- Email: [email protected]
Cyclic bcl-2 gene expression in human uterine endometrium during menstrual cycle
Non-A, non-B hepatitis
Controls
Paracetamol overdose
5
6
7
8
Han S, Stuart LA, Degen SJ. Characterization of the DNF15S2 locus on human chromosome 3: identification of a gene coding for four kringle domains with homology to hepatocyte growth factor. Biochemistry 1991; 30: 9768-80. Canalese J, Gove CD, Gimson AES, Wilkinson SP, Wardle EN, Williams R. Reticuloendothelial system and hepatocyte function in fulminant hepatic failure. Gut 1982; 23: 265-69. Rolando N, Harvey F, Brahm J, et al. Prospective study of bacterial infection in acute liver failure: an analysis of fifty patients. Hepatology 1990; 11: 49-53. Hughes RD, Imawari M, Bihari D, Almasio PL, Langley PG, Williams R. Fibronectin replacement in patients with fulminant hepatic failure. Eur J Clin Invest 1986; 16: 352-56.
Department of Molecular Medicine (P Harrison MD, F Farzaneh phD) and Institute of Liver Studies (P Harrison, R Williams MD), King’s College School of Medicine and Dentistry, Bessemer Road, London SE5 9PJ, UK; and Division of Basic Science Research, Children’s Hospital Research Foundation, Cincinnati, Ohio, USA (S J Friezner Degen PhD) Correspondence to:
Figure:
Northern
hybrldlsation
0-05) (figure). In the nine patients, H3 histone mRNA were inversely correlated with those of the 3,0 kb HGFL/MSP mRNA transcript (r,= -0-83 [95% CI 0-23 to - 0-96], p<0°05). This study demonstrates that reduced hepatic HGFL/ MSP synthesis due to hepatocyte necrosis could be the cause of impaired Kupffer cell phagocytosis in fulminant hepatic failure. The early recovery of Kupffer cell function in such cases has a favourable bearing on survival ;6 therefore, administration of HGFL/MSP to promote Kupffer cell phagocytosis might reduce the frequency of infection and improve outcome. p<
levels
Dr
Phillip Harrison
Cyclic bcl-2 gene expression in human uterine endometrium during menstrual cycle
-
On the basis of the localisation of the gene to the tumour on chromosome 3,5 HGFL/ MSP might also be involved in growth suppression. The chalone theory of control of hepatic regeneration proposes that the normal liver is actively prevented from proliferation by a growth inhibitor synthesised by the liver itself: after hepatic necrosis or partial hepatectomy the concentration of the inhibitor falls, enabling the liver to grow until normal inhibitory tone is restored. The pattern of hepatic HGFL/MSP mRNA expression we found supports the hypothesis that HGFL/MSP might be such an inhibitory chalone. suppressor DNF15S2 locus
PH was supported by a training fellowship from the Medical Research Council of Great Britain. SJFD is an Established Investigator of the American Heart Association.
References 1
2 3
4
JA, Witte DP, Aronow BJ, Degen SJF. Hepatocyte-specific expression of the mouse hepatocyte growth factor-like protein. Hepatology 1993; 18: 394-99. Leonard EJ, Skeel A. A serum protein that stimulates macrophage movement, chemotaxis and spreading. Exp Cell Res 1976; 102: 434-38. Skeel A, Yoshimura T, Showalter SD, Tanaka S, Appella E, Leonard EJ. Macrophage stimulating protein: purification, partial amino acid sequence, and cellular activity. J Exp Med 1991; 173: Bezerra
1227-34. Yoshimura T, Yuhki N, Wang M, Skeel A, Leonard EJ. Cloning, sequencing, and expression of human macrophage stimulating protein (MSP, MST1) confirms MSP as a member of the family of kringle proteins and locates the MSP gene on chromosome 3. J Biol Chem 1993; 268: 15461-68.
To investigate an apoptotic role for bcl-2 in menses, we studied bcl-2 protein production throughout the cycle. Bcl-2 protein was observed in endometrial glandular, stromal and myometrial smooth-muscle cells. Glandular cells expressed bcl-2 at proliferative through early secretory phases but not at late secretory through menstrual phases. This cyclic expression of bcl-2 gene was similar to that of oestrogen and progesterone receptors. Thus bcl-2 expression in glandular cells may be regulated by ovarian hormones. The disappearance of bcl-2 expression in glandular cells at late secretory phase was consistent with the appearance of apoptotic cells in the same phase. Lancet 1994; 344: 28-29
Menses is functional
generally regarded as ischaemic necrosis of a layer caused by contraction of spiral arteries,
depending on sex hormone concentrations. Electron microscopy showed apoptotic bodies mainly in the late secretory phase.’ bcl-2 protein can block apoptosis in various cell lines. 2.3 Therefore, uterine endometrium is an attractive tissue for studying the roles of bcl-2 during tissue reconstruction and also regulation of bcl-2 gene expression by hormones. We have studied the distribution of bcl-2 protein in human uterine samples throughout the menstrual cycle in relation to the distribution of oestrogen receptor (ER) and progesterone receptor (PgR), and the role of bcl-2 protein in apoptosis during menstruation. Uterine
samples were collected from 21 women undergoing hysterectomy with informed consent and were obtained from the fundus within the uterine cavity, and contained endometrium and underlying myometrium. Monoclonal antibodies against human bcl-2 protein (MoAb 100, a gift from Dr David Mason, John Radcliffe Hospital, Oxford, UK), against human ER and PgR
We have demonstrated that bcl-2 expression of human endometrial glandular cells changes periodically during the menstrual cycle. This cyclic bcl-2 expression pattern in endometrial glandular cells was related to those in ER and PgR, suggesting regulation of bcl-2 expression by ovarian hormones. Several motif-like sequences for oestrogen hormone receptor binding are found within the bcl-2
promoter.7 The phase when bcl-2 expression disappears corresponded to that of appearance of apoptotic cells in
Figure: Production of bcl-2, ER, and PgR In endometrial epithelia 21 cases were stained with bcl-2, ER, and PgR antibodies and scored blind. Numbers of cases tested in each phase were: menses 5; early proliferative 3; late proliferative 6; early secretory 3; and late secretory 4.
(Abbott, North Chicago) were used. Tissue samples were prepared immunohistochemistry: serial cryo-sections lightly fixed in 4% paraformaldehyde were immunostained with an indirect immunoperoxidase technique for light microscopy; and samples fixed in a solution containing 005% glutaraldehyde and 4% paraformaldehyde were cut into serial sections (40-60 pm) on a microslicer. After immunostaining with bcl-2 antibody, ultrathin sections were prepared for electron microscopy. The remaining uterine tissues were isolated into endometrial glandular, stromal, and myometrial cells for western blot analysis4 with bcl-2 antibody. for
Strong bcl-2 immunoreactivity was observed in endometrial glandular, stromal, and myometrial smoothmuscle cells. In particular, glandular cells produced bcl-2 at proliferative through early secretory phases (figure). The bcl-2 immunoreactivity of glandular epithelium increased as uterine glands extended from the functional to the basal layer. Vascular smooth-muscle cells did not show immunoreactivity for bcl-2, ER, or PgR throughout the menstrual cycle. Endometrial samples in the late secretory and menstrual phases did not stain for bcl-2 in all specimens examined (figure). At high magnificaton, bcl-2 was detected throughout the cell cytoplasm, whereas ER and PgR were seen in the nucleus. This cyclic expression pattern of bcl-2 in endometrial glandular cells was related to changes in ER and PgR throughout the cycle (figure). Western blot confirmed these immunohistochemical data; bcl-2 protein was detected in endometrial and myometrial cells at late proliferative phase. Moreover, bcl-2 protein detected in endometrium was mainly contributed by endometrial glandular cells and not by stromal cells. Electron microscopy showed that some epithelial cells were reduced in volume and rounded in shape, and nuclear chromatin condensation and fragmentation were often observed at late secretory phase. These apoptotic cells were gradually pushed toward the lumen and removed from the epithelium into the lumen. The bcl-2 protein was localised on multiple membrane sites, such as the outer nuclear envelope, inner and outer mitochondrial membranes, and endoplasmic reticular membrane, which confirms previous observations.5.6
endometrium. This observation suggests that bcl-2 has an important role in regulating death of glandular cells, mainly in the functional layer at late secretory phase. We have shown bcl-2 protein in myometrial smooth-muscle cells by immunohistochemistry and immunoblotting, and this finding agrees with another report.8 Another study with human fetal tissues9 revealed that bcl-2 protein was expressed in the myometrium but not in vascular smoothmuscle cells. Thus, smooth-muscle cells in myometrium may exert a specific function controlled by bcl-2 expression, which may not be shared by vascular smoothmuscle cells. Our results highlight the relation between the physiological events in menstrual cycle and bcl-2 expression, and the possibility of regulation of bcl-2 expression by ovarian hormones, especially oestrogen. This work was funded in part by the comprehensive 10-Year Strategy for Cancer Control for the Ministry of Health and Welfare and by the Ministry of Education, Science and Culture in Japan.
References 1 2 3
Hopwood D, Levison DA. Atrophy and apoptosis in the cyclical human endometrium. J Pathol 1975; 119: 159-66. Tsujimoto Y. Stress-resistance conferred by high level of bcl-2a protein in human B lymphoblastoid cell. Oncogene 1989; 4: 1331-36. Garcia I, Martinou I, Tsujimoto Y, Martinou J-C. Prevention of programmed cell death of sympathetic neurones by the bcl-2 protooncogene. Science
4
1992; 258: 303-04.
Satyaswaroop PG, Bressler RS, Pena MM, Gurpide E. Isolation and culture of human endometrial glands. J Clin Endocrinol Metab 1979; 48: 639-41.
5
6
7
8 9
Monaghan P, Robertson D, Amos TAS, Dyer MJS, Mason DU, Greaves MF. Ultrastructural localization of Bcl-2 protein. J Histochem Cytochem 1992; 40: 1819-25. Akao Y, Otsuki Y, Kataoka S, Ito Y, Tsujimoto Y. Multiple subcellular localization of bcl-2: detection in nuclear outer membrane, endoplasmic reticulum and mitochondrial inner and outer membranes. Cancer Res 1994; 84: 2468-71. Adachi M, Tsujimoto Y. Potential Z-DNA elements surrounded the breakpoints of chromosome translocation within the 5’ flanking region of bcl-2 gene. Oncogene 1990; 5; 1653-57. Lu QL, Poulson R, Wong L, Hanby MA. Bcl-2 expression in adult and embryonic non-hematopoietic tissues. J Pathol 1993; 169: 431-37. LeBrun DP, Warnke RA, Cleary ML. Expression of bcl-2 in fetal tissues suggests a role in morphogenesis. Am J Pathol 1993; 142: 743-53.
Departments of Anatomy and Biology (Prof Y Otsuki MD, Y Ito MD, Y Akao MD) and of Obstetrics and Gynaecology (O Misaki MD, Prof O Sugimoto MD), Osaka Medical College, Takatsuki, Osaka, Japan; and Biomedical Research Center, Osaka University Medical School, Osaka (Prof Y Tsujimoto PhD) Correspondence to: Prof Yoshinori Otsuki, Department of Anatomy and Biology, Osaka Medical College, 2-7, Diagaku-machi, Takatsuki, Osaka 569, Japan
29
Controls
Paracetamol overdose
5
6
7
8
Han S, Stuart LA, Degen SJ. Characterization of the DNF15S2 locus on human chromosome 3: identification of a gene coding for four kringle domains with homology to hepatocyte growth factor. Biochemistry 1991; 30: 9768-80. Canalese J, Gove CD, Gimson AES, Wilkinson SP, Wardle EN, Williams R. Reticuloendothelial system and hepatocyte function in fulminant hepatic failure. Gut 1982; 23: 265-69. Rolando N, Harvey F, Brahm J, et al. Prospective study of bacterial infection in acute liver failure: an analysis of fifty patients. Hepatology 1990; 11: 49-53. Hughes RD, Imawari M, Bihari D, Almasio PL, Langley PG, Williams R. Fibronectin replacement in patients with fulminant hepatic failure. Eur J Clin Invest 1986; 16: 352-56.
Department of Molecular Medicine (P Harrison MD, F Farzaneh phD) and Institute of Liver Studies (P Harrison, R Williams MD), King’s College School of Medicine and Dentistry, Bessemer Road, London SE5 9PJ, UK; and Division of Basic Science Research, Children’s Hospital Research Foundation, Cincinnati, Ohio, USA (S J Friezner Degen PhD) Correspondence to:
Figure:
Northern
hybrldlsation
0-05) (figure). In the nine patients, H3 histone mRNA were inversely correlated with those of the 3,0 kb HGFL/MSP mRNA transcript (r,= -0-83 [95% CI 0-23 to - 0-96], p<0°05). This study demonstrates that reduced hepatic HGFL/ MSP synthesis due to hepatocyte necrosis could be the cause of impaired Kupffer cell phagocytosis in fulminant hepatic failure. The early recovery of Kupffer cell function in such cases has a favourable bearing on survival ;6 therefore, administration of HGFL/MSP to promote Kupffer cell phagocytosis might reduce the frequency of infection and improve outcome. p<
levels
Dr
Phillip Harrison
Cyclic bcl-2 gene expression in human uterine endometrium during menstrual cycle
-
On the basis of the localisation of the gene to the tumour on chromosome 3,5 HGFL/ MSP might also be involved in growth suppression. The chalone theory of control of hepatic regeneration proposes that the normal liver is actively prevented from proliferation by a growth inhibitor synthesised by the liver itself: after hepatic necrosis or partial hepatectomy the concentration of the inhibitor falls, enabling the liver to grow until normal inhibitory tone is restored. The pattern of hepatic HGFL/MSP mRNA expression we found supports the hypothesis that HGFL/MSP might be such an inhibitory chalone. suppressor DNF15S2 locus
PH was supported by a training fellowship from the Medical Research Council of Great Britain. SJFD is an Established Investigator of the American Heart Association.
References 1
2 3
4
JA, Witte DP, Aronow BJ, Degen SJF. Hepatocyte-specific expression of the mouse hepatocyte growth factor-like protein. Hepatology 1993; 18: 394-99. Leonard EJ, Skeel A. A serum protein that stimulates macrophage movement, chemotaxis and spreading. Exp Cell Res 1976; 102: 434-38. Skeel A, Yoshimura T, Showalter SD, Tanaka S, Appella E, Leonard EJ. Macrophage stimulating protein: purification, partial amino acid sequence, and cellular activity. J Exp Med 1991; 173: Bezerra
1227-34. Yoshimura T, Yuhki N, Wang M, Skeel A, Leonard EJ. Cloning, sequencing, and expression of human macrophage stimulating protein (MSP, MST1) confirms MSP as a member of the family of kringle proteins and locates the MSP gene on chromosome 3. J Biol Chem 1993; 268: 15461-68.
To investigate an apoptotic role for bcl-2 in menses, we studied bcl-2 protein production throughout the cycle. Bcl-2 protein was observed in endometrial glandular, stromal and myometrial smooth-muscle cells. Glandular cells expressed bcl-2 at proliferative through early secretory phases but not at late secretory through menstrual phases. This cyclic expression of bcl-2 gene was similar to that of oestrogen and progesterone receptors. Thus bcl-2 expression in glandular cells may be regulated by ovarian hormones. The disappearance of bcl-2 expression in glandular cells at late secretory phase was consistent with the appearance of apoptotic cells in the same phase. Lancet 1994; 344: 28-29
Menses is functional
generally regarded as ischaemic necrosis of a layer caused by contraction of spiral arteries,
depending on sex hormone concentrations. Electron microscopy showed apoptotic bodies mainly in the late secretory phase.’ bcl-2 protein can block apoptosis in various cell lines. 2.3 Therefore, uterine endometrium is an attractive tissue for studying the roles of bcl-2 during tissue reconstruction and also regulation of bcl-2 gene expression by hormones. We have studied the distribution of bcl-2 protein in human uterine samples throughout the menstrual cycle in relation to the distribution of oestrogen receptor (ER) and progesterone receptor (PgR), and the role of bcl-2 protein in apoptosis during menstruation. Uterine
samples were collected from 21 women undergoing hysterectomy with informed consent and were obtained from the fundus within the uterine cavity, and contained endometrium and underlying myometrium. Monoclonal antibodies against human bcl-2 protein (MoAb 100, a gift from Dr David Mason, John Radcliffe Hospital, Oxford, UK), against human ER and PgR
We have demonstrated that bcl-2 expression of human endometrial glandular cells changes periodically during the menstrual cycle. This cyclic bcl-2 expression pattern in endometrial glandular cells was related to those in ER and PgR, suggesting regulation of bcl-2 expression by ovarian hormones. Several motif-like sequences for oestrogen hormone receptor binding are found within the bcl-2
promoter.7 The phase when bcl-2 expression disappears corresponded to that of appearance of apoptotic cells in
Figure: Production of bcl-2, ER, and PgR In endometrial epithelia 21 cases were stained with bcl-2, ER, and PgR antibodies and scored blind. Numbers of cases tested in each phase were: menses 5; early proliferative 3; late proliferative 6; early secretory 3; and late secretory 4.
(Abbott, North Chicago) were used. Tissue samples were prepared immunohistochemistry: serial cryo-sections lightly fixed in 4% paraformaldehyde were immunostained with an indirect immunoperoxidase technique for light microscopy; and samples fixed in a solution containing 005% glutaraldehyde and 4% paraformaldehyde were cut into serial sections (40-60 pm) on a microslicer. After immunostaining with bcl-2 antibody, ultrathin sections were prepared for electron microscopy. The remaining uterine tissues were isolated into endometrial glandular, stromal, and myometrial cells for western blot analysis4 with bcl-2 antibody. for
Strong bcl-2 immunoreactivity was observed in endometrial glandular, stromal, and myometrial smoothmuscle cells. In particular, glandular cells produced bcl-2 at proliferative through early secretory phases (figure). The bcl-2 immunoreactivity of glandular epithelium increased as uterine glands extended from the functional to the basal layer. Vascular smooth-muscle cells did not show immunoreactivity for bcl-2, ER, or PgR throughout the menstrual cycle. Endometrial samples in the late secretory and menstrual phases did not stain for bcl-2 in all specimens examined (figure). At high magnificaton, bcl-2 was detected throughout the cell cytoplasm, whereas ER and PgR were seen in the nucleus. This cyclic expression pattern of bcl-2 in endometrial glandular cells was related to changes in ER and PgR throughout the cycle (figure). Western blot confirmed these immunohistochemical data; bcl-2 protein was detected in endometrial and myometrial cells at late proliferative phase. Moreover, bcl-2 protein detected in endometrium was mainly contributed by endometrial glandular cells and not by stromal cells. Electron microscopy showed that some epithelial cells were reduced in volume and rounded in shape, and nuclear chromatin condensation and fragmentation were often observed at late secretory phase. These apoptotic cells were gradually pushed toward the lumen and removed from the epithelium into the lumen. The bcl-2 protein was localised on multiple membrane sites, such as the outer nuclear envelope, inner and outer mitochondrial membranes, and endoplasmic reticular membrane, which confirms previous observations.5.6
endometrium. This observation suggests that bcl-2 has an important role in regulating death of glandular cells, mainly in the functional layer at late secretory phase. We have shown bcl-2 protein in myometrial smooth-muscle cells by immunohistochemistry and immunoblotting, and this finding agrees with another report.8 Another study with human fetal tissues9 revealed that bcl-2 protein was expressed in the myometrium but not in vascular smoothmuscle cells. Thus, smooth-muscle cells in myometrium may exert a specific function controlled by bcl-2 expression, which may not be shared by vascular smoothmuscle cells. Our results highlight the relation between the physiological events in menstrual cycle and bcl-2 expression, and the possibility of regulation of bcl-2 expression by ovarian hormones, especially oestrogen. This work was funded in part by the comprehensive 10-Year Strategy for Cancer Control for the Ministry of Health and Welfare and by the Ministry of Education, Science and Culture in Japan.
References 1 2 3
Hopwood D, Levison DA. Atrophy and apoptosis in the cyclical human endometrium. J Pathol 1975; 119: 159-66. Tsujimoto Y. Stress-resistance conferred by high level of bcl-2a protein in human B lymphoblastoid cell. Oncogene 1989; 4: 1331-36. Garcia I, Martinou I, Tsujimoto Y, Martinou J-C. Prevention of programmed cell death of sympathetic neurones by the bcl-2 protooncogene. Science
4
1992; 258: 303-04.
Satyaswaroop PG, Bressler RS, Pena MM, Gurpide E. Isolation and culture of human endometrial glands. J Clin Endocrinol Metab 1979; 48: 639-41.
5
6
7
8 9
Monaghan P, Robertson D, Amos TAS, Dyer MJS, Mason DU, Greaves MF. Ultrastructural localization of Bcl-2 protein. J Histochem Cytochem 1992; 40: 1819-25. Akao Y, Otsuki Y, Kataoka S, Ito Y, Tsujimoto Y. Multiple subcellular localization of bcl-2: detection in nuclear outer membrane, endoplasmic reticulum and mitochondrial inner and outer membranes. Cancer Res 1994; 84: 2468-71. Adachi M, Tsujimoto Y. Potential Z-DNA elements surrounded the breakpoints of chromosome translocation within the 5’ flanking region of bcl-2 gene. Oncogene 1990; 5; 1653-57. Lu QL, Poulson R, Wong L, Hanby MA. Bcl-2 expression in adult and embryonic non-hematopoietic tissues. J Pathol 1993; 169: 431-37. LeBrun DP, Warnke RA, Cleary ML. Expression of bcl-2 in fetal tissues suggests a role in morphogenesis. Am J Pathol 1993; 142: 743-53.
Departments of Anatomy and Biology (Prof Y Otsuki MD, Y Ito MD, Y Akao MD) and of Obstetrics and Gynaecology (O Misaki MD, Prof O Sugimoto MD), Osaka Medical College, Takatsuki, Osaka, Japan; and Biomedical Research Center, Osaka University Medical School, Osaka (Prof Y Tsujimoto PhD) Correspondence to: Prof Yoshinori Otsuki, Department of Anatomy and Biology, Osaka Medical College, 2-7, Diagaku-machi, Takatsuki, Osaka 569, Japan
29