Unr, a cytoplasmic RNA-binding protein with cold-shock domains, is involved in control of apoptosis in ES and HuH7 cells.

Unr (upstream of N-ras) is a cytoplasmic RNA-binding protein involved in the regulation of messenger RNA stability and internal initiation of translation. We have used Unr-deficient murine embryonic stem (ES) cells to analyse Unr role in cell proliferation and response to stress. Disruption of both unr gene copies had no effect on ES cell proliferation. However, after ionizing radiation (IR), clonogenic survival of unr(-/-) ES cells was approximately 3-fold enhanced as compared to unr(+/+) cells. We further determined that IR-induced apoptosis was decreased in unr(-/-) ES cells, and that reintroduction of the unr gene in unr(-/-) cells restored normal IR-induced apoptosis. Three pro-apoptotic genes, p53, caspase-3 and Gadd45gamma, were downregulated in unr(-/-) ES cells, indicating that Unr, as other cytoplasmic RNA-binding proteins, regulates a complex genetic program, promoting cell death after IR. In contrast, in the human hepatoma cell line HuH7, Unr knockdown using unr-specific small interfering RNAs induced apoptosis, both in untreated and gamma-irradiated cells. Thus, our results establish that Unr acts as a positive or negative regulator of cell death, depending on the cell type. Manipulating the level of Unr may constitute a specific approach to sensitize cancer cells to anticancer treatments.

Fatty acid composition transport and metabolism in doxorubicin-sensitive and-resistant rat glioblastoma cells.

We have studied the lipid composition and the acyl group composition, transport, and metabolism of doxorubicin-sensitive and -resistant rat glioblastoma cells in monolayer cultures (C6 clone). No difference in lipid composition was evidenced; the acyl group composition was, in contrast, highly modified in resistant cells, and these modifications appeared progressively during the acquisition of the resistance. Resistant cells were characterized by a decrease of n-9 eicosatrienoic acid and by a 2-3-fold increase of the proportions of the polyunsaturated fatty acids of the n-6 and n-3 families, especially arachidonic acid and n-3 docosahexaenoic acid. These differences were probably due to a 2-fold increase of the uptake of fatty acids by resistant cells as compared to sensitive cells, this increase allowing the suppression of an essential fatty acid deficiency. Only small changes in the transformations of 16 and 18-carbon atoms' fatty acids to higher analogues were evidenced. A small reduction of the desaturation of stearic acid to oleic acid and of linoleic acid to arachidonic acid was the main characteristic of resistant cells; these differences can be explained as a consequence of the suppression of the essential fatty acid deficiency.

Fluorescence anisotropy of cell membranes of doxorubicin-sensitive and -resistant rodent tumoral cells.

We have studied the plasma membrane fluidity of rat C6 glioblastoma cells and simian virus 40-transformed mouse liver cells in culture that had been rendered resistant to doxorubicin. This was done by the evaluation of fluorescence anisotropy of two probes; diphenylhexatriene was used on membrane microsomal fractions, and trimethylammonium-diphenylhexatriene was used on whole cell suspensions as a plasma membrane-specific probe since it does not enter the cells. A higher degree of membrane fluidity was exhibited with both techniques by doxorubicin-resistant glioblastoma cells as compared to the doxorubicin-sensitive strain, but in the transformed liver cells no such alteration was seen in the physical properties of their plasma membranes. A higher degree of acyl group unsaturation was noticed in the glioblastoma cells but not in the transformed liver cells upon acquisition of doxorubicin resistance. A similar simultaneous increase in acyl group unsaturation and membrane fluidity can be obtained easily by growing the sensitive cells with a medium supplemented with exogenous polyunsaturated fatty acids. This alteration does not modify the sensitivity of the cells to doxorubicin. We conclude from our work that the increase in membrane fluidity, which is frequently associated with drug resistance, is neither necessary nor sufficient for the expression of the resistance. The reason for a link between cell resistance to doxorubicin and plasma membrane fluidity remains to be found.

Incorporation and metabolism of exogenous fatty acids by cultured normal and tumoral glial cells.

We have investigated the transformation of exogenous radioactive free fatty acids by cultured glial cells and their incorporation into complex lipids. The cells were either tumor lines (C6 and NN) or primary cultures from newborn rat hemispheres. The tumor lines could undergo morphological differentiation with dibutyryl cyclic AMP or bromodeoxyuridine. The fatty acid precursors used were palmitic, stearic, oleic, linoleic and linolenic acids. Tumor cells presented a higher incorporation of the precursors in the cell lipid acyl groups than did normal cells. Tumor cells desaturated and/or elongated palmitic, stearic and oleic acid to a higher extent than did normal cells. In contrast, tumor cells transformed linoleic and linolenic acids to their polyunsaturated derivatives to a lower extent than did normal cells. In differentiated tumor cells, these patterns of metabolism were shifted toward the patterns of normal cells. Tumor cells did not exhibit delta 4-desaturase activity, but such activity was restored in the C6 line upon dibutyryl cyclic AMP-induced differentiation. Transformation of linoleic and linolenic acid is likely to proceed through initial delta 6 desaturation. Phospholipids were preferentially labelled with the radioactive fatty acids, and only a little radioactivity was found in the neutral lipid fraction, mainly in diacylglycerols. Each fatty acid precursor label was incorporated in individual phospholipids to a proportion which reflected the typical acyl group composition of glycerophospholipids; we observed high levels of incorporation of palmitic acid and its derivatives into choline glycerophospholipids, and high levels of incorporation of linolenic acid and its derivatives into ethanolamine glycerophospholipids. This pattern was more marked in tumor cells than in normal cells, and the differentiation of tumor cells partially restored the normal pattern, mainly in bromodeoxyuridine-treated NN cells. Both types of differentiation of glial cell lines can be useful as models for the understanding of membrane physiology in normal and tumor cells.

Alteration of ganglioside composition and metabolism in doxorubicin-resistant rat tumoral cells.

We have investigated the ganglioside levels, composition and metabolism in two lines of doxorubicin-resistant cells and in the corresponding wild strains, the C6 rat glioblastoma and the HTC rat hepatoma. The only ganglioside present was GM3, and its level was increased 2-fold in C6 resistant cells and decreased nearly 2-fold in HTC resistant cells. A decrease of cytidine 5'-monophospho-N-acetylneuraminic acid:galactosylglucosylceramide sialyltransferase activity was observed in both resistant lines as compared to sensitive ones, and could not, therefore, explain the increase in the GM3 level observed in the C6 resistant line. Alterations of acid neuraminidase activity were also observed; a 5-fold decrease was noticed in the C6 resistant line and could account for the increase in the GM3 level observed in these cells; in contrast, a 2-fold increase of acid neuraminidase activity was noticed in the HTC resistant cells: together, with reduced synthesis, it could explain the decrease in the GM3 level observed in these cells. No alterations of exogenous ganglioside transport was exhibited by the C6 resistant cells.

[Fecal elastase-1: a useful test in pediatric practice]. / Intérêt pratique du dosage de l'élastase-1 fécale chez l'enfant: comparaison au dosage de la chymotrypsine fécale.

AIM: To study fecal elastase-1 (E1F) and chymotrypsin (ChT) in stools for the diagnosis of pancreatic insufficiency in pediatric practice. MATERIALS AND METHODS: E1F and ChT were measured in stools of 198 children divided in 3 groups: 49 children without any digestive disease (group A), 71 children with pancreatic diseases (group B), and 78 children with non-pancreatic digestive diseases (group C). RESULTS: In group B, E1F values were very low in 64 children and normal in 7 children without pancreatic insufficiency (6 children with cystic fibrosis and 1 with chronic pancreatitis). ChT values were normal in children without pancreatic insufficiency but also in half of children treated with pancreatic enzymes. Decreased E1F values were seen in 2 children (4%) in the group A and 22 children (28%) in the group C, especially those with acute gastroenteritis or celiac disease. CONCLUSION: E1F is a simple, non-invasive, useful tool for the diagnosis of pancreatic insufficiency in children with growth failure or chronic diarrhea, and those with cystic fibrosis. Nevertheless, low values may be found in diseases with villous atrophy or very liquid stools.

Lipid composition, lipid fluidity and radioresistance of Deinococcus radiodurans and two mutant strains.

The lipid composition of D. radiodurans strain R1 and of two mutant strains has been studied in relation to membrane fluidity and sensitivity to X-ray radiation. No significant difference in the unsaturation degree of fatty acids was found between parental and mutant strains. An important decrease of carbohydrate-containing lipids was observed in the radiosensitive mutant strain. We also observed a higher fluidity in both mutant strains than in the parental one. Modification of membrane lipid fluidity by growing the parental strain at 39 degrees C did not lead to modified radioresistance. These results suggest that a particular chemical composition of the membrane leading to a special lipid phase may be an important parameter in controlling radiosensitivity.

Doxorubicin-induced alterations of c-myc and c-jun gene expression in rat glioblastoma cells: role of c-jun in drug resistance and cell death.

We studied the effect of doxorubicin on the expression of c-myc and c-jun in the rat glioblastoma cell line C6 and its doxorubicin-resistant variant C6 0.5, at equitoxic exposures. For quantitation, the mRNA levels of these oncogenes were related to those of two domestic genes, beta-actin and glyceraldehyde phosphate dehydrogenase. After a transient overexpression of the genes during the first hour of incubation, there was a selective, dose-dependent down-regulation of both genes by doxorubicin in the sensitive cells. In the resistant cell line, c-myc expression was also decreased in response to doxorubicin incubation, but the expression of c-jun remained unchanged over the whole range of concentrations. In contrast, vincristine had no effect on the amounts of c-myc and c-jun mRNAs in either line. The effect of doxorubicin on the mRNA levels of c-jun was also observed on the JUN proteins by immunoblotting, but the MYC protein levels remained unchanged upon doxorubicin treatment. There was a significant correlation between the levels of c-myc and c-jun gene expression and the degree of growth inhibition induced by doxorubicin. In addition, doxorubicin induced a fragmentation of DNA in sensitive cells, but not in resistant cells, thus revealing a resistance to apoptosis in this line. Doxorubicin-induced cell death did not appear to be mediated by p53 in either cell line.

Chromosomal modifications of a rat glioblastoma cell line during the acquisition and reversal of doxorubicin resistance.

We have studied the cytogenetic alterations occurring during the development and reversal of doxorubicin resistance in a clonal line of rat glioblastoma cells. We have observed during the acquisition of resistance an increase in the modal number of chromosomes, from 42 to 60, and the occurrence, in 90% of the mitoses, or large metacentric markers(s) which were infrequent in the sensitive line. This was associated with a net increase in total DNA amount per cell, from 5.3 to 8.3 pg. During reversal of resistance by 2 years culture without drug of the most resistant line, we observed a rapid decrease of the chromosome number as well as of the DNA content per cell; however, the large metacentric marker(s) were still present in 40% of the mitoses after 9 months of reversal, when the remaining resistance was only 4-fold. In situ hybridization of the chromosomes with a probe for the mdr gene revealed that the average number of stained chromosomes rose from 7% in the sensitive line to 38% in the most resistant line; however, only 9% of the silver grains were detected on the large metacentric markers. We conclude that important chromosome rearrangements occurred during the acquisition of resistance to doxorubicin, leading to a random distribution of the mdr gene in the genome.

Relationships between DNA damage and growth inhibition induced by topoisomerase II-interfering drugs in doxorubicin-sensitive and -resistant rat glioblastoma cells.

We have evaluated the DNA breaks occurring after action of three topoisomerase II-interfering drugs (doxorubicin, etoposide and amsacrine) on a line of rat glioblastoma cells in culture and its doxorubicin-resistant variant. DNA breaks were quantified by alkaline unwinding in the presence of a dye exhibiting a quenching of fluorescence with single stranded DNA. The antiproliferative activity of the three drugs was compared to their ability to damage DNA. We have shown that at low exposure doses (up to the IC50 of the drugs), the same low level of DNA damage determined the same inhibition of cell growth in sensitive and resistant cells, but that at higher exposure doses the resistant cells developed special mechanisms allowing them to tolerate more DNA breaks than sensitive cells without lethal effects. The origin of this tolerance of resistant cells to DNA breaks might be due to special mechanisms of protection of genomic sites hypersensitive to topoisomerase II-mediated drug action, to alterations of topoisomerase II or to alterations of the molecular events leading to cell death after occurrence of DNA breaks.

Detection of DNA-strand breaks in cells treated with F 11782, a catalytic inhibitor of topoisomerases I and II.

F 11782, or 2", 3"-bis pentafluorophenoxyacetyl-4',6'-ethylidene-beta-D glucoside of 4'-phosphate-4'-dimethylepipodophyllotoxin 2N-methyl glucamine salt, is a novel fluorinated lipophylic epipodophylloid which has proven cytotoxic activity in vitro and has shown markedly superior antitumour activity in vivo compared to etoposide in various experimental tumour models. However, the precise mechanism(s) of cytotoxicity of F 11782 remains to be defined. In this study, the DNA damaging activity of F 11782 was investigated in GCT27 and C6S cells using, respectively the fluorescence enhancement assay and the technique of DNA alkaline elution. All the results obtained were consistent with induction of DNA damage by F 11782. No evidence of any stabilisation of DNA-topoisomerase cleavable complexes though was obtained with this catalytic inhibitor. Furthermore, such induction of DNA damage has not been reported with other known catalytic topoisomerase inhibitors and so it appears to be unique to F 11782.

Impact of high-flux/high-efficiency dialysis on folate and homocysteine metabolism.

High-flux/high-efficiency (HF/HE) dialysis may have detrimental effects on micro-nutrients and water-soluble vitamins, such as vitamin B6, whose levels are lowered. Folate deficiency may increase cardiovascular risk through an increase in homocysteine (Hcy) serum levels. We therefore investigated the effects of dialysis with a high-flux (HF) membrane on folate and Hcy metabolism. Twelve patients without any folate supplementation, receiving dialysis with a low-flux membrane prior to the study (TO), were switched to dialysis using a HF triacetate membrane for four months (T1, T2, T3, T4) and received an oral daily folate supplementation during the two last months (T3, T4). Mean predialysis plasma folate levels fell dramatically after one month of HF dialysis (T1) and remained significantly lower than the initial level (p<0.05) at T2. Hcy concentrations were high in all patients at TO (mean 47.3 +/- 17.6 microM, normal range 5 to 15 microM). They did not change during the first two months of the study but dropped steeply after the beginning of oral folate supplementation. Folate supplementation should be used in HF/HE dialysis to avoid folate depletion. The combination of folate supplementation and HF/HE may lower Hcy levels and reduce cardiovascular morbidity and mortality in these patients.

Phospholipid acyl group composition in normal and tumoral nerve cells in culture.

We have studied the fatty acid composition of total phosphoglycerides from various types of nerve cells in culture. Primary cell cultures were compared with tumoral cell strains. Glial cells exhibited no characteristic pattern when compared to neurons. Tumoral cell phosphoglycerides contained much higher levels of octadecenoic acid and lower levels of C-20 to C-22 polyunsaturated fatty acids than normal cell phosphoglycerides. This observation seems to be a general feature in tumoral cell membranes. It could be of interest in respect to the membrane fluidity of cancer cells.

Fluorescence polarization study of lipids and membranes prepared from brain hemispheres of a hibernating mammal.

The physical behavior of total lipids, microsomes and microsomal lipids prepared from brain hemispheres of European Hamsters (Cricetus cricetus) was approached by the measure of the fluorescence polarization of the probe 1,6-diphenyl 1,3,5-hexatriene. We compare in this study the results obtained for two critical periods for a hibernator: winter (torpid state) and summer (active state). An increase in fluidity was noticed in the winter lipid and membrane preparations. The difference was however of very low magnitude, suggesting that only the microenvironment of some proteins was involved, rather than the bulk membrane fluidity.

Fluorescence anisotropy of kidney lipids and membranes of a hibernating mammal.

The fluorescence anisotropy of lipids and membranes isolated from kidneys of European hamsters (Cricetus cricetus L.) has been estimated using 1,6-diphenyl-1,3,5-hexatriene as a probe. We have compared in this study the results obtained for two critical periods for a hibernator: winter (torpid state), and summer (active state). The differences were of very low magnitude. A slight increase in anisotropy was noticed in the kidney lipids and microsomal membrane preparations from torpid animals. In contrast, a small decrease in anisotropy was observed in the microsomal lipid extracts of torpid animals. A difference in triglyceride content of winter and summer total kidney lipids was detected, as well as a difference in microsomal protein content between winter and summer membrane preparations. It is hypothesized that the latter observations may explain why the behavior of kidney total lipids and microsomal preparations were different from that presented by kidney microsomal lipids in respect to fluorescence anisotropy. Therefore, only a little, if any, homeoviscous adaptation is exhibited by kidney membranes during hibernation of this mammal.

Incorporation and turnover of phospholipid precursors in normal and tumoral glial cells in culture.

The incorporation and turnover of phospholipid precursors in cultured normal and tumoral glial cells was investigated during the plateau phase of growth. Glycerol was incorporated similarly by all cell types, and was renewed with a half-life of 19-37 hr. Acetate had a much longer half-life in primary cultures (50-75 hr) than in proliferative tumor cells (20-40 hr). Phosphate had a more rapid turnover rate in primary cultures (25 hr) than in proliferative tumor cells (50 hr). For all precursors, inositol- and choline phosphoglycerides had a faster turnover rate than other phospholipids.

[Membrane polyunsaturated fatty acids of mammalian nerve cells in culture]. / Acides gras polyinsaturés membranaires de cellules nerveuses de mammifères en culture.

We have observed some important changes in the distribution of fatty acid classes in cultured nerve cells according to the normal or tumoral origin of the cells: normal cells exhibit higher levels of polyunsaturated fatty acids and lower levels of monounsaturated fatty acids than tumoral cells do. When the culture medium of neuroblastoma cells is supplemented with polyunsaturated fatty acids, these fatty acids are incorporated into membrane phospholipids and some specific alterations of membrane functions occur: modification of the kinetic parameters of ecto-enzyme activities, modification of amino-acid transport characteristics. A brief review of the literature shows that polyunsaturated fatty acids are not essential for cell life in vitro, provided that monounsaturated fatty acids can be present in the cells. Polyunsaturated fatty acids seem therefore play only a regulatory role of some membrane functions.

[Lipids and lipopolysaccharides of "Micrococcus radiodurans" (author's transl)]. / Lipides et lipopolyosides de Micrococcus radiodurans.

Studies were carried out on lipid composition of Micrococcus radiodurans. The polar lipid components were found to be 4 glycolipids, 3 phospholipids and 2 phosphoglycolipids. The major fraction belongs to the last group. Correlation between these components and lipoteichoic acid was not observed. A lipid-polysaccharidic complex was isolated. It was not of this type.

Development of mechanisms of protection against oxidative stress in doxorubicin-resistant rat tumoral cells in culture.

We have compared some mechanisms involved in the defense against doxorubicin-induced free radical damage in rat hepatoma and glioblastoma cell lines and their doxorubicin-resistant variants presenting an overexpression of the multidrug resistance gene. Immediate in vivo production of malondialdehyde was minor and was not different in sensitive and resistant cells. Alpha-tocopherol was undetectable in all cell lines. Glutathione levels were not different in sensitive and resistant cells and these levels did not vary upon doxorubicin treatment. Resistant cells exhibited either a 50% decrease (hepatoma) or a 25% increase (glioblastoma) of glutathione-S-transferase activity. Glutathione reductase presented no important change upon acquisition of resistance. In contrast, selenium-dependent glutathione peroxidase activity was consistently 2-6-fold increased in the resistant cells, which suggests a magnification of protection mechanisms against hydroxyle radical formation from H2O2 in resistant cells. Depletion of glutathione levels by buthionine sulfoximine sensitized hepatoma resistant cells to doxorubicin, but had no effect on doxorubicin cytotoxicity to glioblastoma cells.