High expression of gabarapl1 is associated with a better outcome for patients with lymph node-positive breast cancer.

BACKGROUND: This study evaluates the relation of the early oestrogen-regulated gene gabarapl1 to cellular growth and its prognostic significance in breast adenocarcinoma. METHODS: First, the relation between GABARAPL1 expression and MCF-7 growth rate was analysed. Thereafter, by performing macroarray and reverse transcriptase quantitative-polymerase chain reaction (RT-qPCR) experiments, gabarapl1 expression was quantified in several histological breast tumour types and in a retrospective cohort of 265 breast cancers. RESULTS: GABARAPL1 overexpression inhibited MCF-7 growth rate and gabarapl1 expression was downregulated in breast tumours. Gabarapl1 mRNA levels were found to be significantly lower in tumours presenting a high histological grade, with a lymph node-positive (pN+) and oestrogen and/or progesterone receptor-negative status. In univariate analysis, high gabarapl1 levels were associated with a lower risk of metastasis in all patients (hazard ratio (HR) 4.96), as well as in pN+ patients (HR 14.96). In multivariate analysis, gabarapl1 expression remained significant in all patients (HR 3.63), as well as in pN+ patients (HR 5.65). In univariate or multivariate analysis, gabarapl1 expression did not disclose any difference in metastasis risk in lymph node-negative patients. CONCLUSIONS: Our data show for the first time that the level of gabarapl1 mRNA expression in breast tumours is a good indicator of the risk of recurrence, specifically in pN+ patients.

Strategy of macromolecular grafting onto a gold substrate dedicated to protein-protein interaction measurements.

Many biotechnology applications use proteins immobilized on surface. For biosensor, the sensing layer is a key component interfacing the transducer and the sample. Strategies employed to activate the bidimensional surface act directly on the performance of the biosensor. In this paper we propose a novel strategy for engineered proteins self-assembly. Our original supramolecular structure allows a direct and fast covalent attachment of proteins onto bare gold substrate through a homobifunctional cross-linker, 1,4-di-([2'-pyridyldithio]propionamido)butane (DPDPB). In this work, engineered proteins and linker-protein complexes were synthesized and characterized by gel electrophoresis, chromatography and spectroscopy experiments. Macromolecular construction "DPDPB-GST tag-GEC1 protein" was conceived in order to guarantee a 2D architecture enhancing the capabilities of the target (tubulin) to recognize its partner (GEC1). Surface plasmon resonance measurements clearly showed potential of this particular self-assembled protein layer compared to a commercial immunosensor interface. At the concentrations tested, the recognition process occurs between tubulin and the immobilized GEC1; moreover enhanced binding was obtained with the home-made 2D sensing layer more than with 3D carboxymethyl dextran matrix.

Expression of the secreted FAD-dependent sulfydryl oxidase (QSOX) in the guinea pig central nervous system.

cpQSOx1 is a member of the QSOx family of proteins, expressed in the guinea pig (Cavia porcellus) and ortholog of the rat rQSOx1. In this study, in vitro experiments were conducted and showed that, as other member of this family, cpQSOx1 has a sulfydryl oxidase activity, and is a secreted protein. Then, the expression of this enzyme was researched in the guinea pig brain, as very little information exists yet on the expression of QSOx family members in the central nervous system. By immunohistochemistry, RT-PCR and in situ hybridization, cpQSOx1 is synthesized by neurons throughout the whole guinea pig central nervous system. Reticular structures as the basal forebrain, reticular thalamic nucleus and reticular nuclei of the brainstem contained the densest labeling. These results are discussed in terms of putative roles of this protein in synaptic strengthening and in redox activities.

Improved TRAP-silver staining versus conventional radioactive TRAP assays: quantification of telomerase activity during immortalization and in pathological human endometrium.

OBJECTIVES: To develop a sensitive telomeric repeat amplification protocol (TRAP)-silver staining assay for telomerase activity quantification. DESIGN AND METHODS: TRAP assays were performed by using a TRAPeze telomerase kit with or without [alpha-32P]-dCTP. Amplification products were electrophoresed in polyacrylamide gels and detected by autoradiography or a modified silver staining protocol. Telomerase activity was quantified from radioactive counts or optical density of telomerase products from test extracts and controls. RESULTS: TRAP-silver staining assay was at least as sensitive as radioactive TRAP assay and quantified telomerase activity within linearity from 10 to 3,000 cell equivalents. Both methods quantified a weak telomerase activity in normal endometrial glandular epithelial cells (GEC) and a strong increase in immortalized GEC. In human pathologic endometria (n=24), telomerase activity was correlated with lesion seriousness and distinguished simple hyperplasias from nonhyperplasic or cancerous lesions. CONCLUSIONS: TRAP-silver staining assay is suitable for cell and tissue telomerase activity routine quantification.

Identification and expression of a new sulfhydryl oxidase SOx-3 during the cell cycle and the estrus cycle in uterine cells.

Using differential hybridization of a guinea pig endometrial cell cDNA library, a potentially negatively estrogen-regulated gene, SOX-3, was isolated. According to the nucleotide and protein sequence similarities, SOx-3 belonged to the FAD-linked sulfhydryl oxidase family containing the egg white sulfhydryl oxidase, the rat seminal vesicle sulfhydryl oxidase-2 SOx-2, the quiescence-inducible protein hQ6. The SOX-3 transcript in the guinea pig as well as 5 different mRNAs in human tissues appeared differentially expressed in the tissues studied. In secondary endometrial cell culture, the SOX-3 mRNA level increased during a serum depletion-induced quiescence, decreased when cells enter the G1 phase after serum stimulation, and was restored during the S and G2/M phases. Thus, SOX-3 could be implicated in the negative cell cycle control. The SOx-3 protein appeared to be specific of epithelial cells in the uterus. Its expression level varied during the estrus cycle in the guinea pig, suggesting a regulation by steroid hormones.

A novel early estrogen-regulated gene gec1 encodes a protein related to GABARAP.

We have isolated, in guinea-pig endometrial cells, an estrogen-induced 1.8 kb RNA called gec1. Screening of a guinea-pig genomic library led to identification of gec1 gene consisting of 4 exons and 3 introns. Exon 1 contains the 5'UTR and the ATG initiation codon. A guinea-pig gec1 cDNA was obtained by 5'-RACE. The 351 bp coding sequence shares 76.8% identity with that of the human GABARAP 924 bp cDNA while UTRs of the two cDNAs differ. A gec1 probe from the 3'UTR revealed a 1.9 kb mRNA in human tissues and a human GEC1 cDNA was isolated from placenta. Its coding sequence shares 93 and 79% identity with that of guinea-pig gec1 and human GABARAP, respectively. The human and guinea-pig GEC1 proteins have 100% identity. GEC1 and GABARAP proteins have 87% identity and N terminus featuring a tubulin binding motif. Thus, estrogen-regulated gec1 is a new gene which could encode a microtubule-associated protein.

Cytotoxic activity of a recombinant GnRH-PAP fusion toxin on human tumor cell lines.

Pokeweed antiviral protein (PAP), a ribosome-inactivating protein isolated from the leaves of Phytolacca americana, reveals potent antiviral activity against viruses or cytotoxic action against cells once inside the cytoplasm. Therefore PAP is a good candidate to be used as an immunotoxin. We constructed a bacterial expression plasmid encoding PAP as a fusion protein with gonadotropin-releasing hormone (GnRH), a neuropeptide with receptor sites on several gynaecologic tumors. The resulting recombinant toxin was produced in Escherichia coli and accumulated in inclusion bodies. After purification under denaturing conditions, renaturated GnRH-PAP shows an IC(50) of 3 nM on in vitro translation assays and selectively inhibits the growth of the GnRH receptor positive Ishikawa cell line (ID(50) of 15 nM); on the other hand, neither GnRH nor PAP alone had any effect.

ERE environment- and cell type-specific transcriptional effects of estrogen in normal endometrial cells.

Our previous results have suggested a repression of E2 (17beta-estradiol) effect on the c-fos gene of cultured guinea-pig endometrial cells. To investigate this repression, the expression of three human c-fos gene recombinants, pFC1-BL (-2250/+41), pFC2-BL (-1400/+41) and pFC2E (-1300/-1050 and -230/+41), known to be E2-responsive in Hela cells, was studied in stromal (SC) and glandular epithelial cells (GEC). In both cellular types, pFC1-BL was not induced by E2, even in the presence of growth factors or co-transfected estrogen receptor. The pattern of pFC2-BL and pFC2E expression was strikingly different and depended on the cellular type: pFC2-BL and pFC2E induction was restricted to the glandular epithelial cells and did not occur in the SCs. We argue for a repression of E2 action which is dependent on the estrogen-responsive cis-acting element (ERE) environment and also cell type-specific involving DNA/protein and/or protein/protein interactions with cellular type-specific factors.

Comparison of the effects of epidermal growth factor and fetal calf serum in human endometrial carcinoma RL95-2 cells.

This study was designed to evaluate if c-myc expression could influence RL95-2 cell proliferation after EGF or fetal calf serum (FCS) stimulation. Upon FCS addition, c-myc mRNAs slightly increased in the first 30 minutes, peaked at 75 minutes (2.2 fold induction) and returned to the control value within 24 hours. This slight and transient FCS effect on c-myc expression contrasted with the marked and long-lasting EGF effect (17 fold induction up to 48 hours). The increase of cell number by FCS was partially but significantly reduced in the presence of c-myc antisense oligodeoxynucleotides (ODNs). The EGF inhibitory effect on cell growth was not reversed by c-myc antisense ODNs whatever the backbone may be (phosphorothioate or phosphodiester). It appears that EGF and FCS have differential effects on c-myc and RL95-2 cell proliferation and that c-myc is necessary but insufficient for proliferation.

Identification of a biological inactive complex form of pokeweed antiviral protein.

Pokeweed antiviral protein (PAP) inactivates both eukaryotic and prokaryotic ribosomes via a specific depurination of rRNA. The sensitivity of pokeweed ribosomes to PAP implies the existence of a mechanism to protect the plant. Using monoclonal antibodies specific to PAP, a protein complex (PAPi) which contained PAP was identified in leaf extract. In this complex, the enzymatic activity of the toxin was strongly inhibited. This protein complex had a pI lower than that of PAP and was separated from free PAP by a preparative native gel electrophoresis. PAPi had an apparent molecular mass of 57 kDa and was dissociated by heating for 5 min at 80 degrees C or by treatment by alkaline or acidic pH or by 7 M urea. The other components involved in the complex remain unknown.

Differential EGF action on nuclear protooncogenes in human endometrial carcinoma RL95-2 cells.

Our aim was to analyze EGF action on nuclear protooncogenes in RL95-2 since it has not been documented so far. Synchronization and partial' growth arrest were obtained by maintaining cells for 15 hours in L-methionine-free medium. After this depletion, EGF transiently increased fos and jun mRNAs: the expression peaked at 45 minutes for c-fos (5.5 fold induction) and at 60 minutes for c-jun and jun-B (3 fold induction) and the mRNA levels returned to the basal value within 3 hours. Upon EGF addition, c-myc mRNAs peaked at 12 hours (7.6 fold induction) and surprisingly remained higher than the control up to 48 hours. Unlike fetal calf serum, EGF did not increase the cell number and this could be linked to steadily induced c-myc expression. These data provide evidence for a differential EGF action on fos/jun and c-myc in RL95-2 cells.

Gene transfer into subcultured endometrial cells using lipofection.

Lipofection using the Lipofectin reagent was optimized to transiently transfect subcultured guinea pig endometrial stromal cells with a beta-galactosidase gene driven by a simian virus 40 promoter. Efficient transfection was obtained in the following conditions: a value of six for the ratio of lipofectin to DNA, a low cellular density (10(5) cells per 35-mm well) at the time of subculture (48 h before lipofection) and a lipofection duration of 12 hours. Lipofection was compared to calcium phosphate precipitation previously optimized in the same culture model. At a low cellular density, the lipofection method was found to be more efficient than the calcium phosphate precipitation. This result gives a great relevance to lipofection since the cultured cells available in an experiment are often limited. Then, using cells at low density and a plasmid containing the chloramphenicol acetyltransferase (cat) gene linked to an estrogen response element, it was shown that the lipofection procedure is a suitable tool for the evaluation of gene regulation by estrogen.

[Effects of 17 beta-estradiol and growth factors on cell proliferation and c-fos gene expression, in vitro, in endometrial cells in normal growth]. / Effets de l'estradiol-17 beta et de facteurs de croissance sur la prolifération cellulaire et l'expression du gène c-fos, in vitro, dans les cellules endométriales en croissance normale.

Glandular epithelial cells (GEC) and stromal cells (SC) were isolated from guinea-pig endometrium and cultured separately. After the cells were made quiescent by serum depletion, cell proliferation and c-fos gene expression were investigated. Estradiol-17 beta (E2) alone had no effect on cell proliferation or c-fos gene expression. Cell proliferation was observed in SC and GEC after stimulation with insulin plus EGF and a supplementary effect was obtained when E2 was associated with these factors only in GEC. In GEC, insulin plus EGF had no effect on c-fos expression but an effect was observed when E2 was associated with these factors or with a protein synthesis inhibitor, cycloheximide (Chx) or puromycine. An E2 effect in the presence of Chx was also observed in SC. These data suggest the presence of a labile protein preventing the in vitro estrogenic action in endometrial cells.

[Regeneration of transplanted human liver: gene expression at the time of graft implantation]. / Régénération du foie humain transplanté: expression génique au moment de l'implantation du greffon.

The liver tissue mRNA expression of protooncogenes c-fos, c-myc, c-Ha-ras, c-met and c-erb B1, and TGF alpha and TGF beta genes is sequentially and temporarily increased in the early stages after partial hepatectomy, ischaemia or other mitogenic stimuli. These gene expressions were studied in 38 samples of liver tissue from 24 patients who underwent orthotopic liver transplantation, at different evolution stages. Eleven samples were obtained from surgical liver biopsies before graft implantation at day 0 (Group A), 14 samples from percutaneous liver biopsies during the post-operative period from day 9 to day 48 (Group B) and 13 samples in the long-term follow-up period from day 102 to day 1,382 (Group C). Gene expression was studied using 32P-labeled cDNA and oligonucleotidic probe hybridization in slot blots. A GAPD gene was used as a control gene. All expression values of protooncogenes were related to those of the GAPD gene. After cold ischaema, the relative gene expression (quantity of specific mRNA/quantity of GAPD mRNA ratio) tended to diminish in most cases. The relative expressions of c-fos, c-myc, c-Ha-ras, c-met and TGF alpha gene were correlated at day 0. During and after the liver transplantation, an overexpression of c-fos, c-myc, c-Ha-ras, c-met and TGF alpha was observed in different pathological conditions such as cold ischaemia and conservation injury, acute rejection, and cytomegalovirus infection. In three cases the relative expression values of c-fos, c-myc and c-Ha-ras increased over long-term follow-up without any associated acute pathology. These results suggest the necessity of an intercellular mediation--by means of graft reperfusion--in induction of hepatocyte proliferation and regeneration of the transplanted liver.

Identification and characterization of an early estrogen-regulated RNA in cultured guinea-pig endometrial cells.

A cDNA library was prepared from quiescent guinea-pig endometrial glandular epithelial cells stimulated for 2 h with estradiol-17 beta (E2) in the presence of cycloheximide. It was screened by differential hybridization for estrogen-regulated sequences. Six recombinants containing E2-regulated sequences were identified. One of them, called gec1 was then characterized by Northern blot hybridization. The gec1 mRNA was 1,800 bases in size. A 2-fold increase in the gec1 mRNA level was achieved at 120 min after E2 treatment. The E2 action on gec1 gene required the presence of cycloheximide. The cloned gec1 cDNA was 1 kb in size. The sequence so far determined did not show similarity with well characterized genes. This is the first report on a cloned cDNA probe of early estrogen-induced mRNA in a primary culture of endometrial epithelial cells.

[Thrombosis of the ending internal carotid artery complicating giant aneurysm]. / Thrombose de la terminaison de l'artère carotide interne compliquant un anévrysme géant.

A 30-year old man suddenly developed left hemiplegia. CT scan and cerebral angiography showed complete thrombosis of a right internal carotid giant aneurysm. Anterograde propagation of the thrombus in the parent artery led to ipsilateral hemispheric infarction, an exceptional presenting symptom of such vascular malformation. The diagnostic and etiopathogenic aspects are briefly discussed.

Transfected endometrial cultured cells: a system to study gene-regulation by estrogens.

Glandular epithelial (GE) and stromal cells were isolated from guinea-pig endometrium, cultured and subcultured separately. At the end of subculture, the purity of each cell population was higher than 95% and cells displayed a high level of estrogen receptors. Calcium phosphate transfection conditions were defined using a control plasmid containing the bacterial CAT gene driven by viral promoter and enhancer sequences. Transfection experiments were performed with other plasmids in which CAT gene was linked to different estrogen response elements (EREs) derived from those of vitellogenin genes. CAT activity was significantly increased by estradiol-17 beta treatment only when GE or stromal cells were transfected with plasmids containing EREs previously reported as functional EREs in other cell types. This induction was abolished by ICI 164,384 diethylstilbestrol was as effective as estradiol-17 beta for CAT induction and estradiol-17 alpha was ineffective. Transiently transfected endometrial cells in subculture are a suitable system to study the estrogen effect on gene regulatory elements.

Superinduction of c-fos gene expression by estrogen in cultured guinea-pig endometrial cells requires priming by a cycloheximide-dependent mechanism.

The c-fos gene expression is rapidly induced by various mitogenic agents and protein synthesis inhibitors in many cell types. Estradiol-17 beta can induce c-fos gene expression in breast cancer cell lines and in the uterus in vivo, but not in cultured guinea-pig endometrial cells. Using this model, we investigated whether a protein synthesis inhibitor, cycloheximide, could induce the c-fos gene and permit a superinduction by estrogens. In the presence of cycloheximide (10 micrograms/ml), protein synthesis was inhibited at 95% within the first hour. From 190 min after the addition of estradiol-17 beta or diethylstilbestrol (10(-8) M) and cycloheximide (10 micrograms/ml), there was a significant increase (ranging from 3- to 5-fold) of the c-fos messenger RNA level (2.2 kilobase in size), compared with the level in cells treated with cycloheximide alone. Nonestrogenic steroid hormones and estradiol-17 alpha were unable to induce c-fos gene expression in the presence of cycloheximide. The effect of estradiol-17 beta observed in the presence of cycloheximide was completely abolished by 4-hydroxy-tamoxifen or by Ly 156758 or by ICI 164384 (10(-6) M). The c-fos mRNAs were rather stable in cells treated with cycloheximide for 2 h (half-life = 51 +/- 6 min) and there was no further increase in the c-fos messenger RNA stability after the addition of cycloheximide plus estradiol-17 beta (half-life = 40 +/- 3 min). The overall results suggest a response at the transcriptional level. In conclusion, cycloheximide transmits activating signals to the c-fos gene which act as priming elements to allow the estrogen action in cultured guinea-pig endometrial cells.