Mol. Hum. Reprod. Advance Access originally published online on April 5, 2006
Molecular Human Reproduction 2006 12(5):321-333; doi:10.1093/molehr/gal036
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9-Tetrahydrocannabinol inhibits cytotrophoblast cell proliferation and modulates gene transcription
1Clinical Division of Obstetrics & Gynaecology, Leicester Royal Infirmary, University Hospitals of Leicester NHS Trust and 2Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, Leicester Medical School, University of Leicester, Leicester, UK
3 To whom correspondence should be addressed at: Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary, P.O. Box 65, Leicester, Leicestershire, LE2 7LX, UK. E-mail: aht3{at}le.ac.uk
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Abstract |
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Cannabis use in pregnancy is associated with a range of obstetrical conditions. The molecular mechanisms underlying these effects have not been elucidated but are attributed to the actions of delta-9-tetrahydrocannabinol (
9-THC). In this study, concentrations of 9-THC equivalent to those found in the serum of cannabis users, i.e. 
20 µM, inhibited proliferation and activated a restricted tight transcriptional programme in the BeWo trophoblast cell line. Employing genome-wide expression profiling methods, we found that the pattern of gene expression differs from that described in the placenta of patients with fetal growth restriction (FGR), associated with either hypoxia or discordant dichorionic twins, or of patients with pre-eclampsia. It was also dissimilar to the patterns obtained from the transcriptome of other tissues, such as the mouse brain, treated with 9-THC. The expression of transcription factors, such as thyroid hormone receptor-ß1 (TRß1), and transcriptional co-repressors, such as histone deactylase 3 (HDAC3), was affected by 9-THC in a dose-dependent manner, whereby 15 µM 9-THC caused a 2.8-fold inhibition of TRß1 expression, but a 3.5-fold increase in HDAC3 expression. These data were confirmed by end-point RT–PCR analyses and underpin the observed 9-THC-induced inhibition of BeWo cell proliferation. Genes encoding for growth, apoptosis, cell morphology and ion exchange pathways were modulated by 15 µM 9-THC. This study may provide insight into the mechanisms underlying the effects of 9-THC and cannabis use upon placental development during pregnancy. Key words: cannabis/cytotrophoblast/marijuana/microarray/tetrahydrocannabinol
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Introduction |
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Marijuana is one of the most commonly used illicit drugs in the UK and USA (Hutchings and Dow-Edwards, 1991
; Parliamentary Office of Science and Technology, 1996), and its use appears to be increasing (Knight et al., 1994; King, 1997). It is difficult to quantify the risks of marijuana use in pregnancy, as there are likely to be many confounding variables such as the use of other drugs and associated lifestyle factors. However, marijuana use has been associated with low birthweight, fetal growth restriction (FGR) (Zuckerman et al., 1989), placental abruption, preterm birth, stillbirths and spontaneous miscarriages (Conner, 1984; Hatch and Bracken, 1986; Felder and Glass, 1998).
The most psychoactive agent in marijuana is delta-9-tetrahydrocannabinol (9-THC). 9-THC is highly lipophilic (Leuschner et al., 1986) and has a half-life of about 8 days in fat deposits, and consequently, it may take up to 1 month for it to be eliminated entirely from the body after a single dose (Hatch and Bracken, 1986). In fact, following inhalation from a single marijuana cigarette, blood levels of 9-THC may remain detectable for up to 30 days (Jones, 1980). These pharmacokinetic characteristics pose a particular risk to the fetus, because maternal tissues may act as a reservoir for 9-THC (Hatch and Bracken, 1986; Leuschner et al., 1986); 9-THC readily crosses the placenta (Hatch and Bracken, 1986), and this, combined with a slow fetal clearance, (Bailey et al., 1987) means prolonged fetal exposure, even when smoking is discontinued. Therefore, 9-THC potentially influences the development of many organs, (Harclerode, 1980) especially in the first trimester.
9-THC has been found to have several cellular-binding sites (Felder and Glass, 1998), including one on DNA (Porcella et al., 1998), and can bind to the G-coupled endocannabinoid receptors CB1 and CB2. The endometrium and myometrium possess the cannabinoid receptors CB1 and CB2 and thus are potential targets for the action of 9-THC during implantation, early pregnancy (Paria et al., 2001) and labour (Dennedy et al., 2004). However, cannabinoid receptors are also expressed by placental tissue at term (Park et al., 2003), and although when they are first acquired during pregnancy is unknown, expression has been detected in early first trimester placental tissues (Helliwell et al., 2004). Interestingly, levels of the endogenous cannabinoid, anandamide, fall progressively during pregnancy (Habayeb et al., 2004), supporting other evidence that low systemic levels are required for normal pregnancy progression (Maccarrone et al., 2002). Therefore, exposure to the exocannabinoid 9-THC could lead to inappropriate activation of the CB-mediated pathways in the placental trophoblast. At least one action of 9-THC upon placental transport, i.e. mediated by the serotonin receptor and of 
-amino isobutyric acid, has been proposed to be through the CB1 receptor (Fisher et al., 1987; Kenney et al., 1999).
The BeWo cell line, derived from human gestational choriocarcinoma, has been widely used as an in vitro model for trophoblast intercellular fusion and differentiation (Burres and Cass, 1986; Hohn et al., 1992; Cohran et al., 1996; Kudo et al., 2004) and in toxicology studies (Burres and Cass, 1987; Thibault et al., 2000; Nomura et al., 2004). Additionally, these cells have been used in genome-wide analyses (Aronow et al., 2001; Saito et al., 2001; Endo et al., 2002; Kudo et al., 2004) that have identified putative novel targets for the mechanism of FGR that were subsequently confirmed in animal studies to be of significant experimental and clinical value (Loiselle et al., 2004; Nomura et al., 2004; Ohara et al., 2004). The BeWo cell line thus provides an appropriate model in which to study some aspects of human trophoblast physiology and pathophysiology without the aspect of inter-patient variability and other confounding variables. In this study, we have therefore employed this cell line to determine whether 9-THC would specifically affect cytotrophoblast cell survival and gene transcription and whether extrapolation of such effects may be used to explain the reported in-vivo effects of 9-THC upon clinical conditions affecting feto-placental development.
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Materials and methods |
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Materials
The human choriocarcinoma cell line BeWo (ECACC 86082803, European Collection of Cell Cultures, Salisbury, Wiltshire, UK) was maintained in Ham’s F12 medium (Invitrogen, Paisley, UK) supplemented with 10% fetal calf serum (FCS) (Invitrogen) and cultured at 37°C in humidified atmosphere of 5% CO2 in air.
9-THC (Sigma-Aldrich, Poole, Dorset, UK) was diluted in ethanol and stored under nitrogen at –20°C. Oligonucleotide primers were synthesized (Sigma-Genosys Ltd., Haverhill, Suffolk, UK) and desalted before reconstitution in sterile dH2O at 200 pmol/µl and stored at –20°C.
Cell culture
BeWo cells were plated into Nunc 6-well multi-well plates in triplicate for each data point (Fisher Scientific, Loughborough, Leicestershire, UK) at a density of 8 x 105 cells per well (Nomura et al., 2004). After 48 h culture, the medium was exchanged with one that contained 5% FCS and 15 µM 9-THC [final concentration of 0.1%(v/v) ethanol]. The control consisted of culture medium containing 0.1%(v/v) ethanol. Cells were cultured with media plus additives for 1 h to allow non-specific binding of 9-THC to plastic to reach equilibrium. The medium was then replaced and culture continued for 48 h, with fresh medium replaced at 24 h. Photomicrographs were obtained using a Nikon Eclipse TE2000-U inverted microscope equipped with a DN-100 digital camera image capture system, and percentage confluency was measured by image analysis of pixels representing cells and substratum.
To examine the effect of 9-THC on cell survival and cell proliferation indices, we plated BeWo cells to Nunc 96-well plates (Fisher Scientific) at either 4 x 104 or 1 x 104 cells per well, respectively, in 200 µl of normal growth medium and allowed them to proliferate for 48 h until the higher density cultures reached confluency. The medium was then changed to one that contained 0–30 µM 9-THC for 1 h to allow for non-specific binding. The medium was then replaced and culture continued for 48 h, with fresh medium replaced at 24 h. After 48 h, cell numbers were assessed using the Cell Proliferation and Apoptosis Kit II (Roche Diagnostics Ltd., Lewes, East Sussex, UK) as per the manufacturer’s instructions with measurements taken on a Multiskan Ascent ELISA plate reader (Labsystems Oy, Helsinki, Finland) with the detection filter set at 420 nm and the reference set at 620 nm. Cell numbers were obtained by calibration against a standard curve of untreated BeWo cell numbers grown in parallel (Taylor et al., 2002). To make direct comparisons between cultures, we then converted the cell numbers to a percentage of the untreated control.
The effect of 9-THC dose on gene expression was obtained by culturing BeWo cells with doses of 9-THC ranging from 0.3 to 30 µM in 0.1% ethanol for 48 h, as described above.
Total RNA extraction, cRNA synthesis and microarray hybridization
Total RNA was extracted using a combination of TRIZOL reagent (Invitrogen), RNeasy mini kit (Qiagen Ltd., Crawley, UK) and ethanol precipitation. Briefly, aqueous total RNA from the TRIZOL reagent procedure was mixed with ethanol applied to the RNeasy mini columns, subsequently purified and concentrated through ethanol precipitation. RNA integrity was assessed using agarose gel electrophoresis and Agilent 2100 Bioanalyser (Agilent Technologies, UK, South Queensferry, UK). RNA from two separate experiments performed in triplicate was pooled and samples submitted to the MRC Geneservice (Babraham, Cambridge, UK) for further purification and production of biotinylated cRNA. Biotinylated cRNA was prepared from 10 µg of total RNA according to Affymetrix protocols. The integrity of the labelled cRNA and fragmentation products were assessed on the Agilent 2100 bioanalyser. Next, 15 µg of biotinylated cRNA fragments were hybridized to human HU133_plus 2 microarray chips overnight and then stringently washed, stained and scanned using a GeneArray scanner (Agilent Technologies, Palo Alto, CA, USA), according to Affymetrix protocols.
Gene expression analysis
Fluorescence data were corrected for background fluorescence, reduced in intensity values before normalization against internal standards. All arrays were scaled to the same target intensity and were normalized to housekeeping genes on the U133_plus 2 arrays and analysed using dChip Analyzer software version 1.4 (Harvard School of Public Health and Dana-Farber Cancer Research Institute, Boston, MA, USA, 2004; http://biosun1.harvard.edu/complab/dchip/) using a 2-fold change in expression with an of 0.90 and a ß of 0.05 being the statistical cut-off points for real expression changes from duplicate chips. Hierarchical clustering was used to obtain gene expression patterns and ontology information.
End-point RT–PCR
To confirm the data from the microarray experiment, we performed end-point RT–PCR upon the samples used in these experiments. One microgram of total RNA was reversed transcribed with avian myeloblastosis virus-reverse transcriptase (AMV-RT; Promega, Southampton, UK) at 42°C for 1 h in the presence of 5 units of RNase inhibitor (RNasin; Promega). A minus RT reaction was obtained by omitting the AMV-RT enzyme. At the end of the reaction, the enzymes were denatured by heating at 95°C for 5 min and the cDNA was stored at –20°C. One microlitre of cDNA was subject to PCR using 10 pmol/µl of specific primers for histone deactylase 3 (HDAC3), thyroid hormone receptor-ß1 (TRß1) and glyceraldehyde-3-phosphatedehydrogenase (GAPDH) using the annealing temperatures given in Table I. The RNA from the dose-ranging study was treated with RNAse-free DNAse 1 (Promega) before reverse transcription and PCR amplification. PCR products were resolved on 3% agarose gels and stained with ethidium bromide (2 µg/ml; ICN Biomedicals, Basingstoke, UK) for 15 min, before being destained with dH2O for 30 min. Gel images were captured using a Syngene GeneGenius system (Syngene, Cambridge, UK) equipped with GeneSnap version 6 gel documentation software. Product densities were assessed using the Scion Image beta version 4.0.2 software (Scion Corporation, Frederick, MD, USA, 1999; http://www.scioncorp.com).
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Data analysis
Subtraction of the densitometry values obtained from the –RT lanes from those from the +RT lanes, and then division by the relative GAPDH transcript levels, was used to correct the PCR data. These data were then normalized to the untreated control and expressed relative to the untreated controls. All data were then analysed for differences using one-way ANOVA with Tukey’s honest significant difference (HSD) test with the InStat version 3.0 software package (GraphPad Software, San Diego, CA, USA, 1998; http://www.graphpad.com). Statistical significance was accepted when P < 0.05.
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Results |
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Effect of
9-THC on gross BeWo cell growth, survival and morphologyBeWo cells were plated such that cultures achieved approximately 70–80% confluency after 48 h, the point at which
9-THC treatments were initiated. Confluency was achieved within 24 h of the subsequent 48 h culture period (data not shown). Cultures treated at 48 h with a range of 9-THC demonstrated an inhibitory effect on the BeWo cell cultures (Figure 1A and B), but only at concentrations in excess of 3 µM 9-THC, where confluency was significantly reduced from 95 to 70% (*P < 0.05; one-way ANOVA with Tukey’s HSD test; n = 4). Cultures treated with 15 and 30 µM 9-THC did not reach confluence (Figure 1A and B). There did not appear to be any increased cell death or failure to attach to the substratum as evidenced by the lack of increase of shedding of cells in the spent medium. Analysis of the effect of 9-THC on BeWo cell survival and proliferation revealed that cultures that reached confluency before treatment remained stable (1.32 ± 0.065 x 105 compared with 1.22 ± 0.063 x 105 cells per well; P = 0.37, n = 8 ANOVA with Tukey’s HSD test) and confirmed the observation that the effect of 9-THC on BeWo cultures was not due to increased cell death. Sub-confluent cultures treated with 9-THC demonstrated a significant dose-dependent inhibition of cell numbers at concentrations above 3 µM 9-THC after 48 h (Figure 1C). These cultures failed to exceed the 30% confluency level (the untreated cultures reached a cell density of 3.7 ± 0.31 x 104 cells per well from an initial density of 1 x 104 cells per well). However, 15 µM 9-THC inhibited cell proliferation so that the final cell density was 2.25 ± 0.19 x 104 cells per well after 9-THC treatment (P < 0.001; n = 8; ANOVA with Tukey’s HSD test). Further morphological examination of cell cultures treated with 15 µM 9-THC indicated that the inhibitory effect resulted from a decrease in growth rather than involution of the culture after achieving confluency (Figure 2).
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Microarray analysis: identification of 9-THC-regulated transcripts
The use of microarray analysis facilitates the global assessment of transcriptional profiles of cells. A number of studies have established the very high degree of reproducibility across replicate GeneChip experiments (Irizarry et al., 2003; Cope et al., 2004). In this study, to ensure consistent data, we adopted both biological (experiments performed four times in triplicate) and technical replicates (hybridizations performed twice) for these studies, whereas others adopt either biological (Hoffman et al., 2004) or technical replicates (Kudo et al., 2004), but seldom both (Kothapalli et al., 2002). Therefore, total RNA from two sample pools of the control and 15 µM 9-THC-treated cultures (n = 12) were used to generate biotinylated target cRNA and hybridized to HU133A_Plus 2 arrays, which represented approximately 56 000 characterized transcripts and expressed sequence tags (ESTs). Scaling factors, noise and percentage of present and absent calls showed only minor variations between arrays, an essential requirement when comparing multiple arrays. These replicate gene chip experiments revealed an overall correlation (r2 = 0.89) with an average of only 0.04% of probe sets showing more than a two-fold change between the replicate experiments. The number of genes induced and repressed on microarray 1 at the two-fold level were 304 and 105, respectively, whereas the number induced and repressed on microarray 2 were 220 and 50, respectively, representing a mean error measurement rate of only 0.5%. When the cut-off was reduced to 1.6-fold, the number of genes induced and repressed on microarray 1 were 1504 and 1127, respectively, whereas the number induced and repressed on microarray 2 were 2291 and 5670, respectively, representing a mean error measurement rate of 26.3%. Under these conditions, it was considered that 26.3% error lacked precision for detailed transcriptome analysis and the conventional two-fold cut-off point was used (Cope et al., 2004). Focusing on genes showing an increased level of expression on both arrays and using a two-fold change cut-off, we identified 134 genes of which the top 20 genes are mentioned in Table II. Focusing on genes showing a decreased level of expression on both arrays with a two-fold (50%) change cut-off, we identified only 18 genes (Table II).
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Confirmation of microarray data
To validate the microarray analysis data, we chose two candidate genes, histone deacetylase 3 (also known as MALAT-1; HDAC3) and TRß1, for further study as they were represented by several probe sets on the HU133_plus 2 microarray chip and were significantly modulated more than two-fold and <50% respectively on both arrays. TRß1 was of significant interest because it is associated with placental function and development (Kilby et al., 1998; Yen, 2001; Barber et al., 2005), and direct perturbation of the maternal hypothalamic–pituitary–thyroid–placental axis is a direct cause of multiple obstetrical problems (Nickel and Cattini, 1991; Ohara et al., 2004). HDAC3 is of potential interest because it is associated with transcriptional regulation, cell cycle progression and developmental events, and perturbation of its activity is potentially linked with placental development (Jho et al., 2005).
The cycle numbers for each set of primers were obtained empirically with an untreated BeWo cell extract (Figure 3), and subsequent reactions were performed at 35, 34 and 26 cycles for HDAC3, TRß1 and GAPDH, respectively, with the extension step for HDAC3 and TRß1 starting at 1 min at 72°C and increasing by 5 s/cycle. End-point RT–PCR with the pooled RNA extracts from the control and 15 µM 9-THC-treated BeWo cells showed a 2.34-fold change in HDAC3 transcript levels from experiment 1 and 2.85-fold change from experiment 2 (Figure 4A); the mean was 2.58-fold (Figure 4B). By contrast, the mean fold change for HDAC3 on microarray 1 was 6.51-fold and on microarray 2 was 2.45-fold with a median fold change of 3.47-fold (Table II). The levels of transcripts for TRß1 (Lazar, 1993), found by combining the levels for 5'-mRNA variants ABCF (short form; 168 bp) and variants DE (long form; 314 bp) (Mannavola et al., 2004), were shown to be decreased by 4.61-fold (78.3%) in experiment 1 and by 3.36-fold (70.2%) in experiment 2 (Figure 4C); the mean was 3.90-fold (74.3%), which compared favourably with the 5.28-fold (81.1%) and 2.98-fold (66.5%) reductions in the two microarrays, respectively. The median change in TRß1 levels was 2.79-fold (64.1%; Table II). Variant G (Frankton et al., 2004) was not observed in any BeWo extract.
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Effect of 9-THC dose on HDAC3 and TRß1 mRNA levels
Treatment of BeWo cells with 9-THC did not show a dose-dependent increase on HDAC3 mRNA expression, but a threshold effect at 3 µM (Figure 5). At this dose, HDAC3 mRNA levels increased by 1.55-fold and reached a maximum of 1.70-fold change at 15 µM (Figure 5). By contrast, there was a dose-like effect on the repression of TRß1 that only reached a significant 75.3% decrease at 15 µM 9-THC (Figure 5) that was not further suppressed by 30 µM 9-THC.
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Grouping of genes on the microarray by function
K-means and hierarchical clustering of the 134 up-regulated and 18 down-regulated genes identified by the microarray analysis identified nine major groupings: growth and apoptosis genes; cell morphology and contraction genes; transcription factors; transcription regulation genes; RNA processing and translation genes; protein trafficking genes; ion channels and transducers genes; lipid metabolism genes; and hypothetical proteins and EST genes. These groups are identified where possible in Table III. Because morphologically the BeWo cell cultures appeared to be affected by either failure to proliferate, or increased apoptosis, or contraction of cultures, and because the microarray is a measure of the transcriptional activity of the BeWo treated cultures, analyses of the modulation of transcripts implicated in these pathways were performed.
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The analyses revealed that several genes implicated in the control of cell cycle progression were affected by 48 h 9-THC treatment. These included insulin-like growth 1 receptor, which has been implicated in cancer cell survival through p53-dependent pathways (Girnita et al., 2003), cyclin-dependent kinase 11, which associates with cyclin type L and initiates pre-mRNA splicing events (Hu et al., 2003), and HDAC1-associated repressor protein (also known as SMART/Msx1/SHARP/SMART/SPEN), which is involved in zebrafish neurogenesis (Cunliffe, 2004) and murine neuromusculoskeletal development (Ishii et al., 2003). By contrast, transcripts that are involved with apoptosis tended to be pro-apoptopic rather than anti-apoptopic, e.g. death-associated protein kinase (DAPK)-interacting protein 1 (Dip1), which antagonizes the anti-apoptotic function of DAPK in Hela cells to promote caspase-dependent apoptosis (Jin et al., 2002) and cell division and apoptosis regulator 1 (CARP-1), which promotes retinoid-stimulated cell apoptosis (Rishi et al., 2003) (Table III).
Although the major cell contraction genes actin and myosin were not regulated by 9-THC, non-muscle myosin (heavy polypeptide 10) and protein phosphatase 1, which are predicted to function in a similar manner to myosin, were both up-regulated (Table III), as were several genes that regulate cell morphology by providing or destroying cytoskeletal frameworks, e.g. plectin (trophoblast-derived non-coding RNA/Hemidesmosomal protein 1 intermediate filament-binding protein 500 kDa) (Uitto and Pulkkinen, 1996), whereas intersectin 2 and dynamin-binding protein (McGavin et al., 2001) were down-regulated.
The main effect of 9-THC was to regulate transcription factors and regulators of transcription with 50 up-regulated and five down-regulated genes (Table III). Transcripts for the ‘orphan’ nuclear receptors (NR1D2/EAR-1R/Rev-erb-beta/RVR and Nor-1) and the ß-isoform of the TR that interact with receptors for retinoic acid to control gene transcription (Wolf, 2002) were up-regulated as were several transcriptional co-regulators, such as histone deacetylase 3 (MALAT-1/HDAC3/PRO1073) that interacts with TRs and other nuclear receptors to enhance transcriptional activity and interacts with Phox2 to regulate the dopamine ß hydroxylase promoter (Xu et al., 2003). Ribosomal subunit expression was also represented on the microarray chips and several of these genes such as U3 small nucleolar interacting protein 2 and RNA motif-binding protein 25 (Table III) were regulated. Additionally, the transcripts for several ‘initiation of translation’ proteins were increased.
A number of other transcripts for genes involved in trophoblast function were noted to be affected by 9-THC treatment, e.g. solute carrier family 40 also known as ferroportin 1 (Wallace et al., 2002), which is involved in iron transport and solute carrier family 4 member 7 (SLC4A7) which is involved in bicarbonate and small anion transport (Loiselle et al., 2004). The most down-regulated membrane protein was the orphan G-protein coupled receptor family-related retinoic acid-induced protein, RAIG3/GPRC5C (Robbins et al., 2000), that may be involved in calcium sensing (Brauner-Osborne et al., 2001).
Interestingly, an enzyme involved in the synthesis of the endocannabinoid, anandamide, N-acyl-phosphatidylethanolamine-hydrolyzing Phospholipase D (NAPE-PLD; Habayeb et al. 2002) and an uncharacterized lipoprotein lipase, associated with fatty acid transport (Garnica and Chan, 1996), were both down-regulated.
The microarray analysis also revealed the regulation of several transcripts for which the predicted protein has no known function (Table III).
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Discussion |
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Employing concentrations of
9-THC that are detected in patients using cannabis for recreational use (Cherlet and Scott, 2002), we have demonstrated that 3–30 µM 9-THC inhibited BeWo cell proliferation in a dose-dependent manner. Conversely, the same doses of 9-THC showed no significant effect on confluent cell cultures indicating no toxic or pro-apoptopic effect of 9-THC upon BeWo cells. Through microarray analysis, we have also demonstrated distinct changes in the pattern of transcription by the BeWo cells in response to 15 µM 9-THC. However, only a relatively low number of transcripts were consistently modulated that suggested a tight transcriptional control be manifested in the 9-THC-treated BeWo cultures. The reason for this is not readily apparent but may be part-way due to the conventional two-fold cut-off point used in microarray analyses. However, although ‘raising the bar’ to 1.6-fold increased the number of transcripts that were regulated by 9-THC in the BeWo cell, an unacceptable significant increase in the amount of experimental variability from 0.5 to 26.3% was also introduced which would lead to false-positive identification of non-regulated transcripts. These data mean that several transcripts that might be considered relevant could have been omitted from these analyses in an attempt to obtain precision and identify the most likely mediators involved in 9-THC-induced BeWo cell-growth inhibition. The tight regulation of the transcriptome is not limited to this study, as similar tight transcriptional control mechanisms have been demonstrated in neurons undergoing apoptosis that do not modulate ‘classical’ apoptosis proteins (Desagher et al., 2005).
The effects of 9-THC upon BeWo cells also appeared to be cell specific in that although expression of CPNE6 (copine VI), the lipid metabolizing, neuronal form of the copine vesicle transport family of calcium-sensing proteins, and LlGI 1, a cytoskeletal protein that associates with non-muscle myosin II heavy chain to control asymmetrical cellular polarization in neuroblasts (Klezovitch et al., 2004), was repressed in both BeWo cells and the brain of 9-THC-treated mice (Parmentier-Batteur et al., 2002), no overlap in the expression pattern of other genes was noted. The altered transcripts belonged to seven functional groups with the largest number being transcriptional regulators, such as TRß1, their co-regulators, such as HDAC3 (Table III), suggesting that one of the major effects of 9-THC on the BeWo cell is the regulation of gene transcription and RNA processing.
As cannabis use during pregnancy is associated with FGR, the present microarray data were compared with such data derived from placental tissue obtained from conditions associated with FGR. Interestingly, expressions of genes that were altered in placental tissue from hypoxia-induced FGR, such as adipophilin and vascular endothelial growth factor (VEGF) (Roh et al., 2005), or from discordant dichorionic twins-associated FGR, (Endo et al., 2002) such as SMAD4 and CDC46 (Endo et al., 2002), were not altered in 9-THC-stimulated BeWo cells. Similarly, many of the changes found in our analyses were not present or found on cDNA arrays used in these related genome-wide studies, providing an incomplete analysis. Although expressions of genes that were altered in association with in vitro syncytial formation, such as integrin-1 (Reimer et al., 2002), or in placental tissue in association with pre-eclampsia, such as hCG (Kudo et al., 2004), were elevated by 9-THC-stimulation, this increase did not achieve the two-fold cut-off value used for selection (data not shown) but could be of biological significance in that several genes only require a modest increase in transcript levels to provide a more substantial increase in protein product (Storey and Storey, 2004). These data, if reflective of the in vivo situation, may suggest that the mechanisms underlying FGR associated with hypoxia and/or pre-eclampsia are fundamentally different to the mechanisms involved in the FGR associated with in vivo 9-THC exposure.
In addition to the robust approach to microarray analysis (Li et al., 2004), we validated the results from the microarray analysis by examining the expression of two identified transcripts, TRß1 and HDAC3, that are both regulators of transcriptional programming and are implicated in cell growth and development. The effect of 9-THC on HDAC3 mRNA levels was not dose-dependent but appeared to show a hormetic effect (Calabrese, 2005) that exceeded control levels at 3 µM 9-THC. HDAC3 is a transcriptional co-regulator protein that interacts with other members of the histone deacetylase family of genes, such as HDAC7 or HDAC10. It is a subfamily 1 member which contains proteins that regulate the G1-phase of the cell cycle and is known to complex with NCOR1 and NCOR2, TBL1X, TBL1R, CORO2A and GPS2 to form large multi-protein complexes that constitute the N-Cor repressor complex, responsible for the deacetylation of lysine residues on the N-terminal part of the core histones (H2A, H2B, H3 and H4) (Guenther and Lazar, 2003). Histone deacetylation in turn gives a tag for epigenetic repression and plays an important role in transcriptional regulation, cell cycle progression and developmental events. The observed increase of HDAC3 expression in response to 9-THC may underlie its effects upon BeWo cell cultures (Figure 3) and if present in vivo suggests it may be linked to an inhibition of cytotrophoblast cell-cycle progression and subsequent placental development.
Of significance in this study was the observed 9-THC suppression of TRß1 expression, which was repressed in a dose-dependent manner. Linear-trend analysis of the TRß1 mRNA levels (data not shown) indicated that this effect reached significance at 15 µM 9-THC. Tri-iodothyronine (T3) plays a key role in the developing placenta and fetus (Ohara et al., 2004), and loss of its action by maternal hypothyroidism, placental deiodinase deficiency or mutations in the TRs may lead to FGR and placental insufficiency (Ohara et al., 2004). TR is involved in the normal proliferation and function of the trophoblast cell (Ohara et al., 2004), and although all four TR isoforms, TR1, TR2, TRß1 and TRß2 are found in the human placenta (Kilby et al., 1998), expression of only TRß1 has been consistently reported (Chan et al., 2004), indicating that TRß1 may be the most important TR in the placenta. Although there are multiple TRß1 5'-untranslated region (UTR) transcripts (Frankton et al., 2004), they produce two main transcripts, a short form and a long form (Mannavola et al., 2004). We designed PCR primers that would distinguish between these two isoforms and thus determined whether there was any differential regulation of the long and short forms and found that both the long and short forms of the TRß1 mRNA transcripts were repressed by 9-THC (Figure 5), indicating a common mechanism of action, presumably through the TRß1 promoter, although other intermediary molecules may be involved.
Other functions proposed for T3 in the placenta have included stimulation of placental lactogen production as demonstrated in normal trophoblast (Stephanou and Handwerger, 1995) and BeWo cell cultures (Nickel and Cattini, 1991) and increased progesterone production by early placental tissue (Maruo et al., 1991). Therefore, repression of TRß1 expression may lead to loss of T3-regulated functions in the trophoblast. Additionally, any loss of TRß1 would allow its promiscuous partners RXR and RAR (Stephanou and Handwerger, 1995) to interact with retinoid-induced genes such as the two pro-apoptotic genes, retinoic acid induced 3 (RAIG3/GPRC5C) and CARP-1, the expression of which were both found to be up-regulated by 9-THC in this study. If caused in vivo, this may manifest as increased apoptosis, and hence compromised placental growth and function in patients.
Of note was the down-regulation in expression of N-acyl-phosphatidylethanolamine-hydrolyzing Phospholipase D, the enzyme responsible for the production of anandamide, the principal endogenous cannabinoid with major effects on human reproductive function (Wenger et al., 1999; Habayeb et al., 2002; Maccarrone et al., 2002). As anandamide and the exocannabinoid 9-THC exhibit different affinities for the cannabinoid and vanilloid receptors, in vivo exposure to 9-THC may further exacerbate its effects by switching from anandamide to 9-THC action. Also of note was the suppression of expression of an uncharacterized lipoprotein lipase (Garnica and Chan, 1996), because recently it has been suggested that lipoprotein lipase deficiency can lead to FGR (Magnusson et al., 2004) or fetal death (Tsai et al., 2004).
We have identified a restricted alteration in the expression of genes in BeWo cells exposed to 9-THC. The alterations in the expression of a number of characterized genes are consistent with its effects upon the morphology of cells in culture, whereby the cultures were prevented from achieving confluency not through increased syncytial formation or culture involution, but through inhibition of cell proliferation. Because BeWo cells are considered an appropriate model for human in vivo trophoblast action and function (Sullivan, 2004) and similar effects are observed in vivo, these data suggest that 9-THC use during human pregnancy may similarly inhibit trophoblast proliferation and that placentae during early development when the cytotrophoblastic populations predominate may be more sensitive to the adverse effects of marijuana use and lead to a failure to achieve full placental development, and hence fetal growth. The lack of cell proliferation and migration associated with early placental development might therefore go some way to explain some of the clinical observations of miscarriage and placental abruption associated with marijuana use in early pregnancy and also explain why marijuana use in late pregnancy is not anti-gestational (Conner, 1984; Hatch and Bracken, 1986; Zuckerman et al., 1989). Although the regulatory network that underlies the temporal control of transcript expression and gene function remains to be determined, the identification of 9-THC-modulated trophoblast transcripts is likely to shed light on the mechanisms underlying trophoblast response to marijuana use in pregnancy.
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The authors thank H. Longland and E. Beighton of the RNA Laboratory, MRC Geneservice, Babraham Bioincubator, Babraham, Cambridge, for the production of cRNA, hybridization of probes to Affymetrix genechips and the production of scanned images and Dr E. Halligan, Genomic Instability Research Group, Department of Cancer Studies and Molecular Medicine, for assistance with the dChip software.
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Submitted on May 25, 2005; resubmitted on December 15, 2005; accepted on March 1, 2006.
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