Immunohistochemical study of the expression of MUC6 mucin and co-expression of other secreted mucins (MUC5AC and MUC2) in human gastric carcinomas.

To investigate the expression of MUC6 mucin in gastric carcinomas, we generated a novel monoclonal antibody (MAb CLH5) using an MUC6 synthetic peptide. MAb CLH5 reacted exclusively with the MUC6 peptide and with native and deglycosylated mucin extracts from gastric tissues. MAb CLH5 immunoreactivity was observed in normal gastric mucosa restricted to pyloric glands of the antrum and mucopeptic cells of the neck zone of the body region. In a series of 104 gastric carcinomas, 31 (29.8%) were immunoreactive for MUC6. The expression of MUC6 was not associated with histomorphological type or with clinicopathological features of the carcinomas. Analysis of the co-expression of MUC6 with other secreted mucins (MUC5AC and MUC2) in 20 gastric carcinomas revealed that different mucin core proteins are co-expressed in 55% of the cases. MUC6 was co-expressed and co-localized with MUC5AC in 45% and with MUC2 in 5% of the cases. Expression of MUC2 alone was observed in 25% of the cases. All carcinomas expressing MUC2 mucin in more than 50% of the cells were of the mucinous type according to the WHO classification. The co-expression of mucins was independent of the histomorphological type and stage of the tumors. In conclusion, we observed, using a novel well-characterized MAb, that MUC6 is a good marker of mucopeptic cell differentiation and is expressed in 30% of gastric carcinomas, independent of the clinicopathological features of the cases. Furthermore, we found that co-expression and co-localization of mucins in gastric carcinomas is independent of histomorphology and staging. Finally, we observed that intestinal mucin MUC2 is expressed as the most prominent mucin of the mucins tested in mucinous-type gastric carcinomas.     

C-Mannosylation of MUC5AC and MUC5B Cys subdomains

We expressed recombinant Cys subdomains in COS-7 cells to examine the role of this highly conserved protein domain in mucin biosynthesis. The entire Cys1 and Cys5 and Cys1 and Cys3 subdomains in MUC5AC and MUC5B, respectively, each with six carboxyl terminal histidine residues, were pulse-labeled with [(35)S]cysteine/methionine, and the labeled proteins were examined in the culture medium. Under nonreducing conditions, secreted Cys subdomains were monomers, indicating the absence of interchain disulfide bonds. Cross-linking studies suggested the domains are able to interact through very weak noncovalent interactions. Though the domains had apparent M(r) consistent with the absence of N- and O-glycans, they could be purified with mannose-specific lectins. Lectin binding was prevented by mutation of the first tryptophan residue in the putative C-mannosylation acceptor motif WXXW, indicating that C-mannosylation is responsible for lectin binding. As judged by pulse-chase experiments, C-mannosylation occurred very early during the domain biosynthesis, likely in the endoplasmic reticulum (ER). Mutation of the WXXW motif or expression of the unmutated domain in CHO-Lec35.1 cells, a C-mannosylation-defective cell line, resulted in reduced secretion of the corresponding Cys subdomains. Live cell imaging of green fluorescent protein fused to the Cys subdomains clearly revealed increased presence of Cys subdomains in the ER of CHO-Lec35.1 cells when compared to the same domains expressed in CHO-K1 cells. Considered together, these studies suggest that the Cys subdomains of MUC5AC and MUC5B are C-mannosylated in their respective WXXW motifs. C-mannosylation is likely required for proper folding of the Cys subdomains and/or for some aspect of ER export during mucin biosynthesis.     

Degradation of MUC7 and MUC5B in Human Saliva

Background

Two types of mucins, MUC7 and MUC5B constitute the major salivary glycoproteins, however their metabolic turnover has not been elucidated in detail to date. This study was conducted to examine turnover of MUC7 and MUC5B in saliva, by focusing on the relationship between their deglycosylation and proteolysis.

Methodology/Principal Findings

Whole saliva samples were collected from healthy individuals and incubated at 37°C in the presence of various protease inhibitors, sialidase, or a sialidase inhibitor. General degradation patterns of salivary proteins and glycoproteins were examined by SDS-polyacrylamide-gel-electrophoresis. Furthermore, changes of molecular sizes of MUC7 and MUC5B were examined by Western blot analysis. A protein band was identified as MUC7 by Western blot analysis using an antibody recognizing an N-terminal epitope. The MUC7 signal disappeared rapidly after 20-minutes of incubation. In contrast, the band of MUC7 stained for its carbohydrate components remained visible near its original position for a longer time indicating that the rapid loss of Western blot signal was due to the specific removal of the N-termimal epitope. Pretreatment of saliva with sialidase facilitated MUC7 protein degradation when compared with samples without treatment. Furthermore, addition of sialidase inhibitor to saliva prevented proteolysis of N-terminus of MUC7, suggesting that the desialylation is a prerequisite for the degradation of the N-terminal region of MUC7. The protein band corresponding to MUC5B detected in both Western blotting and glycoprotein staining showed little sign of significant degradation upon incubation in saliva up to 9 hours.

Conclusions/Significance

MUC7 was highly susceptible to specific proteolysis in saliva, though major part of MUC5B was more resistant to degradation. The N-terminal region of MUC7, particularly sensitive to proteolytic degradation, has also been proposed to have distinct biological function such as antibacterial activities. Quick removal of this region may have biologically important implication.     

Immunohistochemical expression of androgen receptor and prostate-specific antigen in breast cancer

AR (androgen receptor) and PSA (prostate-specific antigen) are involved in the pathogenesis of breast cancer, but their role is not clearly defined. The purpose of this study was to analyze by immunohistochemistry the AR and PSA (prostate-specific antigen) expression in 156 female breast carcinomas and to correlate the results with some histopathological parameters, like ER (estrogen receptor), PR (progesterone receptor), HER2/neu, nodal and metastasis status, histological type and grade. ARs and PSA were expressed in 112/156 (72%) and respectively in 61/156 (39%) of cases and we found a positive correlation between AR and PSA expression in breast carcinomas (p<0.0002). We also found an association between the histological type of the tumor and AR (p<0.001), respectively PSA (p=0.01) and between AR and the grade of differentiation (p=0.007) and the nodal status (p=0.02). No correlations were found between the metastasis status and AR or PSA. 47.3% (53/112) of AR-positive cases and 46% (28/61) of PSA-positive cases were ER-negative. High frequency of AR (87.5%) and PSA (75%) expression was found in medullary carcinomas and 53% of lobular invasive carcinomas co-expressed AR and PSA. We found an inverse correlation between HER2/neu and PSA (p=0.05). Although most of the PSA-positive carcinomas were lymph node-negative, well and moderately differentiated, we did not find any statistically significant correlations between these parameters and PSA expression. Our study confirms that ARs are commonly expressed in breast cancer and the expression of PSA and AR are highly correlated. Moreover, all the lobular carcinomas and the majority of medullary carcinomas co-expressed AR and PSA, the majority of AR-positive carcinomas were lymph node-negative, well and moderately differentiated, and large number of ER-negative carcinomas expressed AR and PSA.     

GLUT 5 is not over-expressed in breast cancer cells and patient breast cancer tissues

F18 2-Fluoro 2-deoxyglucose (FDG) has been the gold standard in positron emission tomography (PET) oncologic imaging since its introduction into the clinics several years ago. Seeking to complement FDG in the diagnosis of breast cancer using radio labeled fructose based analogs, we investigated the expression of the chief fructose transporter-GLUT 5 in breast cancer cells and human tissues. Our results indicate that GLUT 5 is not over-expressed in breast cancer tissues as assessed by an extensive immunohistochemistry study. RT-PCR studies showed that the GLUT 5 mRNA was present at minimal amounts in breast cancer cell lines. Further knocking down the expression of GLUT 5 in breast cancer cells using RNA interference did not affect the fructose uptake in these cell lines. Taken together these results are consistent with GLUT 5 not being essential for fructose uptake in breast cancer cells and tissues.     

Identification of UDP-glucuronosyltransferase 1A10 in non-malignant and malignant human breast tissues

UGT1A10 was recently identified as the major isoform that conjugates estrogens. In this study, real-time PCR revealed high levels of UGT1A10 and UGT2B7 mRNA in human breast tissues. The expression of UGT1A10 in breast was a novel finding. UGT1A10 and UGT2B7 mRNAs were differentially expressed among normal and malignant specimens. Their overall expression was significantly decreased in breast carcinomas as compared to normal breast specimens (UGT1A10: 68 ± 26 vs. 252 ± 86, respectively; p < 0.05) and (UGT2B7: 1.4 ± 0.7 vs. 12 ± 4, respectively; p < 0.05). Interestingly, in African American women, UGT1A10 expression was significantly decreased in breast carcinomas in comparison to normals (57 ± 35 vs. 397 ± 152, respectively; p < 0.05). Among Caucasian women, UGT2B7 was significantly decreased in breast carcinomas in comparison to normals (1.1 ± 0.5 vs. 13.5 ± 6, respectively; p < 0.05). Glucuronidation of 4-hydroxylated estrone (4-OHE₁) was significantly reduced in breast carcinomas compared to normals (30 ± 15 vs. 106 ± 31, respectively; p < 0.05). Differential down-regulation of UGT1A10 and UGT2B7 mRNAs, protein, and activity in breast carcinomas compared to the adjacent normal breast specimens from the same donor were also found. These data illustrate the novel finding of UGT1A10 in human breast and confirm the expression of UGT2B7. Significant individual variation and down-regulation of expression in breast carcinomas of both isoforms were also demonstrated. These findings provide evidence that decreased UGT expression and activity could result in the promotion of carcinogenesis.     

Immunohistochemical analysis of nerve growth factor receptor expression in normal and malignant human tissues

Nerve growth factor (NGF) is a polypeptide important for normal development of the nervous system and promotion of survival and differentiation of sensory and sympathetic neurons in culture. The cellular effects of NGF are mediated by a specific cell surface molecule, nerve growth factor receptor (NGF-R). In the present study we have used a monoclonal antibody against human NGF-R to examine, by the avidin-biotin-immunoperoxidase method, the receptor distribution in a wide range of normal tissues and in more than 200 malignant tumors. Our results show that (a) human NGF-R is expressed in the peripheral nervous system but not in any of the central nervous system areas tested; (b) NGF-R expression is not restricted to neural tissues but is also found in a number of normal epithelial, mesenchymal, and lymphoid tissues; (c) NGF-R expression changes during normal development; and (d) NGF-R expression in malignant tumors generally parallels its normal tissue distribution. Thus, NGF-R is detected in a proportion of neuroectoderm-derived tumors, carcinomas, and lymphomas, and also in a characteristic group of small round-cell tumors (Ewing's sarcomas and embryonal rhabdomyosarcomas). These findings suggest a normal regulatory role for NGF in both neuronal and non-neuronal cells and identify a range of human tumors in which the NGF/NGF-R system may contribute to the malignant phenotype.     

Comparative proteome analysis of breast cancer and adjacent normal breast tissues in human

Two-dimensional polyacrylamide gel assisted laser desorption/ionization electrophoresis （2D-PAGE） and matrixtandem time-of-flight mass spectrometry （MALDI-TOF/TOF-MS）, incorporated with online database searching, were performed to investigate differential proteins of breast cancer and adjacent normal breast tissues. Considering that serum albumin is abundantly presented in normal control samples, 15 differential spots detected in 11 out of 12 （91.7%） breast cancer samples were identified by online SIENA-2DPAGE database searching and MALDI-TOF/TOF-MS analysis. The results indicate that pathological changes of breast cancer are concerned with augmentation of substance metabolism, promotion of proteolytic activity, decline of activity of some inhibitors of enzymes, and so on. Some important proteins involved in the pathological process of breast cancer with changed expression may be useful biomarkers, such as alpha-l-antitrypsin, EF- 1-beta, cathepsin D, TCTP, SMT3A, RPS12, and PSMA1, among which SMT3A, RPSl2, and PSMA1 were first reported for breast cancer in this study.     

Immunohistochemical analysis of human neuronectin expression in normal, reactive, and neoplastic tissues

Neuronectin (NEC1) is a human extracellular matrix protein of central nervous system (CNS) parenchyma found throughout the white matter of rostral brain segments (telencephalon, diencephalon, some areas of mesencephalon), but not in rostral CNS gray matter, most areas of mesencephalon, pons, cerebellum, medulla, spinal cord, or peripheral nerves. The present immunohistochemical study, using two monoclonal antibodies to distinct epitopes on the NEC1 molecule, examined NEC1 expression in normal non-neural tissues, malignant tumors of diverse histological types, non-malignant skin lesions, and dermal incision wounds. We show that (a) NEC1 is expressed in normal fetal precartilage blastemas and fetal and adult vascular and visceral smooth muscle, but not in most loose connective tissues and skeletal or cardiac muscle; (b) NEC1 is found along epithelial-mesenchymal junctions, with marked differences in prevalence and histological patterns in different organs; and (c) mesenchymal activation associated with wound healing, actinic keratosis, psoriasis, and neoplasia leads to strong induction of NEC1 expression. Parallel studies with cultured human cells suggest that region-specific NEC1 expression in normal developing tissues and localized induction in wound healing and disparate diseases is under the control of extrinsic signals provided by regulatory polypeptides.     

乳腺癌及良性乳腺组织p185和p16蛋白表达的免疫组织化学研究

应用免疫组化S P法检测了 37例良性乳腺组织 (非上皮增生组 17例、上皮增生组 2 0例 )和 5 9例乳腺癌组织中癌基因蛋白 p185和抑癌基因 p16蛋白的表达状况。结果显示非上皮增生组、上皮增生组和癌的p185阳性率分别为 0 %、15 %和 47%(p <0 0 1) ;p16阳性率分别为 41%、30 %和 34%。p185和p16的表达无明显相关性。乳腺癌早期的 p185过表达和p16失表达率高于浸润性导管癌。两者的阳性率均随组织学级别的增高和瘤体的增大而呈上升趋势 ,但 p >0 0 5。淋巴结转移组的p185阳性率 ( 6 4%)明显高于无淋巴结转移者 ( 32 %) ,p <0 0 5。表明 p185过表达和p16失表达在乳腺癌的发生发展中各自发挥独立的作用。p185是乳腺癌重要的肿瘤标志物。     

Analysis of 5-methylcytosine in DNA of breast and colon cancer tissues

5-methylcytosine (m(5)C) can be used as a sensitive marker of progress of the tumor formation induced by the oxidative damage reactions. We have analyzed the amount of m(5)C in DNA of patients with breast and colon cancers. Two dimensional thin layer chromatography (TLC) has been used to monitor 5-methylcytosine level in DNA extracted from cancer tissues. The level of methylation of cytosine at C-5 position in DNA from breast cancer patients correlates well with the malignancy of tumors. Interestingly higher amount of m(5)C in DNA for the breast cancer patients treated with different chemotherapeutics was observed. It suggests an activation of DNA methyltransferase as well as a genomic suppression of the DNA repair genes expression. These differences clearly reflect the health condition of patients and support the global analysis of m(5)C in DNA as a good marker for diagnosis of neoplasia in clinical practice.     

Coxsackievirus and adenovirus receptor expression in non-malignant lung tissues and clinical lung cancers

Adenoviral vector mediated gene delivery has been applied in clinical trials and mechanistic studies to explore new treatment approaches for lung cancers. The expression of coxsackievirus adenovirus receptor (CAR), the primary receptor for the most commonly used adenovirus serotype 5 (Ad5)-based vectors, predominantly determines the permissiveness of lung cancer cells. CAR expression is also suggested to modulate tumor cell proliferation capacity. Here, we studied CAR expression in archival lung cancer specimens by using well-characterized CAR 72 antibodies. High levels of CAR expression were observed in most of the 32 cases of squamous cell carcinoma lung cancers and in all the five cases of small cell lung cancers investigated. In contrast, high levels of CAR expression were detected only in 6 of 22 adenocarcinoma lung cancers. The relative levels of CAR expression did not correlate with the pathologic grade in lung cancers, and was thus inconsistent with a role of modulating cancer cell proliferation. Of note, CAR expression was not detected in non-malignant alveolar cells. Our data suggest a preferred utility of Ad5 vector mediated gene delivery to squamous cell carcinoma lung cancers, small cell lung cancers, but not to the majority of adenocarcinoma lung cancers.     

Immunohistochemical staining patterns of MUC1, MUC2, MUC4, and MUC5AC mucins in hyperplastic polyps, serrated adenomas, and traditional adenomas of the colorectum.

We studied the distribution of the four human apomucins MUC1, MUC2, MUC4, and MUC5AC in hyperplastic polyps, serrated adenomas, and traditional adenomas of the colorectum using immunohistochemical techniques, with the aim of comparing and contrasting their patterns of expression. A series of 12 hyperplastic polyps, 27 serrated adenomas, and 20 traditional adenomas was studied. No significant change in apomucin expression was observed in traditional adenomas compared with normal colorectal epithelium, except for MUC5AC, which was present in 12 of the adenomas (60%) and only 20% of the normal samples. In both hyperplastic polyps and serrated adenomas, MUC2 and MUC5AC mucin expression was consistently and markedly increased. In 50% of the hyperplastic polyps, MUC4 was reduced but in the remaining cases was similar to normal. Loss of MUC4 expression was observed in all serrated adenomas. MUC1 was not increased in the hyperplastic polyps but increased expression was seen in 17 of the serrated adenomas (63%). Similar altered distribution patterns of MUC2, MUC4, and MUC5AC were seen in hyperplastic polyps and serrated adenomas, whereas traditional adenomas showed little change from normal patterns of expression. Although hyperplastic polyps are commonly defined as benign lesions without neoplastic potential, the similar phenotypes of hyperplastic and serrated adenomas and the existence of mixed polyps suggest that these lesions may represent a histogenetic continuum.     

Immunohistochemical analysis of paraoxonases-1, 2, and 3 expression in normal mouse tissues

The paraoxonase (PON) enzyme family, comprising PON1, PON2, and PON3, are antioxidant enzymes that degrade oxidised phospholipids. We describe the immunohistochemical localisation of the PON proteins in the normal mouse. Antibodies were obtained by inoculating rabbits with peptides derived from specific sequences of mature PONs. PON1 and PON3 were detected in the skin external epithelium, acini of the sebaceous glands, tongue epithelium, acini of the submandibular gland, surface epithelia of the stomach and the intestine, hepatocytes, exocrine pancreas acini, fibre tracts of the encephalon and the spinal cord, skeletal and cardiac muscle, eye lens epithelium and retinal layers, adipocytes, chondrocytes, epithelial cells of the trachea and bronchiole, ovary follicular fluid, seminiferous tubules, spermatozoa, and kidney proximal tubules. PON2 expression was weaker than that of PON1 and PON3, and was absent in some of the tissues studied, such as submandibular gland, nerve cells, and adipocytes. In muscle cells, PON2 expression was restricted to the endomysium. Apolipoprotein A-I did not colocalise with PONs, suggesting local synthesis. This study provides an experimental model to investigate the role played by these enzymes as antioxidants and their relationship with the development of a variety of diseases.     

Immunohistochemical analysis of secretoglobin SCGB 2A1 expression in human ocular glands and tissues

Human secretoglobin (SCGB) 2A1 (or lipophilin C, lacryglobin, mammaglobin B) is a small protein of unknown function that forms heterodimers with secretoglobin 1D1 (lipophilin A) in tears. SCGB 2A1 is homologous to mammaglobin (mammaglobin A) and the C3 component of prostatein, the major secretory protein of the rat ventral prostate. Androgen-dependent expression of SCGB 2A1 has been observed in the prostate. Besides identification of SCGB 2A1 in the tear proteome only its mRNA had been detected in the lacrimal gland. Here, we report expression of SCGB 2A1 in all ocular glands and in the keratinized stratified squamous epithelium of the eyelid as well as in the stratified epithelium of the conjunctiva and in the orbicularis oculi muscle. Almost all of these tissues are also known to express the androgen receptor. Therefore, we conclude that presence of the androgen signalling machinery could be the main general determinant of SCGB 2A1 expression. Implications of the presence in tear fluid of an androgen-regulated secretoglobin, which most likely binds hydrophobic ligands, for tear film lipid layer formation and function is discussed.