Ion channels are important in controlling cell cycle progression and proliferation in a variety of cell types. Using the whole-cell recording mode of the patch-clamp technique, functional ion channels were electrophysiologically characterized in PANC-1 (Kras G12D (ϩ/Ϫ), p53 R273C, ⌬p16), BxPC-3 (smad4 Ϫ

The present study compared the diagnostic accuracy of fluorine-18 2-deoxy-2-fluoro-D-glucose positron emission tomography (FDG-PET) with conventional staging techniques. The differentiation between malignant and benign lesions and the detection of multifocal disease, axillary and internal lymph node involvement, and distant metastases were evaluated. One hundred and seventeen female patients were prospectively examined using FDG-PET and conventional staging methods such as chest X-ray, ultrasonography of the breast and liver, mammography and bone scintigraphy. All patients were examined on a modern full-ring PET scanner. Histopathological analysis of resected specimens was employed as the reference method. The readers of FDG-PET were blinded to the results of the other imaging methods and to the site of the breast tumour. The sensitivity and specificity of FDG-PET in detecting malignant breast lesions were 93% and 75% respectively. FDG-PET was twofold more sensitive (sensitivity 63%, specificity 95%) in detecting multifocal lesions than the combination of mammography and ultrasonography (sensitivity 32%, specificity 93%). Sensitivity and specificity of FDG-PET in detecting axillary lymph node metastases were 79% and 92% (41% and 96% for clinical evaluation). FDG-PET correctly indicated distant metastases in seven patients. False-positive or false-negative findings were not encountered with FDG-PET. Chest X-ray was false-negative in three of five patients with lung metastases. Bone scintigraphy was false-positive in four patients. Three patients were upstaged since FDG-PET detected distant metastases missed with the standard staging procedure. It is concluded that, compared with the imaging methods currently employed for initial staging, FDG-PET is as accurate in interpreting the primary tumour and more accurate in screening for lymph node metastases and distant metastases. Due to a false-negative rate of 20% in detecting axillary lymph node metastases, FDG-PET cannot replace histological evaluation of axillary status.

Transition from a sessile epithelial phenotype to a migrating mesenchymal phenotype is a crucial step in transforming growth factor-B (TGF-B)-induced pancreatic cancer cell migration and invasion. These profound morphologic and functional alterations are associated with characteristic changes in TGF-B-regulated gene expression, defined by rapid repression of epithelial markers and a strong and sustained transcriptional induction of mesenchymal markers such as the intermediate filament vimentin. In this study, we have analyzed the role of the transcription factor Sp1 in TGF-B-induced and Smad-mediated gene regulation during epithelial to mesenchymal transition (EMT) and migration of pancreatic cancer cells. Here, we show that Sp1 is required for TGF-B-induced EMT, and that this function is especially mediated through transcriptional induction of vimentin. Our results emphasize the functional relevance of vimentin in TGF-B-induced EMT because prevention of its induction strongly reduces cell migration. Altogether, this study helps to better understand the role of Sp1 in TGF-B-induced progression of pancreatic cancer. It suggests that Sp1, via transcriptional induction of vimentin, cooperates with activated Smad complexes in mesenchymal transition and migration of pancreatic cancer cells upon TGF-B stimulation.

No abstract

The transcription factor Sp1 has been implicated in cell-type-specific activation of transforming growth factor-beta (TGFbeta) target genes in normal epithelial cells as well as in aberrant gene activation by TGFbeta in epithelial tumor cells. Here, we have examined the interaction of Sp1 with components of the Smad signaling cascade and its role in TGFbeta-induced early gene expression in pancreatic cancer cells. Gene expression profiling was carried out in mithramycin-A-treated cells to identify Sp1-regulated TGFbeta early response genes. We found that in pancreatic cancer cells Smad proteins and Sp1 cooperatively regulate expression of a distinct set of TGFbeta target genes potentially involved in tumor progression, including MMP-11, cyclin D1 and Smad7. Mechanistically, TGFbeta rapidly induces nuclear translocation of Smad proteins and subsequently stimulates Smad-Sp1 complex formation. Using the Smad7 promoter as a model for Smad-/Sp1-induced early gene activation, we demonstrated that this interaction increases Sp1 binding to GC-rich promoter boxes and results in superinduction of Sp1-mediated transcription. Moreover, inhibition of Sp1-DNA binding or transfection of Sp1-specific siRNA prevents TGFbeta-induced Smad7 expression and consequently enhances Smad signaling in pancreatic cancer cells, as indicated by increased receptor-mediated phosphorylation of Smad3. We thus conclude that Sp1 strongly contributes to the aberrant transcriptional response of transformed epithelial cells to TGFbeta stimulation.

c-myc promoter silencing is a key step in epithelial cell growth inhibition by transforming growth factor B (TGFB). During carcinogenesis, however, epithelial cells escape from c-myc repression and consequently become refractory to TGFB-mediated antiproliferation. Here, we assessed the role of the repressor, KLF11, in TGFB-induced growth inhibition in normal epithelial as well as pancreatic carcinoma cells. Endogenous KLF11 was stably down-regulated by RNA interference technology, and the functional consequences were studied by proliferation assays, reporter assays, DNA binding studies, and expression analyses. Coimmunoprecipitation and glutathione S-transferase pulldown assays were conducted to define KLF11-Smad3 interaction and U0126 was administered to examine the effects of the extracellular signal-regulated kinase (ERK) -mitogen-activated protein kinase on complex formation and c-myc promoter binding of KLF11 and Smad3 in pancreatic cancer cells. In TGFB-stimulated normal epithelial cells, nuclear KLF11, in concert with Smad3, binds to and represses transcription from the core region of the TGFB-inhibitory element (TIE) of the c-myc promoter. Disruption of KLF11-Smad3 interaction or small interfering RNA -mediated knockdown of endogenous KLF11 strongly diminishes Smad3-TIE promoter binding and repression, and consequently impairs TGFB-mediated growth inhibition. In pancreatic cancer cells with oncogenic Ras mutations, hyperactive ERK counteracts TGFB-induced c-myc repression and growth inhibition through at least two mechanisms, i.e., via disruption of KLF11-Smad3 complex formation and through inhibition of KLF11-Smad3 binding to the TIE element. Together, these results suggest a central role for KLF11 in TGFB-induced c-myc repression and antiproliferation and identifies a novel mechanism through which ERK signaling antagonizes the tumor suppressor activities of TGFB in pancreatic cancer cells with oncogenic Ras

The aggregation process of beta-amyloid peptide Abeta into amyloid is strongly associated with the pathology of Alzheimer's disease (AD). Aggregation may involve a transition of an alpha helix in Abeta(1-28) into beta sheets and interactions between residues 18-20 of the "Abeta amyloid core." We applied an i, i+4 cyclic conformational constraint to the Abeta amyloid core and devised side chain-to-side chain lactam-bridged cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28). In contrast to Abeta(1-28) and [Lys(17), Asp(21)]Abeta(1-28), cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) was not able to form beta sheets and cytotoxic amyloid aggregates. Cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) was able to interact with Abeta(1-28) and to inhibit amyloid formation and cytotoxicity. Cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) also interacted with Abeta(1-40) and interfered with its amyloidogenesis. Cyclo(17, 21)-[Lys(17), Asp(21)]Abeta(1-28) or similarly constrained Abeta sequences may find therapeutic and diagnostic applications in AD.

Expression profiling analyses were used to elucidate the functional relevance of RAS proteins in mediating the effect of TGFB1 on the transcriptional phenotype of the pancreatic cancer cell line PANC-1. Despite the presence of one mutated KRAS2 allele in parental PANC-1 pancreatic cancer cells, RAS-dependent signal transduction remained susceptible to stimulation by EGF and TGFB1. To analyze the impact of RAS proteins on the TGFB1-induced transcriptional phenotype, we used PANC-1 cells stably transfected with a dominant negative HRAS(S17N) mutant or with a constitutively active KRAS2(G12V) mutant. TGFB1 treatment of mock-transfected PANC-1 cells led to an expression profile suggestive of epithelial-mesenchymal transdifferentiation (EMT). Profiling of the HRAS(S17N)-expressing clone demonstrated that induction of endogenous RAS activity by TGFB1 is required for the development of the TGFB1-induced transcriptional phenotype of PANC-1 cells. The expression of the KRAS2(G12V) mutant by itself repressed transcription of markers of epithelial differentiation and induced transcription of several extracellular matrix-associated genes. This effect was not enhanced further by TGFB1 treatment. In contrast, transcript levels of genes associated with proliferation and cell cycle progression did not appear to be the primary targets of the synergism between the RAS- and TGFB1-dependent cascades. The introduction of the dominant negative and the constitutively active RAS mutants induced partly overlapping and partly inverse effects on the TGFB1-induced expression profile of PANC-1 cells. Additional mechanisms such as the induction of autocrine loops and the use of different RAS isoforms or alternate, ERK-independent signaling pathways may be involved in the interaction between the RAS- and the TGFB1-dependent signaling cascades.