HMGA1 protein overexpression in human breast carcinomas: correlation with ErbB2 expression.

We measured, by immunohistochemistry, HMGA1 protein expression in 212 breast tissue specimens: 6 normal samples, 28 hyperplastic lesions (13 with cellular atypia), 11 fibroadenomas, 10 in situ ductal carcinomas, 144 ductal carcinomas, and 13 lobular carcinomas. HMGA1 was not expressed in normal breast tissue; HMGA1 staining was intense in 40% of hyperplastic lesions with cellular atypia and in 60% of ductal carcinomas and weak in fibroadenomas and in hyperplastic lesions without cellular atypia. Because HMGA1 expression was similar among ductal breast carcinomas with different histologic grading, we evaluated the association between HMGA1 expression and that of other markers of breast carcinoma invasion (estrogen and progesterone receptors, Ki-67 antigen, and ErbB2) in 21 cases of grade 3 breast ductal carcinomas and 7 cases of breast lobular carcinomas. We found that HMGA1 expression tended to be associated only with c-erbB-2 expression (Spearman rho: 0.36; P=0.065). Taken together, these results suggest that HMGA1 expression might be a novel indicator for the diagnosis and prognosis of human breast cancer.     

Analysis of polypeptide expression in benign and malignant human breast lesions: down-regulation of cytokeratins.

Malignant progression of tumour cells is caused by the accumulation of genetic defects, which when combined will generate a large phenotypic diversity. Simultaneous quantitation of a large number of gene products in tumour cells is desirable, but difficult to achieve. We have here quantitated the levels of a number of abundant polypeptides in human breast carcinoma cells using two-dimensional gel electrophoresis (2-DE; PDQUEST). For this purpose, tumour cells were prepared from the tissue of 17 breast carcinomas. Fibroadenoma tissue was used as reference for benign cells. An increase of the spot density of the PCNA polypeptide was observed in rapidly proliferating tumour cells, confirming the validity of the procedures used. In the set of 24 polypeptide spots with known identity, decreases in cytokeratin and tropomyosin levels were observed. The levels of all cytokeratin forms resolved (CK7, CK8, CK15 and CK18) were significantly lower in carcinomas than in fibroadenomas. The levels of tropomyosin 2 and 3 were lower in carcinomas than in fibroadenomas. In contrast, the levels of some members of the stress protein family (pHSP60, HSP90 and calreticulin) were higher in carcinomas. Furthermore, changes in the expression of lactate dehydrogenase and GT-pi, but not in nm23, were observed. We conclude that simultaneous analysis of multiple polypeptides in human carcinomas can be achieved by 2-DE and may be useful in prognostic studies, and that malignant progression of breast carcinomas results in the decreased expression of cytokeratin polypeptides. This phenomenon must be considered in studies where cytokeratins are used as markers to identify the epithelial cell compartment in breast carcinomas.     

Immunohistologic c-myc protein in benign breast disease and cancer

We have studied the histopathology and differential distribution of the c-myc protein (Myc) in human breast tissues including 17 cases of infiltrating mammary carcinoma, 4 cases of fibroadenoma, 5 cases with fibrocystic changes, and 1 case of reduction mammoplasty (as a control). Using a sensitive immunohistochemical method on frozen tissue sections, both a rabbit polyclonal anti-c-myc antibody and a mouse monoclonal anti-c-myc antibody, H51C116, produced high levels of Myc staining in the nuclei of epithelial cells of infiltrating mammary carcinomas (30-90% of cells stained). In contrast, the nuclei of epithelial cells of fibroadenomas, and breast tissues with fibrocystic changes stained infrequently. We studied benign tissue surrounding the tumors in four cases; three were essentially negative, and one showed nuclear epithelial cell staining throughout the lobules. Sixteen of the tumors were examined in parallel, using formalin-fixed, paraffin-embedded samples. Immunohistological procedures for Myc produced uniform, intense epithelial cell cytoplasmic staining (8 cases); light epithelial cell cytoplasmic staining (5 cases) or were unstained (3 cases). We argue that the differences between frozen and paraffin sections are incompatible with the notion of simple displacement of nuclear Myc to the cytoplasm during fixation. Elevated levels of nuclear Myc in tumor cells and subsets of benign tissue are consistent with a role for Myc in mammary cell proliferation and tumorigenesis.     

Expressions of beta-catenin, APC Protein, C-myc and Cyclin D1 in Ovarian Epithelial Tumor and Their Implication

Objective： To investigate the expressions of beta-catenin, protein APC （adenomatous polyposis coil protein）, c-myc and cyclin D1 and their implication in ovarian epithelial tumor. Methods： Immunohistochemical staining with SP method was conducted to identify the expressions of beta-catenin, APC protein, c-myc and cyclin D1 in ovarian epithelial tumor in 48 cases. Results： The abnormal expression rate of beta-catenin in malignant and borderline ovarian epithelial tumors was higher than that in benign epithelial tumors （P〈0.01）. The expression rates of c-myc and cyclin-D1 in ovarian malignant and borderline epithelial tumors were higher than those in benign epithelial tumors too（P〈0.05）. The prevalence of APC protein positive expression in benign epithelial tumors were significantly greater than that in malignant epithelial tumors （P〈0.05）. A significant negative correlation was found between beta-catenin and APC protein in ovarian epithelial tumors; while a significant positive correlation was found between beta-catenin, c-myc and cyclin-D1 in ovarian epithelial tumor （P〈0.05）. Conclusion： The abnormal expressions of Beta-catenin, APC protein, c-myc and cyclin-D1 might be used to indicate the malignance transform of ovarian epithelial tumors.     

Expression of 70-kDa heat shock protein in oral lesions: marker of biological stress or pathogenicity

We showed differential expression of HSP70 during oral tumorigenesis. The precise functional role of HSP70 overexpression in the pathogenesis of betel and tobacco related oral cancer remains to be determined. To evaluate the utility of HSP70 as an indicator of the biological stress experienced by tumour cells or the malignant potential of oral epithelial lesions and predicting clinical outcome, its expression was assessed in different stages of oral carcinogenesis by immunohistochemical analysis and correlated with clinicopathological parameters. Overexpression of HSP70 protein was observed in 38 of 64 (59%) dysplastic lesions and 92 of 125 (74%) oral squamous cell carcinomas (SCCs) which included 76 of 105 cases (72%) of primary oral SCCs and 16 of 20 (80%) of recurrent oral SCCs. A significant correlation of HSP70 expression was observed with severity of dysplasia (P=0.0006767), poor histological differentiation of primary tumours (P=0.0184348), increase primary tumour size (P=0.0221103) and consumption of betel and tobacco (P<0.01). Follow-up studies showed that in patients with premalignant lesions the median transition time (premalignancy to malignancy) was significantly shorter in HSP70 overexpressing cases than those showing basal level of HSP70 (P=0.012). Oral cancer patients with elevated levels of HSP70 showed decreased median disease-free survival time (no recurrence/metastasis) than those showing basal HSP70 immunoreactivity (P=0.0246). The results suggest that HSP70 expression may not be a mere marker of biological stress but may also be implicated in the pathogenesis of oral cancer.     

Enhancing the treatment effects of tumor cell purified autogenous heat shock protein 70-peptide complexes on HER-3-overexpressing breast cancer

Objective The aim of this study was to enhance the treatment effect of tumor purified autogenous heat shock protein 70-peptide complexes (HSP70-PCs) on HER-3-overexpressing breast cancer. Methods In this study, we first studied the expression of HER-3 in breast cancer tissues and its relationship with patient characteristics. We then purified HSP70-PCs from primary breast cancer cells with different HER-2 and HER-3 expression profiles and determined the cytotoxicity of autogenous dendritic cells (DCs) and CD8+ T cells induced by these complexes. Third, recombinant human HSP70-HER-3 protein complexes were used to inhibit the autogenous HSP70-PCs purified from HER-3–overexpressing breast cancer cells, and the resulting immunological response was examined. Results The results show that HSP70-PCs can be combined with recombinant HSP70-HER-3 protein complexes to induce stronger immunological responses than autogenous HSP70-PCs alone and that these treatments induce autogenous CD8+ T cell killing of HER-3-positive breast cancer cells. Conclusion These findings provide a new direction for HSP70-DC-based immunotherapy for patients with HER-3-overexpressing breast cancer.     

Expression of the c-myc oncoprotein in human metaplastic epithelial cells of fibrocystic disease

Sections derived from fibrocystic disease in human breast tissue were examined for the presence of the c-myc oncoprotein by using the c-myc specific monoclonal antibody 9E10 (1). The results obtained revealed that the c-myc gene was not expressed in normal epithelial cells either of ducts or lobules. It was expressed mainly in mucous metaplastic cells and very rarely in apocrine cells. The c-myc protein was observed at a higher level in mucous metaplastic cells of epitheliosis and multiple papillomas. The number and intensity of positive mucous metaplastic cells was significantly increased after pretreating the sections with neuraminidase. We suggest that elevated expression of the c-myc nuclear oncoprotein in human breast metaplastic epithelial cells play a role in the early stages of malignant cell transformation.     

Fra-1在乳腺肿瘤细胞质和细胞核同时定位相关性的研究

侵袭转移是恶性肿瘤最重要也是最本质的生物学特征,是许多恶性肿瘤患者治疗失败和死亡的主要原因。最近研究发现Fra-1在细胞的运动、侵袭,维持转化细胞的恶性表型方面具有重要的作用,但是有关Fra-1在乳腺组织中的表达及Fra-1在细胞中的定位研究很少,本研究探讨转录因子Fra-1在人乳腺良、恶性肿瘤组织中的表达及细胞内的定位。方法:采用免疫组织化学方法检测良、恶性乳腺肿瘤组织Fra-1表达及细胞定位;并分析恶性乳腺肿瘤组织Fra-1表达与乳腺癌预后指标ER、PR和HER-2表达的相关性。结果:61例良、恶性肿瘤组织都存在Fra-1的细胞核表达。在20例良性肿瘤组织中,17例（85.0%）Fra-1主要定位于上皮细胞核内,3例（15.0%）可见Fra-1细胞核及细胞质共表达。而37例（90.2%）恶性肿瘤组织存在Fra-1细胞核和细胞质共表达,Fra-1在恶性肿瘤细胞质中的表达明显高于良性肿瘤（P<0.001）。恶性肿瘤Fra-1细胞质表达与ER、PR和HER-2的表达无明显相关性。结论:Fra-1蛋白的表达强度与方式与乳腺癌上皮细胞的癌变有关;其在乳腺癌细胞细胞质的滞留与乳腺癌的发生发展具有一定的相关性。      

Immunological analysis of 17 beta-hydroxysteroid dehydrogenase in benign and malignant human breast tissue.

The expression of 17 beta-hydroxysteroid dehydrogenase (17-HSD) enzyme protein was studied in benign and malignant human breast tissue using the time-resolved immunofluorometric assay (IFMA), immunoblotting and immunohistochemistry. The presence and distribution of estrogen and progestin receptors was also analyzed immunohistochemically. Cytosolic 17-HSD concentrations in malignant breast specimens were highly variable (less than or equal to 0.2-311 ng/mg protein). As was previously found for the placental enzyme, the molecular weight of the 17-HSD expressed in malignant breast tissue was 35 kDa, estimated following polyacrylamide gel electrophoresis and immunoblotting. The cellular distribution of 17-HSD was further studied by immunohistochemistry. Immunostaining for 17-HSD was observed in 71% of the benign breast lesions (fibroadenomas and cases of mastopathia chronica) and in 47% of the cancer specimens (intra-ductal carcinomas, invasive ductal carcinomas). In benign lesions, the staining was exclusively localized in the cytoplasm of epithelial cells, with no immunoreactivity in the stromal cells. The staining in the cancer specimens was also detected only in the cytoplasm of malignant epithelial cells. A strong or moderate expression of 17-HSD was related to the presence of PR in the specimen (chi 2 = 4.657, p = 0.031). However, the expression of PR was not a prerequisite for expression of 17-HSD in all the cancer specimens. Our data suggest that, in addition to the reported regulation of 17-HSD by progestins, other factors are also involved in this process in breast tissue.     

Ionizing radiation induces alterations in cellular proliferation and c-myc, c-jun and c-fos protein expression in breast epithelial cells

The identification of genes involved in breast cancer is of critical importance in understanding the pathogenesis of the disease. Expression of the nuclear proto-oncogenes, c-myc, c-jun and c-fos, are indicative of early response events during cellular proliferation. Among them, the c-myc oncogene has been found frequently over-expressed in breast cancer. In vitro systems allow us to test the sensitivity of human breast epithelial cells to different carcinogens, including ionizing radiation. The aim of this work was to define whether these oncogenes play a functional role in radiation-induced transformation of human breast epithelial cells. We examined: a) the spontaneously immortalized MCF-10F cell line, b) clones derived from these cells treated with the carcinogen benzo(a)pyrene (BP) and then transfected with c-Ha-ras-oncogene, followed, c) by a single 3 Gy dose of gamma-rays. Protein expressions were analysed by Western immunoblot assays. Results indicated that 3 Gy dose of gamma-ray decreased the expression of these oncoproteins in the MCF-10F cells (ranging from 23 to 80%). In BP1, non-tumorigenic MCF-10F cells, radiation induced an even sharper decrease in the oncoprotein levels (ranging from 50 to 100%) relative to their non-irradiated controls. In contrast, in BP1-E tumorigenic cell line radiation increased the expression in 68-80% of c-myc, c-jun and c-fos protein expression relative to non-irradiated control. Furthermore, radiation increased c-my, c-jun and c-fos protein expression in the c-Ha-ras-3 Gy cell line relative to non-irradiated control cell line (ranging from 45 and 120%). Interesting, among the tumorigenic MCF-10F cells previously exposed to both BP and c-Ha-ras (BP1-Tras-3 Gy cell line), radiation increased the c-myc, c-jun, c-fos protein expression by more than 120% relative to the non-irradiated controls. In can be concluded that the MCF-10F model of breast carcinogenesis allows us to examine various aspects of regulations in gene expression and can provide us the basis for understanding the process of breast cancer.     

Molecular mediators of tumor angiogenesis: enhanced expression and activation of vascular endothelial growth factor receptor KDR in primary breast cancer.

The progression of breast cancer growth and its ability to metastasize are associated with the process of angiogenesis. In this study, we examined the protein expression of vascular endothelial growth factor (VEGF) and its specific and functional receptor KDR in human breast tissue. We investigated a total of 13 mammary carcinomas, 3 fibroadenomas, 5 specimens with fibrocystic breast disease as well as normal (adjacent to malignant) breast tissue using immunohistochemistry and Western blot analysis. In all carcinomas examined, functional KDR protein was present independent of tumor type, tumor stage and histological grade as demonstrated by tyrosine phosphorylation analysis of KDR. When malignant tissues were compared with their neighboring non-neoplastic regions, activated KDR was found to be expressed to a much higher extent within the malignant tissue samples. In fibroadenomas, KDR was barely detectable, whereas in fibrocystic breast disease KDR expression was variable. Immunostaining of KDR was localized to endothelium and epithelium of mammary ducts in malignant and benign breast tissue, while VEGF immunoreactivity was primarily found in the endothelium and also in tumor cells and macrophages. Our data demonstrate that KDR activation is enhanced in breast cancer in vivo and emphasize the functional role of VEGF and KDR in the development of malignant breast disease.     

Elevated expression of the myc gene in human benign and malignant breast lesions compared to normal tissue

Expression of the c-myc gene in human breast lesions and in adjacent normal tissue was studied by immunohistochemical analysis. The previously described monoclonal antibody Myc1-9E10 (1) which recognizes the p62 c-myc protein was used in paraffin tissue sections. A total of 101 cases of breast disease examined included 38 simple and complex cystic disease, 18 simple and hyperplastic fibroadenomas, 36 ductal and lobular carcinomas and 9 in situ carcinomas. Whereas the adjacent normal tissue was slightly positive, 25 out of 38 cystic disease, 7 out of 18 fibroadenoma, 36 out of 36 carcinoma and 9 out of 9 in situ carcinoma specimens showed moderate to high levels of p62 c-myc expression as indicated by staining intensity. These results suggest that the c-myc protein may play a role in breast neoplasia.     

FoxM1、c-myc蛋白在基底细胞样型乳腺癌中的表达及其临床意义

目的 探讨FoxM1和c-myc蛋白在基底细胞样型乳腺癌（BLBC）、非基底细胞样型乳腺癌（non-BLBC）及癌旁正常乳腺组织中的表达情况及二者之间的相互关系.方法 采用免疫组织化学法检测66例BLBC及其癌旁正常乳腺组织、70例non-BLBC组织FoxM1和c-myc蛋白的表达情况,采用Spearman相关分析法分析其与病理临床特征的关系.结果 BLBC、non-BLBC及癌旁正常乳腺组织中FoxM1蛋白阳性表达率分别为77.3％（51/66）、60.0％（42/70）和13.6％（9/66）,c-myc蛋白阳性表达率分别为71.2％（47/66）、54.3％（38/70）和22.7％（15/66）,FoxM1和c-myc蛋白在BLBC和non-BLBC组织中表达率均明显高于癌旁正常乳腺组织,且在BLBC组织中的表达明显高于non-BLBC组织,差异均有统计学意义（均P＜ 0.05）.在BLBC中FoxM1和c-myc蛋白表达呈正相关（r=0.294,P＜ 0.05）,且FoxM1和c-myc蛋白的表达与BLBC淋巴结转移及临床分期有关（P＜0.05）.结论 FoxM1和c-myc蛋白参与了BLBC的发生、发展,并且FoxM1和c-myc蛋白有一定的协同作用.     

Correlation of heat shock protein (HSP70) expression with cell proliferation (MIB1), estrogen receptors (ER) and clinicopathological variables in invasive ductal breast carcinomas

The aim of our study was to evaluate the relationship between the expression of HSP70 protein, cell proliferation, the expression of ER receptors and the clinicopathological variables Grade and LNS in breast invasive human tumors along with the role of HSP70 protein in the prognosis of human breast cancer. A strong association between HSP70 expression and ER content, in agreement with previous data, was found which revealed a statistically significant association between HSP70 positivity and ER expression (p<0.008) in 50 cases of invasive primary human breast cancers. We also found a strong correlation between HSP70 expression, Grade and LNS of invasive ductal breast carcinomas. This suggests that the expression of HSP70 plays a significant role in the progression of human breast cancer, and might prove useful in many other malignancies as an important marker for the outcome of the disease.     

人胃癌组织中热休克蛋白70和糖调节蛋白94的表达及其意义

目的：探讨休克蛋白70（HSP70）和糖调节蛋白94（grp94）在人胃癌组织中的表达与临床病理的关系。方法：应用免疫组织化学和病理学图像分析方法研究60例人胃癌组织及其癌旁组织、发生及未发生转移的癌组织中HSP70和grp94的表达。结果：在胃癌组织中HSP70和grp94的表达明显高于癌旁组织（93.3%,81.7%vs36.7%,25.0%,P〈0.01）。HSP70和grp94在低分化和发生转移的胃癌组织中的表达明显高于非转移癌和癌旁组织（90.0%,85.0%;100%,84.6%vs36.7%,25.0%;P〈0.01）。结论：HSP70和grp94在发生转移的低分化胃癌组织和未发生转移的高分化癌组织中表达存在明显不同,可以作为胃癌的诊断或预后指标。     

Snail mRNA与HSP70mRNA在宫颈鳞癌中的表达及其与生物学特征的关系

背景与目的:Snail和热休克蛋白70(heat shock protein 70,HSP70)可能参与多种恶性肿瘤的侵袭转移,但关于两者与宫颈癌的关系报道较少.本研究探讨Snail,HSP70 mRNA在宫颈癌变过程中的表达及其与生物学特征的关系.方法:应用原位分子杂交方法,检测Snail,HSP70 mRNA在70例宫颈鳞癌、35例宫颈鳞状上皮内瘤变和15例宫颈慢性炎症组织中的表达.结果:(1)70例宫颈鳞状细胞癌组织中,Snail、HSP70mRNA阳性率分别为80.0%(56/70)、85.7%(60/70),分别高于宫颈鳞状上皮瘤变组织的60.0%(21/35)、45.7%(16/35)和宫颈慢性炎症组织的20.0%(3八5)、26.7%(4八5),差异均有统计学意义(P<0.05).(2)Snai1、HSP70 mRNA的表达在淋巴结转移组为95.8%(23/24)、91.7%(22/24),明显高于无转移组的71.7%(33/46)、82.6%(38/46)、Snail mRNA表达与淋巴结转移有关(P<0.05),而HSP70 mRNA的表达与淋巴结转移无关(P>O.05);Snail mRNA与HSP70 mRNA的表达呈正相关(r=0.306,PO.05).结论:Snail mRNA与HSP70 mRNA表达呈正相关,均与宫颈癌的发生和转移密切相关,是宫颈癌的重要生物学标志,联合检测有助于宫颈癌早期诊断和预后评价.     

Differential expression of 70–kDa heat shock-protein in human oral tumorigenesis

To understand the biological events underlying the multistep process of oral tumorigenesis we have studied the expression of 70–kDa heat-shock protein, HSP70, in human normal, pre-malignant and malignant oral tissues. Expression of HSP70 was assessed in oral-tissue specimens using a mouse monoclonal antibody against HSP70 by immunostaining and immunoblot-ting analysis. Strong nuclear and cytoplasmic HSP70 immunostaining was observed in oral squamous-cell carcinomas (30/ 38). Mild to moderate HSP70 expression was observed in oral dysplastic lesions (19/30) and basal low level of HSP70 was observed in normal oral tissues. The results were corroborated by immunoblotting analysis. The wide variation in HSP70 expression in normal, pre-malignant and malignant oral lesions suggests that it is differentially expressed during oral carcinogen-esis and may be implicated in tumor development. © 1995 Wiley-Liss, Inc.     

Immunohistochemical study of the ras oncogene expression in human breast lesions

An immunohistochemical study of ras oncogene expression in human breast lesions was carried out using a monoclonal antibody, Y13 259, to the ras encoded p21 protein. A total of 75 cases of breast disease examined included: 33 simple and complex cystic disease; 22 simple and hyperplastic fibroadenomas; 18 ductal, lobular and mixed carcinomas and 2 in situ carcinomas. Most of the complex cystic disease, hyperplastic fibroadenomas and all types of carcinomas showed high p21 expression as indicated by staining intensity. These results suggest that elevated ras expression may play an important role in the development of some premalignant and malignant breast lesions.     

Myoepithelial expression of Fas and strong nuclear expression of FasL in epithelial cells: A marker for risk stratification of breast cancer

Aim: The clinical significance of Fas and FasL in hormone-sensitive carcinomas has been reported. Our objective was to evaluate the expression of apoptosis-regulating genes Fas and FasL in Indian breast cancer and fibroadenoma patients in relation to hormone receptor status. Study Design: Retrospective. Materials and Methods: Paraffin-embedded tissue samples from 63 untreated female patients with invasive ductal carcinoma (IDC) and 32 female patients with fibroadenoma were studied. Expression of Fas and FasL was evaluated using immunohistochemical staining method. Statistical Analysis: Fisher's exact test and nonparametric correlation test (Spearman rank correlation test) were performed. Result: Fas was detected in 97% of the fibroadenomas and there was a slight decrease in levels of expression with histological grades of IDC. The expression of FasL was detected in 75% fibroadenomas and its expression increased in IDC. There was no correlation between Fas, FasL expression and hormone receptor status. Strong expression of Fas in myoepithelial cells was noted in 12 out of 32 fibroadenoma cases. Conclusion: Expression of Fas and FasL alone is unlikely to be important as a predictive factor as they express in both normal and malignant breast epithelium. But strong expression of Fas in myoepithelial cells along with strong nuclear expression of FasL in epithelial cells of fibroadenoma could be useful as an early predictive factor for onset of malignancy.