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ORIGINAL RESEARCH |
From the Department of Pathology, University of Colorado Health Sciences Center, Denver, Colorado; Geron Corp., Menlo Park, California; and VA Division of Cardiac Research, Veterans Affairs Medical Center, Denver, Colorado.
Address reprint requests to: Kenneth R. Shroyer, MD, PhD Department of Pathology University of Colorado Health Sciences Center Box B-216 4200 East Ninth Avenue Denver, CO 80262 E-mail: ken.shroyer{at}uchsc.edu
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Abstract |
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Methods: Formalin-fixed placental tissues were selected from collections of the Department of Pathology at the University of Colorado. Five specimens of each trimester, five each of preeclamptic and post-term placentas, and 23 molar pregnancies were selected. The telomerase catalytic protein hTERT was localized in placental tissues using the catalyzed signal amplification system, and survivin was localized by conventional immunoperoxidase method. Staining was graded on a scale of zero to 4.
Results: hTERT staining was detected in sections of 42 of 48 specimens (23 of 23 hydatidiform moles, 19 of 25 non-neoplastic placental tissues). The intensity of staining for hTERT was higher in hydatidiform moles (mean 3.3, median 3) compared with levels in non-neoplastic placental tissues (mean 0.92, median 1) (P < .001). Survivin was detected in 39 of 48 specimens (22 of 23 hydatidiform moles, 17 of 25 non-neoplastic placental tissues). Compared with non-neoplastic tissues (mean 0.88, median 1), survivin levels were elevated in hydatidiform moles (mean 1.35, median 1) (P = .031).
Conclusion: Survivin and telomerase were increased in hydatidiform moles, suggesting that regulation of apoptosis and stabilization of telomere length might be involved in neoplastic transformation of the placenta. The patterns of expression observed for survivin and telomerase in non-neoplastic placental tissues suggest that the control of apoptosis and stabilization of telomeric DNA might also be involved in normal gestational development.
Development of the trophoblast is regulated by the coordinated balance of cell proliferation and apoptosis. The expression of telomerase and survivin might be related to normal placental development and dysfunctional development in molar pregnancy. Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA, resulting in stabilization of chromosomal ends. Although expression of telomerase is suppressed in normal somatic human cells, it is reactivated during tumorogenesis and in germ cells of reproductive organs. Telomerase expression has been detected in cancer cells, tissues that undergo continuous cell renewal, and most primary tumors.1–3
The functional telomerase holoenzyme comprises two major subunits, including the RNA component (hTR) and the catalytic protein subunit, hTERT.2,4,5 In most tissues, the expression of hTERT mRNA parallels the level of telomerase enzyme activity, suggesting that induction of hTERT is necessary and perhaps sufficient for expression of telomerase.6,7 Although expression of telomerase has been evaluated by sensitive molecular assays, cellular distribution of hTERT in placental tissues has not been described.
Survivin, a member of the inhibitor of apoptosis protein gene family, is expressed during development and in human cancer.8 At the beginning of mitosis, survivin associates with microtubules of the mitotic spindle in a specific and saturable reaction that is regulated by microtubule dynamics.9 Survivin has been shown in vitro to inhibit effector cell death proteases caspase 3 and 7 and is a mediator and regulator of the antiapoptotic activity of v-Rel and NF-kappa B transcription factor families.9,10
The primary objective of this study was to find if a difference in telomerase or survivin levels exists between non-neoplastic tissues and hydatidiform moles. The secondary objective was to explore the expression of telomerase and survivin during normal placental development, in post-term gestation, and in preeclampsia.
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Materials and Methods |
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hTERT immunohistochemical localization was done with a monoclonal antibody, designated 4B1, using the tyramide-based immunoperoxidase CSA detection system (DAKO, Carpinteria, CA), as described by Frost et al.11 Negative controls included substitution of 4B1 with a subclass-matched monoclonal immunoglobulin that was generated against unrelated antigens.
Survivin immunohistochemical localization was done with a rabbit antibody to the aminoterminal portion of survivin, using an indirect immunoperoxidase method.12 Control slides were reacted with a matched dilution of the pre-immune serum from the same animal that was used to generate the primary antibody.
The evaluation of the staining score was based on review of the entire histologic section by three observers, excluding endometrial components of the tissue specimens. Staining patterns were generally consistent throughout sections, and a final score was assigned based on overall impression, rather than by averaging the results of discrete microscopic fields. The intensity of survivin and telomerase staining was scored on a scale of negative to 4+, where a score of 1+indicated faintly perceptible staining, and a score of 4+corresponded to staining intensity similar to that seen in positive control tissue sections (colonic adenocarcinoma for survivin and normal testis for hTERT). The cellular (syncytiotrophoblast versus cytotrophoblast) and sub-cellular (cytoplasmic versus nuclear) patterns of localization were not considered in intensity scores. A Fisher exact test was done for dichotomous or categoric variables to compare molar and nonmolar specimens. Given the nature of the survivin expression and hTERT staining variable distributions, Wilcoxon rank-sum test (the nonparametric equivalent of the independent two sample t test) was used to evaluate differences in median levels of hTERT and survivin markers between neoplastic and non-neoplastic placental tissues. P 
.05 was considered statistically significant. A qualitative assessment of patterns of survivin and telomerase staining levels was reviewed across progression of placental development, post-term gestation, and preeclampsia. Those observations are reported for descriptive purposes only.
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Results |
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In first-trimester placental tissue, staining for hTERT protein was localized to the inner cytotrophoblastic layer of the chorionic villi and was less intense in the syncytiotrophoblastic layer (Figure 1
). Cells morphologically consistent with Hofbauer cells also stained for hTERT. Most specimens of first-trimester placental tissue and molar tissue included disrupted fragments of endometrial stroma and glandular epithelium. Those endometrial fragments also showed strong staining of decidualized stroma and glands, but that was not considered for statistical evaluation of differences between various histologic categories of tissues. In the second trimester of development, the trophoblastic cells and vascular endothelial cells of the chorionic villi were stained. The overall intensity of hTERT staining of the trophoblast was relatively stable throughout all stages of placental development, in preeclampsia, and in post-term placental tissues.
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). In 19 of 23 of molar placental tissues, both cytoplasmic and nuclear localization of hTERT was observed. The pattern and intensity of staining was similar in partial and complete moles. The mean intensity score for hTERT in hydatidiform moles was 3.30 (standard deviation [SD] 0.70, median 3), but was 0.92 (SD 0.64, median 1) in non-neoplastic placental tissue (Table 1). The difference in proportion in hTERT levels in molar pregnancies (23 of 23, 100% of intensity 2+or higher) versus nonmolar pregnancies (four of 25, 16% of intensity 2+or higher) showed significant differences by the Fisher exact test (P < .001), and the differences in median hTERT intensity (molar pregnancies median score 3 versus nonmolar pregnancies median score 1) were significant by Wilcoxon rank-sum test (P < .001). By dichotomizing the telomerase variable into negative and positive staining categories, there was a difference in the proportion of telomerase-positive cases in neoplastic placental development compared with non-neoplastic placental development (23 of 23; 100% versus 19 of 25; 76%, P = .023).
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Strong staining for survivin was seen in the syncytiotrophoblastic layer of first-trimester chorionic villi (Figure 3) and in the trophoblast of molar pregnancies (Figure 4). Intense staining also occurred in cells morphologically consistent with Hofbauer cells, nucleated hematologic precursor cells, and in villous stromal fibroblasts. In addition, moderate to intense staining was detected in decidualized endometrial stroma and glandular epithelium, but that was not considered for statistical evaluation of the differences between the various histologic categories of tissues. In the second trimester of development, staining for survivin was detected in the trophoblastic layer and vascular endothelial cells of the chorionic villi. Based on visual assessment of the data distributions, intensities of survivin staining of the trophoblast in third-trimester tissues, preeclampsia, and post-term placentas appear to be decreased compared with levels that were seen in first- and second-trimester placentas. Again using a review of the data, the range in intensity of staining for survivin appears greater in hydatidiform moles than was seen in non-neoplastic placental development (Table 1
). Ranked assessment of the survivin levels of molar pregnancies versus nonmolar pregnancies was statistically significantly different by Wilcoxon rank-sum test (P = .031). The mean intensity score for survivin in hydatidiform moles was 1.35 (SD 0.65, median level 1) but was 0.88 (SD 0.72, median 1) in non-neoplastic placental development. Dichotomizing the survivin variable into negative versus positive staining categories, there was also a significant difference in the proportion of survivin-positive cases in neoplastic placental development (22 of 23; 95.7%) versus non-neoplastic placental development (17 of 25; 68.0%) (P = .024).
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Discussion |
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Telomerase and survivin were evaluated by immunohistochemical methods, to permit the determination of the histologic distribution of expression. Previous solution-based studies have shown that normal cycling endometrial tissues express high levels of telomerase, but the expression of telomerase in decidualized endometrium has not been addressed.16,17 The current study found that survivin is present in fragments of decidualized endometrium that were present in the placental tissue samples. Clinical samples of immature placental tissue specimens are almost always contaminated by variable amounts of endometrial tissue, so solution-based assays might not permit precise evaluation of cellular origin of telomerase or survivin expression. Thus, the current study used histochemical approaches for evaluation of telomerase and survivin expression in placental development and neoplasia. Survivin and telomerase were detected in most tissue samples, in all diagnostic categories, but few specimens were not stained. Failure to detect hTERT or survivin in those cases could result from expression levels that were below the threshold of detection by the immunohistochemical assays or could indicate that some cases did not express those proteins. However, lack of staining in those cases could also be a false–negative result, caused by methodologic problems or issues of suboptimal tissue fixation in clinical samples. No large variability in intensity of staining for telomerase was seen throughout placental development, in preeclampsia, or in post-term placental tissues. That result was somewhat unexpected because telomerase is believed to affect gestational development and maturation. The telomerase hTERT protein was localized to the trophoblastic cells of developing and molar placental tissue and other components of the placental villi, including nucleated hematologic precursors of first-trimester placenta and scattered villous stromal fibroblasts. Telomerase staining was markedly increased in hydatidiform molar tissue compared with non-neoplastic tissue specimens. Thus, telomerase expression might be involved in histogenesis of hydatidiform moles by contributing to maintenance of genomic stability in the hyperplastic trophoblastic cell component.
Previous studies concentrated on using sensitive telomeric repeat amplification protocol assay to evaluate the correlation of telomerase expression with the proliferation of the trophoblast.18 Telomerase expression was decreased in placental tissues from cases with fetal growth restriction19 but appeared to be increased over normal developmental levels in molar pregnancies.20,21 An in situ-TRAP approach indicated that telomerase activity was present in trophoblastic cells during normal placental development, but was not detectable at term.22 However, the current study showed that the hTERT protein could be detected at low levels in term and post-term placental tissue. Thus, hTERT activity could, in part, be regulated by post-translational mechanisms.
Ambrosini et al8 reported the immunohistochemical localization of survivin in many fetal tissues, including proximal tubular epithelium of the kidney, pneumocytes, epidermis, vertebral chondroblasts, and endometrial glands. Survivin mRNA was detected by Northern blot analysis in normal placental tissue.8 The immunohistochemical localization of survivin protein in placental tissue has not been reported. It was observed that survivin levels were lower in third-term placental development than in first- and second-term. That might suggest that inhibition of apoptosis has less effect on third-trimester placental maturation. Similar to qualitative observations for telomerase expression, levels of survivin expression in preeclamptic and post-term placental tissues appeared similar to levels that were seen in normal third-term development. Thus, survivin expression might be determined by gestational age of the tissue and is not influenced by maternal hypertension or post-term state. Survivin staining was higher in hydatidiform moles than in non-neoplastic tissues. Thus, survivin expression also might affect histogenesis of hydatidiform moles by inhibition of apoptotic cell death.
The molecular mechanisms that determine the sub-cellular localization of telomerase and survivin have not yet been fully defined. Suzuki et al23 reported that Fas stimulation and cell proliferation cause nuclear translocation of survivin in HepG2 cells. It was also suggested that nuclear translocation of survivin causes an accelerated formation of the Cdk4/Cyclin D1 complex, leading to phosphorylation of Rb and entry into the cell cycle. That might cause the release of p21 from the Cdk4/Cyclin D1 complex and translocation to the mitochondria, followed by interaction with procaspase 3, to suppress Fas-mediated cell death. Although the subcellular localization of telomerase has not yet been specifically addressed, the site of action of hTERT is in the nucleus. There could be disruption of the normal nuclear translocation mechanisms in neoplasia, and previous studies from our laboratory found evidence for nuclear and cytoplasmic localization of hTERT in squamous dysplasia and carcinoma.11,24 Further research might be warranted to evaluate the molecular mechanisms underlying the increased expression of telomerase and survivin in placental neoplasia.
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Footnotes |
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Received September 18, 2000. Received in revised form December 11, 2000. Accepted January 12, 2001.
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2. Nakayama J, Tahara H, Tahara E, Saito M, Ito K, Nakamura H, et al. Telomerase activation by hTRT in human normal fibroblasts and hepatocellular carcinomas. Nat Genet 1998;18:65–8.[Medline]
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7. Counter CM, Meyerson M, Eaton EN, Ellisen LW, Caddle SD, Haber DA, et al. Telomerase activity is restored in human cells by ectopic expression of hTERT (hEST2), the catalytic subunit of telomerase. Oncogene 1998;16:1217–22.[Medline]
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11. Frost M, Bobak JB, Gianani R, Kim N, Weinrich S, Spalding DC, et al. Localization of telomerase hTERT protein and hTR in benign mucosa, dysplasia, and squamous cell carcinoma of the cervix. Am J Clin Pathol 2000;114:726–34.[Medline]
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21. Nishi H, Ohyashiki K, Fujito A, Yahata N, Ohyashiki JH, Isaka K, et al. Expression of telomerase subunits and localization of telomerase activation in hydatidiform mole. Placenta 1999;20:317–23.[Medline]
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