under ACTH stimulation the ratio between aldosterone and 18-hydroxycorticosterone production was 1:3. The transformation of corticosterone under angiotensin II stimulation yielded up to 41% of 18-hydroxycorticosterone (4.7 μg/mg of cell protein per 24h) and 4.4% of aldosterone (0.5 μg/mg of cell protein per 24h) in a low potassium concentration medium (6 mmol/l). To our knowledge this is the first report of continuous proliferative adrenocortical cells producing aldosterone.

High density lipoprotein (HDL) has been shown to induce the cellular accumulation of cholesterol esters and the biosynthetis of 21-hydroxysteroids (corticosteroids) newborn rat adrenocortical cells cultivated in serum-free medium. In order to identify the component(s) of HDL responsible for these effects, we investigated the ability of rat HDL subfractions and HDL with or without apolipoprotein E to deliver cholesterol to cells and to stimulate the steroid biosynthetic pathways in adrenal cultured cells. The total cholesterol uptake from HDL2 was greater than that observed with HDL rich in apolipoprotein E (HDL1 and HDLc). Furthermore, the increase of the ratio between 21-hydroxysteroids and reductive metabolites of progesterone was higher with HDL2 than with HDL1 or HDLc. The results of competitive studies between LDL and HDL subfractions indicate that adrenal cells take up cholesterol from HDL2 and LDL by separate mechanisms but that LDL and HDL containing apolipoprotein E share the same uptake processes. In experiments with various concentrations of HDLc or HDL without apolipoprotein E, the adrenal cells displayed a higher affinity for rat HDLc than for rat HDL without apolipoprotein E. However, HDL without apolipoprotein E produced a higher enhancement of the cholesterol cell content and was 3-fold more effective in stimulating 21-hydroxylated steroid production than rat HDLc. Although these findings suggest a participation of HDL with apolipoprotein E in the HDL interaction with rat adrenal cells, the predominant effect on these cells is devoluted to HDL containing mainly apolipoprotein A.

The development of a method for the serum-free culture of the Y-1 mouse adrenocortical tumor cell line has permitted a detailed search for factors regulating cellular growth and steroidogenesis. The serum-free medium (SFM) was made of Ham's F10 basal medium supplemented with free fatty acids adsorbed on albumin. The SFM complemented with calcium, arachidonic acid and cholesterol, i.e. SFM-S, induced cell proliferation to a density at confluency higher than that obtained with 1% serum-supplemented medium (1%-SSM) and allowed to sustain cell growth for more than six passages. When albumin was replaced by a dextran polymer (Mr = 2 x 10(6)) used as a carrier of lipids instead of albumin (which resulted in a serum-free and protein-free medium, SPFM), the cell number was 75% of that observed with the SFM-S. The addition to SPFM of beta-globulin alone or combined with insulin caused a 2- or 3-fold increase in the final cell density, respectively. The ability of the Y-1 cell line to produce steroids in response to ACTH was found to be higher in SFM-S or SPFM than in 1%-SSM. Furthermore, the addition of beta-globulin to SPFM stimulated steroid hormone biosynthesis with a marked increase in 11 beta-hydroxylated steroid production. These studies demonstrate that the use of a defined mixture of nutriments and of few growth factors permits to sustain not only the cellular proliferation of the Y-1 cell line but also its differentiated function of ACTH-induced steroidogenesis.

Newborn rat adrenal cells in primary culture produce corticosteroid hormones and (20S)-20α-reduced progesterone metabolites in amounts which depend on ACTH concentrations and stimulation time. Eight (20S)-20α-reduced progesterone metabolites, including 18-hydroxy-(20S)-20α-dihydroprogesterone, were identified by comparison of their data in high performance liquid chromatography and in gas chromatography-mass spectrometry to those of existing or newly synthesized reference steroids. Quantitative studies of individual steroid biosynthesis were also performed using high performance liquid chromatography and gas chromatography. Several experiments were made without ACTH and with different concentrations of ACTH for periods of more than 3 weeks. The importance of the two main steroidogenic pathways, corticosteroid biosynthesis and progesterone reductive metabolism was modified by ACTH stimulation of the cultured cells. The progesterone reductive metabolism, important without ACTH and in the first days of ACTH stimulation, was decreased by 6.6 mU of ACTH/ml or higher concentrations but remained active throughout the life span of the stimulated cell cultures.

Mouse adrenal cortex Y-1 cell line was cultured on microcarriers in cell reactors containing 250 ml or 1500 ml culture medium; the same cell density as in the smaller vessel (0.9 × 106 cells ml−1) was obtained in the larger reactor by controlling pH and supplying the medium with O2. Consumption of glucose, oxygen, production of lactate and variation of 19 amino acids were determined with and without oxygen supply. Synthetic ACTH 1–24 induced steroidogenesis in a serum poor medium supplemented with cholesterol and albumin. Steroid production was similar to that of cell growth in usual culture dishes (2 μg 106 cells−1 24 h−1).

[3,4-13C]cholesterol-albumin complex incubated in serum-free medium allowed to evaluate quantitatively the transfer of cholesterol in newborn rat adrenocortical cultured cells, its accumulation as free cholesterol or cholesterol esters and its transformation into steroids which were also originated (47%) from intracellular unlabelled cholesterol. Increasing concentrations of albumin up to 5 g/l enhanced the production of total steroids but in the meantime decreased the 21-hydroxylated steroid fraction. Internalization of albumin shown by using [methyl-14C]methylated-albumin as a tracer accounted only for a minor part in the cholesterol uptake but strikingly affected the steroidogenic pathways by favoring the reductive metabolism of progesterone over the corticosteroid biosynthesis.

In newborn rat adrenal cells in primary culture, the level of activity of the 11 beta/18-steroid hydroxylase system involved in the last step of the corticosteroid biosynthesis is increased by ACTH. A parallel study of 11 beta- and 18-hydroxylation showed the same apparent Km values (64 microM) for both hydroxylations. The Vmax values differed: 11.5 micrograms/10(6) cells/h for corticosterone and 6.9 micrograms/10(6) cells/h for 18-hydroxyDOC. A dose response study of the ACTH effect, measured by the bioconversion of deoxycorticosterone to corticosterone and 18-hydroxyDOC, showed maximum hydroxylation with a dose of 2.2 mU of ACTH/ml. Addition of ACTH after several weeks in culture produced a smaller increase in 11 beta/18-hydroxylation. Removal of ACTH after several weeks of treatment produced an immediate decrease in corticosteroid production; readdition of ACTH produced an increase to the previous level in the case of the 22 mU/ml dose, but not in the case of the 2.2 mU/ml dose. The use of actinomycin D demonstrated that ACTH affects mainly the biosynthesis of protein which must be renewed approximately every 24 h. Finally, the effect of pretreatment or co-treatment with various concentrations of the end products of the reaction showed no inhibition or destruction of the 11 beta/18-hydroxylating enzyme system. Therefore, the regulation of the 11 beta/18-steroid hydroxylase system in these cell cultures seems to be accomplished through the induction by ACTH of the transcription involved in the biosynthesis of cytochrome P450(11) beta and the amount of available precursor furnished by endogenous steroidogenesis.