Differential gene expression profile reveals deregulation of pregnancy specific beta1 glycoprotein 9 early during colorectal carcinogenesis.

BACKGROUND: APC (Adenomatous polyposis coli) plays an important role in the pathogenesis of both familial and sporadic colorectal cancer. Patients carrying germline APC mutations develop multiple colonic adenomas at younger age and higher frequency than non-carrier cases which indicates that silencing of one APC allele may be sufficient to initiate the transformation process. METHODS: To elucidate the biological dysregulation underlying adenoma formation we examined global gene expression profiles of adenomas and corresponding normal mucosa from an FAP patient. Differential expression of the most significant gene identified in this study was further validated by mRNA in situ hybridization, reverse transcriptase PCR and Northern blotting in different sets of adenomas, tumours and cancer cell lines. RESULTS: Eighty four genes were differentially expressed between all adenomas and corresponding normal mucosa, while only seven genes showed differential expression within the adenomas. The first group included pregnancy specific beta-1 glycoprotein 9 (PSG9) (p < 0.006). PSG9 is a member of the carcinoembryonic antigen (CEA)/PSG family and is produced at high levels during pregnancy, mainly by syncytiotrophoblasts. Further analysis of sporadic and familial colorectal cancer confirmed that PSG9 is ectopically upregulated in vivo by cancer cells. In total, deregulation of PSG9 mRNA was detected in 78% (14/18) of FAP adenomas and 75% (45/60) of sporadic colorectal cancer cases tested. CONCLUSION: Detection of PSG9 expression in adenomas, and at higher levels in FAP cases, indicates that germline APC mutations and defects in Wnt signalling modulate PSG9 expression. Since PSG9 is not found in the non-pregnant adult except in association with cancer, and it appears to be an early molecular event associated with colorectal cancer monitoring of its expression may be useful as a biomarker for the early detection of this disease.

Characterization of pregnancy-specific beta 1-glycoprotein synthesized by human placental fibroblasts.

We have previously demonstrated that human placental fibroblasts produce a pregnancy-specific beta 1-glycoprotein (PS beta G) immunologically indistinguishable from placental PS beta G. This was confirmed by the immunocytochemical localization of PS beta G in these fibroblasts. In addition, placental fibroblasts contain all three PS beta G mRNAs of 2.3, 2.2, and 1.7 kilobases which hybridize with the three PS beta G cDNAs (PSG16, PSG93, and PSG95) identified, although at 1.4-2.5% of the levels in human term placenta. The major PS beta G species synthesized by placental fibroblasts is a 62K glycopolypeptide formed from a 58K intracellular precursor polypeptide. However, the PS beta G species found in human placenta are one major glycoprotein of 72K and two minor ones of 64K and 54K. Poly(A)+ RNA from placental fibroblasts directed the synthesis of two polypeptides of 48K and 46K (major), whereas, poly(A)+ RNA from human placenta directed the synthesis of higher levels of four polypeptides of 50 K, 48 K (major), 46 K, and 36 K. Thus, the major PS beta G species found in fibroblasts and human placenta differ. The carbohydrate side-chains are essential for the stability of fibroblast PS beta G, because PS beta G synthesis in these fibroblasts could not be detected in the presence of tunicamycin, a protein glycosylation inhibitor which did not affect PS beta G mRNA expression. Our finding that a variant PS beta G species is produced in placental fibroblasts raises the possibility that the authentic placental PS beta G species may have different functions.

Identification of proteins immunochemically related to human pregnancy-specific beta 1-glycoprotein in the rat placenta.

Human pregnancy-specific beta 1-glycoprotein (hPS beta G) consists of a set of glycoproteins present in placenta and maternal serum. This study characterized proteins in rat placenta that show immunological cross-reactivity with antisera to hPS beta G. Immunocytochemical studies using two independent preparations of anti-hPS beta G showed intense specific staining within basophilic cytotrophoblast cells of the basal zone of the gestation day 15 rat placenta. In contrast, basophilic cytotrophoblasts located in the labyrinth did not stain. Subsequent experiments used gel electrophoresis and immunoblot analysis to compare PS beta G in human placenta and serum with immunoreactive proteins in rat placenta and serum. A set of two or three proteins was detected in human villous tissue and pregnancy serum with apparent mol wt (Mr) ranging from 54,000-76,000. In contrast rat placenta showed a major immunoreactive protein with 120,000 Mr, while rat serum contained bands of 48,000 64,000 and 69,000 Mr. Explant cultures of rat basal zone tissue secreted two [35S]methionine-labeled proteins that were immunoreactive, a major 120,000 Mr species and a minor 76,000 Mr form, with pI values of 4.6-5.5; tunicamycin inhibited the secretion of both species. Thus, a 120,000 Mr glycoprotein appears to be the major tissue and secreted form of rat PS beta G analog in day 15 placenta. Finally, the cytochemical localization of PS beta G-like proteins in rat placenta showed a progressive gestational shift from giant trophoblast cells in the parietal yolk sac placenta on day 12 to the basal zone cytotrophoblast cells by day 15. Data indicate that the pregnant rat may provide an animal model for investigation of the biological function of PS beta G during late gestation.

Expression of the pregnancy-specific beta 1-glycoprotein gene in cultured human trophoblasts.

Human pregnancy-specific beta 1-glycoprotein (PS beta G), the major placental glycoprotein, shares strong sequence similarity with carcinoembryonic antigen and is a member of the immunoglobulin superfamily. To understand the role of PS beta G in placental ontogeny during pregnancy, we examined its synthesis and regulation in primary cultures of trophoblast cells. Freshly plated (12 h) cytotrophoblasts expressed little PS beta G transcripts; however, within 24 h of culture, PS beta G mRNAs became detectable. PS beta G synthesis and mRNA expression increased with time in culture, and maximal synthesis was achieved at 4 days, indicating that primary trophoblasts continue differentiating in vitro. Molecular cloning revealed that PS beta G is an extremely polymorphic protein. Most PS beta G cDNAs identified to date, including the three cDNAs (PSG16, PSG93, and PSG95) isolated in this laboratory, share strong sequence similarity in the 5' (designated PSG-5') and coding regions, but differ in sequences at the 3' region. The PSG-5', PSG93-specific, PSG16/PSG93-3', and PSG95-3' probes, which identify the majority of PS beta G mRNAs, hybridized with three mRNAs of 2.3, 2.2, and 1.7 kilobases in primary trophoblasts and human term placental tissue. Ribonuclease protection analysis demonstrated that primary trophoblasts expressed most of the placental PS beta G transcripts. However, culturing in vitro altered PS beta G gene expression, and the level of PS beta G transcripts containing a PSG95-3' sequence was preferentially increased in primary trophoblasts. Moreover, primary trophoblasts synthesized a 64K PS beta G polypeptide in variable amounts and three PS beta Gs of 72K, 62K, and 54K in roughly equal amounts, whereas purified human term placental PS beta G consists of a major polypeptide of 72K and two minor ones of 64K and 54K. PS beta G gene expression in primary trophoblasts was slightly reduced by 8-bromo-cAMP, but was markedly inhibited by sodium butyrate.

Isolation and characterization of a human amnion epithelial cell line that expresses the pregnancy-specific beta 1-glycoprotein gene.

Human pregnancy-specific beta 1-glycoprotein (PSG) is a family of closely related glycoproteins of 72K, 64K, 62K, and 54K. Together with the carcinoembryonic antigen, they form new members of the immunoglobulin superfamily. To study the molecular mechanisms that regulate expression of the PSG gene, we established a human amnion cell line, HAA58OD-8C, immortalized with an origin-defective simian virus-40 (SV40) temperature-sensitive A58 mutant virus. HAA58OD-8C cells were temperature sensitive for maintenance of transformation and expressed genes encoding PSG and the alpha- and beta-subunits of hCG. At the permissive temperature (33 C; transformed phenotype), they expressed low levels of PSG, hCG alpha, and hCG beta mRNAs and synthesized low levels of a 48K PSG polypeptide. At the nonpermissive temperature (39.5 C), HAA58OD-8C cells exhibited a differentiated phenotype, expressed increased levels of PSG, hCG alpha, and hCG beta mRNAs, and produced high levels of PSG polypeptides of 72K and 48K. Sodium butyrate induced PSG mRNA expression, and in the presence of butyrate, HAA58OD-8C cells produced high amounts of PSG polypeptides of 72K, 62K, and 48K. Ribonuclease protection analysis indicated that similar PSG transcripts were expressed by HAA58OD-8C cells and human term placenta. However, these amnion cells expressed selectively a certain population of PSG transcripts. Our results show that this amnion cell line provides a suitable model for studies of PSG gene expression and regulation.

Pregnancy-specific beta-1-glycoprotein (SP1) in cultured amniotic fluid cells.

The synthesis of pregnancy-specific beta-1-glycoprotein (SP1) was studied in amniotic fluid cell cultures using RIA, immunoperoxidase, and immunofluorescence techniques. SP1 was found by RIA in all 11 sonicates and in 21 of 26 culture media. The SP1-immunoreactive material was immunologically similar to maternal serum SP1. Immunoperoxidase and indirect immunofluorescence staining were positive in large cells identified as epithelial amniotic cells by labeling with antikeratin antibodies. Fibroblast-like cells were occasionally found in cultures, but they did not contain demonstrable amounts of SP1. The physiological significance of the findings presented remains unclear.

Ectopic production of pregnancy-specific beta 1-glycoprotein by a nontrophoblastic tumor in vitro.

To demonstrate the ectopic production of pregnancy-specific beta 1-glycoprotein (PSbetaG) by a nontrophoblastic tumor, the in vitro secretion of this glycoprotein was evaluated in an hCG-producing ovarian cystadenocarcinoma cell line maintained in long term cell culture. Parallelism was demonstrated between the immunoreactive material present in the tissue culture media and highly purified PSbetaG measured by a sensitive and specific RIA. The immunoreactive material was shown to cochromatograph with purified PSbetaG present in normal term pregnancy serum on a Sephadex G-150 column. The tumor PSbetaG was adsorbed to concanavalin A-Sepharose and eluted with alpha-D-methyl-glucoside in a manner similar to purified PSbetaG. The indirect immunoperoxidase method with anti-PSbetaG sera localized PSbetaG in secretory vesicles and in the endoplasmic reticulum of the tumor cells by light and electron microscopy. The addition of sodium butyrate to the tissue culture media stimulated PSbetaG production in a fashion quantitatively similar to that seen with hCG. The number of PSbetaG-staining intracellular vesicles also increased after exposure to butyrate. These studies provide direct evidence for the ectopic production of PSbetaG by a nontrophoblastic tumor cell line.

Production of pregnancy-specific beta 1-glycoprotein by cultured placental cells.

The synthesis of pregnancy-specific beta 1-glycoprotein (PS beta G) was studied in simian virus 40 temperature-sensitive A mutant-transformed human first trimester and term placental cells. At the permissive temperature (33 C, transformed phenotype), they produced low levels of PS beta G. At the restrictive temperature (40 C), the transformed phenotype was lost, and the production of PS beta G was greatly enhanced. The PS beta G produced by these transformed placental cells resembled the purified placental PS beta G by several criteria. Both cell and placental PS beta G bound to Concanavalin A-Sepharose and were, therefore, glycoproteins. The cell PS beta G cochromatographed with placental PS beta G on a Bio-Gel A-0.5m column. Furthermore, the slopes of the dose-response curves for the cell PS beta G were indistinguishable from that for placental PS beta G. The synthesis of PS beta G at both 33 and 40 C in these placental cells was greatly induced by sodium butyrate and 5-bromo-2'-deoxyuridine. Sodium butyrate was a more effective inducer at 33 C, whereas BrdUrd appeared to be a better inducer at 40 C.

Effects of retinoic acid on differentiation of choriocarcinoma cells in vitro.

Choriocarcinoma cells maintain multiple hormonal functions in culture. We have found that these cells secreted no immunoreactive pregnancy-specific beta 1-glycoprotein (PS beta G), a placental protein. Choriocarcinoma cells can be induced to synthesize low levels of PS beta G by retinoic acid, 8-bromo-cAMP (8BrcAMP), cholera toxin, methyl-isobutylxanthine (MIX), and 5-bromo-2'-deoxyuridine (BrdUrd). The simultaneous addition of retinoic acid along with 8BrcAMP, cholera toxin, or MIX gave synergistic induction of PS beta G. The simultaneous addition of retinoic acid and BrdUrd failed to give even additive induction. In addition to stimulating PS beta G production, retinoic acid increased the production of hCG and its alpha-subunit (hCG alpha) by choriocarcinoma cells. The simultaneous addition of retinoic acid along with 8BrcAMP, cholera toxin, or MIX gave additive induction for hCG and hCG alpha. Passage of choriocarcinoma cells in medium containing retinoic acid induced a stable altered phenotype characterized by elevated levels of PS beta G, hCG, and hCG alpha. These retinoid-treated choriocarcinoma cells remained responsive to 8BrcAMP or compounds that increase intracellular cAMP concentrations and to BrdUrd; the production to PS beta G and hCG was greatly stimulated by 8BrcAMP, cholera toxin, or MIX, and the production of hCG and hCG alpha was greatly inhibited by BrdUrd. However, the production of hCG alpha was only slightly induced by these cAMP modulators, and the production of PS beta G was not increased by BrdUrd.

Enzymatic isolation of human trophoblast and culture on various substrates: comparison of first trimester with term trophoblast.

A simple method is described for the isolation of trophoblast cells from both first trimester and term placenta. Trophoblast preparations were characterized by light microscopy, scanning and transmission election miscroscopy and immunohistochemistry to distinguish these cells from mesenchyme and endothelium. Trophoblast cells were cultured on various substrates and a comparison made of their ability to attach, proliferate and function. A collagen gel substrate produced by repolymerization of an acid soluble collagen fraction from chorionic villi allowed rapid attachment of trophoblast cells and maintainance of their original morphology. Term trophoblast cells were shown to become fully functional in short term (three day) cultures by virtue of their increased immunocytochemical staining for the presence of beta hCG, hPL and SPI. beta hCG increased significantly by day three thus demonstrating functional activation. Trophoblast cells from first trimester placenta formed proliferating colonies of hormone producing cells while those from term placenta reaggregated into clusters and closely resembled syncytiotrophoblast both morphologically and functionally. This short term culture system for term trophoblast will allow further studies into the biology of trophoblast polypeptide hormone synthesis and secretion.

Differential gene expression in the amnion, chorion, and trophoblast of the human placenta.

Human chorionic gonadotrophin (hCG), placental alkaline phosphatase (PLAP), and pregnancy-specific glycoprotein (PSG) are three major proteins produced by the trophoblast of the human placenta. Immunocytochemical studies suggest that PSG and hCG are also present in the human amnion. In this study, we examined whether amniotic and chorionic membranes were capable of expressing trophoblastic-specific genes. As previously reported, trophoblasts express high levels of hCG beta, hCG alpha, PLAP, and PSG. Both amnion and chorion were found to express PLAP and hCG beta mRNA. However, the hCG alpha transcript was expressed only by the amnion, but not by the chorion in the term placenta. Recent molecular cloning studies indicate that human PSGs are a group of closely related placental proteins that, together with the carcinoembryonic antigen family members, comprise a subfamily within the immunoglobulin superfamily. To demonstrate that amnion and chorion also express PSG transcripts, we employed ribonuclease protection analysis using probes specific to the 5' and 3' region of PSG mRNAs. Our data indicate that while amniotic as well as chorionic membrane expressed low levels of the PSG genes, only a certain subpopulation of PSG transcripts were expressed. Furthermore, the amnion and chorion demonstrated differences in PSG species expression from each other and from trophoblastic tissue. Thus, human amnion, chorion and trophoblast selectively express several placental genes.

In vivo and in vitro studies on the production of placental proteins (human chorionic gonadotropin, human placental lactogen, and pregnancy-specific beta 1-glycoprotein) in an adrenal choriocarcinoma.

The ectopic production of placental proteins (human chorionic gonadotropin [hCG], human placental lactogen, and pregnancy-specific beta 1-glycoprotein) by an adrenal choriocarcinoma was investigated experimentally in vivo and in vitro. By an immunohistochemical method, the choriocarcinoma tissues obtained from the right adrenalectomy were found to react with hCG, human placental lactogen, and pregnancy-specific beta 1-glycoprotein antibodies. The concentrations of hCG-beta, human placental lactogen, and pregnancy-specific beta 1-glycoprotein in the tumor fluid were 1480, 100, and 47 ng/mL, respectively. On incubation of the tumor slices in vitro, the concentration of hCG-beta in the incubation medium increased markedly with time. Serial sections of the removed uterus and right ovary did not reveal any primary trophoblastic lesions. The present tumor responded well to double chemotherapy with actinomycin D and methotrexate, resulting in a decrease of the level of serum hCG-beta to less than 10 ng/mL after four courses of the chemotherapy.

The Production of beta 1-specific pregnancy glycoprotein (SP1) by the amnion.

Using indirect immunofluorescence, beta 1-specific pregnancy glycoprotein (SP1) was localised to the amnion cells, as well as to trophoblastic cells. Cultures of fragments of amnion incubated with 3H-leucine for up to 72 h demonstrated that protein was synthesized by the amnion. Using antisera specific to SP1 it was shown that after 72 h incubation about 50% of the total protein could be precipitated. These studies demonstrate that the amnion cells are actively involved in the synthesis of SP1 and could account for the high level of this glycoprotein in amniotic fluid.

[Establishment of cell lines of human germinal tumors].

Human germinal tumor cells have been studied in cancer research because of their characteristics of the multi-potentiality and the ability to produce marker proteins such as AFP and SP1. Although human germinal tumor cell colonies had usually been maintained by serial transplantation into nude mice, we established in vitro culture method of cells derived from human malignant germ cell tumors. Thirty-two cell lines were established in vitro, and AFP and SP1 produced in culture medium of those cell line were demonstrated immunochemically.

Expression and transcriptional regulation of individual pregnancy-specific glycoprotein genes in differentiating trophoblast cells.

Human pregnancy-specific glycoproteins (PSGs), encoded by eleven highly conserved genes, are the major placental polypeptides. Low PSG levels in maternal circulation have been associated with complicated pregnancies. However, expression of each PSG gene and their regulation during cytotrophoblast cell differentiation remain poorly explored. Herein, we analyze the expression of five PSG genes and demonstrate that they are almost undetectable in undifferentiated trophoblast, but are all transcribed in differentiated cells. Among them, PSG1, PSG3 and PSG5 genes achieve high mRNA levels while PSG7 and PSG9 are poorly expressed. In addition, total PSG proteins and transcripts markedly increase during trophoblast differentiation, preceding morphological syncytialization and betahCG expression. The 5' regulatory region contributes to the transcriptional control of PSG gene induction in trophoblast cells undergoing differentiation. This responsive region in PSG3 maps within a 130 bp promoter sequence, which overlaps the transcription start site and requires a functional Retinoic Acid Responsive Element (RARE) and a GA-binding protein (GABP) consensus site for basal and differentiation-dependent promoter activity, respectively. Present findings provide novel data for understanding the control of PSG gene expression and demonstrate that their proteins and transcripts represent early markers of trophoblast differentiation.

Spatial expressions of fibronectin and integrins by human and rodent dermal fibroblasts.

BACKGROUND: Human skin shows various morphological characteristics, depending on the body site. As these distinct phenotypes have been explained on the basis of the variance in epidermal keratinocytes and the presence of skin appendages, the spatial distinction of the dermal components has not been fully elucidated. OBJECTIVES: To identify and characterize the profiles of mRNAs that are abundantly or specifically expressed by fibroblasts derived from trunk skin, but not from palmoplantar skin or oral mucosa. METHODS: In order to identify the distinct mRNA expression by trunk skin fibroblasts, a subtraction cDNA screening was performed first, followed by Northern blotting, Western blotting and immunohistochemistry for cultured human and rat dermal fibroblasts and those skin tissues. Finally, whole mount in situ hybridization (WISH) was performed to examine the differences in the expression of the corresponding gene during the developmental stage of mouse embryos. RESULTS: We identified three cDNA clones encoding fibronectin (FN), pregnancy-specific beta1-glycoprotein 5 and beta-actin, respectively, whose mRNAs were abundantly or specifically expressed by trunk skin fibroblasts. FN and some integrins were further confirmed to be expressed more selectively in human and rat trunk skin fibroblasts, both in terms of the RNA and the protein levels, compared with the fibroblasts derived from plamoplantar skin and oral mucosa. WISH demonstrated that FN was localized around the hair follicles of mouse embryos. CONCLUSIONS: FN, one of most potent extracellular matrix molecules, was demonstrated to be spatially transcribed depending on the body sites. The distinct expression of FN was suggestive of the essential commitment in the process of cutaneous development and morphogenesis of appendages originated from hair germ. The paucity of FN in palmoplantar skin and oral mucosa might explain the characteristics of these skin phenotypes.

Synthesis of placental proteins by the human placenta perfused in vitro. Preliminary report.

Fresh human placental tissue from deliveries at term was perfused in closed circulation on both the maternal and fetal side. At given times during the 4-hour perfusion period, the amount of human chorionic gonadotropin, human placental lactogen, pregnancy-specific beta 1-glycoprotein and pregnancy-associated plasma protein A was determined in the perfusing buffer. These placental proteins were also quantified in the placental tissue before and after perfusion. Calculations showed that all placentae (n = 3) were able to synthesize all 4 proteins tested.