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F Müller-Pillasch

Publications and source records attributed to F Müller-Pillasch.

At least 19 recordsLinked to original sources
PubMed2000

A novel transmembrane serine protease (TMPRSS3) overexpressed in pancreatic cancer.

We report the characterization of a novel serine protease of the chymotrypsin family, recently isolated by cDNA-representational difference analysis, as a gene overexpressed in pancreatic cancer. The 2.3-kb mRNA of the gene, named TMPRSS3, is strongly expressed in a subset of pancreatic cancer and various other cancer tissues, and its expression correlates with the metastatic potential of the clonal SUIT-2 pancreatic cancer cell lines. The deduced polypeptide sequence consists of 437 amino acids and exhibits all of the structural features characteristic of serine proteases with trypsin-like activity. TMPRSS3 is membrane bound with a NH2-terminal signal-anchor sequence and a glycosylated extracellular region containing the serine protease domain. Thus, TMPRSS3 is a novel membrane-bound serine protease overexpressed in cancer, which may be of importance for processes involved in metastasis formation and tumor invasion.

Amino Acid Sequence↗
PubMed1999

Strategies for the detection of disease genes in pancreatic cancer.

The present review summarizes our strategies aimed at identifying and characterizing genetic alterations occurring at the transcriptional and chromosomal level in pancreatic cancer. To study transcriptional alterations we have used a number of techniques including modified versions of differential hybridizations and cDNA RDA (representational difference analysis). These approaches have led to the identification of more than 500 genes with differential expression in pancreatic cancer. To study chromosomal aberrations occurring in pancreatic cancer tissues we used comparative genomic hybridization (CGH). This allowed the identification of a number of chromosomal regions containing putative tumor suppressor genes or oncogenes. Genes isolated in both approaches represent potential new disease genes for pancreatic cancer and are at present being characterized by individual or serial analysis.

Amino Acid Sequence↗
PubMed1999

Use of representational difference analysis to study the effect of TGFB on the expression profile of a pancreatic cancer cell line.

It has been shown that TGFBs, their receptors, or downstream targets show genetic alterations in pancreatic cancer. This study was designed to identify transcriptional alterations induced by prolonged treatment of pancreatic cancer cell lines with TGFB. The TGFB-responsive PANC-1 cell line was treated with 10-ng/ml TGFB1 for 24 hr. cDNA representational difference analysis was used to generate subtracted hybridization probes enriched for TGFB regulated genes. These probes were hybridized on gridded arrays of cDNA clones containing genes differentially expressed in pancreatic cancer. Twenty-seven distinct cDNA clones were shown to be TGFB target genes. Eleven genes were upregulated by TGFB and were associated with extracellular matrix composition and formation, including genes usually transcribed by cells of mesenchymal origin only. Transcript levels of 16 genes were downregulated by TGFB and could mainly be classified into markers of epithelial differentiation and genes involved in the transcriptional and translational machinery. In conclusion, a 24-hr treatment of PANC-1 cells with TGFB induced a loss of epithelial and a gain of mesenchymal markers. As in other tumors, this epithelial-mesenchymal transdifferentiation may be of general importance during pancreatic carcinogenesis, and may participate, e.g., in the development of the desmoplastic reaction or the acquisition of an invasive phenotype of pancreatic tumor cells. This study demonstrates the usefulness of cDNA RDA and gridded clone libraries to study the effect of signaling cascades on the expression profile of tumor cells. Similar approaches may be helpful in the context of the genome project for the characterization of novel genes. Genes Chromosomes Cancer 26:70-79, 1999.

Cell Division↗
PubMed1999

Novel technology for detection of genomic and transcriptional alterations in pancreatic cancer.

AIM: The present review summarizes our strategies aimed at identifying and characterizing genetic alterations occurring at the transcriptional and chromosomal level in pancreatic cancer. METHODS: To study transcriptional alterations we have used a number of techniques including modified versions of differential hybridizations and cDNA-RDA (representational difference analysis). Comparative genomic hybridization (CGH) was used to study chromosomal aberrations occurring in pancreatic cancer tissues. RESULTS: The study of transcriptional alterations led to the identification of more than 500 genes with differential expression in pancreatic cancer. The sum of these alterations represented the first expression profile characteristic for pancreatic tumors. The CGH analysis allowed the identification of a number of chromosomal regions containing putative tumor suppressor genes or oncogenes. These regions are presently being characterized at the molecular level. In a first approach the myb-oncogene was identified as the relevant oncogene of an amplification on 6q occurring in up to 10% of pancreatic cancer patients. CONCLUSIONS: Genes isolated in both approaches represent potential new disease genes for pancreatic cancer and are at present being characterized by individual or serial analysis.

Chromosome Aberrations↗
PubMed1998

Identification of a new tumour-associated antigen TM4SF5 and its expression in human cancer.

In a previous large-scale screening for differentially expressed genes in pancreatic cancer, a gene was identified that was highly overexpressed in pancreatic cancer encoding a novel putative tetraspan transmembrane protein highly homologous to the tumour-associated antigen L6. Using a radiation hybrid panel the identified human gene named TM4SF5 (transmembrane 4 superfamily member 5) was localized to chromosome 17 in the region 17p13.3. The cloned TM4SF5 cDNA has a 32bp 5'-untranslated region (UTR), a 591bp open reading frame (ORF) and a 85bp 3'UTR. The predicted TM4SF5 protein with 197 amino acids contains three NH2-terminal hydrophobic transmembrane regions, followed by an extracellular hydrophilic domain containing two potential N-linked glycosylation sites and a COOH-terminal hydrophobic transmembrane region. These structural features are shared by the L6 antigen and a number of related cell surface proteins associated with cell growth. TM4SF5 was overexpressed in pancreatic cancer tissues as compared to both normal pancreas and chronic pancreatitis tissues, and was detected at high levels in other tumour tissues. Although the precise function of TM4SF5 remains to be elucidated it may be useful in a clinical setting for tumour diagnosis and/or therapy. This hypothesis is supported by the strong homology to the L6 antigen, which has proved promising in immunological, therapeutic and diagnostic approaches.

Amino Acid Sequence↗
PubMed1998

Cloning of a new Kunitz-type protease inhibitor with a putative transmembrane domain overexpressed in pancreatic cancer.

In a previous large scale screen for differentially expressed genes in pancreatic cancer, we identified a gene highly overexpressed in cancer encoding a novel putative transmembrane protein with two Kunitz-type serine protease inhibitor domains. The identified gene named kop (Kunitz domain containing protein overexpressed in pancreatic cancer) was assigned to chromosome 19 in the region 19q13.1. Kop was detected at high levels in pancreatic cancer cell lines and was overexpressed in pancreatic cancer tissues as compared to both, normal pancreas and chronic pancreatitis tissues. Being a member of the Kunitz-type serine protease inhibitor family, this new gene may participate in tumour cell invasion and metastasis and in the development of the marked desmoplastic reaction typical for human pancreatic cancer tissues. In this context, the fact that kop has a putative transmembrane domain may have functional implications of particular interest.

Adenocarcinoma↗
PubMed1998

Isolation of differentially expressed genes by combining representational difference analysis (RDA) and cDNA library arrays.

The difference products (DP) of representational difference analyses (RDA) were used as hybridization probes on cDNA arrays. The effectivity of RDA products obtained with increasing driver/tester ratios (DP 1 = 100:1, DP 2 = 800:1 and DP 3 = 400,000:1) to isolate differentially expressed genes was compared with the effectivity of conventional differential hybridizations. Pacreatic cancer and control tissues were used as a test system to isolate differentially expressed genes. The use of RDA products as hybridization probes showed two major advantages: (i) a reliable identification of true differential signals; and (ii) only one autoradiograph had to be analyzed, which eliminated the need for a laborious subtraction of signal intensities obtained with different cDNA probes. Increasing driver/tester ratios in iterative rounds of RDA delivered more specific results, though the total yield of differential clones was gradually reduced. In this situation, the intermediate RDA product DP 2 provided the best compromise.

Blotting, Northern↗
PubMed1997

Cloning of novel transcripts of the human guanine-nucleotide-exchange factor Mss4: in situ chromosomal mapping and expression in pancreatic cancer.

In a previous large-scale analysis of gene expression in pancreatic cancer using gridded arrays of cDNA libraries and differential hybridizations, a gene that was a homolog to human mss4 was identified. Mss4 is a guanine-nucleotide-exchange factor for the Sec4/ Ypt1/Rab family of small GTP-binding proteins involved in the regulation of intracellular vesicular transport. By fluorescence in situ hybridization the human mss4 gene was assigned to chromosome 1q32-q41. Northern blot analysis revealed that three mss4 mRNA species are transcribed in human tissues of 780, 1200, and 2800 bp in length, respectively. Cloning and sequencing of the human mss4 transcripts from a pancreatic cancer cDNA library revealed that these mRNA species differ in the length of the 3-untranslated region and are probably due to the alternate use of polyadenylation sites. All mRNA species were detected at moderate to high levels in pancreatic cancer cell lines and were overexpressed in pancreatic cancer tissue compared to both normal pancreas and chronic pancreatitis tissue. However, the 1200-bp transcript was the Mss4 mRNA species with the highest level of expression in more than 50% of tumor cells and tissues. High levels of expression were found as well in other human tumor tissues. Mss4 as guanine-exchange factor required in the regulation of intracellular transport may be of importance for the function and growth of human tumor cells. However, the precise role of mss4 in human tumor cells is unknown and remains to be elucidated.

Amino Acid Sequence↗
PubMed1997

Characterization of a high copy number amplification at 6q24 in pancreatic cancer identifies c-myb as a candidate oncogene.

In a recent study designed to identify chromosomal aberrations in pancreatic cancer tissues using comparative genomic hybridization, a high copy number amplification on 6q was detected. To identify the most likely candidate oncogene, the extension of the amplification in pancreatic cancer tissues and cell lines was determined by Southern blot analysis. Exon trapping was performed with DNA from a yeast artificial chromosome clone containing the complete minimally amplified region. Only fragments from two genes, namely, the c-myb oncogene and a novel gene, were shown to be amplified. The c-myb proto-oncogene was amplified in 10% of the pancreatic carcinoma tissues and in the pancreatic cancer cell line PC2. Interestingly, the c-myb oncogene was overexpressed not only in the amplified samples but also in the majority of the examined pancreatic cancer tissues and cell lines, suggesting that amplification is only one of the mechanisms leading to overexpression. In contrast, the novel gene, which was called human eRF3b (eukaryotic release factor 3b), seems to be only coamplified with c-myb. Genetic alterations of c-myb were mainly found in advanced tumors, indicating a possible correlation to tumor progression and aggressive tumor phenotypes.

Adenocarcinoma↗
PubMed1997

Identification of genes with specific expression in pancreatic cancer by cDNA representational difference analysis.

cDNA representational difference analysis (cDNA-RDA) is a polymerase-chain-reaction-coupled subtractive and kinetic enrichment procedure for the isolation of differentially expressed genes. In this study, the technique was used to isolate novel genes specifically expressed in pancreatic cancer. cDNA-RDA was done on cDNA reverse transcribed from a poly(A)+ mRNA pool made from 10 cancer tissues (tester) by using as a driver a cDNA from a poly(A)+ mRNA pool made from a combination of 10 tissues of chronic pancreatitis and 10 healthy pancreatic tissues. The use of chronic pancreatitis in addition to healthy pancreas mRNA in the driver preparation eliminated the influence of stromal tissue components present as contamination in the cancer-specific preparations. Such cDNA-RDA led to the isolation of 16 distinct, cancer-specific gene fragments. These were confirmed to be overexpressed in pancreatic cancer tissues by Northern blot analysis. Sequence analysis revealed homologies to five genes previously implicated in the carcinogenesis of the pancreas or other tissues. Eleven fragments had no significant homology to any known gene and thus represent novel candidate disease genes. The experiments demonstrate that cDNA-RDA is a reproducible and highly efficient method for the identification of novel genes with cancer-specific expression.

Annexin A3↗
PubMed1997

Dissociation and reassembly of adherens junctions during experimental acute pancreatitis.

BACKGROUND & AIMS: The initial pathophysiological events that characterize acute pancreatitis include the formation of pancreatic edema. An interstitial accumulation of fluid, however, is incompatible with the presence of intact intercellular junctions between acinar cells. This study examined the major components of adherens junctions, E-cadherin, alpha-catenin, beta-catenin, and actin, during the initial phase of experimental pancreatitis. METHODS: Pancreatitis was induced in rats by 10 micrograms.kg-1.h-1 intravenous cerulein for up to 12 hours. Adherens junction proteins were localized by immunocytochemistry for fluorescence microscopy or electron microscopy, their expression was studied by slot blot analysis, and their association was investigated by immunoprecipitation and Western blot. RESULTS: During a rapid increase of E-cadherin-encoding RNA, E-cadherin protein declined only moderately and, unlike its cytoskeletal binding partner actin, was not proteolytically cleaved during pancreatitis. Morphologically, E-cadherin and beta-catenin were localized at the basolateral cell membrane from where they rapidly dissociated early in pancreatitis and to where they slowly relocalized during the subsequent course. E-cadherin/beta-catenin complexes disintegrated and reassembled completely in parallel on immunoprecipitation experiments. CONCLUSIONS: The dissociation of adherens junctions and the internalization, relocalization, and reassembly of their major components seem to represent the critical biochemical event at cell-cell contacts during edema formation and resolution in acute pancreatitis.

Actins↗
PubMed1997

The influence of transforming growth factor beta 1 on the expression of genes coding for matrix metalloproteinases and tissue inhibitors of metalloproteinases during regeneration from cerulein-induced pancreatitis.

Enhanced synthesis and deposition of extracellular matrix (ECM) components is a characteristic feature during regeneration from acute cerulein-induced pancreatitis in rats. Transforming growth factor beta 1 (TGF beta 1) has been suggested to be an important modulator of the ECM by interfering with a number of essential processes such as the synthesis of ECM components. To study the involvement of the ECM degrading proteases (matrix metalloproteinases; MMPs) and their specific inhibitors in the process of pancreatic regeneration, we examined the expression of these genes on the transcript level and the activation of the corresponding enzymes by use of zymographies. Pancreatic RNA and protein were extracted from rats sacrificed 1, 2, 3, 5, and 7 days after induction of cerulein pancreatitis. To investigate the influence of TGF beta on gene expression of ECM proteases and their specific inhibitors, we blocked the activity of TGF beta 1 during regeneration from acute pancreatitis by use of neutralizing antibodies against TGF beta 1. Steady levels of 72-kD type IV collagenase (MMP-2), stromelysin (MMP-3), and tissue inhibitor of metalloproteinase 2 (TIMP-2) mRNA were significantly increased 2 days after induction of pancreatitis. MMP-9 and MMP-3 enzyme activity was elevated 12 h after induction of pancreatitis, whereas MMP-2 activity increased 12 h later. Inhibition of TGF beta 1 by neutralizing antibodies only reduced the amount of stromelysin transcripts throughout pancreatic regeneration. In summary, ECM degrading proteases, in particular stromelysin, appear to be involved in ECM remodeling during pancreatic regeneration. TGF beta 1 may be responsible for regulation of stromelysin transcription.

Animals↗
PubMed1996

A pancreatic cancer-specific expression profile.

We present an approach making use of technology established in the context of the genome project to describe a pancreatic cancer-specific expression profile and to identify new potential disease genes or disease-associated-genes. By use of gridded arrays of pancreatic cancer cDNA libraries and differential hybridizations we show that 4% the gridded cDNA library clones contain sequences preferentially expressed in pancreatic cancer. EST-sequencing of 369 distinct (408 total), differentially expressed sequences identified novel genes (32.5%) or homologs to EST-sequences with unknown function (26.3%). Homologies to known genes allow to determine a pancreatic cancer-specific expression profile, which provides for the first time evidence for complex primary and secondary alterations of gene expression responsible for the development of the phenotype of pancreatic cancer cells. In addition this has led to the identification of novel differentially expressed genes, which represent potential oncogenes or disease-associated markers and may be helpful for the development of therapeutic or diagnostic modalities.

Animals↗
PubMed1996

Mapping of chromosomal imbalances in pancreatic carcinoma by comparative genomic hybridization.

To identify recurrent chromosomal imbalances in pancreatic adenocarcinoma, 27 tumors were analyzed by using comparative genomic hybridization. In 23 cases chromosomal imbalances were found. Gains of chromosomal material were much more frequent than losses. The most common overrepresentations were observed on chromosomes 16p (eight cases), 20q (seven cases), 22q (six cases), and 17q (five cases) and under-representations on a subregion of chromosome 9p (eight cases). Distinct high-level amplifications were found on 1p32-p34, 6q24, 7q22, 12p13, and 22q. These data provide evidence for a number of new cytogenetically defined recurrent aberrations which are characteristic of pancreatic carcinoma. The overrepresented or underrepresented chromosomal regions represent candidate regions for potential oncogenes and tumor suppressor genes, respectively, possibly involved in pancreatic tumorigenesis.

Adenocarcinoma↗
PubMed1995

Expression and in-situ localization of genes coding for extracellular matrix proteins and extracellular matrix degrading proteases in pancreatic cancer.

Pancreatic cancer shows a strong desmoplastic reaction characterized by a remarkable proliferation of interstitial connective tissue (collagens type I and III, fibronectin). In this study we have analyzed the balance of expression of mRNAs encoding extracellular matrix components (collagens I, III and IV, laminin, fibronectin), extracellular matrix-degrading metalloproteinases (MMP-1, -2, -3 and -9) and tissue inhibitors of metalloproteinases (TIMP-1 and -2) in pancreatic cancer and control pancreatic tissue by Northern-blot analysis and mRNA in situ hybridization. Transcripts for MMP-1 (interstitial collagenase) and MMP-3 (stromelysin-1) were not detectable in pancreatic cancer and control tissues. Steady-state levels of transcripts encoding extracellular matrix proteins, MMP-2 (72-kDa collagenase IV), MMP-9 (92-kDa collagenase type IV), TIMP-1 and TIMP-2 were elevated in the majority of pancreatic-cancer tissue samples as compared to control pancreatic tissue. A good correlation was seen between overexpression of these MMPs and TIMPs and the steady-state levels of transcripts coding for extracellular matrix proteins, the amount of collagen protein and the severity of the desmoplastic reaction. In situ hybridization studies localized transcripts coding for collagens type I and III to spindle-shaped stromal cells, whereas transcripts for MMP-2, MMP-9, TIMP-1 and TIMP-2 were found in both stromal and tumor cells. However, MMP-2 transcripts appeared to be more abundant in stromal cells, TIMP-1 and TIMP-2 transcripts were evenly distributed over tumor and stromal cells and relatively more MMP-9 transcripts were found in tumor cells. We conclude that, in human pancreatic cancer, MMP-2, MMP-9, TIMP-1 and TIMP-2 may be involved in processes leading to the strong desmoplastic reaction observed in these tumors. Both stromal and tumor cells appear to be the source of MMPs and TIMPs in human pancreatic cancer.

Blotting, Northern↗
PubMed1995

Supramaximal secretagogue stimulation enhances heat shock protein expression in the rat pancreas.

Heat shock or stress proteins (HSPs) are synthesized by various cell types in response to different metabolic insults (e.g., hyperthermia). Although the function of HSPs is not fully understood, they are believed to be an evolutionary conserved intracellular defense mechanism. In an attempt to characterize the autoprotective potential of pancreatic acinar cells, we investigated the regulation of HSPs of the 70-kD family and the small HSP ubiquitin in vitro and in vivo during supramaximal cerulein stimulation. Infusion of the secretagogue cerulein induces a mild edematous form of pancreatitis in vivo and is characterized by a marked disturbance of the intracellular transport and segregation of enzymes. Synthesis of HSP70 mRNA is upregulated in isolated pancreatic lobules by either cerulein (100 nM) or hyperthermia (42 degrees C for 60 min). In contrast, expression of ubiquitin mRNA was not altered by either secretagogue treatment or hyperthermia. This heat shock-like response of pancreatic acinar cells could be reproduced in vivo: Pancreatitis was induced in male Wistar rats by intravenous infusion of supramaximal doses of cerulein (10 micrograms/kg/h). Analysis of mRNA expression revealed a significant upregulation of HSP70 RNA during supramaximal secretagogue stimulation. mRNA levels encoding for ubiquitin remained unchanged. Western blot analysis demonstrated that the transcriptional upregulation of HSP70 in vivo was reflected on the protein level. This study demonstrates that the marked intracellular disturbance observed in secretagogue-induced pancreatitis is associated with enhanced expression and synthesis of a major stress protein. Given the autoprotective potential of HSPs, this upregulation may indicate a self-defense mechanism of pancreatic acinar cells in experimental pancreatitis.

Animals↗
PubMed1994

Differential expression of heat shock proteins in pancreatic carcinoma.

In the present study we sought to determine by Northern blot analysis and mRNA in situ hybridization whether gene expression of heat shock proteins (HSPs) (HSP 89 alpha, HSP 89 beta, HSP 70, and ubiquitin) is altered in pancreatic carcinoma, compared to control tissues (normal pancreas and chronic pancreatitis tissue). HSP 89 alpha was selectively overexpressed in pancreatic carcinoma, and tumor cells were shown to contain the largest amount of HSP 89 alpha mRNA. Steady state levels of HSP 70 mRNA were increased in pancreatic carcinoma (tumor and connective tissue cells) and in chronic pancreatitis (connective tissue cells and residues of exocrine acinar cells). HSP 89 beta and ubiquitin B were constitutively expressed at high levels in pancreatic tissue from all three groups; HSP 89 beta mRNA was found in cells of parenchymal and stromal origin. A strong correlation was found between the expression of HSP 70 and the expression of transforming growth factor beta 1. The finding that HSPs are differentially expressed in pancreatic cancer, compared to normal pancreas and chronic pancreatitis tissue, and the cancer specificity of HSP 89 alpha suggest that HSPs play a specific role in the pathogenesis of pancreatic cancer, e.g., by participating in regulatory processes or in tumor immunity, as proposed previously.

Blotting, Northern↗