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ABSTRACT: DUSP4 (MKP-2), a member of the mitogen-activated protein kinase phosphatase (MKP) family and potential tumor suppressor, negatively regulates the MAPKs (mitogen-activated protein kinases) ERK, p38 and JNK. MAPKs play a crucial role in cancer development and progression. Previously, using microarray analyses we found a conspicuously frequent overexpression of DUSP4 in colorectal cancer CRC with high frequent microsatellite instability (MSI-H) compared to microsatellite stable (MSS) CRC. Here we studied DUSP4 expression on mRNA level in 38 CRC (19 MSI-H and 19 MSS) compared to matched normal tissue as well as in CRC cell lines by RT-qPCR. DUSP4 was overexpressed in all 19 MSI-H tumors and in 14 MSS tumors. Median expression levels in MSI-H tumors were significantly higher than in MSS-tumors (p<0.001). Consistently, MSI-H CRC cell lines showed 6.8-fold higher DUSP4mRNA levels than MSS cell lines. DUSP4 expression was not regulated by promoter methylation since no methylation was found by quantitative methylation analysis of DUSP4 promoter in CRC cell lines neither in tumor samples. Furthermore, no DUSP4 mutation was found on genomic DNA level in four CRC cell lines. DUSP4 overexpression in CRC cell lines through DUSP4 transfection caused upregulated expression of MAPK targets CDC25A, CCND1, EGR1, FOS, MYCandCDKN1A in HCT116 as well as downregulation of mismatch repair gene MSH2 in SW480. Furthermore DUSP4 overexpression led to increased proliferation in CRC cell lines. Our findings suggest that DUSP4 acts as an important regulator of cell growth within the MAPK pathway and causes enhanced cell growth in MSI-H CRC. © 2012 Wiley Periodicals, Inc.
International Journal of Cancer 09/2012; · 5.44 Impact Factor
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ABSTRACT: In the course of screening for virus-host systems in extreme thermal environments, we have isolated a strain of the hyperthermophilic archaeaon Acidianus hospitalis producing unusual filamentous particles with a zipper-like appearance. The particles were shown to represent a secreted form of a genuine cellular enzyme, tetrathionate hydrolase, involved in sulfur metabolism.
Applied and environmental microbiology 05/2012; 78(15):5463-5. · 3.69 Impact Factor
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ABSTRACT: Promoter hypermethylation occurs in various tumors and leads to silencing of tumor-relevant genes. Thus, promoter methylation analysis (MA) has been established as an important tool in cancer research and diagnostics. Here we present MethyQESD (methylation-quantification of endonuclease-resistant DNA) as a fast, easy, precise and reliable method for quantitative MA without the need of bisulfite-treatment or fluorescent probes. Though MethyQESD principally works with any gene promoter we established MethyQESD for the mismatch repair gene MLH1 and tested its utility to differentiate between sporadic microsatellite unstable (MSI-H) colorectal cancer and hereditary nonpolyposis colorectal cancer (HNPCC) by quantitative MLH1 MA. We investigated formalin-fixed and paraffin-embedded tissue samples from a previously published, well-characterized tumor collective comprising 25 HNPCC, 14 sporadic MSI-H CRC and 16 sporadic microsatellite stable (MSS) CRC. We found a high accuracy of MethyQESD by spiking experiments with dilution series of methylated (SW48 cancer cell line) and unmethylated (blood) DNA (Pearson's r=0.9997 (proximal MLH1 promoter region), r=0.9976 (distal MLH1 promoter region)). MethyQESD and conventional quantitative MA using of 96 formalin-fixed and paraffin-embedded CRC showed a high degree of concordance of both methods (Pearson's r=0.885). HNPCC tumors showed either null MLH1 methylation or a significantly lower degree of MLH1 methylation than sporadic MSI-H CRC (P<0.001). MLH1 methylation was negative in all MSS tumors. Receiver operating characteristic (ROC) curve analyses defined a cutoff value of 16.5% MLH1 methylation for specific and sensitive identification of sporadic MSI-H CRC (area under ROC curve: 1.000; asymptotic significance: P<0.001). Thus, quantitative MLH1 MA by MethyQESD provides a simple, fast and valuable tool to identify HNPCC candidates. Furthermore, MethyQESD works reliably with formalin-fixed paraffin-embedded tissue and simplifies DNA MA both for research and diagnostic purposes.
Laboratory Investigation 10/2008; 88(12):1367-75. · 3.64 Impact Factor
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ABSTRACT: Promoter hypermethylation occurs frequently in tumors and leads to silencing of tumor-relevant genes like tumor suppressor genes. In a subset of sporadic colorectal cancers (CRC), inactivation of the mismatch repair gene MLH1 due to promoter methylation causes high level of microsatellite instability (MSI-H). MSI-H is also a hallmark of hereditary nonpolyposis colorectal cancer (HNPCC) in which mismatch repair inactivation results from germ-line mutations. For differentiation of sporadic and hereditary MSI-H tumor patients, MLH1 promoter methylation analysis is a promising tool but is not yet used in daily diagnostics because only qualitative techniques without standardization are available. The aim of this study is to establish a reliable and quantitative MLH1 methylation analysis technique and to define valid MLH1 methylation cutoff values for HNPCC diagnostics.
We developed a new real-time PCR-based technique to detect and quantify methylation of both proximal and distal hMLH1 promoter regions. We established and validated this technique in a cohort of 108 CRCs [94 MSI-H and 16 microsatellite stable (MSS) cases] comprising a reference (n = 58) and a tester tumor group (n = 50).
The reference tumor group contained 28 HNPCC with proven germ-line mutations or positive Amsterdam I criteria (median age, 37 years) and loss of MLH1 expression, 14 sporadic MSI-H CRC tumors with loss of MLH1 expression and BRAF V600E mutation (median age, 80.5 years), and 16 sporadic MSS CRC (median age, 76.5 years). No MLH1 promoter methylation could be found in any MSS tumors. HNPCC patients showed no or low level of MLH1 promoter methylation. A cutoff value of 18% methylation extent could be determined in this study to define MLH1 hypermethylation specific for sporadic MSI-H cases. Methylation could also be verified qualitatively by melting point analysis. BRAF V600E mutations were not detected in any HNPCC patients (n = 22 informative cases).
According to the present data, quantitative MLH1 methylation analysis in MSI-H CRC is a valuable molecular tool to distinguish between HNPCC and sporadic MSI-H CRC. The detection of a BRAF V600E mutation further supports the exclusion of HNPCC.
Clinical Cancer Research 07/2007; 13(11):3221-8. · 7.74 Impact Factor
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ABSTRACT: 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.
Pigment Cell Research 05/2007; 20(2):112-9. · 4.29 Impact Factor
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Wolfgang Dietmaier, Marcus Bettstetter,
Peter J Wild,
Matthias Woenckhaus,
Petra Rümmele,
Arndt Hartmann,
Stefan Dechant,
Hagen Blaszyk,
Armin Pauer,
Monika Klinkhammer-Schalke,
Ferdinand Hofstädter
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ABSTRACT: Maspin, a member of the Serpin protease inhibitor family, is overexpressed in poorly differentiated colorectal tumors and more frequently found in tumors with microsatellite instability. Immunohistochemical nuclear Maspin staining is predominantly seen in tumor cells at the invasion front of such cancers, suggesting that this molecule is associated with local tumor cell infiltration and aggressiveness. In a retrospective study, we studied nuclear Maspin expression as a potential prognostic tool in a total of 172 primary stage III colon cancers by immunohistochemistry. Of those 172 patients, 76 were treated by surgery only, and 96 patients received additional adjuvant 5-fluorouracil (5-FU) based chemotherapy. Nuclear Maspin expression was an independent adverse prognostic factor for overall survival in our patient cohort (hazard ratio 2.08; 95% CI, 1.13-3.81; p = 0.018). However, patients with primary tumors expressing Maspin in the nucleus showed a significant treatment benefit from 5-FU chemotherapy (hazard ratio 0.384; 95% CI, 0.188-0.784; p = 0.009) compared to adjuvantly treated patients whose tumors did not express this molecule. Nuclear Maspin expression is highly predictive of 5-FU chemotherapy response in patients with advanced stage colon cancer. Patients with negative immunohistochemical Maspin expression do not benefit from 5-FU treatment and may be candidates for an alternative (non-5-FU based) adjuvant therapy regime.
International Journal of Cancer 06/2006; 118(9):2247-54. · 5.44 Impact Factor
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ABSTRACT: Maspin, a member of the serpin family, has been reported to suppress metastasis and angiogenesis in breast and prostate cancers. Overexpression of maspin was associated with adverse prognostic features in several other tumours. In this study, expression of maspin was analysed in 41 colorectal carcinomas with high-frequency microsatellite instability (MSI-H) and 159 microsatellite stable colorectal cancers (MSS/MSI-L) by immunohistochemistry (IHC) and partly by relative quantitative real-time RT-PCR and western blot analyses. Significant upregulation of maspin expression was found in MSI-H tumours compared to both MSS/MSI-L tumours and matched benign colonic mucosa. Increased maspin expression was also found in three MSI-H colon cancer cell lines, but not in three MSS colon cancer cell lines by RT-PCR and western blot analyses. Regulation of maspin expression depended on promoter methylation as tissue specimens and cell lines expressing maspin showed unmethylated maspin promoters, whereas promoter hypermethylation was found in specimens with loss of maspin expression. Intense nuclear maspin immunostaining was seen specifically in MSI-H tumours (p = 0.013), de-differentiated tumours (p = 0.006), and at the invasion front. These findings provide new insights into the role of maspin in colorectal cancer progression and may be useful for diagnosis and treatment strategies.
The Journal of Pathology 05/2005; 205(5):606-14. · 6.32 Impact Factor
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ABSTRACT: We describe a novel virus, AFV1, of the hyperthermophilic archaeal genus Acidianus. Filamentous virions are covered with a lipid envelope and contain at least five different proteins with molecular masses in the range of 23-130 kDa and a 20.8-kb-long linear double-stranded DNA. The virus has been assigned to the family Lipothrixviridae on the basis of morphotypic characteristics. Host range is confined to several strains of Acidianus and the virus persists in its hosts in a stable carrier state. The latent period of virus infection is about 4 h. Viral DNA was sequenced and sequence similarities were found to the lipothrixvirus SIFV, the rudiviruses SIRV1 and SIRV2, as well as to conjugative plasmids and chromosomes of the genus Sulfolobus. Exceptionally for the linear genomes of archaeal viruses, many short direct repeats, with the sequence TTGTT or close variants thereof, are closely clustered over 300 bp at each end of the genome. They are reminiscent of the telomeric ends of linear eukaryal chromosomes.
Virology 11/2003; 315(1):68-79. · 3.35 Impact Factor
