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1.  The Role for Dickkopf-Homolog-1 in the Pathogenesis of Crohn’s Disease-Associated Fistulae 
PLoS ONE  2013;8(11):e78882.
Background
One of the most challenging conditions in Crohn’s disease (CD) patients is the treatment of perianal fistulae. We have recently shown that epithelial-to-mesenchymal transition (EMT) plays a crucial role during CD-fistulae development. Dickkopf-homolog 1 (DKK-1) is known to play a key role during EMT. Here, we investigated a role for DKK-1 in the pathogenesis of CD-associated fistulae.
Methods
Dkk-1 protein expression in CD-fistula specimens were investigated by immunohistochemistry. Colonic lamina propria fibroblasts (CLPF) were obtained from either non-IBD control patients or patients with fistulizing CD. HT-29 intestinal epithelial cells (IEC) were either grown as monolayers or spheroids. Cells were treated with either TNF-α, TGF-β or IL-13. Knock-down of DKK-1 or β-Catenin was induced in HT-29-IEC by siRNA technique. mRNA expression was determined by real-time-PCR.
Results
Dkk-1 protein was specifically expressed in transitional cells lining the fistula tracts. TGF-β induced DKK-1 mRNA expression in HT-29-IEC, but decreased it in fistula CLPF. On a functional level, DKK-1 knock-down prevented TGF-β-induced IL-13 mRNA expression in HT-29-IEC. Further, loss of β-Catenin was accompanied by reduced levels of DKK-1 and, again, IL-13 in IEC in response to TGF-β. In turn, treatment of HT-29-IEC as well as fistula CLPF with IL-13 resulted in decreased levels of DKK-1 mRNA. Treatment with TNF-α or the bacterial wall component, muramyl-dipeptide, decreased DKK-1 mRNA levels in HT-29-IEC, but enhanced it in fistula CLPF.
Discussion
We demonstrate that DKK-1 is strongly expressed in cells lining the CD-fistula tracts and regulates factors involved in EMT initiation. These data provide evidence for a role of DKK-1 in the pathogenesis of CD-associated perianal fistulae.
doi:10.1371/journal.pone.0078882
PMCID: PMC3826763  PMID: 24250816
2.  Successful Salvage Chemotherapy with FOLFIRINOX for Recurrent Mixed Acinar Cell Carcinoma and Ductal Adenocarcinoma of the Pancreas in an Adolescent Patient 
Case Reports in Oncology  2013;6(3):497-503.
Pancreatic tumors are rare in children and adolescents. Here, we report the case of a 15-year-old boy who presented with a mixed acinar cell carcinoma/ductal adenocarcinoma with blastomatous components. He received multimodal treatment including various chemotherapy regimens and multistep surgery including liver transplantation. Introduction of FOLFIRINOX after relapse repeatedly achieved a durable metabolic and clinical response with good quality of life.
doi:10.1159/000355320
PMCID: PMC3806674  PMID: 24163668
FOLFIRINOX; Acinar cell carcinoma; Ductal adenocarcinoma; Pancreatoblastoma; Pancreatic cancer; Autologous stem cell transplantation; Multimodal treatment
3.  Nodular regenerative hyperplasia of the liver associated with didanosine persists for years even after its interruption 
BMJ Case Reports  2011;2011:bcr0320113928.
The authors describe an HIV-positive patient with nodular regenerative hyperplasia of the liver with non-cirrhotic portal hypertension. Despite stopping the culprit drug, didanosine, the radiologic changes persisted for years. When evaluating liver pathologies, antiretroviral drugs must be included in the differential diagnosis, even when they have been stopped years ago.
doi:10.1136/bcr.03.2011.3928
PMCID: PMC3089926  PMID: 22696691
4.  Toll-Like Receptor 3 Expressing Tumor Parenchyma and Infiltrating Natural Killer Cells in Hepatocellular Carcinoma Patients 
Background
Hepatocellular carcinoma (HCC) is a highly aggressive cancer that is linked to chronically dysregulated liver inflammation. However, appropriate immune responses can control HCC progression. Here we investigated the role and underlying mechanism of toll-like receptor 3 (TLR3) in HCC.
Methods
HCC cell death, and natural killer (NK) cell activation and cytotoxicity were assessed in vitro after treatment with the TLR3 ligand poly(I:C). The effect of TLR3 on the tumor parenchyma and infiltrating immune cells was investigated in a spontaneous liver tumor mouse model and a transplanted tumor mouse model (n = 3–9 mice per group). Immunohistochemistry and quantitative polymerase chain reaction were used to analyze tumor samples from 172 HCC patients. Paired t-tests and analysis of variance tests were used to calculate P-values. The relationship between TLR3 expression and survival was determined by the Kaplan–Meier univariate survival analysis and a log-rank test. All statistical tests were two-sided.
Results
TLR3 activation increased cell death in the TLR3+ SNU182 HCC cell line (30.5% vs 8.5%, P = .03) and promoted NK-cell activation (32.6% vs 19.4%, P < .001) and cytotoxicity (relative fourfold increase, P = .03) in vitro. In vivo, poly(I:C) treatment increased intratumoral chemokine expression, NK-cell activation and tumor infiltration, and proliferation of tumor-infiltrating T and NK cells. Proliferation of tumor parenchyma cells was decreased. Also, expression of chemokines or treatment with poly(I:C) decreased tumor growth. TLR3 expression in patient samples correlated with NK-cell activation, NK- and T-cell tumor infiltration, and inversely correlated with tumor parenchyma cell viability. TLR3 expression was also associated with longer survival in HCC patients (hazard ratio of survival = 2.1, 95% confidence interval = 1.3 to 3.4, P = .002).
Conclusions
TLR3 is an important modulator of HCC progression and is a potential target for novel immunotherapy.
doi:10.1093/jnci/djs436
PMCID: PMC3814220  PMID: 23197495
5.  Fc Gamma Receptor CD64 Modulates the Inhibitory Activity of Infliximab 
PLoS ONE  2012;7(8):e43361.
Background
Tumor necrosis factor (TNF) is an important cytokine in the pathogenesis of inflammatory bowel disease (IBD). Anti-TNF antibodies have been successfully implemented in IBD therapy, however their efficacies differ among IBD patients. Here we investigate the influence of CD64 Fc receptor on the inhibitory activity of anti-TNFs in cells of intestinal wall.
Methods
Intestinal cell lines, monocytes/macrophages and peripheral blood mononuclear cells (PBMCs) were used as models. The efficacies of adalimumab, infliximab and certolizumab-pegol were assessed by RT-PCR for target genes. Protein levels and localizations were examined by Western blotting and immunofluorescence. Antibody fragments were obtained by proteolytic digestion, immunoprecipitation and protein chip analysis. Knock-down of specific gene expression was performed using siRNAs.
Results
Infliximab had limited efficacy towards soluble TNF in cell types expressing Fc gamma receptor CD64. Both adalimumab and infliximab had lower efficacies in PBMCs of IBD patients, which express elevated levels of CD64. Infliximab-TNF complexes were more potent in activating CD64 in THP-1 cells than adalimumab, which was accompanied by distinct phospho-tyrosine signals. Blocking Fc parts and isolation of Fab fragments of infliximab improved its efficacy. IFN-γ-induced expression of CD64 correlated with a loss of efficacy of infliximab, whereas reduction of CD64 expression by either siRNA or PMA treatment improved inhibitory activity of this drug. Colonic mRNA expression levels of CD64 and other Fc gamma receptors were significantly increased in the inflamed tissues of infliximab non-responders.
Conclusions
CD64 modulates the efficacy of infliximab both in vitro and ex vivo, whereas the presence of this receptor has no impact on the inhibitory activity of certolizumab-pegol, which lacks Fc fragment. These data could be helpful in both predicting and evaluating the outcome of anti-TNF therapy in IBD patients with elevated systemic and local levels of Fc receptors.
doi:10.1371/journal.pone.0043361
PMCID: PMC3427356  PMID: 22937039
6.  Chemokine-driven lymphocyte infiltration: an early intratumoural event determining long-term survival in resectable hepatocellular carcinoma 
Gut  2011;61(3):427-438.
Objective
Hepatocellular carcinoma (HCC) is a heterogeneous disease with poor prognosis and limited methods for predicting patient survival. The nature of the immune cells that infiltrate tumours is known to impact clinical outcome. However, the molecular events that regulate this infiltration require further understanding. Here the ability of immune genes expressed in the tumour microenvironment to predict disease progression was investigated.
Methods
Using quantitative PCR, the expression of 14 immune genes in resected tumour tissues from 57 Singaporean patients was analysed. The nearest-template prediction method was used to derive and test a prognostic signature from this training cohort. The signature was then validated in an independent cohort of 98 patients from Hong Kong and Zurich. Intratumoural components expressing these critical immune genes were identified by in situ labelling. Regulation of these genes was analysed in vitro using the HCC cell line SNU-182.
Results
The identified 14 immune-gene signature predicts patient survival in both the training cohort (p=0.0004 and HR=5.2) and the validation cohort (p=0.0051 and HR=2.5) irrespective of patient ethnicity and disease aetiology. Importantly, it predicts the survival of patients with early disease (stages I and II), for whom classical clinical parameters provide limited information. The lack of predictive power in late disease stages III and IV emphasises that a protective immune microenvironment has to be established early in order to impact disease progression significantly. This signature includes the chemokine genes CXCL10, CCL5 and CCL2, whose expression correlates with markers of T helper 1 (Th1), CD8+ T and natural killer (NK) cells. Inflammatory cytokines (tumour necrosis factor α, interferon γ) and Toll-like receptor 3 ligands stimulate intratumoural production of these chemokines which drive tumour infiltration by T and NK cells, leading to enhanced cancer cell death.
Conclusion
A 14 immune-gene signature, which identifies molecular cues driving tumour infiltration by lymphocytes, accurately predicts survival of patients with HCC especially in early disease.
doi:10.1136/gutjnl-2011-300509
PMCID: PMC3273680  PMID: 21930732
Hepatocellular carcinoma; immune-gene signature; tumour microenvironment; chemokines; lymphocytes tumour infiltration; immunology in hepatology; tumour markers; liver immunology; immunohistochemistry; acute hepatitis; cancer; oncogenes; gastric cancer; liver metastases; pancreatic pathology; pancreatic tumours; molecular pathology; gastrointestinal lymphoma; liver biopsy; surgical complications; surgical oncology; surgical resection; immune response
7.  Adjuvant gemcitabine versus NEOadjuvant gemcitabine/oxaliplatin plus adjuvant gemcitabine in resectable pancreatic cancer: a randomized multicenter phase III study (NEOPAC study) 
BMC Cancer  2011;11:346.
Background
Despite major improvements in the perioperative outcome of pancreas surgery, the prognosis of pancreatic cancer after curative resection remains poor. Adjuvant chemotherapy increases disease-free and overall survival, but this treatment cannot be offered to a significant proportion of patients due to the surgical morbidity. In contrast, almost all patients can receive (neo)adjuvant chemotherapy before surgery. This treatment is safe and effective, and has resulted in a median survival of 26.5 months in a recent phase II trial. Moreover, neoadjuvant chemotherapy improves the nutritional status of patients with pancreatic cancer. This multicenter phase III trial (NEOPAC) has been designed to explore the efficacy of neoadjuvant chemotherapy.
Methods/Design
This is a prospective randomized phase III trial. Patients with resectable cytologically proven adenocarcinoma of the pancreatic head are eligible for this study. All patients must be at least 18 years old and must provide written informed consent. An infiltration of the superior mesenteric vein > 180° or major visceral arteries are considered exclusion criteria. Eligible patients will be randomized to surgery followed by adjuvant gemcitabine (1000 mg/m2) for 6 months or neoadjuvant chemotherapy (gemcitabine 1000 mg/m2, oxaliplatin 100 mg/m2) followed by surgery and the same adjuvant treatment. Neoadjuvant chemotherapy is given four times every two weeks. The staging as well as the restaging protocol after neoadjuvant chemotherapy include computed tomography of chest and abdomen and diagnostic laparoscopy. The primary study endpoint is progression-free survival. According to the sample size calculation, 155 patients need to be randomized to each treatment arm. Disease recurrence will be documented by scheduled computed tomography scans 9, 12, 15, 21 and thereafter every 6 months until disease progression. For quality control, circumferential resection margins are marked intraoperatively, and representative histological sections will be centrally reviewed by a dedicated pathologist.
Discussion
The NEOPAC study will determine the efficacy of neoadjuvant chemotherapy in pancreatic cancer for the first time and offers a unique potential for translational research. Furthermore, this trial will provide the unbiased overall survival of all patients undergoing surgery for resectable cancer of the pancreatic head.
Trial registration
clinicalTrials.gov NCT01314027
doi:10.1186/1471-2407-11-346
PMCID: PMC3176241  PMID: 21831266
8.  Protection of epithelial barrier function by the Crohn’s disease associated gene, protein tyrosine phosphatase N2 
Gastroenterology  2009;137(6):2030-2040.e5.
Background & Aims
Protein tyrosine phosphatase N2 (PTPN2) has been identified as a Crohn’s disease (CD) candidate gene. However, a role for PTPN2 in the pathogenesis of CD has not been identified. Increased permeability of the intestinal epithelium is believed to contribute prominently to CD. The aim of this study was to determine a possible role for PTPN2 in CD pathogenesis.
Methods
Intestinal epithelial cell (IEC) lines, T84 and HT29cl.19a, were used in all studies. Protein analysis was performed by Western blotting and protein knock-down was induced by siRNA. Primary samples were from control and CD patients.
Results
Here, we demonstrate increased PTPN2 expression in CD intestinal biopsies and that the pro-inflammatory cytokine, IFNγ, increases PTPN2 expression and activity in IEC. Moreover, IFNγ-induced STAT1 and STAT3 phosphorylation in IEC is enhanced by PTPN2 knock-down. The cellular energy sensor, AMPK, partially regulates the IFNγ-induced effects on PTPN2. Additionally, PTPN2 knock-down potentiates IFNγ-induced increases in epithelial permeability, accompanied by elevated expression of the pore-forming protein, claudin-2.
Conclusions
PTPN2 is activated by IFNγ and limits IFNγ-induced signalling and consequent barrier defects. These data suggest a functional role for PTPN2 in maintaining the intestinal epithelial barrier and in the pathophysiology of CD.
doi:10.1053/j.gastro.2009.07.078
PMCID: PMC2855721  PMID: 19818778
9.  Knock-out of Myeloid cell leukemia-1 induces liver damage and increases apoptosis susceptibility of murine hepatocytes 
Hepatology (Baltimore, Md.)  2009;49(2):627-636.
Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 protein family. It interacts with pro-apoptotic Bcl-2 family members, thereby inhibiting mitochondrial activation and induction of apoptosis. Mcl-1 is essential for embryonal development and the maintenance of B, T and hematopoietic stem cells. We have recently shown that induction of Mcl-1 by growth factors rescues primary human hepatocytes from CD95-mediated apoptosis. This prompted us to further analyze the relevance of Mcl-1 for hepatocellular homeostasis. Therefore, we generated a hepatocyte-specific Mcl-1 knock-out mouse (Mcl-1flox/flox-AlbCre). Deletion of Mcl-1 in hepatocytes results in liver cell damage caused by spontaneous induction of apoptosis. Livers of Mcl-1flox/flox-AlbCre mice are smaller compared to control littermates, due to higher apoptosis rates. As a compensatory mechanism, proliferation of hepatocytes is enhanced in the absence of Mcl-1. Importantly, hepatic pericellular fibrosis occurs in Mcl-1 negative livers in response to chronic liver damage. Furthermore, Mcl-1flox/flox-AlbCre mice are more susceptible towards hepatocellular damage induced by agonistic anti-CD95 antibodies or concanavalin A.
Conclusion
The present study provides in vivo evidence that Mcl-1 is a crucial anti-apoptotic factor for the liver, contributing to hepatocellular homeostasis and protecting hepatocytes from apoptosis induction.
doi:10.1002/hep.22664
PMCID: PMC2753874  PMID: 19127517
Mcl-1; Bcl-xL; CD95; fibrosis; proliferation
10.  The FUSE binding proteins FBP1 and FBP3 are potential c-myc regulators in renal, but not in prostate and bladder cancer 
BMC Cancer  2008;8:369.
Background
The three far-upstream element (FUSE) binding proteins (FBP1, FBP2, and FBP3) belong to an ancient family of single-stranded DNA binding proteins which are required for proper regulation of the c-myc proto-oncogene. Whereas it is known that c-myc alterations play a completely different role in various carcinomas of the urogenital tract, the relevance of FBPs is unclear.
Methods
FBP1, FBP3 and c-myc expression was studied in 105 renal cell, 95 prostate and 112 urinary bladder carcinomas by immunohistochemistry using tissue microarrays.
Results
High rates of FBP1 and FBP3 expression were observed in all cancer types. There was a concomitant up-regulation of FBP1 and FBP3 in renal cell and prostate carcinomas (p < 0.001 both). C-myc expression was detectable in 21% of prostate, 30% of renal and 34% of urothelial carcinomas. Interestingly, strong FBP1 and FBP3 expression was associated with c-myc up-regulation in clear cell renal cell carcinomas (p < 0.001 and 0.09 resp.), but not in bladder or prostate cancer.
Conclusion
The correlation between FBP1/FBP3, c-myc and high proliferation rate in renal cell carcinoma provides strong in vivo support for the suggested role of FBP1 and FBP3 as activators of c-myc. The frequent up-regulation of FBP1 and FBP3 in urothelial and prostate carcinoma suggests that FBPs also have an important function in gene regulation of these tumors.
doi:10.1186/1471-2407-8-369
PMCID: PMC2631590  PMID: 19087307
11.  Bcl-xL and Myeloid cell leukaemia-1 contribute to apoptosis resistance of colorectal cancer cells 
AIM: To explore the role of Bcl-xL and Myeloid cell leukaemia (Mcl)-1 for the apoptosis resistance of colorectal carcinoma (CRC) cells towards current treatment modalities.
METHODS: Bcl-xL and Mcl-1 mRNA and protein expression were analyzed in CRC cell lines as well as human CRC tissue by Western blot, quantitative PCR and immunohistochemistry. Bcl-xL and Mcl-1 protein expression was knocked down or increased in CRC cell lines by applying specific siRNAs or expression plasmids, respectively. After modulation of protein expression, CRC cells were treated with chemotherapeutic agents, an antagonistic epidermal growth factor receptor (EGFR1) antibody, an EGFR1 tyrosine kinase inhibitor, or with the death receptor ligand TRAIL. Apoptosis induction and cell viability were analyzed.
RESULTS: Here we show that in human CRC tissue and various CRC cell lines both Bcl-xL and Mcl-1 are expressed. Bcl-xL expression was higher in CRC tissue than in surrounding non-malignant tissue, both on protein and mRNA level. Mcl-1 mRNA expression was significantly lower in malignant tissues. However, protein expression was slightly higher. Viability rates of CRC cells were significantly decreased after knock down of Bcl-xL expression, and, to a lower extent, after knock down of Mcl-1 expression. Furthermore, cells with reduced Bcl-xL or Mcl-1 expression was more sensitive towards oxaliplatin- and irinotecan-induced apoptosis, and in the case of Bcl-xL also towards 5-FU-induced apoptosis. On the other hand, upregulation of Bcl-xL by transfection of an expression plasmid decreased chemotherapeutic drug-induced apoptosis. EGF treatment clearly induced Bcl-xL and Mcl-1 expression in CRC cells. Apoptosis induction upon EGFR1 blockage by cetuximab or PD168393 was increased by inhibiting Mcl-1 and Bcl-xL expression. More strikingly, CD95- and TRAIL-induced apoptosis was increased by Bcl-xL knock down.
CONCLUSION: Our data suggest that Bcl-xL and, to a lower extent, Mcl-1, are important anti-apoptotic factors in CRC. Specific downregulation of Bcl-xL is a promising approach to sensitize CRC cells towards chemotherapy and targeted therapy.
doi:10.3748/wjg.14.3829
PMCID: PMC2721439  PMID: 18609706
Colorectal carcinoma; Bcl-xL; Myeloid cell leukaemia-1; Epidermal growth factor receptor 1; Apoptosis; 5-fluorouracil; Irinotecan; Oxaliplatin
12.  Suppression of Mcl-1 via RNA interference sensitizes human hepatocellular carcinoma cells towards apoptosis induction 
BMC Cancer  2006;6:232.
Background
Hepatocelluar carcinoma (HCC) is one of the most common cancers worldwide and a major cause of cancer-related mortality. HCC is highly resistant to currently available chemotherapeutic drugs. Defects in apoptosis signaling contribute to this resistance. Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 protein family which interferes with mitochondrial activation. In a previous study we have shown that Mcl-1 is highly expressed in tissues of human HCC. In this study, we manipulated expression of the Mcl-1 protein in HCC cells by RNA interference and analyzed its impact on apoptosis sensitivity of HCC cells in vitro.
Methods
RNA interference was performed by transfecting siRNA to specifically knock down Mcl-1 expression in HCC cells. Mcl-1 expression was measured by quantitative real-time PCR and Western blot. Induction of apoptosis and caspase activity after treatment with chemotherapeutic drugs and different targeted therapies were measured by flow cytometry and fluorometric analysis, respectively.
Results
Here we demonstrate that Mcl-1 expressing HCC cell lines show low sensitivity towards treatment with a panel of chemotherapeutic drugs. However, treatment with the anthracycline derivative epirubicin resulted in comparatively high apoptosis rates in HCC cells. Inhibition of the kinase PI3K significantly increased apoptosis induction by chemotherapy. RNA interference efficiently downregulated Mcl-1 expression in HCC cells. Mcl-1 downregulation sensitized HCC cells to different chemotherapeutic agents. Sensitization was accompanied by profound activation of caspase-3 and -9. In addition, Mcl-1 downregulation also increased apoptosis rates after treatment with PI3K inhibitors and, to a lower extent, after treatment with mTOR, Raf I and VEGF/PDGF kinase inhibitors. TRAIL-induced apoptosis did not markedly respond to Mcl-1 knockdown. Additionally, knockdown of Mcl-1 efficiently enhanced apoptosis sensitivity towards combined treatment modalities: Mcl-1 knockdown significantly augmented apoptosis sensitivity of HCC cells towards chemotherapy combined with PI3K inhibition.
Conclusion
Our data suggest that specific downregulation of Mcl-1 by RNA interference is a promising approach to sensitize HCC cells towards chemotherapy and molecularly targeted therapies.
doi:10.1186/1471-2407-6-232
PMCID: PMC1601962  PMID: 17014711
13.  TFIIH Operates through an Expanded Proximal Promoter To Fine-Tune c-myc Expression 
Molecular and Cellular Biology  2005;25(1):147-161.
A continuous stream of activating and repressing signals is processed by the transcription complex paused at the promoter of the c-myc proto-oncogene. The general transcription factor IIH (TFIIH) is held at promoters prior to promoter escape and so is well situated to channel the input of activators and repressors to modulate c-myc expression. We have compared cells expressing only a mutated p89 (xeroderma pigmentosum complementation group B [XPB]), the largest TFIIH subunit, with the same cells functionally complemented with the wild-type protein (XPB/wt-p89). Here, we show structural, compositional, and functional differences in transcription complexes between XPB and XPB/wt-89 cells at the native c-myc promoter. Remarkably, although the mean levels of c-Myc are only modestly elevated in XPB compared to those in XPB/wt-p89 cells, the range of expression and the cell-to-cell variation of c-Myc are markedly increased. Our modeling indicates that the data can be explained if TFIIH integrates inputs from multiple signals, regulating transcription at multiple kinetically equivalent steps between initiation and promoter escape. This helps to suppress the intrinsic noise of transcription and to ensure the steady transcriptional output of c-myc necessary for cellular homeostasis.
doi:10.1128/MCB.25.1.147-161.2005
PMCID: PMC538784  PMID: 15601838
14.  Transcriptional Consequences of Topoisomerase Inhibition 
Molecular and Cellular Biology  2001;21(24):8437-8451.
In principle, the generation, transmission, and dissipation of supercoiling forces are determined by the arrangement of the physical barriers defining topological boundaries and the disposition of enzymes creating (polymerases and helicases, etc.) or releasing (topoisomerases) torsional strain in DNA. These features are likely to be characteristic for individual genes. By using topoisomerase inhibitors to alter the balance between supercoiling forces in vivo, we monitored changes in the basal transcriptional activity and DNA conformation for several genes. Every gene examined displayed an individualized profile in response to inhibition of topoisomerase I or II. The expression changes elicited by camptothecin (topoisomerase I inhibitor) or adriamycin (topoisomerase II inhibitor) were not equivalent. Camptothecin generally caused transcription complexes to stall in the midst of transcription units, while provoking little response at promoters. Adriamycin, in contrast, caused dramatic changes at or near promoters and prevented transcription. The response to topoisomerase inhibition was also context dependent, differing between chromosomal or episomal c-myc promoters. In addition to being well-characterized DNA-damaging agents, topoisomerase inhibitors may evoke a biological response determined in part from transcriptional effects. The results have ramifications for the use of these drugs as antineoplastic agents.
doi:10.1128/MCB.21.24.8437-8451.2001
PMCID: PMC100007  PMID: 11713279
15.  Nuclear targeting determinants of the far upstream element binding protein, a c-myc transcription factor 
Nucleic Acids Research  2000;28(22):4558-4565.
FUSE binding protein (FBP) binds in vivo and in vitro with the single-stranded far upstream element (FUSE) upstream of the c-myc gene. In addition to its transcriptional role, FBP and its closely related siblings FBP2 (KSRP) and FBP3 have been reported to bind RNA and participate in various steps of RNA processing, transport or catabolism. To perform these diverse functions, FBP must traffic to different nuclear sites. To identify determinants of nuclear localization, full-length FBP or fragments thereof were fused to green fluorescent protein. Fluorescent-FBP localized in the nucleus in three patterns, diffuse, dots and spots. Each pattern was conferred by a distinct nuclear localization signal (NLS): a classical bipartite NLS in the N-terminal and two non-canonical signals, an α-helix in the third KH-motif of the nucleic acid binding domain and a tyrosine-rich motif in the C-terminal transcription activation domain. Upon treatment with the transcription inhibitor actinomycin D, FBP completely re-localized into dots, but did not exit from the nucleus. This is in contrast to many general RNA-binding proteins, which shuttle from the nucleus upon treatment with actinomycin D. Furthermore, FBP co-localized with transcription sites and with the general transcription factor TFIIH, but not with the splicing factor SC-35. Taken together, these data reveal complex intranuclear trafficking of FBP and support a transcriptional role for this protein.
PMCID: PMC113884  PMID: 11071946

Results 1-15 (15)