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.

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.

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.

[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.