Four-And-A-Half LIM-Domain Protein 2 (FHL2) Deficiency Aggravates Cholestatic Liver Injury.

Cholestasis occurs in different clinical circumstances and leads to severe hepatic disorders. The four-and-a-half LIM-domain protein 2 (FHL2) is a scaffolding protein that modulates multiple signal transduction pathways in a tissue- and cell context-specific manner. In this study, we aimed to gain insight into the function of FHL2 in cholestatic liver injury. FHL2 expression was significantly increased in the bile duct ligation (BDL) model in mice. In Fhl2-deficient (Fhl2-ko) mice, BDL caused a more severe portal and parenchymal inflammation, extended portal fibrosis, higher serum transaminase levels, and higher pro-inflammatory and pro-fibrogenic gene expression compared to wild type (wt) mice. FHL2 depletion in HepG2 cells with siRNA resulted in a higher expression of the bile acid transporter Na+-taurocholate cotransporting polypeptide (NTCP) gene. Furthermore, FHL2-depleted HepG2 cells showed higher expression of markers for oxidative stress, lower B-cell lymphoma 2 (Bcl2) expression, and higher Bcl2-associated X protein (BAX) expression after stimulation with deoxycholic acid (DCA). In hepatic stellate cells (HSCs), FHL2 depletion caused an increased expression of TGF-ß and several pro-fibrogenic matrix metalloproteinases. In summary, our study shows that deficiency in FHL2 aggravates cholestatic liver injury and suggests FHL2-mediated effects on bile acid metabolisms and HSCs as potential mechanisms for pronounced hepatocellular injury and fibrosis.

Liver regeneration associated protein (ALR) exhibits antimetastatic potential in hepatocellular carcinoma.

Augmenter of liver regeneration (ALR), which is critically important in liver regeneration and hepatocyte proliferation, is highly expressed in cirrhotic livers and hepatocellular carcinomas (HCC). In the current study, the functional role of ALR in hepatocancerogenesis was analyzed in more detail. HepG2 cells, in which the cytosolic 15 kDa ALR isoform was reexpressed stably, (HepG2-ALR) were used in migration and invasion assays using modified Boyden chambers. Epithelial-mesenchymal transition (EMT) markers were determined in HepG2-ALR cells in vitro and in HepG2-ALR tumors grown in nude mice. ALR protein was quantified in HCC and nontumorous tissues by immunohistochemistry. HepG2-ALR, compared with HepG2 cells, demonstrated reduced cell motility and increased expression of the epithelial cell markers E-cadherin and Zona occludens-1 (ZO-1), whereas SNAIL, a negative regulator of E-cadherin, was diminished. Matrix metalloproteinase MMP1 and MMP3 mRNA expression and activity were reduced. HepG2-ALR cell-derived subcutaneously grown tumors displayed fewer necrotic areas, more epithelial-like cell growth and fewer polymorphisms and atypical mitotic figures than tumors derived from HepG2 cells. Analysis of tumor tissues of 53 patients with HCC demonstrated an inverse correlation of ALR protein with histological angioinvasion and grading. The 15 kDa ALR isoform was found mainly in HCC tissues without histological angioinvasion 0. In summary the present data indicate that cytosolic ALR reduces hepatoma cell migration, augments epithelial growth and, therefore, may act as an antimetastatic and EMT reversing protein.

Aberrant methylation of DAPK in long-standing ulcerative colitis and ulcerative colitis-associated carcinoma.

Death-associated protein kinase (DAPK) has pro-apoptotic functions and participates in various apoptotic systems. DAPK acts as a tumor suppressor, and its inactivation by promoter hypermethylation has been frequently observed in various human cancers. As alterations of pro-apoptotic genes might cause instability in the balance of cell-turnover during chronic inflammatory processes, epigenetic silencing of DAPK might be involved in the carcinogenesis of ulcerative colitis-associated carcinoma (UCC). To evaluate the role of DAPK in the inflammation-driven carcinogenesis of ulcerative colitis (UC), we analyzed promoter hypermethylation and protein expression of DAPK using methylation-specific PCR and immunohistochemistry in 43 UCCs and paired UC-background mucosa, as well as in UC-background mucosa of 50 patients without UCC. The frequency of methylation of DAPK in UCCs was low (27.6%) compared to overall non-neoplastic UC-background mucosa (48.3%; p=0.02) and sporadic colorectal carcinoma (57.4%, p=0.019). The difference in the methylation frequency in UC-background mucosa in patients without UCC (54.2%), compared to those with UCC (40.0%), was not significant (p=0.141). Promoter methylation correlated significantly with decreased DAPK protein expression (p<0.001) and severity of inflammatory activity (p=0.024). In unmethylated UC-background mucosa, DAPK protein expression increased with activity of UC-associated inflammation, suggesting a protective role of the pro-apoptotic DAPK during the chronic inflammatory process of UC. Thus, inactivation of DAPK by promoter hypermethylation might be crucial for accumulation of DNA damage in inflamed mucosa of UC, and might therefore contribute to the initiation of the neoplastic process and development of UC-associated carcinoma.

Methylthioadenosine phosphorylase represents a predictive marker for response to adjuvant interferon therapy in patients with malignant melanoma.

Using tissue microarrays assembling 465 nevi, primary melanomas and metastases, we investigated whether expression of methylthioadenosine phosphorylase (MTAP), a recently suggested biomarker of malignant melanoma, has prognostic significance and may predict responsiveness to adjuvant interferon therapy in patients with melanoma. Because of its association with MTAP activity and interferon signalling pathways, signal transducer and activator of transcription 1 (STAT1) immunohistochemistry was analysed, too. MTAP expression was significantly reduced in melanomas and metastases compared with nevi (P < 0.001); STAT1 expression significantly increased. In melanomas, loss of MTAP expression was significantly related to Clark level (P < 0.05) and tumor thickness (P < 0.01); whereas STAT1 immunoreactivity was significantly related to gender (p < 0.05) and tumor thickness (P < 0.05). Interestingly, subgroup analysis of patients with a tumor thickness of 1.5-4.0 mm revealed a significant survival benefit from adjuvant interferon treatment regarding recurrence-free survival (RFS; P < 0.05) if MTAP expression was observed in the primary melanoma. Patients with STAT1-positive melanomas also tended to benefit from interferon concerning RFS (P = 0.074) and showed a significant benefit concerning overall survival (OS; P < 0.05). According to Cox analysis, MTAP expression in contrast to STAT1 was an independent positive prognostic marker for RFS and OS. In conclusion, MTAP represents a highly promising immunohistochemical marker for prognosis and interferon response of patients with malignant melanoma.

Xanthohumol feeding does not impair organ function and homoeostasis in mice.

Xanthohumol, the major prenylated chalcone found in hops, is known to exert several beneficial effects but only few studies evaluated the safety profile of this natural compound with in part discrepant results. Here, we fed female BALB/c mice with a standard diet supplemented with xanthohumol for 3 weeks, and thus, achieved a daily dose of approximately 1000 mg xanthohumol/kg body weight. There were no significant differences in body weight or food intake between mice on standard diet and animals receiving the same diet supplemented with xanthohumol. Histopathological examination of liver, kidney, colon, lung, heart, spleen and thymus revealed no signs of xanthohumol-toxicity, and biochemical serum analysis confirmed normal organ function. Further, xanthohumol treatment did not affect hepatic glycogen content CYP2E1 and CYP1A2 expression levels, but CYP3A11 mRNA was approximately 30% reduced. Expression of several genes indicative of early hepatic inflammation and fibrosis, a hallmark of chronic liver injury, did not differ between xanthohumol treated and control mice. In summary, these results indicate that oral administration of xanthohumol exhibits no adverse effects on major organ function and homoeostasis in mice. Particularly, hepatotoxic effects could be ruled out confirming a good safety profile of xanthohumol as prerequisite for further studies in humans.

Reduced expression of fibroblast growth factor receptor 2IIIb in hepatocellular carcinoma induces a more aggressive growth.

Fibroblast growth factor receptor 2 isoform b (FGFR2-IIIb) is highly expressed in hepatocytes and plays an important role in liver homeostasis and regeneration. Here, we analyzed the expression and function of FGFR2-IIIb in hepatocellular carcinoma (HCC). FGFR2-IIIb expression in HCC tissues and cell lines was lower than in primary human hepatocytes and nontumorous tissue. FGFR2-IIIb-negative HCCs showed a significantly higher Ki-67 labeling index, and loss of FGFR2-IIIb expression correlated significantly with vascular invasion and more advanced tumor stages. A decrease in FGFR-2IIIb expression in HCC cell lines was not related to promoter hypermethylation. However, PCR analysis indicated that chromosomal deletion at 10q accounted for the loss of FGFR2 expression in a subset of HCC cells. FGFR2-IIIb re-expression in stable transfected HCC cell lines induced a higher basal apoptosis rate and a significantly reduced proliferation and migratory potential in vitro. In nude mice, FGFR2-IIIb re-expressing HCC cells grew significantly slower, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay revealed higher apoptosis rates. The antitumorigenic effects of FGFR2-IIIb expression in HCC cells were not affected by keratinocyte growth factor or an inhibitor of FGFR-phosphorylation, indicating that they are independent of tyrosine kinase activation. In conclusion, our data indicate that FGFR2-IIIb inhibits tumorigenicity of HCC cells. Identification of the molecular mechanisms promoting regeneration in normal tissue while suppressing malignancy may lead to novel therapeutic targets of this highly aggressive tumor.

Cyclooxygenase 2 (COX2) and Peroxisome Proliferator-Activated Receptor Gamma (PPARG) Are Stage-Dependent Prognostic Markers of Malignant Melanoma.

Using tissue microarrays (TMAs) we studied COX2/PPARG immunoreactivity in a broad spectrum of tumors focussing on clinicopathological correlations and the outcome of patients with malignant melanoma (MM). TMA-1 contained normal and tumor tissues (n = 3448) from 47 organs including skin neoplasms (n = 323); TMA-2 88 primary MM, 101 metastases, and 161 benign nevi. Based on a biomodulatory approach combining COX/PPAR-targeting with metronomic low-dose chemotherapy metastases of 36 patients participating in a randomized trial with metastatic (stage IV) melanoma were investigated using TMA-3. COX2/PPARG immunoreactivity significantly increased from nevi to primary MM and metastases; COX2 positivity was associated with advanced Clark levels and shorter recurrence-free survival. Patients with PPARG-positive metastases and biomodulatory metronomic chemotherapy alone or combined with COX2/PPARG-targeting showed a significantly prolonged progression-free survival. Regarding primary MM, COX2 expression indicates an increased risk of tumor recurrence. In metastatic MM, PPARG expression may be a predicitive marker for response to biomodulatory stroma-targeted therapy.

Histopathologic features and microsatellite instability of cancers of the papilla of vater and their precursor lesions.

The prevalence and development of microsatellite instability (MSI) and underlying mismatch repair (MMR) deficiency in the carcinogenesis of adenocarcinomas of the papilla of Vater and their precursor lesions are not well established. We analyzed 120 ampullary adenomas (31 pure adenomas and 89 carcinoma-associated adenomas) and 170 pure adenocarcinomas for MSI, immunohistochemical expression of MMR proteins and specific histopathologic features. The most common histologic subtype was intestinal (46.5%), followed by pancreatobiliary (23.5%), poorly differentiated adenocarcinomas (12.9%), intestinal-mucinous (8.2%), and invasive papillary carcinomas (5.3%). Eight of 89 adenomas (9%) and 15/144 carcinomas (10%) showed high microsatellite instability (MSI-H), 10/89 adenomas (11%) and 5/144 carcinomas (4%) showed low microsatellite instability (MSI-L), and 71/89 adenomas (80%) and 124/144 carcinomas (86%) were microsatellite stable (MSS). MSI analysis from carcinomas contiguous with an adenomatous component (n=54) exhibited concordant results in 6/8 (75%) MSI-H and 42/46 (91.3%) MSS tumors. Of 14 carcinomas with MSI-H, 7 showed loss of MLH1 and 5/6 (83%) MLH1 promoter methylation, and 2 carcinomas showed simultaneous loss of MSH2 and MSH6. Two carcinomas and 3 adenomas with MSI-H revealed exclusive loss of MSH6. MSI-H cancers were significantly associated with intestinal mucinous subtype (P<0.001), high tumor grade (P=0.003), expansive growth pattern (P=0.044), and marked lymphoid host response (P=0.004). Patients with MSI-H carcinoma had a significantly longer overall survival (P=0.0082) than those with MSI-L or MSS tumors. Our findings indicate that the MSI-phenotype is an early event, which develops at the stage of adenoma and is reliably detectable in the precursor lesion. The MMR deficient molecular pathway of carcinogenesis is associated with a histopathologic phenotype in ampullary cancer, similar to the one that has been well described in colon cancer.

Activated hepatic stellate cells promote tumorigenicity of hepatocellular carcinoma.

Liver cirrhosis is the main risk factor for the development of hepatocellular carcinoma (HCC). Activated hepatic stellate cells (HSC) are the effector cells of hepatic fibrosis and also infiltrate the HCC stroma where they might play a critical role in HCC progression. Here we aimed to analyze the effects of activated HSC on the proliferation and growth of HCC cell lines in vitro and in vivo. Conditioned media (CM) collected from HSC significantly induced proliferation and migration of HCC cells cultured in monolayers. In a 3-dimensional spheroid coculture system, HSC promoted HCC growth and diminished the extent of central necrosis. In accordance, in vivo simultaneous implantation of HSC and HCC cells into nude mice promoted tumor growth and invasiveness, and inhibited necrosis formation. As potential mechanism of the tumorigenic effects of HSC we identified activation of NFkappaB and extracellular-regulated kinase (ERK) in HCC cells, two signaling cascades that play a crucial role in HCC progression. In summary, our data indicate that stromal HSC promotes HCC progression and suggest the HSC-HCC interaction as an interesting tumor differentiation-independent target for therapy of this highly aggressive cancer.

Evidence for a role of epithelial mesenchymal transition during pathogenesis of fistulae in Crohn's disease.

BACKGROUND: The pathogenesis of fistulae in Crohn's disease (CD) patients is barely understood. We recently showed that more than two-thirds of CD fistulae are covered with flat, mesenchymal-like cells (transitional cells [TC]) forming a patchy basement membrane. Epithelial-to-mesenchymal transition (EMT) is a process of reprogramming epithelial cells, allowing them to migrate more effectively and giving epithelial cells an "invasive" potential. EMT has been suggested to be crucial in fibrosis found in different tissues and diseases. We therefore investigated whether EMT could be involved in the pathogenesis of fistulae formation in CD. METHODS: In all, 18 perianal fistulae, 2 enteroenteric, and 1 enterovesical fistulae from 17 CD patients were analyzed. In addition 2 perianal fistulae of non-CD patients were studied. Hematoxylin and eosin staining, immunohistochemistry for the expression of cytokeratins 8 and 20, beta6-integrin, E-cadherin, beta-catenin, vimentin, and TGF-beta1 and 2 were performed according to standard techniques. RESULTS: The TC covering perianal or enteroenteric fistulae were strongly positive for cytokeratins 8 and 20 but negative for vimentin, indicating their epithelial origin. beta6-Integrin and TGF-beta had the highest staining intensities in the transitional zone between the epithelium and the TC. Expression of junctional proteins such as E-cadherin was reduced in TC as compared to regular fistulae epithelium. In addition, a translocation of beta-catenin from the membrane to the cytoplasm was observed. CONCLUSIONS: Our data for the first time indicate an expression pattern of epithelial and mesenchymal markers in TC associated with fistulae formation that is characteristic for EMT. Studying the pathways of EMT during intestinal fistulae formation may help to develop new therapeutic strategies.

Blocking lymphotoxin beta receptor signalling exacerbates acute DSS-induced intestinal inflammation--opposite functions for surface lymphotoxin expressed by T and B lymphocytes.

The lymphotoxin beta receptor (LTbetaR) signalling pathway is involved in the development of secondary lymphoid organs and the maintenance of organized lymphoid tissues. Additionally, previous studies clearly demonstrated the involvement of the LTbetaR interaction with its ligands in promoting intestinal inflammation. In order to dissect the role of LTbetaR activation in the mouse model of acute DSS-induced colitis we treated mice with a functional inhibitor of LTbetaR activation (LTbetaR:Ig) and compared it to disease in LTbetaR-deficient and LTalphabeta-deficient mice. All these modes of LTbetaR signalling ablation resulted in significant aggravation of the disease and in release of inflammatory cytokines such as TNF, IL-6, and IFNgamma. Finally, using mice with conditionally ablated expression of membrane bound LTbeta on T or B cells, respectively, distinct and opposite contributions of surface LTbeta expressed on T or B cells was found. Thus, activation of LTbetaR by LTalphabeta mainly expressed on T lymphocytes is crucial for the down regulation of the inflammatory response in this experimental model.

Bone morphogenetic protein 7 (BMP7) expression is a potential novel prognostic marker for recurrence in patients with primary melanoma.

The purpose of this study was to investigate whether protein expression of bone morphogenetic protein 7 (BMP7) is associated with clinico-pathologic characteristics in benign and malignant melanocytic skin tumors. Tissue microarrays (TMAs) were used to analyze BMP7 expression and the Ki-67 labeling index immunohistochemically. Expression was scored semi quantitatively (0-2+). BMP7 protein expression of any intensity (1+-2+) was detected in 50.2% (153/305) of informative cases. In general, BMP7 expression was significantly induced in malignant melanomas (P< 0.001) and melanoma metastases (P< 0.001), compared to benign nevi. Additionally, expression of BMP7 in primary melanomas was associated with Ki-67 labeling index > 5% suggesting that induction of BMP7 expression is associated with proliferation (P=0.028). None of the other clinical and histological factors analyzed was significantly related to BMP7 expression. Interestingly, lymph node metastases demonstrated a significantly higher BMP7 expression compared to skin metastases (P<0.01). Strong BMP7 expression (score 2+) was significantly associated with shorter tumor recurrence (P< 0.05). In summary, induction of BMP7 expression is frequent in melanomas and may serve as a novel prognostic marker of progression in melanoma patients.

Bone morphogenetic protein 7 is elevated in patients with chronic liver disease and exerts fibrogenic effects on human hepatic stellate cells.

Hepatic stellate cells (HSCs) are the main extracellular matrix (ECM)-producing cells in liver fibrogenesis. The excessive synthesis of ECM proteins deteriorates hepatic architecture and results in liver fibrosis and cirrhosis. This study investigated the role of bone morphogenetic protein 7 (BMP7) as a member of the transforming growth factor (TGF)-beta superfamily in chronic liver disease. Plasma levels of BMP7 were significantly elevated in patients with chronic liver disease compared with healthy controls. Immunohistochemistry of cirrhotic human liver demonstrated upregulated BMP7 protein expression in hepatocytes as compared with normal human liver. Because gene expression for all putative BMP7 receptors was induced during the culture activation process of primary human HSCs, we studied the effects of BMP7 on hTERT immortalized human HSCs in vitro. BMP7, as expressed and secreted after infection with adenoviruses encoding BMP7 (AdBMP7), increased proliferation of HSCs. The mRNA and protein expression of type I collagen and fibronectin was increased in BMP7-stimulated HSCs. Elevated systemic and hepatic levels of BMP7 in patients with chronic liver disease may contribute to progression of liver fibrogenesis in vivo.

Loss of maspin expression contributes to a more invasive potential in malignant melanoma.

Deregulation of protease expression and activity is known to play an important role in tumour progression of malignant melanoma. The serpin maspin, a tumour suppressor in breast and prostate cancer was described as an inhibitor of cell migration and inducer of cell adhesion between the basement membrane and extracellular matrix resulting in inhibition of tumour metastasis. In contrast, overexpression of maspin is correlated with poor prognosis in other cancers. However, little is known about expression, regulation and function of maspin in malignant melanoma. In this study, we found loss of maspin expression in malignant melanoma cells compared with normal human epidermal melanocytes, which was analysed by quantitative real-time PCR, Western blot analysis, immunohistochemistry and microarray. For functional studies, melanoma cell clones stably transfected with a maspin expression vector were tested for changes in proliferation, migration and invasion. Although we could not see differences in proliferation and migration, we detected strongly reduced invasive capacity in the melanoma cell clones in which maspin is re-expressed compared with control. Reduced invasive potential was also detected in three different melanoma cell lines transiently transfected with a maspin expression vector. Furthermore, exogenously added maspin alone was sufficient to reduce invasion in MelIm significantly, indicating that maspin directly inhibits invasion on the cell surface. In summary, we believe that maspin is a tumour suppressor in malignant melanoma.

Role of soluble factors and three-dimensional culture in in vitro differentiation of intestinal macrophages.

AIM: To examine the factor(s) involved in differentiation of intestinal macrophages (IMACs) using a recently established in vitro model. METHODS: To test whether soluble or membrane bound factors induce IMAC-differentiation, freshly elutriated monocytes (MO) were incubated with conditioned media or cell membranes of intestinal epithelial cells (IEC) or cultured with IEC in transwell systems. To determine the importance of an active migration of MO, three-dimensional aggregates from a 1:1-mixture of MO and IEC were examined by immunohistochemistry and flow cytometry. Apoptosis was examined by caspase-3 Western blots. Extracellular matrix production in differentiation models was compared by immunohistochemistry. RESULTS: IMAC differentiation was observed in a complex three-dimensional co-culture model (multicellular spheroid, MCS) with IEC after migration of MO into the spheroids. By co-culture of MO with conditioned media or membrane preparations of IEC no IMAC differentiation was induced. Co-culture of MO with IEC in transwell-cultures, with the two cell populations separated by a membrane also did not result in intestinal-like differentiation of MO. In contrast to IEC-spheroids with immigrating MO in mixed MCS of IEC and MO only a small subpopulation of MO was able to survive the seven day culture period. CONCLUSION: Intestinal-like differentiation of MO in vitro is only induced in the complex three-dimensional MCS model after immigration of MO indicating a role of cell-matrix and/or cell-cell interactions during the differentiation of IMACs.

Mammalian target of rapamycin is activated in human gastric cancer and serves as a target for therapy in an experimental model.

The mammalian target of rapamycin (mTOR) has become an interesting target for cancer therapy through its influence on oncogenic signals, which involve phosphatidylinositol-3-kinase and hypoxia-inducible factor-1alpha (HIF-1alpha). Since mTOR is an upstream regulator of HIF-1alpha, a key mediator of gastric cancer growth and angiogenesis, we investigated mTOR activation in human gastric adenocarcinoma specimens and determined whether rapamycin could inhibit gastric cancer growth in mice. Expression of phospho-mTOR was assessed by immunohistochemical analyses of human tissues. For in vitro studies, human gastric cancer cell lines were used to determine S6K1, 4E-BP-1 and HIF-1alpha activation and cancer cell motility upon rapamycin treatment. Effects of rapamycin on tumor growth and angiogenesis in vivo were assessed in both a subcutaneous tumor model and in an experimental model with orthotopically grown tumors. Mice received either rapamycin (0.5 mg/kg/day or 1.5 mg/kg/day) or diluent per intra-peritoneal injections. In addition, antiangiogenic effects were monitored in vivo using a dorsal-skin-fold chamber model. Immunohistochemical analyses showed strong expression of phospho-mTOR in 60% of intestinal- and 64% of diffuse-type human gastric adenocarcinomas. In vitro, rapamycin-treatment effectively blocked S6K1, 4E-BP-1 and HIF-1alpha activation, and significantly impaired tumor cell migration. In vivo, rapamycin-treatment led to significant inhibition of subcutaneous tumor growth, decreased CD31-positive vessel area and reduced tumor cell proliferation. Similar significant results were obtained in an orthotopic model of gastric cancer. In the dorsal-skin-fold chamber model, rapamycin-treatment significantly inhibited tumor vascularization in vivo. In conclusion, mTOR is frequently activated in human gastric cancer and represents a promising new molecular target for therapy.

Reduced expression of CYLD in human colon and hepatocellular carcinomas.

CYLD was originally identified as a tumor suppressor that is mutated in familial cylindromatosis. Recent studies suggested a role for CYLD in nuclear factor-kappaB (NF-kappaB) regulation. NF-kappaB activation has been connected with multiple aspects of oncogenesis but the underlying molecular mechanisms of persistent NF-kappaB activation in tumors remain largely unknown. Thus, we evaluated CYLD transcription in different colon and hepatocellular carcinoma cell lines and tissue samples, respectively. CYLD was downregulated or lost in all tumor cell lines investigated as compared with primary human colonic epithelial cells and hepatocytes, respectively. Further, quantitative PCR analysis revealed reduced CYLD mRNA expression in most tumor samples compared with non-tumorous tissue. Analysis on protein level confirmed these findings. Functional assays with CYLD transfected cell lines revealed that CYLD expression decreased NF-kappaB activity. Thus, functional relevant loss of CYLD expression may contribute to tumor development and progression, and may provide a new target for therapeutic strategies.

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.

Multiparameter immunofluorescence on paraffin-embedded tissue sections.

Immunohistochemical techniques have gained increasing importance in diagnostics and research. While formalin-fixed, paraffin-embedded human tissue retains excellent morphology, the detection of antigens by immunofluorescence in its sections and especially the demonstration of multiple simultaneous antibodies have limitations. Double immunofluorescence labeling of routinely processed paraffin sections has been described previously. The signal intensity observed after triple labeling has been reported to be significantly inferior to that obtained by application of double fluorochromes. The authors show multicolor labeling of three and four primary antibodies in routinely processed paraffin-embedded tissue sections using a standardized immunofluorescence technique. In addition, procedures to reduce background staining and to avoid nonspecific double staining are described.

Tissue microarray analysis of methylthioadenosine phosphorylase protein expression in melanocytic skin tumors.

BACKGROUND: Using tissue microarrays, we investigated whether methylthioadenosine phosphorylase (MTAP) protein expression is associated with clinicopathologic variables in benign and malignant melanocytic skin tumors. OBSERVATIONS: Cytoplasmic MTAP expression was detected in 227 (72.1%) of 315 informative cases. Expression was significantly reduced in primary malignant melanomas and in melanoma metastases compared with benign nevi (P<.001 for both). No difference was noted in MTAP expression between primary malignant melanomas and melanoma metastases. In primary malignant melanomas, a Ki67-labeling index less than 5% was associated with MTAP expression (P = .04), suggesting that loss of MTAP expression is associated with proliferation. No other variables had significant associations with MTAP expression. Lymph node metastases demonstrated significantly higher MTAP expression compared with skin metastases (P = .01). In the overall cohort, MTAP expression was not associated with prognosis. Among 26 patients with MTAP-positive melanomas and tumor recurrence, 18 patients who received interferon therapy had a significant benefit compared with 8 patients who did not receive interferon therapy (P = .009). This was not seen in the patients with MTAP-negative tumors. Conclusion Methylthioadenosine phosphorylase protein expression may be a predictive marker of interferon therapy resistance in patients with melanoma and disease progression.