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1.
Mol Pharmacol ; 95(1): 127-138, 2019 01.
Article in English | MEDLINE | ID: mdl-30409790

ABSTRACT

Lysosomes degrade cellular proteins and organelles and regulate cell signaling by providing a surface for the formation of critical protein complexes, notably molecular target of rapamycin (mTOR) complex 1 (mTORC1). Striking differences in the lysosomes of cancer versus normal cells suggest that they could be targets for drug development. Although the lysomotropic drugs chloroquine (CQ) and hydroxychloroquine (HCQ) have been widely investigated, studies have focused on their ability to inhibit autophagy. We synthesized a novel compound, called EAD1, which is structurally related to CQ but is a 14-fold more potent inhibitor of cell proliferation. Here we find that EAD1 causes rapid relocation, membrane permeabilization (LMP), and deacidification of lysosomes, and it induces apoptosis and irreversibly blocks proliferation of human lung cancer H460, H520, H1299, HCC827, and H1703 cells. EAD1 causes dissociation of mTOR from lysosomes and increases mTOR's perinuclear versus cytoplasmic localization, changes previously shown to inactivate mTORC1. The effect on mTOR was not seen with HCQ, even at >10-fold greater concentrations. Phosphorylation of a downstream target of mTORC1, ribosomal protein S6, was inhibited by EAD1. Although EAD1 also inhibited autophagy, it retained full antiproliferative activity in autophagy-deficient H1650 lung cancer cells, which have a biallelic deletion of Atg7, and in H460 Atg7-knockout cells. As Atg7 is critical for the canonical autophagy pathway, it is likely that inhibition of autophagy is not how EAD1 inhibits cell proliferation. Further studies are needed to determine the relationship of LMP to mTORC1 disruption and their relative contributions to drug-induced cell death. These studies support the lysosome as an underexplored target for new drug development.


Subject(s)
Cell Proliferation/drug effects , Chloroquinolinols/pharmacology , Lung Neoplasms/drug therapy , Lysosomes/drug effects , Membranes/drug effects , Permeability/drug effects , TOR Serine-Threonine Kinases/metabolism , Apoptosis/drug effects , Autophagy/drug effects , Cell Line, Tumor , Chloroquine/pharmacology , Humans , Lung Neoplasms/metabolism , Lysosomes/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Phosphorylation/drug effects , Signal Transduction/drug effects
2.
ACS Med Chem Lett ; 6(2): 134-9, 2015 Feb 12.
Article in English | MEDLINE | ID: mdl-25699157

ABSTRACT

The autophagy inhibitors chloroquine (CQ) and hydroxychloroquine (HCQ) have single agent antiproliferative activity against human cancer cell lines; however, low potency may limit their antitumor efficacy clinically. We synthesized a series of chloroquine analogs that retained the 4-aminoquinoline subunit and incorporated different substituted triazoles into the target structure. These compounds were tested for growth inhibition against H460 and HCC827 human lung cancer and BxPC3 pancreatic cancer cells. The most potent compound, EAD1, had an IC50 of 5.8 µM in the BxPC3 cells and was approximately 8-fold more potent than CQ and HCQ. EAD1 inhibited autophagy, as judged by the cellular accumulation of the autophagy-related autophagosome proteins LC3-II and p62 and induced apoptosis. The increases in LC3-II levels by the analogues were highly correlated with their growth inhibitory IC50s, suggesting that autophagy blockade is closely linked to inhibition of cell proliferation. EAD1 is a viable lead compound for evaluation of the antitumor activity of autophagy inhibitors in vivo.

3.
J Biol Chem ; 288(42): 30445-30453, 2013 Oct 18.
Article in English | MEDLINE | ID: mdl-24022482

ABSTRACT

Cytosolic carboxypeptidase 5 (CCP5) is a member of a subfamily of enzymes that cleave C-terminal and/or side chain amino acids from tubulin. CCP5 was proposed to selectively cleave the branch point of glutamylated tubulin, based on studies involving overexpression of CCP5 in cell lines and detection of tubulin forms with antisera. In the present study, we examined the activity of purified CCP5 toward synthetic peptides as well as soluble α- and ß-tubulin and paclitaxel-stabilized microtubules using a combination of antisera and mass spectrometry to detect the products. Mouse CCP5 removes multiple glutamate residues and the branch point glutamate from the side chains of porcine brain α- and ß-tubulin. In addition, CCP5 excised C-terminal glutamates from detyrosinated α-tubulin. The enzyme also removed multiple glutamate residues from side chains and C termini of paclitaxel-stabilized microtubules. CCP5 both shortens and removes side chain glutamates from synthetic peptides corresponding to the C-terminal region of ß3-tubulin, whereas cytosolic carboxypeptidase 1 shortens the side chain without cleaving the peptides' γ-linked residues. The rate of cleavage of α linkages by CCP5 is considerably slower than that of removal of a single γ-linked glutamate residue. Collectively, our data show that CCP5 functions as a dual-functional deglutamylase cleaving both α- and γ-linked glutamate from tubulin.


Subject(s)
Carboxypeptidases/metabolism , Glutamic Acid/metabolism , Microtubules/metabolism , Tubulin/metabolism , Animals , Carboxypeptidases/chemistry , Carboxypeptidases/genetics , Glutamic Acid/chemistry , Glutamic Acid/genetics , Mice , Microtubules/chemistry , Microtubules/genetics , Paclitaxel/pharmacology , Sf9 Cells , Spodoptera , Tubulin/chemistry , Tubulin/genetics , Tubulin Modulators/pharmacology
4.
J Thorac Oncol ; 8(6): 693-702, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23575415

ABSTRACT

INTRODUCTION: The epidermal growth factor receptor (EGFR) inhibitor erlotinib is much less effective in non-small-cell lung cancer (NSCLC) tumors with wild-type EGFR, than in tumors with activating EGFR mutations. Autophagy is a tightly regulated lysosomal self-digestion process, which may alternatively promote cell survival or type II cell death. This study assessed the role of autophagy in erlotinib-mediated cytotoxicity. METHODS: We used wild-type EGFR erlotinib-sensitive and erlotinib-resistant NSCLC cell lines to determine whether inhibiting autophagy by a therapeutic agent potentiated the antitumor activity of erlotinib in vitro and in vivo. RESULTS: Erlotinib at a clinically relevant concentration (2 µM) induced autophagy in NSCLC cells with wild-type EGFR, and the degree of induction was greater in cells that were resistant than sensitive, suggesting that autophagy is cytoprotective. This was confirmed by knockdown of the autophagy-related gene Atg-5, and by using the autophagy inhibitor chloroquine (CQ), both of which increased the cytotoxicity of erlotinib. The synergistic activity of CQ was not because of the potentiation of erlotinib's effects on autophagy, cell-cycle arrest, and inhibition of both EGFR or downstream signaling of EGFR. Rather, CQ markedly activated apoptosis in the cells. The ability of CQ to potentiate the antitumor activity of erlotinib was also seen in mice bearing NSCLC tumor xenografts. CONCLUSIONS: The ability to adapt to anti-EGFR therapy by triggering autophagy may be a key determinant for resistance to erlotinib in wild-type EGFR NSCLC. Inhibition of autophagy by CQ represents a novel strategy to broaden the spectrum of erlotinib efficacy in wild-type EGFR NSCLC tumors.


Subject(s)
Autophagy/drug effects , Carcinoma, Non-Small-Cell Lung/drug therapy , Chloroquine/pharmacology , Drug Resistance, Neoplasm/drug effects , Lung Neoplasms/drug therapy , Quinazolines/pharmacology , Animals , Antimalarials/pharmacology , Antineoplastic Combined Chemotherapy Protocols , Apoptosis/drug effects , Blotting, Western , Carcinoma, Non-Small-Cell Lung/pathology , Cell Cycle/drug effects , Cell Proliferation/drug effects , ErbB Receptors/antagonists & inhibitors , Erlotinib Hydrochloride , Flow Cytometry , Humans , Lung Neoplasms/pathology , Mice , Mice, Nude , Protein Kinase Inhibitors/pharmacology , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
5.
PLoS One ; 8(4): e60981, 2013.
Article in English | MEDLINE | ID: mdl-23593366

ABSTRACT

Cytosolic carboxypeptidase 1 (CCP1) is a metallopeptidase that removes C-terminal and side-chain glutamates from tubulin. The Purkinje cell degeneration (pcd) mouse lacks CCP1 due to a mutation. Previously, elevated levels of peptides derived from cytosolic and mitochondrial proteins were found in adult pcd mouse brain, raising the possibility that CCP1 functions in the degradation of intracellular peptides. To test this hypothesis, we used a quantitative peptidomics technique to compare peptide levels in wild-type and pcd mice, examining adult heart, spleen, and brain, and presymptomatic 3 week-old amygdala and cerebellum. Contrary to adult mouse brain, young pcd brain and adult heart and spleen did not show a large increase in levels of intracellular peptides. Unexpectedly, levels of peptides derived from secretory pathway proteins were altered in adult pcd mouse brain. The pattern of changes for the intracellular and secretory pathway peptides in pcd mice was generally similar to the pattern observed in mice lacking primary cilia. Collectively, these results suggest that intracellular peptide accumulation in adult pcd mouse brain is a secondary effect and is not due to a role of CCP1 in peptide turnover.


Subject(s)
Mutation , Peptide Fragments/metabolism , Proteomics , Purkinje Cells/pathology , Amino Acid Sequence , Amygdala/metabolism , Animals , Cerebellum/metabolism , Female , GTP-Binding Proteins/deficiency , GTP-Binding Proteins/genetics , Mice , Molecular Sequence Data , Peptide Fragments/chemistry , Purkinje Cells/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Serine-Type D-Ala-D-Ala Carboxypeptidase/deficiency , Serine-Type D-Ala-D-Ala Carboxypeptidase/genetics
6.
PLoS One ; 8(1): e53263, 2013.
Article in English | MEDLINE | ID: mdl-23308178

ABSTRACT

Bortezomib is an antitumor drug that competitively inhibits proteasome beta-1 and beta-5 subunits. While the impact of bortezomib on protein stability is known, the effect of this drug on intracellular peptides has not been previously explored. A quantitative peptidomics technique was used to examine the effect of treating human embryonic kidney 293T (HEK293T) cells with 5-500 nM bortezomib for various lengths of time (30 minutes to 16 hours), and human neuroblastoma SH-SY5Y cells with 500 nM bortezomib for 1 hour. Although bortezomib treatment decreased the levels of some intracellular peptides, the majority of peptides were increased by 50-500 nM bortezomib. Peptides requiring cleavage at acidic and hydrophobic sites, which involve beta-1 and -5 proteasome subunits, were among those elevated by bortezomib. In contrast, the proteasome inhibitor epoxomicin caused a decrease in the levels of many of these peptides. Although bortezomib can induce autophagy under certain conditions, the rapid bortezomib-mediated increase in peptide levels did not correlate with the induction of autophagy. Taken together, the present data indicate that bortezomib alters the balance of intracellular peptides, which may contribute to the biological effects of this drug.


Subject(s)
Antineoplastic Agents/pharmacology , Boronic Acids/pharmacology , Neuroblastoma/drug therapy , Peptides/metabolism , Proteasome Inhibitors/pharmacology , Pyrazines/pharmacology , Amino Acid Sequence , Bortezomib , Cell Line, Tumor , HEK293 Cells , Humans , Molecular Sequence Data , Neuroblastoma/pathology , Peptides/analysis , Proteomics
7.
J Biol Chem ; 287(9): 6503-17, 2012 Feb 24.
Article in English | MEDLINE | ID: mdl-22170066

ABSTRACT

The Purkinje cell degeneration (pcd) mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1). This study tested two proposed functions of CCP1: degradation of intracellular peptides and processing of tubulin. Overexpression (2-3-fold) or knockdown (80-90%) of CCP1 in human embryonic kidney 293T cells (HEK293T) did not affect the levels of most intracellular peptides but altered the levels of α-tubulin lacking two C-terminal amino acids (delta2-tubulin) ≥ 5-fold, suggesting that tubulin processing is the primary function of CCP1, not peptide degradation. Purified CCP1 produced delta2-tubulin from purified porcine brain α-tubulin or polymerized HEK293T microtubules. In addition, CCP1 removed Glu residues from the polyglutamyl side chains of porcine brain α- and ß-tubulin and also generated a form of α-tubulin with two C-terminal Glu residues removed (delta3-tubulin). Consistent with this, pcd mouse brain showed hyperglutamylation of both α- and ß-tubulin. The hyperglutamylation of α- and ß-tubulin and subsequent death of Purkinje cells in pcd mice was counteracted by the knock-out of the gene encoding tubulin tyrosine ligase-like-1, indicating that this enzyme hyperglutamylates α- and ß-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from ß- as well as α-tubulin in vitro and in vivo.


Subject(s)
GTP-Binding Proteins/metabolism , Nerve Degeneration/metabolism , Serine-Type D-Ala-D-Ala Carboxypeptidase/metabolism , Tubulin/metabolism , Animals , Breast Neoplasms , Cell Line, Tumor , Colonic Neoplasms , Cytosol/enzymology , Female , GTP-Binding Proteins/genetics , Glutamic Acid/metabolism , HEK293 Cells , Humans , Mice , Mice, Inbred BALB C , Mice, Mutant Strains , Nerve Degeneration/genetics , Peptide Synthases/genetics , Peptide Synthases/metabolism , Protein Structure, Tertiary , Purkinje Cells/enzymology , Purkinje Cells/pathology , Serine-Type D-Ala-D-Ala Carboxypeptidase/genetics , Swine , Tubulin/chemistry
8.
J Proteome Res ; 10(4): 1583-92, 2011 Apr 01.
Article in English | MEDLINE | ID: mdl-21204522

ABSTRACT

Peptides have been proposed to function in intracellular signaling within the cytosol. Although cytosolic peptides are considered to be highly unstable, a large number of peptides have been detected in mouse brain and other biological samples. In the present study, we evaluated the peptidome of three diverse cell lines: SH-SY5Y, MCF7, and HEK293 cells. A comparison of the peptidomes revealed considerable overlap in the identity of the peptides found in each cell line. The majority of the observed peptides are not derived from the most abundant or least stable proteins in the cell, and approximately half of the cellular peptides correspond to the N- or C- termini of the precursor proteins. Cleavage site analysis revealed a preference for hydrophobic residues in the P1 position. Quantitative peptidomic analysis indicated that the levels of most cellular peptides are not altered in response to elevated intracellular calcium, suggesting that calpain is not responsible for their production. The similarity of the peptidomes of the three cell lines and the lack of correlation with the predicted cellular degradome implies the selective formation or retention of these peptides, consistent with the hypothesis that they are functional in the cells.


Subject(s)
Cell Line/chemistry , Peptides/analysis , Animals , Humans , Mice , Proteins/chemistry , Proteins/metabolism , Proteome/analysis
9.
Clin Cancer Res ; 17(4): 690-9, 2011 Feb 15.
Article in English | MEDLINE | ID: mdl-21148747

ABSTRACT

PURPOSE: Because loss of Nkx2-8 increases lung cancer in the mouse, we studied suppressive mechanisms in human lung cancer. EXPERIMENTAL DESIGN: NKX2-8 is located within 14q13.3, adjacent to its close relative TTF1/NKX2-1. We first analyzed LOH of 14q13.3 in forty-five matched human lung cancer and control specimens. DNA from tumors with LOH was then analyzed with high-density single-nucleotide polymorphism (SNP) arrays. For correlation with this genetic analysis, we quantified expression of Nkx2-8 and TTF1 mRNA in tumors. Finally, suppressive function of Nkx2-8 was assessed via colony formation assays in five lung cancer cell lines. RESULTS: Thirteen of forty-five (29%) tumors had LOH. In six tumors, most adenocarcinomas, LOH was caused by gene amplification. The 0.8-Mb common region of amplification included MBIP, SFTA, TTF1, NKX2-8, and PAX9. In 4 squamous or adenosquamous cancers, LOH was caused by deletion. In three other tumors, LOH resulted from whole chromosome mechanisms (14(-), 14(+), or aneuploidy). The 1.2-Mb common region of deletion included MBIP, SFTA, TTF1, NKX2-8, PAX9, SLC25A21, and MIPOL1. Most tumors had low expression of Nkx2-8. Nevertheless, sequencing did not show NKX2-8 mutations that could explain the low expression. TTF1 overexpression, in contrast, was common and usually independent of Nkx2-8 expression. Finally, stable transfection of Nkx2-8 selectively inhibited growth of H522 lung cancer cells. CONCLUSIONS: 14q13.3, which contains NKX2-8, is subject to both amplification and deletion in lung cancer. Most tumors have low expression of Nkx2-8, and its expression can inhibit growth of some lung cancer cells.


Subject(s)
Adenocarcinoma, Bronchiolo-Alveolar/genetics , Carcinoma, Adenosquamous/genetics , Carcinoma, Squamous Cell/genetics , Chromosomes, Human, Pair 14/genetics , Gene Amplification , Homeodomain Proteins/genetics , Lung Neoplasms/genetics , Transcription Factors/genetics , Adenocarcinoma, Bronchiolo-Alveolar/pathology , Base Sequence , Carcinoma, Adenosquamous/pathology , Carcinoma, Squamous Cell/pathology , Cell Line, Tumor , Cell Proliferation , DNA-Binding Proteins/genetics , Down-Regulation , Gene Deletion , Gene Dosage , Genetic Association Studies , Humans , Loss of Heterozygosity , Lung Neoplasms/pathology , Molecular Sequence Data , Sequence Analysis, DNA
10.
J Neurochem ; 113(4): 871-80, 2010 May.
Article in English | MEDLINE | ID: mdl-20202081

ABSTRACT

Many hemoglobin-derived peptides are present in mouse brain, and several of these have bioactive properties including the hemopressins, a related series of peptides that bind to cannabinoid CB1 receptors. Although hemoglobin is a major component of red blood cells, it is also present in neurons and glia. To examine whether the hemoglobin-derived peptides in brain are similar to those present in blood and heart, we used a peptidomics approach involving mass spectrometry. Many hemoglobin-derived peptides are found only in brain and not in blood, whereas all hemoglobin-derived peptides found in heart were also seen in blood. Thus, it is likely that the majority of the hemoglobin-derived peptides detected in brain are produced from brain hemoglobin and not erythrocytes. We also examined if the hemopressins and other major hemoglobin-derived peptides were regulated in the Cpe(fat/fat) mouse; previously these mice were reported to have elevated levels of several hemoglobin-derived peptides. Many, but not all of the hemoglobin-derived peptides were elevated in several brain regions of the Cpe(fat/fat) mouse. Taken together, these findings suggest that the post-translational processing of alpha and beta hemoglobin into the hemopressins, as well as other peptides, is up-regulated in some but not all Cpe(fat/fat) mouse brain regions.


Subject(s)
Brain/metabolism , Erythrocytes/metabolism , Hemoglobins/metabolism , Myocardium/metabolism , Peptide Fragments/blood , Peptides/blood , Animals , Blotting, Western , Brain Chemistry/physiology , Hemoglobins/analysis , Hemoglobins/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Peptide Fragments/analysis , Peptide Fragments/genetics , Peptides/analysis , Peptides/genetics , Protein Processing, Post-Translational/physiology , Proteome/metabolism , Proteomics/methods , RNA, Messenger/analysis , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction
11.
FASEB J ; 24(6): 1813-23, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20061535

ABSTRACT

Purkinje cell degeneration (pcd) mice have a mutation within the gene encoding cytosolic carboxypeptidase 1 (CCP1/Nna1), which has homology to metallocarboxypeptidases. To assess the function of CCP1/Nna1, quantitative proteomics and peptidomics approaches were used to compare proteins and peptides in mutant and wild-type mice. Hundreds of peptides derived from cytosolic and mitochondrial proteins are greatly elevated in pcd mouse hypothalamus, amygdala, cortex, prefrontal cortex, and striatum. However, the major proteins detected on 2-D gel electrophoresis were present in mutant and wild-type mouse cortex and hypothalamus at comparable levels, and proteasome activity is normal in these brain regions of pcd mice, suggesting that the increase in cellular peptide levels in the pcd mice is due to reduced degradation of the peptides downstream of the proteasome. Both nondegenerating and degenerating regions of pcd mouse brain, but not wild-type mouse brain, show elevated autophagy, which can be triggered by a decrease in amino acid levels. Taken together with previous studies on CCP1/Nna1, these data suggest that CCP1/Nna1 plays a role in protein turnover by cleaving proteasome-generated peptides into amino acids and that decreased peptide turnover in the pcd mice leads to cell death.


Subject(s)
GTP-Binding Proteins/physiology , Nerve Degeneration/metabolism , Nerve Degeneration/pathology , Nerve Tissue Proteins/metabolism , Purkinje Cells/pathology , Serine-Type D-Ala-D-Ala Carboxypeptidase/physiology , Amino Acids/metabolism , Animals , Autophagy , Cell Death , Electrophoresis, Gel, Two-Dimensional , Female , Male , Mice , Mice, Inbred BALB C , Peptide Fragments/metabolism , Proteomics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
12.
J Biol Chem ; 284(21): 14105-16, 2009 May 22.
Article in English | MEDLINE | ID: mdl-19282285

ABSTRACT

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is an intracellular enzyme that has been proposed to metabolize peptides within cells, thereby affecting antigen presentation and G protein-coupled receptor signal transduction. However, only a small number of intracellular substrates of EP24.15 have been reported previously. Here we have identified over 100 peptides in human embryonic kidney 293 (HEK293) cells that are derived from intracellular proteins; many but not all of these peptides are substrates or products of EP24.15. First, cellular peptides were extracted from HEK293 cells and incubated in vitro with purified EP24.15. Then the peptides were labeled with isotopic tags and analyzed by mass spectrometry to obtain quantitative data on the extent of cleavage. A related series of experiments tested the effect of overexpression of EP24.15 on the cellular levels of peptides in HEK293 cells. Finally, synthetic peptides that corresponded to 10 of the cellular peptides were incubated with purified EP24.15 in vitro, and the cleavage was monitored by high pressure liquid chromatography and mass spectrometry. Many of the EP24.15 substrates identified by these approaches are 9-11 amino acids in length, supporting the proposal that EP24.15 can function in the degradation of peptides that could be used for antigen presentation. However, EP24.15 also converts some peptides into products that are 8-10 amino acids, thus contributing to the formation of peptides for antigen presentation. In addition, the intracellular peptides described here are potential candidates to regulate protein interactions within cells.


Subject(s)
Intracellular Space/enzymology , Metalloendopeptidases/metabolism , Amino Acid Sequence , Animals , Cell Extracts , Cell Line , Humans , Intracellular Space/drug effects , Isotope Labeling , Molecular Sequence Data , Peptides/chemistry , Quaternary Ammonium Compounds/pharmacology , Rats , Sequence Analysis, Protein , Spectrometry, Mass, Electrospray Ionization , Substrate Specificity/drug effects
13.
Mol Med ; 14(3-4): 167-74, 2008.
Article in English | MEDLINE | ID: mdl-18224251

ABSTRACT

Fanconi anemia (FA) predisposes to hematopoietic failure, birth defects, leukemia, and squamous cell carcinoma of the head and neck (HNSCC) and cervix. The FA/BRCA pathway includes 8 members of a core complex and 5 downstream gene products closely linked with BRCA1 or BRCA2. Precancerous lesions are believed to trigger the DNA damage response (DDR), and we focused on the DDR in FA and its putative role as a checkpoint barrier to cancer. In primary fibroblasts with mutations in the core complex FANCA protein, we discovered that basal expression and phosphorylation of ATM (ataxia telangiectasia mutated) and p53 induced by irradiation (IR) or mitomycin C (MMC) were upregulated. This heightened response appeared to be due to increased basal levels of ATM in cultured FANCA-mutant cells, highlighting the new observation that ATM can be regulated at the transcriptional level in addition to its well-established activation by autophosphorylation. Functional analysis of this response using gamma-H2AX foci as markers of DNA double-stranded breaks (DSBs) demonstrated abnormal persistence of only MMC- and not IR-induced foci. Thus, we describe a processing defect that leads to general DDR upregulation but specific persistence of DNA crosslinker-induced damage response foci. Underscoring the significance of these findings, we found resistance to DNA crosslinker-induced cell cycle arrest and apoptosis in a TP53-mutant, patient-derived HNSCC cell line, whereas a lymphoblastoid cell line derived from this same individual was not mutated at TP53 and retained DNA crosslinker sensitivity. Our results suggest that cancer in FA may arise from selection for cells that escape from a chronically activated DDR checkpoint.


Subject(s)
Cell Cycle Proteins , DNA Damage , DNA-Binding Proteins , Fanconi Anemia Complementation Group A Protein/metabolism , Fanconi Anemia , Gene Expression Regulation , Neoplasms , Protein Serine-Threonine Kinases , Tumor Suppressor Proteins , Adult , Animals , Apoptosis/physiology , Ataxia Telangiectasia Mutated Proteins , Cell Cycle/physiology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cells, Cultured , DNA Repair , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Fanconi Anemia/complications , Fanconi Anemia/genetics , Fanconi Anemia/metabolism , Fanconi Anemia Complementation Group A Protein/genetics , Fibroblasts/cytology , Fibroblasts/physiology , Humans , Neoplasms/etiology , Neoplasms/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA Interference , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Up-Regulation
14.
Front Biosci ; 13: 240-8, 2008 Jan 01.
Article in English | MEDLINE | ID: mdl-17981542

ABSTRACT

Our previous results that IFI16 is involved in p53 transcription activity under conditions of ionizing radiation (IR), and that the protein is frequently lost in human breast cancer cell lines and breast adenocarcinoma tissues suggesting that IFI16 plays a crucial role in controlling cell growth. Here, we show that loss of IFI16 by RNA interference in cell culture causes elevated phosphorylation of p53 Ser37 and accumulated NBS1 (nibrin) and p21WAF1, leading to growth retardation. Consistent with these observations, doxycyclin-induced NBS1 caused accumulation of p21WAF1 and increased phosphorylation of p53 Ser37, leading to cell cycle arrest in G1 phase. Wortmannin treatment was found to decrease p53 Ser37 phosphorylation in NBS-induced cells. These results suggest that loss of IFI16 activates p53 checkpoint through NBS1-DNA-PKcs pathway.


Subject(s)
Cell Cycle Proteins/biosynthesis , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , Nuclear Proteins/physiology , Phosphoproteins/genetics , Phosphoproteins/physiology , Tumor Suppressor Protein p53/metabolism , Blotting, Western , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Enzyme Inhibitors/pharmacology , Flow Cytometry , Genes, p53 , Humans , Phosphorylation , Radiation, Ionizing , Serine/chemistry
15.
J Biol Chem ; 279(6): 4066-74, 2004 Feb 06.
Article in English | MEDLINE | ID: mdl-14623896

ABSTRACT

STAT1 (signal transducer and activator of transcription 1) has been implicated as a mediator of a variety of biological responses in response to stimulation by specific growth factors and cytokines. To understand better the role of STAT1 in the interferon-gamma (IFN-gamma)-induced phenotype, we generated an active form of STAT1 (STAT1C) by substituting Cys residues for both Arg-656 and Asn-658 within the C-terminal loop of the STAT1 SH2 domain. The IFN-gamma activation site element was stimulated and bound efficiently by STAT1C without IFN-gamma treatment. STAT1C was found to be tyrosine-phosphorylated in the nucleus for more than 30 h after IFN-gamma stimulation. STAT1-negative U3A cells reexpressing STAT1C showed retarded cell growth and underwent apoptosis when treated with IFN-gamma. Further analysis demonstrated that apoptosis was preceded by proteolytic cleavage of caspases 2, 3, and 7, and wild type STAT1 also induced cleavage of caspase 7 when expressed in STAT1-negative U3A cells, indicating that STAT1C augments potential activity of wild type STAT1. Studies with cycloheximide treatment showed that protein synthesis induced in the first 24 h after IFN-gamma treatment was required for apoptosis under these conditions. Finally, we found that STAT1C-induced apoptosis was, in part, mediated by caspase 2, 3, and 7 because benzyloxycarbonyl-valyl-aspartyl-valyl-alanyl-aspartic acid fluoromethyl ketone (Z-VDVAD-FMK) treatment partially blocked apoptosis. These results suggest that prolonged nuclear localization of activated STAT1 results in apoptosis involving specific regulation of caspase pathway.


Subject(s)
Apoptosis/physiology , Caspases/metabolism , DNA-Binding Proteins/metabolism , Trans-Activators/metabolism , Amino Acid Sequence , Base Sequence , Caspase 2 , Caspase 3 , Caspase 7 , Cell Division , Cell Line , Cysteine Proteinase Inhibitors/pharmacology , DNA/genetics , DNA/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , HeLa Cells , Humans , Interferon-gamma/pharmacology , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligopeptides/pharmacology , Phosphorylation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , STAT1 Transcription Factor , Trans-Activators/chemistry , Trans-Activators/genetics , Tyrosine/chemistry
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