Dual over-expression pattern of membrane-type metalloproteinase-1 in cancer and stromal cells in human gastrointestinal carcinoma revealed by in situ hybridization and immunoelectron microscopy

Membrane-type metalloproteinase-l (MTI-MMP) is a transmembrane metalloproteinase, which activates pro-gelatinase A. There has been disagreement as to whether the cell types expressing MTI-MMP are cancer cells or stromal fibroblasts. Using human gastrointestinal carcinomas, the present study disclosed the tissue localization of MTI-MMP mRNA by in situ hybridization and ultrastructural localization of its protein by immunoelectron microscopy. In normal colon and stomach tissues, MTI-MMP mRNA and protein were negative or faintly positive both in epithelial cells and in stromal fibroblasts, except in the fundic gland of the stomach, which showed the positivity for MTI-MMP. In contrast, gastrointestinal cancer tissue showed over-expression of MTI-MMP mRNA and protein both in cancer cells and in stromal cells (fibroblasts). Stromal fibroblasts also expressed mRNA for gelatinase A and type-l procollagen. Double immunohistochemistry revealed that macrophages were also positive for MTI-MMP. Immunoelectron microscopy showed that MTI-MMP was localized along the plasma membrane of cancer cells and macrophages and in rough endoplasmic reticulum of fibroblasts. The present study reveals a dual over-expression pattern of MTI-MMP both in cancer cells and in stromal fibroblasts; the expression in cancer cells may be related to the invasive growth, whereas that in fibroblasts may be related to the tissue remodeling process caused by invasive growth of cancer cells. © 1996 Wiley-Liss, Inc.     

Similarities of in situ mRNA Expression between Gelatinase A (MMP-2) and Type I Procollagen in Human Gastrointestinal Carcinoma: Comparison with Granulation Tissue Reaction

The mRNA localizations of gelatinase A (MMP-2) and type I procollagen in human gastrointestinal carcinoma and non-neoplastic fibrous granulation tissue were compared. By in situ hybridization, gelatinase A was shown to be expressed in fibroblasts not only in cancer stroma but also in the "reactive fibrosis zone," which surrounds the invasive margin of cancer. In most cases, no accentuation of gelatinase A expression was observed in the invasive margin. This localization pattern of gelatinase A was essentially the same as that of type I procollagen. In gastric cancer associated with deep ulceration, gelatinase A was more abundantly expressed in fibroblastic cells in granulation tissue of the ulcer base than in cancer stroma. The same situation was found in non-neoplastic fibrous granulation tissue, in which fibroblasts abundantly expressed mRNAs for both gelatinase A and type I collagen. Scar tissue (old fibrotic lesion) showed diminished expression of mRNAs for both gelatinase A and type I procollagen. The localization patterns suggest another function of gelatinase A in cancer tissue as a turnover enzyme of the extracellular matrix.     

Regulation of Gelatinase Production in Metastatic Renal Cell Carcinoma by Organ-specific Fibroblasts

We have recently established a human renal cell carcinoma KG-2 line that is tumorigenic in the subcutis (ectopic) and kidney (orthotopic) of nude mice but spontaneously metastasizes to the lung only after orthotopic implantation. KG-2 cells growing in the kidney (orthotopic) and lung metastases secreted higher levels of gelatinase than did cells growing in the subcutis (ectopic). We examined whether organ-specific fibroblasts play a role in the regulation of gelatinase production and invasion by renal carcinoma cells. The gelatinase level in the culture supernatants of KG-2 cells was increased by their cultivation with mouse kidney or lung fibroblasts. In contrast, cocultivation of KG-2 cells with mouse skin fibroblasts resulted in a significant reduction of gelatinase activity. Similar results were obtained by culturing KG-2 cells in the media conditioned by the different mouse fibroblasts. We, therefore, investigated effects on KG-2 cells of cytokines and growth factors known to be produced by fibroblasts of various origins. Of ten cytokines and growth factors tested, basic fibroblast growth factor, hepatocyte growth factor, and transforming growth factor-β₁ (TGF-β₁) stimulated gelatinase expression by the cultured KG-2 cells. Parallel immunohistochemical analyses revealed that mouse kidney and lung fibroblasts produced higher levels of TGF-β₁ than did skin fibroblasts. These results indicate that gelatinase production by KG-2 renal cell carcinoma cells is influenced by the organ microenvironment. Specifically, organ-specific fibroblasts regulate the production of degradative enzymes by KG-2 cells and, hence, profoundly influence their invasive and metastatic capacity.     

Coordinate enhancement of gelatinase a mRNA and activity levels in human fibroblasts in response to breast-adenocarcinoma cells

Gelatinases/type-IV collagenases are metalloproteinases involved in some carcinoma invasion and metastatic processes. The exact cellular source of the 72-kDa gelatinase A is controversial. We have analyzed the expression of mRNA coding for gelatinase A in vivo by in situ hybridization on breast-cancer tissues. The mRNA for gelatinase A was present in fibroblasts. We have therefore evaluated the gelatinase-A activity in vitro, in co-cultures of different breast adenocarcinoma cell lines and human fibroblasts. In monoculture, none of the tumor cells tested produced detectable amounts of gelatinase A. The gelatinase-A activity was enhanced in cultures of fibroblasts maintained in the presence of MDA-MB 231 or SKBR3 cells, or their conditioned medium. This increased enzymatic activity was evidenced both in the culture medium and in the membrane fraction and was paralleled by enhancement of the steady-state levels of mRNA. These results are an in vitro demonstration of a regulation of fibroblasts gelatinase-A production by soluble factors secreted by breast-tumor cells.     

Molecular mechanism of black tea polyphenols induced apoptosis in human skin cancer cells: involvement of Bax translocation and mitochondria mediated death cascade

Theaflavins (TF) and thearubigins (TR) are the most exclusive polyphenols of black tea. Even though few previous reports showed the anticancer effects of TF through apoptosis, the potential effect of TR has not been appraised. This study investigated the induction of apoptosis in human skin cancer cells after treatment of TF and TR. We report that both TF and TR could exert inhibition of A431 (human epidermoid carcinoma) and A375 (human malignant melanoma) cell proliferation without adversely affecting normal human epidermal keratinocyte cells. Growth inhibition of A375 cells occurred through apoptosis, as evident from cell cycle arrest at G(0)/G(1) phase, increase in early apoptotic cells, externalization of phosphatidylserine and DNA fragmentation. In our pursuit to dissect the molecular mechanism of TF- and TR-induced apoptosis in A375 cells, we investigated whether cell death is being mediated by mitochondria. In our system, Bax translocation to mitochondria persuaded depolarization of mitochondrial membrane potential, cytochrome c release in cytosol and induced activation of caspase-9, caspase-3 and poly (ADP-ribose) polymerase cleavage. Our intricate investigations on apoptosis also explained that TF and TR augmented Bax:Bcl2 ratio, up-regulated the expression of p53 as well as p21 and inhibited phosphorylation of the cell survival protein Akt. Furthermore, TF and TR elicited intracellular reactive oxygen species generation in A375 cells. These observations raise speculations that TF as well as TR might exert chemopreventive effect through cell cycle arrest and induction of apoptogenic signals via mitochondrial death cascade in human skin cancer cells.     

The Expression of Invasive Behavior of Differentiated Squamous Carcinoma Cell Line Evaluated by an in vitro Invasion Model

In order to elucidate the factors contributory to the expression of invasiveness of oral squamous cell carcinoma, we conducted biochemical and morphological comparisons of well differentiated squamous carcinoma cell line OSC-19 (oral squamous cell carcinoma) and undifferentiated carcinoma cell line KB, both cultured on 3T3 cell-embedded collagen gel ( in vitro invasion model). OSC-19 cells invaded 3T3 cell-embedded collagen gel, while KB cells and OSC-19 cells on 3T3 cell-free gel matrix were less invasive. Cultured OSC-19 cells were characterized by lower proliferating activity, lower secretion of laminin and higher secretion of fibronectin than those of KB cells. Although the basement membrane with deposition of laminin and type IV collagen was formed, it was discontinuous at the invasion front. Gelatin zymography and western blotting showed matrix metalloproteinases (MMP), i.e., 72 kDa gelatinase (MMP-2) and 92 kDa gelatinase (MMP-9). Gelatinolytic activity was assayed, and was higher in OSC-19 cells than in KB cells or OSC-19 cells of the 3T3 cell-free model. By immuno-histochemical analysis, MMP-2-positive cells were found scattered in both cell lines without any preferential localization, and the positivity for MMP-9 was localized in the invasion front of OSC-19 cells. These results strongly suggest that the invasiveness of squamous cell carcinoma is well correlated with cell-matrix adhesion by fibronectin and with focal elaboration of metalloproteinases, especially MMP-9, which play a major role in degrading the extracellular matrix components.     

Extraction of type-IV collagenase/gelatinase from plasma membranes of human cancer cells

Tumor proteinases are considered to be important in the process of cancer invasion and metastasis. We have proposed that the surface membrane localization of these proteinases places them in an optimal site to facilitate the invasion of surrounding extracellular matrix. In this study, we have used the organic solvent, n-butanol, and the detergent, n-octyl-glucoside, to sequentially extract metalloproteinases from crude plasma membranes of human RWP-I pancreatic cancer cells. Anion exchange chromatography and gel permeation chromatography were employed to further purify enzymes with the capacity to degrade gelatin, type-IV collagen, and carboxymethylated transferrin. Gelatin zymography was used to demonstrate proteinase bands of 92, 70 and 62-kDa. Immunoblotting of solubilized, partially purified pancreatic cancer plasma membrane proteins using polyclonal rabbit antibodies, which have specificity for type-IV collagenase/gelatinase, resulted in the recognition of a 70-kDa protein, but not the 92-kDa gelatinase. A type-IV collagenase/gelatinase of 68-kDa was similarly identified in A2058 human melanoma cancer cell plasma membranes.     

Effect of organ-specific fibroblasts on proliferation and differentiation of breast cancer cells

Summary Breast carcinomas contain both tumor cells and stromal cells, including fibroblasts, endothelial cells, and lymphocytes. Proliferation of breast cancer cells may be controlled partly by mesenchymal cells, especially fibroblasts. We studied effects of fibroblasts on tumorigenicity and histologic features of breast cancer cells vivo, and analyzed fibroblast-produced growth-promoting factors in vitro. Breast carcinoma cells from four lines, and fibroblasts from lines obtained from skin and breast tissue of four patients with breast cancer were used. A suitable number of breast tumor cells and fibroblasts were inoculated subcutaneously into nude mice; resulting tumors were examined. Then conditioned medium from fibroblasts was added to cultures of breast cancer cells to study growth effects, and growth-promoting factors from breast fibroblasts were analyzed. Co-inoculation of breast cancer cells with breast fibroblasts into mice significantly increased tumorigenicity and tumor size beyond those obtained with breast cancer cells alone. Histologically, tumors resulting from co-inoculation with breast fibroblasts showed a scirrhous pattern with extensive fibrosis, while those formed by breast cancer cells injected alone or co-inoculation with skin fibroblasts showed a solid pattern. Medium from breast fibroblasts significantly increased breast cancer cell growth in vitro, while the various skin fibroblasts did not all show this effect. Structural and functional interactions between organ-specific fibroblasts and breast cancer cells may importantly regulate breast cancer growth and progression.     

Production of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Human Breast Carcinomas

We examined production and tissue localization of matrix metalloproteinase (MMP)-1 (tissue collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin-1), MMP-9 (gelatinase B), tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in human breast carcinomas. In more than half of the cases, MMP-1, MMP-2, MMP-9, TIMP-1 and TIMP-2 were immunolocalized in carcinoma cells and MMP-2 was on the carcinoma cell membranes as well, whereas MMP-3 was positively stained in less than 15% of the cases. MMP-1 staining in carcinoma cells was significantly higher in scirrhous carcinoma than in other types of carcinoma. MMP-9 expression was remarkably higher in the carcinoma cases with lymphnode metastasis than in the non-metastatic cases. MMP-3 was mainly expressed in T-lymphocytes infiltrated in the tumor stroma. Stromal fibroblasts were positive for all these MMPs except for MMP-3. The TIMP-1 levels released into the culture media by carcinoma tissues were significantly lower than those by fibroadenoma tissues, although there were no significant differences in the levels of MMP-1, MMP-2, MMP-9 and TIMP-2. Gelatin zymographical analyses showed that the activation rate of the zymogen of MMP-2 (proMMP-2) is significantly higher in the more advanced carcinoma group with lymphnode metastasis than in the metastasis-negative and fibroadenoma groups. These data indicate that MMP-1, MMP-2 and MMP-9 are highly expressed in human breast carcinoma tissue and suggest that activation of proMMP-2 may be an indicator of lymphnode metastasis of the breast carcinoma.     

Emmprin in epithelioid sarcoma: expression in tumor cell membrane and stimulation of MMP-2 production in tumor-associated fibroblasts

Emmprin is a transmembrane glycoprotein on tumor cells that stimulates peritumoral fibroblasts to produce matrix metalloproteinases (MMPs). Emmprin and the induced MMPs play a crucial role in tumor progression, invasion and metastasis of human carcinomas (epithelial malignancies). However, only a few reports have addressed its role in soft tissue sarcomas. This study investigated the expression and role of emmprin in epithelioid sarcoma (ES). Immunoblot studies of 2 ES cell lines showed that they express emmprin, and co-culture of these ES cells with dermal fibroblasts resulted in upregulation of gelatinase A (MMP-2) in fibroblasts, as shown by zymography, immunoblotting and enzyme immunoassay. This stimulation was inhibited by an activity-blocking peptide against emmprin and by antiemmprin antibody. In addition, in vivo, immunohistochemical analysis of 5 ES patient cases demonstrated diffuse emmprin expression in ES cells and MMP-2 expression in both ES cells and peritumoral fibroblasts. The histopathological findings that peritumoral fibroblasts that were not in direct contact with emmprin-expressing ES cells exhibit upregulated MMP-2 prompted us to look for a soluble form of emmprin. Soluble full-length emmprin released from ES cells was detected in conditioned medium and shown to stimulate MMP-2 production by fibroblasts. In conclusion, emmprin is expressed in ES in both membrane and soluble forms and stimulates MMP-2 production via interactions with fibroblasts, which could play a role in ES cell stromal invasion and vascular involvement.     

Production of endothelin in human cancer cell lines

Endothelin (ET)-1 is a vasoconstrictor peptide derived from endothelial cells and now known to be a local regulator of vascular tonus. Recent studies, however, have revealed that ET-1 functions also as growth factor in various cells. By using a specific ET-1 radioimmunoassay, immunoreactive (IR) ET-1, ranging from 4.2 to 150 pM (minimum detectable amount, 4.0 pM), was detected in 13 of 42 human cancer cell lines. The frequencies of IR-ET-1 production and its concentrations were high in mammary, pancreatic, and colon carcinoma cell lines. IR-ET-1 produced by cancer cells possessed the same molecular size as synthetic ET-1 and also had ET-1-like biological activity. Moreover, Northern blot analysis revealed bands corresponding to ET-1 mRNA in cancer cell lines, indicating that IR-ET-1 produced by cancer cells is a product of the ET-1 gene. Since ET-1 in the spent media is present in a sufficient amount to stimulate cellular growth, we sought ET-1 receptors in four pancreatic carcinoma cell lines and human skin fibroblasts. No ET-1 receptors were detected in the pancreatic carcinoma cell lines. However, human skin fibroblasts possessed a large number of ET-1 receptors. This finding raises the possibility that ET-1 produced by cancer cells plays a modulatory role in the growth of stromal cells surrounding cancer cells.     

Annexin II expression is reduced or lost in prostate cancer cells and its re-expression inhibits prostate cancer cell migration

While studying Bim, a BH3-only proapoptotic protein, we identified an approximately 36 kDa protein, which was abundantly expressed in all five strains of primary normal human prostate (NHP) epithelial cells but significantly reduced or lost in seven prostate cancer cell lines. The approximately 36 kDa protein was subsequently identified as annexin II by proteomic approach and confirmed by Western blotting using an annexin II-specific antibody. Conventional and 2D SDS-PAGE, together with Western blotting, also revealed reduced or lost expression of annexin I in prostate cancer cells. Subcellular localization studies revealed that in NHP cells, annexin II was distributed both in the cytosol and underneath the plasma membrane, but not on the cell surface. Prostate cancer cells showed reduced levels as well as altered expression patterns of annexin II. Since annexins play important roles in maintaining Ca(2+) homeostasis and regulating the cytoskeleton and cell motility, we hypothesized that the reduced or lost expression of annexin I/II might promote certain aggressive phenotypes of prostate cancer cells. In subsequent experiments, we indeed observed that restoration of annexin II expression inhibited the migration of the transfected prostate cancer cells without affecting cell proliferation or apoptosis. Hence, our results suggest that annexin II, and, likely, annexin I, may be endogenous suppressors of prostate cancer cell migration and their reduced or lost expression may contribute to prostate cancer development and progression.     

Role of IKKα in skin squamous cell carcinomas

Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are two major types of skin cancer derived from keratinocytes. SCC is a more aggressive type of cancer than BCC in humans. One significant difference between SCC and BCC is that SCC development is generally associated with cell dedifferentiation and morphological changes. When SCC is converted to spindle cell carcinoma, the latest stage of cancer, the tumor cells change to a fibroblastic cell morphology (epithelial-to-mesenchymal transition) and lose their differentiation markers. Recently, several laboratories have reported altered IκB kinase α (IKKα) protein localization, downregulated IKKα, and IKKα gene deletions and mutations in human SCCs of the skin, lung, esophagus, and neck and head. In addition, IKKα reduction promotes chemical carcinogen- and ultraviolet B-induced skin carcinogenesis, and IKKα deletion in keratinocytes causes spontaneous skin SCCs, but not BCCs, in mice. Thus, IKKα emerges as a bona fide skin tumor suppressor. In this article, we will discuss the role of IKKα in skin SCC development.     

泛素特异性肽酶22在胃癌细胞系中的表达和定位

目的:研究泛素特异性肽酶22(USP22)蛋白和信使核糖核酸(mRNA)在人胃癌细胞中的表达和定位.方法:分别用蛋白印迹法、实时荧光定量聚合酶链反应检测USP22在人胃癌细胞系中蛋白和基因的表达,激光共聚焦扫描显微镜检测内源USP22在SGC7901胃癌细胞中的定位.结果:USP22在人胃癌细胞系SGC7901中蛋白和基因表达水平增高,激光共聚焦扫描显微镜观察发现USP22主要定位于SGC7901细胞核中.结论:USP22在人胃癌细胞系SGC7901中高表达,主要定位于胃癌细胞核内,可能参与人胃癌发生发展的进程.     

Establishment of an oral squamous cell carcinoma cell line (NOS-1) exhibiting amplification of the erbB-1 oncogene and point mutation of p53 tumor suppressor gene: its biological characteristics and animal model of local invasion by orthotopic transplantation of the cell line

We established a new cancer cell line, NOS-1, which was derived from a human oral primary squamous cell carcinoma. Geneticin treatment was adopted to eliminate contaminating fibroblasts and to enrich tumor cells in the early stage of the culture. The NOS-1 cells showed epithelial morphological features with light and electron microscopy, and immunohistochemical analysis confirmed their epithelial origin. Overexpression of mutant p53 protein, a p53 point mutation at codon 248 with transition from CGG to TGG, and amplification of the erbB-1 oncogene/epidermal growth factor receptor gene were also observed in NOS-1 cells. The NOS-1 cells formed tumors in nude mice when transplanted subcutaneously into their backs. Further, they were transplantable orthotopically in the tongues of nude mice, and the transplanted tumors in the tongue showed diffuse invasion without forming capsules. The NOS-1 cells were useful for elucidating the mechanism involving p53 inactivation and erbB-1 oncogene amplification, as well as treatment of oral cancer.