PubMed Health⌕ Search

Biomedical subjects

L Tseng

Publications and source records attributed to L Tseng.

At least 37 records · Page 2Linked to original sources
PubMed1994

Activation of the human IGFBP-1 gene promoter by progestin and relaxin in primary culture of human endometrial stromal cells.

We have studied the activity for the insulin-like growth factor binding protein-1 (IGFBP-1) gene promoter in human endometrial stromal cells by transient transfection. The promoter activity derived from p3.6CAT or p3.6Luc (3400 bp IGFBP-1 promoter 5' to the reporter gene chloramphenicol acetyltransferase or luciferase) was minimal in unstimulated cells. A time study over 13 days of culture showed that the promoter activity increased exponentially to > 10(4) fold in cells treated with medroxyprogesterone acetate (MPA) and relaxin (RLX). Induction of the IGFBP-1 gene promoter activity by hormones was similar to the secretion pattern of IGFBP-1 in endometrial stromal cells. MPA alone caused a moderate induction, 3-40-fold increase over the control. Deletion analysis showed that two regions in the IGFBP-1 gene promoter were responsible for the activation of the IGFBP-1 gene. The basal promoter region, termed bp1-A (+68 bp to -1.205 kb), contains multiple sections of regulatory sequence including a cis-element CCAAT (-72 bp). A DNase I protection assay in the bp-1A region revealed four distinct binding regions, one of which contained the CCAAT box region. Another promoter region, termed bp1-B (-2.6 to -3.4 kb), mediated 95% of the total promoter activity in endometrial stromal cells. The bp1-B region also contains multiple regulatory sequences. Mutation and DNase I protection assay suggest that Sp1-like binding site at -2.63 kb was a regulatory site responsible for the activation of IGFBP-1 gene promoter.

Adult↗
PubMed1994

Identification of a distal regulatory sequence of the human IGFBP-1 gene promoter and regulation by the progesterone receptor in a human endometrial adenocarcinoma cell line.

The activity of the insulin-like growth factor binding protein-1 (IGFBP-1) gene promoter was studied in the human endometrial adenocarcinoma cell line HEC-1B. Basal promoter activity was directed by the region +68 to -207 bp, similar to observations in the hepatoma HepG2 cell line. A distal regulatory sequence approximately -2.6 kb from the transcription initiation site strongly enhanced the activity of the IGFBP-1 gene promoter in HEC-1B cells, but not in HepG2 cells. Sequence analysis revealed that this active region resides in 105 bp between -2,628 to -2,732 bp (the Rsa I-Cla I fragment). This region contains many putative active motifs homologous to known cis elements. Additional deletion and mutation in the Rsa I-Cla I fragment showed that the activity was confined to a 58-bp DNA fragment. In cells treated with progestin and co-transfected with progesterone receptor vector hPR1, the CAT activity derived from constructs containing the Rsa I-Cla I fragment was reduced in a dose-dependent manner. The active DNA fragment also stimulated the activity of the heterologous TK/CAT promoter in HEC-1B cells, while the PR complex inhibited this activity by 50%. These observations indicate that most of the regulation of the IGFBP-1 gene in HEC-1B cells is derived from the distal promoter region confined to the Rsa I-Cla I fragment and that the same region mediates an inhibitory effect from the progesterone receptor.

Adenocarcinoma↗
PubMed1994

Decidualization of human endometrial stromal cells in vitro: effects of progestin and relaxin on the ultrastructure and production of decidual secretory proteins.

Decidual cells arise by proliferation and differentiation of endometrial stromal cells of the uterus after appropriate stimulation by ovarian hormones. Previously we have shown that progestin and relaxin stimulate the secretion of several decidual-cell-specific secretory proteins in a long-term primary cell culture system. We now report the effects of progestin and relaxin on the morphology of stromal cells in association with the production rate of two major decidual secretory proteins, prolactin and insulin-like growth factor binding protein-1 (IGFBP-1). Stromal cells were cultured in RPMI 1640 and 2% fetal bovine serum for 22 days under control conditions (no hormone), with relaxin or medroxyprogesterone acetate (MPA), or MPA plus relaxin. Cells treated with MPA alone or MPA plus porcine relaxin grew to a high density with many areas of heaping while control cells and cells grown in medium containing relaxin alone formed discontinuous layers. The cytoplasm was distinguished by aggregates of rough endoplasmic reticulum and secretory granules. Surfaces of cells treated with MPA plus relaxin had clusters of short blunt processes containing secretory granules. The processes were rarely seen in cells exposed to MPA alone and completely absent in control cells or cells exposed to relaxin alone. Intercellular space was greatly widened in cells treated with MPA alone or MPA plus relaxin. There were many extended gap junctions in MPA- and relaxin-treated cells in contrast to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Insulin-like growth factor binding protein-1 inhibits the mitogenic effect of insulin-like growth factors and progestins in human endometrial stromal cells.

This study describes the effects of insulin-like growth factors (IGFs), progestins, and IGF binding protein-1 (IGF-BP1) on DNA synthesis in human endometrial stromal cells. Endometrial stromal cells treated with IGF-I or IGF-II or progestins increased their rate of DNA synthesis 3-6-fold. In a time study, IGF-I or IGF-II stimulated DNA synthesis 3-fold in 24 h. An equivalent increase by medroxyprogesterone acetate (MPA) required 48 h. Progestin-stimulated DNA synthesis was inhibited completely by an equimolar concentration of the antiprogesterone RU486. IGF-I-stimulated DNA synthesis was inhibited completely by IGF-BP1. Half-maximal inhibition of IGF-I-stimulated DNA synthesis occurred when IGF-I and IGF-BP1 were at equimolar concentrations. Non-phosphorylated and phosphorylated forms of IGF-BP1 were equipotent inhibitors of IGF-I-stimulated DNA synthesis. IGF-BP1 also inhibited progestin-stimulated DNA synthesis. Half-maximal inhibition of progestin-stimulated DNA synthesis occurred at 100 ng/ml of phosphorylated IGF-BP1. Phosphorylated IGF-BP1 was a 3-4-fold more potent inhibitor of progestin-stimulated DNA synthesis than the nonphosphorylated form. The inhibition by IGF-BP1 of progestin-stimulated DNA synthesis was reversed by an equimolar concentration of IGF-II. This study suggests that progestins regulate an autocrine growth control loop in the endometrium that involves IGFs, IGF receptors, and IGF-BPs.

Adult↗
PubMed1993

Envelope-binding domain in the cationic amino acid transporter determines the host range of ecotropic murine retroviruses.

Infection of rodent cells by ecotropic type C retroviruses requires the expression of a cationic amino acid transporter composed of multiple membrane-spanning domains. By exchanging portions of cDNAs encoding the permissive mouse and nonpermissive human transporters and examining their abilities to specify virus infection upon expression in human 293 cells, we have identified the amino acid residues in the extracellular loop connecting the fifth and sixth membrane-spanning segments of the mouse transporter that are required for both envelope gp70 binding and infection. These findings strongly suggest that the role of the mouse transporter in determining infection is to provide an envelope-binding site. This role is analogous to those of host membrane proteins composed of a single membrane-spanning domain that serve as binding proteins or receptors for other enveloped viruses such as human immunodeficiency virus, Epstein-Barr virus, and murine and human coronaviruses.

3T3 Cells↗
PubMed1992

The human cationic amino acid transporter (ATRC1): physical and genetic mapping to 13q12-q14.

The product of the mouse Rec-1 locus is an integral membrane protein that determines susceptibility to infection by murine ecotropic retroviruses. Recently it has been determined that its role in normal cell metabolism is transport of the cationic amino acids, arginine, lysine, and ornithine across the plasma membrane. Southern blot analysis of genomic DNA from a panel of 48 mouse-human somatic cell hybrids assigned the human version of this gene, ATRC1, to chromosome 13. Chromosomal in situ hybridization localized the gene to 13q12-q14. A restriction fragment length polymorphism (RFLP) was detected with TaqI. There were two alleles with frequencies of 0.29 and 0.71. Pairwise linkage analysis established linkage between ATRC1 and ATP1AL1, D13S1, D13S6, D13S10, D13S11, D13S21, D13S22, D13S33, D13S36, and D13S37. Multilocus linkage analysis of five of the loci indicated that the most likely order of loci in this region was D13S11-ATP1AL1-ATRC1-D13S6-D13S33.

Amino Acid Transport Systems, Basic↗
PubMed1992

Progestin stimulates the biosynthesis of fibronectin and accumulation of fibronectin mRNA in human endometrial stromal cells.

Fibronectin is a major component of decidual basement membrane. In the present study, we have investigated the effect of progestin on the synthesis and secretion of fibronectin in human endometrial stromal cells. Stromal cells were isolated during the menstrual cycle and cultured in RPMI-1640 with 2% fetal calf serum supplemented with progesterone or medroxyprogesterone acetate (MPA) in a long-term culture system. Indirect immunofluorescent staining showed that fibronectin was uniformly distributed in the intracellular and extracellular regions of stromal cells treated with MPA for 14 days. The biosynthesis and secretion of this protein and the accumulation of cellular fibronectin mRNA were studied after various culture periods. Cells were pulse-labelled with [35S]methionine to determine the amount of newly synthesized fibronectin secreted into the culture medium. A monoclonal antibody (Mab) identified human fibronectin on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), showing a predominant band (Mr 230-250 kDa) which migrated with authentic fibronectin run in parallel. In six endometrial specimens, the amount of radioactivity incorporated as [35S]fibronectin was increased by progestin. Maximal stimulation occurred after 6 days treatment with MPA. Culture beyond 16 days reduced the rate of synthesis and secretion to 40% of the maximum. The effect of progestin was dose dependent with 0.02, 0.2 and 1 microM progesterone, producing 2.0, 3.8 and 11-fold increases respectively, over the control. Medroxyprogesterone acetate was more effective than progesterone, the maximal response (10-fold increase) being achieved at 0.02 microM MPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗
PubMed1992

Effect of progestin, antiprogestin, and relaxin on the accumulation of prolactin and insulin-like growth factor-binding protein-1 messenger ribonucleic acid in human endometrial stromal cells.

Prolactin (PRL) and insulin-like growth factor-binding protein (IGFBP-1) are two major secretory proteins of human endometrial/decidual cells. We have characterized the mRNA of PRL and IGFBP-1 and studied the effect of progestin, medroxyprogesterone acetate (MPA), anti-progestin (RU486), and relaxin (RLX) on the levels of these two mRNA transcripts in a long-term culture of human endometrial stromal cells. Northern blot analysis showed that the size of PRL mRNA was 1.15 kb and that of IGFBP-1 mRNA, 1.6 kb. Primer extension of endometrial/decidual IGFBP-1 mRNA showed two transcription initiation sites identical to those found in HepG2 human hepatoma cell line. The levels of mRNA in control samples remained low, approximately 2 pg PRL and approximately 5 pg IGFBP-1/microgram RNA at various times of culture. When stromal cells were treated with MPA for 28 days, PRL mRNA gradually increased 100-fold whereas IGFBP-1 mRNA exponentially increased approximately 1000-fold compared to control values and leveled after 25 days in culture. The timing of maximal stimulation was shortened by withdrawing MPA or by replacing MPA with RU486. After removal of MPA, levels of both mRNAs increased and each peaked after approximately 10 days, with PRL showing a 2-fold and IGFBP-1 a 20-fold increase compared to cells treated with MPA continuously. Replacing MPA by RU486 caused a rapid increase of PRL mRNA (2-3-fold) in 2-3 days followed by a gradual reduction to less than 20% of peak levels over the next 3 days. IGFBP-1 mRNA levels increased 30- and 100-fold in 1-2 days followed by a reduction to less than 20% of peak levels over the next 24 h. The reduction of mRNA levels by RU486 was reversed when cells were rechallenged with MPA. Relaxin alone caused a transient stimulation of PRL and IGFBP-1 mRNA. Maximal stimulation occurred between 10 and 20 days of culture and was 100-fold for PRL and 1000-fold for IGFBP-1 relative to control values. Cells treated with MPA and RLX in sequence had higher mRNA levels than cells treated with MPA continuously or cells subjected to MPA withdrawal. Maximal mRNA levels reached 0.4 ng PRL and approximately 8 ng IGFBP-1/microgram total RNA, approximately 0.04% and 0.8% of cellular RNA. The mRNA levels under various hormonal manipulations were similar to the previously published synthesis and secretion patterns of PRL and IGFBP-1 proteins in this system.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1991

Insulin-like growth factor-binding protein-1 is phosphorylated by cultured human endometrial stromal cells and multiple protein kinases in vitro.

The insulin-like growth factor-binding protein IGF-BP1 is a major secretory protein of human endometrial stromal cells decidualized in culture. Anion exchange chromatography and nondenaturing gel electrophoresis showed IGF-BP1 to exist in five electrophoretically and chromatographically distinct isoforms. IGF-BP1 variants migrated as a quintet on nondenaturing polyacrylamide gels and as a single band (28 kDa) on sodium dodecyl sulfate-polyacrylamide gels. Alkaline phosphatase treatment reduced the IGF-BP1 variants to a single band. Cells incubated with [32P]orthophosphate for 12 h secreted four 32P-labeled IGF-BP1 phosphovariants, and their migration coincided with those bands that were eliminated by alkaline phosphatase treatment. In cells treated with medroxyprogesterone acetate and relaxin, the concentration of phosphorylated IGF-BP1 was increased dramatically as compared with controls. All the phosphovariants were confirmed to be IGF-BP1 by their ability to be supershifted on nondenaturing polyacrylamide gels after binding a monoclonal antibody to IGF-BP1. Thin layer electrophoresis of IGF-BP1 acid hydrolysates showed IGF-BP1 to be phosphorylated exclusively on serine. Non-phosphorylated IGF-BP1 was phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and casein kinase II in vitro. This suggests that IGF-BP1 may be a substrate of multiple protein kinases in vivo.

Adult↗
PubMed1991

Regulation of insulin-like growth factor-binding protein-1 synthesis and secretion by progestin and relaxin in long term cultures of human endometrial stromal cells.

The decidualized endometrium during the first trimester of pregnancy synthesizes and secretes a 32-kDa insulin-like growth factor-binding protein (termed hIGFBP-1) at high levels. IGFBP-1 is the major soluble protein product of this tissue and is principally localized to the differentiated endometrial stromal cell, the decidual cell. In the present study long term culture of stromal cells from the nonpregnant endometrium have been employed to elucidate the hormonal requirements for IGFBP-1 production. Immunoreactive IGFBP-1 was undetectable in control cultures. However, inclusion of medroxyprogesterone acetate (MPA) induced rates of 0.35 +/- 0.09 microgram/0.1 mg cell DNA.day (mean +/- SEM; n = 5) after 20-30 days. In these cultures cells exhibited morphological changes consistent with decidual cell differentiation. In all cultures removal of MPA after exposure for 10-16 days, with or without subsequent inclusion of relaxin (RLX), increased production of IGFBP-1 450- to 4600-fold to rates of 150-710 micrograms/0.1 mg cell DNA.day or 26-131 micrograms/10(6) cells.day on days 24-26. The rates tended to be higher with the inclusion of RLX and were sustained in contrast to cultures without RLX, where rates fell by day 30. Individual cultures responded differently to RLX when added from the initiation of culture, with either a response similar to MPA alone or a cyclical change in production, achieving maximal rates of 190-290 micrograms/0.1 mg cell DNA.day. Cultures in which RLX alone induced high IGFBP-1 high production were obtained from endometrium during the progesterone-dominated luteal phase. In cultures exhibiting high rates of immunoreactive IGFBP-1 production, the protein represented their major secretory protein product. This was confirmed by [35S]methionine incorporation and the presence of IGFBP-1 as the predominant protein in serum-free culture medium. The immunoreactive IGFBP-1 isolated from culture medium was found to be identical, by a number of criteria, with IGFBP-1 derived from decidual tissue. These results were consistent with a primary role of progestin exposure, whether in vivo or in vitro, in converting endometrial stromal cells to cells potentially able to exhibit the high rates of IGFBP-1 production typical of the decidualized endometrium of pregnancy.

Adult↗
PubMed1990

CA 125 is an excretory product of human endometrial glands.

The present investigation was undertaken to study the cellular localization and kinetics of synthesis of CA 125 in the endometrium. CA 125 was localized by immunohistochemistry to the infranuclear region of epithelial cells during the proliferative phase and to the apical luminal border during the secretory phase. In gestational endometrium, both the cytoplasm and the apical luminal border of epithelial cells were intensely positive. No staining was seen in endometrial stromal cells during the normal cycle or in decidualized endometria. Results obtained from in vitro cultures of separated glandular and stromal cells were similar to those obtained by immunohistochemistry. That is, epithelial cells released between 5 and 25 times more CA 125 into the culture medium than did stromal cells. The release of CA 125 was highest in epithelial and stromal cells obtained during the early secretory phase. CA 125 concentrations were markedly elevated in endometrial aspirations obtained during the secretory phase or in endometria with crumbling stroma compared to plasma levels. Plasma levels of CA 125 were slightly elevated during menses. These results suggest that CA 125 is an exocrine product of endometrial epithelial cells. Plasma levels of CA 125 may be of endometrial origin only when the membrane barriers, which normally prevent its entry into the circulation, are damaged.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed1990

Differential effects of progestin and relaxin on the synthesis and secretion of immunoreactive prolactin in long term culture of human endometrial stromal cells.

PRL secretion from human endometrium is a continuous process extending from the luteal phase of the menstrual cycle throughout the entire gestational stage. We have developed a long term primary cell culture system to elucidate the hormonal requirements for this sustained production of PRL. The effects of medroxyprogesterone acetate (MPA), progesterone, and relaxin (RLX) on the production of immunoreactive PRL were investigated. MPA stimulated cell growth and PRL production rate during days 5-20 of culture. Progesterone was 20-40% less effective in stimulating PRL than MPA. Stimulation of PRL was continued 1-2 weeks after MPA withdrawal. Relaxin did not promote cell growth. However, it induced the PRL production which fluctuated during the long term culture. The maximal response to RLX was 2- to 3-fold higher or similar to that of MPA. Only five of nine endometrial specimens examined responded to RLX alone. The effect of MPA plus RLX was significantly greater than that of MPA or RLX alone. The highest production rate was shown in cells treated with MPA and then RLX in sequence. After a month of culture, the production rates (micrograms of PRL per 0.1 mg cell DNA/day) under various culture conditions (A, control; B, MPA; C, MPA for 10-15 days and no hormone afterward; D, both MPA and RLX; and E, MPA and RLX in sequence) were: A, about 0-0.01 (n = 12); B, 2.5 +/- 0.9 (n = 8); C, 4.8 +/- 2.5 (n = 8); D, 5.7 +/- 3.0 (n = 5); and E, 11 +/- 3.7 (n = 7); mean +/- SD; n, number of specimens). Endometrial stromal cells were incubated with [35S]methionine, and [35S]immunoreactive PRL and other secretory proteins were analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to characterize the size and isoforms of immunoreactive PRL. PRL was one of the five major secretory proteins (23-25K, 32K, 42K, 78K, and 150K daltons, sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition) induced by MPA and RLX in endometrial stromal cells. More than 90% of immunoreactive PRL was secreted into the medium. The apparent mol wt of immunoreactive PRL were 21K, 23K (the predominant size), and 25K daltons. Results obtained from the incorporation of [14C]glucosamine into immunoreactive PRL indicated that both 23K and 25K PRL contained glycosylated PRL. A 45K-dalton glycosylated immunoreactive PRL was also present in the culture medium.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1989

A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection.

Murine type C ecotropic retrovirus infection is initiated by virus envelope binding to a membrane receptor expressed on mouse cells. We have identified a cDNA clone that may encode for this receptor through a strategy combining gene transfer of mouse NIH 3T3 DNA into nonpermissive human EJ cells, selection of EJ clones that have acquired susceptibility to infection by retrovirus vectors containing drug resistance genes, and identification of the putative receptor cDNA clone through linkage to a mouse repetitive DNA sequence. Human EJ cells that express the cDNA acquire a million-fold increase in MuLV infectivity. The predicted 622 amino acid sequence of the putative receptor protein is extremely hydrophobic; 14 potential membrane-spanning domains have been identified. A computer-based search of sequence data banks did not identify a protein with significant similarity to the putative receptor. We conclude that a novel membrane protein determines susceptibility to ecotropic MuLV infection by binding and/or fusion with the virus envelope.

Amino Acid Sequence↗
PubMed1989

Immunologic identification of the aromatase enzyme system in human endometrium.

Confluent human endometrial stromal cells were cultured in medium with no hormone or supplemented with medroxyprogesterone acetate (MPA), estradiol (E2), and porcine relaxin (RLX) for 5 days. These stromal cells were then labeled with [35S]methionine for 3 h. The radioactive proteins in the particulate fraction of cell homogenate were extracted by detergent and incubated with antisera to purified placental aromatase cytochrome P-450 (P-450arom) and NADPH-cytochrome P-450 reductase to isolate the radio-labeled aromatase enzyme components. Analysis of the radio-labeled protein, isolated by antibody to the cytochrome P-450arom from different preparations (P45FBIII or R-8-2) showed a major band at molecular weight 54k on SDS polyacrylamide gel electrophoresis (SDS-PAGE). The intensity of 54k band was stronger in hormone treated stromal cells than that of control in parallel with the increase of aromatase activity. The radio-labeled protein isolated by anti-NADPH cytochrome P-450 reductase, REDFBIV, showed a major band at the molecular weight 73k on SDS-PAGE with comparable intensity in control and hormone treated samples. Thus, the apparent molecular weights of endometrial cytochrome P-450arom and cytochrome P-450 reductase were identical to placental aromatase enzyme system. When a secretory endometrium and a decidua were labeled with [35S]methionine, the cytochrome P-450arom was detected only in the decidua. NADPH cytochrome P-450 reductase was detected both in the endometrium and the decidua. These results show that antisera to placental aromatase enzyme system cross reacts with the endometrial aromatase enzyme components. The synthesis of cytochrome P-450arom was stimulated by MPA, E2 and RLX while the synthesis of the NADPH-cytochrome P-450 reductase aromatase component was not affected by the hormone.

Aromatase↗
PubMed1989

Long-term effects of progestin and RU 486 on prolactin production and synthesis in human endometrial stromal cells.

Previous studies have shown that the production of prolactin (PRL) is increased in human endometrial stromal cells cultured with medroxyprogesterone acetate (MPA) for 3-5 days. In the present study, we have investigated the effect of prolonged treatment of progestin and anti-progestin, RU 486, on the production and synthesis of PRL. Stromal cells were isolated from human endometrium obtained from non-pregnant women and cultured for 20-30 days in medium RPMI 1640 with 2% fetal calf serum or supplemented with MPA, RU 486, alone or in sequence. The PRL content in medium was measured by radioimmunoassay. The production rate was estimated from the PRL content in medium accumulated in 24 h. The PRL production rate was progressively increased in stromal cells continuously treated with MPA for 30 days (greater than 100-fold over the control value, i.e. 0-0.01 microgram/0.1 mg cell DNA/day). RU 486 alone had no effect on the production of PRL. However, the production of PRL was increased by MPA in stromal cells pretreated with RU 486 indicating that the effect of RU 486 is reversible. When stromal cells were treated with MPA and RU 486 sequentially, RU 486 stimulated the PRL production (approximately 2-fold over the MPA-treated cells) for 2-3 days and then reduced to basal levels over a 5-day period. The stimulatory and inhibitory effects of RU 486 on PRL production in stromal cells pretreated with progestin was also observed in the rate of synthesis of PRL estimated by incubating the stromal cells with [35S]methionine and immuno-isolating the [35S]PRL with anti-PRL.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗