[Pathogenesis of pancreatic carcinoma].
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Animals↗
Biomedical subjects
R M Schmid
Publications and source records attributed to R M Schmid.
Nuclear factor-kappaB is constitutively active in C-cell carcinoma and required for RET-induced transformation.
Specific point mutations of the RET proto-oncogene have been demonstrated to be responsible for multiple endocrine neoplasia (MEN) types 2A and 2B, for familial medullary thyroid carcinoma (MTC) syndromes, as well as for sporadic MTC. Here we show that nuclear factor (NF)-kappaB is activated in RET-associated C-cell carcinoma specimens. TT cells, a human MTC cell line expressing MEN 2A type RET, display transcriptionally active RelA(p65) in the nucleus. NF-kappaB activity in these cells is attributable to constitutive IkappaB kinase (IKK) activity and high turn over of IkappaBalpha. RET harboring the mutations C634R (MEN 2A) or M918T (MEN 2B), in contrast to wild-type RET, activates a NF-kappaB-dependent reporter construct upon transient transfection in HeLa cells. We show that the prototype RET mutation C634R enhances phosphorylation of IkappaBalpha by IKKbeta but not by IKKalpha. RET-induced NF-kappaB and IKKbeta activity requires Ras function but does neither involve the classical mitogen-activated protein kinase kinase/extracellular signal-regulated kinase nor the phosphoinositide 3-kinase/Akt pathways. In contrast, RET-induced NF-kappaB activity is dependent on Raf and MEKK1. Inhibition of constitutive NF-kappaB activity results in cell death of TT cells and blocks focus formation induced by oncogenic forms of RET in NIH 3T3 cells. These results suggest that RET-mediated carcinogenesis critically depends on IKK activity and subsequent NF-kappaB activation.
3T3 Cells↗
In vivo evaluation of 5-[(18)F]fluoro-2'-deoxyuridine as tracer for positron emission tomography in a murine pancreatic cancer model.
We used a murine tumor progression model for the evaluation of potential proliferation markers using positron emission tomography (PET). 5-[(18)F]-2'-deoxyuridine ([(18)F]FdUrd) was synthesized with >98% radiochemical purity and investigated in a pancreatic cancer model, transforming growth factor alpha transgenic mice crossbred to p53 deficient mice. Thymidylate synthase was increased already in premalignant lesions, whereas thymidine kinase 1 mRNA levels were up-regulated 4-fold in the pancreatic cancer specimen of these mice. PET imaging was performed after injection of 1 MBq of [(18)F]FdUrd and 1 MBq of [(18)F]fluoro-deoxyglucose. Animals with pancreatic cancer displayed focal uptake of both tracers. The [(18)F]FdUrd uptake ratio closely correlated with the proliferation index as evaluated in morphometric and fluorescence-activated cell sorter analysis. These results indicate the potential of our tumor model for the evaluation of PET tracers and suggest [(18)F]FdUrd as a tracer for the assessment of proliferation in vivo.
Animals↗
A murine tumor progression model for pancreatic cancer recapitulating the genetic alterations of the human disease.
This study describes a tumor progression model for ductal pancreatic cancer in mice overexpressing TGF-alpha. Activation of Ras and Erk causes induction of cyclin D1-Cdk4 without increase of cyclin E or PCNA in ductal lesions. Thus, TGF-alpha is able to promote progression throughout G1, but not S phase. Crossbreeding with p53 null mice accelerates tumor development in TGF-alpha transgenic mice dramatically. In tumors developing in these mice, biallelic deletion of Ink4a/Arf or LOH of the Smad4 locus is found suggesting that loci in addition to p53 are involved in antitumor activities. We conclude that these genetic events are critical for pancreatic tumor formation in mice. This model recapitulates pathomorphological features and genetic alterations of the human disease.
Animals↗
Inhibition of nitric oxide synthesis by aminoguanidine increases intestinal damage in the acute phase of rat TNB-colitis.
The role of nitric oxide (NO) in the pathophysiology of inflammatory bowel diseases (IBD) is controversially discussed. The aim of the present study was to investigate the role of NO inhibition in the acute phase of rat 2,4,6-trinitrobenzenesulphonic acid (TNB)-colitis. To inhibit NO synthesis we used aminoguanidine (AG) as a selective inhibitor of inducible nitric oxide synthase (iNOS). TNB-colitis was induced in rats with and without pretreatment with AG (200 mg kg-1 body weight in the drinking water). The severity of colitis was observed over a period of 7 days. On days 1 and 2, AG reduced concentrations of plasma nitrate and nitrite as well as of portal 6-keto-prostaglandin 1alpha. AG pretreatment increased colonic damage and inflammatory response, assessed by colonic myeloperoxidase and serum lactate dehydrogenase activity, macroscopic damage score, tumour necrosis factor-alpha concentration in stool and colonic glutathione content. The AG-treated group showed a higher and prolonged nuclear factor kappaB (NF-kappaB)/Rel binding activity in the colon. We conclude that NOS inhibition by AG is not beneficial in acute intestinal inflammation. With regard to appropriate therapeutic strategies, NF-kappaB/Rel activation might be a more suitable target.
Acute Disease↗
Oxidized low-density lipoproteins bind to the scavenger receptor, CD36, of hepatic stellate cells and stimulate extracellular matrix synthesis.
Cumulating evidence suggests that oxidative stress resulting in lipid peroxidation and protein modification is involved in the pathogenesis of chronic liver injury and fibrogenesis. We investigated the effects of oxidized low-density lipoproteins (oxLDL) on collagen and fibronectin synthesis of cultured human and rat hepatic stellate cells (HSC). As shown on protein and mRNA levels, oxLDL dose-dependently stimulated the synthesis of collagen types I and III and fibronectin of cultured HSC. The effect was biphasic, with a maximum between 5 and 25 microg/mL oxLDL (c-fibronectin concentration in HSC supernatants increased 3.9-fold; collagen type I increased 4-fold). Higher oxLDL concentrations were cytotoxic. LDL modified with malondialdehyde (MDA) was not toxic, but stimulated extracellular matrix synthesis as well. As demonstrated by immunofluorescence microscopy (double staining of CD36 and iso-alpha-smooth muscle actin [iso-alpha-sm actin]), immunoblot, and reverse-transcription polymerase chain reaction (RT-PCR), respectively, cultured human HSC express the oxLDL receptor, CD36 (glycoprotein IIIb). Colocalization of CD36 and iso-alpha-sm actin on sinusoidal lining cells was further demonstrated using sections of human fibrotic liver. Preincubation of cultured human HSC with the monoclonal antibody, OKM5, known to block CD36-mediated oxLDL uptake, resulted in a reduction of the oxLDL-stimulated collagen type I synthesis by 56%. In summary, our results demonstrate that low concentrations of modified lipoproteins (oxLDL and MDA-LDL) represent fibrogenic mediators that bind to CD36 and stimulate matrix synthesis of HSC.
Blotting, Northern↗
p300 acts as a transcriptional coactivator for mammalian Notch-1.
Notch-1 belongs to a family of transmembrane receptor proteins that direct the decisions as to various cell fates. After ligand binding, a proteolytic cleavage step occurs and the intracellular part of Notch-1, Notch-1-IC, translocates into the nucleus, where it targets the DNA binding protein RBP-J kappa/CBF1. RBP-J kappa mediates repression through recruitment of a histone deacetylase-containing complex. The Notch-1-IC/RBP-J kappa complex overcomes repression and activates the transcription of Notch target genes. We have identified a novel domain in Notch-1-IC, the EP domain, which is indispensable for full transcriptional activation. This transactivation domain is localized adjacent to the ankyrin repeats of Notch-1-IC. In cotransfection experiments, Notch-1-IC-mediated transcriptional activation was inhibited by E1A12S and p53, two proteins, which interfere with the function of the common coactivator p300. Protein-protein interaction assays demonstrated the association of Notch-1-IC and the CH3 region of p300. In addition, the interaction of mammalian Notch-1-IC with p300 was destabilized after deletion of the EP domain of Notch-1-IC. Based on physical interaction with Notch-1-IC and coactivator functions of p300, we propose a model for Notch-1-mediated gene regulation via p300.
Adenovirus E1A Proteins↗
Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells.
The aim of this study was to identify fibrogenic mediators stimulating activation, proliferation, and/or matrix synthesis of rat pancreatic stellate cells (PSC). PSC were isolated from the pancreas of normal Wistar rats and from rats with cerulein pancreatitis. Cell activation was demonstrated by immunofluorescence microscopy of smooth muscle alpha-actin (SMA) and real-time quantitative RT-PCR of SMA, fibronectin, and transforming growth factor (TGF)-beta(1). Proliferation was measured by bromodeoxyuridine incorporation. Matrix synthesis was demonstrated on the protein and mRNA level. Within a few days in primary culture, PSC changed their phenotype from fat-storing to SMA-positive myofibroblast-like cells expressing platelet-derived growth factor (PDGF) alpha- and PDGF beta-receptors. TGF-beta(1) and tumor necrosis factor (TNF)-alpha accelerated the change in the cells' phenotype. Addition of 50 ng/ml PDGF and 5 ng/ml basic fibroblast growth factor (bFGF) to cultured PSC significantly stimulated cell proliferation (4.37 +/- 0.49- and 2.96 +/- 0.39-fold of control). Fibronectin synthesis calculated on the basis of DNA was stimulated by 5 ng/ml bFGF (3.44 +/- 1.13-fold), 5 ng/ml TGF-beta(1) (2.46 +/- 0.89-fold), 20 ng/ml PDGF (2.27 +/- 0.68-fold), and 50 ng/ml TGF-alpha (1.87 +/- 0.19-fold). As shown by RT-PCR, PSC express predominantly the splice variant EIII-A of fibronectin. Immunofluorescence microscopy and Northern blot confirmed that in particular bFGF and TGF-beta(1) stimulated the synthesis of fibronectin and collagens type I and III. In conclusion, our data demonstrate that 1) TGF-beta(1) and TNF-alpha accelerate the change in the cell phenotype, 2) PDGF represents the most effective mitogen, and 3) bFGF, TGF-beta(1), PDGF, and, to a lesser extent, TGF-alpha stimulate extracellular matrix synthesis of cultured rat PSC.
Animals↗
Differentiation of chronic pancreatitis from pancreatic cancer: recent advances in molecular diagnosis.
Chronic pancreatitis is an inflammatory disease of the pancreas, characterized by a progressive destruction of the exocrine and endocrine pancreas, leading both to exocrine and endocrine insufficiency. In recent years, our knowledge of this disease has improved, an epidemiological link between chronic pancreatitis and pancreatic cancer has been established, and the molecular alterations underlying their pathogenesis have been partly revealed. Nevertheless, the differentiation of chronic inflammation of the pancreas from cancer of the pancreas remains a great challenge. This overview will point out the present knowledge of the molecular pathogenesis of chronic pancreatitis and pancreatic cancer and will focus on the role of molecular markers for differentiating chronic pancreatitis from pancreatic cancer.
Animals↗
TGF alpha transgenic mice. A model of pancreatic cancer development.
Pancreatic cancer is a devastating disease with a fatal prognosis due to late diagnosis and resistance to radiation and chemotherapy. The average survival after diagnosis is still 3 to 8 months. In the last few years genetic alterations in cancer-causing genes have been identified in tumors and putative premalignant lesions using microdissection techniques. However, the functional consequence of these genetic alterations for pancreatic growth and differentiation is unknown. TGF alpha overexpressed in the pancreas causes the development of tubular structures and fibrosis. Mice older than one year develop ductal pancreatic cancer. Crossbreeding these mice with p53 knockout mice dramatically accelerated tumor development. Moreover, tumors developing in these mice show frequently biallelic deletion of the Ink4a locus or LOH of SMAD4. These mice represent the first model of pancreatic adenocarcinomas with genetic alterations as well as growth characteristics similar to the human disease.
Animals↗
Antiapoptotic bcl-2 and bcl-xL in advanced malignant melanoma.
Apoptosis is an important cofactor in the pathogenesis of a plethora of malignancies. However, little is known about modulation of the expression of bcl gene family in melanocytic tumors. To determine the role of bcl-2, bcl-x and bax in melanocytic tumors we investigated the differential expression of these genes via RT-PCR in tissue samples from human benign nevi, primary melanomas and melanoma metastases in comparison with normal skin. Bcl-2 was strongly expressed in 14/16 metastases (87.5%), whereas only 7/13 primary melanomas (53%), 7/15 nevi (46%) and 7/16 normal tissue samples (43%) showed expression of bcl-2 (P < 0.05). There was a strong indication of a correlation between tumor thickness and bcl-2 expression in nodular malignant melanomas. Expression of bcl-x was found in 16/16 melanoma metastases (100%), 11/13 primary melanomas (84%), 12/15 nevi (80%) and 10/16 normal tissue samples (62%) (P < 0.05). Bcl-xL expression increased from primary melanoma to melanoma metastases, whereas bcl-xS showed a decreasing expression level during melanoma progression. No differences in bax expression were seen between melanoma metastases, primary melanoma, nevi and normal tissue. Immunohistochemical investigations of another 53 tissue samples showed similar results. Our results strongly indicate that bcl-2 and bcl-xL gene expression increases with progression of malignant melanoma. Bcl-2 and bcl-xL expression could reflect an increased malignant potential caused by an inhibition of apoptosis and growth advantage for metastatic melanoma cells.
Apoptosis↗
NF-kappaB/rel/IkappaB: implications in gastrointestinal diseases.
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Animals↗
Disturbed bile secretion and cytochrome P450 function during the acute state of experimental colitis in rats.
BACKGROUND/AIMS: A variety of hepatobiliary abnormalities has been described in patients with inflammatory bowel diseases. However, the pathophysiological mechanisms leading to these liver alterations are poorly understood. The aim of the present study was to investigate parameters of liver function in a trinitrobenzenesulfonic acid (TNB)-induced rat colitis model. METHODS: Glucose output, bile acid secretion, bile acid uptake, and the cytochrome P-450 metabolic capacity during TNB-colitis were studied in the perfused liver model. Furthermore, hepatic bile acid- and glycogen content was measured. To evaluate the inflammatory response in the colon and liver, NF-kappaB/Rel was quantified by electrophoretic mobility shift assays. As an NF-kappaB/Rel regulated gene the inducible NO-synthase (NOS2) was evaluated by Western blot analysis. As possible mediators released from the inflamed colon into the portal vein, endotoxin and the stable metabolite of prostaglandin I2 (6-keto-prostaglandin-F1alpha) were determined. RESULTS: Glucose output, bile acid secretion, bile acid uptake, and cytochrome P-450 metabolic capacity decreased on the first and second day of TNB-colitis. Hepatic bile acid content increased at day 14 of colitis. Glycogen content was reduced, most likely due to an inadequate chow intake of these animals. A low level of portal endotoxin was detectable during the first 2 days of colitis. In addition, 6-keto-prostaglandin-F1alpha was clearly increased in portal blood. NF-kappaB/Rel binding activity and inducible NOS2 were strongly positive in the colon during colitis. Although low levels of portal endotoxin were measured during the first 2 days of colitis, no significant NF-kappaB/Rel activity and NOS2 induction were detected in the liver. CONCLUSION: Our results indicate that during the acute state of the TNB-colitis, bile acid secretion and cytochrome P-450 function are disturbed in the absence of distinct inflammatory changes in the liver.
Animals↗
Helicobacter pylori in the oral cavity: high prevalence and great DNA diversity.
To test the hypothesis that Helicobacter pylori may be transmitted by the oral-oral route, we applied nested PCR and DNA sequencing to detect and analyze H. pylori DNA in the oral cavity of 20 adult patients undergoing endoscopy. Dental plaques of molars, premolars, and incisors and saliva were collected. Additional paraffin-embedded gastric biopsies were analyzed in four patients. Two sets of highly sensitive and specific primers, EHC-U/EHC-L and ET5-U/ET-5L directed to a 860-bp fragment of H. pylori DNA, were used in the nested PCR. Eight patients had an active infection in the stomach determined with the [13C]urea breath test and the other 12 were negative. Nested PCR showed that all 20 subjects (100%) were positive for H. pylori in the oral cavity. DNA sequencing demonstrated that all tested PCR products of the expected size from the oral samples have more than 97% identity with that from H. pylori type strain ATCC 43629. However, sequences differed in oral samples from different subjects as well as between different oral locations and gastric biopsies within the same individuals. In conclusion, the oral cavity may be a permanent reservoir for H. pylori and can harbor multiple H. pylori strains at the same time.
Adult↗
Suppression of NF-kappaB activity by sulfasalazine is mediated by direct inhibition of IkappaB kinases alpha and beta.
BACKGROUND & AIMS: Activation of NF-kappaB/Rel has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Various drugs used in the treatment of IBD, such as glucocorticoids, 5-aminosalicylic acid, and sulfasalazine, interfere with NF-kappaB/Rel signaling. The aim of this study was to define the molecular mechanism by which sulfasalazine inhibits NF-kappaB activation. METHODS: The effects of sulfasalazine and its moieties on NF-kappaB signaling were evaluated using electromobility shift, transfection, and immune complex kinase assays. The direct effect of sulfasalazine on IkappaB kinase (IKK) activity was investigated using purified recombinant IKK-alpha and -beta proteins. RESULTS: NF-kappaB/Rel activity induced by tumor necrosis factor alpha, 12-O-tetradecanoylphorbol-13-acetate, or overexpression of NF-kappaB-inducing kinase, IKK-alpha, IKK-beta, or constitutively active IKK-alpha and IKK-beta mutants was inhibited dose dependently by sulfasalazine. Sulfasalazine inhibited tumor necrosis factor alpha-induced activation of endogenous IKK in Jurkat T cells and SW620 colon cells, as well as the catalytic activity of purified IKK-alpha and IKK-beta in vitro. In contrast, the moieties of sulfasalazine, 5-aminosalicylic acid, and sulfapyridine or 4-aminosalicylic acid had no effect. Activation of extracellular signal-related kinase (ERK) 1 and 2, c-Jun-N-terminal kinase (JNK) 1, and p38 was unaffected by sulfasalazine. The decrease in substrate phosphorylation by IKK-alpha and -beta is associated with a decrease in autophosphorylation of IKKs and can be antagonized by excess adenosine triphosphate. CONCLUSIONS: These data identify sulfasalazine as a direct inhibitor of IKK-alpha and -beta by antagonizing adenosine triphosphate binding. The suppression of NF-kappaB activation by inhibition of the IKKs contributes to the well-known anti-inflammatory and immunosuppressive effects of sulfasalazine.
Adenosine Triphosphate↗
Assignment of the mouse Rbpsuh gene to chromosome 5 and one processed pseudogene Rbpsuh-rs3 to chromosome 6.
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Animals↗
[Colorectal carcinomas in Crohn disease].
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Carcinoma↗
Regulation of cell growth and cyclin D1 expression by the constitutively active FRAP-p70s6K pathway in human pancreatic cancer cells.
The FRAP-p70s6K signaling pathway was found to be constitutively phosphorylated/active in MiaPaCa-2 and Panc-1 human pancreatic cancer cells and a pancreatic cancer tissue sample as judged by the retarded electrophoretic mobility of the two major FRAP downstream targets, p70s6K and 4E-BP1. Treatment of cells with rapamycin, a selective FRAP Inhibitor, inhibited basal p70s6K kinase activity and induced dephosphorylation of p70s6K and 4E-BP1. Moreover, rapamycin inhibited DNA synthesis as well as anchorage-dependent and -independent proliferation in MiaPaCa-2 and Panc-1 cells. Finally, rapamycin strikingly inhibited cyclin D1 expression in pancreatic cancer cells. Thus, inhibitors of the constitutively active FRAP-p70s6K pathway may provide a novel therapeutic approach for pancreatic cancer.
Calcium-Calmodulin-Dependent Protein Kinases↗