Expression of MT-1 MMP, MMP2, MMP9 and TIMP2 mRNAs in ductal carcinoma in situ and invasive ductal carcinoma of the breast

We investigated the expression of membrane type-1 (MT1)-MMP, MMP2, MMP9 and TIMP2 mRNAs and their roles in ductal carcinoma in situ (DCIS) and T1 and T2 invasive ductal carcinoma of the breast. We further compared these two types of carcinomas for differences in microvessel density, and expression of angiogenic factors and CD44std. MT1-MMP, MMP2, MMP9 and TIMP2 mRNA were expressed in both DCIS and invasive ductal carcinomas. Expression rates of MT1-MMP, MMP2, MMP9 and TIMP2 mRNAs were not statistically different between DCIS and invasive ductal carcinomas, nor did they differ statistically when grouped by tumor size, histologic grade or nuclear grade of invasive ductal carcinoma. Microvessel density and expression of VEGF and TGF-beta1 were not statistically different between DCIS and invasive ductal carcinoma. CD44std expression was significantly increased in DCIS compared to invasive ductal carcinoma (p < 0.05) and it was also significantly increased in lower clinical stage, histologic grade and nuclear grade of invasive ductal carcinoma (p < 0.05). Axillary node metastasis was significantly correlated with MT1-MMP mRNA, VEGF and TGF-beta1 expression (p < 0.05) and MT1-MMP mRNA was positively correlated with VEGF expression and TIMP2 mRNA (p < 0.05). In summary, patterns of MMP mRNA expression in DCIS and invasive ductal carcinoma suggest that the invasive potential of breast carcinoma is already achieved before morphologically overt invasive growth is observed. As MT1-MMP mRNA expression is significantly correlated with axillary nodal metastasis, it may be useful as a prognostic indicator of invasive ductal carcinoma. Considering the positive correlation of MT1-MMP mRNA and TIMP2mRNA expression, our finding supports a role for TIMP2 in tumor growth, as well as the utility of CD44std as a prognostic indicator of breast cancer.     

乙型肝炎病毒对HepG2细胞侵袭的影响

目的 研究HBV全长对HepG2细胞侵袭相关基因表达及活性的影响,探讨HBV在整体水平对HepG2细胞侵袭的影响.方法 采用定量PCR分析HBV对HepG2细胞MMP2、9和TIMP1-4基因转录的影响;通过明胶酶谱及反相明胶酶谱检测MMP2、MMP9及TIMPs的活性;应用体外侵袭小室法检测细胞的侵袭能力.结果 HBV的复制可以促进HepG2细胞MMP2、MMP9、TIMP1和TIMP3基因的转录,抑制TIMP4基因转录,增强HepG2细胞MMP2 、MMP9的活性并增强细胞中TIMP1、TIMP3功能,HBV稳定复制的细胞具有更强的体外侵袭能力.结论 HBV可影响HepG2细胞MMPs和TIMPs的基因转录、表达及功能,促进HepG2细胞的体外侵袭,这可能与HBV相关的HCC侵袭转移密切相关.     

Expression and role of matrix metalloproteinases MMP-2 and MMP-9 in human spinal column tumors

Matrix metalloproteinases (MMPs) have been implicated in the process of tumor invasion and metastasis formation. Thus, we determined the expression of MMPs in various primary and metastatic spinal tumors in order to assess the role of these enzymes in spinal invasion. MMP expression was examined by immuno-histochemical localization, and quantitative evaluation of MMP protein content was determined by enzyme-linked immunosorbant assay (ELISA) and Western blotting. MMP enzyme activity was determined by gelatin zymography. Lung carcinomas and melanomas metastatic to the spine were shown to have higher levels of MMP-9 activity than those of breast, thyroid, renal metastases and primary spinal tumors. Immunohistochemical analysis revealed similar difference in expression of MMP-9 in tissue samples. When the tissue samples were subjected to gelatin zymography for examination of MMP-2 and MMP-9 activity and to ELISA and Western blotting for quantitative estimation of protein content, the most striking results were obtained for lung carcinomas and melanomas relative to the other tumors. Lung carcinomas and melanomas metastatic to the spine had considerably higher levels of MMP-9 activity than those of primary spinal tumor or breast, thyroid, and renal carcinoma metastases. Within the metastatic tumor category, neoplasms that are known to be associated with the shortest overall survival rates and most aggressive behavior, such as lung carcinomas and melanomas, had the highest levels of MMP-2 and MMP-9 activity compared to those less aggressive metastatic tumors such as breast, renal cell, and thyroid carcinomas. Our results suggest that MMPs may contribute to the metastases to the spinal column, and overexpression of these enzymes may correlate with enhanced invasive properties of both primary and metastatic spinal tumors.© Kluwer Academic Publishers 1998     

Malignant glandular lesions and glandular differentiation in invasive/noninvasive urothelial carcinoma of the urinary bladder

Although the lumen of the urinary bladder is covered with only urothelial epithelium, malign glandular lesions (eg, nonurachal adenocarcinoma) and benign lesions (eg, cystitis cystica and cystitis glandularis) can also rarely occur in this site due to its characteristic embryologic development. Glandular differentiation is uncommon in urothelial carcinomas and is even less common in noninvasive urothelial cancers. In addition, in situ urothelial carcinomas are more likely to progress in the presence of glandular differentiation toward high-grade urothelial carcinomas and/or aggressive urothelial carcinomas. Pure nonurachal adenocarcinomas and mixed carcinomas (urothelial carcinoma and adenocarcinoma) are very rare, and their pathogenesis is not clear. Most of the nonurachal adenocarcinomas are thought to arise on the grounds of cystitis glandularus with intestinal metaplasia. Here, I present 2 cases with noninvasive urothelial carcinoma with substantial glandular differentiation showing progression to signet ring cell carcinoma and invasive urothelial carcinoma, one case with mixed carcinoma (urothelial carcinoma and adenocarcinoma) and another case with pure adenocarcinoma developing from cystitis glandularis with intestinal metaplasia, and discuss malign glandular lesions in the bladder and invasive/noninvasive urothelial carcinomas with glandular differentiation.     

Localization of MT1-MMP, TIMP-1, TIMP-2, and TIMP-3 messenger RNA in normal, hyperplastic, and neoplastic endometrium. Enhanced expression by endometrial adenocarcinomas is associated with low differentiation

We studied membrane-type matrix metalloproteinase-1 (MT1-MMP), tissue inhibitor of metalloproteinase-1 (TIMP-1), TIMP-2, and TIMP-3 messenger RNA (mRNA) using in situ hybridization to elucidate their temporal and spatial expression patterns in normal, hyperplastic, and neoplastic endometrium. All mRNAs studied were expressed weakly in proliferating endometrium but were induced strongly in late secretory endometrium except MT1-MMP. Endometrial hyperplasia samples did not show increased MT1-MMP or TIMP mRNA expression, indicating that the overall expression patterns in hyperplasia are comparable to those in proliferating endometrium under estrogen effect and that synthesis of extracellular matrix proteins, rather than degradation, predominates in this condition. Exceptionally, stromal cells in areas of desquamation were seen to express focally intense MT1-MMP mRNA in hyperplasia samples. All mRNAs investigated were expressed increasingly in endometrial adenocarcinomas, especially in less differentiated carcinomas. Furthermore, gelatin zymography revealed higher functional degradative activities in carcinoma tissues than in normal endometrium. Our results indicate that MT1-MMP expression, together with that of TIMPs, is involved most notably in normal endometrium under progesterone effect and, without being connected to cyclic hormonal levels, has an important role in the invasive growth of endometrial adenocarcinomas.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in some endocrine organs and their tumors

Matrix metalloproteinases (MMPs) are single-chain zinc-containing metalloenzymes. The MMP gene family currently includes more than 19 endopeptidases. Both MMP-2 and 9 are widely expressed by many stromal and endothelial cells. Tissue inhibitors of metalloproteinases (TIMPs) form complexes with MMPs, which in turn inhibit active MMPs. MMP and TIMP homeostasis has been implicated in many aspects of both physiological and pathological processes. The latter include tumor invasion and metastasis. Although ductal adenocarcinomas of pancreas were immunocytochemically faintly stained for MMPs and TIMPs, normal pancreatic islets in the normal adjacent pancreas were found to be strongly stained for MMPs and TIMPs. Five kinds of islet cell tumors, including insulinomas, gastrinomas, glucagonomas, pancreatic polypeptide- (PP) omas, and nonfunctioning islet cell tumor, were stained for MMPs and TIMPs. The tumor cells were relatively weakly stained for MMPs and TIMPs compared to normal islets. Similarly, weaker staining for MMPs and TIME’s was noted for medullary thyroid carcinomas (MTCs) and pituitary adenomas. There was no correlation between immunostaining intensity of protein hormones and MMPs and TIMPs. However parathyroid hyperplasia, adenoma, and carcinoma that stained for MMPs and TIMPs were weaker, which paralleled the weaker immunostaining for parathyroid hormone and chromogranin. This weaker staining for MMPs and TIMPs in endocrine tumors may imply a less significant role of tumor invasion and metastasis by MMP and TIMP homeostasis. At present, immunocytochemical staining for MMPs and TIMPs may well be used as new markers for neuroendocrine cells and their tumors.     

Expression of MMP2, MMP9, MT1-MMP, TIMP1, and TIMP2 mRNA in valvular lesions of the heart

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play an important role in several diseases. This study was undertaken to investigate the mRNA synthesis of MMP2, MMP9, membrane-type 1 (MT1)-MMP, and matrix metalloproteinase inhibitors TIMP1 and TIMP2 by in situ hybridization in a set of heart mitral and aortic valves operatively removed due to degenerative or inflammatory valvular diseases. The material consisted of 21 valves, eight with endocarditis and 13 with a degenerative valvular disease. The samples were studied by in situ hybridization with specific probes for MMP2, MMP9, MT1-MMP, TIMP1, and TIMP2. Synthesis of MMP2 mRNA was found in seven valves, five with endocarditis and two with degenerative valvular disease. Signals for MMP9 mRNA were found in two cases with endocarditis and five cases with degenerative valvular disease. No signal for MT1-MMP mRNA was found in the lesions. TIMP1 mRNA, on the other hand, was found in 17 cases, both endocarditis and degenerative valvular disease. TIMP2 mRNA was found in three cases of endocarditis. The signals for MMP2, MMP9, TIMP1, and TIMP2 mRNA were localized in endothelial cells and in fibroblast-like cells expressing alpha-smooth muscle actin, thus showing myofibroblast-type differentiation. The results show that matrix metalloproteinases MMP2 and MMP9, and matrix metalloproteinase inhibitors TIMP1 and TIMP2 mRNAs are synthesized in diseased valves and suggest that they may contribute to matrix remodelling in valvular disease.     

Regulation of matrix metalloproteinases and their inhibitor genes in lipopolysaccharide-induced endotoxemia in mice

An imbalance between matrix metalloproteinases (MMPs) and inhibitors of MMPs (TIMPs) may contribute to tissue destruction that is found in various inflammatory disorders. To determine in an in vivo experimental setting whether the inflammatory reaction in the course of lipopolysaccharide (LPS)-induced endotoxemia causes an altered balance in the MMP/TIMP system, we analyzed the expression of a number of MMP and TIMP genes as well as MMP enzymatic activity in the liver, kidney, spleen, and brain at various time points after systemic injection of different doses of LPS in mice. Injection of sublethal doses of LPS led to an organ- and time-specific pattern of up-regulation of several MMP genes and the TIMP-1 gene in the liver, spleen, and kidney, whereas in the brain only TIMP-1 was induced. Injection of a lethal dose of LPS caused similar but more prolonged expression of these MMP genes as well as the induction of additional MMP genes in all organs. In LPS-treated mice in situ hybridization revealed collagenase 3 gene induction in cells resembling macrophages whereas TIMP-1 RNA was detected predominantly in parenchymal cells. Finally, gelatin zymography revealed increased gelatinolytic activity in all organs after LPS treatment. These observations highlight a dramatic shift in favor of increased expression of the MMP genes over the TIMP genes during LPS-induced endotoxemia, and suggest that MMPs may contribute to the development of organ damage in endotoxemia.     

Expression and intracellular localization of matrix metalloproteinases in intraductal papillary mucinous neoplasms of the pancreas

To analyze the expression of matrix metalloproteinases (MMPs) and their relationships with the histological grades of the intraductal papillary mucinous neoplasm (IPMN) of the pancreas, we examined the frequency of expression and intracellular localization of MMP1, MMP2, MMP3, MMP7, and MMP9 in IPMN by immunohistochemistry. A total of 45 IPMN lesions (14 adenomas, 17 borderline lesions, nine noninvasive carcinomas, and five invasive lesions) from 21 patients were examined. MMP1, MMP2, MMP7, and MMP9 were expressed in tumor cells. Frequency of tumor cells expressing MMP7 was low in adenomas (median, 5.0%), higher in borderline lesions (median, 30.0%), in noninvasive carcinomas (median, 50.0%), and in invasive lesions (median, 80.0%), with a significant trend (P < 0.0001). Such a trend was also observed when the lesions were classified into gastric and intestinal subtypes (P < 0.0001 and P = 0.011, respectively). Basolateral expression of MMP7 in tumor cells was more prominent in lesions with higher histological grades (P < 0.0001). The frequency and the localization of MMP1, MMP2, and MMP9 did not correlate to the histological grades. MMP7 may contribute to the process by which IPMN advances from adenoma to carcinoma and to subsequent invasion of tumor cells in IPMN.     

Cathepsin K is selectively expressed in the stroma of lung adenocarcinoma but not in bronchioloalveolar carcinoma. A useful marker of invasive growth

Lung bronchioalveolar carcinomas (BACs) are noninvasive tumors showing lepidic growth and excellent prognosis, whereas all the other variants of adenocarcinoma are invasive tumors with a worse prognosis. The identification of minimal invasive foci in adenocarcinoma, therefore, is of prognostic relevance. A series of 68 pulmonary tumors, including 40 acinar/papillary adenocarcinomas, 18 adenocarcinomas of the mixed subtype, and 10 BACs was tested by immunohistochemical analysis for cathepsin K expression, a proteinase involved in bone and extracellular matrix remodeling. Cathepsin K was produced by epithelial tumor cells in most invasive adenocarcinomas and, interestingly, by macrophages and fibroblasts in the stroma of invasive adenocarcinomas but not of BACs (P < .001). Our findings suggest pathogenetic implications of cathepsin K in the mechanisms of tumor invasiveness in lung carcinoma; in addition, cathepsin K immunodetection may be a valuable adjunct in the correct classification of pulmonary adenocarcinomas, especially in small sclerosing BACs and mixed adenocarcinoma subtypes with minimal infiltrative growth.     

Expression of metalloproteinases and its inhibitor in later stage of rabbit neointima development

Neointima formation after arterial de-endothelialization refers not only to smooth muscle cell (SMC) migration and proliferation, but also involves extracellular matrix (ECM) metabolism. Most studies regarding the role of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in neointima have focused on the early phase of vascular remodeling. In this study, we examined the expression of MMP and TIMP in rabbit aortic neointima at a relatively late stage of lesion development, between 4 and 12 weeks after initial de-endothelialization. Northern blot analysis revealed expression of steady-state MMP-9 mRNA was increased up to the 4th week and MMP-2 mRNA to the 12th week after de-endothelialization. In situ hybridization shown that MMP positive cells were predominantly distributed in arterial neointima. Expression of TIMP-1 mRNA was continuously up-regulated up to the 12th week and TIMP-1 positive cells, primarily SMCs, were also localized to the neointimal tissue. Alteration at mRNA level was accompanied by that at protein level, as assessed by SDS-PAGE zymography for MMPs and immunoblotting for TIMP-1. The profile of alteration at protein level correlated well with that at mRNA level. These data suggest that synthesis of MMPs and TIMP is a prolonged process and arterial SMC is a major source of MMP production in arterial neointima. Enhanced synthesis of MMPs and TIMPs at late stage of neointimal development may contribute to arterial ECM metabolism.     

Kinetics of matrix metalloproteinases and their regulatory factorsin mercuric chloride-induced tubulointerstitial fibrosisin Brown Norway rats

We investigated the kinetics of matrix metalloproteinases (MMPs) and their regulatory factors mRNAs in the kidneys of mercuric chloride-treated Brown Norway rats. The expression of MMP-1 mRNA remained at lower levels than control, while other MMPs mRNAs were upregulated. The expression of tissue inhibitor of matrix metalloproteinase (TIMP)-1 mRNA showed significant upregulation. On the other hand, the expressions of TIMP-2 and TIMP-3 mRNAs were not significantly changed. In the plasmin-dependent pathway, the expression of plasminogen activator inhibitor (PAI-1) mRNA was continuously increased, while the expression of urokinase-type plasminogen activator (uPA) mRNA was not increased. The signals of TIMP-1 and PAI-1 mRNAs examined by in situ hybridization, were localized in the regenerative epithelial cells of the proximal tubules. In conclusion, these findings suggest that the activity of MMPs may bealtered by MMP-1 downregulation and inhibition of MMP activity by PAl-1 and TIMP-1 generated from tubular epithelial cells.     

Hep Par 1 expression in carcinoma of the cervix: implications for diagnosis and prognosis

AIMS: To determine the frequency and pattern of Hep Par 1 expression in cervical carcinomas of various histological types and to correlate expression with prognostic parameters. METHODS: Twenty nine cervical carcinomas were analysed for tumour type, hepatoid and neuroendocrine differentiation, and vascular invasion. A semiquantitative analysis was performed for Hep Par 1, alpha fetoprotein, chromogranin, and synaptophysin immunoreactivity. RESULTS: Hep Par 1 expression was seen in seven of the 29 cervical carcinomas (three of seven adenocarcinomas, one of 17 squamous cell carcinomas, one of two adenocarcinomas with adenocarcinoma in situ, one of two adenocarcinomas in situ, and one of one large cell neuroendocrine carcinoma with adenocarcinoma in situ). Normal looking endocervical epithelium was also positive in one case. Cases expressing Hep Par 1, with or without neuroendocrine coexpression, were associated with a higher rate of vascular invasion and a worse prognosis. Three of the five cases expressing neuroendocrine markers also coexpressed Hep Par 1. CONCLUSIONS: Hep Par 1 expression in carcinoma of the cervix is not uncommon and is present in a variety of histological types. Expression of this marker appears to be associated with more aggressive biological behaviour and a worse prognosis. The uterine cervix is another site that may express Hep Par 1 and hence the use of this antibody in situations of diagnostic difficulty, especially involving lesions within the liver, have to be coupled with the knowledge of the range of tissues it may stain.     

Simultaneous demonstration of gelatinolytic activity, morphology, and immunohistochemical reaction using zymography film

In situ zymography has been used to assess gelatinolytic activity, which is mainly due to matrix metalloproteinases (MMPs) in cancer tissues. MMPs play an important role in cancer invasion and metastasis. Film in situ zymography (FIZ) enables the in situ evaluation of gelatinolytic activity with high reproducibility. In this article, we report a study of FIZ, in a case of breast cancer with an invasive carcinoma component showing clear gelatinolytic activity, and in a non-invasive carcinoma component showing little gelatinolytic activity. Immunohistochemistry on FIZ was also performed. The simultaneous detection of gelatinolytic activity and immunohistochemical reaction was established in a single film. Immunohistochemistry on FIZ may have good potential for the investigation of cancer microenvironment.     

In situ DNA hybridization of cervical small cell carcinoma and adenocarcinoma using biotin-labeled human Papillomavirus probes.

A preferential association of human Papillomavirus (HPV) type 18 with cervical small cell carcinoma and adenocarcinoma has been identified by in situ and blot hybridization analysis using radionucleotide-labeled DNA and RNA probes. We attempted to detect HPV DNA in nine cases each of invasive cervical small cell carcinoma and adenocarcinoma using biotin-labeled probes to HPV types 6/11, 16/31/33/35, and 18 with a peroxidase-conjugated streptavidin detection system. HPV type 18 DNA was detected within four of nine small cell carcinomas and one of nine adenocarcinomas. HPV types 16/31/33/35 were detected in one additional case of cervical adenocarcinoma. All HPV-positive small cell and glandular tumors showed a distinctive, punctate, often juxtanucleolar pattern of nuclear staining which involved the majority of carcinoma cells throughout each neoplasm. This pattern of HPV DNA labeling has not been observed in any of the HPV-positive typical squamous carcinomas or condylomas hybridized at our institution. It is possible that punctate nuclear HPV DNA staining is a marker of viral integration into the host cell genome. We conclude that in situ DNA hybridization with biotinylated probes, although less sensitive than detection of virally transcribed RNA, still allows detection of relatively low copy numbers of HPV DNA in cervical small cell carcinomas and adenocarcinomas. Furthermore, the spatial precision of biotinylated probes may provide morphological information not obtainable using radionucleotide-labeled probes.