Type 1 protein tyrosine kinases in benign and malignant breast lesions

Aims : To determine their significance, we examined the expression pattern of the four epidermal growth factor receptor (EGFR) family members as well as the phosphotyrosine kinase activity in breast tumour tissues.  

Methods and results

Fifty-three malignant breast tumours, four breast cancer cell lines, and 10 benign breast tumours were investigated. Fifty-three per cent (28/53) of the malignant tumours expressed EGFR protein, and the majority of these positive tumours were strongly positive. Eighty per cent (8/10) of the benign tumours also expressed EGFR protein, but all in a lower or moderate level. An association between EGFR expression and increasing malignancy grade was found in the group of infiltrating ductal carcinomas. Of the malignant tumours, 35.8% (19/53) expressed c-erbB-2 protein and 17% (9/53) c-erbB-3 protein, while no expression of c-erbB-2 and c-erbB-3 proteins was found in the benign tumours. Contrary to previous reports, we observed c-erbB-4 receptor protein to be less expressed in the malignant breast tumours. The 'normal' breast epithelial cells adjacent to the malignant tumours and the benign tumours demonstrated intensified membrane staining for c-erbB-4, while a number of the malignant tumours demonstrated a weak cytoplasmic staining or were negative. However, several malignant tumours with strong membrane staining for the c-erbB-4 protein were also found. No simple association between the expression of the four receptors and phosphotyrosine kinase activity was found.  

Conclusion

Our study has revealed a complex expression pattern of the EGFR family members in breast tumour cells. While the data about EGFR, c-erbB-2, c-erbB-3 and phosphotyrosine are largely in line with what has been reported, we found the c-erbB-4 protein expression to be decreased in the malignant tumours.     

Human Secretory IgM Antibodies Activate Human Complement and Offer Protection at Mucosal Surface

IgM molecules circulate in serum as large polymers, mainly pentamers, which can be transported by the poly‐Ig receptor (pIgR) across epithelial cells to mucosal surfaces and released as secretory IgM (SIgM). The mucosal SIgM molecules have non‐covalently attached secretory component (SC), which is the extracellular part of pIgR which is cleaved from the epithelial cell membrane. Serum IgM antibodies do not contain SC and have previously been shown to make a conformational change from ‘a star’ to a ‘staple’ conformation upon reaction with antigens on a cell surface, enabling them to activate complement. However, it is not clear whether SIgM similarly can induce complement activation. To clarify this issue, we constructed recombinant chimeric (mouse/human) IgM antibodies against hapten 5‐iodo‐4‐hydroxy‐3‐nitro‐phenacetyl (NIP) and in addition studied polyclonal IgM formed after immunization with a meningococcal group B vaccine. The monoclonal and polyclonal IgM molecules were purified by affinity chromatography on a column containing human SC in order to isolate joining‐chain (J‐chain) containing IgM, followed by addition of excess amounts of soluble SC to create SIgM (IgM J+ SC+). These SIgM preparations were tested for complement activation ability and shown to be nearly as active as the parental IgM J+ molecules. Thus, SIgM may offer protection against pathogens at mucosal surface by complement‐mediated cell lysis or by phagocytosis mediated by complement receptors present on effector cells on mucosa.     

Modulation of glioma cell invasion and motility by adenoviral gene transfer of PAI-1

A number of studies have emphasized the role of PAI-1 as an important regulator of tumor cell invasion and metastasis. The hallmark of primary tumors of the central nervous system and glioblastomas in particular is the diffuse invasion into the normal brain tissue. Since PAI-1 is expressed in such tumors, we studied the effect of adenoviral-mediated transfer of the PAI-1 gene in regulating the in vitro invasiveness of D54Mg glioma cells into Matrigel, and into fetal rat brain aggregates. Treatment of D54Mg cells with 50 MOI (multiplicity of infection) of the replication defective vector AdCMVPAI-1 increased PAI-1 expression 23-fold compared to control vectors, and the invasion through Matrigel was reduced by 67%. The motility of the cells was reduced by 58% compared to controls (indicating that inhibition of motility was the principal effect of PAI-1 in these cells). The ability of D54Mg tumor spheroids to invade fetal rat brain aggregates was not reduced by the PAI-1 gene transfer. The results show that overexpression of PAI-1 can inhibit glioma cell motility and invasion through extracellular matrix (ECM) components, like laminin and collagen, but does not inhibit tumor cell invasion in a three-dimensional invasion assay, simulating normal brain tissue having a different ECM and interstitial composition. The different results obtained in the two invasion assays reflect the complex biological effects of the uPA/PAI-1 system, and questions a simplistic view of PAI-1 as an inhibitor of brain tumor invasion. This revised version was published online in July 2006 with corrections to the Cover Date.     

Invasion potential and N-acetylgalactosamine expression in a human melanoma model

Reactivity of the N-acetylgalactosamine-binding Helix pomatia agglutinin (HPA) in tumours has been associated with poor prognosis and metastasis development. In our LOX/FEMX-I human melanoma model, the binding of HPA correlates with experimental lung metastasis formation in athymic nude mice. In the present study, the metastatic potential of 2 human melanoma cell lines (LOX and FEMX-I) was assessed in relation to carbohydrate and invasive phenotype. Immuno-cytological and invasion assays highlighted significant differences between these 2 cell lines. Immuno-cytochemical analysis confirmed the widespread expression of HPA-binding glycoconjugates on LOX but not FEMX-I cells. One of these HPA-binding glycoconjugates, the Tn antigen, was expressed highly on the surface of LOX cells but only weakly in the cytoplasm of FEMX-I cells. The sialyl Tn antigen was expressed in FEMX-I but not in LOX cells. There was no difference between the cell lines in adhesion/rate of trapping in athymic nude mouse lung tissues. In Matrigel invasion assays, LOX cells demonstrated an invasion potential more than 6 times greater than that observed with FEMX-I cells. Matrigel invasion of LOX cells was inhibited after incubation with HPA (89%) compared to controls with HPA and GalNAc blocking sugar or without HPA (p < 0.0005 at 5 df). In contrast, there was no inhibitory effect with the anti-Tn antibody IE3. Invasion of FEMX-I cells was not affected by the lectin and the IE3 antibody. Immuno-cytochemical analysis revealed expression of the terminal galactose- and polylactosamine-binding lectin galectin 3 (Mac-2) in these melanoma cell lines. Expression of both the lectin and its receptor may be a contributory feature in the pulmonary invasion of LOX melanoma cells. Overall, our findings suggest that HPA-binding glycoconjugates other than the αGalNAc-O-Ser/Thr of the Tn antigen may be important in the extracellular matrix invasion of LOX melanoma cells. Int. J. Cancer 75:609–614, 1998. © 1998 Wiley-Liss, Inc.     

Representations of cycling in metropolitan newspapers - changes over time and differences between Sydney and Melbourne,Australia

Background  

Cycling is important for health, transport, environmental and economic reasons. Newspaper reporting of cycling reflects and can influence public and policy maker attitudes towards resource allocation for cycling and cycling infrastructure, yet such coverage has not been systematically examined.     

Cultured anaplastic cell lines as immunocytochemistry controls: a comparison of ThinPrep-processed smears and conventional air-dried cytospins.

Cultured anaplastic cell lines with previously characterized phenotypes are considered to be the best positive controls for immunocytochemistry. We assessed the validity of using anaplastic cell line cytospins as positive controls for immunocytochemistry performed on ThinPrep-processed clinical samples. We compared ThinPrep-processed slides and air-dried cytospins from cultured anaplastic cell lines for intensity and pattern of staining. Also, antigen preservation was assessed over a 3-mo period, using a panel of 16 primary antibodies and 12 anaplastic cell lines. A three-step alkaline phosphatase procedure was used except when in a single instance the EnVision method was employed. If appropriately stored, both preparations showed excellent correlation with no decrease in antigenicity during the 3-mo testing period. ThinPrep-processed slides from clinical samples are ideal for immunocytochemistry, because internal negative controls can be performed for each test. We recommend the use of cytospins for positive controls because of the lower cost.     

Detection of cancer cells in effusions from patients diagnosed with gynaecological malignancies

The detection of malignant cells in pleural, peritoneal, and pericardial fluids of cancer patients marks the presence of metastatic disease and is associated with a grave prognosis. We evaluated five epithelial markers for the detection of cancer cells in 94 fresh pleural, peritoneal and pericardial effusions. Eighty-four of the samples were regarded as adequate for analysis after evaluation of cytological smears, including 61 samples from patients known to have gynaecological neoplasms. The other 23 samples were from patients with various non-gynaecological malignancies or tumours of unknown origin. Our control cases were 10 fallopian tubes not affected by any malignancy and 12 malignant mesotheliomas. Cell blocks from all cases were stained for CA-125, BerEP4, carcinoembryonic antigen (CEA), BG8 (Lewis Y blood antigen), and B72.3 (TAG-72). Fifty-one of 84 cases were diagnosed as malignant or suggestive of malignancy in cytological smears and/or cell block sections. However, staining for epithelial markers highlighted the presence of malignant cells in 7 additional cases. When membrane staining was evaluated, the sensitivity of the markers studied in detecting malignant cells was as follows: CA-125: 88%, BerEP4: 78%, CEA: 26%, BG8: 86%, B72.3: 79%. Membrane positivity for CEA, B72.3 and BerEP4 was not detected in reactive mesothelial cells. However, membranous staining in mesothelial cells was evident in 13% and 31% of cases with the use of BG8 and CA-125, respectively. Weak cytoplasmic staining for CEA was observed in mesothelial cells in 2 cases. When Ber-EP4, B72.3, and BG8 staining results in cancer cells were combined, the following sensitivity levels were observed: BG8+B72.3: 91%; BG8+Ber-EP4: 90%; B72.3+Ber-EP4: 93%; BG8+ Ber-EP4+B72.3: 95%. The detection of malignant cells in effusions is facilitated by the use of immunocytochemistry using a wide panel of antibodies. BerEP4 and B72.3 appear to be the best markers when both sensitivity and specificity are considered, followed by BG8, while CEA and CA-125 have a limited role in the detection of metastases from gynaecological tumours owing to the low sensitivity of the former and the low specificity of the latter. Analysis of all staining results should be based on a thorough morphological examination. Received: 14 December 1998 / Accepted: 23 February 1999     

Role of the multidrug resistance protein 1 gene in the carcinogenicity of aflatoxin B1: investigations using mrp1-null mice

The multidrug resistance protein 1 (MRP1) protects cells from xenobiotics by extruding from the intracellular compartment glutathione (GSH)-S-conjugates, glucuronyl conjugates and sulfate conjugates and by the co-export of xenobiotic(s) and GSH. An ATP-dependent transport of aflatoxin B1 (AFB1) and its GSH conjugates by MRP1 has been previously demonstrated in vitro. In the present study, we have sought to investigate the in vivo role of MRP1 in AFB1 carcinogenicity, by comparing the incidence of tumors occurring in mrp1 (+/+) and mrp1 (-/-) mice 12 months after an 8 weeks exposure to AFB1. The carcinogen induced a similar number of lung and liver tumors in both strains. Most lung tumors were of the solid type and showed a moderate degree of differentiation in both mrp1 (+/+) and mrp1 (-/-) mice. These data provide direct evidence that in vivo MRP1 does not protect from AFB1 carcinogenicity. Due to the redundancy of transmembrane export pumps, other pump(s) may effectively vicariate for MRP1-mediated transport of AFB1 and its glutathione conjugates.     

A novel grid polymerase chain reaction (G-PCR) approach at ultrastructural level to detect target DNA in cell cultures and tissues

A novel grid polymerase chain reaction (G-PCR) method has been developed to be used at the ultrastructural level and with a high degree of resolution. Samples applied to test the method were fresh cell lines (CaSki, SiHa) and HPV-16 DNA-containing tissues rescued from routine paraffin blocks. The specimens were embedded in Epon-Araldite and/or hydrophilic-resin LRWhite. Ultrathin sections mounted on grids were subjected to G-PCR using an HPV-16-specific primer set. The amplified products were identified by auro-immunohistochemical labelling of the biotinylated nucleotide. The results indicated successful amplification of target DNA in both cell and tissue samples, being confined to the intranuclear region. The negative controls [HeLa cells, isolated mammary carcinoma cell cultures (MCF 7, and T47-D) (ATCC) (U.S.A.), normal thyroid tissue and steroid-producing tumour tissue] failed to exhibit any amplification of the target DNA sequences. The sensitivity of the G-PCR system was evaluated by performing a parallel in situ hybridization (ISH) of serial sections. The signals obtained from G-PCR were more intense than those of ISH and more informative as to the precise subcellular localization of amplicons. © 1997 John Wiley & Sons, Ltd.     

MGST1 expression in serous ovarian carcinoma differs at various anatomic sites, but is unrelated to chemoresistance or survival

Objective

To investigate the expression of MGST1 in primary tumors, solid metastases and metastatic effusions in advanced-stage serous ovarian carcinoma (OC) and analyze the association with clinicopathologic parameters, including chemotherapy resistance and survival.

Methods

MGST1 mRNA expression was investigated in 178 tumors (88 effusions, 38 primary carcinomas, 52 solid metastases) from 144 patients using real-time quantitative PCR (qRT-PCR). Forty-two of the 88 effusions were additionally analyzed for MGST1 protein expression by Western blotting.

Results

mRNA expression of MGST1 was higher in primary carcinomas and solid metastases compared to effusions (p = 0.008 and p = 0.012, respectively). In patient-matched samples, mRNA expression of MGST1 was higher in solid metastases compared to effusions (p = 0.023), and a trend for higher MGST1 levels in solid metastases compared to primary tumors was observed (p = 0.06). Biopsies from primary carcinomas obtained from patients with > 200 ml ascites at diagnosis had higher mRNA expression of MGST1 compared to samples from patients with < 200 ml ascites (p = 0.037). MGST1 mRNA expression was not associated with age, histological grade, tumor stage, residual disease volume, response to chemotherapy, chemotherapy resistance or survival. Western blot analysis of patient-matched effusions showed high concordance between MGST1 protein and mRNA levels measured by qRT-PCR (p < 0.001).

Conclusions

The present study documents frequent MGST1 mRNA and protein expression in OC. The data suggest increased activity of oxidative response pathways, reflected by higher mRNA expression, in solid OC tumors compared to metastatic effusions. Additionally, a tumor microenvironment consisting of ascites may induce antioxidant activity.