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1.
Oncogene ; 37(7): 912-923, 2018 02 15.
Article in English | MEDLINE | ID: mdl-29059173

ABSTRACT

SRY (sex determining region Y)-box 9 (SOX9) is required for oncogenic Kras-mediated acinar-to-ductal metaplasia (ADM), pancreatic intraepithelial neoplasias (PanINs) and ultimately pancreatic ductal adenocarcinoma (PDAC). However, how oncogenic Kras affects SOX9 activity is not yet understood, and SOX9-associated genes in PDAC are also unknown at all. Here, we investigated the mechanistic link between SOX9 and oncogenic Kras, studied biological function of SOX9, and identified SOX9-related genes and their clinical significance in patients with PDAC. Our studies reveal that oncogenic Kras induces SOX9 mRNA and protein expression as well as phosphorylated SOX9 expression in human pancreatic ductal progenitor cells (HPNE) and pancreatic ductal cells (HPDE). Moreover, oncogenic Kras promoted nuclear translocation and transcriptional activity of SOX9 in these cells. TAK1/IκBα/NF-κB pathway contributed to induction of SOX9 by oncogenic Kras, and SOX9 in turn enhanced NF-κB activation. SOX9 promoted the proliferation of HPNE and PDAC cells, and correlated with minichromosome maintenance complex components (MCMs) and mediator of DNA damage checkpoint 1 (MDC1) expression. The overexpressive MDC1 was associated with less perineural and lymph node invasion of tumors and early TNM-stage of patients. Our results indicate that oncogenic Kras induces constitutive activation of SOX9 in HPNE and HPDE cells, and Kras/TAK1/IκBα/NF-κB pathway and a positive feedback between SOX9 and NF-κB are involved in this inducing process. SOX9 accelerates proliferation of cells and affects MCMs and MDC1 expression. MDC1 is associated negatively with invasion and metastasis of PDAC.


Subject(s)
Carcinoma, Pancreatic Ductal/pathology , Minichromosome Maintenance Proteins/metabolism , Nuclear Proteins/metabolism , Pancreatic Ducts/pathology , Pancreatic Neoplasms/pathology , Proto-Oncogene Proteins p21(ras)/metabolism , SOX9 Transcription Factor/metabolism , Trans-Activators/metabolism , Adaptor Proteins, Signal Transducing , Animals , Apoptosis , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Case-Control Studies , Cell Cycle Proteins , Cell Movement , Cell Proliferation , Female , Follow-Up Studies , Gene Expression Regulation, Neoplastic , Humans , Male , Mice , Mice, Inbred NOD , Mice, SCID , Middle Aged , Minichromosome Maintenance Proteins/genetics , Neoplasm Invasiveness , Nuclear Proteins/genetics , Pancreatic Ducts/metabolism , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Prognosis , Proto-Oncogene Proteins p21(ras)/genetics , SOX9 Transcription Factor/genetics , Trans-Activators/genetics , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
2.
J Prev Alzheimers Dis ; 4(4): 255-263, 2017.
Article in English | MEDLINE | ID: mdl-29181491

ABSTRACT

The amyloid hypothesis has been the dominant framework for Alzheimer's disease (AD) research, including the development of anti-AD therapies. However, none of the phase III clinical trials conducted to date that targeted amyloid ß (Aß) production, aggregation, or clearance demonstrated a statistically significant treatment effect in patients with AD. This includes the approach of using monoclonal antibodies that recognize various Aß epitopes and display different binding selectivity. While some monoclonal antibodies have failed in phase III trials, several are still in development. Aducanumab (BIIB037) is a human antibody that selectively targets aggregated forms of Aß, including soluble oligomers and insoluble fibrils. In PRIME (NCT01677572), an ongoing phase Ib trial (N=196 patients dosed), aducanumab was shown to reduce Aß plaques and slow decline in clinical measures in patients with prodromal or mild AD, with acceptable safety and tolerability. The main safety finding was amyloid-related imaging abnormalities (ARIA), a side effect associated with removal of Aß, which was dose-dependent and occurred more often in ApoE ε4 carriers than non-carriers. ENGAGE (NCT02477800) and EMERGE (NCT02484547), the ongoing phase III trials of aducanumab in early AD, have been designed based on the outcomes of PRIME and on lessons from past clinical trials in patients with AD. Those study design features include patient selection with confirmed Aß pathology, ensuring sufficient target engagement, and conducting clinical trials in patients at earlier symptomatic stages of AD.


Subject(s)
Alzheimer Disease/therapy , Antibodies, Monoclonal, Humanized/therapeutic use , Immunologic Factors/therapeutic use , Antibodies, Monoclonal, Humanized/adverse effects , Clinical Trials as Topic , Humans , Immunologic Factors/adverse effects
3.
Clin Pharmacol Ther ; 98(1): 47-60, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25869938

ABSTRACT

Advances in multimodality fusion imaging technologies promise to accelerate the understanding of the systems biology of disease and help in the development of new therapeutics. The use of molecular imaging biomarkers has been proven to shorten cycle times for central nervous system (CNS) drug development and thereby increase the efficiency and return on investment from research. Imaging biomarkers can be used to help select the molecules, doses, and patients most likely to test therapeutic hypotheses by stopping those that have little chance of success and accelerating those with potential to achieve beneficial clinical outcomes. CNS imaging biomarkers have the potential to drive new medical care practices for patients in the latent phases of progressive neurodegenerative disorders by enabling the detection, preventative treatment, and tracking of disease in a paradigm shift from today's approaches that have to see the overt symptoms of disease before treating it.


Subject(s)
Biomarkers, Pharmacological , Central Nervous System Agents , Drug Discovery , Molecular Imaging/methods , Biomarkers, Pharmacological/metabolism , Central Nervous System Agents/pharmacology , Humans , Magnetic Resonance Imaging , Positron-Emission Tomography , Precision Medicine , Tomography, Emission-Computed, Single-Photon
4.
Int J Oncol ; 17(2): 381-5, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10891550

ABSTRACT

Telomerase activation in differentiated cells is associated with cell immortalization. Detection of telomerase expression in cancer provides a potential biomarker to be evaluated in the diagnosis and prognosis of cancer patients. Analysis of telomerase activity in biopsy specimens may serve as a useful marker for the diagnosis of pancreatic adenocarcinoma. This study was conducted to determine if telomerase could be detected in fine needle aspirates from patients with pancreatic carcinoma. The telomeric repeat amplification protocol (TRAP) assay was used to determine telomerase activity from pancreatic cancer cell lines, paired tumor/normal human pancreatic tissues and specimens obtained at the time of diagnostic fine-needle aspiration (FNA) biopsies. In this report, we demonstrate that: i) telomerase activity is present in pathologically confirmed pancreatic cancer specimens, but absent in matched adjacent areas of normal pancreatic tissues; ii) telomerase activity is present in FNA samples obtained at the time of CT-guided FNA of pancreatic adenocarcinomas; and iii) this activity can be detected in material normally discarded during FNA slide preparation. Activation of telomerase can be identified in FNA specimens obtained at the time of routine cytologic diagnosis. These findings may be useful for further diagnostic or therapeutic investigations of telomerase activity in pancreatic carcinoma.


Subject(s)
Adenocarcinoma/enzymology , Neoplasm Proteins/metabolism , Pancreatic Neoplasms/enzymology , Telomerase/metabolism , Adenocarcinoma/pathology , Biopsy, Needle/methods , Humans , Pancreatic Neoplasms/pathology , Tumor Cells, Cultured
5.
Invest New Drugs ; 18(1): 43-56, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10830140

ABSTRACT

Pancreatic cancer is a lethal disease characterized by local invasion and early dissemination. It is resistant to conventional surgical, radiotherapeutic, and chemotherapeutic modalities. These interventions have had minimal impact on overall survival with very few patients enjoying long term survival. Over the past few years, 2'difluoro-2'deoxycytidine (gemcitabine) has demonstrated modest activity in this disease and investigations are proceeding to expand its role in combination with radiotherapy and other chemotherapeutic agents. In addition, the identification of the molecular defects underlying this disease has suggested molecular targets for the design of rational systemic therapy. These targets include matrix metalloproteinases, K-ras, HER2/neu, p53, and the epidermal growth factor receptor. Current and future clinical trials designed to improve the survival of patients with pancreatic cancer will be discussed.


Subject(s)
Pancreatic Neoplasms/therapy , Animals , Antineoplastic Agents/therapeutic use , Genetic Therapy , Humans , Neoplasm Metastasis , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/etiology
6.
Oncogene ; 18(32): 4554-63, 1999 Aug 12.
Article in English | MEDLINE | ID: mdl-10467400

ABSTRACT

The Rel/NF-kappaB transcription factors regulate the expression of many genes. The activity of RelA, a member of the Rel/NF-kappaB transcription factor family, is constitutively activated in the majority of pancreatic adenocarcinomas and cell lines. We report that the urokinase-type plasminogen activator (uPA), one of the critical proteases involved in tumor invasion and metastasis, is overexpressed in pancreatic tumor cells and its overexpression is induced by constitutive RelA activity. The uPA promoter contains an NF-kappaB binding site that directly mediates the induction of uPA expression by RelA. Expression of a dominant-negative IkappaBalpha mutant inhibits kappaB site-dependent transcriptional activation of a uPA promoter-CAT reporter gene. Treating the pancreatic tumor cell lines with the known NF-kappaB inhibitors, dexamethasone and n-tosylphenyalanine chloromethyl ketone (TPCK), abolishes constitutive RelA activity and uPA overexpression. These results show that uPA is one of the downstream target genes induced by constitutively activated RelA in human pancreatic tumor cells, and suggests that constitutive RelA activity may play a critical role in tumor invasion and metastasis. Inhibition of constitutive RelA in pancreatic tumor cells may reduce their invasive and metastatic potential.


Subject(s)
Adenocarcinoma/enzymology , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , NF-kappa B/metabolism , Pancreatic Neoplasms/enzymology , Urokinase-Type Plasminogen Activator/genetics , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Cell Adhesion , Dexamethasone/pharmacology , Humans , NF-kappa B/antagonists & inhibitors , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Tosylphenylalanyl Chloromethyl Ketone/pharmacology , Transcription Factor RelA , Transcription, Genetic , Tumor Cells, Cultured , Urokinase-Type Plasminogen Activator/biosynthesis
7.
J Gastrointest Surg ; 3(3): 263-77, 1999.
Article in English | MEDLINE | ID: mdl-10481119

ABSTRACT

The p53 tumor suppressor gene and the Bcl-2 proto-oncogene regulate cell cycle progression and apoptosis. We evaluated the expression of these molecular markers with standard pathologic prognostic variables in patients who received multimodality therapy for resectable adenocarcinoma of the pancreas to study the effect of p53 and Bcl-2 on survival duration. Immunohistochemical staining of archival material was performed to determine levels of expression of p53 and Bcl-2 proteins in 70 patients with adenocarcinoma of pancreatic origin. All patients underwent a potentially curative pancreaticoduodenectomy and standardized pathologic analysis of resected specimens. Potential pathologic and molecular prognostic variables were assessed for their effect on survival duration. Nuclear staining for p53 was observed in 33 (47%) of 70 specimens. Immunostaining for Bcl-2 was observed in 23 specimens (33%). A trend toward improved survival duration was seen in patients whose tumors stained positive for either p53 or Bcl-2. Negative staining for both markers predicted short survival (P = 0.01). By univariate and multivariate analyses, no single pathologic factor was associated with survival duration. Immunohistochemical staging using both p53 and Bcl-2 significantly predicted survival duration by univariate and multivariate analysis; patients whose tumors stained positively for p53 and/or overexpressed Bcl-2 had a significantly longer survival than those whose tumors stained negative for both proteins.


Subject(s)
Adenocarcinoma/surgery , Biomarkers, Tumor/analysis , Pancreatic Neoplasms/surgery , Proto-Oncogene Proteins c-bcl-2/analysis , Tumor Suppressor Protein p53/analysis , Adenocarcinoma/pathology , Analysis of Variance , Apoptosis/genetics , Cell Cycle/genetics , Cell Nucleus/ultrastructure , Coloring Agents , Combined Modality Therapy , Female , Forecasting , Gene Expression Regulation, Neoplastic/genetics , Genes, bcl-2/genetics , Genes, p53/genetics , Humans , Immunohistochemistry , Male , Middle Aged , Multivariate Analysis , Neoplasm Staging , Pancreatic Neoplasms/pathology , Pancreaticoduodenectomy , Prognosis , Prospective Studies , Proto-Oncogene Mas , Survival Rate
8.
Surgery ; 126(2): 399-405, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10455913

ABSTRACT

BACKGROUND: The activation of transcription factor nuclear factor-kappa B (NF-kappa B) by extracellular stimuli has been shown to protect cells from apoptotic cell death. Inhibition of NF-kappa B activity should result in increased tumor cell killing in response to apoptotic stimuli. This study evaluated the effect of inhibition of NF-kappa B on a series of sarcoma and normal cell lines. METHODS: Human sarcoma cell lines (HT1080, SKLMS-1, and MFH) and normal cell lines (NLF and BSMC) were infected with an adenoviral dominant-negative mutant Ad5I kappa B alpha M in vitro. Control cells were infected with the empty adenoviral vector and mock-infected with media alone. Viable cell counts were determined by microscopic evaluation on days 1 to 6 after infection. Cell proliferation was determined at 48 hours by MTT (1-[4,5-dimethylthiazol-2-yl]-3,5-dephenylformazan) assay. RESULTS: All cell lines showed evidence of successful adenoviral infection as evidenced by the infection of all cell lines with the adenoviral marker gene Ad5 LacZ. All the tumor cells were found to have a significant decrease in cell viability and proliferation after treatment with the Ad5I kappa B alpha M gene compared with both mock-infected cells and cells infected with empty vector (P < .0001). The normal cell lines, although able to be successfully infected, did not show a significant decrease in cell viability or proliferation with adenoviral-mediated I kappa B alpha M infection. CONCLUSIONS: Inhibition of NF-kappa B through adenoviral-mediated infection of the dominant-negative inhibitor I kappa B alpha M resulted in a significant decrease in tumor cell viability and proliferation while having no deleterious effect on normal cell lines. The Ad5I kappa B alpha M gene therefore could be potentially used as a clinical treatment for patients with soft-tissue sarcoma.


Subject(s)
Adenoviridae/genetics , Apoptosis , DNA-Binding Proteins/physiology , I-kappa B Proteins , NF-kappa B/antagonists & inhibitors , Sarcoma/therapy , Cell Division , Cell Survival , DNA-Binding Proteins/genetics , Humans , NF-KappaB Inhibitor alpha , Sarcoma/pathology , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/pharmacology
9.
Ann N Y Acad Sci ; 880: 31-7, 1999 Jun 30.
Article in English | MEDLINE | ID: mdl-10415848

ABSTRACT

The tumor suppressor gene deleted in pancreatic cancer locus 4 (Smad4/DPC4) is inactivated in about 50% of pancreatic adenocarcinomas. The role of DPC4 in the transforming growth factor-beta (TGF-beta) receptor-mediated signal transduction cascade in human pancreatic, colon, and breast carcinoma cell lines has been investigated by a number of laboratories. The results demonstrate that Smad4/DPC4 protein functions as a key transcription factor required in regulation of TGF-beta inducible gene expression and subsequent growth inhibition. Many transcription regulators that are involved in cell growth, differentiation, and oncogenesis have been identified and cloned. Yet paradoxically, it is much more difficult to identify the important downstream target genes responsible for the biological effects elicited by these transcription factors. Although numerous attempts have been made and different approaches have been used to identify the target genes, only limited success has been achieved. Our data show that p21waf1 is one of the Smad4/DPC4-regulated downstream target genes and suggest that overexpression of the Smad4/DPC4 gene can bypass TGF-beta receptor activation and reestablish one of the key regulatory controls of cell proliferation. Identification of the Smad-regulated downstream target genes responsible for diverse biological processes that they control will extend our understanding of the mechanism for cell cycle regulation and cell differentiation.


Subject(s)
Adenocarcinoma/genetics , Cyclins/genetics , DNA-Binding Proteins/physiology , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , Pancreatic Neoplasms/genetics , Signal Transduction , Trans-Activators/physiology , Transforming Growth Factor beta/metabolism , Cell Cycle/physiology , Cell Division/drug effects , Cyclin-Dependent Kinase Inhibitor p21 , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Smad4 Protein , Trans-Activators/genetics , Trans-Activators/metabolism , Transforming Growth Factor beta/pharmacology , Tumor Cells, Cultured
10.
J Biol Chem ; 274(31): 21495-8, 1999 Jul 30.
Article in English | MEDLINE | ID: mdl-10419449

ABSTRACT

The adenovirus E1A protein has been implicated in increasing cellular susceptibility to apoptosis induced by tumor necrosis factor (TNF); however, its mechanism of action is still unknown. Since activation of nuclear factor kappaB (NF-kappaB) has been shown to play an anti-apoptotic role in TNF-induced apoptosis, we examined apoptotic susceptibility and NF-kappaB activation induced by TNF in the E1A transfectants and their parental cells. Here, we reported that E1A inhibited activation of NF-kappaB and rendered cells more sensitive to TNF-induced apoptosis. We further showed that this inhibition was through suppression of IkappaB kinase (IKK) activity and IkappaB phosphorylation. Moreover, deletion of the p300 and Rb binding domains of E1A abolished its function in blocking IKK activity and IkappaB phosphorylation, suggesting that these domains are essential for the E1A function in down-regulating IKK activity and NF-kappaB signaling. However, the role of E1A in inhibiting IKK activity might be indirect. Nevertheless, our results suggest that inhibition of IKK activity by E1A is an important mechanism for the E1A-mediated sensitization of TNF-induced apoptosis.


Subject(s)
Adenovirus E1A Proteins/metabolism , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Adenovirus E1A Proteins/genetics , Apoptosis , Female , Humans , I-kappa B Kinase , Kinetics , Male , NF-kappa B/antagonists & inhibitors , Ovarian Neoplasms , Prostatic Neoplasms , Protein Serine-Threonine Kinases/antagonists & inhibitors , Transfection , Tumor Cells, Cultured
11.
Clin Cancer Res ; 5(1): 119-27, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9918209

ABSTRACT

Pancreatic adenocarcinoma is a leading cause of adult cancer mortality in the United States. Recent studies have revealed that point mutation of the K-ras oncogene is a common event in pancreatic cancer, and oncogenesis mediated by Ras may also involve activation of Rel/nuclear factor (NF)-kappa B transcription factors. Furthermore, the c-rel member of Rel/NF-kappa B transcription factor family was first identified as a cellular homologue of the v-rel oncogene, suggesting that other members of the Rel/NF-kappa B family are potentially oncogenes. We therefore investigated the possibility that Rel/NF-kappa B transcription factors are activated in pancreatic cancer. Immunohistochemical analysis, Western blot and Northern blot analysis, electrophoretic mobility shift assays, and chloramphenicol acetyltransferase assays were performed to determine RelA activity in human pancreatic adenocarcinomas and normal tissues and nontumorigenic or tumorigenic cell lines. RelA, the p65 subunit of NF-kappa B, was constitutively activated in approximately 67% (16 of 24) of pancreatic adenocarcinomas but not in normal pancreatic tissues. Constitutive RelA activity was also detected in 9 of 11 human pancreatic tumor cell lines but not in nontumorigenic Syrian golden hamster cell lines. I kappa B alpha, a previously identified NF-kappa B-inducible gene, was overexpressed in human pancreatic tumor tissues and cell lines, and RelA activation could be inhibited by curcumin and dominant-negative mutants of I kappa B alpha, raf, and MEKK1. This is the first report demonstrating constitutive activation of RelA in nonlymphoid human cancer. These data are consistent with the possibility that RelA is constitutively activated by the upstream signaling pathway involving Ras and mitogen-activated protein kinases in pancreatic tumor cells. Constitutive RelA activity may play a key role in pancreatic tumorigenesis through activation of its downstream target genes.


Subject(s)
Adenocarcinoma/metabolism , NF-kappa B/metabolism , Pancreatic Neoplasms/metabolism , Adenocarcinoma/genetics , Adult , Amino Acid Sequence , Animals , Cricetinae , DNA, Neoplasm/metabolism , Gene Expression Regulation, Neoplastic , Genes, ras , Humans , Immunohistochemistry , Mesocricetus , Molecular Sequence Data , Pancreatic Neoplasms/genetics , Point Mutation , Transcription Factor RelA , Tumor Cells, Cultured
12.
Ann Surg Oncol ; 5(8): 681-8, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9869513

ABSTRACT

BACKGROUND: The p53 tumor suppressor gene is mutated in up to 70% of pancreatic adenocarcinomas. We determined the effect of reintroduction of the wild-type p53 gene on proliferation and apoptosis in human pancreatic cancer cells using an adenoviral vector containing the wild-type p53 tumor suppressor gene. METHODS: Transduction efficiencies of six p53-mutant pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, CFPAC-1, MIA PaCa-2, and PANC-1) were determined using the reporter gene construct Ad5/CMV/beta-gal. Cell proliferation was monitored using a 3H-thymidine incorporation assay, Western blot analysis for p53 expression was performed, and DNA laddering and fluorescence-activated cell sorter analysis were used to assess apoptosis. p53 gene therapy was tested in vivo in a subcutaneous tumor model. RESULTS: The cell lines varied in transduction efficiency. The MIA PaCa-2 cells had the highest transduction efficiency, with 65% of pancreatic tumor cells staining positive for beta-galactosidase (beta-gal) at a multiplicity of infection (MOI) of 50. At the same MOI, only 15% of the CFPAC-1 cells expressed the beta-gal gene. Adenovirus-mediated p53 gene transfer suppressed growth of all human pancreatic cancer cell lines in a dose-dependent manner. Western blot analysis confirmed the presence of the p53 protein product at 48 hours after infection. DNA ladders demonstrated increased chromatin degradation, and fluorescence-activated cell sorter analysis demonstrated a four-fold increase in apoptotic cells at 48 and 72 hours following infection with Ad5/CMV/p53 in the MIA PaCa-2 and PANC-1 cells. Suppression of tumor growth mediated by induction of apoptosis was observed in vivo in an established nude mouse subcutaneous tumor model following intratumoral injections of Ad5/CMV/p53. CONCLUSIONS: Introduction of the wild-type p53 gene using an adenoviral vector in pancreatic cancer with p53 mutations induces apoptosis and inhibits cell growth. These data provide preliminary support for adenoviral mediated p53 tumor suppressor gene therapy of human pancreatic cancer.


Subject(s)
Adenocarcinoma/therapy , Apoptosis , Genes, p53 , Genetic Therapy , Pancreatic Neoplasms/therapy , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Adenoviridae/genetics , DNA Fragmentation , Flow Cytometry , Humans , Immunoblotting , In Situ Nick-End Labeling , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Transduction, Genetic , Tumor Cells, Cultured
13.
Cancer Res ; 58(24): 5656-61, 1998 Dec 15.
Article in English | MEDLINE | ID: mdl-9865717

ABSTRACT

The Smad4/DPC4 protein functions as a key transcription factor in transforming growth factor beta (TGF-beta) signaling pathways. However, the downstream target genes regulated by Smad4/DPC4 have not been identified until now. We previously demonstrated that the loss of TGF-beta-induced p21waf1 expression and growth inhibition correlates with inactivation of the Smad4/DPC4 gene. Now we show that transient overexpression of Smad4/DPC4 can induce p21waf1 expression, specific Smad4 DNA binding activity, SBE4-luc reporter gene activity, and subsequent growth inhibition in Smad4/DPC4-null cells and other carcinoma cells in the presence or absence of TGF-beta. Taken together, these data show that p21waf1 is one of the Smad4/DPC4-regulated downstream target genes and suggest that overexpression of the Smad4/DPC4 gene can bypass TGF-beta receptor activation and reestablish one of the key regulatory controls of cell proliferation.


Subject(s)
Carcinoma/genetics , Cyclins/metabolism , DNA-Binding Proteins/genetics , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor/physiology , Trans-Activators/genetics , Carcinoma/pathology , Cell Division , Cyclin-Dependent Kinase Inhibitor p21 , Humans , Signal Transduction , Smad4 Protein , Transforming Growth Factor beta/pharmacology , Tumor Cells, Cultured
14.
Ann Surg Oncol ; 5(4): 342-9, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9641456

ABSTRACT

BACKGROUND: Clinical trials have suggested a survival advantage for selected patients with metastatic pancreatic cancer treated with tamoxifen. We sought to identify the molecular mechanism by which tamoxifen inhibits human pancreatic cancer cell (HPCC) growth. METHODS: HPCCs were grown in tamoxifen and growth inhibition was determined by 3H-thymidine uptake and by the MTT assay; changes in cell viability were determined by cell counts. Cell cycle alterations were evaluated by FACS, and the induction of apoptosis was evaluated using the TUNEL assay. Total cellular RNA was isolated after tamoxifen treatment, and Northern blot analysis was performed for p21waf1. RESULTS: Tamoxifen inhibited HPCC growth as measured by inhibition of 3H-thymidine incorporation and by the MTT assay. However, there was no decrease in the total number of viable cells after 6 days of treatment with 10 microM of tamoxifen and no evident apoptosis, confirming the absence of a cytotoxic effect. Cell cycle analysis revealed cellular arrest in the G0/G1 phase, which correlated with p21waf1 mRNA upregulation in response to tamoxifen treatment. CONCLUSIONS: Tamoxifen inhibits HPCC growth by inducing G0/G1 arrest with an associated increase in p21waf1 mRNA expression. Tamoxifen is an effective inhibitor of HPCC growth in vitro and warrants further in vivo study.


Subject(s)
Antineoplastic Agents, Hormonal/therapeutic use , Cell Cycle/drug effects , Cyclins/biosynthesis , Gene Expression Regulation/drug effects , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/pathology , Tamoxifen/therapeutic use , Antineoplastic Agents, Hormonal/pharmacology , Apoptosis , Cell Division/drug effects , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/genetics , Flow Cytometry , Humans , RNA, Messenger/analysis , RNA, Neoplasm/analysis , Tamoxifen/pharmacology , Tumor Cells, Cultured
15.
DNA Cell Biol ; 17(5): 437-47, 1998 May.
Article in English | MEDLINE | ID: mdl-9628587

ABSTRACT

Contact is a vital mechanism used by cells to interact with their environment. Contact with living and nonliving elements adjacent to a cell is the basis for many common biological events ranging from growth regulation to metastasis to embryonic pattern formation. We describe the cloning and characterization of a novel density-regulated protein (drp) whose expression is increased in cultured cells at high density compared with cells at low density. A drp cDNA was isolated from the human teratocarcinoma cell line PA-1. Northern analysis with a drp probe revealed transcripts of 2.8 and 3.2 kb. The drp RNA was expressed in a variety of tissues, with the highest amounts in skeletal and cardiac muscle. Using antipeptide antisera, increasing amounts of a 70-kDa protein were detected using several experimental approaches in several cells lines as cell density is increased. Conditioned medium from high-density cells was unable to induce expression of drp in cells growing at low density. Similarly, growth arrest by serum starvation or transforming growth factor-beta (TGF-beta) treatment failed to elicit drp expression. We conclude that drp is a novel protein whose expression is increased at high cell density but not growth arrest.


Subject(s)
Cell Cycle Proteins/biosynthesis , Neoplasm Proteins/biosynthesis , Amino Acid Sequence , Base Sequence , Cell Count , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cloning, Molecular , Culture Media, Conditioned/pharmacology , Eukaryotic Initiation Factors , G1 Phase/genetics , Gene Expression Regulation/drug effects , Humans , Molecular Sequence Data , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Teratocarcinoma , Transforming Growth Factor beta/pharmacology , Tumor Cells, Cultured
16.
Cancer Res ; 58(7): 1538-43, 1998 Apr 01.
Article in English | MEDLINE | ID: mdl-9537261

ABSTRACT

Previous studies have shown that the negative cell cycle regulator WAF1/Cip1 is often overexpressed in human gliomas and that WAF1/Cip1 overexpression may be a factor in cancer chemoresistance. We established a doxycycline-inducible WAF1/Cip1 expression system in two glioblastoma cell lines and examined the role of WAF1/Cip1 in their response to the chemotherapy agents 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and cis-diamminedichloroplatinum (cisplatin), in an isogeneic background. Our results showed that the induction of WAF1/Cip1 expression rendered glioma cells resistant to cell death induced by BCNU and cisplatin. Using an in vivo host-cell reactivation DNA repair assay, we demonstrated that WAF1/Cip1 enhances the repair of BCNU-induced DNA damage. We conclude that WAF1/Cip1 allows repair of BCNU- and cisplatin-damaged DNA and protects glioma cells from chemotherapy agent-induced apoptosis. Thus, blocking WAF1/Cip1 production or function may serve as a useful chemosensitization regimen for glioma.


Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , Antineoplastic Agents/pharmacology , Carmustine/pharmacology , Cisplatin/pharmacology , Cyclins/biosynthesis , Drug Resistance, Multiple , Glioblastoma/drug therapy , Glioblastoma/metabolism , Cell Death/drug effects , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/genetics , DNA Damage , DNA Repair , DNA, Neoplasm/drug effects , DNA, Neoplasm/metabolism , Doxorubicin/pharmacology , Doxycycline/pharmacology , Drug Resistance, Neoplasm , Gene Expression Regulation, Neoplastic/drug effects , Glioblastoma/genetics , Humans , Transfection , Tumor Cells, Cultured/drug effects
17.
Surg Oncol Clin N Am ; 7(1): 183-97, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9443995

ABSTRACT

Pancreaticoduodenectomy is performed on carefully selected patients as part of a protocol-based clinical research program emphasizing the importance of multimodality management for patients with potentially resectable adenocarcinoma of the pancreatic head. Treatment schemas emphasize the importance of minimizing toxicity and treatment duration, while attempting to improve therapeutic efficacy. Cytotoxicity is enhanced by combining radiation therapy with more potent radiation-sensitizing agents. Because of the high incidence of liver metastases, systemic therapy is continued after chemoradiation and surgery with systemic agents of low toxicity directed at specific molecular events involved in pancreatic tumorigenesis such as inhibition of angiogenesis, induction of apoptosis, or arrest of the cell cycle.


Subject(s)
Adenocarcinoma/therapy , Pancreatic Neoplasms/therapy , Antineoplastic Agents/therapeutic use , Chemotherapy, Adjuvant , Humans , Pancreaticoduodenectomy , Radiation-Sensitizing Agents/therapeutic use , Radiotherapy, Adjuvant
18.
Cancer Res ; 58(1): 38-41, 1998 Jan 01.
Article in English | MEDLINE | ID: mdl-9426054

ABSTRACT

Pancreas cancer is the fourth and fifth leading cause of cancer death for men and women, respectively, in the United States. Although the etiology of this cancer is poorly understood, smoking and dietary fat have been implicated by epidemiological studies. To test the hypothesis that DNA damage derived from carcinogen exposure and diet is involved in pancreatic carcinogenesis, aromatic and lipid peroxidation-related DNA adducts in 13 normal tissues adjacent to tumor and 20 tumors from pancreatic cancer patients were analyzed by 32P-postlabeling. Normal pancreatic tissues from 5 nonpancreatic cancer patients and 19 healthy organ donors served as controls. To correlate the DNA adduct level with patients' characteristics, information on age, sex, body mass index, and smoking status of pancreatic cancer patients were collected from medical records. A significantly higher level of total DNA adducts was detected in pancreatic cancer patients as compared with controls. The mean level of adducts/10(8) nucleotides in adjacent normal pancreatic tissues from pancreatic cancer patients (A tissues) was 102 +/- 21 compared with 39 +/- 6 and 13 +/- 1 in pancreatic tumor tissues (T tissues) and normal pancreatic tissues from controls (C tissues), respectively. Among the adducts observed, one single aromatic adduct (spot 1) was present in 100, 90, and 0% of the A, T, and C tissues, respectively. Two novel clusters of adducts (spots 2 and 3) were observed in 11 of 13, 12 of 20, and 2 of 24 of A, T, and C tissues, respectively, and the presence of these adducts was positively correlated with smoking status. In addition, the previously defined smoking-related diagonal radioactive zone was detected in three A samples only, although 50% (10 of 20) of the patients with pancreatic cancers in this study were ever smokers. Putative lipid peroxidation-related adducts were detected in all samples examined and were significantly higher in A than in T and C samples. Multiple regression analyses showed that body mass index was positively correlated to the levels of spot 1 and the lipid peroxidation-related adducts in A tissues and the total aromatic adducts in tumors. Smoking was also positively correlated to the level of total adducts. These observations are consistent with previous epidemiological findings and support the hypothesis that DNA damage related to carcinogen exposure and lipid peroxidation is involved in human pancreatic carcinogenesis.


Subject(s)
DNA Adducts/analysis , Pancreas/chemistry , Pancreatic Neoplasms/chemistry , Adult , Age Factors , Aged , Aged, 80 and over , Body Mass Index , Female , Humans , Lipid Peroxidation , Male , Middle Aged , Regression Analysis , Smoking/adverse effects
19.
Cancer Res ; 57(18): 3929-34, 1997 Sep 15.
Article in English | MEDLINE | ID: mdl-9307274

ABSTRACT

The tumor suppressor gene deleted in pancreatic cancer locus 4 (DPC4) is inactivated in about 50% of pancreatic adenocarcinomas. DPC4 was found to be homologous to Smad4 and may function as a transcription factor in the transforming growth factor beta (TGF-beta) receptor-mediated signal transduction pathway. We have investigated the role of DPC4 in the TGF-beta receptor-mediated signal transduction cascade in five human pancreatic cancer cell lines (Panc-1, MDAPanc-28, HS766T, Capan-1, and MiaPaCa-2). Our results demonstrate that the loss of responsiveness to TGF-beta-induced growth inhibition correlates with the loss of expression of DPC4. We have shown that TGF-beta induces p21waf1 expression in Panc-1 cells, whereas no induction of p21waf1 expression by TGF-beta was detected in the other four cell lines lacking either DPC4 expression or the TGF-beta type II receptor. No increase in p21waf1 mRNA stability was observed after treatment with TGF-beta, which suggests that the induction of p21waf1 in Panc-1 cells is transcriptionally regulated by TGF-beta. Our data also demonstrate that the expression of DPC4 is directly involved in TGF-beta-mediated induction of the 3TP-lux reporter gene, which contains a known TGF-beta-inducible plasminogen activator inhibitor promoter. These data suggest that: (a) TGF-beta-mediated induction of p21waf1 and subsequent growth inhibition require the expression of DPC4; (b) p21waf1 is a downstream target gene of DPC4; and (c) transfection of the DPC4 gene restores the TGF-beta-inducible gene expression. Inactivation of the tumor suppressor gene DPC4 and other components of the TGF-beta signal cascades may abolish one of the key negative controls of cell proliferation in pancreatic adenocarcinomas.


Subject(s)
Adenocarcinoma/genetics , Cyclins/genetics , DNA-Binding Proteins , Genes, Tumor Suppressor , Pancreatic Neoplasms/genetics , Trans-Activators/physiology , Transforming Growth Factor beta/physiology , Adenocarcinoma/pathology , Cell Division , Cyclin-Dependent Kinase Inhibitor p21 , Gene Expression Regulation, Neoplastic , Humans , Pancreatic Neoplasms/pathology , RNA, Messenger/genetics , RNA, Neoplasm/genetics , Smad4 Protein , Tumor Cells, Cultured
20.
Ann Surg Oncol ; 4(2): 149-55, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9084852

ABSTRACT

BACKGROUND: In a small pilot study, thymostimulin (TP-1) produced tumor regression in almost 50% of patients with hepatocellular cancer (HCC) who were treated with TP-1 alone. However, the mechanism of the TP-1-mediated antitumor effect against HCC is unknown. METHODS: Human hepatocytes and Kupffer cells were isolated from liver biopsy specimens by collagenase infusion and counterflow elutriation. Hepatocytes and Kupffer cells were incubated in vitro with clinically relevant doses of TP-1. Cell-free supernatant levels for a panel of growth factors and monokines were determined by enzyme-linked immunosorbent assay. The cytotoxic activity of TP-1 alone of TP-1-stimulated hepatocyte and Kupffer cell supernatants against Hep G2 and Hep 3B human HCC cells in vitro was measured by MIT assay. RESULTS: Doses of TP-1 up to 100 micrograms/ml produced no cytotoxicity against Hep G2 or Hep 3B cells. Furthermore, supernatants from TP-1-treated hepatocytes produced no cytotoxicity against Hep G2 or Hep 3B cells, and TP-1 did not stimulate the release of transforming growth factor (TGF)-alpha, TGF-beta, or hepatocyte growth factor. TP-1-treated Kupffer cell supernatants produced significant cytotoxicity against Hep G2 cells but produced no cytotoxicity against Hep 3B cells. Kupffer cells stimulated by TP-1 released significant amounts of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 alpha, and IL-6 compared with control Kupffer cells (p < 0.01). The activity of TP-1-treated Kupffer cell supernatants against Hep G2 cells was blocked by anti-TNF-alpha antibodies, whereas neither anti-IL-1 alpha nor anti-IL-6 antibodies blocked cytotoxicity. CONCLUSIONS: These results demonstrate that TP-1 cytotoxicity against human HCC cells is not mediated directly or through hepatocytes, but occurs through activation of Kupffer cells and release of TNF-alpha. Understanding the mechanism of TP-1 cytotoxicity against human HCC has been used to plan a phase 1 trial of TP-1 combined with regional infusion of doxorubicin to treat unresectable HCC.


Subject(s)
Adjuvants, Immunologic/pharmacology , Carcinoma, Hepatocellular/pathology , Kupffer Cells/drug effects , Kupffer Cells/immunology , Liver Neoplasms, Experimental/pathology , Thymus Extracts/pharmacology , Animals , Carcinoma, Hepatocellular/immunology , Cells, Cultured , Cytotoxicity, Immunologic/drug effects , Humans , Liver Neoplasms, Experimental/immunology , Monokines/biosynthesis
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