Raf kinase inhibitor protein is downregulated in hepatocellular carcinoma.

The Ras/Raf/MEK/ERK signalling cascade is frequently deregulated in tumourigenic diseases and known to be involved in proliferation and transformation of cells. Also in hepatocellular carcinoma (HCC) increased ERK levels are observed and known to correlate with tumour progression, but the underlying molecular mechanism are unknown. We analyzed expression of Raf-1 kinase inhibitory protein (RKIP) in HCC. Expression of RKIP mRNA and protein was downregulated in HCC cell lines and tissue as compared to primary human hepatocytes (PHH) or non-tumorous liver tissue, respectively. Transfection of an HCC cell line with an RKIP expression construct blocked the Raf kinase pathway resulting in decreased activity of ERK1/2 and AP-1. In contrast, downregulation of RKIP by transfection with an antisense RKIP construct led to increased ERK1/2 and AP-1 activity. Since HCC develop in the majority of cases in cirrhotic liver tissue and cirrhosis is the main risk factor for HCC development, we analyzed RKIP expression also in non-cancerous cirrhotic liver tissues by immunohistochemistry. In contrast to normal liver tissue, where the staining was equally distributed within the cytoplasm, hepatocytes in cirrhotic liver revealed an intense RKIP staining of the membrane. It can be speculated that this changed RKIP expression pattern parallels impaired protein function in PHH in cirrhotic livers that may predispose PHH to malignant transformation. In addition, our study demonstrates functional relevance of downregulation of RKIP in HCC that may play an important role in HCC development and progression.

Induction of Raf kinase inhibitor protein contributes to macrophage differentiation.

Differential gene expression analysis of human blood monocytes has identified the Raf kinase inhibitor protein (RKIP) as a continuously upregulated gene in macrophage and dendritic cell maturation. Using realtime RT-PCR and Western blot analysis we were able to confirm the initial DNA-microarray findings of RKIP induction on mRNA and protein levels. RKIP upregulation in primary cells and overexpression in THP-1 cells did not alter ERK activity but strongly reduced the amount of the NFkappaB subunit p65 in the nucleus. mRNA levels and cell surface expression of maturation markers including the integrin CD11c and the scavenger receptor CD36 were significantly increased in RKIP transfected THP-1 cells. Our data show for the first time that RKIP is upregulated during macrophage and dendritic cell differentiation on mRNA and protein levels and we conclude that RKIP contributes to the monocytic differentiation process via inhibition of the NFkappaB signaling cascade independent from the canonical Ras/Raf/MEK/ERK pathway.

Reduced expression of beta-catenin inhibitor Chibby in colon carcinoma cell lines.

AIM: To analyse the Chibby expression and its function in colon carcinoma cell lines and colorectal carcinoma (CRC). METHODS: Chibby expression levels were investigated by quantitative RT-PCR in a panel of seven different colon carcinoma cell lines. By sequencing, we analysed mutational status of Chibby. To test whether Chibby exhibited effects on beta-catenin signalling in colon carcinoma cells, we transfected SW480 cells with Chibby expression plasmid and, subsequently, analysed activity of beta-catenin and tested for alterations in cellular phenotype. In addition, we examined Chibby mRNA levels in samples of colorectal carcinomas and adjacent normal tissues by using quantitative RT-PCR and hybridised gene chips with samples from CRC and normal tissues. RESULTS: Chibby mRNA expression was strongly down-regulated in colon carcinoma cell lines in comparison to normal colon epithelial cells and no mutation in any of the examined colon carcinoma cell lines was found. Further, we could show that Chibby inhibited beta-catenin activity in TOPflash assays when over-expressed in SW480 cells. Proliferation and invasion assays with Chibby transfected SW480 cells did not reveal profound differences compared to control cells. In contrast to these in vitro data, quantitative RT-PCR analyses of Chibby mRNA levels in CRC tumor samples did not show significant differences to specimens in adjacent non-cancerous tissue. Consistent with these findings, gene chips analysing tissue samples of tumors and corresponding normal tissue did not show altered Chibby expression. CONCLUSION: Altered Chibby expression might be observed in vitro in different colon carcinoma cell lines. However, this finding could not be confirmed in vitro in CRC tumors, indicating that Chibby is not likely to promote CRC tumor development or progression. As Chibby is an important inhibitor of beta-catenin signalling, our data implicate that the usability of colon carcinoma cell lines for in vitro studies analysing the Wnt/beta-catenin pathway in colorectal carcinoma needs extensive verification.

Promoter-hypermethylation is causing functional relevant downregulation of methylthioadenosine phosphorylase (MTAP) expression in hepatocellular carcinoma.

The methylthioadenosine phosphorylase (MTAP) gene is localized in the chromosomal region 9p21. Here, frequently homozygous deletions occur in several kinds of cancer associated with the loss of tumour suppressor genes as p16 and p15. The aim of this study was to analyse MTAP expression in hepatocellular carcinoma (HCC) and to get an insight into the regulation and functional role of MTAP in hepatocancerogenesis. Compared with primary human hepatocytes MTAP expression was markedly downregulated in three different HCC cell lines as determined by real-time PCR and western blotting. This was not due to genomic losses or mutations but to promoter-hypermethylation. Reduced MTAP-expression was confirmed in vivo in HCC compared with non-cancerous liver tissue on both mRNA and protein levels. To study the functional relevance of the downregulated MTAP expression in HCC, MTAP expression was re-induced in HCC cell lines by stable transfection. In these MTAP re-expressing cell clones the invasive potential was strongly reduced, whereas no effects on cell proliferation were observed in comparison with mock transfected cell clones. Furthermore, in MTAP re-expressing cells interferon (IFN)-alpha and IFN-gamma induced a significantly stronger inhibition of cell proliferation than in mock transfected cells. In conclusion, our results suggest a functional role of MTAP inactivation in HCC development and invasiveness. Furthermore, in the light of a recent report revealing an association between MTAP activity and IFN sensitivity, our findings may have clinical significance for therapeutic strategies.

Microsatellite analysis of pleural supernatants could increase sensitivity of pleural fluid cytology.

Pleural effusions may result from various inflammatory, hemodynamic, or neoplastic conditions. A common diagnostic problem lies in distinguishing malignant from benign pleural effusions using routine cytological evaluation. We studied pleural fluid samples obtained from 14 patients with histologically confirmed malignancy and from 6 patients with benign pleural effusions using 12 microsatellite markers from 8 different chromosomal regions. Supernatants and cellular sediments of all 20 pleural fluid samples were analyzed. Routine cytological examination was 100% specific for malignancy but was only 57% sensitive. Microsatellite analyses of pleural fluid supernatants showed genetic alterations in tumor patients only. However, 50% of pleural effusions that were considered negative for malignancy by routine cytological analysis showed either loss of heterozygosity or microsatellite instability. The sensitivity of pleural fluid examination rose to 79% when routine cytological assessment was supplemented by molecular studies. Our data suggest that microsatellite analysis increases the sensitivity of cytological pleural fluid examination in assessing potential malignancy and that combining cytological and molecular methods may improve yield and certainty in diagnostically challenging cases.

Molecular diagnosis of ATP-binding cassette transporter-related diseases.

ATP-binding cassette (ABC) transporters are involved in a variety of physiologic processes such as xenobiotic defense, lipid metabolism, ion homeostasis and immune functions. A large number of ABC proteins have been causatively linked to rare and common human genetic diseases including familial high-density lipoprotein deficiency, retinopathies, cystic fibrosis, diabetes and cardiomyopathies. Furthermore, genetic variations in ABC transporter genes and dysregulated expression patterns of these molecules significantly contribute to drug resistance in human cancer cells and alter the pharmacokinetic properties of a variety of drugs. In order to analyze DNA sequence alterations or define disease-associated mRNA expression patterns of the complete ABC transporter superfamily, novel high-throughput molecular methods such as quantitative real-time PCR and DNA microarray analysis are emerging. The aim of this review is to provide an overview and to present some examples of human ABC transporters involved in monogenic diseases, cancer and pharmacogenetics. Methodologic aspects of molecular diagnostics applied to analyze genetic variations, mRNA and protein expression levels and functional characteristics of ABC transporters are discussed.

Analyses of the differentiation potential of satellite cells from myoD-/-, mdx, and PMP22 C22 mice.

BACKGROUND: Sporadic and sometimes contradictory studies have indicated changes in satellite cell behaviour associated with the progressive nature of human Duchenne muscular dystrophy (DMD). Satellite cell proliferation and number are reportedly altered in DMD and the mdx mouse model. We recently found that satellite cells in MSVski transgenic mice, a muscle hypertrophy model showing progressive muscle degeneration, display a severe ageing-related differentiation defect in vitro. We tested the hypothesis that similar changes contribute to the gradual loss of muscle function with age in mdx and PMP22 mice, a model of human motor and sensory neuropathy type 1A (HMSN1A). METHODS: Single extensor digitorum longus muscle fibres were cultured from mdx and PMP22 mice and age- and genetic background-matched controls. Mice at several ages were compared with regard to the differentiation of satellite cells, assayed as the proportion of desmin-expressing cells that accumulated sarcomeric myosin heavy chain. RESULTS: Satellite cells of 2 month, 6 month, and 12 month old mdx mice were capable of differentiating to a similar extent to age-matched wild type control animals in an in vitro proliferation/differentiation model. Strikingly, differentiation efficiency in individual 6 month and 12 month old mdx animals varies to a much higher extent than in age-matched controls, younger mdx animals, or PMP22 mice. In contrast, differentiation of myoblasts from all myoD null mice assayed was severely impaired in this assay system. The defect in satellite cell differentiation that occurs in some mdx animals arises from a delay in differentiation that is not overcome by IGF-1 treatment at any phase of cultivation. CONCLUSION: Overall, a defect in satellite cell differentiation above that arising through normal ageing does not occur in mdx or PMP22 mouse models of human disease. Nonetheless, the impaired differentiation of satellite cells from some mdx animals suggests that additional factors, environmental or epigenetic, may lead to deteriorating muscle repair through poor differentiation of satellite cells in genetically predisposed individuals.

Strong expression of methylthioadenosine phosphorylase (MTAP) in human colon carcinoma cells is regulated by TCF1/[beta]-catenin.

Methylthioadenosine phosphorylase (MTAP) is known as a ubiquitously expressed house keeping gene important in biochemical salvage processes. The MTAP gene is localized on the human chromosomal region 9p21, a region often deleted in cancer. Recently, several groups including our own have shown that MTAP serves as a tumour suppressor gene. The aim of this study was to analyse the role of MTAP in colon carcinoma and normal colon epithelium and the regulation of gene expression. To examine MTAP RNA and protein expression, we screened six colon carcinoma cell lines and human primary colon epithelial cells by RT-PCR and immunoblotting. MTAP expression was confirmed in vivo by immunohistochemical staining of normal colon tissue compared to adenoma and colon carcinoma. Interestingly, we found strong MTAP mRNA and protein expression by colon carcinoma cell lines but no expression by colonic epithelial cells. To analyse the regulation of MTAP expression, promoter studies were performed and revealed control of MTAP expression by LEF/TCF/beta-catenin. Furthermore, we demonstrated a significant correlation between MTAP protein expression and tumour progression as the intensity of MTAP protein staining increased from normal tissue to carcinoma. In addition, the recently postulated association between MTAP activity and interferon (IFN) sensitivity was confirmed in colon epithelial cells showing only little response to IFN-gamma, in contrast to the carcinoma cell lines. In summary, these data indicate for the first time that MTAP is not expressed in normal human colonic epithelium but is strongly upregulated in colon carcinoma. This finding may be of clinical significance concerning the homeostasis of normal colon epithelium and potential treatment of colon carcinoma.

Reduction in Raf kinase inhibitor protein expression is associated with increased Ras-extracellular signal-regulated kinase signaling in melanoma cell lines.

Mutations in the Raf signaling pathway are known to play a pivotal role in the progression of malignant melanoma. In this study, we provide evidence that the Raf-1 kinase inhibitory protein (RKIP) and its effects on Raf-1-mediated activation of mitogen-activated protein/extracellular signal-regulated kinase kinase are important for the metastatic potential of malignant melanoma. Screening nine melanoma cell lines at mRNA and protein levels, we detected significant down-regulation of RKIP expression in comparison with normal melanocytes. Loss of RKIP expression in transformed cells in vivo was confirmed in immunohistochemical analyses demonstrating reduction of RKIP expression already in primary melanoma and even stronger down-regulation or complete loss in melanoma metastases. Stable transfection of the melanoma cell line Mel Im with an RKIP expression plasmid blocked the Raf kinase pathway, resulting in down-regulation of extracellular signal-regulated kinase 1/2 and activator protein 1 activity. In very good agreement with the in vivo finding that down-regulation of RKIP expression is most obvious in melanoma metastasis, overexpression of RKIP in the highly invasive Mel Im cell line leads to a significant inhibition of invasiveness in vitro. Taken together, our results suggest that loss of RKIP in malignant melanoma contributes to enhanced invasiveness of transformed cells and therefore to progression of the disease.

Characterization of methylthioadenosin phosphorylase (MTAP) expression in malignant melanoma.

Homozygous deletions of human chromosomal region 9p21 occur frequently in malignant melanoma and are associated with the loss of the tumor suppressor genes p16(INK4a) and p15(INK4b). In the same chromosomal region the methylthioadenosine phosphorylase (MTAP) gene is localized and therefore may also serve as a tumor suppressor gene. The aim of this study was to analyze MTAP mutations and expression patterns in malignant melanomas. To examine the MTAP gene and expression of MTAP protein we screened 9 human melanoma cell lines and primary human melanocytes by reverse transcriptase-polymerase chain reaction, sequencing, and immunoblotting. Analyzing the melanoma cell lines we found significant down-regulation of MTAP mRNA expression. In only one cell line, HTZ19d, this was due to homozygous deletion of exon 2 to 8 whereas in the other cell lines promoter hypermethylation was detected. MTAP expression was further analyzed in vivo by immunohistochemical staining of 38 tissue samples of benign melanocytic nevi, melanomas, and melanoma metastases. In summary, we demonstrate significant inverse correlation between MTAP protein expression and progression of melanocytic tumors as the amount of MTAP protein staining decreases from benign melanocytic nevi to metastatic melanomas. Our results suggest an important role of MTAP inactivation in the development of melanomas. This finding may be of great clinical significance because recently an association between MTAP activity and interferon sensitivity has been suggested.