Proteinase inhibitors reduce basement membrane degradation by human breast cancer cell lines.

The relative importance of different proteinases, and their inhibition, in the breakdown of human endothelial basement membrane (BM) by MDA-MB-231 and MCF7ADR human breast cancer cell lines has been studied using 35S-labelled BM-coated 96-well culture plates. Basement membrane degradation (BMD) was independent of cell proliferation above the seeding density. Inhibitors of aspartic (pepstatin and PD 134678-0073) and cysteine proteinases (E64) had little effect on BMD under normal culture conditions, suggesting that cathepsins D, B and L have only a minor role. In contrast, inhibitors of urokinase-type plasminogen activator (uPA) and/or plasminogen activation to plasmin (aprotinin, amiloride, EACA, tranexamic acid, anti-uPA antibody) all reduced BMD by MDA-MB-231 cells by approximately 30-40%, but only in the presence of serum or plasminogen. BB94, an inhibitor of matrix metalloproteinases (MMPs), also reduced BMD by about 30% under these conditions but was similarly effective in serum-free medium. Combinations of BB94 with any of the uPA/plasminogen activation inhibitors in serum-containing medium had additive effects, while BB94 with pepstatin and E64 under serum-free conditions reduced BMD to 16% of control. Serum-containing conditioned medium exhibited appreciable BMD, largely due to aprotinin-inhibitable activity. Although small reductions in cell proliferation were seen with some inhibitors, the combination of BB94 with E64 or E64d reduced the cell population by about 60% under serum-containing conditions. These in vitro observations suggest that combinations of proteinase inhibitors, particularly of uPA/plasminogen activation and MMPs, may merit clinical evaluation as potential antimetastatic therapy for breast cancer.     

Distribution of basement membrane components in human hepatocellular carcinoma

The distribution of fibronectin (FN), laminin (LAM), and collagen IV (Coll IV), three components of the basement membranes (BM), was investigated in human hepatocellular carcinoma (HCC) and the surrounding uninvolved liver and was compared with the grade of differentiation of the tumor. The following three patterns of BM antigens were observed in HCC: (1) peritrabecular or periacinar, (2) pericellular, and (3) stromal-vascular. In the more differentiated tumors, FN, LAM, and Coll IV were observed in a peritrabecular or periacinar pattern whereas a pericellular pattern was only seen with anti-FN antisera that occasionally stained the content of acini. Double staining showed that the four antigens were usually codistributed. Occasionally, however, there was a different distribution along the BM suggesting an heterogeneity in the composition of BM. In the more anaplastic tumors and in the intrahepatic metastasis, BM components were seen around vessels and in the stroma and they were usually fragmented. The finding that FN can be located pericellularly or within acini supports the concept that FN is synthesized, at least in part, by hepatoma cells. The peritrabecular and periacinar location of Coll IV and LAM suggests a sinusoidal cell derivation of these two antigens. The immunohistochemical staining patterns for BM in HCC reflect the differentiation of the tumor, with differentiated tumors showing a relatively intact BM and poorly differentiated tumors showing a sharply defective BM.     

Distribution of individual components of basement membrane in human colon polyps and adenocarcinomas as revealed by monoclonal antibodies.

Double-label immunofluorescence was used to monitor basement-membrane composition and integrity in 22 human colon polyps, 36 adenocarcinomas and 2 metastases. Cryostat sections were stained with polyclonal anti-laminin anti-serum combined with monoclonal antibodies (MAbs) to all major basement-membrane components (laminin, entactin/nidogen, collagen type IV and large heparan sulfate proteoglycan), as well as to keratin 8. In all adenocarcinomas, including mucinous, basement membranes were altered more at the invasive front than in the parenchyma. The degree of this alteration was inversely correlated with the level of tumor differentiation. An uncoordinated loss of basement membrane components (dissociation of markers), previously described by us in rat colon adenocarcinomas, was also found in human tumors. In the great majority of adenocarcinomas a pronounced stromal reaction was seen. It was manifested by the presence of fibrillar deposits of basement-membrane components, mainly of collagen type IV and/or heparan sulfate proteoglycan. This reaction was never observed in polyps and may be derived from myofibroblasts reported to accumulate in colon cancer stroma. The combined use of antibodies to basement-membrane components and to a specific keratin may constitute an adequate immunohistochemical test for the presence of invasion, and may be useful in the histologic analysis of polyps, especially in dubious cases.     

Occurrence of components of fibrinolysis pathways in situ in neoplastic and nonneoplastic human breast tissue

The occurrence and distribution of components of fibrinolysis pathways were determined using immunohistochemical techniques applied to 10 cases of primary carcinoma of the breast, normal breast tissue obtained from two patients undergoing reductive mammoplasty, and three cases of benign breast tumors. Tumor cells stained for urokinase- and tissue-type plasminogen activators, plasminogen activation inhibitor-1, plasminogen, and plasmin-antiplasmin complex neoantigen. The tumor connective tissue stained for fibrinogen and its D fragment plasmin digestion product. By contrast, only occasional nonneoplastic duct epithelial cells stained for urokinase- and tissue-type plasminogen activators and there was little or no staining for the other antigens tested. These results are consistent with the existence of local amplification of expression of enzymatically active plasminogen activators, and particularly of urokinase-type plasminogen activator, in situ in primary breast cancer tissue. These features distinguish malignant from benign breast tissue and may modulate neoplastic progression through an effect on tumor cell proliferation, invasion, and metastatic dissemination.     

An immunoperoxidase study of the components of the basal membrane in cancers of the cervix]

An immunohistochemical study of samples from 12 cervical carcinomas was carried out using monoclonal antibodies against laminin (L) and heparan sulfate proteoglycan (HSP). Cryostat sections of operative and biopsy specimens were processed by the immunoperoxidase method. L and HSP were identified in the basal membrane of normal and neoplastic epithelium. Basal membrane disruptions were found in in situ, microinvasive and invasive carcinomas. However, in the first group they were less common and occurred only in the presence of lymphohistiocytic infiltration of the underlying stroma. An inverse relationship was established between depth of invasion and amount of L and HSP in the basal membrane of tumor epithelium.     

Epithelial mesenchymal transition during the neoplastic transformation of human breast epithelial cells by estrogen

Epithelial-mesenchymal transition (EMT) in epithelial cells has been indicated as an important component of neoplastic transformation although, the genetic mechanism involved in this process has not been defined. The aim of this study was to evaluate the expression of different genes related to EMT such as E-cadherin, TGFbeta1, TGFbeta2, h-RAS, TWIST1, SNAIL2, SMAD5, FN1, CEACAM1 and JAG1 using the in vitro-in vivo model of the estrogen induced cell transformation developed in our laboratory. The E2-transformed MCF-10F (E2 70) cells and the tumorigenic cell line C5-A8-T8 (C5-T8) exhibit progressive loss of ductulogenesis as demonstrated by growth in collagen matrix. MCF-10F cells form ductal structures while E2 70 cells form solid spherical masses that in histological sections exhibit a pattern of growth resembling ductal hyperplasia or carcinoma in situ. The tumorigenic cells C5-T8 did not form structures on collagen acquiring an invasive pattern with spindle like features. We have observed a reduction in E-cadherin expression in E2 70 cells and a complete loss in C5-T8 cells. TGFbeta1, TGFbeta2, CEACAM1 and JAG1 were down-regulated in E2 70 and C5-T8 cells. SMAD5 and h-RAS were up-regulated in the tumorigenic C5-T8 cells whereas FN1, Twist1 and Snail2 were up-regulated in C5-T8 and down-regulated in E2 70. We conclude that the loss of expression of TGFbeta1, TGFbeta2, CEACAM1 and JAG1 are related to ductulogenesis and branching and the overexpression of h-RAS with loss of E-cadherin expression and up-modulation of TWIST1, SNAIL2 and SMAD5 expressions are involved in the EMT modulation.     

Laminin isoforms 8 and 10 are primary components of the subendothelial basement membrane promoting interaction with neoplastic lymphocytes

To determine whether subendothelial laminins (LNs) could be implicated in the extravasation of neoplastic lymphocytes, we have examined the distribution of a number of LN isoforms in human vascular structures of adult individuals and have assayed the ability of the isolated LN molecules to promote adhesion of lymphoma and leukemic cells in vitro using a novel cell adhesion assay, CAFCA, Centrifugal Assay for Fluorescence-based Cell Adhesion (E. Giacomello et al., Biotechniques, 26: 758-762, 1999; P. Spessotto et al., Methods Mol. Biol., 139: 321-343, 2000). The use of previously characterized LN chain-specific antibodies showed that the vast majority of the smaller vascular compartments, known to correspond to sites of lymphocyte transmigration, expressed the subunits involved in the structuring of 9 of the 12 LN isoforms known to date. Eight LN isoforms (i.e., LN-1, -2, -4, -5, -8, -9, -10, and -11) and four naturally occurring LN complexes were isolated from various tissues and cultured cells by combined gel filtration, ion exchange, and immunoaffinity chromatographies, and the identity/composition of the isolated LNs/LN complexes was asserted by immunochemical means and amino-acid sequencing. Notwithstanding the widespread colocalization of LN isoforms, a panel of neoplastic B- and T-cell lines and lymphocytes isolated from patients affected by chronic lymphocytic B-cell leukemia attached preferentially and with high avidity to purified LN-8, purified LN-10, and LN-10-containing protein complexes, whereas lymphocytes derived from patients diagnosed with acute lymphocytic leukemia failed to bind to these LNs. All of the tested neoplastic lymphocytes failed to adhere to the isolated LN-1, LN-4, LN-9, and LN-11 and attached moderately well to purified LN-2 and LN-5. The interaction of transformed lymphocytes with LNs was cation-dependent and interchangeably mediated by the alpha3beta1 and alpha6beta1 integrins. The degree of engagement of the two LN receptors was dependent upon their relative levels of cell surface expression, whereas, irrespective of the phenotype, lymphocytes deprived of either of these receptors were incapable of LN binding. The findings suggest that LN-8 and LN-10 may act in an independent or complementary fashion as primary components of the endothelial basement membrane favoring the interaction of extravasating neoplastic lymphocytes. Thus, our results would demonstrate that different LN isoforms may evoke diverse cellular responses in different cell types and that this divergence may be the basis for the redundancy of LN distribution in a number of vascular structures.     

C-ha-ras enhances the neoplastic transformation of human breast epithelial-cells treated with chemical carcinogens

The present study was carried out with the purpose of analyzing the additive effect of c-Ha-ras oncogene on tumorigenesis in human breast epithelial cells (HBEC) treated with chemical carcinogens. A human breast epithelial cell (HBEC) line, MCF-10F, previously treated with dimethylbenz(a) anthracene (DMBA) and benzo(a)pyrene (BP) was used in these studies. The MCF-10F cells, DMBA and/or BP-transformed cells originated from the clones D3-1 and BP1 which were transfected with the plasmid pH06T1 containing the human T24 mutated c-Ha-ras oncogene and termed MCF-10F-Tras, D3-1-Tras and BP1-Tras, respectively. Whereas the c-Ha-ras transfected cells presented altered morphology, increased anchorage independent growth in agar-methocel, invasiveness and tumorigenicity, the MCF-10F cells, the clones D3 and BP1 were not tumorigenic. Importantly, whereas MCF-10F-Tras was slightly tumorigenic, the D3-1-Tras and BP1-Tras transfected cells were 100% tumorigenic in the SCID mice; and the tumors thus obtained were poorly differentiated carcinomas. DNA fingerprinting confirmed that the tumors derived originated from the cell lineage used. It was concluded that c-Ha ras induces an additive effect on the expression of tumorigenesis in human breast epithelial cell line MCF-10F treated with chemical carcinogens. Our work provide a model for analyzing the role of c-Ha-ras in human breast cancer.     

Quantitative determination of water-soluble scavengers in neoplastic and non-neoplastic human breast tissue

The water-soluble scavengers ascorbic acid (Asc), cysteine (Cys), glutathione (GSH) and uric acid (UA) as well as DNA content were determined in 40 breast tissue samples (neoplastic and non-neoplastic tissues from 20 patients). To allow proper homogenization to take place, a fixed number of sections was cut from a tissue cylinder of known diameter. Adjacent sections were stained with hematoxylin-eosin and the fractions of epithelium, fat and connective tissue were estimated as a percentage of the section area. Protein-free extracts were injected into a reverse-phase high-pressure liquid chromatography system and scavengers quantified with 2 electrochemical detectors (gold and glassy carbon). DNA and all scavengers, except UA, were greatly increased in cancer tissues in nearly all cases. Amounts of Asc and GSH in neoplastic tissue correlated closely with DNA values and percentage of epithelium, those of Cys not so closely and those of UA not at all. We assume that Asc and GSH were located mainly in the epithelium, UA mainly in the extracellular space and Cys in both spaces. When values were expressed as mumol/g DNA, a parameter related to content per cell, values were higher in neoplastic than in non-neoplastic tissue for Asc (18/20 cases), GSH (17/20) and Cys (14/20) and lower in neoplastic tissue for UA (19/20). It is known that increased GSH protects cells against certain drugs in tissue cultures. For in vivo treatment the presence of increased Asc (and to a lesser extent Cys) in addition to GSH could be of importance.     

Stages in neoplastic transformation of adult epithelial cells by 7,12-dimethylbenz(a)anthracene in vitro.

Five tumor-producing cell lines were established from explant cultures of adult C57BL mouse submandibular gland. Four lines were from cultures treated for 24 hr on Day 4 of culture with 7,12-dimethylbenz(a)anthracene. Three of these gave rise to adenocarcinomas after transplantation into syngeneic mice; the fourth produced tumors with carcinomatous and sarcomatous areas. The fifth cell line was derived from an untreated culture and gave rise to adenocarcinomas. A series of four well-defined morphological stages occurred in the cultures before tumor-producing cell lines were established. In Stage I (0 to 30 days) there was an outgrowth of epithelium; in Stage II (30 to 70 days) ductal differentiation occurred in some epithelium; in Stage III (70 to 100 days) small, slowly proliferating foci developed either from the ducts or from flat epithelial areas. In Stage IV (over 100 days) the proliferation rate in some of the foci increased, and the cells became more irregular. The cells could not be transferred easily until about 150 days, after which time they were tumor producing. Neoplastic transformation occurred between 158 and 240 days in the treated cultures and at 325 days in the untreated culture.     

Effect of retinol on radiation- and estrogen-induced neoplastic transformation of human breast epithelial cells

Clinical, epidemiological and experimental findings have provided evidence supporting a role of free radicals in the etiology of cancer. Free radical production is enhanced in many disease states, by carcinogen exposure, and under conditions of stress contributing widely to cancer development in humans. We have established an experimental breast cancer model to examine the effects of all-trans-retinol (retinol/vitamin A) on the production of free radicals in human breast epithelial cells induced by high linear energy transfer (LET)-radiation in the presence of 17beta estradiol. The following cell lines were used in these studies: the MCF-10F cell line, a spontaneously immortalized human breast epithelial cell line. Alpha 5 derived from MCF-10F cells irradiated with two separated doses of 60 cGy alpha particles in the presence of estrogens (60E/60E). Tumor 2, from a tumor formed in nude mice after injection with the cell line alpha 5. Tumor 3, from secondary tumor formed from injecting tumor 2 cells into nude mice. Each of the cell types examined had significantly elevated H(2)O(2) production levels compared to MCF-10F control cells (p<0.001). Retinol (1 microl/ml) significantly (p<0.05) decreased H(2)O(2) production in all cell types examined. Retinol significantly decreased (p<0.05) invasive capabilities of cells across matrigel coated invasion chambers and significantly reduced (p<0.05) PCNA, Fra-1, mutant p53 and increased Rb protein expression levels in comparison to non-retinol-treated ones when assayed using immunofluorescent staining coupled with confocal microscopy. The reduced H(2)O(2) production, decrease in cell invasive capabilities and alterations in protein expression levels suggest that retinol can be used as a chemopreventive agent in human breast cancer.     

Comparison between concentrations of trace elements in normal and neoplastic human breast tissue

Histologically normal and neoplastic human breast tissues obtained from 25 patients at the time of mastectomy were homogenized (200 mg/ml) in distilled water and 5-microliter aliquots dried on Formvar films for trace element analysis by energy-dispersive X-ray fluorescence. The elements measured were calcium, vanadium, copper, zinc, iron, chromium, manganese, nickel, selenium, molybdenum, bromine, rubidium, strontium, mercury, arsenic, and lead. In general, significantly large increases (p less than 0.001) in calcium, vanadium, copper, zinc, selenium, and rubidium were found in breast tumors, with a less significant increase (p less than 0.05) for nickel. When a comparison was made between histologically normal and neoplastic tissues from the same individual, zinc and rubidium were found to be consistently higher in the tumor, whereas calcium, copper, and vanadium levels varied from normal to high. In no instance were the tissue changes in calcium, copper, zinc, or rubidium reflected in the blood levels, which were within normal limits. The distribution of calcium, copper, and zinc in urine varied among individuals with primary tumors; however, rubidium levels tended to be consistently elevated. An attempt is being made to correlate these various differences with the extent of the primary disease at the time of surgery, the postoperative tumor-free interval, and subsequent therapy.     

Molecular and cellular analysis of basement membrane invasion by human breast cancer cells in Matrigel-basedin vitro assays

Summary In vitro analyses of basement membrane invasiveness employing Matrigel (a murine tumor extract rich in basement membrane components) have been performed on human breast cancer model systems. Constitutive invasiveness of different human breast cancer (HBC) cell lines has been examined as well as regulation by steroid hormones, growth factors, and oncogenes. Carcinoma cells exhibiting a mesenchymal-like phenotype (vimentin expression, lack of cell border associated uvomorulin) show dramatically increased motility, invasiveness, and metastatic potential in nude mice. These findings support the hypothesis that epithelial to mesenchymal transition (EMT)-like events may be instrumental in the metastatic progression of human breast cancer. The MCF-7 subline MCF-7_ADR appears to have undergone such a transition. The importance of such a transition may be reflected in the emergence of vimentin expression as an indicator of poor prognosis in HBC. Matrix degradation and laminin recognition are highlighted as potential targets for antimetastatic therapy, and analyses of laminin attachment and the matrix metalloproteinase (MMP) family in HBC cell lines are summarized. Matrigel-based assays have proved useful in the study of the molecular mechanisms of basement membrane invasiveness, their regulation in HBC cells, and their potential as targets for antimetastatic therapy.     

In vitro stimulation of human breast tissue by human prolactin.

Normal human breast tissue was cultured with defined media plus hormones. The epithelium survived at least 4 days in culture but did not grow in the absence of hormones. Both insulin and human prolactin stimulated growth, but ovine prolactin did not.     

Alterations of the basement membrane and connective tissue antigens in human metastatic lymph nodes

Antigens of the basement membrane (type-IV collagen and laminin) and the connective tissue (type-Ill collagen and fibronectin) were studied by immunofluorescence in 16 lymph nodes draining colorectal carcinomas and 6 lymph nodes draining breast carcinomas. A comparison was also made between 7 primary colorectal carcinomas and 9 lymph nodes draining these tumors. Anti-type-IV collagen and anti-laminin rarely stained the basement membrane of metastatic tumors. In contrast, we detected type-IV collagen in the peritumoral stroma, although similar images were rarely seen in primary tumors. When tumoral cells were in the vicinity of lymphoid cells, they were occasionaly separated by a barrier stained by the four antisera, or only by antifibronectin and anti-type-lll collagen. In other cases no barrier was observed between both types of cells which were in close contact. On the whole the above alterations were more marked in the lymph nodes draining breast carcinomas, in comparison to those draining colorectal carcinomas. Tumor cells were never stained by antitype-IV collagen or antilaminin serum. Some cells found either in the lymphoid or in the tumor area of metastatic lymph nodes were stained not only by these antisera, but also by a monoclonal antibody against Willebrand Factor, which is a marker of endothelial cells. Thus the labelled cells were characterized as being derived from the capillary wall.     

Quantitative determination of water- and lipid-soluble antioxidants in neoplastic and non-neoplastic human breast tissue

Ascorbic acid, cysteine, glutathione and uric acid were determined by reverse-phase high-pressure liquid chromatography (HPLC) in 46 breast tissue samples [neoplastic (C) and non-neoplastic (N) from the same patient]. Cholesterol, alpha-tocopherol and gamma-tocopherol were quantified in 64 similar samples by extraction into heptane followed by direct-phase HPLC. DNA was measured in all samples and the percentages of epithelium, fat and connective tissue were estimated in sections adjacent to the sample. Results confirm previous findings that ascorbic acid and glutathione, expressed as mumol/g DNA, were greatly increased in the epithelium of neoplastic tissue. Similar increases in cysteine could be accounted for by the presence of inflammatory cells. Although values of alpha- and gamma-tocopherol correlated with the percentage of fat in both types of tissue, these compounds were also present in the epithelium. Because of the varying amounts of fat in the samples, no significant difference could be found between N and C values. Cholesterol correlated with fat in N and epithelium in C. Consideration of 10 cases with equal amounts of fat in C and N tissue suggests that cholesterol is reduced in C in the epithelial cells.     

Identification of lymphocyte subpopulations in human breast cancer tissue and its significance: an immunoperoxidase study with anti-human T- and B-cell sera

Subpopulations of the infiltrating lymphocytes in breast cancer tissue from 31 patients were identified by indirect immunoperoxidase technique with antihuman T- and B-cell sera. In all noncancerous lesions examined (seven cases), B-cells were predominant and T-cells were scarcely found. In contrast, T-cells were predominant in breast cancer tissues (17 in 21 cases). T-cells tended to contact closely with cancer cells or cancer cell nests and accumulated around and in the walls of venules draining the cancer, while B-cells tended to cluster focally apart from cancer cell nests. T-cell infiltration was scanty in scirrhus carcinoma, whereas it was ample in infiltrating papillotubular carcinoma which had a better prognosis. There was a significant reverse correlation between the intensity of the T-cell infiltration and the clinical stages. The intensity of the T cell infiltration was significantly high in patients without lymph node metastasis. These facts suggest the possibility that the infiltrating T-cells in cancer tissue represent host resistance against cancer and that the intensity of the T cell infiltration correlates with the clinical prognosis of the breast cancer patients.     

Mutations in mismatch repair genes are involved in the neoplastic transformation of human breast epithelial cells

Mismatch repair (MMR) genes play a fundamental role in the correction of replication errors, leading to cancer development when mutated. In order to test the hypothesis that the MMR system was compromised in the initiation and progression of breast cancer, we used an in vitro-in vivo model for analyzing the mRNA levels of the MMR genes hMLH1, hMSH2, hPMS1, hPMS2 and hMSH6. MCF-10F, immortalized human breast epithelial cells, BP-1, benz(a)pyrene (BP)-transformed cells, BP-1Tras, a tumorigenic cell line derived from c-Ha-ras transfected BP-1 cells, and seven tumor-derived cell (TDC) lines obtained from BP-1Tras-induced tumors were tested. hMLH1, hMSH2, hPMS1, hPMS2 and hMSH6 mRNA expression were similar in MCF-10F, BP-1, and BP-1Tras cells; hMLH1 and hPMS1 were also equally expressed in TDC. An exception was hPMS2, whose mRNA level was decreased from BP-1Tras and from all TDC. hMSH2 and hMSH6 mRNA were also decreased in most TDC. DNA sequencing revealed mutations in hMSH2, which in MCF-10F cells had one frameshift mutation and one polymorphism in exons 12 and 13, respectively. Two mutations in exon 13, and three in exon 14 were detected in BP-1 and TDC, which had, in addition, three missense mutations in exon 14. hPMS2 had four mutations in exon 10 in MCF-10F cells, and BP-1 cells had three missense mutations in exon 9, four missense and one non-sense mutations in exon 10, codon 675 (Arg x Stop signal). BP-1Tras and TDC shared three missense mutations with BP-1 cells, and in addition had seven missense and one non-sense mutations in exon 9. hMSH6 had three frameshift and three missense mutations in exons 4 and 5 in BP-1, and 12 mutations in the same exons in BP-1Tras and TDC, which had three additional mutations in exons 4 and 5. This is the first report demonstrating mutations of MMR genes during the neoplastic transformation of human breast epithelial cells.