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Induction of Manganese Superoxide Dismutase in Cultured Human Trophoblast during in Vitro Differentiation’ SUSANL. CHURCHFDONNA R. FARMER,AND D. Department

of

MICHAELNELSONS

Pediatrics, St. Louis Children’s Hospital, Department of Obstetrics and Gynecobgy, The Jew&h Hospital of St. Lab, Washington University School of Medicine, St. Louis, Missmwi 63110 Accepted September 17, 1991

The antioxidant responses of human cell differentiation and membrane fusion are not known and may be important in understanding cellular response to injury in the human placenta. We studied the regulation of antioxidant enzymes in human trophoblasts which differentiate from mononucleated cellular trophoblasts to synctium in tiwo and in culture. We characterized morphological and biochemical differentiation of cultured trophoblasts from term placenta in the presence or absence of serum, on different growth surfaces, and with a range of plating densities. Culture of cellular trophoblasts consistently and transiently induced the mRNAs of the mitochondrial antioxidant manganese superoxide dismutase (Mn SOD) but not the mRNAs for the antioxidant enzymes copper zinc SOD or catalase. Fibrin and type I collagen substrates modulated only the expression of the placental specific proteins, human chorionic gonadotropin, and human placental lactogen. Both Mn SOD induction and terminal differentiation, as reflected by human chorionic gonadotropin expression, were dependant on trophoblastic plating density. Increased levels of a smaller Mn SOD mRNA species correlated temporally with an increase in Mn SOD enzyme activity in cultured trophoblasts. These results demonstrate that Mn SOD gene expression and enzyme activity precede or are coordinately regulated with morphological and biochemical trophoblastic differentiation. o lwz Academic Press, Inc. INTRODUCTION

It has been hypothesized that bursts of oxidant production modulate gene expression (Allen, 1991) and ac.tive oxygen species are widely believed to play fundamental roles in cellular aging and tumorigenesis (Duschesne, 1977; Oberley, 1983). Under conditions of normal oxygenation, active oxygen species are produced in the mitochondria, endoplasmic reticulum, cellular and nuclear membranes, phagosomes and peroxisomes, and in the ground cytoplasm (Fisher, 1988; Gutteridge and Halliwell, 1990; Katusic and Vanhoutte, 1939). Cells are protected from reactive oxygen species by an antioxidant defense system. The initial enzymes in this system are the superoxide dismutases (SOD) which in eukaryotic cells are characterized by their metal requirements and by their subcellular localization. The manganesecontaining SOD (Mn SOD) is found in the mitochondria (Weisiger and Fridovich, 19’73), while the two copperzinc-containing SOD enzymes (CuZn SOD) are found throughout the cytoplasm (McCord and Fridovich, 1969) and in plasma (Marklund, 1934). Catalase, which is

1 Portions of this work were presented at the scientific sessions of the AAP in May 1990 and at the Society for Gynecologic Investigation in March 1991. * Recipient of Clinical Investigator Award K08HD99885 from the National Institutes of Health. 8 Recipient of NIH HD-22913.

found in plasma and in peroxisomes, protects cells from hydrogen peroxide (Deisseroth and Dounce, 1970). Normally, the human placenta develops from the trophoblast component of the products of conception and performs both transport and secretory functions in supporting fetal development in, normal pregnancy. The differentiated syncytial trophoblast on the villous surface is bathed in maternal blood and is in a position to regulate nutrient transfer and waste disposal for the fetus (Jones and Fox, 1991). An adjacent discontinuous layer of mitotically active cellular trophoblast shares a basement membrane with the syncytial trophoblast of term villi. These cellular trophoblasts differentiate to form new syncytium by a process of cell fusion (Jones and Fox, 1991) in viva and in culture. Following ischemia in the preeclamptic human placenta, damaged syncytial tissue is replaced and repaired by proliferating cellular trophoblasts which rapidly differentiate into syncytial tissue (Jones and Fox, 1980). The study of antioxidant enzymes in human trophoblast would provide insight into the antioxidant defenses of the placenta and responses of antioxidant defenses during active cell fusion and differentiation in a human cell population. In the present study, human trophoblasts were cultured on growth surfaces coated with type I collagen or fibrin and on uncoated plastic in the presence or absence of serum to determine whether trophoblastic differentiation could be altered. Morphological differentiation of trophoblasts was studied by immu177

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nofluorescence microscopy. Biological differentiation of these cells was examined by assay of human chorionic gonadotropin in the media and by RNA blot analysis of placental specific mRNAs of p human chorionic gonadotropin, human placental lactogen and cytoplasmic actin. Gene expression of Mn SOD, CuZn SOD, and catalase and the enzyme activity of Mn SOD and CuZn SOD were then examined in term human placenta and in cultured human trophoblast during in vitro differentiation. The purpose of this study was twofold: (1) to examine morphological and biochemical differentiation of trophoblasts in vitro under conditions which might alter trophoblast terminal commitment and fusion, and (2) to determine the antioxidant response of term human placenta and of a differentiating human cell population under defined conditions. MATERIALS

AND METHODS

Tissue Preparation and Cell Isolation

Placentas (n = 12) from uncomplicated term pregnancies at 38-41 weeks of gestation were obtained immediately after vaginal delivery or repeat cesarean section. All women were nomotensive throughout pregnancy, were not taking any medications, denied use of tobacco and alcohol, and had no medical illnesses. Tissue free of visible infarct, calcification, or hematoma was dissected from several cotyledons. The basal plate surface of these cotyledons was separated from the remaining villous tissue, and both specimens were frozen in liquid nitrogen and stored at -70°C until used for tissue RNA extraction. The basal plate from other cotyledons was removed and 30-35 g of the villous tissue was used to isolate cellular trophoblast by the method of Kliman et al (1986) with modifications previously described (Nelson et al., 1990). Ninety to 95% of the cells were immunoreactive for cytokeratin intermediate filaments indicating their trophoblastic origin. Cell viability was >95% by trypan blue exclusion. Culture of Cellular Trophoblasts

Cells were cultured from 3 hr to 6 days in modified Eagle’s minimum essential medium containing a 1.5 fold excess of essential amino acids, 2.0-fold excess of nonessential amino acids, and a 1.5-fold excess of vitamins (MEMB; Sigma, St. Louis, MO) and 15% (vol/vol) chemically defined fetal bovine serum (FBS; Hyclone, Logan, UT). Cells were routinely seeded at 10’ cells per 60-mm polystyrene culture dish (Corning, Corning, NY) in 4.6 ml of MEMB with 15% (vol/vol) FBS. For cell density experiments, 9.1 X 103, 3.6 X 104, 1.8 x lo’, and 3.5 X 10’ cells/cm’ were seeded on enough dishes to provide 20 million total cells at each seeding density for RNA

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extraction and analysis. For serum-free experiments, cells were isolated in the presence of serum as usual, but seeded in MEMB without FBS. For the matrix experiments, plastic dishes were coated with a fibrin substrate as previously described (Nelson et al., 1990) or with type I collagen (Sigma). Five milligrams of type I collagen was dissolved in 2.5 ml of 0.1% acetic acid in doubly distilled water (vol/vol) at 4°C overnight, diluting this to a final volume of 25 ml with MEMB (0.2 mg collagen/ ml). An aliquot of this solution was dried onto polystyrene dishes in a laminar flow hood under ultraviolet lighting, providing a uniform coating of the plastic surface with 20 gg of type I collagen/cm2. Dishes with or without substrate coatings were rinsed twice with MEMB containing 5% FBS (voVvo1) prior to cell seeding. All cultures were grown in humidified 5% CO, atmosphere at 37°C in a Lunaire incubator (Lunaire Environmental Inc., Williamsport, PA) and were examined by inverted phase microscopy daily. Media were changed daily and media from selected cultures were stored at -20°C until they were assayed for human chorionic gonadotropin (hCG)4 in the clinical chemistry laboratory of the Jewish Hospital of St. Louis using a kit purchased from Baxter Healthcare Corp. (Miami, FL) according to the manufacturer’s instructions. Immuno&orescence

The formation of syncytial trophoblast is a morphologic feature indicative of trophoblastic differentiation in situ (Jones and Fox, 1991). Douglas and King (1990) have described a cytochemical method to identify desmosomes on the surface membranes interfacing cultured trophoblasts. The combination of fluorescent anti-desmosomal staining and fluorescent antinuclear staining allows for the identification of aggregated nuclei in a syncytium or of closely apposed mononucleated cells. We used this double fluorescent staining method to follow morphologic differentiation of trophoblasts in culture. Cells grown 1,3, or 6 days on 35-mm Corning culture dishes (Corning) were rinsed twice with warm MEMB, fixed, and permeabilized in methanol at -20°C for 25 min and rinsed with two changes of phosphate buffered saline (PBS), pH 7.2, for 5 min each. Nonspecific immunoglobulin binding sites were blocked by incubating cells with goat serum (1:50 dilution) for 30 min at 37°C. All antibodies were diluted in 0.2% (wt/vol) BSA containing 0.1% (wt/vol) sodium azide. Excess serum was drained and the cells were incubated with affinity-purified mouse anti-desmosomal antibodies (Sigma) diluted 4 Abbreviations used: hCG, human chorionic gonadotropin, human placental lactogen; SOD, superoxide dismutase.

hPL,

CHURCH, FARMER, AND NELSON

Gene

1:400 (vol/vol) and human antinuclear antibodies (Disease Detection International, Irvine, CA) diluted 1:4 (vol/vol) for 30 min at 37°C. The cells were then rinsed 5 min each with three changes of PBS, the secondary antibodies were added, and the cells were incubated for 35 min at 37°C. FITC-labeled goat anti-mouse IgG diluted 1:lOO (vol/vol) were used to detect anti-desmosomal antibodies while TRITC-labeled goat anti-human IgG diluted 1:200 (vol/vol) were used for detection of nuclear staining. The washing procedure was repeated and the sides of the culture dishes were removed. The remaining bottom portions were mounted using an aqueous, glycerol based mounting medium, and glued to glass slides. The cells were examined using a Zeiss microscope equipped with epifluorescence optics. Photomicrographs of specimens were made from double exposures of Kodak Ektachrome P800/1600 (Kodak, Rochester, NY) with the green and red filters sequentially inserted. RNA Analyses

Total RNA was isolated by the acid guanidinium thiocyanate-phenol-chloroform extraction method described by Belyavsky et al. (1989) with modifications. Using 1 g of dissected placental tissue, crushed under liquid nitrogen, or 2 X lo’cells, homogenates were resuspended in 4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0,0.5% sarkosyl, and 0.1 M2-mercaptoethanol before shearing and sequentially extracted with 0.625 vol of 3 M sodium acetate at pH 5.0, an equal volume of phenol-equilibrated 0.3 M sodium acetate at pH 5.0, and 0.2 vol of chloroform. After centrifugation, the aqueous phase was ethanol precipitated three times and resuspended. RNA blot analyses were performed by size fractionation of 10 or 15 pg of total cellular RNA on a 1% agarose, 200 mM 4-morpholinepropanesulfonic acid, pH 7.4, 1 mM EDTA, and 18% formaldehyde gel. RNA was transferred to nylon (Hybond, Amersham Arlington Heights, IL), uv cross-linked, and hybridized to a2P-labeled antisense Mn SOD RNA transcribed from Sp6 or T7 (Promega, Enc.) or 32P-random-primer-labeled (Pharmacia U G, Uppsala, Sweden) catalase, copperzinc SOD, human growth hormone (hGH), hCG, /3r actin, or mitochondrial medium chain acyl dehydrogenase cDNAs. Mn SOD has been previously isolated and characterized by our laboratory (Church, 1990). The B hCG cDNA probe was the kind gift of Dr. I. Boime (Washington University). Catalase and CuZn SOD cDNAs were obtained from American Tissue Culture Center (Rockville, Maryland). The actin cDNA was obtained from Dr. L. Kedes (Stanford University). The hGH was obtained from Nicholls Diagnostic (San Juan Capistrano, CA) and has 92% nucleotide similarity with hPL and cross-

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hybridizes under stringent conditions (Fitzpatrick et aL, 1988). Mitochondrial medium chain acyl dehydrogenase cDNA was obtained from Dr. D. Kelly (Washington University). Assay for Mn SOD and CuZn SOD Activity

Approximately 2 g of the basal plate and villous tissues were homogenized separately according to the procedure of Salin and McCord (1974) and the supernatant was used for protein determination and for electrophoresis. In addition, 10’ cells for each sample were scraped from dishes into 0.05 M potassium phosphate with 0.1 mM EDTA, at pH 7.4, were collected by centrifugation, and were resuspended in 150 ~1 of the same phosphate buffer at 4°C. Resuspended cells were freeze-thawed three times and centrifuged to remove cell debris, and the supernatants were dialyzed for 24-48 hr against the same phosphate buffer. Protein concentrations were determined by a protein assay kit using bovine serum albumin as standard (Sigma Diagnostics). Samples containing equivalent amounts of total protein (50 1.18)were electrophoresed on the 10% native acrylamide resolving gels of Davis (1964) and were stained for activity as described by Salin and McCord (1974) with modifications. The gels were soaked in solutions prepared in the dark immediately before use. Following removal from the solution, the gels were exposed to light for 1-2 hr. The photogenerated reactive oxygen species reduced the dye to the insoluble purple formazan except at those areas where SODS scavenged the 0, radical, resulting in achromatic bands. The addition of 1 mM potassium cyanide to the staining solution allowed for the detection of the cyanide-insensitive Mn SOD by the inactivation of the CuZn SOD. RESULTS

of Trophoblasts in Culture By phase microscopy, cellular trophoblasts cultured in serum-containing medium on either plastic or fibrin substrates dispersed uniformly and remained rounded for the 3 to 5 hr required for attachment. Cells cultured on type I collagen dispersed uniformly, but clumped during attachment. Cellular trophoblasts in all cultures subsequently flattened and established extensive cellcell contact with cell aggregation over the next 24 hr. A nearly confluent cell layer developed by 72-96 hr in culture at the standard 3.5 X lo6 cells/cm2 or 10’ cells per 60-mm dish. Cellular trophoblasts cultured without serum or matrices flattened, extended long cytoplasmic processes, and formed small aggregates and apparent multinucleated masses by 96 hr in culture. Cells cultured at the lowest seeding density (9.1 x lo3 cells/cm’) were dispersed on the culture dishes with many isolated single cells and small cell aggregates present. Cultures at each of the higher seeding densities exhibited progresMorphological D$krentiation

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FIG. 1. Epifluorescence micrographs of human trophoblast cells cultured 1 day in serum-containing medium (a), or 3 days in serum-free medium (b). Cells were seeded at 350,000 cells/cm’ in MEM in the presence or absence of serum, grown 1 or 3 days, fixed in methanol, and stained sequentially with anti-desmosomal and antinuclear antibodies as described under Materials and Methods. Cells were predominantly mononucleated on Day 1 (a). The fluorescence of desmosomes localized on surface membranes appeared as green lines (e.g., closed arrows) interfacing individual nuclei in separate cells. Parts of the surface membrane of these mononucleated cells not in contact with an adjacent surface membrane (e.g., open arrow) generally did not have staining since desmosomes on free surfaces are infrequent. In cultures grown 3 days with or without serum, multiple nuclei (S) were present within the same cytoplasmic mass without intervening lines of anti-desmosomal fluorescence, indicating differentiation into syncytiotrophoblasts occurred. Other nuclei were isolated or bordered by green fluorescence indicating they were still in mononucleated eytotrophoblasts (C). Bar, 5 pm.

sively larger cell aggregates between 24 and ‘72 hr. Thus, as seeding density was increased, cultures were closer to confluence and presumably had more opportunity for cell-cell interaction. The combined fluorescent staining of cell nuclei and desmosomes on surface membranes allows for the determination of whether adjacent nuclei are in separate cells or are in a syncytium (Douglas and King, 1990). Trophoblast cultures were fixed and stained after 1 (Fig. la) or 3 days (Fig. lb) in culture in the presence or absence of serum. Cells were predominantly mononucleated on Day 1 under all conditions. Green fluorescence of desmosomes stained on surface membranes was at the interface between nuclei in separate cells and outlined parts of the lateral cell borders of the mononucleated cells (Fig. la). Cultures grown 3 days in the presence or absence (Fig. lb) of serum had multiple nuclei within the same cytoplasmic mass without intervening lines of anti-desmosomal fluorescence. This appearance indicated that the nuclei were in a syncytium and that the cultured cells were differentiating into syncytial trophoblasts, even in the absence of serum. Approximately one-half of the nuclei present in cultures at this time were located in multinucleated masses. Biochemical

Ll$erentiation

of Trophoblasts

in Culture

The gene expression patterns of /3 hCG, hPL, and /3r actin were determined in the basal plate or villous tissue

from term placenta, in trophoblasts grown on uncoated plastic in the presence or absence of serum, and on extracellular matrix of type I collagen or fibrin to establish that our cultured trophoblasts were differentiating biochemically (Fig. 2), As can be seen by RNA blot analysis in Fig. 2, p hCG mRNA was not detected in the basal plate, in the villous tissue of human term placenta, in freshly isolated cellular trophoblasts, or in cells grown 24 hr in the presence or absence of serum, type I collagen, or fibrin. However, a marked increase in the level of fl hCG mRNA occurred by Day 3 in culture which was followed by a decrease in 0 hCG mRNA by Day 6 under all culture conditions. As expected, hPL mRNA was abundant in term placental basal plate and villous tissue due to the abundant syncytial trophoblast (Fig. 2). The hPL mRNA was absent from freshly isolated trophoblastic cells and cells grown 24 hr but all cultures had detectable hPL mRNA by Day 3. Abundant hPL mRNA was maintained through Day 6 in trophoblasts cultured on either a substrate of type I collagen with serum-containing media or in serum-free media on an uncoated plastic surface. Trophoblasts grown on a fibrin substrate also expressed hPL mRNA on Days 3 and 6 in culture but in a much lower amount than cells grown on either type I collagen or uncoated plastic. The expression of cytoplasmic actin mRNA was examined to verify that differences in placental gene expression occurring with time in culture were due to changes in

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FIG. 2. Term placental tissue and cultured trophoblast RNA blots. The position of 28 S and 18 S RNAs are shown to the right. The position of designated mRNA populations are highlighted by arrows to the left of each panel. Ten micrograms of total cellular RNA was prepared from basal plate (B) and villous tissue from human term placenta or from freshly isolated trophoblasts (0) or after 1, 3, or 6 days in culture on uncoated plastic in the presence of serum (Plastic), on uncoated plastic in the absence of serum (Serum Free plastic), or in the presence of serum on a matrix of Type I Collagen or Fibrin. The RNA was electrophoresed through agarose, transferred to nylon, and hybridized to the indicated probes. MnSOD: RNA blots hybridized with a MnSOD cRNA probe. CuZn SOD, catalase, p hCG, hPL, and ,6r actin duplicate or RNA blots rehybridized with respective cDNA probes. An artifact of rehybridization resulting in a nonspecific signal is seen with the CuZn cDNA probe in the Day 6 serum-free lane.

growth conditions and not caused by RNA sample variability. Despite equal quantities of RNA in all lanes, cytoplasmic actin (&r) mRNA, present in cells grown on plastic in the presence of serum, was down-regulated between Days 3 and 6 in cells grown on substrates of type I collagen or fibrin (Fig. 2). Trophoblasts grown on plastic also decreased the abundance of ,#r actin mRNA after 3 days in culture if they were cultured in the absence of serum (Fig. 2). These results reflect a selective decrease in trophoblastic cellular content of ,6 y actin mRNA, since on Day 6 in culture, trophoblasts yielding equal amounts of RNA express abundant hPL mRNA. We conclude that /3 y actin mRNA content cannot serve as a control with time in culture because culture conditions also modulate its expression and that changes in cytoskeletal gene expression accompany morphologic differentiation of trophoblasts. At the standard seeding density of 10’cells per 60-mm dish, media hCG levels were low at 12 mIU/ml after 1 day in culture, rose to 287 mIU/ml by Day 3, and to 856 mIU/ml by Day 6, indicating that the cells were actively secreting hCG. All of these results confirm the biochemical differentiation of cellular trophoblasts in vitro and suggest that growth surfaces and the presence or absence of serum are able to modulate the gene expression of placental specific hormones /3 hCG and hPL and cytoplasmic actin.

Eflect of Isolatiolz and Culture of Cellular Trophoblasts Enxgmes on mRNA Levels of Antioxidant Having confirmed that these cells differentiate morphologically and biochemically in vitro, we studied the gene expression patterns of the antioxidant enzymes. A single 4.2-kb transcript of the mitochondrial antioxidant enzyme MnSOD was present in both term placental basal plate and villous tissue total cellular RNAs (Fig. 2). This was compared to the Mn SOD gene expression pattern in purified cellular trophoblasts studied immediately after cell isolation and after 1,3, or 6 days of culture where two different-sized Mn SOD MRNAs were present (Fig. 2, MnSOD). A smaller Mn SOD transcript of 1 kb was markedly induced with the isolation of the cells. Both Mn SOD transcripts were further induced during the first day in culture. By the third day in culture, the smaller molecular weight MnSOD mRNA was reduced in amount and this mRNA was almost undetectable by 6 days in culture. A similar pattern of Mn SOD gene induction was present in trophoblasts plated in the presence or absence of serum on uncoated plastic or on matrices of fibrin or type I collagen (Fig. 2). The levels of other antioxidant defense enzyme mRNAs during trophoblast isolation and differentiation were examined in order to study the specificity of Mn SOD induction. Term human placental basal plate and villous tissue

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hours

0

3

I I 18 25

,l8S

Catalase : Pyactin

FIG. 3. RNA blot analysis of Mn SOD, catalase, and & actin in trophoblasts cultured over 25 hr. The positions of 28 S and 18 S RNAs are depicted to the right. The positions of Mn SOD, catalase, and 07 actin are depicted by arrows. Ten micrograms of total cellular RNA was prepared from trophoblasts at the time of isolation (0), or after 3, 11,18, or 25 hr in culture on uncoated plastic in the presence of serum. This RNA was electrophoresed through agarose, transferred to nylon, and hybridized to a Mn SOD cRNA. The blot was then rehybridized to cDNA probes of catalase and P-y actin. A photograph of the ethidiumstained gel depicts RNA loading.

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duction. The mitochondrial medium chain acyl dehydrogenase mRNA remained constitutively expressed (data not shown) in contrast to the marked induction of Mn SOD mRNAs, suggesting that not all mitochondrial enzyme mRNAs are altered by trophoblastic isolation or differentiation. The cell density dependence of Mn SOD mRNA expression is illustrated by Fig. 4. While Mn SOD mRNAs remained induced in freshly isolated cellular trophoblasts (Fig. 4, Day 0), their abundance after 1 day in culture was markedly reduced when cellular trophoblasts were plated at 3.5 X lo4 cells/cm’ (Fig. 4, Day 1, 4X) and increased further in trophoblasts after 1 day in culture at the usual plating density of 3.5 x 105 cells/cm2 or lo7 cells per 60-mm dish (Fig. 4, Day 140X). The effect of trophoblast plating density on @hCG mRNA expression was also examined (Fig. 4). There was no detectable fi hCG mRNA in freshly isolated cellular trophoblast or trophoblast cultured 24 hr (Fig. 4, Day 0 and Day 1,4x and 40X). With the lowest seeding density of 9.1 X lo3 cells/cm2, there was minimal detectable p hCG mRNA after 3 days in culture (Fig. 4, Day 3,1X). However, with each of the three higher seeding densities there was a

day

0

density have detectable mRNAs for catalase and CuZn SOD (Fig. 2). Both the 500 and 700 base CuZn mRNAs were present. Neither were affected by the process of isolation and both CuZn SOD mRNAs remained in equal abundance with time in each culture condition. The 2.4kb catalase mRNA remained detectable after cellular trophoblast isolation, but this mRNA was barely detectable in cells during the first day in culture. Catalase mRNA abundance was not affected by the growth conditions, remaining undetectable after 24 h of culture in the presence or absence of serum or on matrices of fibrin or type I collagen. A time course for Mn SOD mRNA induction (Fig. 3) showed that following the initial induction of Mn SOD mRNA with cellular isolation, a further increase in Mn SOD RNA abundance occurs between 11 and 18 hr in culture. This contrasts with the lack of induction and decreasing abundance of catalase mRNA and with the steady-state levels of cytoplasmic actin mRNA. Trophoblastic differentiation changes mitochondrial morphology (Kliman et aL, 1987) and Mn SOD is a nuclear-encoded, mitochondrial enzyme. We examined the expression pattern of mitochondrial medium chain acyl dehydrogenase, another nuclear-encoded mitochondrial enzyme to verify the specificity of Mn SOD mRNA in-

I 4x 40x

3 Ix 4x 20x 40x -28s

MnSOD

PhCG

-18s

)

FIG. 4. RNA blot analysis of Mn SOD and fl hCG expression in the trophoblasts cultured at different seeding densities. The positions of 28 S and 18 S are shown to the right. The positions of Mn SOD, hCG, and 4 S RNAs are depicted by arrows. Ten micrograms of total cellular RNA was prepared from trophoblasts at the time of isolation (Day 0), from trophoblasts on uncoated plastic in the presence of serum after 1 day in culture at a plating density of 3.6 X 10’ cells/cm2 (Day 1,4X), or 3.5 X 10 cells/cm2 (Day 1,40X), or from trophoblasts after 3 days in culture at a plating density of 9.1 X l@ cells/cm2 (Day 3,1X), 3.6 X 10' cells/cm’ (Day 3, 4X), 1.8 x lo5 cells/cm’ (Day 3, 20X), and 3.5 X 106 cells/cm2 (Day 3, 40X). The RNA was electrophoresed through agarose, transferred to nylon, and hybridized to a Mn SOD cRNA probe. A duplicate blot was hybridized to a @hCG cDNA probe and cross hybridization with 4 S is included to illustrate RNA loading.

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(Fig. 5), paralleling the constitutive mRNA expression pattern of this enzyme. The CuZn SOD enzyme activity was minimally influenced by the cell isolation procedure or the differentiation of cellular trophoblast in culture. A comparison of Figs. 2 and 5 suggests that the induction of the activity of Mn SOD enzyme follows induction of the Mn SOD mRNAs and that the presence of a smaller Mn SOD transcript is temporally related to the increase in Mn SOD enzyme activity. DISCUSSION

FIG. 5. Results of polyacrylamide gel electrophoresis stained for superoxide dismutase activity. Electrophoresis and staining procedures were performed as described under Materials and Methods. Gels labeled Tissue (B for basal plate and V for villous tissue of term human placenta) received 50 pg of total protein. Gels received 50 pg of total protein derived from trophoblasts cultured 1,3, or 6 days in the presence of serum on uncoated plastic. The gels designated + were stained in the presence of 1 mM potassium cyanide.

progressive increase in the amount of fl BCG mRNA (Fig. 4, Day 3 4x, 20x, 40X). The differences in Mn SOD and /3 hCG mRNA levels contrast with trophoblastic 4 S ribosomal RNA content which does not vary with time in culture or with changes in plating densities, reflecting equal RNA content in all lanes. These results suggest that induction of Mn SOD mRNAs precedes or is coordinately regulated with morphological and biochemical trophoblastic differentiation and that either cellcell interaction or a component concentrated in the media of densely plated cellular trophoblasts is associated with maximal expression of the Mn SOD gene and with maximal expression of p hCG mRNA in this differentiating cell system. Eflect of Isolation and Culture of Cellulur on Antioxidant Enzyme Activity

Trophdast

The selective induction of Mn SOD mRNAs was compared to the enzyme activity of the Mn SOD enzyme. An equal amount of protein was taken from term placental basal plate, term villous tissue, and cultured trophoblasts. Each was subjected to native gel electrophoresis and activity staining. There was little detectable Mn SOD enzyme activity in the basal plate or villous tissue of human term placenta (Fig. 5). In contrast, marked Mn SOD activity was present in trophoblast cells cultured 1, 3, or 6 days. The cyanide sensitive CuZn-containing SOD enzyme activity was present in basal and villous tissue

In this report we have demonstrated that Mn SOD mRNAs are transiently induced during the culture of human trophoblasts and that this induction is dependent on cellular trophoblast plating density. Induction of Mn SOD is specific in contrast to the mRNAs for other antioxidant or mitochondrial enzymes which are expressed in term placental tissue but are not induced during the isolation or culture of human trophoblasts. The Mn SOD mRNA induction and the increase in enzyme activity both occur while the cells are predominantly mononucleated and precede morphological and biochemical evidence of syncytial formation. In the present investigation, mRNA analysis of placental specific hormones demonstrates that differentiating cultured trophoblasts mimic on a molecular level the gene expression patterns of proliferating cellular and fusing syncytial trophoblasts in vivo as described by Otani et aZ. (1988). Previous studies have shown that trophoblast morphology and activity are affected by different growth conditions. Culture of trophoblasts on fibrin decreased trophoblast proliferation (D. R. Farmer and D. M. Nelson, unpublished data) and facilitated trophoblast differentiation (Nelson et aL, 1990). Increased thickness of a matrigel substrate altered trophoblast morphology, extracellular matrix proteolysis, and invasion (Kliman and Feinberg, 1990). Douglas and King (1990) recently demonstrated that morphological and biochemical differentiation were induced during culture of trophoblasts in keratinocyte media. Our results demonstrate that in contrast to the modulation of placental specific and cytoplasmic actin mRNA levels by fibrin and type I collagen substrates, antioxidant enzyme mRNA expression patterns are unaffected. We also found that trophoblast differentiate morphologically and biochemically in serum-free medium without a change in gene expression of the antioxidant enzymes. These results suggest that changes in antioxidant mRNA levels during differentiation of trophoblasts are not mediated nor influenced by epithelial-matrix interactions or the presence of serum. Our results demonstrate that the increase in abundance of a smaller Mn SOD is accompanied by an in-

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crease in Mn SOD enzyme activity. The Mn SOD mRNAs are distinguished by different alternatively spliced 3’-untranslated regions (Church, 1990). Recent evidence that structural elements in the 3’-untranslated regions of several mRNAs regulate translational efficiency (Kruys et aL, 1989) makes this temporal relationship between Mn SOD message selection and increased enzyme activity of interest and we are currently investigating the potential role of Mn SOD 3’untranslated region selection and enzyme availability. In this report we have found that Mn SOD gene expression and its post-transcriptional processing either precede or are coordinately regulated with the process of trophoblastic differentiation. There are many examples where cellular transformation is associated with a loss in SOD activity (Allen, 1991; Cerutti, 1985; Oberley, 1983) and where the exogenous administration of SOD to tumor cells including Friend erythroleukemia (Beckman et aL, 1989) and neuroblastoma (Oberley, 1983) and a mutant slime mold induce cellular differentiation (Allen et al, 1988). Increases in total SOD activity are observed during in vitro differentiation of human monocytes (Nakagawara et aL, 1981) and induction of Mn SOD activity occurs with the differentiation of the normal P. polycephalum slime mold (Allen et ab, 1988). Our data temporally associate the selective induction of Mn SOD with the differentiation of nonmalignant human primary trophoblast cells; however, further definitive experiments will be required to understand the mechanism of Mn SOD induction and whether or not this induction is linked or necessary for trophoblastic differentiation. We thank Drs. H. R. Colten and J. W. Grant for their many helpful discussions and advice and Drs. A. W. Strauss and D. Perlmutter for their critical review of the manuscript. REFERENCES Allen, R. G. (1991). Oxygen-reactive species and antioxidant responses during development: The metabolic paradox of cellular differentiation. Proe. Sot. Exp. Biol. Med. 196,117129. Allen, R. G., Balin, A. K., Reimer, R. J., Sohal, R. S., and Nations, C. (1988). Superoxide dismutase induces differentiation in the slime mold. Physarum polycephalum. Arch B&hem Biophys. 261, 205211. Beckman, B. S., Balm, A. K., and Allen, R. G. (1989) Superoxide dismutase induces differentiation in Friend erythroleukemia cells. J. Cell Physiol 139,370-376. Belyavsky, A., Vinogradova, T., and Rajewsky, K. (1989). PCR-based cDNA library construction: General cDNA libraries at the level of a few cells. Nucleic Acids Res. 17,2920-2932. Cerutti, P. A. (1985). Prooxidant states and tumor promotion. Science 22,375-381. Church, S. L. (1990). Manganese superoxide dismutase: Nucleotide and deduced amino acid sequence of a cDNA encoding a new human transcript. Biochim. Biophys. Acta 1087,250-252.

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