1 Oncogenetic laboratory, Department of Medicine A and 2 Department of Obstetrics & Gynecology, Sapir Medical Center, Kfar-Saba 44281 Tel-Aviv University, Tel-Aviv, Israel and 3 Laboratory of Teratology, Department of Anatomy and Cell Biology, Hebrew University Hadassah Medical School, Jerusalem, Israel
4 To whom correspondence should be addressed at: Oncogenetic Laboratory, Sapir Medical Center, 45 Tshernehovski street, Kfar-Saba, 44281, Tel-Aviv University, Tel Aviv, Israel. Email: matalon.shelly{at}clalit.org.il
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Abstract |
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Key words: 6-mercaptopurine/migration/placenta/pregnancy/proliferation
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Introduction |
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Human placenta contains cytotrophoblast (CT) stem cells. These cells retain the ability to proliferate, and can differentiate by fusion to form syncytiotrophoblast cells (SCT) that perform gas and nutrient exchange for the developing embryo and fetus and are responsible for hormonal secretion (Malassine and Cronier, 2002). Alternatively, they may give rise to the extravillous trophoblast cells (EVT cells) (Malassine and Cronier, 2002
). EVT cells are released from the CT column, progressively migrate and diffusely infiltrate the decidua, and invade maternal uterine blood vessels (Genbacev et al., 1992
). Migration, proliferation and apoptotic processes in the trophoblast cells play an important role in determining the structure and function of the placenta in the first trimester of pregnancy (Haanen and Vermes, 1996
; Barnea et al., 2000
). Migration is performed by EVT cells (Genbacev et al., 1992
). Proliferation of trophoblast cells in the villi is observed in the CT cells as well as in the EVT cells before they migrate out of the villi (Malassine and Cronier, 2002
). All trophoblast cells may undergo apoptosis (Chan et al., 1999
; Levy and Nelson, 2000
). Cytotoxic drugs may adversely affect these processes and their regulation and may cause damage to placental structure and function.
6-mercaptopurine (6-MP), an inhibitor of purine metabolism is an antineoplastic and immunosuppressive drug, which is increasingly used in a wide spectrum of immune related disorders (Estlin, 2001; Nielsen et al., 2001
). 6-MP may adversely affect the embryo and fetus directly and indirectly (Githens et al., 1965
; Schmid, 1984
; Norgard et al., 2003
). It was reported that 6-MP may cross the human placenta, but that its fetal blood concentration represents 12% of its respective maternal blood levels (Saarikoski and Seppala, 1973
). Thus, it is reasonable to assume that the adverse effects of 6-MP on the embryo may result from indirect damage to the fetus through placental malfunction (Kolonin et al., 2002
). Since nowadays an increasing number of women tend to delay pregnancy until later reproductive age and older age is associated with malignant diseases (Zemlickis et al., 1992
), it is expected that the incidence of cancer in pregnancy will rise. Furthermore, the use of immunosuppressive drugs for autoimmune diseases and following organ transplantation is rising as well (Prevot et al., 2002
). Cases of women exposed to azathioprine or its metabolite 6-MP during pregnancy are described in the literature and it appears that in the future, women, embryos and fetuses will be exposed more frequently to cytotoxic and/or immunosuppressive agents such as 6-MP during pregnancy (Polifka and Friedman, 2002
; Norgard et al., 2003
; Tartakover Matalon et al., 2004
). We studied the direct effects of 6-MP on trophoblastic cells, by the use of an in vitro system of human placental explants in culture.
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Materials and methods |
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The culture techniques were based upon methods developed previously (Yacobi et al., 2002). Briefly, the placental tissue (villi) was dissected from the fetal membranes. Explants of 10 mg wet weight were transferred into MillicellCM culture dish inserts (Millipore Corporation, Bedford, USA), which had been layered with polymerized Matrigel (BD Biosciences, Bedford, USA) (Figure 1A). Medium (Dulbecco's modified Eagle's medium supplemented with L-glutamine (2 mM), sodium pyruvate (1 mM), antibiotics, fetal calf serum (10%); Biological Industries, Beit, Haemek, Israel) was added to the lower well of the culture dish (bottom medium). Cultures were pre-incubated overnight in 5% CO2, 20% O2 incubator in the presence of the bottom medium (Figure 1B). No medium was present above the explants. On the following day, medium was added above (top medium) and replaced below the placental explants (Figure 1C). During that time 6-MP (0.1 mM and 0.01 mM, Sigma M7000) was added to the upper medium of the placental culture (these concentrations are similar to those found in the sera, and 10-fold higher). 6-MP was dissolved in 1 M NaOH (50 mg/ml), and diluted with the medium to the appropriate concentration. Medium with NaOH alone served as control. In the first three experiments (out of five experiments) we included an additional control of the same medium without NaOH, to eliminate the possibility of any adverse effect of the 6-MP solvents. Indeed, no differences were observed in the migration and proliferation of EVT cells between explants that were incubated in the presence of medium with or without NaOH (data not shown). Both media from the insert (top, with/without the drug), and from the well (bottom) were changed 24, 72 and 96 h following the beginning of the experiment, and the collected media were stored at 80 °C until processing. Villous explants were inspected daily using an inverted phase-contrast microscope for general cellular integrity, cellular migration and outgrowth. Hexaplicates of each treatment for each placenta were preformed. Ninety-six hours following the beginning of the experiments, villi with supporting matrigel were dissected out. The specimens were fixed in 4% buffered formaldehyde and paraffin embedded for immunohistochemical examination.
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Apoptotic events in the human trophoblast
Apoptosis was detected by two assays: Tunnel and anti-caspase 3 analysis.
Tunnel
Apoptosis was detected on paraffin sections by ApopTag peroxides labeling of fragmented DNA (KIT S7101, Intergen Company, USA), according to the manufacturer's instructions. In short, paraffin-embedded sections were deparaffinized, pretreated with proteinase K, rinsed and quenched in 1% H2O2. Samples were then incubated with terminal deoxynucleotidyl transferase (TdT), in the presence of nucleotides. (TdT catalyzes a template-independent addition of nucleotide triphosphates to the 3'-OH ends of double- or single-stranded DNA, and forms an oligomer composed of digoxigenin.) The slides were then incubated with stop-wash buffer, incubated with anti-dioxigenin peroxidase, rinsed and stained with AECchromogen. Sections were counterstained with Mayers hematoxilin. Slides that were treated in the same way, but without exposure to TdT, served as negative control.
Activated caspase 3 analysis
Activated caspase 3 stained cells were detected on paraffin sections in a similar way to the proliferation assay without proteinase K pretreatment, and by using rabbit anti-caspase 3 polyclonal antibody (Biocare medical, CA) primary antibody instead of anti-Ki67. Purified normal rabbit immunoglobulin (Zymed, San Francisco, CA) served as isotype control.
Counting the cells
Quantitative analysis of cell proliferation and apoptosis was done by enumerating the stained cells out of the total trophoblast cells present in the villi. Proliferation and apoptosis analysis were performed on three explants for each treatment in five experiments. They were assessed in the trophoblast cells in the villi, excluding the non-proliferating cells (i.e. SCT and EVT cells that migrated into the matrigel). Twelve slices from each treatment in every experiment were analyzed (four slices from each of three randomly chosen wells in every treatment). In every slice, cells were counted in three/four villi (thus a minimum of 36 villi were counted). The average number of cells that were counted in each villous during proliferation analysis was 81±50, 84±55 and 71±35 cells for control, 6-MP (0.01 mM) and 6-MP (0.1 mM) treated explants, respectively. The average number of cells that were counted in each villous during apoptosis analysis was 79±43, 77±49 and 88±49 cells for control, 6-MP (0.01 mM) and 6-MP (0.1 mM) treated explants, respectively. Counting was performed at x400 magnification.
Screening the upper fluid of the human trophoblast for hormones
Twenty-four and 72 h following culture, media were collected from the inserts of control and 6-MP (0.01 mM) treated explants for the analysis of hormone levels. Estradiol (E2), progesterone and HCG were measured by enzyme-linked immunosorbent assay using ICN Pharmaceuticals kits (beta HCG 07B56102, estradiol 07B38102, progesterone 07B70102; ICN Pharmaceuticals, NY), according to the manufacturer's instructions. Media of three explants were analyzed from each treatment in every experiment. All tests were performed in duplicates.
Statistical analysis
Student's t-tests were employed in analysis of differences between cohorts. An effect was considered significant when the P-value was 0.05.
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Results |
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Discussion |
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We found that 6-MP adversely affected trophoblast cells in several ways. 6-MP inhibited migration of EVT cells from the villi to the matrigel. Fewer villi, from fewer explants, released fewer EVT migrating cells in the 6-MP treated explants in comparison with the controls. Moreover, we observed that 6-MP inhibited proliferation of trophoblast cells. The number of apoptotic events increased in the 6-MP treated explants in comparison to control. No significant effect of 6-MP on hormone levels was observed.
The effect of 6-MP on placental explants was similar in both drug concentrations, the one that is similar to the level in the sera (0.01 mM) and the 10-fold higher concentration (0.1 mM).
6-MP is a source of thioguanine nucleotides, which may be incorporated into DNA, yielding abnormal DNA, thus interfering with the function of DNA polymerases, ligases and endonucleases (Platzek et al., 1994; Estlin, 2001
). Moreover, 6-MP is catalyzed to inhibitors of enzymes that are important in de novo purine synthesis (Estlin, 2001
). Nucleoside metabolism probably plays an important role in the early post-implantation stages of the placenta, since enzymes of the degradation pathways of purine metabolism are enriched at the maternalfetal interface of mice (Blackburn et al., 1997
), and are highly expressed in human maternally-derived decidual cells and in trophoblast cells at that time (Lupidi et al., 1998
). Thus, it is not surprising that 6-MP, which affects nucleoside metabolism, will adversely affect the placenta. Inhibition of migration and proliferation, and increased apoptosis of other cell types by 6-MP was already observed in the past. Azathioprine, the mother drug of 6-MP was shown to inhibit leukocyte migration (Szczepanska et al., 1988
; Elferink et al., 1997
) probably through a cGMP dependent mechanism. This mechanism may have a significant role in EVT cell migration (Cartwright et al., 1999
). Inhibition of proliferation by 6-MP was also observed in the past, when 6-MP led to decreased endothelial and T-cells proliferation, by inhibiting the purine/pyrimidine nucleotides in the T-cells and altering endothelial nucleotide balance (Weigel et al., 1999
; Quemeneur et al., 2003
). Moreover, 6-MP inhibited proliferation of mature B-cells after being triggered with mitogen (Hortelano and Bosca, 1997
). In addition to its anti proliferative effect, 6-MP evoked apoptotic cell death in activated cycling T- and activated B-cells (Hortelano and Bosca, 1997
; Quemeneur et al., 2003
).
In our study, 6-MP's most vigorous effect was on EVT migration. EVT cells differentiate in vitro by proliferation of cells in the tip of the anchoring villi which later migrate into the surrounding matrix (Genbacev et al., 1993). As a result of cell proliferation and migration, the number of EVT cells in the matrigel increases during the culture period. It is possible that as a consequence of lower proliferative rate, fewer EVT cells with migration potential were formed and therefore fewer migrating cells were observed. However, it is important to emphasize that different regulators control proliferation and migration of EVT cells (Athanassiades et al., 1998
; Xu et al., 2002
). Thus, it is possible that two different mechanisms of action of 6-MP are responsible for the inhibition of migration and proliferation.
EVT cells play an important role during implantation, when they migrate into the decidua, by remodeling the pregnant endometrium and its vasculature (Malassine and Cronier, 2002). Since 6-MP inhibits EVT cells migration, it is reasonable to assume that this drug may affect the implantation process. Indeed, it was found that azathioprine (Gross et al., 1977
) injected daily into pregnant rats has a detrimental effect on the trophoblast of the implanted blastocyst by impeding its penetration, restraining the placental formation with subsequent degeneration of the conceptus (Gross et al., 1977). Moreover, implantation traces of abortion were smaller in pregnant rats that were injected with 6-MP on days 7 and 8 of gestation in comparison with control (Yamada et al., 1985
). Thus, our findings suggest a mechanism by which 6-MP may impede implantation and normal placental function not only in animals but also in human.
Interestingly, the highest inhibition of EVT migration occurred in the first 24 h, and was much less vigorous during the rest of the experimental period. It seems that the placenta developed resistance to the effect of 6-MP on migration. Resistance to drugs may result from expression of ATP-dependent efflux pumps (Gottesman et al., 2002) and by other proteins such as heat shock proteins (Vargas-Roig et al., 1998
). We are currently studying the mechanisms by which the placental resistance was induced.
No significant differences were observed in the medium hormone levels of 6-MP treated explants in comparison to the medium of the controls. However, although not significant, a higher level of beta HCG was observed in the medium of the control explants 72 h following the beginning of the experiment in comparison with the 6-MP treated explants. The receptor for HCG is found on intermediate trophoblasts (Tao et al., 1995), and HCG increased matrigel invasion of a choriocarcinoma cell line (JEG-3) (Zygmunt et al., 1998
). Thus, decreased levels of hCG may contribute to the adverse migration of trophoblast cells following 6-MP exposure.
Our results demonstrate that 6-MP adversely affects the placenta. These effects may contribute to failure of the implantation process. The observation that the effect of 6-MP on migration of EVT cells was mainly evident at 24 h after exposure, and substantially decreased with time, suggests that the placenta develops resistance to the drug. This may explain the non-lethal effects of the drug on pregnancy outcome. Further studies will clarify the mechanism of the effect of 6-MP on placental function, and of the placental resistance to the drug.
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Acknowledgements |
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Submitted on May 4, 2004; resubmitted on September 8, 2004; accepted on December 7, 2004.
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