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(A) Normal myometrium stained with mAbH. High expression of epitope H in smooth muscle cells (closed asterisk). Unstained bundle of collagen (open asterisk). Nuclei of unstained fibroblasts (arrows). Mag  250 (B) Leiomyoma stained with mAbH. Moderate cytoplasmic expression of epitope H. Mag  400 (C) Leiomyoma stained with mAbH. High cytoplasmic expression of epitope H. Mag  400 (D) Leiomyosarcoma stained with mAbH. Moderate cytoplasmic expression of epitope H. Mitoses (arrow heads). Mag  400 (E) Leiomyosarcoma stained with mAbH. High cytoplasmic expression of epitope H. Mitosis (arrow head). Mag  400 (F) Leiomyosarcoma stained with mAbH. Moderate cytoplasmic expression of epitope H. Abnormal mitosis (arrow). Mag  400 (G) Leiomyoma stained with AbCK8. Absence of staining. Mag  400 (H) Leiomyosarcoma stained with AbCK8. Absence of staining. Mitoses (arrow heads). Mag  400.
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Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAc) residue in a specific conformation and/or environment recognized by monoclonal antibody H (mAbH). We have previously shown that epitope H is present in more than one polypeptide and in various types of normal and pathological cells. In the present study, we focused on uterine smooth musc...
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... that it may play a role in the normal histophysiology and in the pathology of many diseases, including cancer [12,35]. In this respect, we have recently generated the monoclonal antibody H (mAbH), which recognizes epitope H, consisting of an O-GlcNAc and neighboring amino acids [1,2]. The mAbH stains two bands with Mr  10 À 3 of 209 and 62 in lysates of cultured rat astrocytes [2]. Using mAbH, we found that epitope H is shared by cytokeratin 8—which contains single O-GlcNAc residues [10]—and five additional polypeptides with Mr  10 À 3 of 232, 67, 51, 50, and 37 lysates of cultured MCF-7 human breast carcinoma cell line cells [1]. To investigate the expression levels and the cellular distribution of epitope H in human tissues, we used mAbH for the immunostaining of various normal and pathological tissues, including infiltrating ductal breast carcinomas, fibroadenomas, normal human brains, human brains with a variety of lesions, astrocytic tumors and mitochondria-rich normal, metaplastic and neoplastic cells [1–4,19]. In normal human brains, epitope H was absent from the overwhelming majority of normal astrocytes, and only sparse immunoreactivity was observed, confined mostly to fibrous astrocytes [2]. Upregulation of epitope H immunoexpression was found in reactive astrocytes observed in pathological specimens from a variety of brain lesions, including anisomorphic and isomorphic gliosis [2]. Epitope H immunoexpression was higher in astrocytomas compared to anaplastic astrocytomas and glioblastomas [3]. Infiltrating ductal breast carcinomas showed moderate cytoplasmic immunostaining of the malignant cells with mAbH. In addition, small groups of tumor cells displayed intense cytoplasmic mAbH immunostaining [1]. The intensity of mAbH ultrastructural immunostaining in the mitochondria, nucleoli, and cytoplasmic vesicles was decreased in infiltrating ductal breast carcinomas when compared to fibroadenomas [19], and mitochondria-rich normal, metaplastic and neoplastic cells showed overexpression of epitope H [4]. The above-mentioned findings prompted us to investigate the expression pattern of epitope H in uterine smooth muscle cell tumors and their adjacent myometrium. Therefore, we used mAbH to investigate the immunohistochemical expression of epitope H in 50 cases of uterine leiomyomas with their adjacent myometrium and in five cases of uterine leiomyosarcomas with their adjacent myometrium. Furthermore, as our previous Western immunoblotting data showed that epitope H is shared by cytokeratin 8—which contains single O-GlcNAc residues [10]—and five additional polypeptides in the MCF-7 human breast carcinoma cell line [1], we attempted to clarify the relations between epitope H expression and CK8 expression in uterine smooth muscle cell tumors and their adjacent normal myometrium. The material of the present study consisted of 50 cases of typical uterine leiomyomas and their adjacent myometrium, and of five cases of uterine leiomyosarcomas, four of which belonged to Group II A and one to Group III according to Bell et al. [6] and their adjacent myometrium. For immunodetection of epitope H, the indirect immunoperoxidase procedure was applied as described in detail previously [2]. Briefly, tissue sections about 4 m m were cut from formalin-fixed, paraffin-embedded tumor blocks. After deparaffinization and blocking of endogenous peroxidase activity by immersing the sections in 3% H 2 O 2 in Tris–Saline buffer pH 7.6, the sections were incubated in 10% normal rabbit serum in buffer for 30 min to inhibit non-specific binding of antibodies. The sections were then incubated in undiluted supernatant containing the mouse monoclonal antibody H [2] for 2 h at room temperature. After washing for 3  10 min in buffer, the sections were incubated with peroxidase- conjugated rabbit anti-mouse antibody diluted 1:50 in buffer for 1 h. After washing for 3  10 min, the color was developed by incubating the sections in DAB-H 2 O 2 in buffer for 8 min; then, after washing, counterstaining in hematoxylin, and dehydrating, the sections were covered with permount. In the negative control sections, the primary antibody was omitted. The staining pattern was graded as negative ( À ) when no stained cells were present, as weak (+) when less than 30% of the cells were stained, as moderate (++) when 30–75% of the cells were stained, and as intense (+++) when 75–100% of the cells were stained. Cytokeratin 8 immunostaining was performed using the monoclonal anti-cytokeratin 8 antibody (AbCK8) from Sigma Chemical according to the previously described protocol [19]. Western immunoblotting was performed using mAbH and monoclonal anti- cytokeratin 8 antibody (AbCK8) from Sigma Chemical according to the previously described protocol [1]. The main results were as follows: (1) epitope H showed intense cytoplasmic immunohistochemical ex- pression in 46% (23/50) and moderate immunohistochemical expression in 54% (27/50) of uterine leiomyomas (Fig. 1), (2) epitope H showed intense cytoplasmic immunohistochemical expression in 40% (2/5) and moderate immunohistochemical expression in 60% (3/5) of uterine leiomyosarcomas (Fig. 1), (3) epitope H showed no difference in cytoplasmic immunohistochemical expression between leiomyomas and their adjacent myometrium, and between leiomyosarcomas and their adjacent myometrium, (4) immunohistochemical expression of cytokeratin 8 was detected in no case of leiomyomas, leiomyosarcomas, or their adjacent myometrium (glandular epithelium of the endometrium was stained by monoclonal anti-cytokeratin 8 antibody (AbCK8) and served as an internal positive control), (5) Western immunoblotting showed that in the smooth muscle cells of the myometrium and the leiomyomas, epitope H is localized in four polypeptides, with molecular weights of 100, 61, 59, and 54 kDa (Fig. 2), and (6) Western immunoblotting did not detect cytokeratin 8 in the normal and neoplastic smooth muscle cells. In the present study, epitope H showed (1) intense immunohistochemical expression in 46% (27/50) and moderate immunohistochemical expression in 54% (23/50) of uterine leiomyomas and (2) intense immunohistochemical expression in 40% (2/5) and moderate immunohistochemical expression in 60% (3/5) of uterine leiomyosarcomas. These results indicate that epitope H expression fluctuates in normal and neoplastic human myometrium, as it is expressed either moderately or strongly in uterine leiomyosarcomas, uterine leiomyomas, and their adjacent myometrium. Taken together, our present and previous results [1–4,18] provide further evidence that epitope H expression fluctuates in normal and neoplastic tissues. Indeed, (1) epitope H is absent in the overwhelming majority of normal astrocytes and becomes upregulated in reactive astrocytes, (2) epitope H expression is significantly higher in astrocytomas than in anaplastic astrocytomas and glioblastomas, (3) the intensity of mAbH immunostaining was moderate mainly in malignant cells of infiltrating ductal breast carcinomas, but small groups of the malignant cells displayed intense mAbH immunostaining, (4) the intensity of mAbH ultrastructural immunostaining in the mitochondria, nucleoli, and cytoplasmic vesicles was decreased in infiltrating ductal breast carcinomas when compared to fibroadenomas, and (5) mitochondria-rich normal, metaplastic, and neoplastic cells showed overexpression of epitope H [1–4,19]. As epitope H contains an O-GlcNAc residue [2], it is possible that the fluctuations of the epitope H expression we found in the present and previous studies [1–4,19] reflect differences in the expression of a O-GlcNAc-glycosylated cellular protein or proteins. These fluctuations may be of interest for gaining insight into the pathology of uterine smooth muscle cell tumors, as O-GlcNAc glycosylation may modify proteins involved in important biological functions such as cytoskeletal proteins, transcription factors, heat-shock proteins, chromatin proteins, tumor suppressor proteins, and oncoproteins [9,12,15–18,22, 24,26,27,29,31,35,36]. Indeed, c -myc is modified by O-GlcNAc at threonine 58, a known phosphorylation site and a mutational hot spot in human lymphomas [12]. In addition, there is evidence that O-GlcNAc modification at the carboxy-terminus of p53 may have a role in the regulation of specific DNA binding by p53 [29]. It has been suggested that the putative involvement of O-GlcNAc modifications in oncogenesis may be mediated by addition/removal of O-GlcNAc on oncoproteins, tumor suppressor proteins, and other tumor- related proteins [12]. Further studies using biochemical methods and immunoelectron microscopy will help to clarify the nature of the polypeptides bearing epitope H and their accurate localization on cells of uterine smooth muscle cell tumors. It is noteworthy that O-GlcNAc is involved in apoptosis and cell cycle pathways [7,14,20,23,32–34]. Indeed, O-GlcNAcase is cleaved by caspase 3, and elevation/reduction of O-GlcNAc levels inhibits/en- hances activation of the anti-apoptotic AKT [7,32–34]. Of particular interest are the findings that in smooth muscle cells, O-GlcNAc glycosylation might have a profound effect on cell cycle transitions that regulate the heterodimerization of the YY1 zinc finger DNA-binding transcription factor with the Retinoblastoma protein (Rb) [20]. Indeed, O-GlcNAc-glycosylated YY1 no longer binds the Rb protein; upon dissociation from Rb, the O-GlcNAc-glycosylated YY1 is free to bind DNA; thus, O-GlcNAc glycosylation promotes the activity of the YY1 transcription factor [20]. As epitope H contains an O-GlcNAc residue [2], one might suggest that the fluctuations of epitope H expression may reflect differences in the expression of O-GlcNAc-glycosylated cellular proteins involved in the cell cycle and apoptosis regulation in uterine smooth muscle cell tumors and normal myometrium. A working model is emerging that O-GlcNAc may be a nutrient metabolic sensor ...
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... antibody H (mAbH), which recognizes epitope H, consisting of an O-GlcNAc and neighboring amino acids [1,2]. The mAbH stains two bands with Mr  10 À 3 of 209 and 62 in lysates of cultured rat astrocytes [2]. Using mAbH, we found that epitope H is shared by cytokeratin 8—which contains single O-GlcNAc residues [10]—and five additional polypeptides with Mr  10 À 3 of 232, 67, 51, 50, and 37 lysates of cultured MCF-7 human breast carcinoma cell line cells [1]. To investigate the expression levels and the cellular distribution of epitope H in human tissues, we used mAbH for the immunostaining of various normal and pathological tissues, including infiltrating ductal breast carcinomas, fibroadenomas, normal human brains, human brains with a variety of lesions, astrocytic tumors and mitochondria-rich normal, metaplastic and neoplastic cells [1–4,19]. In normal human brains, epitope H was absent from the overwhelming majority of normal astrocytes, and only sparse immunoreactivity was observed, confined mostly to fibrous astrocytes [2]. Upregulation of epitope H immunoexpression was found in reactive astrocytes observed in pathological specimens from a variety of brain lesions, including anisomorphic and isomorphic gliosis [2]. Epitope H immunoexpression was higher in astrocytomas compared to anaplastic astrocytomas and glioblastomas [3]. Infiltrating ductal breast carcinomas showed moderate cytoplasmic immunostaining of the malignant cells with mAbH. In addition, small groups of tumor cells displayed intense cytoplasmic mAbH immunostaining [1]. The intensity of mAbH ultrastructural immunostaining in the mitochondria, nucleoli, and cytoplasmic vesicles was decreased in infiltrating ductal breast carcinomas when compared to fibroadenomas [19], and mitochondria-rich normal, metaplastic and neoplastic cells showed overexpression of epitope H [4]. The above-mentioned findings prompted us to investigate the expression pattern of epitope H in uterine smooth muscle cell tumors and their adjacent myometrium. Therefore, we used mAbH to investigate the immunohistochemical expression of epitope H in 50 cases of uterine leiomyomas with their adjacent myometrium and in five cases of uterine leiomyosarcomas with their adjacent myometrium. Furthermore, as our previous Western immunoblotting data showed that epitope H is shared by cytokeratin 8—which contains single O-GlcNAc residues [10]—and five additional polypeptides in the MCF-7 human breast carcinoma cell line [1], we attempted to clarify the relations between epitope H expression and CK8 expression in uterine smooth muscle cell tumors and their adjacent normal myometrium. The material of the present study consisted of 50 cases of typical uterine leiomyomas and their adjacent myometrium, and of five cases of uterine leiomyosarcomas, four of which belonged to Group II A and one to Group III according to Bell et al. [6] and their adjacent myometrium. For immunodetection of epitope H, the indirect immunoperoxidase procedure was applied as described in detail previously [2]. Briefly, tissue sections about 4 m m were cut from formalin-fixed, paraffin-embedded tumor blocks. After deparaffinization and blocking of endogenous peroxidase activity by immersing the sections in 3% H 2 O 2 in Tris–Saline buffer pH 7.6, the sections were incubated in 10% normal rabbit serum in buffer for 30 min to inhibit non-specific binding of antibodies. The sections were then incubated in undiluted supernatant containing the mouse monoclonal antibody H [2] for 2 h at room temperature. After washing for 3  10 min in buffer, the sections were incubated with peroxidase- conjugated rabbit anti-mouse antibody diluted 1:50 in buffer for 1 h. After washing for 3  10 min, the color was developed by incubating the sections in DAB-H 2 O 2 in buffer for 8 min; then, after washing, counterstaining in hematoxylin, and dehydrating, the sections were covered with permount. In the negative control sections, the primary antibody was omitted. The staining pattern was graded as negative ( À ) when no stained cells were present, as weak (+) when less than 30% of the cells were stained, as moderate (++) when 30–75% of the cells were stained, and as intense (+++) when 75–100% of the cells were stained. Cytokeratin 8 immunostaining was performed using the monoclonal anti-cytokeratin 8 antibody (AbCK8) from Sigma Chemical according to the previously described protocol [19]. Western immunoblotting was performed using mAbH and monoclonal anti- cytokeratin 8 antibody (AbCK8) from Sigma Chemical according to the previously described protocol [1]. The main results were as follows: (1) epitope H showed intense cytoplasmic immunohistochemical ex- pression in 46% (23/50) and moderate immunohistochemical expression in 54% (27/50) of uterine leiomyomas (Fig. 1), (2) epitope H showed intense cytoplasmic immunohistochemical expression in 40% (2/5) and moderate immunohistochemical expression in 60% (3/5) of uterine leiomyosarcomas (Fig. 1), (3) epitope H showed no difference in cytoplasmic immunohistochemical expression between leiomyomas and their adjacent myometrium, and between leiomyosarcomas and their adjacent myometrium, (4) immunohistochemical expression of cytokeratin 8 was detected in no case of leiomyomas, leiomyosarcomas, or their adjacent myometrium (glandular epithelium of the endometrium was stained by monoclonal anti-cytokeratin 8 antibody (AbCK8) and served as an internal positive control), (5) Western immunoblotting showed that in the smooth muscle cells of the myometrium and the leiomyomas, epitope H is localized in four polypeptides, with molecular weights of 100, 61, 59, and 54 kDa (Fig. 2), and (6) Western immunoblotting did not detect cytokeratin 8 in the normal and neoplastic smooth muscle cells. In the present study, epitope H showed (1) intense immunohistochemical expression in 46% (27/50) and moderate immunohistochemical expression in 54% (23/50) of uterine leiomyomas and (2) intense immunohistochemical expression in 40% (2/5) and moderate immunohistochemical expression in 60% (3/5) of uterine leiomyosarcomas. These results indicate that epitope H expression fluctuates in normal and neoplastic human myometrium, as it is expressed either moderately or strongly in uterine leiomyosarcomas, uterine leiomyomas, and their adjacent myometrium. Taken together, our present and previous results [1–4,18] provide further evidence that epitope H expression fluctuates in normal and neoplastic tissues. Indeed, (1) epitope H is absent in the overwhelming majority of normal astrocytes and becomes upregulated in reactive astrocytes, (2) epitope H expression is significantly higher in astrocytomas than in anaplastic astrocytomas and glioblastomas, (3) the intensity of mAbH immunostaining was moderate mainly in malignant cells of infiltrating ductal breast carcinomas, but small groups of the malignant cells displayed intense mAbH immunostaining, (4) the intensity of mAbH ultrastructural immunostaining in the mitochondria, nucleoli, and cytoplasmic vesicles was decreased in infiltrating ductal breast carcinomas when compared to fibroadenomas, and (5) mitochondria-rich normal, metaplastic, and neoplastic cells showed overexpression of epitope H [1–4,19]. As epitope H contains an O-GlcNAc residue [2], it is possible that the fluctuations of the epitope H expression we found in the present and previous studies [1–4,19] reflect differences in the expression of a O-GlcNAc-glycosylated cellular protein or proteins. These fluctuations may be of interest for gaining insight into the pathology of uterine smooth muscle cell tumors, as O-GlcNAc glycosylation may modify proteins involved in important biological functions such as cytoskeletal proteins, transcription factors, heat-shock proteins, chromatin proteins, tumor suppressor proteins, and oncoproteins [9,12,15–18,22, 24,26,27,29,31,35,36]. Indeed, c -myc is modified by O-GlcNAc at threonine 58, a known phosphorylation site and a mutational hot spot in human lymphomas [12]. In addition, there is evidence that O-GlcNAc modification at the carboxy-terminus of p53 may have a role in the regulation of specific DNA binding by p53 [29]. It has been suggested that the putative involvement of O-GlcNAc modifications in oncogenesis may be mediated by addition/removal of O-GlcNAc on oncoproteins, tumor suppressor proteins, and other tumor- related proteins [12]. Further studies using biochemical methods and immunoelectron microscopy will help to clarify the nature of the polypeptides bearing epitope H and their accurate localization on cells of uterine smooth muscle cell tumors. It is noteworthy that O-GlcNAc is involved in apoptosis and cell cycle pathways [7,14,20,23,32–34]. Indeed, O-GlcNAcase is cleaved by caspase 3, and elevation/reduction of O-GlcNAc levels inhibits/en- hances activation of the anti-apoptotic AKT [7,32–34]. Of particular interest are the findings that in smooth muscle cells, O-GlcNAc glycosylation might have a profound effect on cell cycle transitions that regulate the heterodimerization of the YY1 zinc finger DNA-binding transcription factor with the Retinoblastoma protein (Rb) [20]. Indeed, O-GlcNAc-glycosylated YY1 no longer binds the Rb protein; upon dissociation from Rb, the O-GlcNAc-glycosylated YY1 is free to bind DNA; thus, O-GlcNAc glycosylation promotes the activity of the YY1 transcription factor [20]. As epitope H contains an O-GlcNAc residue [2], one might suggest that the fluctuations of epitope H expression may reflect differences in the expression of O-GlcNAc-glycosylated cellular proteins involved in the cell cycle and apoptosis regulation in uterine smooth muscle cell tumors and normal myometrium. A working model is emerging that O-GlcNAc may be a nutrient metabolic sensor that attenuates the response of a cell to extracellular stimuli based on the energy state of the cell [34]. The model proposes that cells do not blindly respond to extracellular ...
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... main results were as follows: (1) epitope H showed intense cytoplasmic immunohistochemical ex- pression in 46% (23/50) and moderate immunohisto- chemical expression in 54% (27/50) of uterine leiomyomas ( Fig. 1), (2) epitope H showed intense cytoplasmic immunohistochemical expression in 40% (2/5) and moderate immunohistochemical expression in 60% (3/5) of uterine leiomyosarcomas (Fig. 1), (3) epitope H showed no difference in cytoplasmic immu- nohistochemical expression between leiomyomas and their adjacent myometrium, and between ...
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... epitope H showed intense cytoplasmic immunohistochemical ex- pression in 46% (23/50) and moderate immunohisto- chemical expression in 54% (27/50) of uterine leiomyomas ( Fig. 1), (2) epitope H showed intense cytoplasmic immunohistochemical expression in 40% (2/5) and moderate immunohistochemical expression in 60% (3/5) of uterine leiomyosarcomas (Fig. 1), (3) epitope H showed no difference in cytoplasmic immu- nohistochemical expression between leiomyomas and their adjacent myometrium, and between leiomyosarco- mas and their adjacent myometrium, (4) immunohisto- chemical expression of cytokeratin 8 was detected in no case of leiomyomas, leiomyosarcomas, or their adjacent myometrium ...
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Trophinin and tastin form a cell adhesion molecule complex that potentially mediates an initial attachment of the blastocyst to uterine epithelial cells at the time of implantation. Trophinin and tastin, however, do not directly bind to each other, suggesting the presence of an intermediary protein. The present study identifies a cytoplasmic protei...
Citations
... express the epitope, whereas the epitope is greatly upregulated in reactive astrocytes (13); O-GlcNAcH is upregulated in hypoxia in human ependymal cells (14) and is greatly up-regulated in endometrial decidual cells compared to stromal cells of the proliferative endometrium (15). Epitope H occurs in several polypeptides (7,10,13), including keratin 8 (7) and vimentin in cells under stress (16). ...
... The stromal cells of the polyps showed induction of O-GlcNAcH, since all of them showed strong cytoplasmic staining for the epitope. This strong induction may be attributed either to O-GlcNAcylation of vimentin under the stress condition of inflammation, or to the conversion of fibroblasts to myofibroblasts, thus acquiring some polypeptides bearing O-GlcNAcH, as has been shown to occur in normal smooth muscle and leiomyoma cells (10). Further biochemical characterization of the polypeptides which bear the O-GlcNAcH epitope might shed light on all these processes. ...
Background/aim:
Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAcH) residue in a specific conformation or environment, recognized by a site-specific monoclonal mouse IgM antibody H. O-GlcNAcH occurs in several normal and pathological cells and in several polypeptides, including keratin-8 and vimentin, on the latter in cells under stress.
Materials and methods:
In this work, we studied the distribution of O-GlcNAcH on cells of endocervical mucosa in 60 specimens of endocervical curettings, 10 of which contained 15 inflamed polyps.
Results:
In our results, expression of O-GlcNAcH was weak in the mucosa with <5% mucin-secreting cells and up to 30% of the polyps staining positively. All non-ciliated, non-mucin-secreting cells, normal and hyperplastic 'reserve' cells, as well as the cells of immature squamous metaplasia, showed strong diffuse cytoplasmic staining for O-GlcNAcH. In mature squamous epithelium, fewer than 5% of basal cells and all the intermediate and superficial cells showed cytoplasmic staining for O-GlcNAcH, whereas parabasal cells were negative. All ciliated cells showed patchy or diffuse cytoplasmic staining. Nuclear staining for O-GlcNAcH was weak with fewer than 5% of hyperplastic 'reserve' and ciliated cells staining positively. Moreover, mucosal fibroblasts were negative, whereas all stromal cells of the polyps showed strong cytoplasmic staining for O-GlcNAcH.
Conclusion:
O-GlcNAcH is: a) differentially expressed among the cellular elements of mucosa and polyps, b) upregulated in mucin-secreting cells of polyps, c) induced in stromal cells of inflamed polyps, and d) can be used as a marker to differentiate between 'reserve' (positive) and parabasal (negative) cells, which have similar morphology using conventional cytological stains.
... In that context, mouse monoclonal IgM antibody H (mabH) is a site-specific antibody that recognizes epitope H. Epitope H contains an O-linked Νacetylglucosamine (O-GlcNAcH) residue in a specific conformation and/or environment (13). O-GlcNAcH is present in several types of normal, metaplastic and neoplastic cells (14)(15)(16)(17) and in several polypeptides including cytokeratin 8 (14) and vimentin, on the latter in cells under stress (18). Modifications of the expression of O-GlcNAcH have been found in several conditions, such as O-GlcNAcH is present in a minority of fibrous astrocytes in normal human brains and is upregulated greatly in reactive astrocytes (13). ...
... Indeed in the work of Howerton et al. (22), where material was used from normal human term placentas, with Western blot by using the pan-specific monoclonal antibody MMS-248R; covance, a greater number of O-GlcNAcylated polypeptides were recognized, (b) the vimentin with MW 57 existing in the fibroblastic and endothelial cells (23) is not included in the 5 polypeptides, which bear the O-GlcNAcH epitope since no polypeptide near the range of 57 kD is present. This finding in conjunction with previous immunohistochemical work which showed that the normal fibroblasts in the fibrous septa of the myometrium were negative for the O-GlcNAcH (17), as well as the fibroblasts of the (7), heat stress (8,9), endoplasmic reticulum stress (25), hypoxia (26), and ischemia-reperfusion injury (27). This increase is also proportional to the intensity of the stressors and to the degree of cell damage (8). ...
... At present, we do not have any biochemical explanation for this finding. However, the results of this work show for the first time that there is modulation of the O-GlcNAcH in the fibroblastic cells under the oxidative stress conditions of the miscarriage since previous work have shown that the fibroblasts under normal conditions such as in the fibrous septa of the myometrium (17) and in the normal endocervical human uterine mucosa (24) are negative for the O-GlcNAcH. Modulation of the O-GlcNAcH in fibroblastic cells has been reported previously, as in the fibroblastic type stromal endometrial cells in the proliferative phase there was low expression of O-GlcNAcH, since there was either negative or weak cytoplasmic stain, whereas in the decidual cells there was high expression since practically all decidual cells showed strong cytoplasmic stain for O-GlcNAcH (19). ...
Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAcH) residue in a specific conformation and/or environment recognized by the mouse monoclonal antibody H. O-GlcNAcH is present in several types of cells and in several polypeptides, including cytokeratin 8 and vimentin, on the latter in cells under stress. In the present work, we examined the expression of the O-GlcNAcH in 60 cases of endometrial curettings from missed miscarriage cases containing normal and simple hydropic degenerated chorionic villi in each case, using monoclonal antibody H and indirect immunoperoxidase and Western blot immunoblot. In all cases examined the expression of the O-GlcNAcH was cytoplasmic as follows: (1) syncytiotrophoblastic cells showed very low expression in chorionic villi (CV) with nonhydropic degeneration (NHD) and high expression in hydropic degenerated (HD) CV; (2) cytotrophoblastic cells showed low expression in CV with NHD and high expression in HD CV; (3) fibroblastic cells showed high expression in CV with NHD and very low expression in HD CV; (4) histiocytes showed very low expression in both types of CV; (5) endothelial cells showed high expression in both types of CV. An immunoblot of CV from one case of a legal abortion from a normal first-trimester pregnancy showed 5 polypeptides with 118.5, 106.3, 85, 53, and 36.7 kD bearing the epitope H and the 53 kD corresponded to cytokeratin 8. The expression of the O-GlcNAcH is upregulated in the trophoblastic cells and downregulated in the fibroblastic cells in the HD CV in comparison to the NHD CV.
... mRNA expression levels of O-GlcNAc cycling enzymes OGT and OGA have been found to be significantly higher in undifferentiated endometrial tumors compared to welldifferentiated ones (Krzeslak et al. 2012b). However, Sgantzos et al. (2007) showed that immunohistochemical expression of O-GlcNAc fluctuates in normal and neoplastic uterine tissues, which may reflect differences in the expression of O-GlcNAcylated cellular proteins. Their work implies that the role of O-GlcNAc in oncogenesis may be mediated by addition or removal of this modification on tumor-related proteins and sheds light on the relevance of this modification in normal histophysiology and in the pathogenesis of uterine muscle cell tumors. ...
O-GlcNAcylation is emerging as a critical regulatory post-translational modification, impacting proteins that regulate cell division, apoptosis, metabolism, cell signaling, and transcription. O-GlcNAc also affects biological homeostasis by integrating information coming from the environment, such as nutrient conditions and extracellular stimuli, with cellular response. Aberrant O-GlcNAc modulation has been linked to metabolic and neurodegenerative diseases, as well as cancers. While many studies have highlighted the significance of O-GlcNAc in cancer, a specific function for O-GlcNAc during tumorigenesis remains unclear and seems to differ according to cancer type. Herein, we review the impact of altered O-GlcNAcylation in breast, ovarian and uterine cancers.
... Since mAbH recognizes an O-GlcNAc residue, the authors concluded that O-GlcNAc-glycosylated polypeptides are present in mitochondria where the components of the respiratory chain and energy transduction are localized (Arvanitis et al. 2005). The mAbH also recognized elevated O-GlcNAcylation of cytokeratin 8 and other cytokeratins suggesting it possesses broad specificity for recognizing O-GlcNAc in cancer (Havaki et al. 2006;Sgantzos et al. 2007). Subsequent work has shown that epitope H, like the O-GlcNAc modification, is elevated by hypoxia (Arvanitis et al. 2011 Additional molecular evidence for mOGT targets came from in-gel detection of O-GlcNAc modified proteins (Clark et al. 2008). ...
Cancer cells exhibit unregulated growth, altered metabolism, enhanced metastatic potential and altered cell surface glycans. Fueledby oncometabolism and elevated uptake of glucose and glutamine, the hexosamine biosynthetic pathway (HBP) sustains glyco-sylation in the endomembrane system. In addition, the elevated pools of UDP-GlcNAc drives the O-GlcNAc modification of keytargets in the cytoplasm, nucleus and mitochondrion. These targets include transcription factors, kinases, key cytoplasmic enzymesof intermediary metabolism, and electron transport chain complexes. O-GlcNAcylation can thereby alter epigenetics, transcription,signaling, proteostasis, and bioenergetics, key ‘hallmarks of cancer’. In this review, we summarize accumulating evidence thatmany cancer hallmarks are linked to dysregulation of O-GlcNAc cycling on cancer-relevant targets. We argue that onconutrientand oncometabolite-fueled elevation increases HBP flux and triggers O-GlcNAcylation of key regulatory enzymes in glycolysis,Kreb’s cycle, pentose-phosphate pathway, and the HBP itself. The resulting rerouting of glucose metabolites leads to elevated O-GlcNAcylation of oncogenes and tumor suppressors further escalating elevation in HBP flux creating a ‘vicious cycle’.Downstream, elevated O-GlcNAcylation alters DNA repair and cellular stress pathways which influence oncogenesis. Theelevated steady-state levels of O-GlcNAcylated targets found in many cancers may also provide these cells with a selectiveadvantage for sustained growth, enhanced metastatic potential, and immune evasion in the tumor microenvironment
... Since mAbH recognizes an O-GlcNAc residue, the authors concluded that O-GlcNAc-glycosylated polypeptides are present in mitochondria where the components of the respiratory chain and energy transduction are localized (Arvanitis et al. 2005). The mAbH also recognized elevated O-GlcNAcylation of cytokeratin 8 and other cytokeratins suggesting it possesses broad specificity for recognizing O-GlcNAc in cancer (Havaki et al. 2006;Sgantzos et al. 2007). Subsequent work has shown that epitope H, like the O-GlcNAc modification, is elevated by hypoxia (Arvanitis et al. 2011 Additional molecular evidence for mOGT targets came from in-gel detection of O-GlcNAc modified proteins (Clark et al. 2008). ...
Cancer cells exhibit unregulated growth, altered metabolism, enhanced metastatic potential and altered cell surface glycans. Fueledby oncometabolism and elevated uptake of glucose and glutamine, the hexosamine biosynthetic pathway (HBP) sustains glyco-sylation in the endomembrane system. In addition, the elevated pools of UDP-GlcNAc drives the O-GlcNAc modification of keytargets in the cytoplasm, nucleus and mitochondrion. These targets include transcription factors, kinases, key cytoplasmic enzymesof intermediary metabolism, and electron transport chain complexes. O-GlcNAcylation can thereby alter epigenetics, transcription,signaling, proteostasis, and bioenergetics, key ‘hallmarks of cancer’. In this review, we summarize accumulating evidence thatmany cancer hallmarks are linked to dysregulation of O-GlcNAc cycling on cancer-relevant targets. We argue that onconutrientand oncometabolite-fueled elevation increases HBP flux and triggers O-GlcNAcylation of key regulatory enzymes in glycolysis,Kreb’s cycle, pentose-phosphate pathway, and the HBP itself. The resulting rerouting of glucose metabolites leads to elevated O-GlcNAcylation of oncogenes and tumor suppressors further escalating elevation in HBP flux creating a ‘vicious cycle’.Downstream, elevated O-GlcNAcylation alters DNA repair and cellular stress pathways which influence oncogenesis. Theelevated steady-state levels of O-GlcNAcylated targets found in many cancers may also provide these cells with a selectiveadvantage for sustained growth, enhanced metastatic potential, and immune evasion in the tumor microenvironment.
... Chronic lymphocytic leukemia is characterized by aberrant O-GlcNAcylation [103] Thyroid OGA enzyme activity is increased in thyroid cancer [63] Breast OGT regulates oncogenesis through FoxM1 [14] Erwing sarcoma O-GlcNAc regulates transcriptional activity of transcription factor FLI1 [6] Uterus O-GlcNAc containing epitope H expressed in smooth muscle cell tumors [101] Breast O-GlcNAc-containing epitope H are localized in the nucleus of epithelial cells [42] Lymphoma Role of O-GlcNAc modification and subcellular distribution of transcription factor Sp1 [27] Lymphoma c-Myc is O-GlcNAcylated at Thr 83, a mutational hot spot in lymphoma [21] Cancer Lett. Author manuscript; available in PMC 2016 January 28. ...
... In the context of O-GlcNAcylation, the monoclonal mouse IgM H antibody (mAbH) recognizes epitope H, which contains an O-linked N-acetylglucosamine (O-GlcNAc) residue in a specific conformation and/or environment, thus recognizing a subpopulation of polypeptides which bear the residue O-GlcNAc [3]. Epitope H is present in several normal and pathological cells and in several cellular polypeptides outside the central nervous system (CNS) [2,4,5,27,38,42]. In normal human brains, epitope H showed sparse immunoreactivity confined to a minority of fibrous astrocytes [3]. ...
... To date, several different types of cells have produced different sets of polypeptides which bear epitope H (e.g. cortical rat astrocytes contain two polypeptides with 209 kDa and 62 kDa [3], smooth muscle cells of human uterus, and leiomyomas contain 4 polypeptides with molecular weights of 100, 61, 59, and 54 kDa, which are recognized by mAbH [42], and the MCF-7 breast carcinoma cell line contains 6 polypeptides with 232, 67, 51, 50, 37 kDa, and cytokeratin 8 which are recognized by mAbH) [2]. From these data, it appears that different types of cells contain different sets of polypeptides bearing epitope H. Therefore, one possible explanation might be that the neurons, for example, and the other cells which remain negative do not produce polypeptides which bear epitope H. ...
Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAc) residue in a specific conformation and/or environment recognized by mouse IgM monoclonal antibody H (mabH). Epitope H is present in several types of cells and in several polypeptides outside the CNS. Previous results have shown that in the adult human brains, epitope H is confined mostly to a minority of fibrous astrocytes, and it is greatly upregulated in the reactive astrocytes. Post-translational modification with O-GlcNAc occurs on many proteins involved in several cell processes, such as cell cycle progression, apoptosis, proteasome degradation pathways, and modulation of cellular function in response to nutrition and stress. Hypoxia is one of the major causes of cellular stress. Therefore, in this study, we used the mAbH and the indirect immunoperoxidase method to investigate the expression of epitope H in ependymal cells in brains of persons who died with signs of hypoxic encephalopathy. The results of the present study showed that practically all ependymal cells showed cytoplasmic staining for epitope H in supranuclear cytoplasm in the brain of two premature neonates and in ten infants who died with signs of hypoxic encephalopathy. However, the overwhelming majority of ependymal cells of the nine human embryos taken from legal abortions, ranging from 26 days until 13 weeks of gestational age, and of the ten infants' brains without any sign of hypoxic encephalopathy remained negative. Only occasionally did the ependymal cells show weak cytoplasmic staining in some foci. In addition, the reactive astrocytes in the hypoxic brains showed strong cytoplasmic staining, confirming previous results.
... C D E F G in pre-decidualized, decidualized and non-decidualized normal endometrial stromal cells, in normal myometrium and uterine smooth muscle cell tumors, in normal human brains, in human brains with a variety of lesions and in astrocytic tumors (8,(21)(22)(23)(24)(25)(26). ...
The epitope H contains an O-linked N-acetylglucosamine residue in a specific conformation and/or environment recognized by the monoclonal antibody H. It has previously been shown that the epitope H is present in several types of normal and pathological cells and in several polypeptides. In normal human brains the epitope H is present mostly to a minority of fibrous astrocytes, whereas it is greatly up-regulated in reactive astrocytes and is increased in well differentiated fibrillary astrocytomas compared to anaplastic astrocytomas and glioblastomas. In this study the expression of the epitope H was investigated in thirty cases of gemistocytic (WHO grade II), pilocytic (WHO grade I), and subependymal giant cell (WHO grade I) astrocytomas using the mAbH with the indirect immunoperoxidase method. The ten cases of gemistocytic astrocytomas revealed an overall high expression pattern. The ten cases of pilocytic astrocytomas revealed a biphasic pattern of epitope H expression. The dense tumor areas composed of elongated pilocytic cells revealed high expression of the epitope H. The loose cystic tumor areas composed of stellate cells revealed low expression of the epitope H. The ten cases of subependynal giant cell astrocytomas occurring in tuberous sclerosis revealed an overall high expression pattern. This study shows that there is high expression of the epitope H in gemistocytic, pilocytic and subependymal giant cell astrocytomas. Collectively considering, the present and our previous data, it appears that there is a spectrum of the expression levels of the epitope H ranging from the high expression in the reactive astrocytes and low grade astrocytomas to the low/null expression in the normal astrocytes and glioblastomas.
... Tumor diameter was measured preoperatively by ultrasound scanning and postoperatively in the pathology laboratory. Core biopsy specimens were stained by H/E for histological diagnosis and evaluated immunocytochemically for HER-2/neu, estrogen and progesterone receptor content [6,7] . All participants signed an informed consent form and the ethics committee of Thessalia University Hospital approved this protocol. ...
Radiofrequency (RF) thermal ablation is a minimally invasive technique of local mass elimination with variable efficiency.
Ten patients with small primary breast cancer diagnosed preoperatively by core needle biopsy were ablated percutaneously by an RF (Radionics Cool-tip) device operating on impedance control mode. The percent fat-containing area was calculated in each slide of a total of 47 slides introduced to IQ materials software image analysis.
Seven of 10 tumors with tumor diameter less than 2.8 cm and fat content less than 12.47% were totally ablated (score 3). One of 10 with 3 cm tumor diameter and 5.45% fat content showed an intermediate degree of ablated tissue (score 2), and the last 2 with 2 cm and 2.2 cm tumor diameter and more than 19.74% tumor fat content were minimally ablated (score 1). Our present exploratory study on 10 patients suggests dependence of the degree of thermal damage on tumor fat content.
We conclude that the fat content of small primary breast cancer could serve as a 'heat sink' and should be considered as a preventing factor of complete local tumor destruction by RF thermal ablation.
Aneurysm is associated to a complex remodeling of arteries that affects all their layers. Although events taking place in the intima and the media have received a particular attention, molecular and cellular events taking place in the adventitia have started to be deciphered only recently. In this study, we have precisely described the composition and distribution of stromal and hematopoietic cells in human arterial adventitia, both at steady state and in the setting of aortic aneurysm. Using polychromatic immunofluorescent and flow cytometry analyses, we observed that unlike the medial layer (which comprises mostly macrophages and T cells among leukocytes), the adventitia comprises a much greater variety of leukocytes. We observed an altered balance in macrophages subsets in favor of M2-like macrophages, an increased proliferation of macrophages, a greater number of all stromal cells in aneurysmal aortas. We also confirmed that in this pathological setting, adventitia comprised blood vessels and arterial tertiary lymphoid organs (ATLOs), which contained also M-DC8(+) dendritic cells (slanDCs) that could participate in the induction of T-cell responses. Finally, we showed that lymphatic vessels can be detected in aneurysmal adventitia, the functionality of which will have to be evaluated in future studies. All together, these observations provide an integrative outlook of the stromal and hematopoietic cell network of the human adventitia both at steady state and in the context of aneurysm.
