Identification of Multiple Differentially Expressed Messenger RNAs in Normal and Pathological Trophoblast

Placenta (2003), 24, 209–218 doi:10.1053/plac.2002.0885

Identification of Multiple Differentially Expressed Messenger RNAs in Normal and Pathological Trophoblast S. Durandb, P. Abadieb, S. Angeletti and S. Genti-Raimondia Departamento de Bioquı´ mica Clı´ nica, Facultad de Ciencias Quı´ micas, Universidad Nacional de Co´rdoba, Pabello´n Argentina, Ala Oeste, Ciudad Universitaria, 5000 Co´rdoba, Argentina Paper accepted 3 September 2002

In an attempt to assess the molecular basis of phenotypic alterations present in the gestational trophoblastic diseases (GTDs) and to identify genes whose expression is specifically associated to these placental proliferative disorders we performed differential display techniques. Initially 19 candidate gene fragments were identified and differential expression was confirmed in eight of these fragments by Northern blot analysis. At the mRNA level ribosomal L26 (rL26), ribosomal L27 (rL27), a new Kru¨ppel type zinc finger protein and TIS11d were preferentially expressed in normal early placenta (NEP) relative to complete hydatidiform mole (CHM), persistent gestational trophoblastic disease (PGTD) and choriocarcinoma JEG-3 cell line. In contrast, heterogeneous ribonucleoprotein A1 (hnRNPA1), the ferritin light chain mRNA, and the uncharacterized protein KIAA0992 were predominantly expressed in JEG-3 cell line. Finally, decorin, a prototype member of an expanding family of small leucine-rich proteoglycans, showed high expression in CHM. In addition we demonstrated by immunohistochemistry analysis that increased decorin mRNA in CHM reflected a genuine augmentation in average steady state mRNA levels within cells. Taken together, these findings provide several interesting candidates for regulation of tumorigenic expression as well as early placentation development, including those involved in protein synthesis (rL26 and rL27), metabolism (ferritin light chain), intercellular communication (decorin) and regulation of gene expression (Kru¨ppel-like zinc finger, TIS11d and hnRNPA1). Information about such alterations in gene expression could be useful for elucidating the genetic events associated to gestational trophoblastic pathogenesis, developing new diagnostic markers, or determining novel therapeutic targets.  2003 Elsevier Science Ltd. All rights reserved. Placenta (2003), 24, 209–218

INTRODUCTION Blastocyst implantation and early placentation development, considered as examples of physiological invasiveness, involve cell proliferation and differentiation. In contrast to these normal trophoblastic processes, gestational trophoblastic diseases (GTDs) are characterized by an abnormal proliferation of different types of trophoblastic epitheliums (Horn and Bilek, 1997). Therefore elucidation of the molecular mechanisms that control trophoblast migration and differentiation is important in understanding these clinical problems. GTDs comprise a heterogeneous group of interrelated lesions that, including partial hydatidiform mole, complete hydatidiform mole (CHM), placental site trophoblastic tumour and choriocarcinoma are derived from a fertilization event (Ohlsson et al., 1993; Redline and Abdul-Karim, 1995). CHM represents a noninvasive placental disease that is characterized a

To whom correspondence should be addressed at: E-mail: [email protected] b S.D. and P.A contributed equally to this work. 0143–4004/03/$-see front matter

by hydropic swelling of the chorionic villi with marked trophoblastic proliferation. The partial mole contains two populations of villi: one of normal size, the other hydropic with less marked trophoblastic hyperplasia. Placental site trophoblastic tumour and choriocarcinoma involve a malignant avillous invasive proliferation of trophoblastic cells. Activation of some proto-oncogenes such as p53 (Lee, 1995), epidermal growth factor receptor (Balaram et al., 1997) and c-erbB-2 (Bauer et al., 1997); increased expression of proliferative markers such as Ki-67 and PCNA (Cheung et al., 1998; Ostrzega et al., 1998), down-regulation of Ras GTPase activating protein (Stahle-Backdhal et al., 1995), and DOC-2/ hDab2 (Fulop et al., 1998), and dysregulation in the imprinted genes IGF2, H19 (Arima et al., 1997) and p57kip2 (Chilossi et al., 1998), extracellular matrix proteins (Crescimanno et al., 1999) and the basic helix loop helix Hand-1 gene (Kno¨fler et al., 1998) have been reported in GTDs. More recently, studies using human cDNA expression array (Vegh et al., 1999) and differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) (Huch et al., 1998; Xu et al.,  2003 Elsevier Science Ltd. All rights reserved.

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1999) have begun to define groups of genes that are altered in trophoblastic tumours. Previously, we have reported some biochemical and molecular alterations found in CHM in comparison to normal early placenta (NEP) and normal term placenta. Thus, we have shown a low aromatase activity in microsomes from molar tissue (Genti-Raimondi et al., 1993) and down-regulation of pregnancy specific glycoproteins genes in CHM in comparison with what is seen in both NEP and normal term placenta (Bocco et al., 1989). Furthermore, we have isolated by differential screening techniques a novel member of the tumour necrosis factor receptor family preferentially expressed in CHM (Dumur et al., 1998, 2001). Recently, using DDRTPCR we have identified four mitochondrial transcripts downregulated in benign, as well as in malignant trophoblastic diseases encoding the cytochrome oxidase subunit I, the ATPase subunit 6, the 12S ribosomal RNA and the transfer RNA for phenylalanine (Durand et al., 2001). In the present report we extended those analyses and identified eight transcripts differentially expressed between normal and pathological trophoblastic samples involved in protein synthesis, metabolism, intercellular communication and regulation of gene expression.

MATERIALS AND METHODS Tissue collection and cell lines The tissues employed in this study were obtained after therapeutic abortion from patients with amenorrhea of about a 120-day duration. They include three NEPs and nine CHMs. The diagnosis of the CHM was established before the evacuation time according to biochemical (
-hCG value), echographic and clinical parameters (Tham and Ratnam, 1998). Two of the CHMs collected had persistently raised serum concentrations of hCG during 8 to 10 weeks of subsequent follow-up, therefore they were defined as persistent gestational trophoblastic diseases (PGTDs) and single-agent chemotherapy was given. NEPs and CHMs were identified on the basis of gross morphology and histopathology. The tissues were repeatedly washed with ice-cold 137 m NaCl, cut into small pieces, and stored at
70C within 30 min after the material was available. It is unusual to have appropriate availability of malignant trophoblastic tissues (choriocarcinoma, invasive mola or placental site trophoblastic tumour) chiefly due to the fact that hysterectomies are not commonly performed in this condition as it was recommended by several authors (Goldstein, 1972; Sheridan et al., 1993). Consequently, and for comparative purpose we include in the experiments the human choriocarcinoma JEG-3 cell line which maintains several of the biochemical parameters characteristic of the trophoblastic tissues such as the synthesizes of hCG, hCS and progesterone (Kohler et al., 1971). JEG-3 cells (obtained from ATCC, Rockville, MD, USA) were grown in Dulbecco’s modified Eagle’s medium (Sigma, Buenos Aires, Argentina), supplemented with

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10 per cent foetal calf serum, streptomycin (0.1 mg/ml), and penicillin (100 U/ml). Differential display Total RNA was isolated from one NEP, two CHMs and the JEG-3 cell line using the acid guanidinium thiocyanate-phenol-chloroform extraction method described by Chomczynski and Sacchi (1987), treated with RNase-free DNase (Sigma) and used as samples for RT and PCRs. A reaction mixture containing 250 ng of DNA-free total RNA and 1  3 oligo(dT)-degenerate primers (Bauer et al., 1993; Linskens et al., 1995) was incubated at 65C for 10 min. After the addition of single-strength first strand buffer [50 m Tris-HCl (pH 8.3), 40 m KCl, 6 m MgCl2], 10 m of DTT (Sigma), 1 U/l of RNasin Ribonuclease Inhibitor (Promega, Madison, WI, USA), 500  of dNTPs (Sigma) and 1 l of 200 U/l SuperScript II RNase H Reverse Transcriptase (Life Technologies, Buenos Aires, Argentina), the reaction mixture was incubated at 42C for 90 min followed by boiling for 5 min. PCRs were carried out in 50 l of reaction mixture containing 2 l of reverse-transcribed products, single-strength PCR buffer [50 m KCl, 10 m Tris-HCl (pH 9.0 at 25C) and 1.0 per cent Triton X-100], 1.5 m MgCl2, 1  3 oligo(dT)degenerate primers, 0.2  5 arbitrary primers (Bauer et al., 1993; Linskens et al., 1995), 2  of dNTPs, 1 l of 35Slabelled dATP (1250 Ci/mmol; DuPont NEN, Boston, MA, USA) and 1 U of Taq DNA Polymerase (Promega). The optimal reaction conditions were as follows: 5 min of incubation at 95C, and 40 cycles of sequential incubations at 94C for 30 sec, at 40C for 2 min, and at 72C for 30 sec, followed by 5 min incubation at 72C. A portion (7 l) of the PCR products was then mixed with 3 l of stop solution containing loading dyes, incubated at 80C for 2 min, and subsequently electrophoresed on a 6 per cent polyacrylamide-8  urea DNA sequencing gel. Differences in the banding patterns among these radiolabelled amplicons were revealed by exposing the dried gel on Kodak X-Omat-K films. Bands representing differentially expressed cDNAs were excised from the gel, soaked in 100 l of deionized H2O (dH2O) for 10 min, boiled for 15 min and centrifuged for 2 min to clarify particulate material. After extracting with chloroform, the DNA was precipitated from the supernatant with 10 l of 3  sodium acetate (pH 6.0), 3 l of glycogen (10 mg/ml) and 400 l of 100 per cent ethanol for 15 min at
70C. Then, the samples were centrifuged for 5 min; the pellet was rinsed with 100 l of ice-cold 85 per cent ethanol and dissolved in 10 l of dH2O. For each band, 3 l of extracted DNA was reamplified in a total reaction volume of 50 l using the same set of primers and the initial RT-PCR conditions. Cloning and sequencing of cDNA fragments Reamplified PCR products were electrophoresed on a 1.2 per cent agarose low melting point gel and visualized using

Durand et al.: Differential Display in Trophoblastic Diseases

ethidium bromide staining. These amplified cDNA fragments were then cloned into pGEM-T Easy Vector (Promega) as recommended by the manufacturer. The ligation products (10 l) were then subjected to transformation using a heat shock reaction carried out at 42C for 60 sec. Both strands of the cloned cDNA fragments were sequenced by the dideoxy chain termination method using the Sequenase Version 2.0 DNA Sequencing Kit (United States Biochemical Corp., Amersham Life Science Inc., Arlington Heights, IL, USA). The sequences were analysed using the BLAST program (Altschul et al., 1997).

Northern blot analysis Total RNA was isolated from three NEPs, seven CHMs, two PGTDs and the JEG-3 cell line as described below. Approximately 15 g of total RNA from each sample were fractionated by electrophoresis through 1.2 per cent agarose/ formaldehyde gels in 20 m 3-(N-Morpholino) propanesulfonic acid (MOPS) buffer (pH 6.8) at a constant rate of 20 V/cm for 4 h at room temperature. RNA was denatured, neutralized and then transferred overnight to nylon membranes in 20-strength SSC (single-strength SSC is 150 m sodium chloride and 15 m sodium citrate, pH 7.0). The nylon membranes were subjected to UV-cross-linking for 4 min on a Bio-Rad (Richmond, CA, USA) 300 nm UV transilluminator and then prehybridized for at least 4 h at 42C in prehybridization solution [50 per cent formamide, 5-strength SSPE (750 m NaCl, 50 m NaH2PO4 · H2O, 5 m Na2EDTA, pH 7.4), 10-strength Denhardt’s solution (1 per cent Ficoll, 1 per cent polyvinylpyrrolidone, 1 per cent BSA), 2 per cent SDS and 100 g/ml heat-denatured salmon sperm DNA]. The membranes were subsequently hybridized overnight at 42C with 5 ng/ml [-32P]dATP random primerlabelled cDNA probes prepared with the Prime-a-Gene Labeling System kit (Promega) according to the protocol recommended by the manufacturer. After hybridization, membranes were washed in a buffer containing 0.2 strength SSC and 0.2 per cent SDS at 65C for 20 min (three times) and then exposed for autoradiography. The membranes were subsequently stripped and rehybridized with 32P-labelled cDNA for
-actin. Autoradiographs were densitometric scanned using dual wavelength chromatoscanner (Shimadzu CS-930).

Immunohistochemistry Human trophoblastic tissues were frozen in Tissue-Tek OCT embedding medium (Miles, Buenos Aires, Argentina) and stored at
70C. Cryostat sections of trophoblastic tissues (7 m thickness) and a JEG-3 cells grown on coverslips were mounted on poly--lysine (Sigma) coated slides and air-dried, followed by fixation for 5 min in 4 per cent (v/v) methanolfree p-formaldehyde, 10 m CaCl2, 0.1 m phosphate buffered saline (PBS, pH 7.4). To inhibit endogenous peroxidase

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activity, the sections were incubated with 0.6 per cent H2O2 in 80 per cent (v/v) of methanol for 30 min at room temperature. Afterwards the sections were pre-incubated for 10 min at room temperature in PBS supplemented with 1 per cent (w/v) BSA and then incubated for 2 h with rabbit anti-decorin antibody kindly provided by Dr E. Ruoslahti (Krusius and Ruoslahti, 1986). Bound antibodies were detected with biotinylated anti-rabbit immunoglobulins secondary antibodies and streptavidin-peroxidase complex using 3-amino-9-ethyl carbazole as substrate (Histostain-Plus kit, Zymed Laboratories, Buenos Aires, Argentina). Subsequently the sections were counterstained with haematoxylin, dehydrated and mounted with an aqueous mounting solution. Controls were performed by replacing the primary antibody by non-immune serum. Further controls were performed by omitting the secondary antibody. The controls were always negative.

RESULTS Gene expression of cells isolated from normal and pathological trophoblastic tissues was compared by DDRT-PCR of mRNA transcripts using three different set of primers. In general, 50–100 bands of sizes between 200 bp and 600 bp were displayed in each line. Several cDNA fragments exhibiting distinct intensities among NEP, CHM and JEG-3 choriocarcinoma cell line were detected (data not shown). Preliminarily, 19 bands were isolated, nine of them appeared to be upregulated in NEP, eight in the JEG-3 cell line and two in CHM. To confirm the different expression pattern of these genes, the isolated bands were reamplified, cloned and used as probes in Northern blot assays. The data shown represent the results of three independent experiments performed with total RNA extracted from three NEPs, seven CHMs, two PGTDs and JEG-3 cell line. Eight clones were found to hybridize with genes which were differentially expressed between normal and pathological trophoblastic samples. The constitutively expressed
-actin gene was used for the normalization of samples. Differential gene expression was defined as 2.0-fold difference in the mRNA/
-actin mRNA level between at least 2 of the trophoblastic samples. As shown in Figure 1, four transcripts of 2 kb, 0.5 kb, 2 kb and 3.7 kb probed by clones 1, 2, 3 and 4 respectively, were highly expressed in NEP in comparison to CHM, PGTD and JEG-3 cell line. In contrast, two transcripts (1.4 and 1.8 kb) probed by clone 5, one transcript (0.8 kb) probed by clone 11 and one transcript (4 kb) probed by clone 19 were preferentially expressed in JEG-3 cell line (Figure 2). When clone 10 was used as probe one transcript of 1.8 kb was predominantly expressed in CHM (Figure 3). Despite of the fact that differential display revealed contrasting intensities in the bands representing clones 6, 7, 8 and 12 to 18, Northern blot analysis failed to detect signals. We attribute this to very low expression of the corresponding genes. Finally, no difference in the mRNA expression was seen for clone 9 (data not shown).

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Placenta (2003), Vol. 24

Figure 1. Transcript level of upregulated genes in NEP. Northern blot analysis of total RNA from NEP, CHM, PGTD and the JEG-3 cells probed with P-labelled clone 1, 2, 3 and 4 cDNAs. The expression of respective clones was confirmed as 2 kb, 0.5 kb, 2 kb and 3.7 kb transcripts. The membranes were rehybridized with
-actin probe as control (A). Relative intensity of the ribosomal L26, ribosomal L27, Kru¨ppel zinc finger and TIS11d mRNA bands were assessed by scanning densitometry and corrected for loading as determined from the
-actin signals (B). The experiment was repeated three times with three NEPs, seven CHMs, two PGTDs and JEG-3 cell line cultures with similar results. Data shown are each from a representative experiment. 32

Durand et al.: Differential Display in Trophoblastic Diseases

213

Figure 2. Transcript level of upregulated genes in JEG-3 choriocarcinoma cell line. Northern blot analysis of total RNA from NEP, CHM, PGTD and the JEG-3 cells probed with 32P-labelled clone 5, 11 and 19 cDNAs. The expression of the respective clones was confirmed as 1.8 and 1.4 kb, 0.8 kb and 4 kb transcripts. The membrane was rehybridized with
-actin probe as control (A). Relative intensity of the heterogeneous nuclear ribonucleoprotein A1, ferritin light chain and KIAA0992 (AN AB023209) mRNA bands were assessed by scanning densitometry and corrected for loading as determined from the
-actin signals (B). The experiment was repeated three times with three NEPs, seven CHMs, two PGTDs and JEG-3 cell line cultures with similar results. Data shown are each from a representative experiment.

All cDNA fragments were sequenced, and a homology search of them against GenBank and EMBL DNA data bases was performed (Table 1). The results revealed that 10 clones showed complete identity to known genes, i.e. one clone to 406–658 bp of ribosomal (rL26) transcript (AN AF083248), one of them being homologous to the complete 465 bp cDNA fragment of ribosomal L27 (rL27) gene (AN L19527), one clone to the 912–1300 bp of Kru¨ppel type zinc finger (AN AJ245587), one clone to the 5235–5488 bp of human TIS11d

(AN U07802), one clone to the 4796–5238 bp 3 end of heterogeneous nuclear ribonucleoprotein A1 (AN X12671), one clone to the 2239–2417 bp of TFII-I protein (AN AF015553), one fragment being homologous to the 1611– 1864 bp of the LMO4 protein (AN U24576), one clone to the 2036–2173 bp fragment of subunit alpha-1 of collagen type VI (AN X15880), one clone was homologous to 245–693 bp of decorin transcript (AN M14219) and one fragment being homologous to 240–639 bp of ferritin light chain mRNA (AN

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Figure 3. Transcript level of upregulated genes in CHM. Northern blot analysis of total RNA from NEP, CHM, PGTD and the JEG-3 cells probed with P-labelled clone 10 cDNA. The expression of clone 10 was confirmed as a 1.8 kb transcript (top). The membrane was rehybridized with
-actin probe as control (bottom) (A). Relative intensity of the decorin mRNA bands were assessed by scanning densitometry and corrected for loading as determined from the
-actin signals (B). The experiment was repeated three times with three NEPs, seven CHMs, two PGTDs and JEG-3 cell line cultures with similar results. Data shown are each from a representative experiment. 32

NM000146). Finally, two fragments (clone 13 and 14) had no significant homology to any registered DNA sequences and their sequences are deposited in GenBank as AW342137 and AW342138. Six clones were homologous to expressed sequence tag (EST) sequences (clone 7, 12, 15, 16, 17 and 18). Two of them contain Alu repetitive elements (clone 16 and 18) and two of them present LINE 1 repetitive element (clone 15 and 17). One clone was homologous to the uncharacterized protein KIAA0992, accession number AB023209. Translation of the 375 bp sequence of clone 7 revealed an open reading frame of 86 amino acids with 75 per cent of identity and 85 per cent of similarity to a novel helicase C-terminal domain and SNF2 N-terminal domains containing protein (AN AL031667). In addition, we analysed the decorin protein synthesis by immunohistochemistry analysis. Immunostaining of CHM section tissues revealed strong decorin immunoreactivity localized in the extracellular matrix and villi syncytiotrophoblast cells (Figure 4B). In contrast, a very slight staining in NEP and PGTD tissues was observed (Figure 4A and C). JEG-3 cells did not exhibite intracellular immunostaining (Figure 4D). In brief, these data demonstrate a close correlation with those found in the Northern blot experiments (Figure 3).

DISCUSSION Using DDRT-PCR, we have previously reported the isolation of four mitochondrial transcripts downregulated in benign as well as in malignant trophoblastic diseases encoding the cytochrome oxidase subunit I, the ATPase subunit 6, the 12S ribosomal rRNA and the transfer RNA for phenylalanine (Durand et al., 2001). In the present report, we isolated four genes that were highly expressed in NEP relative to CHM, PGTD and JEG-3 cell line; three genes that were predomi-

nantly expressed in JEG-3 cell line and one gene with increased expression in CHM. All of the preferentially expressed genes in NEP were identified as known genes that may reflect different functions of trophoblast cell. Two of them were identified as rL26 and rL27 transcripts. Protein L26 is proposed to be at the interface of the large and small ribosome subunits (Villareal Jr and Lee, 1998); whereas rL27 protein has been implicated as a constituent of the peptidyl transferase center of the 50S ribosomal subunit by a variety of experimental observations (Wower et al., 1998). Upregulated rL26 transcript was observed in the marine gastropod, Littorina littorea, during anoxia exposure displaying a role in stabilizing ribosomal subunits (Larade et al., 2001). In this regard, normal blastocyst implantation and early placentation occur under relatively low oxygen levels (Yedwab et al., 1976) and oxygenmediated changes in gene expression were demonstrated in first trimester trophoblast cells (Pak et al., 1998). Similarly, accumulation of rL27 protein is correlated with cell proliferation (Gallagher et al., 1994) and the phorbol ester induced shut off cell division of leukemia cell line K562 associated with a decrease of rL27 protein (Lin et al., 1994). One cDNA fragment with increased mRNA levels in NEP corresponds to a new member of the Kru¨ppel type zinc finger proteins recently described (Dang et al., 2000). Zinc fingers are structural motifs found in many transcription factors that bind DNA and regulate gene expression. Genes containing this motif have been shown to function in regulating a diverse range of biological processes, including cell growth, differentiation, embryogenesis and tumourigenesis. The last gene found strongly expressed in NEP was TIS11d gene, a member of TIS11 early response gene family which contains unique cysteine-histidine motifs that is rapidly and transiently induced by both 12-O-tetradecanoylphorbol-13-acetate and growth factors (Varnum et al., 1991). Also, it was suggested that this protein is involved in regulating mRNA stability in

KIAA0992 (AB023209)

Clone 19

ND, no difference in expression; NA, no blot result available.

Clone 18 (AW342143)

Clone 17 (AW342139)

EST78824 (AA367704) Wo20d06.x1 NCI-CGAP Pan1 Homo sapiens (AI925869.1) hd32d11x1 Soares-NFL-T-GBC-S1 Homo sapiens (AW469659) DKFZp762J0911-r1 (AL135018.1)

Clone 15 (AW342140) Clone 16 (AW342142)

Unclassified

JEG-3

NA

NA

NA NA

Light ferritin (NM000146) JEG-3 MR4-UM0021-090300-002-d07 UM0021 NA Homo sapiens (AW795664.1) Clone 13 (AW342137)) None NA Clone 14 (AW342138) None NA

ND CHM

NA

NEP JEG-3 NA NA

NEP

NEP NEP

4

0.8

4.5 1.8

3.7 1.4 and 1.8

2

2 0.5

364

305

253

471 270

498 251

400 378

138 448

254

253 443 178 375

388

250 465

Last exon and 3 ut

Last exon and 3 ut Coding

Overlap

3 ut Coding

96 per cent from EST 198–305 bp 100 Coding

100 EST 90 per cent from EST 1–124 pb 100 EST

100 240–639 bp 98 per cent from EST 106–209 bp

100 100

100 Coding 100 Exon 10 100 Coding 97 per cent from EST 163 to 261 100 3 ut

100

100 100

Increased MRNA size expression by observed Fragment size Identity Northern (kb) (bp) (%)

Clone 11 Clone 12 (AW342136)

Collagen VI alpha-1 (X15880) Decorin (M14219)

Kru¨ppel type zinc finger (AJ245587) TIS11d (U07802) HnRNP A1 (X12671) TFII-I (AF015553) Novel Helicase C-ter BF950394 (AL031667) LMO4 (U24576)

Ribosomal L26 (AF083248) Ribosomal L27 (L19527)

Database homology (accession number)

Metabolism

Intercellular communication Clone 9 Clone 10

Clone 8

4 5 6 7 (AW342141)

Clone 3

Gene expression regulation

Clone Clone Clone Clone

Clone 1 Clone 2

Protein synthesis

Functional role

cDNA fragments (accession number)

Table 1. Isolation of genes differentially expressed and homology of cDNA fragments to NCBI database files

Durand et al.: Differential Display in Trophoblastic Diseases 215

216

Figure 4. Representative pictures of immunostaining for decorin in normal and pathological samples. A, NEP. B, CHM. C, PGTD. D, JEG-3 cells. Original magnifications: (400). Bar=25 m.

Placenta (2003), Vol. 24

oocyte maturation and early embryogenesis in Xenopus (De et al., 1999). Two of the genes with increased mRNA expression in choriocarcinoma JEG-3 cell line correspond to the hnRNP A1 and ferritin light chain mRNAs. Protein A1 is one of the major components of mammalian ribonucleoprotein particles (hnRNP). hnRNP A1 shuttles between the cytoplasm and nucleus and plays important roles in RNA metabolism. It has been shown that the hnRNP A1 participates in telomere biogenesis. Telomere attrition is associated with chromosome instability and cell senescence, whereas stabilization of telomere length correlates with the immortalization of somatic cells (LaBranche et al., 1998). Also, it was demonstrated that hnRNP A1 binds to the transcription-regulatory sequences of mouse hepatitis virus leader RNA suggesting a probable participation in its transcription (Li et al., 1999). Ferritin light chain transcript was preferentially expressed in JEG-3 cell line respect to NEP, CHM and PGTD samples. In this regard, a potential role of the increased ferritin light chain expression in the control of cell proliferation and invasiveness in murine adenocarcinoma (Monitto et al., 2001) and human epithelioid sarcoma cell line (Weber et al., 2001) was suggested. The remaining transcript predominantly expressed in JEG-3 cell line corresponds to the predicted KIAA0992 cDNA sequence obtained from human brain cDNA libraries (Nagase et al., 1999). The predicted protein sequence (ANo PN0689) has 30.8 per cent identity in 448 amino acids sequence overlap with connectin 1 highly expressed in spinal cord. The functional role of this protein and its implications in the control of proliferation remain to be determined. Decorin, a prototype member of an expanding family of small leucine-rich proteoglycans, showed increased expression in CHM assayed by Northern as well as immunohistochemistry techniques. In contrast, this proteoglycan showed a marked decrease expression related to the increase of severity of trophoblastic disease, in particular in PGTD and choriocarcinoma. In concordance with our results, Lysiak et al. (1995) hypothesized that normal human extravillous trophoblast cell proliferation, migration and invasiveness are limited by at least two major decidual cell products, transforming growth factor beta (TGF
) and TGF
binding proteoglycan decorin. Furthermore, it was recently suggested that decorin plays a key role in cancer biology, not only as modulator of growth factor activity, but also as a physical and bioactive barrier to the invading neoplastic cells (Santra et al., 2000). In addition, we have identified and cloned fragments of eleven genes, for ten of them Northern blot analysis failed to detect signals. This is probably the result of scarcity of the respective mRNAs. In summary these observations suggest that, high mRNA levels of rL26 and rL27, a Kru¨ppel type zinc finger and TIS11d are more general feature associated to normal trophoblast phenotype. In contrast, increased gene expression of hnRNP A1, ferritin light chain and KIAA0992 may contribute to a dysregulation in the trophoblast cell metabolism.

Durand et al.: Differential Display in Trophoblastic Diseases

Finally, increased decorin expression was associated to benign trophoblastic disease in which a normal regulated invasiveness is observed. Taken together, these findings provide several interesting candidates for regulation of tumourigenic expression as well as early placentation development, including those involved in protein synthesis (rL26 and rL27), metabolism (ferritin light

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chain), intercellular communication (decorin) and regulation of gene expression (Kru¨ppel type zinc finger, TIS11d and hnRNP A1). Information about such alterations in gene expression could be useful for elucidating the genetic events associated to gestational trophoblastic pathogenesis, developing new diagnostic markers, or determining novel therapeutic targets.

ACKNOWLEDGEMENTS We are grateful to Dr E. Ruoslahti for generously providing anti-decorin rabbit antiserum. We are also grateful to Dr L. Patrito, Dr A. Flury and Dr G. Panzetta-Dutari for discussions and critical reading of the manuscript. This work was supported by Ministerio de Salud Pu´blica de la Nacio´n, CONICOR, SECYT, ANPCYT and CONICET grants. The results are derived from Ph.D. thesis by SD and PA.

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