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1.
Oncogene ; 36(41): 5661-5667, 2017 10 12.
Article in English | MEDLINE | ID: mdl-28604750

ABSTRACT

To date, a large number of long non-coding RNAs (lncRNAs) have been recently discovered through functional genomics studies. Importantly, lncRNAs have been shown, in many cases, to function as master regulators for gene expression and thus, they can play a critical role in various biological functions and disease processes including cancer. Although the lncRNA-mediated gene expression involves various mechanisms, such as regulation of transcription, translation, protein modification, and the formation of RNA-protein or protein-protein complexes, in this review, we discuss the latest developments primarily in important cell signaling pathways regulated by lncRNAs in cancer.


Subject(s)
Neoplasms/genetics , RNA, Long Noncoding/genetics , Signal Transduction/genetics , Gene Expression Regulation, Neoplastic , Humans
2.
Oncogenesis ; 5(12): e279, 2016 Dec 12.
Article in English | MEDLINE | ID: mdl-27941930

ABSTRACT

Acidic microenvironment is a common feature of solid tumors. We have previously shown that neuron specific acid-sensing ion channel 1 (ASIC1) is expressed in breast cancer, and it is responsible for acidosis-induced cellular signaling through AKT, leading to nuclear factor-κB (NF-κB) activation, and cell invasion and metastasis. However, AKT is frequently activated in cancer. Thus, a key question is whether ASIC1-mediated cell signaling still takes place in the cancer cells carrying constitutively active AKT. In the present study, we show that among four prostate cancer cell lines tested, 22Rv1 cells express the highest level of phosphorylated AKT that is not impacted by acidosis. However, acidosis can still induce NF-κB activation during which extracellular signal-regulated kinase (ERK) serves as an alternative pathway for ASIC-mediated cell signaling. Inhibition of ERK by chemical inhibitors or small interfering RNAs suppresses the acidosis-induced NF-κB activity through regulation of the inhibitory subunit IκBα phosphorylation. Furthermore, suppression of ASIC1-mediated generation of reactive oxygen species (ROS) by ROS scavengers, such as glutathione or N-acetyl-cysteine causes a decrease in ERK phosphorylation and degradation of IκBα. Finally, ASIC1 is upregulated in a subset of prostate cancer cases and ASIC1 knockout by CRISPR/Cas9 significantly suppresses cell invasion, and castration resistance both in vitro and in vivo. Together, these results support the significance of ASIC1-ROS-ERK-IκBα-NF-κB axis in prostate tumorigenesis, especially in the constitutively active AKT background.

3.
Cell Death Dis ; 7(6): e2262, 2016 06 09.
Article in English | MEDLINE | ID: mdl-27277684

ABSTRACT

BC200 is a long non-coding RNA (lncRNA) that has been implicated in the regulation of protein synthesis, yet whether dysregulation of BC200 contributes to the pathogenesis of human diseases remains elusive. In this study, we show that BC200 is upregulated in breast cancer; among breast tumor specimens there is a higher level of BC200 in estrogen receptor (ER) positive than in ER-negative tumors. Further experiments show that activation of estrogen signaling induces expression of BC200. To determine the significance of ER-regulated BC200 expression, we knockout (KO) BC200 by CRISPR/Cas9. BC200 KO suppresses tumor cell growth in vitro and in vivo by expression of the pro-apoptotic Bcl-xS isoform. Mechanistically, BC200 contains a 17-nucleotide sequence complementary to Bcl-x pre-mRNA, which may facilitate its binding to Bcl-x pre-mRNA and recruitment of heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, a known splicing factor. Consequently, hnRNP A2/B1 interferes with association of Bcl-x pre-mRNA with the Bcl-xS-promoting factor Sam68, leading to a blockade of Bcl-xS expression. Together, these results suggest that BC200 plays an oncogenic role in breast cancer. Thus, BC200 may serve as a prognostic marker and possible target for attenuating deregulated cell proliferation in estrogen-dependent breast cancer.


Subject(s)
Alternative Splicing/genetics , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Carcinogenesis/pathology , RNA, Long Noncoding/metabolism , bcl-X Protein/genetics , Alternative Splicing/drug effects , Apoptosis/drug effects , Apoptosis/genetics , Base Sequence , Carcinogenesis/drug effects , Carcinogenesis/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cell Survival/drug effects , Cell Survival/genetics , Estrogens/pharmacology , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Knockout Techniques , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Humans , Promoter Regions, Genetic/genetics , Protein Binding , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA Precursors/metabolism , RNA, Long Noncoding/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription, Genetic/drug effects , Up-Regulation/drug effects , Up-Regulation/genetics , bcl-X Protein/metabolism
4.
Oncogene ; 35(31): 4102-11, 2016 08 04.
Article in English | MEDLINE | ID: mdl-26686084

ABSTRACT

The low extracellular pH in the microenvironment has been shown to promote tumor growth and metastasis; however, the underlying mechanism is poorly understood. Particularly, little is known how the tumor cell senses the acidic signal to activate the acidosis-mediated signaling. In this study, we show that breast cancer cells express acid-sensing ion channel 1 (ASIC1), a proton-gated cation channel primarily expressed in the nervous system. RNA interference, knockout and rescue experiments demonstrate a critical role for ASIC1 in acidosis-induced reactive oxidative species and NF-κB activation, two key events for tumorigenesis. Mechanistically, ASIC1 is required for acidosis-mediated signaling through calcium influx. We show that as a cytoplasmic membrane protein, ASIC1 is also associated with mitochondria, suggesting that ASIC1 may regulate mitochondrial calcium influx. Importantly, interrogation of the Cancer Genome Atlas breast invasive carcinoma data set indicates that alterations of ASIC1 alone or combined with other 4 ASIC genes are significantly correlated with poor patient survival. Furthermore, ASIC1 inhibitors cause a significant reduction of tumor growth and tumor load. Together, these results suggest that ASIC1 contributes to breast cancer pathogenesis in response to acidic tumor microenvironments, and ASIC1 may serve as a prognostic marker and a therapeutic target for breast cancer.


Subject(s)
Acid Sensing Ion Channels/physiology , Breast Neoplasms/etiology , Acidosis/metabolism , Animals , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Calcium/metabolism , Cell Line, Tumor , Extracellular Signal-Regulated MAP Kinases/physiology , Female , Humans , Hydrogen-Ion Concentration , Lung Neoplasms/secondary , Mice , Neoplasm Invasiveness , Proto-Oncogene Proteins c-akt/physiology , Reactive Oxygen Species/metabolism
5.
Oncogene ; 34(37): 4890-900, 2015 Sep 10.
Article in English | MEDLINE | ID: mdl-25659578

ABSTRACT

The median survival time of breast cancer patients with brain metastasis is less than 6 months, and even a small metastatic lesion often causes severe neurological disabilities. Because of the location of metastatic lesions, a surgical approach is limited and most chemotherapeutic drugs are ineffective owing to the blood brain barrier (BBB). Despite this clinical importance, the molecular basis of the brain metastasis is poorly understood. In this study, we have isolated RNA from samples obtained from primary breast tumors and also from brain metastatic lesions followed by microRNA profiling analysis. Our results revealed that the miR-509 is highly expressed in the primary tumors, whereas the expression of this microRNA is significantly decreased in the brain metastatic lesions. MicroRNA target prediction and the analysis of cytokine array for the cells ectopically expressed with miR-509 demonstrated that this microRNA was capable of modulating the two genes essential for brain invasion, RhoC and TNF-α that affect the invasion of cancer cells and permeability of BBB, respectively. Importantly, high levels of TNF-α and RhoC-induced MMP9 were significantly correlated with brain metastasis-free survival of breast cancer patients. Furthermore, the results of our in vivo experiments indicate that miR-509 significantly suppressed the ability of cancer cells to metastasize to the brain. These findings suggest that miR-509 has a critical role in brain metastasis of breast cancer by modulating the RhoC-TNF-α network and that this miR-509 axis may represent a potential therapeutic target or serve as a prognostic tool for brain metastasis.


Subject(s)
Brain Neoplasms/secondary , Breast Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , MicroRNAs/physiology , Tumor Necrosis Factor-alpha/genetics , rho GTP-Binding Proteins/genetics , Animals , Blood-Brain Barrier/metabolism , Blood-Brain Barrier/pathology , Brain Neoplasms/genetics , Breast Neoplasms/genetics , Cells, Cultured , Female , HEK293 Cells , Humans , MCF-7 Cells , Mice , Mice, Nude , MicroRNAs/genetics , rhoC GTP-Binding Protein
6.
Cell Death Dis ; 5: e1008, 2014 Jan 23.
Article in English | MEDLINE | ID: mdl-24457952

ABSTRACT

Functional genomics studies have led to the discovery of a large amount of non-coding RNAs from the human genome; among them are long non-coding RNAs (lncRNAs). Emerging evidence indicates that lncRNAs could have a critical role in the regulation of cellular processes such as cell growth and apoptosis as well as cancer progression and metastasis. As master gene regulators, lncRNAs are capable of forming lncRNA-protein (ribonucleoprotein) complexes to regulate a large number of genes. For example, lincRNA-RoR suppresses p53 in response to DNA damage through interaction with heterogeneous nuclear ribonucleoprotein I (hnRNP I). The present study demonstrates that hnRNP I can also form a functional ribonucleoprotein complex with lncRNA urothelial carcinoma-associated 1 (UCA1) and increase the UCA1 stability. Of interest, the phosphorylated form of hnRNP I, predominantly in the cytoplasm, is responsible for the interaction with UCA1. Moreover, although hnRNP I enhances the translation of p27 (Kip1) through interaction with the 5'-untranslated region (5'-UTR) of p27 mRNAs, the interaction of UCA1 with hnRNP I suppresses the p27 protein level by competitive inhibition. In support of this finding, UCA1 has an oncogenic role in breast cancer both in vitro and in vivo. Finally, we show a negative correlation between p27 and UCA in the breast tumor cancer tissue microarray. Together, our results suggest an important role of UCA1 in breast cancer.


Subject(s)
Breast Neoplasms/physiopathology , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p27/genetics , Gene Expression Regulation, Neoplastic , RNA, Long Noncoding/metabolism , Apoptosis , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Down-Regulation , Female , Humans , Protein Binding , RNA, Long Noncoding/genetics
7.
Cell Death Differ ; 20(11): 1558-68, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23933812

ABSTRACT

In addition to protein-coding genes, the human genome makes a large amount of noncoding RNAs, including microRNAs and long noncoding RNAs (lncRNAs). Both microRNAs and lncRNAs have been shown to have a critical role in the regulation of cellular processes such as cell growth and apoptosis, as well as cancer progression and metastasis. Although it is well known that microRNAs can target a large number of protein-coding genes, little is known whether microRNAs can also target lncRNAs. In the present study, we determine whether miR-21 can regulate lncRNA expression. Using the lncRNA RT-PCR (reverse transcription-polymerase chain reaction) array carrying 83 human disease-related lncRNAs, we show that miR-21 is capable of suppressing the lncRNA growth arrest-specific 5 (GAS5). This negative correlation between miR-21 and GAS5 is also seen in breast tumor specimens. Of interest, GAS5 can also repress miR-21 expression. Whereas ectopic expression of GAS5 suppresses, GAS5-siRNA increases miR-21 expression. Importantly, there is a putative miR-21-binding site in exon 4 of GAS5; deletion of the miR-21-binding site abolishes this activity. Experiments with in vitro cell culture and xenograft mouse model suggest that GAS5 functions as a tumor suppressor. We further show that the biotin-labeled GAS5-RNA probe is able to pull down the key component (AGO2) of the RNA-induced silencing complex (RISC) and we subsequently identify miR-21 in this GAS5-RISC complex, implying that miR-21 and GAS5 may regulate each other in a way similar to the microRNA-mediated silencing of target mRNAs. Together, these results suggest that miR-21 targets not only tumor-suppressive protein-coding genes but also lncRNA GAS5.


Subject(s)
MicroRNAs/metabolism , RNA, Long Noncoding/metabolism , Animals , Cell Line, Tumor , Female , Genes, Tumor Suppressor , Heterografts , Humans , In Situ Hybridization , MCF-7 Cells , Mice , Mice, Nude , MicroRNAs/genetics , Plasmids/genetics , RNA, Long Noncoding/genetics , Transfection , Up-Regulation
8.
Oncogene ; 32(42): 5111-22, 2013 Oct 17.
Article in English | MEDLINE | ID: mdl-23208501

ABSTRACT

Upregulation of lipogenesis is a hallmark of cancer and blocking the lipogenic pathway is known to cause tumor cell death by apoptosis. However, the exact role of lipogenesis in tumor initiation is as yet poorly understood. We examined the expression profile of key lipogenic genes in clinical samples of ductal carcinoma in situ (DCIS) of breast cancer and found that these genes were significantly upregulated in DCIS. We also isolated cancer stem-like cells (CSCs) from DCIS.com cell line using cell surface markers (CS24(-)CD44(+)ESA(+)) and found that this cell population has significantly higher tumor-initiating ability to generate DCIS compared with the non-stem-like population. Furthermore, the CSCs showed significantly higher level of expression of all lipogenic genes than the counterpart population from non-tumorigenic breast cancer cell line, MCF10A. Importantly, ectopic expression of SREBP1, the master regulator of lipogenic genes, in MCF10A significantly enhanced lipogenesis in stem-like cells and promoted cell growth as well as mammosphere formation. Moreover, SREBP1 expression significantly increased the ability of cell survival of CSCs from MCF10AT, another cell line that is capable of generating DCIS, in mouse and in cell culture. These results indicate that upregulation of lipogenesis is a pre-requisite for DCIS formation by endowing the ability of cell survival. We have also shown that resveratrol was capable of blocking the lipogenic gene expression in CSCs and significantly suppressed their ability to generate DCIS in animals, which provides us with a strong rationale to use this agent for chemoprevention against DCIS.


Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Carcinoma, Intraductal, Noninfiltrating/metabolism , Carcinoma, Intraductal, Noninfiltrating/pathology , Lipogenesis/genetics , Sterol Regulatory Element Binding Protein 1/genetics , Animals , Apoptosis/drug effects , Breast Neoplasms/prevention & control , Carcinoma, Intraductal, Noninfiltrating/prevention & control , Cell Line, Tumor , Cell Proliferation , Cell Survival , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lipogenesis/drug effects , Mice , Mice, Nude , Resveratrol , Stem Cells/pathology , Sterol Regulatory Element Binding Protein 1/metabolism , Stilbenes , Xenograft Model Antitumor Assays
9.
Oncogene ; 30(39): 4075-86, 2011 Sep 29.
Article in English | MEDLINE | ID: mdl-21499308

ABSTRACT

Notch signaling is often and aberrantly activated by hypoxia during tumor progression; however, the exact pathological role of hypoxia-induced Notch signaling in tumor metastasis is as yet poorly understood. In this study, we aimed to define the mechanism of Notch-ligand activation by hypoxia in both primary tumor and bone stromal cells in the metastatic niche and to clarify their roles in tumor progression. We have analyzed the expression profiles of various Notch ligands in 779 breast cancer patients in GEO database and found that the expression of Jagged2 among all five ligands is most significantly correlated with the overall- and metastasis-free survival of breast cancer patients. The results of our immunohistochemical (IHC) analysis for Jagged2 in 61 clinical samples also revealed that both Jagged2 and Notch signaling were strongly upregulated at the hypoxic invasive front. Activation of Jagged2 by hypoxia in tumor cells induced EMT and also promoted cell survival in vitro. Notably, a γ-secretase inhibitor significantly blocked Notch-mediated invasion and survival under hypoxia by promoting expression of E-cadherin and inhibiting Akt phosphorylation. Importantly, Jagged2 was also found to be upregulated in bone marrow stroma under hypoxia and promoted the growth of cancer stem-like cells by activating their Notch signaling. Therefore, hypoxia-induced Jagged2 activation in both tumor invasive front and normal bone stroma has a critical role in tumor progression and metastasis, and Jagged2 is considered to be a valuable prognostic marker and may serve as a novel therapeutic target for metastatic breast cancer.


Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Hypoxia , Intercellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Neoplasm Metastasis , Neoplastic Stem Cells/metabolism , Receptors, Notch/metabolism , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Breast Neoplasms/genetics , Cadherins/biosynthesis , Cadherins/genetics , Cell Line, Tumor , Cell Survival , Female , Humans , Intercellular Signaling Peptides and Proteins/genetics , Jagged-2 Protein , Membrane Proteins/genetics , Neoplastic Stem Cells/pathology , Oncogene Protein v-akt/metabolism , Phosphorylation , Receptors, Notch/genetics , Stromal Cells
10.
Oncogene ; 30(7): 822-31, 2011 Feb 17.
Article in English | MEDLINE | ID: mdl-20956939

ABSTRACT

MicroRNAs are gene regulators that work through a posttranscriptional repression mechanism. Dysregulation of microRNA expression could lead to a variety of disorders, in particular, human cancer, and has also been implicated in antihormone therapy resistance. However, little is known whether microRNAs have a role in estrogen-independent growth, leading to tamoxifen resistance in estrogen receptor (ER)-positive tumors. In this study, we use an in vivo selection system against a microRNA library using the MCF-7 model and demonstrate that miR-101 promotes estrogen-independent growth and causes the upregulation of phosphorylated Akt (pAkt) without impacting the ER level or activity. Importantly, although miR-101 suppresses cell growth in normal estradiol (E2)-containing medium, it promotes cell growth in E2-free medium. Moreover, estrogen deprivation greatly enhances miR-101-mediated Akt activation. Finally, we show that MAGI-2 (membrane-associated guanylate kinase), a scaffold protein required for PTEN (phosphatase and tensin homolog) activity, is a direct target for miR-101; suppression of MAGI-2 by miR-101 reduces PTEN activity, leading to Akt activation. Taken together, these results not only establish a role for miR-101 in estrogen-independent signaling but also provide a mechanistic link between miR-101 and Akt activation.


Subject(s)
Estrogens/metabolism , MicroRNAs/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Adaptor Proteins, Signal Transducing , Adenocarcinoma/drug therapy , Adenocarcinoma/metabolism , Antineoplastic Agents, Hormonal/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Carrier Proteins/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Resistance, Neoplasm , Female , Guanylate Kinases , Humans , PTEN Phosphohydrolase/metabolism , Receptors, Estrogen/metabolism , Tamoxifen/pharmacology , Up-Regulation
11.
Oncogene ; 29(12): 1763-72, 2010 Mar 25.
Article in English | MEDLINE | ID: mdl-20023705

ABSTRACT

Ubc9 is an E2-conjugating enzyme that transfers the activated small ubiquitin-like modifier (SUMO) to protein substrates, and thus it has an important function in sumoylation-mediated cellular pathways. We have earlier reported that Ubc9 promotes tumor growth in the xenograft mouse model using breast cancer cell line MCF-7 in part through regulation of Bcl-2 expression. In this study, we show that ectopic expression of wild-type Ubc9 (Ubc9-WT) promotes cell invasion and metastasis. Surprisingly, the dominant negative mutant Ubc9 (Ubc9-DN) also causes the same phenotype, indicating that the ability of Ubc9 to promote invasion and metastasis is distinct from its ability to conjugate SUMO to protein substrates. Of considerable interest, several microRNAs such as miR-224 are regulated by Ubc9. Although ectopic expression of Ubc9 causes downregulation of miR-224, suppression of Ubc9 by Ubc9-siRNAs leads to its upregulation. We further show that miR-224 can inhibit cell invasion and directly targets CDC42 and CXCR4, and that suppression of CDC42 and CXCR4 by RNAi causes inhibition of Ubc9-mediated invasion. Together, these results show a molecular link between Ubc9 and the metastasis genes such as CDC42 and CXCR4, and thus provide new insight into the mechanism by which Ubc9 promotes tumor invasion and metastasis.


Subject(s)
Neoplasm Invasiveness/pathology , Neoplasm Metastasis/pathology , Ubiquitin-Conjugating Enzymes/physiology , Adenocarcinoma/genetics , Animals , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Female , Genetic Vectors , Humans , Lung Neoplasms/genetics , Mice , MicroRNAs/genetics , Myotonin-Protein Kinase , Neoplasm Invasiveness/genetics , Neoplasm Metastasis/genetics , Oligonucleotide Array Sequence Analysis , Protein Serine-Threonine Kinases/genetics , RNA, Messenger/genetics , RNA, Neoplasm/genetics , Receptors, CXCR4/genetics , Transfection , Ubiquitin-Conjugating Enzymes/genetics , Up-Regulation
12.
Oncogene ; 26(19): 2799-803, 2007 Apr 26.
Article in English | MEDLINE | ID: mdl-17072344

ABSTRACT

MicroRNAs (miRNAs) are approximately 22 nucleotide non-coding RNA molecules that regulate gene expression post-transcriptionally. Although aberrant expression of miRNAs in various human cancers suggests a role for miRNAs in tumorigenesis, it remains largely unclear as to whether knockdown of a specific miRNA affects tumor growth. In this study, we profiled miRNA expression in matched normal breast tissue and breast tumor tissues by TaqMan real-time polymerase chain reaction miRNA array methods. Consistent with previous findings, we found that miR-21 was highly overexpressed in breast tumors compared to the matched normal breast tissues among 157 human miRNAs analysed. To better evaluate the role of miR-21 in tumorigenesis, we transfected breast cancer MCF-7 cells with anti-miR-21 oligonucleotides and found that anti-miR-21 suppressed both cell growth in vitro and tumor growth in the xenograft mouse model. Furthermore, this anti-miR-21-mediated cell growth inhibition was associated with increased apoptosis and decreased cell proliferation, which could be in part owing to downregulation of the antiapoptotic Bcl-2 in anti-miR-21-treated tumor cells. Together, these results suggest that miR-21 functions as an oncogene and modulates tumorigenesis through regulation of genes such as bcl-2 and thus, it may serve as a novel therapeutic target.


Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/pathology , Gene Expression Regulation, Neoplastic , MicroRNAs/physiology , Oligonucleotides, Antisense/pharmacology , Animals , Apoptosis , Breast/metabolism , Breast Neoplasms/metabolism , Cell Proliferation , Female , Humans , Mice , Mice, Nude , MicroRNAs/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA Processing, Post-Transcriptional , RNA, Messenger/genetics , RNA, Messenger/metabolism , Xenograft Model Antitumor Assays
13.
Biochem Soc Trans ; 29(Pt 6): 702-3, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11709058

ABSTRACT

DNA topoisomerase (topo) II inhibitors either stabilize DNA-topo II complexes by blocking DNA religation (e.g. etoposide) or block the enzyme's catalytic activity (e.g. dexrazoxane). The former class of drugs causes direct DNA damage through topo II, while the latter class does not, but both classes cause apoptosis. We cloned the Fas ligand (FasL) promoter and coupled it to the luciferase gene. Treatment of cells transfected with this construct revealed that complex-stabilizing (DNA-damaging) agents induce FasL expression, but the catalytic inhibitors do not, suggesting that the FasL pathway may not be involved in all cases of topoisomerase-mediated apoptosis. Some topo II inhibitors activate a pathway involving stress-activated protein kinases, which include c-Jun N-terminal kinase-1 (JNK-1). We will discuss the effects of these agents on components of this pathway. Our earlier work revealed that topo IIalpha interacts with the cell cycle regulatory protein, retinoblastoma protein (Rb). This interaction and the subcellular distribution of these proteins are altered by topo II inhibitory drugs and lead to apoptosis. In addition, agents that affect Rb, such as E1A and E2F1/DP-1, when transfected into cells, also alter topo IIalpha-Rb localization, activate jun kinase pathways and cause apoptosis. This paper discusses current studies that are designed to determine the contributions of these signalling events to the alterations in subcellular protein distribution and apoptosis. We suggest that protein-protein interactions are important for mediation of cytotoxic signalling by anticancer drugs.


Subject(s)
Enzyme Inhibitors/pharmacology , Signal Transduction , Topoisomerase II Inhibitors , Animals , Apoptosis , Fas Ligand Protein , Humans , Membrane Glycoproteins/metabolism , Protein Binding , Tumor Cells, Cultured
14.
J Biol Chem ; 275(52): 41107-13, 2000 Dec 29.
Article in English | MEDLINE | ID: mdl-11016921

ABSTRACT

DNA topoisomerase (topo) I is a nuclear enzyme that plays an important role in DNA metabolism. Based on conserved nuclear targeting sequences, four classic nuclear localization signals (NLSs) have been proposed at the N terminus of human topo I, but studies with yeast have suggested that only one of them (amino acids (aa) 150-156) is sufficient to direct the enzyme to the nucleus. In this study, we expressed human topo I fused to enhanced green fluorescent protein (EGFP) in mammalian cells and demonstrated that whereas aa 150-156 are sufficient for nuclear localization, the nucleolar localization requires aa 157-199. More importantly, we identified a novel NLS within aa 117-146. In contrast to the classic NLSs that are rich in basic amino acids, the novel NLS identified in this study is rich in acidic amino acids. Furthermore, this novel NLS alone is sufficient to direct not only EGFP into the nucleus but also topo I; and the EGFP.topo I fusion driven by the novel NLS is as active in vivo as the wild-type topo I in response to the topo I inhibitor topotecan. Together, our results suggest that human topo I carries two independent NLSs that have opposite amino acid compositions.


Subject(s)
Cell Nucleus/enzymology , DNA Topoisomerases, Type I/chemistry , Amino Acid Sequence , Cell Nucleolus/enzymology , DNA Topoisomerases, Type I/metabolism , HeLa Cells , Humans , Molecular Sequence Data , Topoisomerase I Inhibitors , Topotecan/pharmacology
15.
J Biol Chem ; 275(51): 39907-13, 2000 Dec 22.
Article in English | MEDLINE | ID: mdl-10986282

ABSTRACT

Antioxidant response element (ARE) regulates the induction of a number of cellular antioxidant and detoxifying enzymes. However, the signaling pathways that lead to ARE activation remain unknown. Here, we report that the expression of mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1), transforming growth factor-beta-activated kinase (TAK1), and apoptosis signal-regulating kinase (ASK1) in HepG2 cells activated the ARE reporter gene, whereas the expression of their dominant-negative mutants impaired ARE activation by the chemicals sodium arsenite and mercury chloride. Coexpression of downstream kinases, MAP kinase kinase 4, MAP kinase kinase 6, and c-Jun NH(2)-terminal kinase-1, but not MAP kinase kinase 3 and p38, augmented ARE activation by MEKK1, TAK1, and ASK1. The coexpression of a basic leucine zipper transcription factor Nrf2 but not c-Jun also greatly enhanced the activation of reporter gene by MEKK1, TAK1, and ASK1; however, a dominant-negative mutant of Nrf2 (NF-E2-related factor 2) blocked this event. Furthermore, when overexpressed, MEKK1, TAK1, and ASK1 induced the expression of heme oxygenase-1, a gene regulated by ARE, and the cotransfection with the dominant-negative mutant of Nrf2 abolished the induction. Taken together, these results suggest that MAP kinase pathways that are activated by MEKK1, TAK1, and ASK1 may link chemical signals to Nrf2, leading to the activation of ARE-dependent genes.


Subject(s)
Antioxidants/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation , MAP Kinase Signaling System , Trans-Activators/metabolism , Base Sequence , DNA Primers , Enzyme Activation , Genes, Reporter , NF-E2-Related Factor 2
16.
Exp Cell Res ; 256(2): 480-90, 2000 May 01.
Article in English | MEDLINE | ID: mdl-10772820

ABSTRACT

DNA topoisomerase (topo) I plays an important role in DNA metabolism by relieving the torsional restraints of DNA topology through ATP-independent single-strand DNA breakage. In the present study, we expressed human topo I in HeLa cells by fusing it to enhanced green fluorescent protein (EGFP). The EGFP-topo I fusion protein is functionally active in that it relaxes supercoiled plasmid DNA; forms complexes with DNA, as revealed by band depletion assays; and increases the sensitivity of cells to topo I inhibitors such as topotecan, as determined by growth inhibition assays. In contrast, a mutant form of the EGFP-topo I fusion protein, in which the active Tyr has been replaced by Phe (Y723F), has no such activities. Furthermore, the fusion protein localizes to the nucleus at interphase and completely associates with chromatids at every stage of mitosis. Of importance, the mutant fusion protein (Y723F) displays a pattern of subcellular localization identical to that of the wild-type fusion protein, although the mutant fusion protein is catalytically inactive. These results suggest that in addition to its role in DNA metabolism, topo I might also play a structural role in chromosomal organization; moreover, the association of topo I with chromosomal DNA is independent of its catalytic activity. Finally, the fusion constructs may provide a useful tool to study drug action in tumor cells, as demonstrated by nucleolar delocalization of the fusion proteins in response to treatment with the topo I inhibitor topotecan.


Subject(s)
DNA Topoisomerases, Type I/metabolism , Amino Acid Substitution , Cell Nucleus/enzymology , Chromosomes, Human , DNA/metabolism , DNA Topoisomerases, Type I/genetics , Enzyme Inhibitors/pharmacology , Green Fluorescent Proteins , HeLa Cells , Humans , Immunoblotting , Interphase , Luminescent Proteins/genetics , Microscopy, Fluorescence , Mitosis , Mutation , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Subcellular Fractions/enzymology , Topoisomerase I Inhibitors , Topotecan/pharmacology
17.
Exp Cell Res ; 252(1): 50-62, 1999 Oct 10.
Article in English | MEDLINE | ID: mdl-10502399

ABSTRACT

DNA topoisomerase (topo) II is an essential nuclear enzyme that plays an important role in DNA metabolism and chromosome organization. In the present study, we expressed human topo IIalpha in mammalian cells by fusion to an enhanced green fluorescent protein (EGFP). Decatenation assays indicated that the EGFP-topo IIalpha is catalytically active in vitro. Assays for band depletion, growth inhibition, and cytotoxicity by topo II inhibitors suggested that the fusion protein is also functional in vivo. By following its subcellular localization throughout the cell cycle in living cells, we found that the fusion protein is localized to the nucleus and nucleolus at interphase, and it is bound to chromosomal DNA at every stage of mitosis. Of importance, a mutant EGFP-topo IIalpha, in which the active Tyr 805 is replaced by Phe (Y805F) and is catalytically inactive, still binds to chromosomal DNA throughout the cell cycle like the wild-type enzyme. Together, our results suggest that the ability of topo IIalpha to bind to chromosomal DNA in the cell, a presumed requirement for its structural role, can be separated from its catalytic activity.


Subject(s)
Chromosomes/enzymology , DNA Topoisomerases, Type II , DNA Topoisomerases, Type II/metabolism , Isoenzymes/metabolism , Mitosis/physiology , Antigens, Neoplasm , Base Sequence , Cell Line , Cell Nucleus/enzymology , Cytoplasm/enzymology , DNA Primers/genetics , DNA Topoisomerases, Type II/genetics , DNA-Binding Proteins , Enzyme Inhibitors/pharmacology , Gene Expression , Green Fluorescent Proteins , HeLa Cells , Humans , Interphase/physiology , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mutagenesis, Site-Directed , Point Mutation , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Teniposide/pharmacology , Topoisomerase II Inhibitors
18.
Mol Pharmacol ; 55(2): 216-22, 1999 Feb.
Article in English | MEDLINE | ID: mdl-9927611

ABSTRACT

Many anticancer agents exert their cytotoxicity through DNA damage and induction of apoptosis. Fas ligand (FasL), a key component of T lymphocytes, has been shown to be induced by some of those agents. To address what is an early signal for this induction, we constructed a FasL promoter-luciferase reporter gene to investigate effects of DNA topoisomerase (Topo) II inhibitors on FasL promoter activity. Transient transfection assays in HeLa and other tumor cell lines demonstrated that induction of FasL promoter activity in response to Topo II inhibitors such as VM-26 mimicked endogenous FasL expression under the same conditions. The ability of these agents to induce FasL expression correlated with their ability to cause DNA damage. For instance, complex-stabilizing Topo II inhibitors such as etoposide, teniposide, and doxorubicin, which cause DNA damage, strongly induce FasL expression; by contrast, non-DNA-damaging catalytic Topo II inhibitors such as ICRF-187 and merbarone do not do this. In support of the notion that DNA damage triggers FasL induction, we found that DNA-damaging irradiation also induced FasL promoter activity in a dose-dependent manner. Finally, the catalytic Topo II inhibitor ICRF-187 suppressed VM-26-induced-FasL expression. This suppression correlated with the ability of this drug to inhibit VM-26-induced DNA strand breaks. Together, our results suggest that DNA damage in response to agents such as etoposide and teniposide might serve as an early signal to induce FasL expression.


Subject(s)
DNA Damage/physiology , Membrane Glycoproteins/genetics , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , CHO Cells , Cricetinae , DNA/chemistry , DNA/drug effects , DNA Damage/genetics , Doxorubicin/pharmacology , Enzyme Inhibitors/pharmacology , Etoposide/pharmacology , Fas Ligand Protein , Gene Expression Regulation, Neoplastic/drug effects , HeLa Cells , Humans , Jurkat Cells , Nucleic Acid Synthesis Inhibitors/pharmacology , Promoter Regions, Genetic/genetics , Razoxane/pharmacology , Recombinant Fusion Proteins/genetics , Signal Transduction/drug effects , Teniposide/pharmacology , Thiobarbiturates/pharmacology , Topoisomerase II Inhibitors , Tumor Cells, Cultured , Up-Regulation
19.
Biotechniques ; 25(6): 1052-7, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9863061

ABSTRACT

DNA topoisomerase (topo) II alpha is a major target for many anticancer agents. However, progress towards understanding how these agents interact with this enzyme in human cells and how resistance to these agents arises is greatly impeded by difficulties in expressing this gene. Here, we report on achieving a high level of expression of a full-length human topo II alpha gene in human cells. We started with the topo II alpha cDNA driven by a strong cytomegalovirus promoter and transiently transfected HeLa cells. Although topo II alpha mRNA was consistently detected in transfected cells, no exogenous topo II alpha protein was detected. By contrast, when the same cDNA was fused to an enhanced green fluorescent protein (EGFP), we detected a high level of expression at both mRNA and protein levels. The exogenous topo II alpha was localized to cell nuclei as expected, indicating that the fusion protein is properly folded. Furthermore, overexpression of the EGFP-topo II alpha fusion protein increased the sensitivity of the transfected cells to teniposide, suggesting that it functions as the endogenous counterpart. Thus, in addition to being used as a gene tag, the GFP fusion approach may be generally applicable for expressing genes, such as topo II alpha, that are difficult to express by conventional methods.


Subject(s)
DNA Topoisomerases, Type II/genetics , Gene Expression Regulation, Viral , Indicators and Reagents/metabolism , Luminescent Proteins/genetics , Recombinant Fusion Proteins/genetics , Base Sequence , Cell Nucleus/chemistry , DNA Topoisomerases, Type II/analysis , Enzyme Inhibitors/pharmacology , Genes, Reporter , Green Fluorescent Proteins , HeLa Cells , Humans , Immunoblotting , Microscopy, Fluorescence , Molecular Sequence Data , RNA, Messenger/analysis , Recombinant Fusion Proteins/analysis , Teniposide/pharmacology , Topoisomerase II Inhibitors , Transfection
20.
Biochem J ; 335 ( Pt 1): 67-77, 1998 Oct 01.
Article in English | MEDLINE | ID: mdl-9742214

ABSTRACT

FK506-binding proteins (FKBPs) have been identified in a variety of eukaryotic and prokaryotic organisms. Macrophage infectivity potentiator (CbMip, 23.5 kDa) protein of the obligate intracellular bacterium, Coxiella burnetii, was shown previously to belong to the family of FKBPs based on sequence homology and peptidyl-prolyl cis/trans isomerase (PPIase) activity. Further characterization of the cbmip gene has identified two additional proteins with molecular masses of 15.5 and 15.0 kDa that are synthesized, in addition to the 23.5 kDa CbMip, when expressed in Escherichia coli. Amino acid sequencing at the N-terminus combined with transcription and translation fusion expression revealed that the two proteins were synthesized from the same open reading frame of the cbmip gene, but starting at different internal translation start codons, probably by translational reinitiation. When the internal methionines serving as start sites were replaced with lysine by site-directed mutagenesis, the synthesis of 15.5 and 15.0 kDa proteins was abolished even though the synthesis of 23.5 kDa CbMip was intact. This confirmed that the 15.5 and 15.0 kDa proteins are indeed generated by translational reinitiation and are not degradation products of the 23.5 kDa protein. Like other FKBPs, both 15.5 and 15.0 kDa proteins exhibit PPIase activity. Because they share significant sequence homology with FKBPs and have a similar PPIase activity, 15.5 and 15. 0 kDa proteins are designated as C. burnetii FKBP (Cb-FKBP) analogues I and II, respectively. TnphoA mutagenesis demonstrated that whereas the large protein (CbMip) is secreted, Cb-FKBP analogues I and II are cytoplasmic, indicating that structural variations could allow for different subcellular compartmentalization of similar proteins. Western-blot analysis of lysates of purified C. burnetii using a CbMip-specific monoclonal antibody revealed the presence of a protein migrating at approximately 15 kDa, indicating the presence of smaller Cb-FKBP analogue(s) in C. burnetii, although at much lower levels compared with 23.5 kDa CbMip. This unique gene organization seen with cbmip may provide the organism with a mechanism of efficient use of its limited genetic information to synthesize proteins that are structurally different yet functionally similar.


Subject(s)
Escherichia coli/metabolism , Immunophilins/chemistry , Immunophilins/genetics , Membrane Proteins/chemistry , Membrane Proteins/genetics , Open Reading Frames , Amino Acid Sequence , Animals , Coxiella burnetii , Escherichia coli Proteins , Humans , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptidylprolyl Isomerase/metabolism , RNA, Messenger/metabolism , Tacrolimus Binding Proteins
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