Zsuzsa Schaff

Claudin-1, -3 and -4 proteins and mRNA expression in benign and malignant breast lesions: a research study.

IntroductionWe compared levels of protein and mRNA expression of three members of the claudin (CLDN) family in malignant breast tumours and benign lesions.MethodsAltogether, 56 sections from 52 surgically resected breast specimens were analyzed for CLDN1, CLDN3 and CLDN4 expression by immunohistochemistry. mRNA was also analyzed using real-time PCR in 17 of the 52 cases.ResultsCLDNs were rarely observed exclusively at tight junction structures. CLDN1 was present in the membrane of normal duct cells and in some of the cell membranes from ductal carcinoma in situ, and was frequently observed in eight out of nine areas of apocrine metaplasia, whereas invasive tumours were negative for CLDN1 or it was present in a scattered distribution among such tumour cells (in 36/39 malignant tumours). CLDN3 was present in 49 of the 56 sections and CLDN4 was present in all 56 tissue sections. However, CLDN4 was highly positive in normal epithelial cells and was decreased or absent in 17 out of 21 ductal carcinoma grade 1, in special types of breast carcinoma (mucinous, papillary, tubular) and in areas of apocrine metaplasia. CLDN1 mRNA was downregulated by 12-fold in the sample (tumour) group as compared with the control group using GAPDH as the reference gene. CLDN3 and CLDN4 mRNA exhibited no difference in expression between invasive tumours and surrounding tissue.ConclusionsThe significant loss of CLDN1 protein in breast cancer cells suggests that CLDN1 may play a role in invasion and metastasis. The loss of CLDN4 expression in areas of apocrine metaplasia and in the majority of grade 1 invasive carcinomas also suggests a particular role for this protein in mammary glandular cell differentiation and carcinogenesis.

Viral Hepatitis: New Data on Hepatitis C Infection

Viral hepatitis (VH) is almost as old as human beings, at least as old as known human history. However, the natural history and the epidemiology of the disease has undergone changes during the centuries and even recently we have been facing several new aspects. The estimated global prevalence is around 3–5%, which means that approximately 400 million patients are infected with hepatitis B virus49 and that there are 170 million infections with hepatitis C virus.62’63 The mortality figures are projected to show a 2-to 3-fold increase over the next two decades as hepatitis C virus-infected patients develop cirrhosis, which makes this the leading indication for liver transplantation.4’6 These data point to the importance of VH being a significant public health problem worldwide.65 The list of hepatotropic viruses is well known, including hepatitis A (HAV), B (HBV), C (HCV), D (HDV), E (HEV), G (HGV) and F (HFV). HGV and HFV are excluded from the present review, mainly because they are questionable in relation to the causation of liver disease. Our knowledge of HAV, HBV, HDV and HEV has accumulated over the last decade, so the present discussion is focused on HCV, which is currently generating considerable concern and controversy, and is the leading cause of chronic liver disease worldwide. The main questions to be discussed, are: the characterization of the agents′ viral genotypes/ subtypes, the viral-cell interaction, the pathogenesis of VH, the extrahepatic manifestations of viral infection and hepatocarcinogenesis.

The role of TTF-1 in differentiating primary and metastatic lung adenocarcinomas.

Thyroid transcription factor-1 (TTF-1) is a sensitive marker for pulmonary and thyroid adenocarcinomas. The aim of this work was to determine its usefulness in distinction between primary and metastatic lung adenocarcinomas. We have examined the expression of TTF-1 in 100 solitary pulmonary nodules. They included 50 stage I peripheral primary bronchial adenocarcinomas (30 men, 20 women, mean age: 60 years) and 50 metastatic pulmonary adenocarcinomas (21 men, 29 women, mean age: 57 years) of different origins, such as breast (13), colon (13), rectum (13), kidney (7), stomach (2), and thyroid gland (2). TTF-1 immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissues. In primary bronchial adenocarcinomas we found immunopositivity in 46/50 cases, among them 30 cases showed strong nuclear immunostaining. In four primary adenocarcinoma cases the observed immunopositivity was localized to the cytoplasm. Out of the metastatic adenocarcinomas all but the 2 thyroid cancers were negative. Both thyroid tumors showed strong immunopositivity. Our results confirm that TTF-1 immunohistochemistry is a very sensitive and highly specific method in the differential diagnosis of primary and metastatic lung adenocarcinomas and should be used in the everyday clinical practice.

Similarities and differences in hepatitis B and C virus induced hepatocarcinogenesis

Hepatocellular carcinoma (HCC), the major manifestation of primary liver cancer, is one of the most frequent and malignant diseases worldwide. Among other environmental factors, hepatitis viruses, as the hepatitis B (HBV) and hepatitis C (HCV) viruses, are to be listed in the etiology of HCC. Both of these viruses cause a wide spectrum of clinical manifestations, ranging from healthy carrier state to acute and chronic hepatitis, cirrhosis and HCC. HBV and HCV are different viruses in structure: HBV contains a DNA genome which replicates through an RNA intermediate and requires an active viral reverse transcriptase (RT) polymerase enzyme, while HCV is an RNA virus which has no RT activity and replicates on the cellular membrane by RNA replication. In this review we discuss how these two biologically diverse viruses use common pathways to induce hepatocarcinogenesis despite their significant structural and viral cycle differences. A summary is also given of several observable common and different features. Direct integration of HBV viral sequences into the host genome increases the genomic instability, which does not occur in HCV infection. However, viral proteins may directly play a significant role in the induction of carcinogenesis by both viruses.

Claudin-4 differentiates biliary tract cancers from hepatocellular carcinomas.

The recently identified claudins are dominant components of tight junctions, responsible for cell adhesion, polarity and paracellular permeability. Certain claudins have been shown to have relevance in tumor development, with some of them, especially claudin-4, even suggested as future therapeutic target. The aim of the present study was to analyze the expression of claudin-4 in the biliary tree, biliary tract cancers and hepatocellular carcinomas. A total of 107 cases were studied: 53 biliary tract cancers, 50 hepatocellular carcinomas, 10 normal liver and 10 normal extrahepatic biliary duct samples. Immunohistochemical analysis was performed on conventional specimens and on tissue microarrays as well. Claudin-4 was further investigated by Western blot analysis and real-time RT-PCR. Intense membranous immunolabeling was found for claudin-4 in all biliary tract cancers unrelated to the primary site of origin, namely intrahepatic, extrahepatic or gallbladder cancers. Normal biliary epithelium showed weak positivity for claudin-4. In contrast, normal hepatocytes and tumor cells of hepatocellular carcinomas did not express claudin-4. The results of Western immunoblot analysis and real-time RT-PCR were in correlation with the immunohistochemical findings. Cytokeratins, as CK7 (92%) and CK19 (83%) were mostly positive in biliary tract cancers, however, one-third of hepatocellular carcinomas also expressed CK7 (34%). HSA antibody (HepPar1) reacted with the majority of hepatocellular carcinomas (86%), while being positive in a low percentage of the biliary tract cancers (8%). In conclusion, this is the first report of a significantly increased claudin-4 expression in biliary tract cancers, which represents a novel feature of tumors of biliary tract origin. Claudin-4 expression seems to be a useful marker in differentiating biliary tract cancers from hepatocellular carcinomas and could well become a potential diagnostic tool.

Effect of formalin, acetone, and RNAlater fixatives on tissue preservation and different size amplicons by real-time PCR from paraffin-embedded tissue.

RNA recovered from paraffin-embedded tissue has been reported to be a suitable substrate for polymerase chain reaction. During tissue fixation and paraffin embedding, RNA undergoes degradation, but with certain restrictions, it can be used for gene expression studies. At the same time, formalin-fixed, paraffin embedded histopathology archives contain an unestimable collection, which could be analyzed to investigate changes in mRNA expression in pathologic processes. To decide for future tissue conservation of pathology samples, it would be reasonable to satisfy both histologic and molecular biologic needs. The effect of three different fixation methods, RNAlater (SIGMA R 0901, St Louis, MO), acetone, and formalin, were compared by histology, immunohistochemistry, and real-time PCR. To assess tissue structure preservation and antigenicity, hematoxylin-eosin staining and immunohistochemistry were performed; to assess RNA quality, RNA was extracted and the transcription of different amplicon sizes (121, 225, 406 bp for GAPDH; 166, 310, 536 bp for β globin) were examined on human endometrium samples. The most adequate tissue preservation was found in case of formalin fixation, while there were no significant differences in the three fixatives’ yields for various size real-time PCR amplicons. Longer amplicons (above ∼225 bp) have limited use for gene expression studies, while shorter amplicons could give more reliable results.

Expression of tight junction protein claudin-4 in Basal-Like breast carcinomas

Claudins (CLDN) are tight junction proteins which contribute to the paracellular transport and ionic permeability of various epithelia. In recent years they came into focus for their suggested role in carcinogenesis and possible role in cancer therapy. According to our previous studies, in breast tissue CLDN4 is also related to the level of cellular differentiation. Thirty-eight estrogen (ER) and progesterone receptor (PgR) negative, HER2/neu negative, but cytokeratin 5/6 positive basal-like—mainly grade 3—breast carcinomas were compared with twenty-one grade 1, twenty-five grade 2 and twenty grade 3 non-basal-like invasive breast carcinomas, in respect to their CLDN4 expression. The immunohistochemical reactions were evaluated both semiquantitatively and by morphometrical analysis. Statistically significant difference (p = 0.001) was observable regarding CLDN4 expression in the basal-like group as compared to grade 1 and 2 cancers. Further, CLDN4 expression was significantly higher (p = 0.017) in the basal-like compared with the non-basal-like grade 3 carcinomas. Our results suggest that basal-like carcinomas are a subset of breast cancer with high level of CLDN4 protein expression. The finding is in accordance with our former observation that CLDN4 is indeed related to cellular differentiation. This observation may be seen as a further proof that basal-like carcinomas represent a separable group amongst grade 3 breast carcinomas.

Ablation of the decorin gene enhances experimental hepatic fibrosis and impairs hepatic healing in mice

Accumulation of connective tissue is a typical feature of chronic liver diseases. Decorin, a small leucine-rich proteoglycan, regulates collagen fibrillogenesis during development, and by directly blocking the bioactivity of transforming growth factor-β1 (TGFβ1), it exerts a protective effect against fibrosis. However, no in vivo investigations on the role of decorin in liver have been performed before. In this study we used decorin-null (Dcn−/−) mice to establish the role of decorin in experimental liver fibrosis and repair. Not only the extent of experimentally induced liver fibrosis was more severe in Dcn−/− animals, but also the healing process was significantly delayed vis-à-vis wild-type mice. Collagen I, III, and IV mRNA levels in Dcn−/− livers were higher than those of wild-type livers only in the first 2 months, but no difference was observed after 4 months of fibrosis induction, suggesting that the elevation of these proteins reflects a specific impairment of their degradation. Gelatinase assays confirmed this hypothesis as we found decreased MMP-2 and MMP-9 activity and higher expression of TIMP-1 and PAI-1 mRNA in Dcn−/− livers. In contrast, at the end of the recovery phase increased production rather than impaired degradation was found to be responsible for the excessive connective tissue deposition in livers of Dcn−/− mice. Higher expression of TGFβ1-inducible early responsive gene in decorin-null livers indicated enhanced bioactivity of TGFβ1 known to upregulate TIMP-1 and PAI-1 as well. Moreover, two main axes of TGFβ1-evoked signaling pathways were affected by decorin deficiency, namely the Erk1/2 and Smad3 were activated in Dcn−/− samples, whereas no significant difference in phospho-Smad2 was observed between mice with different genotypes. Collectively, our results indicate that the lack of decorin favors the development of hepatic fibrosis and attenuates its subsequent healing process at least in part by affecting the bioactivity of TGFβ1.

Expression of decorin, transforming growth factor-beta1, tissue inhibitor metalloproteinase 1 and 2, and type IV collagenases in chronic hepatitis

Decorin is a small extracellular matrix proteoglycan. It binds and modulates transforming growth factor (TGF)-beta 1 action, the major stimulator of fibrogenesis. Its role in the pathogenesis of human liver cirrhosis is unknown. Therefore, we studied the relationship of the 2 proteins in normal human liver and in 43 chronic hepatitis and liver cirrhosis specimens. To understand the mechanism that maintains matrix deposition in stage IV hepatitis, we studied expression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2, as well as the activities of type IV collagenases. Gene expression was analyzed on messenger RNA and protein level by morphologic and biochemical approaches. Decorin proved to be an early marker of fibrogenesis, and its deposition increased parallel to that of TGF-beta 1 and to inflammatory activity. Liver fibrosis progressed despite high temporospatial expression of decorin with TGF-beta 1. Neither decorin nor TGF-beta 1 protein deposition increased further in cirrhosis with low inflammatory activity, suggesting that impaired extracellular matrix catabolism rather than active production plays a role in this stage. This possibility was supported by high message levels of metalloproteinase inhibitors, no 72-kd collagenase activities, and low 92-kd collagenase activities.

Different Expression of Occludin and ZO-1 in Primary and Metastatic Liver Tumors

Tight junction (TJ) components were found to be correlated with carcinogenesis and tumor development. TJs are composed of three main integral membrane proteins; occludin, claudins and JAMs. Alteration of the TJ protein expression may play an important role in the process of cell dissociation, which is among the first steps of tumor invasion and metastasis. Reduced expression of ZO-1 has been reported to be associated with invasion of several tumors. The aim of the present study was to detect differences between occludin and ZO-1 expression in normal liver samples, HCCs and colorectal liver metastases. Expression of occludin and ZO-1 was analysed in 25 surgically removed human hepatocellular carcinomas (HCC) and 25 human colorectal liver metastases. Gene expression levels were measured by real-time RT PCR, protein expression was determined by immunohistochemistry, comparing tumors with the surrounding nontumorous parenchyma and with seven normal liver samples. Occludin and ZO-1 mRNAs showed significant downregulation in HCCs in comparison with normal liver and were also downregulated in the metastases when compared with normal liver. Occludin and ZO-1 proteins were weakly expressed on hepatocytes in normal liver, while strong expression was found on bile canaliculi. In HCCs occludin and ZO-1 did not show immunopositivity on tumor cells, while colorectal metastatic tumors revealed high levels of these molecules. HCCs and metastases are characterized by markedly different protein expression pattern of occludin and ZO-1, which phenomenon might be attributed to the different histogenesis of these tumors.