Human Reproduction, Vol. 16, No. 10, 2211-2218,
October 2001
© 2001 European Society of Human Reproduction and Embryology
Apoptosis and the expression of Bax and Bcl-2 in hyperplasia and adenocarcinoma of the uterine endometrium
Department of Obstetrics and Gynecology, Wakayama Medical University, Wakayama 641-0012, Japan
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BACKGROUND: Apoptosis plays a crucial role in carcinogenesis in various tumours. This study was designed to investigate the occurrence of apoptosis and the expression of Bcl-2 and Bax proteins in endometrial tumours of corpus uteri. METHODS: Endometrial tissues were obtained from 20 patients with endometrioid adenocarcinoma, 16 patients with endometrial hyperplasia, and 4 patients with myoma uteri (which were used as controls). The occurrence of apoptosis was examined by using molecular biochemical techniques. The expression of Bcl-2 and Bax proteins was also investigated using immunohistochemical staining with appropriate antibodies. RESULTS: The labelling of DNA in situ indicated that apoptotic cells were sporadically seen in postmenopausal endometrium (5.2 ± 2.1, n = 4) and endometrial hyperplasia without atypia (2.6 ± 0.5, n = 9). In contrast, labelled cells were detected in atypical endometrial hyperplasia (15.9 ± 2.2, n = 7), and their numbers increased intensely in adenocarcinoma (29.3 ± 3.7, n = 20). Autoradiographic analysis revealed DNA laddering in many cases of carcinoma. Bcl-2 was highly immunopositive in hyperplasia without atypia (36.2 ± 6.5%, n = 9), but was decreased in the atypical endometrial hyperplasia (16.3 ± 4.8%, n = 7). Large fractions of the carcinoma (6.3 ± 1.8%, n = 20) and normal endometrium (2.8 ± 1.4%, n = 4) were immunonegative or slightly immunopositive to Bcl-2. In contrast, Bax immunoreactivity was more frequent and stronger in adenocarcinoma (43.6 ± 4.1%, n = 20) than that in normal endometrium (17.6 ± 6.7%, n = 4) and hyperplasia (7.2 ± 2.2%, n = 16). CONCLUSIONS: These results suggest that cells in hyperplasia expressing Bcl-2 might have prolonged survival ability. Neoplastic cells in adenocarcinoma might show apoptosis in association with a decreased expression of Bcl-2 and an increased expression of Bax. Therefore, the frequency of apoptosis and the expression of Bcl-2 and Bax might be correlated with carcinogenesis in the uterine endometrium of humans.
Key words: Apoptosis/Bax/Bcl-2/endometrial carcinoma/endometrial hyperplasia
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Introduction |
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It has become clear that carcinogenesis cannot be explained simply in terms of the enhanced stimulation of cell growth, but may be caused by the loss of growth suppression and changes in apoptotic cell death (Williams, 1991
; Kerr et al., 1994). The key biochemical mechanism that is unique to apoptosis is the internucleosomal cleavage of the DNA (Arends et al., 1990). This cleavage of the DNA results in the appearance, on agarose gels, of a ladder-like pattern of fragments (Tilly and Hsueh, 1993). The labelling of the DNA in situ allows the detection of apoptotic cells in histological sections (Gavrieli et al., 1992). In the female reproductive tissues, it has been demonstrated that apoptosis occurs in the ovaries (Shikone et al., 1997), the uterine endometrium (Tabibzadeh et al., 1995; Kokawa et al., 1996), and the cervical carcinomas (Kokawa et al., 1999a, b). Some investigators have found apoptotic cells in endometrial carcinomas, using only morphological changes (Saegusa et al., 1996).
The orderly expression of several gene products is necessary for the completion of the apoptosis. The bcl-2 gene belongs to a group of proto-oncogenes that prolong cell survival by counteracting the process of apoptosis (Reed, 1994). The bax gene is an apoptosis-promoting member of the bcl-2 gene family (Oltvai et al., 1993). It is thought that the Bcl-2 protein forms heterodimers with the Bax protein in vivo, and that the molar ratio of Bcl-2 to Bax determines whether apoptosis is induced or inhibited in several tissues. Previous studies have demonstrated the expression of Bcl-2 in endometrial hyperplasia and adenocarcinoma (Saegusa et al., 1996; Nakamura et al., 1997). However, the expression of Bax has not been investigated in endometrial carcinomas. This study examined the occurrence of apoptosis by molecular biochemical techniques, and the expression of Bax and Bcl-2 by immunohistochemical staining.
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Materials and Methods |
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Materials
Endometrial tissues were obtained from 20 patients with endometrioid adenocarcinoma and from 16 patients with endometrial hyperplasia, who had received abdominal hysterectomies at an affiliate hospital of Wakayama Medical College (Wakayama, Japan) from 1988–2000. The histological subtype of the endometrial hyperplasia was divided into atypical endometrial hyperplasia (n = 7) and endometrial hyperplasia without atypia (n = 9). The histological differentiation of the endometrioid adenocarcinoma was divided into well differentiated (G1, n = 10), moderately differentiated (G2, n = 5), and poorly differentiated (G3, n = 5). The histological grading and clinical staging was based on the criteria of the International Federation of Gynecology and Obstetrics (FIGO stages, 1989). Normal postmenopausal endometria were obtained from 4 patients after simple hysterectomies for treatment of myoma uteri. The project was approved by the Committee on Investigations Involving Human Subjects of Wakayama Medical College. Informed consent was obtained from each subject after the purpose and nature of the study had been fully explained. After 3 washes in saline, each sample was rapidly frozen and stored at –70 °C and/or fixed in Bouin's solution.
In-situ analysis of the DNA fragmentation in the histological sections
For the analysis in situ, Bouin's-fixed, paraffin-embedded samples were subjected to 3'-end labelling of the DNA with digoxigenin-dideoxyUTP [(dig-ddUTP); Boehringer Mannheim, Indianapolis, IN, USA], and was performed as described previously (Kokawa et al., 1996). Terminal transferase (TdT; 25 IU; Boehringer Mannheim), dig-ddUTP and dideoxyATP were used at concentrations of 1 IU/µl, 1 µmol/l and 49 µmol/l, respectively. The standard substrates (337.5 µg/ml nitroblue tetrazolium and 175 µg/ml 5-bromo-4-chloro-3-inolyl-phosphate) were used for staining. After the colour reaction, the sections were counterstained with eosin. In this method, the apoptotic cells are indicated by a blue coloration of the nuclei. Positive and negative controls were included. The addition of DNase-1 led to the positive staining of all the nuclei, whereas the omission of either TdT or dig-ddUTP led to a complete absence of staining (data not shown).
Isolation and analysis of the apoptotic fragmentation of the DNA
The DNA was isolated from some frozen endometrial tissues and quantitated spectrophotometrically at 260 nm. Autoradiographic analyses of the apoptotic fragmentation of the DNA were performed as described previously (Kokawa et al., 1996). 1 µg of the DNA from each sample was labelled at the 3' end with [
32P]dideoxyATP (3000 Ci/mmol; Amersham, Arlington Heights, IL, USA) and TdT (25 U).
Immunohistochemical analyses of the Bcl-2 and Bax proteins
For the immunostaining, the avidin-biotin-peroxidase complex technique, using a Vectastain ABC kit (Vector Labs, Burlingame, CA, USA), was performed as described previously (Kokawa et al., 1999a, b). Bouin's-fixed, paraffin-embedded samples were incubated with monoclonal mouse anti-human Bcl-2 (clone 124, isotype IgG1; Dako, Glostrup, Denmark) or polyclonal rabbit anti-human Bax (isotype IgG; Calbiochem, Cambridge, MA, USA). Immunoreactivity specific to Bcl-2 and Bax was expressed as the percentage (%) of the cells exhibiting specific staining. The intensities of staining were assessed as negative (-), faintly positive (±), positive (+), or intensely positive (++). In addition, normal cyclical endometrium was used as a positive control during the proliferative phase for Bcl-2 (Figure 3D) and during the late secretory phase for Bax (Figure 4D), and specific stainings were detected. The negative controls were performed by replacing the primary antibody with the same dilution of preimmune mouse serum for Bcl-2 (Figure 3E) and rabbit serum for Bax (Figure 4E), and no immunoreaction product was observed. In addition, non-specific IgG was also used as a negative control, and no immunoreaction product was detected (data not shown).
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Analysis of data
For the in-situ analysis, an apoptotic index (AI) was calculated. This index corresponded to the number of positive cells per 100 cells. The cells in 3 microscopic fields per slide, and of 3 different slides of each specimen were counted. In addition, two of the authors (K.K. and T.S.) blindly enumerated the positive cells, and the variability between observers was <5 cells. The results obtained from the two authors did not differ significantly. A statistical analysis was performed by using an analysis of variance (ANOVA) and the unpaired Student's t-test, and a P value of < 0.05 reflected a significant difference from the corresponding control group. To check the reproducibility of the value of AI and the immunostaining for the Bcl-2 and Bax proteins, another gynaecologist, who was unaware of the original assessment, re-evaluated the same histological sections, and there was no difference between the two observers.
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Results |
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Analysis in situ of the DNA fragmentation in the histological sections
To identify the cells involved in the apoptotic fragmentation of DNA in the tumours of uterine endometrium, we labelled the DNA in the specimens at the 3' ends. There were some labelled cells in the postmenopausal endometrium (data not shown). Few labelled cells were seen in the patients with endometrial hyperplasia without atypia (Figure 1A
). The apoptotic cells increased in cases of atypical endometrial hyperplasia (Figure 1B), and increased intensely in endometrioid adenocarcinoma (Figure 1C). The values of AI in the postmenopausal endometrium, and the endometrial hyperplasia with and without atypia, are shown in Table I. The values of AI in the endometrioid adenocarcinoma are shown in Table II. The AI (mean ± SEM) in the atypical endometrial hyperplasia (15.9 ± 2.2, n = 7) was higher than that in the postmenopausal endometrium (5.3 ± 2.1, n = 4) and the endometrial hyperplasia without atypia (2.6 ± 0.5, n = 9, P < 0.05). There were significant differences in AI between the samples of the atypical endometrial hyperplasia (15.9 ± 2.2, n = 7) and the adenocarcinoma (29.3 ± 3.7, n = 20, P < 0.05). The AI in G3 was significantly elevated (47.1 ± 7.3 n = 5, P < 0.05) compared with that in G1 (23.8 ± 3.8, n = 10) and G2 (22.7 ± 6.2, n = 5). The value of AI did not differ significantly in each FIGO staging (Stage I, 26.7 ± 4.6; Stage II, 20.1 ± 4.2; Stage III, 37.7 ± 7.5). There were no significant differences between the endometrioid adenocarcinoma stage Ia (32.7 ± 9.2, n = 5) and the endometrioid adenocarcinoma progressed stages (28.2 ± 4.1, n = 15). However, the value of AI was significantly increased (45.4 ± 6.6) in patients who had metastasis of the lymph nodes (patients 32, 35, 36, 37 and 40), compared with the other 15 cases (24.0 ± 3.5).
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Analysis of the apoptotic fragmentation of the DNA
To verify that apoptosis, not necrosis, had occurred, we further examined the DNA ladder formation in some cases. Autoradiographic analysis revealed that high molecular weight DNA was predominant in all the samples of the postmenopausal endometrium and the endometrial hyperplasia with and without atypia. In contrast, a ladder pattern, characteristic of the apoptotic cleavage of DNA, was generated in 4 out of 7 patients (57%) with endometrioid adenocarcinoma (Figure 2
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Immunohistochemical staining of Bcl-2
Immunostaining specific for Bcl-2 was found in the cytoplasm of the cells. The localization of Bcl-2 was rare in the postmenopausal endometrium. In contrast, the Bcl-2 immunoreactivity was strong in the glandular epithelium of the endometrial hyperplasia with and without atypia (Figure 3
, A and B). However, many cancer cells in the endometrioid adenocarcinoma were negative or slightly positive (Figure 3C). In addition, the normal cyclical endometrium during the proliferative phase was used as a positive control for Bcl-2 (Figure 3D). The details of the Bcl-2 immunoreactivity for each patient are shown in Tables I and II. The expression of Bcl-2 in cases of hyperplasia (27.5 ± 4.8%, n = 16, P < 0.05) was stronger than that in the postmenopausal endometrium (2.8 ± 1.4%, n = 4) and the endometrioid adenocarcinoma (6.3 ± 1.8%, n = 20). The immunostaining of Bcl-2 in the endometrial hyperplasia without atypia (36.2 ± 6.5%, n = 9) was significantly elevated compared with that in the atypical endometrial hyperplasia (16.3 ± 4.8%, n = 7). The accumulation of Bcl-2 was 4.6 ± 2.0% for G1, 8.6 ± 5.2% for G2 and 7.3 ± 3.4% for G3, and there was no significant difference.
Immunohistochemical staining of Bax
Immunostaining specific for Bax was also cytoplasmic. The expression of Bax was rarely seen in the postmenopausal endometrium or the endometrial hyperplasia without atypia (Figure 4A). The localization of Bax was detected in the atypical endometrial hyperplasia (Figure 4B). Strongly positive staining was detected in all cases of the endometrioid adenocarcinoma (Figure 4C). In addition, the normal cyclical endometrium during the late secretory phase was used as a positive control for Bax (Figure 4D). The accumulation of Bax protein is summarized in Tables I and II. Overall, Bax immunoreactivity was more frequent and stronger in cases of adenocarcinoma (43.6 ± 4.1%, n = 20) than that in the cases of postmenopausal endometrium (17.6 ± 6.7%, n = 4) and hyperplasia (7.2 ± 2.2%, n = 16). The expression of Bax in the atypical endometrial hyperplasia (11.4 ± 4.1%, n = 7) tended to increase the value in the endometrial hyperplasia without atypia (3.9 ± 1.9%, n = 9), but there were no significant differences. The immunostaining for Bax was stronger in G3 (55.7 ± 2.0%, n = 5) than that in G1 (45.3 ± 6.0%, n = 15) and G2 (29.4 ± 8.2%, n = 5).
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Discussion |
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Saegusa and Okayasu reported that the number of apoptotic cells increased in endometrioid adenocarcinoma compared with those in endometrial hyperplasia, and the increase of AI was correlated with the histological differentiation (Saegusa and Okayasu, 1998
). Our findings support their data. However, the method for labelling the DNA in situ does not prove conclusively that apoptotic cell death occurs, because this analysis does not discriminate between apoptotic and necrotic cells. We further examined the occurrence of apoptosis in the patients with endometrial tumours using end labelling of the DNA gel fractionation. Autoradiographic analysis revealed that high molecular weight DNA was predominant in three cases of hyperplasia, whereas a ladder pattern that is characteristic of the apoptotic cleavage of DNA was observed in patients with adenocarcinoma. Together, the results of the in-situ and electrophoretic analyses indicate that the cells in the endometrioid adenocarcinoma underwent apoptotic cell death. In the clinical stage, some patients with endometrial hyperplasia revert to normal, others persist with the hyperplasia, and a few advance to endometrial adenocarcinoma. It is thought that the atypical endometrial hyperplasia includes the adenocarcinoma in situ. In the present study, we detected an increase in the apoptotic cells in the atypical endometrial hyperplasia compared with those in the endometrial hyperplasia without atypia. There is a positive relationship between the occurrence of apoptosis and the grade of atypia in human colorectal adenoma (Arai and Kino, 1995) and furthermore, cells undergoing apoptosis increase in the neoplastic lesion compared with those in normal or premalignant tissues (Baretton et al., 1996). Therefore, it is suggested that the occurrence of apoptosis might be linked with carcinogenesis of endometrioid adenocarcinoma.
It is commonly accepted that prolonged cell survival with inhibition of apoptosis is associated with prognosis. However, a positive correlation between the frequency of apoptosis and prognosis has been noted in several carcinomas, for example, in adenocarcinoma of the prostate (Aihara et al., 1995), in colorectal carcinoma (Baretton et al., 1996), and in non-Hodgkin's lymphomas (Leoncini et al., 1993). In cervical carcinoma, it is suggested that a high incidence of spontaneous apoptosis might predict poor prognosis and less responsiveness to radiation (Levine et al., 1994; Kokawa et al., 1999b). In endometrial carcinoma, clear cell carcinoma and serous adenocarcinoma are poorer prognoses than endometrioid adenocarcinoma, and the occurrence of apoptosis is more frequent in clear cell carcinoma and serous adenocarcinoma than in endometrioid adenocarcinoma (Kokawa et al., 2001). Our results have demonstrated that the appearance of apoptosis increases in G3 compared with G1 and G2 in endometrioid adenocarcinoma. The histological differentiation is one of the important prognostic factors in endometrial carcinoma. The value of AI was elevated in stage III compared with that in stages I and II, but there was no statistically significant difference. Five patients (patients 32, 35, 36, 37 and 40) had metastasis of the lymph nodes and three died within 1 year of primary therapy. The value of AI of these five patients had a significant increase compared with the other 15 cases. It is suggested that the frequency of apoptosis might be related to the prognosis and the ability of metastasis. However, there were no significant differences between the cases of stage Ia and the other advanced stages. Therefore, it seems likely that apoptosis may not be associated with local progressive potential in endometrioid adenocarcinoma.
The overexpression of the Bcl-2 protein can block apoptosis and prolong cell survival, and it can, therefore, play an important role in carcinogenesis (Reed, 1994). Some investigators have shown that Bcl-2 expression is localized in many cases of endometrioid adenocarcinoma (Taskin et al., 1997; Sakuragi et al., 1998). In our study, 9 out of 20 cases were immunopositive for Bcl-2 in endometrioid adenocarcinoma. Other earlier reports, however, have shown that Bcl-2 immunoreactivity is relatively high in endometrial hyperplasia without atypia, but is markedly down-regulated in the atypical endometrial hyperplasia and endometrioid adenocarcinoma (Henderson et al., 1996; Nakamura et al., 1997). Our results demonstrate that the expression of Bcl-2 decreases in the atypical endometrial hyperplasia compared to that in the endometrial hyperplasia without atypia, and only slight immunostaining is detected in endometrioid adenocarcinoma. There were no significant differences between the histological differentiation, the clinical staging and the Bcl-2 expression. These results suggest that Bcl-2 expression might be essential to generate endometrial hyperplasia without atypia, and that it might be associated with the progression of atypical endometrial hyperplasia to endometrioid adenocarcinoma. Moreover, it has been shown that some patients with endometrioid adenocarcinoma show loss of heterozygosity at several chromosome 18q loci (Gima et al., 1994; Ronnett et al., 1997). It is speculated that this Bcl-2 down-regulation in endometrioid adenocarcinoma might be associated with genetic errors.
Bax protein forms homodimers and heterodimers with Bcl-2 in vivo and can induce Bcl-2-inhibited apoptosis. However, Bax expression in patients with carcinoma of the uterus is not entirely understood (Kokawa et al., 1999a, b). In our study, the immunoreactivity of Bax was significantly higher in endometrioid adenocarcinoma than that in postmenopausal endometrium, and endometrial hyperplasia with and without atypia. There was an inverse correlation between the expression of Bcl-2 and Bax, and the ratio of Bcl-2 to Bax declined in endometrioid adenocarcinoma. It is possible that these increased expressions of Bax might induce apoptosis in endometrioid adenocarcinoma, and might be correlated with the progression of atypical endometrial hyperplasia to endometrioid adenocarcinoma.
Using molecular biochemical techniques, we have demonstrated the distribution of apoptosis in endometrial tumours of corpus uteri. Our results indicate that the incidence of apoptosis increases in atypical endometrial hyperplasia, and markedly increases in endometrioid adenocarcinoma, especially G3. It suggests that the occurrence of apoptosis may play a critical role in carcinogenesis and differentiation in endometrioid adenocarcinoma. The immunohistochemical analysis revealed that Bcl-2 expression is stronger in endometrial hyperplasia with and without atypia than postmenopausal endometrium and endometrioid adenocarcinoma. It is possible that the overexpression of Bcl-2 may be associated with the generation of endometrial hyperplasia. In contrast, a widespread expression of Bax was observed in endometrioid adenocarcinoma compared with that in the endometrial hyperplasia with and without atypia. This indicates that the overexpression of Bax may be correlated with the progression from hyperplasia to carcinoma. We speculate that the proportion of Bcl-2 and Bax proteins might be linked to the regulation of apoptosis in endometrial tumours.
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This work was supported by a research grant from the 2000 Wakayama Medical Award for Young Researchers.
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1 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Wakayama Medical University,811–1 Kimiidera, Wakayama 641-0012, Japan. E-mail: katsu{at}wakayama-med.ac.jp
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Submitted on December 13, 2000; accepted on July 9, 2001.
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