1 Department of Obstetrics and Gynecology, National Taiwan University Hospital, 2 Department of Microbiology, 3 Center for Optoelectronic Biomedicine, College of Medicine, National Taiwan University, Taipei, Taiwan
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Abstract |
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Key words: abortion/complete hydatidiform mole/pregnancy/telomerase activity
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Introduction |
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Gestational trophoblastic disease (GTD) refers to a category of neoplasm that includes complete hydatidiform mole, partial hydatidiform mole and choriocarcinoma (World Health Organization, 1983; Kohorn et al., 2000
). All of these tumours are pregnancy-related and possess a proliferative neoplastic trophoblast. In normal pregnancies, a villous trophoblast in the placenta consists of a population of proliferating cytotrophoblasts that differentiate and individually fuse into a syncytiotrophoblast (Huppertz et al., 1998
). In the first trimester, the cytotrophoblast proliferation rate is high (Castellucci et al., 2000
). The proportion of proliferative villous cytotrophoblasts among the total trophoblast population decreases after the first trimester. In the third trimester, the overall proliferation rate for the trophoblasts falls to nearly 10% of the first trimester value (Castellucci and Kaufmann, 2000
). In spontaneous abortions, the trophoblasts are less hyperplastic (van Lijnschoten et al., 1994
) and degenerative lesions are frequently found in the placental tissue (Larsen and Graem, 1999
; Benirschke and Kaufmann, 2000
). Therefore, trophoblasts from the placentas of normal pregnancies, abortions, and cases of intrauterine death, and from tissue samples from cases of GTD, seem to differ in terms of their respective rates of proliferation and degeneration. At present, no study has specifically compared these several kinds of tissue with regards to the presence of telomerase activity.
The aim of our study was to evaluate and compare the occurrence of telomerase activity in tissue samples from normal pregnancies, spontaneous abortions, cases of intrauterine fetal death (IUFD) and cases of GTD.
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Materials and methods |
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We began by washing these tissue samples in an ice-cold wash buffer [10 mmol/l Hepes-KOH (pH 7.5), 1.5 mmol/l MgCl2, 1 mmol/l EGTA, 10 mmol/l KCl, 1 mmol/l dithiothreitol] and then used liquid nitrogen to shock freeze the samples into smaller pieces. Afterwards, we used disposable surgical knife blades to slice flakes from these frozen tissue specimens, which had previously been prepared on sterile Petri dishes; these flakes were immediately transferred to homogenization tubes containing 200 µl ice-cold lysis buffer [10 mmol/l Tris-HCl (pH 7.5), 1 mmol/l MgCl2, 1mmol/l EGTA, 0.1 mmol/l phenylmethylsulphonyl fluoride, 5 mmol/l ß-mercaptoethenol, 0.5% CHAPS (Sigma, St Louis, MO, USA) and 10% glycerol]. The flakes were then homogenized on ice with a motorized pestle until they reached a uniform consistency. After incubating the lysate on ice for 30 min, it was centrifuged at 16 000 g for 20 min at 4°C using Eppendorf tubes. We then carefully removed the supernatant and measured the protein concentration by Bradford assay (Bradford, 1976). Finally, we used liquid nitrogen to shock freeze the tissue extracts into aliquot parts, and then stored them at 80°C.
Telomerase repeat amplification protocol (TRAP) reaction
We performed a telomerase repeat amplification protocol (TRAP) assay using the Telomerase PCR ELISA assay in a 50 µl reaction mixture according to the manufacturer's protocol (Boehringer Mannheim Biochemicals, Mannheim, Germany). In this primer-extension based assay for detecting telomerase activity, the telomerase reaction product is amplified by PCR (Kim et al., 1994) and a photometric enzyme immunoassay is used. We began by mixing a 25 µl reaction solutionwhich contained a Tris buffer, a biotin-labelled P1-TS primer, a P2 primer, nucleotides and a Tag polymerasewith 3 µl of cell extract. We then added sterile water to the result, until a final volume of 50 µl was reached. After a 30 min incubation period at 25°C for telomerase-mediated extension of the P1-TS primer, the reaction mixture was heated to 94°C for 5 min and immediately subjected to 33 PCR cycles at 94°C for 30 s, 50°C for 30 s, and 72°C for 90 s. After adding 5 µl of the amplification product and 20 µl of a denaturation reagent containing sodium hydroxide (<0.5%) to 225 µl of hybridization buffer (digoxigenin-labelled detection probe), we mixed the resulting combination thoroughly.
Hybridization and ELISA procedure
We then transferred 100 µl of the mixture to a well made from a streptavidin-coated microtitre plate. We incubated the microtitre plate at 37°C on a shaker for 2 h, and then washed it three times with 250 µl of washing buffer. After adding 100 µl of anti-digoxigenin-peroxidase and incubating at room temperature for 30 min, while shaking, we removed the solution. We then added 100 µl of a substrate solution containing 3,3',5,5'-tetramethyl benzidine and incubated at room temperature for 1020 min for colour development while still shaking. Finally, we added 100 µl of 5% sulphuric acid to stop the reaction. Using a microtitre reader, we measured the absorbance of the samples at 450 nm. Absorbance values were reported as the A450 nm reading against blank (reference wavelength A690 nm). We regarded samples as telomerase-positive if the difference in absorbance (A) was higher than 0.25 units. For negative controls, we incubated 5 µl of cell extract with DNase-free RNase at a concentration of 1 µg/µl for 20 min at 37°C. The maximum value of absorbance for the negative control should be 0.25 units; if the value was higher, the whole test, including TRAP, was repeated. A cell extract prepared from immortalized telomerase-expressing human kidney cells was used as a positive control. Since the absorbance readings of the positive controls should be >1.5 units, the test was repeated if the values were lower.
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Results |
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For patients with GTD, an initial work-up was carried out which included chest roentgenography and liver and renal function tests. Patient data included age, gravidity, parity, uterine size at the time of evacuation, method of termination, pre-evacuation serum ß-HCG level, radiological findings, histologic evaluation and laboratory findings. Seventeen of the 18 patients with complete hydatidiform mole were treated with suction curettage. The resulting evacuated specimens were all found to have translucent molar vesicles. Microscopic analysis showed marked hydropic change and oedema and cisterna formation on the villi, a villous core that lacked fetal capillaries, and marked hyperplastic trophoblasts on the villous surface. The patients were tested weekly after evacuation until three consecutive negative serum ß-HCG levels were obtained, and afterwards were tested at 1 month intervals for at least 1 year. Patients with a ß-HCG plateau that lasted >3 weeks, who had an elevated level for >2 weeks, or who had any sign of metastasis were diagnosed as having persistent GTD. Among the 18 cases of complete hydatidiform mole, two developed persistent GTD and 16 spontaneously regressed. Both of the patients with persistent GTD underwent chemotherapy and achieved remission: one underwent single agent chemotherapy with methotrexate for three courses and was then shifted to a combination chemotherapy with EMACO (etoposide, actinomycin-D, methotrexate, vincristine and cyclophosphamide), while the other underwent seven courses of single agent chemotherapy.
Of the three patients with choriocarcinoma, two underwent an initial surgical operation before chemotherapy and one underwent surgery during the course of chemotherapy. In one case, that of a nulliparous woman, there was an initial clinical impression of ectopic pregnancy and myoma uteri. During her operation, a 7x5x5 cm myoma was found on the uterine fundus, while on the myoma there was a 5x4.5x4 cm haemorrhagic tumour mass. Local excision of the myoma together with the haemorrhagic tumour was performed. The other patient underwent an emergency laparotomy because of heavy intra-abdominal bleeding. During her operation, a subtotal hysterectomy was performed after a 6x5.5x5 cm actively bleeding mass was discovered on the uterine fundus. Microscopically, the haemorrhagic tumours from both cases showed a choriocarcinoma composed of clusters of cytotrophoblasts that were separated by streaming syncytiotrophoblast masses. Evident haemorrhage and necrosis were also noted, but no chorionic villus was seen. In both cases, remission was achieved after post-operative chemotherapy with EMACO. The third patient had a choriocarcinoma that had metastasized to her lungs, intestines and brain. This patient underwent seven courses of chemotherapy with EMACO and two courses with Ifosfamide and cisplatin. During the treatment period, severe intestinal bleeding due to jejunum metastasis occurred. A segmental resection of the intestine was performed for treatment of the intestinal bleeding. Histopathological examination revealed a picture of metastatic choriocarcinoma. Irradiation therapy to the whole brain for 3000 cGy was also performed for the brain metastasis. Unfortunately, she eventually died.
Telomerase activity in cases of normal pregnancy, spontaneous abortion, IUFD and GTD is shown in Table II. Fifteen of the 18 (83%) cases of complete hydatidiform mole and all three cases of choriocarcinoma tested positive for telomerase activity. Of the 45 placental tissue samples from early pregnancies that we examined using the TRAP assay, 33 (73%) tested positive for telomerase. In contrast, only 11 of the 37 (30%) late pregnancy samples expressed telomerase activity. Samples from early spontaneous abortions also exhibited telomerase activity, but at a low level; only nine of the 27 (33%) tested positive for telomerase activity. Telomerase activity was not detected in the placental tissue from the two cases of late-pregnancy intrauterine fetal death.
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Discussion |
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In our study, we found that telomerase activity was at its highest during the early period of normal pregnancy. We also found that this activity decreases significantly after the first trimester. Our findings suggest that in normal pregnancies, telomerase was down-regulated as the pregnancy progressed. However, the mechanism that underlies this phenomenon is not known. As for placental tissue from normal pregnancies, although there is a subpopulation of trophoblasts (cytotrophoblasts) that may be proliferative, other cytotrophoblasts and the syncytiotrophoblast are not proliferative. The proportion of proliferative villous cytotrophoblasts among the total trophoblast population decreases as the pregnancy progresses from the first trimester to term (Castellucci and Kaufmann, 2000). Whether isolated proliferating cytotrophoblast cells possess a higher level of telomerase activity than non-proliferating cytotrophoblast cells or the syncytiotrophoblast needs further study.
Maintenance of telomerase activity has been associated with increased cellular resistance to apoptosis (Holt et al., 1999). Apoptosis (programmed cell death) leads to the elimination of old, unnecessary and unhealthy cells. In recent studies of first trimester placental tissue by means of either haematoxylin or eosin staining (Smith et al., 1997
) or by electron microscopic assessment (Smith et al., 2000
), apoptosis was discovered in the human placenta. These studies also found that the incidence of placental apoptosis increased with increasing gestational age.
To our knowledge, there have been no reports of telomerase activity in cases of spontaneous abortion or IUFD. In this study, we found that rates of positive telomerase activity are low for cases of fetal death no matter when they occurred. Telomerase activity was rarely present in cases of spontaneous abortion, and not present at all in cases of fetal demise. In recent reports that evaluated telomerase activity in the placenta in cases with or without fetal growth retardation, telomerase activity was detected more often in cases without fetal growth retardation (Isuzu et al., 1999; Kudo et al., 2000
). Furthermore, increased apoptosis has also been found in the villi from spontaneous abortions (Qumsiyeh et al., 2000
). From both these reports and our data, we hypothesize that the down-regulation of telomerase activity in normal pregnancies may play a role in placental senescence and may be connected to placental apoptosis.
Complete hydatidiform mole is an abnormal conceptus. Whether neoplastic trophoblasts from molar tissue samples and normally proliferative trophoblasts from early pregnancy placental tissue samples possess the same degree of telomerase activity has rarely been investigated. In our study, we detected telomerase activity in the majority of the tissue samples from both normal early pregnancies and from cases of complete hydatidiform mole. In fact, during the early stages of gestation, there was no significant difference between them. This finding suggests that placental tissue that has a tumour-like character during early pregnancy and neoplastic trophoblastic tissue both possess the same proliferative ability.
In addition to these findings, we discovered that in cases of complete hydatidiform mole, telomerase activity remained constant as the pregnancy progressed. This contrasts with what we found in normal pregnancies, where telomerase activity was present less often in the later stages of pregnancy, and indicates that the down-regulation of telomerase activity that occurs in normal pregnancies is not present in complete hydatidiform mole. These results support the hypothesis that complete hydatidiform mole is essentially a neoplasm, and that telomerase activity provides a mechanism for avoiding the proliferative limitation due to telomere loss.
Furthermore, in our study three cases of choriocarcinoma tested positive for telomerase activity. To our knowledge, in previous studies only six cases of choriocarcinoma had been tested for the presence of telomerase; all tested positive for telomerase activity (Bae and Kim, 1999; Cheung et al., 1999
). The high rates of telomerase activity for choriocarcinoma suggest that this tumour is highly oncogenic, and that such activity may be a critical step in the oncogenesis of this malignancy.
In our study, we found that telomerase activity occurs similarly in both placental tissue from normal early pregnancies and neoplastic trophoblastic tissue. Thus, our findings suggest that proliferative normal trophoblasts and neoplastic trophoblasts possess similar levels of telomerase activity. In normal pregnancies, telomerase activity is down-regulated over the course of gestation, while in cases of hydatidiform mole it is not. The fact that telomerase activity decreases in cases of fetal demise, and as pregnancy progresses, also suggests that placental senescence may play a role in the development and ageing of the placenta, but not in the appearance and growth of trophoblasts in cases of complete hydatidiform mole.
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Acknowledgements |
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Notes |
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Submitted on April 17, 2001; resubmitted on August 13, 2001
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References |
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Bae, S.N. and Kim, S.J. (1999) Telomerase activity in complete hydatidiform mole. Am. J. Obstet. Gynecol., 180, 328333.[ISI][Medline]
Bamberger, A., Sudahl, S., Bamberger, C.M. et al. (1999) Expression patterns of the cell-cycle inhibitor p27 and the cell-cycle promoter cyclin E in the human placenta throughout gestation: implications for the control of proliferation. Placenta, 20, 401406.[ISI][Medline]
Benirschke, K. and Kaufmann, P. (2000) Abortion, placentas of trisomies, and immunological considerations of recurrent reproductive failure. In Benirschke, K. and Kaufmann, P. (eds) Pathology of the Human Placenta, 4th edition. Springer-Verlag, New York, pp. 685717.
Bischof, P. and Campana, A. (2000) A putative role for oncogenes in trophoblast invasion? Hum. Reprod., 15 (Suppl. 6), 5158.[Medline]
Bradford, M.M. (1976) A rapid and sensitive method for the quantification of microgram quantities. Anal. Biochem., 72, 248254.[ISI][Medline]
Castellucci, M. and Kaufmann, P. (2000) Basic structure of the villous trees. In Benirschke, K. and Kaufmann, P. (eds) Pathology of the Human Placenta, 4th edition. Springer-Verlag, New York, pp. 50115.
Castellucci, M., Kosanke, G., Verdenelli, F. et al. (2000) Villous sprouting: fundamental mechanisms of human placental development. Hum. Reprod. Update, 6, 485494.
Cheung, A.N., Zhang, D.K., Liu, Y. et al. (1999) Telomerase activity in gestational trophoblastic disease. J. Clin. Pathol., 52, 588592.[Abstract]
Collins, K., Kobayashi, R. and Grieder, C.W. (1995) Purification of Tetrahymena telomerase and cloning of genes encoding the two protein components of the enzyme. Cell, 81, 677686.[ISI][Medline]
Counter, C.M., Hirte, H.W., Bacchetti, S. et al. (1994) Telomerase activity in human ovarian carcinoma. Proc. Natl Acad. Sci. USA, 91, 29002904.[Abstract]
Harley, C.B. (1991) Telomere loss: mitotic clock or genetic time bomb? Mutat. Res., 256, 271282.[ISI][Medline]
Hemberger, M., Himmelbauer, H., Ruschmann, J. et al. (2000) cDNA subtraction cloning reveals novel genes whose temporal and spatial expression indicates association with trophoblast invasion. Dev. Biol., 222, 158169.[ISI][Medline]
Holt, S.E., Glinsky, V.V., Ivanova, A.B. et al. (1999) Resistance to apoptosis in human cells conferred by telomerase function and telomere stability. Mol. Carcinog., 25, 241248.[ISI][Medline]
Huppertz, B., Frank, H.G., Kingdom, J.C. et al. (1998) Villous cytotrophoblast regulation of the syncytial apoptotic cascade in the human placenta. Histochem. Cell Biol., 110, 495508.[ISI][Medline]
Isuzu, T., Kudo, T., Sato, T. et al. (1999) Telomerase and proliferative activity in placenta from women with and without fetal growth restriction. Obstet. Gynecol., 93, 124129.
Khoo, N.K., Bechberger, J.F., Shepherd, T. et al. (1998) SV40 Tag transformation of the normal invasive trophoblast results in a premalignant phenotype. I. Mechanisms responsible for hyperinvasiveness and resistance to anti-invasive action of TGFbeta. Int. J. Cancer, 77, 429439.[ISI][Medline]
Kim, N.W., Piatyszek, M.A., Prowse, K.R. et al. (1994) Specific association of human telomerase activity with immortal cells and cancer. Science, 226, 20112015.
Kohorn, E.I., Goldstein, D.P., Hancock, B.W. et al. (2000) Combining the staging system of the International Federation of Gynecology and Obstetrics with the scoring system of the World Health Organization for trophoblastic neoplasia. Report of the Working Committee of the International Society for the Study of Trophoblastic Disease and the International Gynecologic Cancer Society. Int. J. Gynecol. Cancer, 10, 8488.[ISI][Medline]
Kudo, T., Izutsu, T. and Sato, T. (2000) Telomerase activity and apoptosis as indicators of ageing in placenta with and without intrauterine growth retardation. Placenta, 21, 493500.[ISI][Medline]
Larsen, L.G. and Graem, N. (1999) Morphological findings and value of placental examination at fetal and perinatal autopsy. APMIS, 107, 337345.[ISI][Medline]
Lopata, A. (1996) Blastocyst-endometrial interaction: an appraisal of some old and new ideas. Mol. Hum. Reprod., 2, 519525.[Abstract]
Qumsiyeh, M.B., Kim, K.R., Ahmed, M.N. et al. (2000) Cytogenetics and mechanisms of spontaneous abortions: increased apoptosis and decreased cell proliferation in chromosomally abnormal villi. Cytogenet. Cell Genet., 88, 230235.[ISI][Medline]
Smith, S.C., Baker, P.N. and Symonds, E.M. (1997) Placental apoptosis in normal human pregnancy. Am. J. Obstet. Gynecol., 177, 5765.[ISI][Medline]
Smith, S.C., Leung, T.N., To, K.F. et al. (2000) Apoptosis is a rare event in first-trimester placental tissue. Am. J. Obstet. Gynecol., 183, 697699.[ISI][Medline]
van Lijnschoten, G., Arends, J.W. and Geraedts, J.P. (1994) Comparison of histological features in early spontaneous and induced trisomic abortions. Placenta, 15, 765773.[ISI][Medline]
World Health Organization (1983) Gestational trophoblastic diseases. Report of a WHO Scientific Group. Technical Report Series 692. World Health Organization, Geneva, pp. 781.
accepted on October 15, 2001.