Department of Obstetrics and Gynecology, Wakayama Medical University, Wakayama 641-0012, Japan
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Abstract |
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Key words: Apoptosis/Bax/Bcl-2/endometrial carcinoma/endometrial hyperplasia
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Introduction |
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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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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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Results |
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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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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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Acknowledgements |
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Notes |
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References |
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Submitted on December 13, 2000; accepted on July 9, 2001.