26 results on '"Helfrich BA"'
Search Results
2. Eribulin inhibits the growth of small cell lung cancer cell lines alone and with radiotherapy.
- Author
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Helfrich BA, Gao D, and Bunn PA Jr
- Subjects
- Apoptosis, Caspase 3 metabolism, Cell Cycle Checkpoints, Cell Line, Tumor, Cell Proliferation, Chemoradiotherapy, Humans, Lung Neoplasms pathology, Lung Neoplasms radiotherapy, Small Cell Lung Carcinoma pathology, Small Cell Lung Carcinoma radiotherapy, Antineoplastic Agents therapeutic use, Furans therapeutic use, Ketones therapeutic use, Lung Neoplasms drug therapy, Small Cell Lung Carcinoma drug therapy, Tubulin Modulators therapeutic use
- Abstract
Objectives: Small cell lung cancer (SCLC) patients of all stages are treated with etoposide and cisplatin or carboplatin with or without surgery or chest radiotherapy. Initial response rates are ≥70% however the majority of patients relapse and are resistant to additional therapies due to pan-resistance to these salvage therapies. Therefore, new treatments are urgently needed. The non-taxane microtubule inhibitor eribulin has produced responses in heavily pretreated breast cancer patients. We evaluated the efficacy of eribulin alone and in combination with radiation in a panel of SCLC cell lines established from patients prior to or after receiving chemotherapy and or radiation., Material and Methods: Growth inhibition by eribulin alone, radiation alone and the combination was assessed by MTS assay and clonogenic survival. Eribulin induced cell cycle arrest was evaluated by FACS. Apoptosis was evaluated by using the Caspase-GLO 3/7 luminescent plate assay and by the Vybrant apoptosis assay with analysis by FACS., Results: Eribulin mesylate inhibited the growth of all 17-SCLC lines at concentrations of ≤10 nM which is a clinically achievable dose. Growth inhibition was not significantly different between cell lines established prior to or after chemotherapy (p = .5). Concurrent eribulin + radiation induced a greater G2-M arrest, an increase in apoptotic cells and increased growth inhibition over radiation alone., Conclusions: Eribulin was highly active alone and in combination with radiation in treatment naïve SCLC lines and lines established from previously treated patients. In vivo pre-clinical studies of eribulin alone and in combination with radiation should be considered in SCLC cell lines., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2018
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3. AZ1366: An Inhibitor of Tankyrase and the Canonical Wnt Pathway that Limits the Persistence of Non-Small Cell Lung Cancer Cells Following EGFR Inhibition.
- Author
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Scarborough HA, Helfrich BA, Casás-Selves M, Schuller AG, Grosskurth SE, Kim J, Tan AC, Chan DC, Zhang Z, Zaberezhnyy V, Bunn PA, and DeGregori J
- Subjects
- Animals, Carcinoma, Non-Small-Cell Lung genetics, Carcinoma, Non-Small-Cell Lung pathology, Cell Line, Tumor, Cell Proliferation drug effects, Drug Resistance, Neoplasm genetics, Drug Synergism, Gefitinib, Humans, Mice, Neoplasm Recurrence, Local drug therapy, Neoplasm Recurrence, Local genetics, Neoplasm Recurrence, Local pathology, Quinazolines administration & dosage, Wnt Signaling Pathway drug effects, Xenograft Model Antitumor Assays, Carcinoma, Non-Small-Cell Lung drug therapy, Enzyme Inhibitors administration & dosage, ErbB Receptors antagonists & inhibitors, Tankyrases antagonists & inhibitors
- Abstract
Purpose: The emergence of EGFR inhibitors such as gefitinib, erlotinib, and osimertinib has provided novel treatment opportunities in EGFR-driven non-small cell lung cancer (NSCLC). However, most patients with EGFR-driven cancers treated with these inhibitors eventually relapse. Recent efforts have identified the canonical Wnt pathway as a mechanism of protection from EGFR inhibition and that inhibiting tankyrase, a key player in this pathway, is a potential therapeutic strategy for the treatment of EGFR-driven tumors. Experimental Design: We performed a preclinical evaluation of tankyrase inhibitor AZ1366 in combination with multiple EGFR-inhibitors across NSCLC lines, characterizing its antitumor activity, impingement on canonical Wnt signaling, and effects on gene expression. We performed pharmacokinetic and pharmacodynamic profiling of AZ1366 in mice and evaluated its therapeutic activity in an orthotopic NSCLC model. Results: In combination with EGFR inhibitors, AZ1366 synergistically suppressed proliferation of multiple NSCLC lines and amplified global transcriptional changes brought about by EGFR inhibition. Its ability to work synergistically with EGFR inhibition coincided with its ability to modulate the canonical Wnt pathway. Pharmacokinetic and pharmacodynamic profiling of AZ1366-treated orthotopic tumors demonstrated clinically relevant serum drug levels and intratumoral target inhibition. Finally, coadministration of an EGFR inhibitor and AZ1366 provided better tumor control and improved survival for Wnt-responsive lung cancers in an orthotopic mouse model. Conclusions: Tankyrase inhibition is a potent route of tumor control in EGFR-dependent NSCLC with confirmed dependence on canonical Wnt signaling. These data strongly support further evaluation of tankyrase inhibition as a cotreatment strategy with EGFR inhibition in an identifiable subset of EGFR-driven NSCLC. Clin Cancer Res; 23(6); 1531-41. ©2016 AACR ., (©2016 American Association for Cancer Research.)
- Published
- 2017
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4. Barasertib (AZD1152), a Small Molecule Aurora B Inhibitor, Inhibits the Growth of SCLC Cell Lines In Vitro and In Vivo.
- Author
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Helfrich BA, Kim J, Gao D, Chan DC, Zhang Z, Tan AC, and Bunn PA Jr
- Subjects
- Animals, Aurora Kinase B antagonists & inhibitors, Cell Line, Tumor, Cell Proliferation drug effects, Cluster Analysis, Disease Models, Animal, Dose-Response Relationship, Drug, Gene Amplification, Gene Expression Profiling, Gene Expression Regulation, Neoplastic drug effects, Genes, myc, Histones metabolism, Humans, Lung Neoplasms drug therapy, Lung Neoplasms metabolism, Lung Neoplasms pathology, Mice, Phosphorylation, Polyploidy, Small Cell Lung Carcinoma drug therapy, Small Cell Lung Carcinoma metabolism, Small Cell Lung Carcinoma pathology, Transcriptome, Tumor Burden drug effects, Xenograft Model Antitumor Assays, Antineoplastic Agents pharmacology, Aurora Kinase B metabolism, Organophosphates pharmacology, Protein Kinase Inhibitors pharmacology, Quinazolines pharmacology
- Abstract
Small-cell lung cancer (SCLC) cells have rapid proliferation, universal Rb inactivation, and high rates of MYC family amplification, making aurora kinase inhibition a natural target. Preclinical studies have demonstrated activity for Aurora A and pan-Aurora inhibitors with some relationship to MYC family expression. A clinical trial showed activity for an Aurora kinase A inhibitor, but no biomarkers were evaluated. We screened a panel of 23 SCLC lines with and without MYC family gene amplification or high MYC family gene expression for growth inhibition by the highly potent, selective aurora kinase B inhibitor barasertib. Nine of the SCLC lines were very sensitive to growth inhibition by barasertib, with IC
50 values of <50 nmol/L and >75% growth inhibition at 100 nmol/L. Growth inhibition correlated with cMYC amplification (P = 0.018) and cMYC gene expression (P = 0.026). Sensitive cell lines were also enriched in a published MYC gene signature (P = 0.042). In vivo, barasertib inhibited the growth of xenografts established from an SCLC line that had high cMYC gene expression, no cMYC amplification, and was positive for the core MYC gene signature. Our studies suggest that SCLC tumors with cMYC amplification/high gene expression will frequently respond to Aurora B inhibitors and that clinical studies coupled with predictive biomarkers are indicated. Mol Cancer Ther; 15(10); 2314-22. ©2016 AACR., Competing Interests: AstraZeneca provided partial funding for these studies through a sponsored research agreement. The authors have no stock or commercial involvement with AstraZeneca., (©2016 American Association for Cancer Research.)- Published
- 2016
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5. FGFR1 mRNA and protein expression, not gene copy number, predict FGFR TKI sensitivity across all lung cancer histologies.
- Author
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Wynes MW, Hinz TK, Gao D, Martini M, Marek LA, Ware KE, Edwards MG, Böhm D, Perner S, Helfrich BA, Dziadziuszko R, Jassem J, Wojtylak S, Sejda A, Gozgit JM, Bunn PA Jr, Camidge DR, Tan AC, Hirsch FR, and Heasley LE
- Subjects
- Adenocarcinoma drug therapy, Adenocarcinoma genetics, Adenocarcinoma metabolism, Blotting, Western, Carcinoma, Non-Small-Cell Lung drug therapy, Carcinoma, Squamous Cell drug therapy, Carcinoma, Squamous Cell genetics, Carcinoma, Squamous Cell metabolism, Cell Proliferation, Cohort Studies, Follow-Up Studies, Gene Amplification, Humans, Imidazoles pharmacology, Immunoenzyme Techniques, Lung Neoplasms drug therapy, Lung Neoplasms genetics, Lung Neoplasms metabolism, Neoplasm Staging, Pyridazines pharmacology, RNA, Messenger genetics, Real-Time Polymerase Chain Reaction, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction, Tumor Cells, Cultured, Carcinoma, Non-Small-Cell Lung genetics, Carcinoma, Non-Small-Cell Lung metabolism, Drug Resistance, Neoplasm genetics, Gene Dosage, Protein Kinase Inhibitors pharmacology, Receptor, Fibroblast Growth Factor, Type 1 genetics, Receptor, Fibroblast Growth Factor, Type 1 metabolism
- Abstract
Purpose: FGFR1 gene copy number (GCN) is being evaluated as a biomarker for FGFR tyrosine kinase inhibitor (TKI) response in squamous cell lung cancers (SCC). The exclusive use of FGFR1 GCN for predicting FGFR TKI sensitivity assumes increased GCN is the only mechanism for biologically relevant increases in FGFR1 signaling. Herein, we tested whether FGFR1 mRNA and protein expression may serve as better biomarkers of FGFR TKI sensitivity in lung cancer., Experimental Design: Histologically diverse lung cancer cell lines were submitted to assays for ponatinib sensitivity, a potent FGFR TKI. A tissue microarray composed of resected lung tumors was submitted to FGFR1 GCN, and mRNA analyses and the results were validated with The Cancer Genome Atlas (TCGA) lung cancer data., Results: Among 58 cell lines, 14 exhibited ponatinib sensitivity (IC50 values ≤ 50 nmol/L) that correlated with FGFR1 mRNA and protein expression, but not with FGFR1 GCN or histology. Moreover, ponatinib sensitivity associated with mRNA expression of the ligands, FGF2 and FGF9. In resected tumors, 22% of adenocarcinomas and 28% of SCCs expressed high FGFR1 mRNA. Importantly, only 46% of SCCs with increased FGFR1 GCN expressed high mRNA. Lung cancer TCGA data validated these findings and unveiled overlap of FGFR1 mRNA positivity with KRAS and PIK3CA mutations., Conclusions: FGFR1 dependency is frequent across various lung cancer histologies, and FGFR1 mRNA may serve as a better biomarker of FGFR TKI response in lung cancer than FGFR1 GCN. The study provides important and timely insight into clinical testing of FGFR TKIs in lung cancer and other solid tumor types., (©2014 American Association for Cancer Research.)
- Published
- 2014
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6. A mechanism of resistance to gefitinib mediated by cellular reprogramming and the acquisition of an FGF2-FGFR1 autocrine growth loop.
- Author
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Ware KE, Hinz TK, Kleczko E, Singleton KR, Marek LA, Helfrich BA, Cummings CT, Graham DK, Astling D, Tan AC, and Heasley LE
- Abstract
Despite initial and often dramatic responses of epidermal growth factor receptor (EGFR)-addicted lung tumors to the EGFR-specific tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, nearly all develop resistance and relapse. To explore novel mechanisms mediating acquired resistance, we employed non-small-cell lung cancer (NSCLC) cell lines bearing activating mutations in EGFR and rendered them resistant to EGFR-specific TKIs through chronic adaptation in tissue culture. In addition to previously observed resistance mechanisms including EGFR-T790M 'gate-keeper' mutations and MET amplification, a subset of the seven chronically adapted NSCLC cell lines including HCC4006, HCC2279 and H1650 cells exhibited marked induction of fibroblast growth factor (FGF) 2 and FGF receptor 1 (FGFR1) mRNA and protein. Also, adaptation to EGFR-specific TKIs was accompanied by an epithelial to mesenchymal transition (EMT) as assessed by changes in CDH1, VIM, ZEB1 and ZEB2 expression and altered growth properties in Matrigel. In adapted cell lines exhibiting increased FGF2 and FGFR1 expression, measures of growth and signaling, but not EMT, were blocked by FGFR-specific TKIs, an FGF-ligand trap and FGFR1 silencing with RNAi. In parental HCC4006 cells, cell growth was strongly inhibited by gefitinib, although drug-resistant clones progress within 10 days. Combined treatment with gefitinib and AZD4547, an FGFR-specific TKI, prevented the outgrowth of drug-resistant clones. Thus, induction of FGF2 and FGFR1 following chronic adaptation to EGFR-specific TKIs provides a novel autocrine receptor tyrosine kinase-driven bypass pathway in a subset of lung cancer cell lines that are initially sensitive to EGFR-specific TKIs. The findings support FGFR-specific TKIs as potentially valuable additions to existing targeted therapeutic strategies with EGFR-specific TKIs to prevent or delay acquired resistance in EGFR-driven NSCLC.
- Published
- 2013
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7. Tankyrase and the canonical Wnt pathway protect lung cancer cells from EGFR inhibition.
- Author
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Casás-Selves M, Kim J, Zhang Z, Helfrich BA, Gao D, Porter CC, Scarborough HA, Bunn PA Jr, Chan DC, Tan AC, and DeGregori J
- Subjects
- Adenocarcinoma, Bronchiolo-Alveolar drug therapy, Adenocarcinoma, Bronchiolo-Alveolar enzymology, Adenocarcinoma, Bronchiolo-Alveolar genetics, Adenocarcinoma, Bronchiolo-Alveolar metabolism, Animals, Antineoplastic Agents pharmacology, Carcinoma, Non-Small-Cell Lung drug therapy, Carcinoma, Non-Small-Cell Lung enzymology, Carcinoma, Non-Small-Cell Lung genetics, Carcinoma, Non-Small-Cell Lung metabolism, Cell Line, Tumor, ErbB Receptors metabolism, Female, Gefitinib, Humans, Lung Neoplasms enzymology, Lung Neoplasms genetics, Mice, Mice, Nude, Quinazolines pharmacology, RNA, Small Interfering administration & dosage, RNA, Small Interfering genetics, Signal Transduction, Xenograft Model Antitumor Assays, ErbB Receptors antagonists & inhibitors, Lung Neoplasms drug therapy, Lung Neoplasms metabolism, Protein Kinase Inhibitors pharmacology, Tankyrases metabolism, Wnt Proteins metabolism
- Abstract
Lung cancer is the leading cause of death worldwide. Adenocarcinomas, the most common histologic subtype of non-small cell lung cancer (NSCLC), are frequently associated with activating mutations in the epidermal growth factor receptor (EGFR) gene. Although these patients often respond clinically to the EGFR tyrosine kinase inhibitors erlotinib and gefitinib, relapse inevitably occurs, suggesting the development of escape mechanisms that promote cell survival. Using a loss-of-function, whole genome short hairpin RNA (shRNA) screen, we identified that the canonical Wnt pathway contributes to the maintenance of NSCLC cells during EGFR inhibition, particularly the poly-ADP-ribosylating enzymes tankyrase 1 and 2 that positively regulate canonical Wnt signaling. Inhibition of tankyrase and various other components of the Wnt pathway with shRNAs or small molecules significantly increased the efficacy of EGFR inhibitors both in vitro and in vivo. Our findings therefore reveal a critical role for tankyrase and the canonical Wnt pathway in maintaining lung cancer cells during EGFR inhibition. Targeting the Wnt-tankyrase-β-catenin pathway together with EGFR inhibition may improve clinical outcome in patients with NSCLC., (©2012 AACR.)
- Published
- 2012
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8. ZEB1-responsive genes in non-small cell lung cancer.
- Author
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Gemmill RM, Roche J, Potiron VA, Nasarre P, Mitas M, Coldren CD, Helfrich BA, Garrett-Mayer E, Bunn PA, and Drabkin HA
- Subjects
- Blotting, Western, Cadherins genetics, Carcinoma, Non-Small-Cell Lung genetics, Cell Line, Tumor, Epithelial-Mesenchymal Transition, Homeodomain Proteins analysis, Homeodomain Proteins genetics, Humans, Lung Neoplasms genetics, Repressor Proteins analysis, Reverse Transcriptase Polymerase Chain Reaction, Serine Endopeptidases analysis, Transcription Factors analysis, Transcription Factors genetics, Zinc Finger E-box Binding Homeobox 2, Zinc Finger E-box-Binding Homeobox 1, Carcinoma, Non-Small-Cell Lung pathology, Homeodomain Proteins physiology, Lung Neoplasms pathology, Transcription Factors physiology
- Abstract
The epithelial to mesenchymal transition (EMT) is a developmental process enabling epithelial cells to gain a migratory mesenchymal phenotype. In cancer, this process contributes to metastases; however the regulatory signals and mechanistic details are not fully elucidated. Here, we sought to identify the subset of genes regulated in lung cancer by ZEB1, an E-box transcriptional repressor known to induce EMT. Using an Affymetrix-based expression database of 38 non-small cell lung cancer (NSCLC) cell lines, we identified 324 genes that correlated negatively with ZEB1 and 142 that were positively correlated. A mesenchymal gene pattern (low E-cadherin, high Vimentin or N-cadherin) was significantly associated with ZEB1 and ZEB2, but not with Snail, Slug, Twist1 or Twist2. Among eight genes selected for validation, seven were confirmed to correlate with ZEB1 by quantitative real-time RT-PCR in a series of 22 NSCLC cell lines, either negatively (CDS1, EpCAM, ESRP1, ESRP2, ST14) or positively (FGFR1, Vimentin). In addition, over-expression or knockdown of ZEB1 led to corresponding changes in gene expression, demonstrating that these genes are also regulated by ZEB1, either directly or indirectly. Of note, the combined knockdown of ZEB1 and ZEB2 led to apparent synergistic responses in gene expression. Furthermore, these responses were not restricted to artificial settings, since most genes were similarly regulated during a physiologic induction of EMT by TGF-β plus EGF. Finally, the absence of ST14 (matriptase) was linked to ZEB1 positivity in lung cancer tissue microarrays, implying that the regulation observed in vitro applies to the human disease. In summary, this study identifies a new set of ZEB-regulated genes in human lung cancer cells and supports the hypothesis that ZEB1 and ZEB2 are key regulators of the EMT process in this disease., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)
- Published
- 2011
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9. Rapidly acquired resistance to EGFR tyrosine kinase inhibitors in NSCLC cell lines through de-repression of FGFR2 and FGFR3 expression.
- Author
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Ware KE, Marshall ME, Heasley LR, Marek L, Hinz TK, Hercule P, Helfrich BA, Doebele RC, and Heasley LE
- Subjects
- Blotting, Western, Carcinoma, Non-Small-Cell Lung genetics, Carcinoma, Non-Small-Cell Lung metabolism, Carcinoma, Non-Small-Cell Lung pathology, Cell Line, Tumor, Coculture Techniques, Drug Resistance, Neoplasm genetics, ErbB Receptors antagonists & inhibitors, Extracellular Signal-Regulated MAP Kinases metabolism, Fibroblast Growth Factor 2 pharmacology, Fibroblast Growth Factor 7 pharmacology, Fibroblasts cytology, Fibroblasts drug effects, Fibroblasts metabolism, Gefitinib, Humans, Lung Neoplasms genetics, Lung Neoplasms metabolism, Lung Neoplasms pathology, Receptor, Fibroblast Growth Factor, Type 2 metabolism, Receptor, Fibroblast Growth Factor, Type 3 metabolism, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction drug effects, Gene Expression Regulation, Neoplastic drug effects, Protein Kinase Inhibitors pharmacology, Quinazolines pharmacology, Receptor, Fibroblast Growth Factor, Type 2 genetics, Receptor, Fibroblast Growth Factor, Type 3 genetics
- Abstract
Despite initial and sometimes dramatic responses of specific NSCLC tumors to EGFR TKIs, nearly all will develop resistance and relapse. Gene expression analysis of NSCLC cell lines treated with the EGFR TKI, gefitinib, revealed increased levels of FGFR2 and FGFR3 mRNA. Analysis of gefitinib action on a larger panel of NSCLC cell lines verified that FGFR2 and FGFR3 expression is increased at the mRNA and protein level in NSCLC cell lines in which the EGFR is dominant for growth signaling, but not in cell lines where EGFR signaling is absent. A luciferase reporter containing 2.5 kilobases of fgfr2 5' flanking sequence was activated after gefitinib treatment, indicating transcriptional regulation as a contributing mechanism controlling increased FGFR2 expression. Induction of FGFR2 and FGFR3 protein as well as fgfr2-luc activity was also observed with Erbitux, an EGFR-specific monoclonal antibody. Moreover, inhibitors of c-Src and MEK stimulated fgfr2-luc activity to a similar degree as gefitinib, suggesting that these pathways may mediate EGFR-dependent repression of FGFR2 and FGFR3. Importantly, our studies demonstrate that EGFR TKI-induced FGFR2 and FGFR3 are capable of mediating FGF2 and FGF7 stimulated ERK activation as well as FGF-stimulated transformed growth in the setting of EGFR TKIs. In conclusion, this study highlights EGFR TKI-induced FGFR2 and FGFR3 signaling as a novel and rapid mechanism of acquired resistance to EGFR TKIs and suggests that treatment of NSCLC patients with combinations of EGFR and FGFR specific TKIs may be a strategy to enhance efficacy of single EGFR inhibitors.
- Published
- 2010
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10. Fibroblast growth factor (FGF) and FGF receptor-mediated autocrine signaling in non-small-cell lung cancer cells.
- Author
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Marek L, Ware KE, Fritzsche A, Hercule P, Helton WR, Smith JE, McDermott LA, Coldren CD, Nemenoff RA, Merrick DT, Helfrich BA, Bunn PA Jr, and Heasley LE
- Subjects
- Cell Line, Tumor, Fibroblast Growth Factors genetics, Humans, RNA, Small Interfering metabolism, Receptors, Fibroblast Growth Factor genetics, Carcinoma, Non-Small-Cell Lung genetics, Fibroblast Growth Factors metabolism, Lung Neoplasms genetics, Receptors, Fibroblast Growth Factor metabolism, Signal Transduction genetics
- Abstract
Despite widespread expression of epidermal growth factor (EGF) receptors (EGFRs) and EGF family ligands in non-small-cell lung cancer (NSCLC), EGFR-specific tyrosine kinase inhibitors (TKIs) such as gefitinib exhibit limited activity in this cancer. We propose that autocrine growth signaling pathways distinct from EGFR are active in NSCLC cells. To this end, gene expression profiling revealed frequent coexpression of specific fibroblast growth factors (FGFs) and FGF receptors (FGFRs) in NSCLC cell lines. It is noteworthy that FGF2 and FGF9 as well as FGFR1 IIIc and/or FGFR2 IIIc mRNA and protein are frequently coexpressed in NSCLC cell lines, especially those that are insensitive to gefitinib. Specific silencing of FGF2 reduced anchorage-independent growth of two independent NSCLC cell lines that secrete FGF2 and coexpress FGFR1 IIIc and/or FGFR2 IIIc. Moreover, a TKI [(+/-)-1-(anti-3-hydroxy-cyclopentyl)-3-(4-methoxy-phenyl)-7-phenylamino-3,4-dihydro-1H-pyrimido-[4,5-d]pyrimidin-2-one (RO4383596)] that targets FGFRs inhibited basal FRS2 and extracellular signal-regulated kinase phosphorylation, two measures of FGFR activity, as well as proliferation and anchorage-independent growth of NSCLC cell lines that coexpress FGF2 or FGF9 and FGFRs. By contrast, RO4383596 influenced neither signal transduction nor growth of NSCLC cell lines lacking FGF2, FGF9, FGFR1, or FGFR2 expression. Thus, FGF2, FGF9 and their respective high-affinity FGFRs comprise a growth factor autocrine loop that is active in a subset of gefitinib-insensitive NSCLC cell lines.
- Published
- 2009
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11. EGFR regulation by microRNA in lung cancer: correlation with clinical response and survival to gefitinib and EGFR expression in cell lines.
- Author
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Weiss GJ, Bemis LT, Nakajima E, Sugita M, Birks DK, Robinson WA, Varella-Garcia M, Bunn PA Jr, Haney J, Helfrich BA, Kato H, Hirsch FR, and Franklin WA
- Subjects
- Carcinoma, Non-Small-Cell Lung drug therapy, Carcinoma, Non-Small-Cell Lung mortality, Cell Line, Tumor, Gefitinib, Gene Expression, Humans, Lung Neoplasms drug therapy, Lung Neoplasms mortality, MicroRNAs, Survival Analysis, Antineoplastic Agents therapeutic use, Carcinoma, Non-Small-Cell Lung genetics, Genes, erbB-1 genetics, Lung Neoplasms genetics, Quinazolines therapeutic use
- Abstract
Background: Allelic loss in chromosome 3p is one of the most frequent and earliest genetic events in lung carcinogenesis. We investigated if the loss of microRNA-128b, a microRNA located on chromosome 3p and a putative regulator of epidermal growth factor receptor (EGFR), correlated with response to targeted EGFR inhibition. Loss of microRNA-128b would be equivalent to losing a tumor suppressor gene because it would allow increased expression of EGFR., Patients and Methods: We initially showed that microRNA-128b is a regulator of EGFR in non-small-cell lung cancer (NSCLC) cell lines. We tested microRNA-128b expression levels by quantitative RT-PCR, genomic copy number by quantitative PCR, and mutations in the mature microRNA-128b by sequencing. We determined whether microRNA-128b loss of heterozygosity (LOH) in 58 NSCLC patient samples correlated with response to gefitinib and evaluated EGFR expression and mutation status., Results: We determined that microRNA-128b directly regulates EGFR. MicroRNA-128b LOH was frequent in tumor samples and correlated significantly with clinical response and survival following gefitinib. EGFR expression and mutation status did not correlate with survival outcome., Conclusion: Identifying microRNA regulators of oncogenes could have far-reaching implications for lung cancer patients including improving patient selection for targeted agents, development of novel therapeutics, or development as early biomarkers of disease.
- Published
- 2008
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12. Combining halogen bonds and hydrogen bonds in the modular assembly of heteromeric infinite 1-D chains.
- Author
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Aakeröy CB, Desper J, Helfrich BA, Metrangolo P, Pilati T, Resnati G, and Stevenazzi A
- Abstract
Hydrogen bonds and halogen bonds operate in concert in the directed assembly of infinite 1-D chains in binary co-crystals of iodine iso-nicotinamide (2 : 2), 1 and tetrafluorodiiodobenzene iso-nicotinamide (1 : 2) 2.
- Published
- 2007
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13. Epithelial to mesenchymal transition predicts gefitinib resistance in cell lines of head and neck squamous cell carcinoma and non-small cell lung carcinoma.
- Author
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Frederick BA, Helfrich BA, Coldren CD, Zheng D, Chan D, Bunn PA Jr, and Raben D
- Subjects
- Animals, Antineoplastic Agents pharmacology, Cell Division drug effects, Cell Line, Tumor, Female, Gefitinib, Mice, Mice, Nude, Oligonucleotide Array Sequence Analysis, Carcinoma, Non-Small-Cell Lung pathology, Carcinoma, Squamous Cell pathology, Drug Resistance, Neoplasm, Epithelial Cells pathology, Head and Neck Neoplasms pathology, Lung Neoplasms pathology, Mesoderm pathology, Quinazolines pharmacology
- Abstract
The modest response of patients with head and neck squamous cell carcinoma (HNSCC) and non-small cell lung carcinoma (NSCLC) to epithelial growth factor receptor tyrosine kinase inhibitors such as gefitinib and erlotinib indicates the need for the development of biomarkers to predict response. We determined gefitinib sensitivity in a panel of HNSCC cell lines by a 5-day 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and confirmed these responses with analysis of downstream signaling by immunoblotting and cell cycle arrest. Basal gene expression profiles were then determined by microarray analysis and correlated with gefitinib response. These data were combined with previously reported NSCLC microarray results to generate a broader predictive index. Common markers of resistance between the two tumor types included genes associated with the epithelial to mesenchymal transition. We confirmed that increased protein expression of vimentin combined with the loss of E-cadherin, claudin 4, and claudin 7 by immunoblotting was associated with gefitinib resistance in both HNSCC and NSCLC cell lines. In addition, the loss of the Ca(2+)-independent cell-cell adhesion molecules EpCAM and TROP2 in resistant lines was confirmed by immunofluorescence. Tumor xenografts derived from the gefitinib-sensitive UM-SCC-2 were growth-delayed by gefitinib, whereas the gefitinib-resistant 1483 xenografts were unaffected. These data support a role for epithelial to mesenchymal transition in establishing gefitinib resistance for both HNSCC and NSCLC, and indicate that clinical trials should address whether these biomarkers will be useful for patient selection.
- Published
- 2007
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14. Antitumor activity of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib (ZD1839, Iressa) in non-small cell lung cancer cell lines correlates with gene copy number and EGFR mutations but not EGFR protein levels.
- Author
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Helfrich BA, Raben D, Varella-Garcia M, Gustafson D, Chan DC, Bemis L, Coldren C, Barón A, Zeng C, Franklin WA, Hirsch FR, Gazdar A, Minna J, and Bunn PA Jr
- Subjects
- Animals, Antineoplastic Agents therapeutic use, Apoptosis drug effects, Carcinoma, Non-Small-Cell Lung enzymology, Cell Cycle drug effects, Cell Line, Tumor, Cell Proliferation drug effects, DNA, Neoplasm analysis, Dose-Response Relationship, Drug, Drug Screening Assays, Antitumor, Female, Flow Cytometry, Gefitinib, Gene Expression Regulation, Neoplastic drug effects, Humans, In Situ Hybridization, Fluorescence, Lung Neoplasms enzymology, Male, Mice, Mice, Nude, Mutation, Predictive Value of Tests, Quinazolines therapeutic use, Signal Transduction drug effects, Structure-Activity Relationship, Transplantation, Heterologous, Antineoplastic Agents pharmacology, Carcinoma, Non-Small-Cell Lung drug therapy, ErbB Receptors antagonists & inhibitors, ErbB Receptors genetics, Gene Expression Regulation, Neoplastic genetics, Lung Neoplasms drug therapy, Quinazolines pharmacology
- Abstract
Purpose: Recognition that the epidermal growth factor receptor (EGFR) was a therapeutic target in non-small cell lung cancer (NSCLC) and other cancers led to development of the small-molecule receptor tyrosine kinase inhibitors gefitinib and erlotinib. Clinical trials established that EGFR tyrosine kinase inhibitors produced objective responses in a minority of NSCLC patients. We examined the sensitivity of 23 NSCLC lines with wild-type or mutated EGFR to gefitinib to determine genes/proteins related to sensitivity, including EGFR and HER2 cell surface expression, phosphorylated EGFR expression, EGFR gene copy number, and EGFR mutational status. Downstream cell cycle and signaling events were compared with growth-inhibitory effects., Experimental Design: We determined gefitinib sensitivity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, EGFR expression by fluorescence-activated cell sorting and immunohistochemistry, phosphorylated EGFR by Western blotting, EGFR gene copy number by fluorescence in situ hybridization, and EGFR mutation by sequencing. The cellular effects of gefitinib on cell cycle were determined by flow cytometry and the molecular effects of gefitinib EGFR inhibition on downstream signal proteins by Western blotting. Gefitinib in vivo effects were evaluated in athymic nude mice bearing sensitive and resistant NSCLC xenografts., Results: There was a significant correlation between EGFR gene copy number, EGFR gene mutations, and gefitinib sensitivity. EGFR protein was necessary but not sufficient for predicting sensitivity. Gefitinib-sensitive lines showed a G(1) cell cycle arrest and inactivation of downstream signaling proteins; resistant cell lines had no changes. The in vivo effects mirrored the in vitro effects., Conclusions: This panel of NSCLC lines characterized for gefitinib response was used to identify predictive molecular markers of response to gefitinib. Several of these have subsequently been shown to identify NSCLC patients likely to benefit from gefitinib therapy.
- Published
- 2006
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15. Baseline gene expression predicts sensitivity to gefitinib in non-small cell lung cancer cell lines.
- Author
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Coldren CD, Helfrich BA, Witta SE, Sugita M, Lapadat R, Zeng C, Barón A, Franklin WA, Hirsch FR, Geraci MW, and Bunn PA Jr
- Subjects
- Cadherins metabolism, Cluster Analysis, Drug Screening Assays, Antitumor, ErbB Receptors genetics, Flow Cytometry methods, Gefitinib, Gene Expression, Gene Expression Profiling classification, Humans, Inhibitory Concentration 50, Multigene Family, Mutation drug effects, Polymerase Chain Reaction methods, Protein Kinase Inhibitors, Proteome drug effects, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins p21(ras), Treatment Outcome, Tumor Cells, Cultured, ras Proteins, Antineoplastic Agents therapeutic use, Carcinoma, Non-Small-Cell Lung drug therapy, Lung Neoplasms drug therapy, Quinazolines therapeutic use
- Abstract
Tyrosine kinase inhibitors (TKI) of the epidermal growth factor receptor (EGFR) produce objective responses in a minority of patients with advanced-stage non-small cell lung cancer (NSCLC), and about half of all treated patients progress within 6 weeks of instituting therapy. Because the target of these agents is known, it should be possible to develop biological predictors of response, but EGFR protein levels have not been proven useful as a predictor of TKI response in patients and the mechanism of primary resistance is unclear. We used microarray gene expression profiling to uncover a pattern of gene expression associated with sensitivity to EGFR-TKIs by comparing NSCLC cell lines that were either highly sensitive or highly resistant to gefitinib. This sensitivity-associated expression profile was used to predict gefitinib sensitivity in a panel of NSCLC cell lines with known gene expression profiles but unknown gefitinib sensitivity. Gefitinib sensitivity was then determined for members of this test panel, and the microarray-based sensitivity prediction was correct in eight of nine NSCLC cell lines. Gene and protein expression differences were confirmed with a combination of quantitative reverse transcription-PCR, flow cytometry, and immunohistochemistry. This gene expression pattern related to gefitinib sensitivity was independent from sensitivity associated with EGFR mutations. Several genes associated with sensitivity encode proteins involved in HER pathway signaling or pathways that interrelate to the HER signaling pathway. Some of these genes could be targets of pharmacologic interventions to overcome primary resistance.
- Published
- 2006
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16. A high-yielding supramolecular reaction.
- Author
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Aakeröy CB, Beatty AM, and Helfrich BA
- Abstract
The X-ray crystal structure determinations of twelve cocrystals involving iso-nicotinamide and a variety of carboxylic acids have revealed a very consistent pattern of hydrogen-bond preferences. The combination of a monocarboxylic acid, an amide, and a pyridine moiety leads, in every case, to discrete "supermolecules" (consisting of two molecules of iso-nicotinamide and two molecules of the relevant carboxylic acid) with well-defined and robust connectivity. The two dominant (regularly occurring) supramolecular synthons in these crystal structures are (1) the heteromeric carboxylic acid.pyridine hydrogen bond and (2) a self-complementary amide.amide hydrogen-bond interaction, both of which prevail in the presence of widely differing chemical functionalities. In four of these cocrystals, a dicarboxylic acid is employed, which alters the structural outcome from discrete entities to infinite assemblies (or to a hexameric complex in a "U-shaped" dicarboxylic acid), which is fully expected since the two primary supramolecular synthons remain intact. This structural study shows that iso-nicotinamide is a supramolecular reagent that can produce well-defined supermolecules (containing carboxylic acids) in very high yields.
- Published
- 2002
- Full Text
- View/download PDF
17. Solution and solid-state models of peptide CH...O hydrogen bonds.
- Author
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Baures PW, Beatty AM, Dhanasekaran M, Helfrich BA, Pérez-Segarra W, and Desper J
- Subjects
- Crystallography, X-Ray, Dimethyl Sulfoxide chemistry, Hydrogen Bonding, Models, Molecular, Nuclear Magnetic Resonance, Biomolecular, Protein Structure, Secondary, Solutions, Amides chemistry, Peptides chemistry
- Abstract
Fumaramide derivatives were analyzed in solution by (1)H NMR spectroscopy and in the solid state by X-ray crystallography in order to characterize the formation of CH...O interactions under each condition and to thereby serve as models for these interactions in peptide and protein structure. Solutions of fumaramides at 10 mM in CDCl(3) were titrated with DMSO-d(6), resulting in chemical shifts that moved downfield for the CH groups thought to participate in CH...O=S(CD(3))(2) hydrogen bonds concurrent with NH...O=S(CD(3))(2) hydrogen bonding. In this model, nonparticipating CH groups under the same conditions showed no significant change in chemical shifts between 0.0 and 1.0 M DMSO-d(6) and then moved upfield at higher DMSO-d(6) concentrations. At concentrations above 1.0 M DMSO-d(6), the directed CH...O=S(CD(3))(2) hydrogen bonds provide protection from random DMSO-d(6) contact and prevent the chemical shifts for participating CH groups from moving upfield beyond the original value observed in CDCl(3). X-ray crystal structures identified CH...O=C hydrogen bonds alongside intermolecular NH...O=C hydrogen bonding, a result that supports the solution (1)H NMR spectroscopy results. The solution and solid-state data therefore both provide evidence for the presence of CH...O hydrogen bonds formed concurrent with NH...O hydrogen bonding in these structures. The CH...O=C hydrogen bonds in the X-ray crystal structures are similar to those described for antiparallel beta-sheet structure observed in protein X-ray crystal structures.
- Published
- 2002
- Full Text
- View/download PDF
18. Syntheses and bioactivities of substituted 9,10-dihydro-9,10-[1,2]benzenoanthracene-1,4,5,8-tetrones. Unusual reactivities with amines.
- Author
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Hua DH, Tamura M, Huang X, Stephany HA, Helfrich BA, Perchellet EM, Sperfslage BJ, Perchellet JP, Jiang S, Kyle DE, and Chiang PK
- Subjects
- Animals, Antimalarials chemistry, Antimalarials pharmacology, Antineoplastic Agents chemistry, Antineoplastic Agents pharmacology, Benz(a)Anthracenes chemistry, Benz(a)Anthracenes pharmacology, Catalysis, Crystallography, X-Ray, Drug Screening Assays, Antitumor, Hydrocarbons, Brominated chemical synthesis, Hydrocarbons, Brominated chemistry, Hydrocarbons, Brominated pharmacology, Inhibitory Concentration 50, Leukemia, Mice, Molecular Conformation, Molecular Structure, Plasmodium falciparum drug effects, Structure-Activity Relationship, Tumor Cells, Cultured drug effects, Amines chemistry, Antimalarials chemical synthesis, Antineoplastic Agents chemical synthesis, Benz(a)Anthracenes chemical synthesis
- Abstract
A number of substituted 9,10-dihydro-9,10-[1,2]benzenoanthracene-1,4,5,8-tetrones have been synthesized and their anticancer and antimalarial activities evaluated. A one-pot synthesis of 2,5,8-trimethoxy-9,10-dihydro-9,10-[1,2]benzenoanthracene-1,4-dione (4) was achieved by heating a mixture of 1,4-dimethoxyanthracene, methoxyhydroquinone, silver oxide, and zinc iodide in toluene. Regioselective bromination of 4 and 2-methoxy-9,10-dihydro-9,10-[1,2]benzenoanthracene-1,4,5,8-tetrone (7) with N-bromosuccinimide provided 2-bromo-3,5,8-trimethoxy-9,10-dihydro-9,10-[1,2]benzenoanthracene-1,4-dione and 2-bromo-3-methoxy-9,10-dihydro-9,10-[1,2]benzenoanthracene-1,4,5,8-tetrone (1), respectively. The reactions of 1 with aliphatic primary amines and secondary amines, respectively, produced different products, a result most likely attributed to the different basicities (or nucleophilicities) and steric effects of the two kinds of amines. The structure of the displacement product, 2-bromo-3-[2-(tert-butoxycarbonyl)ethylamino]-9,10-dihydro-9,10-[1,2]benzenoanthracene-1,4,5,8-tetrone, from the reaction of 1 with tert-butyl 3-aminopropanoate was unequivocally determined by a single-crystal X-ray analysis. IC(50) values of triptycene bisquinones for the inhibition of L1210 leukemia cell viability are in the 0.11-0.27 microM range and for the inhibition of Plasmodium falciparum 3D7 are in the 4.7-8.0 microM range.
- Published
- 2002
- Full Text
- View/download PDF
19. Epidermal growth factor receptor family in lung cancer and premalignancy.
- Author
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Franklin WA, Veve R, Hirsch FR, Helfrich BA, and Bunn PA Jr
- Subjects
- Carcinoma, Non-Small-Cell Lung drug therapy, Carcinoma, Non-Small-Cell Lung genetics, Cell Cycle, Clinical Trials as Topic, ErbB Receptors physiology, Humans, Loss of Heterozygosity, Lung Neoplasms drug therapy, Lung Neoplasms genetics, Phosphorylation, Signal Transduction, Smoking adverse effects, Up-Regulation, Carcinoma, Non-Small-Cell Lung physiopathology, Cell Transformation, Neoplastic, ErbB Receptors biosynthesis, Gene Expression Regulation, Neoplastic, Lung Neoplasms physiopathology, Precancerous Conditions physiopathology, Receptor Protein-Tyrosine Kinases metabolism
- Abstract
Lung cancer, like many other epithelial malignancies, is thought to be the outcome of genetic and epigenetic changes that result in a constellation of phenotypic abnormalities in bronchial epithelium. These include morphologic epithelial dysplasia, angiogenesis, increased proliferative rate, and changes in expression of cell surface proteins, particularly overexpression of epidermal growth factor receptor (EGFR) family proteins. The EFGR family is a group of four structurally similar tyrosine kinases (EGFR, HER2/neu, ErbB-3, and ErbB-4) that dimerize on binding with a number of ligands, including EGF and transforming growth factor alpha. Epidermal growth factor receptor overexpression is pronounced in virtually all squamous carcinomas and is also found in > or = 65% of large cell and adenocarcinomas. It is not expressed in situ by small cell lung carcinoma. Overexpression of EGFR is one of the earliest and most consistent abnormalities in bronchial epithelium of high-risk smokers. It is present at the stage of basal cell hyperplasia and persists through squamous metaplasia, dysplasia, and carcinoma in situ. Recent studies of the effect of inhibitors of receptor tyrosine kinases suggest that patterns of coexpression of multiple members of the EGFR family could be important in determining response. Intermediate endpoints of such trials could include monitoring of phosphorylation levels in signal transduction molecules downstream of the receptor dimers. These trials represent a new targeted approach to lung cancer treatment and chemoprevention that will require greater attention to molecular endpoints than required in past trials.
- Published
- 2002
- Full Text
- View/download PDF
20. ZD1839, a selective epidermal growth factor receptor tyrosine kinase inhibitor, alone and in combination with radiation and chemotherapy as a new therapeutic strategy in non-small cell lung cancer.
- Author
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Raben D, Helfrich BA, Chan D, Johnson G, and Bunn PA Jr
- Subjects
- Administration, Oral, Animals, Antineoplastic Agents administration & dosage, Carcinoma, Non-Small-Cell Lung radiotherapy, Cell Cycle drug effects, Chemoprevention, Clinical Trials as Topic, Combined Modality Therapy, Disease Models, Animal, ErbB Receptors antagonists & inhibitors, ErbB Receptors drug effects, Gefitinib, Head and Neck Neoplasms radiotherapy, Humans, Lung Neoplasms radiotherapy, Quinazolines administration & dosage, Signal Transduction drug effects, Transplantation, Heterologous, Up-Regulation, Antineoplastic Agents pharmacology, Carcinoma, Non-Small-Cell Lung drug therapy, ErbB Receptors biosynthesis, ErbB Receptors metabolism, Head and Neck Neoplasms drug therapy, Lung Neoplasms drug therapy, Quinazolines pharmacology
- Abstract
The epidermal growth factor receptor is overexpressed in a majority of non-small cell lung cancers and has been associated with a poor prognosis. Preclinical studies have shown that ZD1839, an oral anilinoquinazoline, targets the epidermal growth factor receptor-associated tyrosine kinase, reversibly inhibiting critical downstream signaling and resulting in cancer cell growth arrest. Potent antitumor effects have been observed in human lung tumor xenograft models. Preclinical studies have shown additive to synergistic effects when ZD1839 is combined with radiation or chemotherapy in colon, head and neck, and non-small cell lung cancers. Phase I clinical trials have shown modest dose-related toxicity, and antitumor activity has been reported in a variety of malignancies including lung cancer. Future studies will certainly combine ZD1839 with chemotherapy or radiation. ZD1839 also may be effective as a chemoprevention agent because premalignant lesions often overexpress epidermal growth factor receptor.
- Published
- 2002
- Full Text
- View/download PDF
21. "Total Synthesis" Supramolecular Style: Design and Hydrogen-Bond-Directed Assembly of Ternary Supermolecules.
- Author
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Aakeröy CB, Beatty AM, and Helfrich BA
- Abstract
Getting the right balance between intermolecular interactions is crucial for the synthesis of supermolecules in a preconceived manner. The three-component supermolecule in the ternary cocrystal of 3,5-dinitrobenzoic acid, isonicotinamide, and 4-(dimethylamino)benzoic acid (1:1:1) assembles through a "primary" (between the stronger acid and pyridine) and a "secondary" hydrogen-bonding interaction (between the weaker acid and amide)., (© 2001 WILEY-VCH Verlag GmbH, Weinheim, Fed. Rep. of Germany.)
- Published
- 2001
- Full Text
- View/download PDF
22. Effects of recombinant neutral endopeptidase (EC 3.4.24.11) on the growth of lung cancer cell lines in vitro and in vivo.
- Author
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Bunn PA Jr, Helfrich BA, Brenner DG, Chan DC, Dykes DJ, Cohen AJ, and Miller YE
- Subjects
- Animals, Carcinoma, Non-Small-Cell Lung drug therapy, Carcinoma, Non-Small-Cell Lung pathology, Carcinoma, Small Cell drug therapy, Carcinoma, Small Cell pathology, Cell Division drug effects, Cell Line, Dose-Response Relationship, Drug, Fibroblasts drug effects, Humans, Lung drug effects, Lung pathology, Lung Neoplasms pathology, Mice, Mice, Nude, Neoplasm Transplantation, Neprilysin biosynthesis, Neprilysin metabolism, Neuropeptides metabolism, Neuropeptides pharmacology, Recombinant Proteins metabolism, Recombinant Proteins pharmacology, Recombinant Proteins therapeutic use, Signal Transduction drug effects, Tumor Cells, Cultured, Lung Neoplasms drug therapy, Neprilysin therapeutic use
- Abstract
Many lung cancers are stimulated by an autocrine/paracrine system of neuroendocrine peptide hormones. Attempts to block this autocrine growth pathway by interactions with specific ligand-receptor binding using monoclonal antibodies and peptide-specific antagonists have been largely unsuccessful because of the heterogeneity of hormone production and receptor expression. In the normal lung, neutral endopeptidase (NEP; CD10, CALLA, enkephalinase, and EC 3.4.24.11) plays a physiological role in degrading biologically active peptides, including all peptides implicated in autocrine growth stimulation of lung cancer. Cigarette smoke decreases the activity of NEP, indicating that the lack of NEP contributes to the dysregulation of the peptide autocrine system. The cloning of the human NEP gene allowed for production of sufficient quantities of recombinant NEP (rNEP) to evaluate its role in inhibiting the growth of lung cancer cells. In this study, we evaluated the ability of rNEP to inactivate the peptides involved in lung cancer signal transduction and to inhibit the growth of lung cancer cells as well as normal lung cells in vitro and in vivo in athymic nude mice. We showed that the growth inhibition of lung cancer cells by rNEP was related to the dose and schedule. Continuous exposure to high doses was required for growth inhibition. These studies confirm the importance of NEP in this autocrine pathway.
- Published
- 1998
23. Identification of a novel CD56- lymphokine-activated killer cell precursor in cancer patients receiving recombinant interleukin 2.
- Author
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McKenzie RS, Simms PE, Helfrich BA, Fisher RI, and Ellis TM
- Subjects
- CD56 Antigen, Carcinoma, Renal Cell drug therapy, Cell Separation, Hematopoietic Stem Cells drug effects, Hematopoietic Stem Cells immunology, Humans, Kidney Neoplasms drug therapy, Killer Cells, Lymphokine-Activated drug effects, Killer Cells, Lymphokine-Activated immunology, Killer Cells, Natural drug effects, Killer Cells, Natural immunology, Killer Cells, Natural physiology, Lymphocyte Subsets drug effects, Lymphocyte Subsets immunology, Lymphocytes drug effects, Lymphocytes immunology, Lymphocytes physiology, Melanoma drug therapy, Phenotype, Receptors, IgG immunology, Recombinant Proteins therapeutic use, Antigens, CD immunology, Antigens, Differentiation, T-Lymphocyte immunology, Carcinoma, Renal Cell blood, Hematopoietic Stem Cells physiology, Interleukin-2 therapeutic use, Kidney Neoplasms blood, Killer Cells, Lymphokine-Activated physiology, Melanoma blood
- Abstract
Circulating lymphokine-activated killer (LAK) cell activity in cancer patients receiving recombinant interleukin 2 (rIL-2) therapy is confined to cells expressing the CD56- surface marker. However, CD56- cells from these patients but not normal individuals have been reported to exhibit LAK cytotoxicity only following in vitro activation with rIL-2. Studies were performed to document the existence of CD56- LAK precursor cells and to phenotypically characterize this population in patients receiving rIL-2 therapy using fluorescence-activated cell sorter-purified CD56- cell subsets. Initial studies confirmed that CD56- cells exhibit NK activity [20 +/- 7 (SE) LU/10(6) cells] but not LAK activity (0 +/- 0 LU/10(6) cells) when evaluated directly from peripheral blood of patients receiving rIL-2. CD56- cells from patients but not normal individuals developed significant LAK cytolytic activity against NK-resistant COLO 205 targets (16 +/- 3 LU/10(6) cells) when cultured for 3 days with 1500 units/ml rIL-2. The CD56- LAK precursor activity was confined to cells expressing a CD56-CD16+ phenotype and a large granular lymphocyte morphology; little or no NK or LAK precursor activity was detectable in CD56-CD5+ T-cells from patients. Phenotypic characterization of CD16+CD56- cells revealed that this population is uniformly CD11a+,CD18+, and CD38+ and is heterogeneous in its expression of CD11b, CD11c, and CD16/Leu 11c. These results indicate that rIL-2 administration induces enhanced LAK precursor activity in a novel population of CD5-CD16+CD56- cells.
- Published
- 1992
24. Radiant heat-induced hyperthermia in mice: in vivo effects on the immune system.
- Author
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Greeley EH, Helfrich BA, Feuerman LL, Cain CA, and Segre M
- Subjects
- Animals, Antibody Formation, In Vitro Techniques, Lymphocyte Activation, Lymphocyte Subsets immunology, Mice, Mice, Inbred BALB C, T-Lymphocytes immunology, Hyperthermia, Induced, Immunity
- Abstract
Whole-body hyperthermia (WBH) in mice was induced by 2-4-h exposure to radiant heat resulting in core body temperatures of 38.5-40.4 degrees C, and correlated directly with the magnitude and duration of heat treatment. Two-hour heat treatments in this temperature range did not consistently affect generation of antibody-forming cells in vivo, while 4-h treatments at temperatures greater than or equal to 40 degrees C significantly suppressed the antibody-forming cell response. The capacity of lymphocytes from similarly heated mice to generate antibody-forming cells in vitro was not affected, suggesting that the observed in vivo suppression may be mediated by circulating factors rather than by some heat-induced alteration in the cells themselves. In vivo treatment did not alter T-cell responsiveness to the mitogen Con-A or delayed-type hypersensitivity responses to sheep red blood cells. Quantitative and flow cytometric assessment of splenic and thymic lymphocyte numbers showed that WBH did not alter absolute numbers of lymphocytes but did temporarily change the proportions of lymphocyte subsets. An immediate increase in splenic L3T4+ cells was observed, followed within 18 h by an overall decrease in Lyt 2+ and Thy 1.2+ T-cells. In the thymus the percentages of mature T cells increased. In general, only minimal effects of heat on the immune responses of normal mice could be demonstrated.
- Published
- 1992
- Full Text
- View/download PDF
25. Age-related changes in the degeneracy of the mouse T-cell repertoire.
- Author
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Helfrich BA, Segre M, and Segre D
- Subjects
- Animals, Female, H-2 Antigens immunology, Haptens immunology, Immunity, Cellular, Isoantigens immunology, Lymph Nodes immunology, Lymphocyte Activation, Male, Mice, Mice, Inbred BALB C immunology, Mice, Inbred C57BL immunology, T-Lymphocytes classification, Aging immunology, T-Lymphocytes immunology
- Abstract
Aging is associated with a decline in T-cell-dependent immune responses. As the number of T-cells remains relatively constant throughout life, the observed decline in T-cell function may reflect qualitative changes in the T-cells themselves or in the composition of the T-cell population. In this investigation, the quality of the T-cell response was studied in young-adult and aged B6C3F1 mice by assessing the degeneracy of the hapten-specific proliferative response to the antigen-MHC complex. Degeneracy was defined as the ability of lymph node cells from mice contact-sensitized to the hapten TNP to proliferate in response to in vitro stimulation with haptenated allogeneic antigen-presenting cells, thus escaping strict MHC restriction. It was found that, although degeneracy occurred in both age groups, it was more prevalent and of a greater magnitude in the young than in the old lymphocytes. These results suggest that there are qualitative differences in T-cell populations derived from young-adult and aged mice.
- Published
- 1989
- Full Text
- View/download PDF
26. Induction of human lymphokine-activated killer cells by IFN-alpha and IFN-gamma.
- Author
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Ellis TM, McKenzie RS, Simms PE, Helfrich BA, and Fisher RI
- Subjects
- Dose-Response Relationship, Immunologic, Humans, Interleukin-2 physiology, Kinetics, Phenotype, Tumor Cells, Cultured, Cytotoxicity, Immunologic drug effects, Interferon Type I pharmacology, Interferon-gamma pharmacology, Killer Cells, Lymphokine-Activated immunology, Lymphocyte Activation drug effects
- Abstract
By traditional definitions, NK cells can be activated by cytokines to exhibit two functionally distinct levels of cytotoxicity. Whereas IL-2-mediated activation of NK cells leads to the development of lymphokine-activated killer (LAK) cytotoxicity, characterized by the acquisition of cytolytic activity against NK-resistant targets, IFN-treated NK cells become activated without the acquisition of novel cytolytic specificities. In this study we show that NK cells activated by 18 to 24 h of stimulation with either IFN-alpha or IFN-gamma do acquire LAK cytolytic activity, demonstrated by the ability of IFN-treated PBMC to lyse NK-resistant COLO 205 cells as well as fresh tumor targets. The level of IFN-alpha-induced LAK activity was significantly greater than that induced by IFN-gamma, although IL-2-induced LAK activity was considerably greater than IFN-alpha-induced LAK cytotoxicity. Maximal IFN-induced LAK cytotoxicity occurred after 24 h of culture, and occurred with the use of IFN-alpha at 500 U/ml and IFN-gamma at 1000 U/ml. Whereas neutralizing antibody experiments demonstrated that IFN-alpha-induced LAK activation did not involve the participation of endogenously produced IL-2, the partial inhibition (63%) of IFN-gamma-induced LAK cytotoxicity by anti-IL-2 and of IL-2-induced LAK by anti-IFN-gamma (33.3%) indicates that the induction of LAK cytotoxicity by either of these individual cytokines involves the endogenous production and participation of the other cytokine. Similar to IL-2-induced LAK cells, phenotypic analysis revealed that IFN-alpha/gamma LAK cells were Leu-19+, although the Leu 19"dim"+ subset exhibited greater IFN-induced LAK activity than the Leu-19"bright"+ subset. The results of this study clearly demonstrate that IFN-alpha and IFN-gamma induce classic LAK activity and IFN-gamma plays a participatory role in the optimal induction of LAK cells by IL-2.
- Published
- 1989
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