VEGF-D promotes tumor growth and lymphatic spread in a mouse model of hepatocellular carcinoma.

Lymphatic spread is an important clinical determinant for the prognosis of hepatocellular carcinoma (HCC), but little is known about the control of lymphangiogenesis in HCC. We addressed expression and biological role of the pro-(lymph), angiogenic protein VEGF-D in this tumor entity. Using immunohistochemistry and in situ hybridization on specimens of HCC, cirrhotic and normal liver we found abundant expression of VEGF-D exclusively in the tumor cells. The cognate receptor VEGFR-3 was detected on blood and lymphatic vessels. By clinicopathological analysis VEGF-D expression was correlated with pT-stage of the primary, lymph node metastasis and lymphangiosis carcinomatosa. Three out of 4 human HCC cell lines expressed and secreted VEGF-D. To approach its biological function, VEGF-D deficient SKHep-1 cells were stably transfected with VEGF-D cDNA and effects on tumor progression were determined in vivo. Compared to mock-transfected controls, subcutaneous tumors derived from VEGF-D expressing cells were larger and more frequently metastasized to regional lymph nodes. VEGF-D expressing tumors exhibited increased microvessel density and increased abundance of peri- and intratumoral lymphatics, as assessed by immunostaining for CD31 and for LYVE-1 and/or podoplanin, respectively. Furthermore, coexpression of the soluble extracellular VEGFR-3 domain blocked VEGF-D-induced tumor growth and lymphatic spread via reduction of angiogenesis and lymphangiogenesis. In the orthotopic approach, VEGF-D expression resulted in an increased rate of intra- and extrahepatic as well as lymph node metastasis. In conclusion, our study suggests that expression of VEGF-D is involved in growth and lymphatic spread of HCC. Therefore, VEGF-D might represent a therapeutic target in HCC.

Tumor suppressor p16INK4a--modulator of glycomic profile and galectin-1 expression to increase susceptibility to carbohydrate-dependent induction of anoikis in pancreatic carcinoma cells.

Expression of the tumor suppressor p16(INK4a) after stable transfection can restore the susceptibility of epithelial tumor cells to anoikis. This property is linked to increases in the expression and cell-surface presence of the fibronectin receptor. Considering its glycan chains as pivotal signals, we assumed an effect of p16(INK4a) on glycosylation. To test this hypothesis for human Capan-1 pancreatic carcinoma cells, we combined microarray for selected glycosyltransferase genes with 2D chromatographic glycan profiling and plant lectin binding. Major differences between p16-positive and control cells were detected. They concerned expression of beta1,4-galactosyltransferases (down-regulation of beta1,4-galactosyltransferases-I/V and up-regulation of beta1,4-galactosyltransferase-IV) as well as decreased alpha2,3-sialylation of O-glycans and alpha2,6-sialylation of N-glycans. The changes are compatible with increased beta(1)-integrin maturation, subunit assembly and binding activity of the alpha(5)beta(1)-integrin. Of further functional relevance in line with our hypothesis, we revealed differential reactivity towards endogenous lectins, especially galectin-1. As a result of reduced sialylation, the cells' capacity to bind galectin-1 was enhanced. In parallel, the level of transcription of the galectin-1 gene increased conspicuously in p16(INK4a)-positive cells, and even figured prominently in a microarray on 1996 tumor-associated genes and in proteomic analysis. The cells therefore gain optimal responsiveness. The correlation between genetically modulated galectin-1 levels and anoikis rates in engineered transfectants inferred functional significance. To connect these findings to the fibronectin receptor, galectin-1 was shown to be co-immunoprecipitated. We conclude that p16(INK4a) orchestrates distinct aspects of glycosylation that are relevant for integrin maturation and reactivity to an endogenous effector as well as the effector's expression. This mechanism establishes a new aspect of p16(INK4a) functionality.

Galectin-1 interacts with the {alpha}5{beta}1 fibronectin receptor to restrict carcinoma cell growth via induction of p21 and p27.

Surface binding of galectin family members has the potential to link distinct glycan structures to growth regulation. Therefore, we addressed the antiproliferative potential of galectin-1 (Gal-1) in a panel of carcinoma cell lines. We discovered growth inhibition by Gal-1 in epithelial tumor cell lines from different origins and provide evidence that this effect requires functional interaction with the alpha5beta1 integrin. Antiproliferative effects result from inhibition of the Ras-MEK-ERK pathway and consecutive transcriptional induction of p27. We have further identified two Sp1-binding sites in the p27 promoter as crucial for Gal-1 responsiveness. Inhibition of the Ras-MEK-ERK cascade by Gal-1 increased Sp1 transactivation and DNA binding due to reduced threonine phosphorylation of Sp1. Furthermore, Gal-1 induced p21 transcription and selectively increased p27 protein stability. Gal-1-mediated accumulation of p27 and p21 inhibited cyclin-dependent kinase 2 activity and ultimately resulted in G(1) cell cycle arrest and growth inhibition. These data define a novel mechanism whereby Gal-1 regulates epithelial tumor cell homeostasis via carbohydrate-dependent interaction with the alpha5beta1 integrin.

Vascular endothelial growth factor-D induces lymphangiogenesis and lymphatic metastasis in models of ductal pancreatic cancer.

The presence of lymphatic metastases is a strong indicator for poor prognosis in patients with ductal pancreatic cancer. In order to better understand the mechanisms controlling lymphatic growth and lymph node metastasis in human ductal pancreatic cancer, we analyzed the expression pattern of the vascular endothelial growth factor-D (VEGF-D), its receptor VEGF-receptor-3 (VEGFR-3) and the lymphatic endothelium-specific hyaluronan receptor LYVE-1 in a panel of 19 primary human ductal pancreatic tumors and 10 normal pancreas specimens. We further addressed the biological function of VEGF-D for induction of lymphatic metastasis in a nude mouse xenograft model using two human ductal pancreatic cancer cell lines with overexpression of VEGF-D. Compared to normal human pancreas, pancreatic cancer tissue showed overexpression of VEGF-D and VEGFR-3 in conjunction with a high lymphatic vascularization as determined by immunohistochemistry and in situ hybridization. Tumors derived from VEGF-D-overexpressing cells had a higher microvessel density compared to their mock-controls, as determined based on CD31 immunohistochemistry. Importantly, these tumors also revealed a significant induction of intra- and peritumoral lymphatics, as judged from immunohistochemical detection of LYVE-1 expression. This was associated with a significant increase in lymphatic vessel invasion by tumor cells and an increased rate of lymphatic metastases, as indicated by pan-cytokeratin reactive cells in lymph nodes. Our results suggest that VEGF-D plays a pivotal role in stimulating lymphangiogenesis and lymphatic metastasis in human ductal pancreatic cancer, and therefore represents a novel therapeutic target for this devastating disease.

Evaluation of gas-filled microparticles and sonoporation as gene delivery system: feasibility study in rodent tumor models.

PURPOSE: To evaluate the feasibility of gene delivery mediated with diagnostic ultrasound and plasmid DNA (pDNA) encapsulated in gas-filled microparticles (GFMP) in rodent tumor models. MATERIALS AND METHODS: This study was performed according to a protocol approved by the regional animal research committee. The model plasmid UT651 (pUT651) that contained the Escherichia coli LacZ gene for beta-galactosidase was used to demonstrate the feasibility of ultrasound-mediated gene delivery in CC531 liver tumors in rats. In preliminary experiments, a single injection of pUT651-containing GFMP was administered intraarterially (n=4) or intravenously (n=6) with simultaneous sonication (color Doppler mode, maximum mechanical index) of the GFMP passing through the capillaries of the tumors. All animals were sacrificed 2-5 days later, and liver tumors were examined for beta-galactosidase expression histochemically. Subsequently, potential medical usefulness of this delivery system was tested in nude mice bearing Capan-1 tumors (adenocarcinoma of the human pancreas) by using the plasmid RC/CMV-p16 (pRC/CMV-p16), which contains tumor suppressor gene p16. The tumor suppressor gene p16 is deleted in Capan-1 cells. Twenty-five tumor-bearing mice were classified into five groups (four to six mice per group, one treatment group, four control groups) at random. All mice were treated once weekly for 5 weeks with intravenous infusion of p16-containing GFMP or control substances with simultaneous tumor sonication with color Doppler mode ultrasound and maximum mechanical index or without ultrasound treatment. The therapeutic effect of p16 was measured as an increase in tumor volume doubling time. Data were analyzed with analysis of variance. Results were considered significant at the 5% critical level (P < .05). RESULTS: A clear expression of pDNA was found in tumors in rats treated with a combination of pUT651-containing GFMP and ultrasound; relevant controls showed a significantly lower expression of marker gene. The controlled ultrasound-triggered release of pRC/CMV-p16 from GFMP leads to a strong tumor growth inhibition, which is significant (P < .002), compared with that in controls. CONCLUSION: A combination of GFMP and ultrasound provides an effective approach for nonviral gene therapy-based cancer treatment.

Interferon-alpha: regulatory effects on cell cycle and angiogenesis.

In the current study, we investigated the effects of interferon-alpha (IFN-alpha) on proliferation and angiogenesis in neuroendocrine tumor disease. Using a panel of human neuroendocrine tumor cell lines, we confirmed functionally active IFN-alpha signaling by STAT activation and nuclear translocation as well as transactivation. IFN-alpha results in anchorage-dependent and -independent growth inhibition due to a delayed progression from S-phase to G2 phase of the cell cycle. This was due to substantial reduction in cellular cyclin B levels resulting in the inhibition of Cdc2 kinase activity. In parallel to growth inhibition, we observed a profound inhibition of VEGF gene transcription by IFN-alpha in human neuroendocrine tumor cells due to an Sp1/Sp3-dependent inhibition of VEGF promoter activity. Treatment of neuroendocrine tumors with IFN-alpha in nude mice resulted in growth inhibition and inhibition of angiogenesis. Furthermore, treatment of neuroendocrine tumor patients with IFN-alpha resulted in decreased VEGF expression as well as tumor angiogenesis in liver metastases. In summary, IFN-alpha acts via direct antiproliferative effects as well as inhibition of tumor angiogenesis mediated by suppression of VEGF gene expression in neuroendocrine tumor disease.

Human galectin-2: novel inducer of T cell apoptosis with distinct profile of caspase activation.

Galectin-2 is structurally closely related to galectin-1, but has a distinct expression profile primarily confined to the gastrointestinal tract. Prominent differences in the proximal promoter regions between galectins-2 and -1 concern Sp1-, hepatocyte NF-3, and T cell-specific factor-1 binding sites. Of note, these sequence elements are positioned equally in the respective regions for human and rat galectins-2. Labeled galectin-2 binds to T cells in a beta-galactoside-specific manner. In contrast to galectin-1, the glycoproteins CD3 and CD7 are not ligands, while the shared affinity to beta1 integrin (or a closely associated glycoprotein) accounts for a substantial extent of cell surface binding. The carbohydrate-dependent binding of galectin-2 induces apoptosis in activated T cells. Fluorogenic substrate and inhibitor assays reveal involvement of caspases-3 and -9, in accordance with cleavage of the DNA fragmentation factor. Enhanced cytochrome c release, disruption of the mitochondrial membrane potential, and an increase of the Bax/Bcl-2 ratio by opposite regulation of expression of both proteins add to the evidence that the intrinsic apoptotic pathway is triggered. Cell cycle distribution and expression of regulatory proteins remained unaffected. Notably, galectins-1 and -7 reduce cyclin B1 expression, defining functional differences between the structurally closely related galectins. Cytokine secretion of activated T cells was significantly shifted to the Th2 profile. Our study thus classifies galectin-2 as proapoptotic effector for activated T cells, raising a therapeutic perspective. Of importance for understanding the complex galectin network, it teaches the lesson that selection of cell surface ligands, route of signaling, and effects on regulators of cell cycle progression are markedly different between structurally closely related galectins.

Inhibition of ghrelin action in vitro and in vivo by an RNA-Spiegelmer.

Employing in vitro selection techniques, we have generated biostable RNA-based compounds, so-called Spiegelmers, that specifically bind n-octanoyl ghrelin, the recently discovered endogenous ligand for the type 1a growth hormone secretagogue (GHS) receptor. Ghrelin is a potent stimulant of growth hormone release, food intake, and adiposity. We demonstrate that our lead compound, L-NOX-B11, binds ghrelin with low-nanomolar affinity and inhibits ghrelin-mediated GHS-receptor activation in cell culture with an IC(50) of 5 nM. l-NOX-B11 is highly specific for the bioactive, n-octanoylated form of ghrelin. Like the GHS receptor, it does not recognize the inactive unmodified peptide and requires only the N-terminal five amino acids for the interaction. The i.v. administration of polyethylene glycol modified l-NOX-B11 efficiently suppresses ghrelin-induced growth hormone release in rats. These results demonstrate that the neutralization of circulating bioactive ghrelin leads to inhibition of ghrelin's secretory effects in the CNS.

Retrospective digital image fusion of multidetector CT and 18F-FDG PET: clinical value in pancreatic lesions--a prospective study with 104 patients.

UNLABELLED: Differential diagnosis of pancreatic lesions still remains a problem. Whereas CT provides high spatial resolution, PET detects malignant lesions with high sensitivity. The objective of this study was to evaluate the clinical benefit of PET/CT image fusion in the diagnostic workup of pancreatic cancer. METHODS: One hundred four patients with suspected pancreatic lesion underwent triple-phase multidetector CT and (18)F-FDG PET scanning. Voxel-based retrospective registration and fusion of CT and PET were performed with recently developed software. CT, PET, and fused images were assessed by 2 radiologists with regard to the detection of malignancies, possible infiltration of adjacent tissue or lymph nodes, or distant metastases. RESULTS: Fusion of CT and PET images was technically successful in 96.2%. In 2 cases, paraaortic lymph node infiltration was detected only by image fusion; in a further 8 cases, lymph node metastases were confirmed with improved localization. In 5 patients, additional pancreatic tumors or distant metastases only suspected during PET scanning were confirmed. Image fusion improved the sensitivity of malignancy detection from 76.6% (CT) and 84.4% (PET) to 89.1% (image fusion). Compared with CT alone, image fusion increased the sensitivity of detecting tissue infiltration to 68.2%, but at the cost of decreased specificity. CONCLUSION: The most important supplementary finding supplied by image fusion is a more precise correlation with focal tracer hot spots in PET. Image fusion improved the sensitivity of differentiating between benign and malignant pancreatic lesions with no significant change in specificity. All image modalities failed to stage lymph node involvement.

Activin A stimulates vascular endothelial growth factor gene transcription in human hepatocellular carcinoma cells.

BACKGROUND & AIMS: Up-regulation of vascular endothelial growth factor is known to play a critical role in hepatocellular tumor biology. In an attempt to identify factors responsible for vascular endothelial growth factor induction in human hepatocellular carcinoma, we evaluated the effects of activin A, a member of the transforming growth factor-beta cytokine superfamily, on vascular endothelial growth factor gene expression. METHODS: Expression of vascular endothelial growth factor, activin A, and its receptors was analyzed by immunohistochemistry, polymerase chain reaction, and enzyme-linked immunosorbent assay. Functional vascular endothelial growth factor promoter analysis and gel shift assays were performed to define minimal promoter requirements and potential transcription factors. Nuclear expression and biochemical modifications of Sp1, as well as subcellular distribution, expression, and physical interaction of Smad proteins with Sp1, were assessed with immunoprecipitation and Western blot analysis. RESULTS: Hepatocellular carcinoma tumors and cell lines expressed activin A and its receptors. Activin A stimulated vascular endothelial growth factor gene transcription through Sp1-dependent induction of vascular endothelial growth factor promoter activity. Furthermore, activin A stimulated the DNA-binding and transactivation potential of Sp1. Immunoprecipitation showed activin A-dependent nuclear translocation of Smad2 and induction of Sp1/Smad2 interaction. The functional relevance of Sp1/Smad2 interaction was confirmed by transient transfection experiments, which showed that overexpression of Smad2 increased vascular endothelial growth factor promoter activity and endogenous vascular endothelial growth factor protein expression, whereas dominant negative Smad2 blocked activin A responsiveness. CONCLUSIONS: This study identifies activin A as a novel stimulus of vascular endothelial growth factor gene expression in hepatocellular carcinoma and delineates physical and functional cooperation of Sp1 and Smad2 as the underlying mechanism.

Nonsteroidal anti-inflammatory drugs inhibit growth of human neuroendocrine tumor cells via G1 cell-cycle arrest.

Therapeutic options to inhibit growth of human NETs of the GEP system are limited. Since NSAIDs might provide an antiproliferative treatment alternative with acceptable toxicity, we examined the effects of different NSAIDs on growth and survival in a representative set of human GEP NET cell lines. Growth and apoptosis were determined based on cell numbers, cell-cycle analyses, kinase assays, DNA fragmentation and PARP cleavage. Expression of COX and cell cycle-regulatory molecules was examined by immunoblotting and reporter gene assays. Depending on the drug and cell line investigated, NSAID treatment resulted in profound growth inhibition of GEP NET cells. Growth-inhibitory effects were achieved with either COX-2 selective (NS398) or unselective (indomethacin, sulindac) compounds. Cell-cycle analyses documented a G1 arrest in NSAID-treated GEP NET populations. In addition, 100 microM sulindac or indomethacin induced apoptosis. All 3 COX inhibitors prevented CDK-2 activation. In parallel to the NSAID-mediated reduction of CDK-2 activity, p21(cip-1) promoter activity and cellular p21(cip-1) levels increased and p21(cip-1) was sequestered into CDK-2 complexes. Thus, the G1 arrest likely resulted from p21(cip-1)-dependent inhibition of CDK-2 activity. At therapeutically relevant concentrations, sulindac significantly reduced GEP NET cell numbers, whereas IFN-alpha and octreotide remained ineffective. The extent of growth inhibition in GEP NETs was comparable to the antiproliferative effects of sulindac in established NSAID-sensitive cell models. NSAIDs acted as potent antiproliferative agents in GEP NET cells via G1 cell-cycle arrest and might therefore offer a therapeutic alternative to current treatment modalities.

Activated signal transducer and activator of transcription 3 (STAT3) supports the malignant phenotype of human pancreatic cancer.

BACKGROUND & AIMS: Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been implicated in regulation of growth and malignant transformation. We therefore analyzed the expression and biologic significance of STAT3 in human pancreatic cancer cells. METHODS: Expression and activation of STAT3 were investigated by immunohistochemistry and immunoblotting. Functional inactivation of STAT3 was achieved by stable transfection of dominant-negative STAT3 constructs in 2 pancreatic cancer cell lines and confirmed by electrophoretic mobility shift assay and immunoblotting. Cell proliferation and tumorigenicity were evaluated by cell counting, colony formation in soft agar, and xenotransplantation in nude mice. STAT3-dependent cell cycle distribution was monitored by flow cytometry, immunoprecipitation, immunoblotting, and histone H1 and GST-Rb kinase assays. RESULTS: Compared with nontransformed human pancreas, activated STAT3 is overexpressed in ductal carcinoma cells but not in ducts from chronic pancreatitis. Constitutive activation was also observed in all human pancreatic cancer cell lines examined. Functional inactivation of STAT3 resulted in significant inhibition of anchorage-dependent and -independent proliferation in vitro and reduced tumor growth in vivo. Cell cycle analysis showed a delay of G(1)/S-phase progression due to inhibition of cyclin-dependent kinase 2 activity based on increased expression of p21(WAF1) in vitro and in vivo. Blocking of the STAT3 upstream activator Janus kinase 2 by tyrphostin also resulted in growth arrest because of delayed G(1)/S-phase progression and increased expression of p21(WAF1). CONCLUSIONS: On malignant transformation, activated STAT3 promotes cellular proliferation by acceleration of G(1)/S-phase progression and thereby contributes to the malignant phenotype of human pancreatic cancer.

Effects of interferon alpha on vascular endothelial growth factor gene transcription and tumor angiogenesis.

BACKGROUND: Interferon alpha (IFN-alpha) has antiangiogenic activity, although the underlying mechanism of action is unclear. Because human neuroendocrine (NE) tumors are highly vascularized and sensitive to IFN-alpha, we investigated whether the therapeutic effects of IFN-alpha result from an inhibition of angiogenesis mediated by a decrease in vascular endothelial growth factor (VEGF) gene expression. METHODS: VEGF gene and protein expression was analyzed in NE tumors by immunohistochemistry and in NE tumor cell lines by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). VEGF promoter-reporter gene constructs containing various deletions or mutations and gel shift assays were used to identify minimal promoter requirements and potential transcription factors. A xenograft nude mouse model (five mice per group) was used to determine the effect of IFN-alpha on tumor growth (NE Bon cells and pancreatic Capan-1 cells) and microvessel density. Liver metastases from eight patients with NE tumors were analyzed for microvessel density, VEGF mRNA content, and VEGF plasma levels before and after initiation of IFN-alpha therapy. RESULTS: NE tumors and cell lines expressed VEGF mRNA and secreted VEGF protein. In vitro, IFN-alpha decreased transcription of VEGF gene expression through an Sp1- and/or Sp3-dependent inhibition of VEGF promoter activity. Compared with vehicle treatment in mice, IFN-alpha inhibited tumor growth by 36% and reduced microvessel density from 56 (95% confidence interval [CI] = 49 to 69) to 37 per x400 Field (95% CI = 32 to 41, P =.015). Patients with NE tumors had lower VEGF plasma levels and reduced VEGF mRNA levels and microvessel density in liver metastasis biopsy material after IFN-alpha treatment. CONCLUSION: IFN-alpha confers its antitumor activity, at least in part, by its antiangiogenic activity, which results from Sp1- and/or Sp3-mediated inhibition of VEGF gene transcription.

Transforming growth factor beta 1 stimulates vascular endothelial growth factor gene transcription in human cholangiocellular carcinoma cells.

The expression pattern and functional interaction of proangiogenic factors in human cholangiocellular carcinoma (CCC) have not been fully defined. We therefore investigated the expression of vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-beta 1 as well as their respective receptors in human CCC tumor samples and further analyzed their functional interaction in vitro. Expression of VEGF, TGF-beta 1, and their receptors was examined by immunohistochemistry, in situ hybridization, quantitative competitive reverse transcription-PCR, and ELISA. VEGF promoter analysis and identification of transcription factors involved in promoter regulation were investigated using transient transfection and electrophoretic mobility shift assays. We observed strong expression of VEGF in CCC tumor cells and localization of VEGF receptors 1 and 2 in endothelial cells; in addition, coexpression of TGF-beta 1 and its receptors in tumor cells suggests a possible functional interaction between both cytokines. In vitro studies confirmed a paracrine/autocrine stimulation of VEGF by TGF-beta 1 at a transcriptional level. Additional molecular studies using 5' deletion and mutational analysis of the human VEGF promoter revealed that TGF-beta 1 stimulates VEGF through Sp1-dependent transcriptional activation. These data suggest that overexpression and functional interaction of TGF-beta 1 and VEGF might contribute to the "angiogenic switch" and the malignant phenotype in human CCC.

Downregulation of p21(waf/cip-1) mediates apoptosis of human hepatocellular carcinoma cells in response to interferon-gamma.

There is no effective treatment for advanced hepatocellular carcinoma (HCC). We therefore explored the molecular mechanisms of interferon-gamma (IFN-gamma)-mediated growth regulation in human HCC cell lines. IFN-gamma receptor expression, signal transduction, and regulation of effectors were examined by RT-PCR, immunoprecipitation, immunoblotting, and reporter gene assays. Growth and apoptosis were determined based on cell numbers, cell cycle analyses, kinase assays, DNA fragmentation, and PARP cleavage. HCC cell lines express functionally intact IFN-gamma receptors and downstream effectors. IFN-gamma profoundly inhibited growth of HCC cells via two different mechanisms: inhibition of G1 cell cycle progression and induction of apoptosis. Analyses in SK-Hep-1 cells revealed a deficient cyclin D induction in IFN-gamma-treated cells, resulting in reduced activity of CDK4 and CDK2 kinases and pRB hypophosphorylation. In contrast, apoptosis prevailed in IFN-gamma-treated HepG2 cultures. A survey of apoptosis relevant IFN-gamma effectors including IRF-1, caspase-1, caspase-3, and p21(waf/cip-1) documented a dramatic transcriptional downregulation of p21(waf/cip-1) exclusively in apoptosis-susceptible HepG2 cells. Reconstitution of p21(waf/cip-1) rescued HepG2 cells from IFN-gamma-induced apoptosis, indicating that p21(waf/cip-1) reduction was required for apoptosis execution. Inversely, downregulation of p21(waf/cip-1) sensitized SK-Hep-1 cells to IFN-gamma-induced apoptosis. Thus, downregulation of p21(waf/cip-1) in HCC cells functions as a novel, critical determinant of alternative growth inhibitory pathways in response to IFN-gamma.

Interaction of early growth response protein 1 (Egr-1), specificity protein 1 (Sp1), and cyclic adenosine 3'5'-monophosphate response element binding protein (CREB) at a proximal response element is critical for gastrin-dependent activation of the chromogranin A promoter.

Recently, binding of specific protein 1 (Sp1) and cAMP response element binding protein (CREB) to a GC-rich element at -92/-62 has been identified as a critical step in gastrin-dependent regulation of the chromogranin A (CgA) gene in gastric epithelial cells. Here we demonstrate that binding of early growth response protein 1 (Egr-1) to the distal part of the -92/-62 site is also required for gastrin-dependent CgA transactivation. Gastrin elevated cellular and nuclear Egr-1 levels in a time-dependent manner and also increased Egr-1 binding to the CgA -92/-73 region. Disruption of this site reduced gastrin responsiveness without influencing basal promoter activity, while loss of Sp1 and/or CREB binding sites diminished basal and gastrin-stimulated CgA promoter activity. Ectopic Egr-1 overexpression potently stimulated the CgA promoter, whereas coexpression of Egr-1 with Sp1 and/or CREB resulted in additive effects. Functional analysis of Sp1-, Egr-1-, or CREB-specific promoter mutations in transfection studies confirmed the tripartite organization of the CgA -92/-62 element. Signaling studies revealed that MAPK kinase 1 (MEK1)/ERK1/2 cascades are critical for gastrin-dependent Egr-1 protein accumulation as well as Egr-1 binding to the CgA promoter. Our studies for the first time identify Egr-1 as a nuclear target of gastrin and show that functional interplay of Egr-1, Sp1, and CREB is indispensable for gastrin-dependent CgA transactivation in gastric epithelial cells.

Prospective evaluation of pancreatic tumors: accuracy of MR imaging with MR cholangiopancreatography and MR angiography.

PURPOSE: To prospectively assess accuracy of magnetic resonance (MR) imaging, MR cholangiopancreatography (MRCP), and MR angiography in patients suspected of having pancreatic tumors. MATERIALS AND METHODS: Sixty-six patients suspected of having pancreatic tumors underwent MR imaging (unenhanced and contrast material-enhanced MR, MRCP, and contrast-enhanced MR angiography). Two blinded readers prospectively analyzed the images by consensus, and results were correlated with surgery, biopsy, or follow-up findings. Results were tabulated in two-by-two tables. RESULTS: MR assessment of pancreatic lesion status (differentiation of benign vs malignant) resulted in 60 correct diagnoses (accuracy, 91%), and six (10%) false diagnoses. Among histologically proved malignant tumors, MR imaging yielded correct diagnoses in 42 of 44 patients (sensitivity, 95%; 95% CI: 85%, 99%), whereas 18 of 22 patients with benign findings were classified correctly. At MR imaging, findings in four patients with chronic pancreatitis were wrongly categorized as malignant tumors (specificity, 82%; 95% CI: 60%, 95%), and in one patient, a distal common bile duct carcinoma was not detected. In no patient with pancreatic adenocarcinoma was this tumor misdiagnosed as benign. In patients with malignant tumors who underwent resection, local-regional tumor growth and vascular infiltration were accurately classified in 89% and 94%, respectively. MR imaging depicted histologically proved synchronous hepatic metastases in 82%. The positive and negative predictive values for cancer nonresectability were 90% and 83%, respectively, and the accuracy, sensitivity, and specificity were 85%, 69%, and 95%, respectively. CONCLUSION: Unenhanced and contrast-enhanced MR imaging with MRCP and MR angiography offers potential as a noninvasive tool for assessment of patients suspected of having pancreatic tumors.

Overexpression of pRB in human pancreatic carcinoma cells: function in chemotherapy-induced apoptosis.

BACKGROUND: Human pancreatic adenocarcinomas are highly resistant to chemotherapy. The p16 tumor-suppressor protein is inactivated in more than 90% of human pancreatic cancers. The p16 protein transcriptionally inhibits expression of retinoblastoma tumor-suppressor gene pRB. The pRB protein transcriptionally inhibits expression of the p16 gene. Because pRB normally prevents apoptosis, we investigated whether pRB is involved in resistance to chemotherapy-induced apoptosis in pancreatic cancer cells. METHODS: pRB expression was examined by immunohistochemistry in 106 human pancreatic tissue specimens. The human pancreatic tumor cell line Capan-1 (pRB+/p16-) was stably transfected with p16 to functionally inactivate pRB. pRB gene expression was examined by western and northern blot analyses, and pRB function was assessed by electrophoretic mobility shift assays and promoter transactivation studies for the transcription factor E2F. Changes in cell sensitivity to chemotherapy were measured by assays for cytotoxicity and apoptosis. RESULTS: pRB was overexpressed in pancreatic ductal adenocarcinomas but was hardly detectable in other pancreatic malignancies, chronic pancreatitis, or nontransformed human pancreatic tissue. Expression of p16 in Capan-1 cells resulted in the loss of pRB gene and protein expression concomitant with increased activity of the transcription factor E2F, which was not detected in wild-type or control-transfected Capan-1 cells. Wild-type and control-transfected Capan-1 cells were resistant to chemotherapy-induced apoptosis, but pRB-depleted (i.e., p16-transfected) Capan-1 cells were highly sensitive. The effect was specific to pRB depletion because two other human pancreatic cancer cell lines that retained high pRB expression after p16 transfection were resistant to chemotherapy-induced apoptosis. CONCLUSIONS: Overexpression of pRB is associated with human pancreatic duct-cell cancer and may allow pancreatic cancer cells to evade chemotherapy-induced apoptosis.