ABSTRACT
Glioblastoma multiforme (GBM) is the most common primary malignant brain cancer in adults. However, the molecular events underlying carcinogenesis and their interplay remain elusive. Here, we report that the stability of Ubiquitin-conjugating enzyme E2S (UBE2S) is regulated by the PTEN/Akt pathway and that its degradation depends on the ubiquitin-proteasome system. Mechanistically, Akt1 physically interacted with and phosphorylated UBE2S at Thr 152, enhancing its stability by inhibiting proteasomal degradation. Additionally, accumulated UBE2S was found to be associated with the components of the non-homologous end-joining (NHEJ) complex and participated in the NHEJ-mediated DNA repair process. The association of Ku70 with UBE2S was enhanced, and the complex was recruited to double-stranded break (DSB) sites in response to etoposide treatment. Furthermore, knockdown of UBE2S expression inhibited NHEJ-mediated DSB repair and rendered glioblastoma cells more sensitive to chemotherapy. Overall, our findings provide a novel drug target that may serve as the rationale for the development of a new therapeutic approach.
Subject(s)
Biomarkers, Tumor/metabolism , DNA Repair/genetics , Drug Resistance, Neoplasm/genetics , Glioblastoma/pathology , Ku Autoantigen/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Animals , Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Biomarkers, Tumor/genetics , Cell Proliferation/drug effects , DNA Damage/drug effects , DNA Damage/genetics , DNA Repair/drug effects , Etoposide/pharmacology , Female , Glioblastoma/drug therapy , Glioblastoma/genetics , Humans , Ku Autoantigen/genetics , Mice , Mice, Inbred BALB C , Mice, Nude , Phosphorylation , Proto-Oncogene Proteins c-akt/genetics , Tumor Cells, Cultured , Ubiquitin/metabolism , Ubiquitin-Conjugating Enzymes/genetics , Xenograft Model Antitumor AssaysABSTRACT
Immune escape describes a critical event whereby tumor cells adopt an immunoresistant phenotype to escape adaptive surveillance. We show that expression of a pivotal negative regulator of T-cell function, B7-H1, correlates with PI(3) kinase activation in breast and prostate cancer patients. B7-H1-mediated immunoresistance can be attenuated by inhibitors of the PI(3) kinase pathway, and is dependent on S6K1-mediated translational regulation of B7-H1 protein. Breast and prostate carcinoma cells with activated PI(3) kinase lose the immunoresistant phenotype after treatment with B7-H1 siRNA. Conversely, breast and prostate carcinoma cells with minimal PI(3) kinase activation adopt an immunoresistant phenotype when engineered to overexpress B7-H1 protein. These observations describe a mechanism for immune escape from tumor dormancy in humans that relates to oncogenesis.
Subject(s)
Adenocarcinoma/enzymology , Antigens, CD/physiology , Breast Neoplasms/enzymology , Neoplasm Proteins/physiology , Phosphatidylinositol 3-Kinases/physiology , Prostatic Neoplasms/enzymology , Tumor Escape/immunology , Adenocarcinoma/immunology , Adenocarcinoma/pathology , Antigens, CD/genetics , B7-H1 Antigen , Breast Neoplasms/immunology , Breast Neoplasms/pathology , Cell Line, Tumor/drug effects , Cell Line, Tumor/enzymology , Cell Line, Tumor/immunology , Female , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Humans , Male , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , PTEN Phosphohydrolase/physiology , Phenotype , Phosphoinositide-3 Kinase Inhibitors , Prostatic Neoplasms/immunology , Prostatic Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , RNA, Small Interfering/pharmacology , Recombinant Fusion Proteins/physiology , Ribosomal Protein S6 Kinases/physiology , T-Lymphocytes, Cytotoxic/immunology , Tumor Escape/drug effects , Tumor Escape/geneticsABSTRACT
Although autophagy enhances cell survival in nutrient-deprived cells by increasing adenosine triphosphate (ATP) production, it remains unclear if autophagy functions similarly in cells treated with cytotoxic chemotherapy agents. To address this issue, we measured both the ability of DNA damaging agents (Temozolomide, and Etoposide) to induce an autophagy-dependent production of ATP, and the effects of modulation of autophagy on drug-induced cell death. Both drugs induced an autophagy-associated increase in ATP production in multiple glioma cell lines. The drug-induced ATP surge could not be blocked by glucose starvation, but could be blocked by preincubation with the autophagy inhibitor 3-methyladenine (3-MA), an siRNA targeting beclin 1, or the mitochondrial inhibitor oligomycin. Inhibition of autophagy-induced ATP production increased non-apoptotic cell death associated with micronucleation, while restoration of the 3-MA-inhibited ATP surge by addition of pyruvate suppressed cell death. These results show that DNA damaging agents induce an autophagy-associated ATP surge that protects cells and may contribute to drug resistance.
Subject(s)
Adenosine Triphosphate/metabolism , Autophagy , DNA Damage , Dacarbazine/analogs & derivatives , Glioma/metabolism , Adenosine Triphosphate/physiology , Antineoplastic Agents, Alkylating/pharmacology , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Beclin-1 , Cell Death , Cell Line, Tumor , Dacarbazine/pharmacology , Etoposide/pharmacology , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Oxidation-Reduction , Phosphorylation , TemozolomideABSTRACT
Human malignant gliomas are thought to develop as the result of stepwise accumulations of multiple genetic alterations. Recently, we showed that E6/E7-mediated inactivation of p53/pRb, ras pathway activation (initiated by expression of mutant H-Ras), and expression of human telomerase reverse transcriptase (hTERT) in combination converted normal human astrocytes into cells that formed intracranial tumors resembling human anaplastic astrocytoma (AA). In this study, we created human astrocytes that, in addition to expressing E6/E7, hTERT, and Ras, also expressed a constitutive activated form of Akt intended to mimic the Akt activation noted in grade IV glioblastoma multiforme (GBM). Although these cells grew no differently than astrocytes expressing E6, E7, and H-Ras in vitro or in the first 28 days following s.c. implantation, they ultimately formed tumors four to six times larger than those formed by the E6/E7/hTERT/Ras cells. Unlike the poorly vascularized, necrosis-free AA formed by E6/E7/hTERT/Ras cells, the tumors formed by s.c. or intracranial injection of Akt-expressing cells had large areas of necrosis surrounded by neovascularization and were consistent in appearance with grade IV human GBM. These results show that activation of the Akt pathway is sufficient to allow conversion of human AA to human GBM.
Subject(s)
Astrocytoma/enzymology , Astrocytoma/pathology , Brain Neoplasms/enzymology , Brain Neoplasms/pathology , Glioblastoma/enzymology , Glioblastoma/pathology , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/physiology , Animals , Astrocytes/enzymology , Astrocytes/pathology , Astrocytes/physiology , Astrocytoma/genetics , Brain Neoplasms/genetics , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Disease Progression , Enzyme Activation , Glioblastoma/genetics , Humans , Mice , Mice, Nude , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-akt , Rats , Signal Transduction/physiology , TransfectionABSTRACT
Temozolomide (TMZ) produces O(6)-methylguanine in DNA, which in turn mispairs with thymine, triggering futile DNA mismatch repair (MMR) and ultimately cell death. We found previously that in p53-proficient human glioma cells, TMZ-induced futile DNA MMR resulted not in apoptosis but rather in prolonged, p53- and p21-associated G(2)-M arrest and senescence. Additionally, p53-deficient cells were relatively more TMZ resistant than p53-deficient glioma cells, which underwent only transient G(2)-M arrest before death by mitotic catastrophe. These results suggested that prolonged G(2)-M arrest might protect cells from TMZ-induced cytotoxicity. In the present study, we therefore focused on the mechanism by which TMZ induces G(2)-M arrest and on whether inhibition of such G(2)-M arrest might sensitize glioma cells to TMZ-induced toxicity. U87MG glioma cells treated with TMZ underwent G(2)-M arrest associated with Chk1 activation and phosphorylation of both cdc25C and cdc2. These TMZ-induced effects were inhibited by the Chk1 kinase inhibitor UCN-01. Although not in itself toxic, UCN-01 increased the cytotoxicity of TMZ 5-fold, primarily by inhibiting cellular senescence and increasing the percentage of cells bypassing G(2)-M arrest and undergoing mitotic catastrophe. In addition to enhancing TMZ-induced cytotoxicity in p53-proficient cells, UCN-01 also blocked TMZ-induced Chk1 activation and transient G(2)-M arrest in p53-deficient U87MG-E6 cells and similarly enhanced TMZ-induced mitotic catastrophe and cell death. Taken together, these results indicate that Chk1 links TMZ-induced MMR to G(2)-M arrest. Furthermore, inhibition of the cytoprotective G(2) arrest pathway sensitizes cells to TMZ-induced cytotoxicity and may represent a novel, mechanism-based means of increasing TMZ efficacy in both p53 wild-type and p53 mutant glioma cells.
Subject(s)
Antineoplastic Agents, Alkylating/toxicity , Dacarbazine/toxicity , G2 Phase/physiology , Glioblastoma/drug therapy , Protein Kinase Inhibitors , Protein Kinases , Tumor Suppressor Protein p53/physiology , Alkaloids/pharmacology , CDC2 Protein Kinase/metabolism , Cell Cycle Proteins/metabolism , Checkpoint Kinase 1 , Dacarbazine/analogs & derivatives , Drug Synergism , Enzyme Inhibitors/pharmacology , G2 Phase/drug effects , Glioblastoma/enzymology , Glioblastoma/pathology , Humans , Mitosis/drug effects , Mitosis/physiology , Phosphorylation/drug effects , Staurosporine/analogs & derivatives , Temozolomide , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolism , cdc25 Phosphatases/metabolismABSTRACT
The formation of human malignant gliomas is thought to involve the accumulation of multiple genetic alterations. To define the function of specific alterations in glioma formation, we serially introduced genetic alterations functionally equivalent to those noted in human malignant gliomas into normal human astrocytes (NHAs). We then monitored the ability of each of these alterations to contribute to the growth of otherwise genetically stable NHAs into intracranial malignant gliomas. Using this model, we show that expression of human telomerase catalytic component (hTERT), but not E7-mediated inactivation of pRb or E6/E7-mediated inactivation of p53/pRb, was sufficient to initiate the tumorigenic process by circumventing cellular senescence in astrocytes. hTERT expression, even in combination with inactivation of p53/pRb, did not transform astrocytes. These alterations together, however, cooperated with ras pathway activation (initiated by expression of mutant H-Ras), but not with phosphatidylinositol 3-kinase pathway activation (initiated by expression of myristoylated Akt) or epidermal growth factor receptor activation, to allow for the formation of intracranial tumors strongly resembling p53/pRb pathway-deficient, telomerase-positive, ras-activated human grade III anaplastic astrocytomas. These results identify four pathways as key in the development of human anaplastic astrocytomas.
Subject(s)
Astrocytes/physiology , Astrocytoma/genetics , Brain Neoplasms/genetics , Cell Transformation, Neoplastic/genetics , RNA , Repressor Proteins , Animals , Astrocytes/pathology , DNA-Binding Proteins , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Oncogene Proteins, Viral/biosynthesis , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/physiology , Papillomavirus E7 Proteins , Retinoblastoma Protein/antagonists & inhibitors , Signal Transduction/genetics , Telomerase/biosynthesis , Telomerase/genetics , Telomerase/physiology , Transfection , Tumor Suppressor Protein p53/antagonists & inhibitors , ras Proteins/physiologyABSTRACT
Temozolomide (TMZ) is a DNA-methylating agent that has recently been introduced into Phase II and III trials for the treatment of gliomas. TMZ produces O6-methylguanine in DNA, which mispairs with thymine during the next cycle of DNA replication. Subsequent futile cycles of DNA mismatch repair can lead to a p53-associated apoptotic cell death, although this mechanism has been described mostly in hematopoietic neoplasms. We studied the action of TMZ in gliomas and the role p53 might play by using U87 glioma cells that were either p53-wild-type or p53-deficient (by virtue of expression of the viral oncoprotein E6). LN-Z308 cells, in which p53 gene is deleted, were also used. p53-proficient U87 MG cells underwent a prolonged, p53- and p21(Waf1/Cip1)-associated G2-M arrest beginning 2 days after TMZ treatment. Although very few of these cells underwent apoptosis, most underwent senescence over a 10-day period. p53-deficient (E6-transfected U87 and LN-Z308) cells similarly underwent G2-M arrest in response to TMZ, but this arrest was accompanied by only minor changes in p53 or p21(Waf1/Cip1) and was reversed within 7 days of TMZ treatment in association with the appearance of cells with either 8n or subG1 DNA content. These results suggest that glioma cells respond to TMZ by undergoing G2-M arrest. p53 is not necessary for this G2-M arrest to occur but is important in the duration of G2-M arrest and in the ultimate fate of TMZ-treated cells. Therefore, the integrity of the G2-M cell cycle checkpoint may be important in the cytotoxicity of TMZ in glioma cells.
Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , Dacarbazine/analogs & derivatives , Dacarbazine/pharmacology , G2 Phase/drug effects , Glioblastoma/pathology , Mitosis/drug effects , Tumor Suppressor Protein p53/physiology , Base Pair Mismatch , Cell Survival/drug effects , Cell Survival/physiology , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/metabolism , G2 Phase/physiology , Glioblastoma/drug therapy , Humans , Mitosis/physiology , O(6)-Methylguanine-DNA Methyltransferase/deficiency , Temozolomide , Tumor Cells, Cultured/drug effects , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/metabolismABSTRACT
Normal human fibroblasts have been shown to undergo a p16(Ink4a)-associated senescence-like growth arrest in response to sustained activation of the Ras/Raf/MEK/ERK pathway. We noted a similar p16(Ink4a)-associated, senescence-like arrest in normal human astrocytes in response to expression of a conditional form of Raf-1. While HPV16 E7-mediated functional inactivation of the p16(Ink4a)/pRb pathway in astrocytes blocked the p16(Ink4a)-associated growth arrest in response to activation of Raf-1, it also revealed a second p21(Cip1)-associated, senescence-associated, beta-galactosidase-independent growth arrest pathway. Importantly, the p21(Cip1)-associated pathway was present not only in normal astrocytes but also in p53-, p14(ARF)-, and p16(Ink4a)/pRb-deficient high grade glioma cells that lacked the p16(Ink4a)-dependent arrest mechanism. These results suggest that normal human cells have redundant arrest pathways, which can be activated by Raf-1, and that even tumors that have dismantled p16(Ink4a)-dependent growth arrest pathways are potentially regulated by a second p21(Cip1)-dependent growth arrest pathway.
Subject(s)
Astrocytes/metabolism , Brain Neoplasms/metabolism , Glioma/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Blotting, Western , Cell Division , Cellular Senescence , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/metabolism , Enzyme Activation , Humans , Oncogene Proteins, Viral/metabolism , Papillomavirus E7 Proteins , Phenotype , Phosphorylation , Protein Binding , Retinoblastoma Protein/metabolism , Retroviridae/genetics , Time Factors , Transfection , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolism , beta-Galactosidase/metabolismABSTRACT
Apoptotic cells display signals that trigger phagocytic removal by macrophages or neighboring cells. To better understand the signals triggering phagocytosis of apoptotic glioma cells, and to identify the cells that might be involved in the phagocytic process, U-251 MG glioma cells were made apoptotic by etoposide (25 microg/ml) treatment and were incubated with normal human astrocytes (NHA), glioma cells, or microglia. Extent of phagocytosis was assessed by an in vitro phagocytosis assay. After 3 h of incubation with apoptotic cells, phagocytes tested were washed to remove nonengulfed cells, then fixed, stained, and counted to determine phagocytosis index (PI). NHA, glioma cells, and microglia all phagocytosed apoptotic, but not nonapoptotic, glioma cells. Microglia, however, had a PI approximately 4-fold higher than did either NHA or glioma cells. Binding of phosphatidylserine (PS) on apoptotic glioma cell membranes by annexin-V inhibited phagocytosis by 90% in both microglia and NHA. The activity of an enzyme (scramblase) that moves PS from the inner cell membrane to the outer cell membrane was also increased in apoptotic glioma cells. These results suggest that a variety of cells present in and near gliomas in vivo can remove glioma cells in a PS-dependent scramblase-mediated fashion. Manipulation of scramblase and/or PS exposure in glioma cells may therefore be a means of triggering phagocytic removal of glioma cells.
Subject(s)
Apoptosis , Astrocytes/physiology , Glioma/physiopathology , Microglia/physiology , Phagocytosis/physiology , Phosphatidylserines/physiology , Phospholipid Transfer Proteins , Brain/cytology , Carrier Proteins/metabolism , Cell Line , Glioma/enzymology , Glioma/pathology , Humans , Membrane Proteins/metabolism , Phagocytosis/drug effects , Phosphatidylserines/pharmacology , Reference ValuesABSTRACT
GADD45 has been suggested to coordinate cell cycle regulation with the repair of DNA damage following ionizing radiation (IR). Although the GADD45 gene is transcriptionally up-regulated in response to IR, alterations in in vivo transcription factor (TF) binding or chromatin structure associated with up-regulation have not been defined. To understand how chromatin structure might influence TF binding and GADD45 up-regulation, key regulatory regions of the gene were identified by in vivo DNase I hypersensitivity (HS) analysis. Chromatin structure and in vivo TF binding in these regions were subsequently monitored in both non-irradiated and irradiated human ML-1 cells. In non-irradiated cells expressing basal levels of GADD45, the gene exhibited a highly organized chromatin structure with distinctly positioned nucleosomes. Also identified in non-irradiated cells were DNA-protein interactions at octamer binding motifs and a CCAAT box in the promoter and at consensus binding sites for AP-1 and p53 within intron 3. Upon irradiation and a subsequent 15-fold increase in GADD45 mRNA levels, neither the chromatin structure nor the pattern of TF binding in key regulatory regions was altered. These results suggest that the GADD45 gene is poised for up-regulation and can be rapidly induced independent of gross changes in chromatin structure or TF binding.
Subject(s)
Chromatin , Proteins/genetics , Transcription Factors/metabolism , Up-Regulation , CCAAT-Enhancer-Binding Proteins , DNA-Binding Proteins/metabolism , Deoxyribonuclease I , Host Cell Factor C1 , Humans , Intracellular Signaling Peptides and Proteins , Introns , Nuclear Proteins/metabolism , Nucleosomes , Octamer Transcription Factor-1 , Octamer Transcription Factor-2 , Polymerase Chain Reaction/methods , Transcription, Genetic , Tumor Cells, Cultured , GADD45 ProteinsABSTRACT
Growth constraint of bacterial and human cells has been shown to trigger genetic mutation. We questioned whether growth constraint might also trigger epigenetic mutation in the form of CpG island methylation. Logarithmically growing normal human fibro-blasts (NHF) displayed little (0-15%) CpG methylation in select regions of three CpG islands [estrogen receptor (ER), E-cadherin (ECAD) and O (6)-methylguanine-DNA methyltransferase (MGMT)] examined. NHF grown to and left at confluence for 2-21 days showed little (<10%) CpG methylation in the ER and ECAD CpG islands. These confluent, growth-arrested cells, however, displayed extensive ( approximately 50%) methylation of the MGMT CpG island. CpG methylation in the MGMT CpG island was not associated with cellular senescence. The methylation was, however, heritable, but not permanent, as the level of CpG methylation in the MGMT CpG island of cells 4 population doublings following replating after confluence were no different from those in confluent cultures, but returned to levels noted in logarithmically growing cells by 10 population doublings following replating. These results suggest that growth constraint can trigger transient epigenetic change even in normal non-senescent human cells.
Subject(s)
CpG Islands/genetics , DNA Methylation , Fibroblasts/cytology , Fibroblasts/metabolism , Cadherins/genetics , Cell Count , Cell Division/genetics , Cell Line , Cell Line, Transformed , Cellular Senescence/genetics , DNA/biosynthesis , DNA/metabolism , Humans , O(6)-Methylguanine-DNA Methyltransferase/genetics , Promoter Regions, Genetic/genetics , Receptors, Estrogen/genetics , Simian virus 40 , Sulfites , Time FactorsABSTRACT
Tumor-associated aberrant silencing of CpG island-containing genes has been correlated with increased cytosine methylation, a "closed" chromatin structure, and exclusion of transcription factor binding in the CpG island/promoter regions of affected genes. Given the lack of understanding of what constitutes a closed chromatin structure in CpG islands, however, it has been difficult to assess the relationship among cytosine methylation, chromatin structure, and inappropriate gene silencing. In this study, nuclease accessibility analysis was used to more clearly define the chromatin structure in the CpG island of the human O6-methylguanine DNA methyltransferase (MGMT) gene. Chromatin structure was then related to in vivo DNA-protein interactions and cytosine methylation status of the MGMT CpG island in human glioma cells varying in MGMT expression. The results of these studies indicated that the "open" chromatin structure associated with the MGMT CpG island in MGMT+ cells consisted of an approximately 250-bp transcription factor-binding, nuclease-accessible, nucleosome-free region of DNA, whose formation was associated with at least four flanking, precisely positioned nucleosome-like structures. In MGMT- cells, this precise nucleosomal array was lost and was replaced by randomly positioned nucleosomes (i.e., the closed chromatin structure), regardless of whether methylation of the CpG island was spread over the entire island or limited to regions outside the transcription factor binding region. These results suggest that CpG islands facilitate the expression of housekeeping genes by facilitating nucleosomal positioning and that the conditions that alter the formation of this array (such as perhaps methylation) may indirectly affect CpG island-containing gene expression.
Subject(s)
CpG Islands/physiology , Gene Expression Regulation, Neoplastic/genetics , Methyltransferases/genetics , Nucleosomes/metabolism , Base Sequence , Cell Line , Chromatin/metabolism , Cloning, Molecular , Cytosine/metabolism , DNA Methylation , Endodeoxyribonucleases , Fibroblasts , Glioma/metabolism , Humans , Molecular Sequence Data , O(6)-Methylguanine-DNA Methyltransferase , T-Lymphocytes , Tumor Cells, CulturedABSTRACT
O6-Methylguanine DNA methyltransferase (MGMT) repairs the mutagenic and cytotoxic O6-alkylguanine lesions produced by environmental carcinogens and the chemotherapeutic nitrosoureas. As such, MGMT-mediated repair of O6-alkylguanine lesions constitutes a major form of resistance to nitrosourea chemotherapy and makes control of MGMT expression of clinical interest. The variability of expression in cell lines and tissues, along with the ease with which the MGMT phenotype reverts under various conditions, suggests that MGMT is under epigenetic control. One such epigenetic mechanism, 5-methylation of cytosines, has been linked to MGMT expression. We have used an isogenic human multiple myeloma tumor cell line model composed of an MGMT-positive parent cell line, RPMI 8226/S, and its MGMT-negative variant, termed 8226/V, to study the control of MGMT expression. The loss of MGMT activity in 8226/V was found to be due to the loss of detectable MGMT gene expression. Bisulfite sequencing of the MGMT CpG island promoter revealed large increases in the levels of CpG methylation within discrete regions of the 8226/V MGMT CpG island compared to those in 8226/S. These changes in CpG methylation are associated with local heterochromatinization of the 8226/V MGMT transcription start site and provide a likely mechanism for the loss of MGMT transcription in 8226/V.
Subject(s)
Chromatin/metabolism , CpG Islands , DNA Methylation , Methyltransferases/metabolism , Transcription, Genetic , Calcium Channel Blockers/pharmacology , Cells, Cultured , Cytosine/metabolism , DNA Repair/drug effects , Gene Expression/drug effects , Humans , Methyltransferases/genetics , O(6)-Methylguanine-DNA Methyltransferase , Restriction Mapping , Verapamil/pharmacologyABSTRACT
O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that transfers methyl and alkyl lesions from the O6 position of guanine to a cysteine in its structure. The ability of MGMT to also remove precytotoxic O6-alkylguanine lesions induced by chemotherapeutic chloroethylnitrosoureas has made down-regulation of MGMT expression the key component in strategies designed to sensitize tumors to the cytotoxic potential of chloroethylnitrosoureas. The study of how to regulate MGMT expression at the gene, mRNA, and protein levels has contributed not only to the development of effective inhibitors of MGMT action, but also, in a broader sense, to a better understanding of gene regulation and protein structure/function.
Subject(s)
Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/physiology , Nitrosourea Compounds/pharmacology , O(6)-Methylguanine-DNA Methyltransferase/genetics , O(6)-Methylguanine-DNA Methyltransferase/physiology , Animals , DNA Repair , Down-Regulation/drug effects , HumansABSTRACT
Aberrant transcriptional inactivation of the non-X-linked human O-6-methylguanine DNA methyltransferase (MGMT) gene has been associated with loss of open chromatin structure and increases in cytosine methylation in the Sp1-binding region of the 5'-CpG island of the gene. To examine the necessity of these events for gene silencing, we have isolated and characterized a subline of human MGMT+ T98G glioma cells. The subline, T98Gs, does not express MGMT activity or MGMT mRNA, and exhibits no in vivo DNA-protein interactions at Sp1-like binding sites in the MGMT 5'-CpG island. While the MGMT CpG island is less accessible to exogenously added restriction enzymes in T98Gs nuclei than in T98G nuclei, it is similarly methylated in both T98G and T98Gs cell lines 5' and 3' to the transcription factor binding sites, and similarly unmethylated in the region encompassing the binding sites. Inappropriate transcriptional inactivation of MGMT, therefore, does not require methylation of transcription factor binding sites within the 5'-CpG island. Rather, MGMT gene silencing and transcription factor exclusion from T98Gs MGMT CpG island binding sites is most closely associated with condensed chromatin structure, which is in turn indirectly influenced by distant sites of methylation.
Subject(s)
CpG Islands , Methyltransferases/genetics , Transcription Factors/metabolism , Base Sequence , Binding Sites/genetics , Cytosine/chemistry , DNA, Neoplasm/chemistry , DNA, Neoplasm/genetics , Gene Expression , Genetic Linkage , Glioma/enzymology , Glioma/genetics , Humans , Methylation , Molecular Sequence Data , O(6)-Methylguanine-DNA Methyltransferase , Promoter Regions, Genetic , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tumor Cells, Cultured , X Chromosome/geneticsABSTRACT
N,N'-Bis(2-chloroethyl)-N-nitrosourea (BCNU) and its derivatives are chemotherapeutic DNA-damaging agents that generate a variety of monoadducts, intrastrand cross-links, and interstrand cross-links. The cytotoxic potential of the compounds has been linked to their ability to form DNA interstrand cross-links, which presumably inhibit subsequent DNA replication. To address the possibility that BCNU-induced lesions may also influence other DNA-directed actions such as transcription, and to identify the DNA lesions involved, a synthetic DNA template containing phage RNA polymerase promoters at both ends was incubated with BCNU and, after drug removal, transcribed in vitro. For comparison, similar studies were carried out with cis-diammine-dichloroplatinum(II) and trans-diamminedichloroplatinum(II), which are known to induce defined transcription-terminating lesions. The results suggest that BCNU, like platinum compounds, can induce lesions resulting in termination of transcription in vitro, although the predominant transcription-terminating lesions, unlike those produced by cis-diamminedichloroplatinum(II), most likely represent interstrand DNA cross-links.
Subject(s)
Carmustine/pharmacology , DNA/drug effects , Terminator Regions, Genetic , Transcription, Genetic , Base Sequence , DNA Damage , Molecular Sequence DataABSTRACT
There is considerable interest in identifying factors responsible for expression of the O-6-methylguanine DNA methyltransferase (MGMT) gene, as MGMT is a major determinant in the response of glioma cells to the chemotherapeutic agent 1,3 bis(2-chloroethyl)-1-nitrosourea. Recently we have shown that MGMT expression is correlated in a direct, graded fashion with methylation in the body of the MGMT gene and in an inverse, graded fashion with promoter methylation in human glioma cell lines. To determine if promoter methylation is an important component of MGMT expression, this study addressed the complex interactions between methylation, chromatin structure, and in vivo transcription factor occupancy in the MGMT promoter of glioma cell lines with different levels of MGMT expression. Our results show that the basal promoter in MGMT-expressing glioma cell lines, which is 100% unmethylated, was very accessible to restriction enzymes at all sites tested, suggesting that this region may be nucleosome free. The basal promoter in glioma cells with minimal MGMT expression, however, which is 75% unmethylated, was much less accessible, and the basal promoter in nonexpressing cells, which is 50% unmethylated, was entirely inaccessible to restriction enzymes. Despite the presence of the relevant transcription factors in all cell lines examined, in vivo footprinting showed DNA-protein interactions at six Sp1 binding sites and one novel binding site in MGMT-expressing cell lines but no such interactions in nonexpressors. We conclude that in contrast to findings of previous in vitro studies, Sp1 is an important component of MGMT transcription. These correlations also strongly suggest that methylation and chromatin structure, by determining whether Sp1 and other transcription factors can access the MGMT promoter, set the transcriptional state of the MGMT gene.
Subject(s)
Gene Expression Regulation, Neoplastic , Glioma/genetics , Methyltransferases/genetics , Nervous System Neoplasms/genetics , Promoter Regions, Genetic/genetics , Base Sequence , Chromatin/metabolism , Deoxyribonuclease HpaII , Deoxyribonucleases, Type II Site-Specific/metabolism , Glioma/enzymology , Glioma/metabolism , Humans , Methylation , Models, Genetic , Molecular Sequence Data , Nervous System Neoplasms/enzymology , Nervous System Neoplasms/metabolism , O(6)-Methylguanine-DNA Methyltransferase , Protein Binding , Restriction Mapping , Sp1 Transcription Factor/metabolism , Transcription, Genetic , Tumor Cells, CulturedABSTRACT
To assess the possibility that two conserved amino acids (glutamine 90 and asparagine 137) in O6-methylguanine-DNA methyltransferase (MGMT) are involved in protein-substrate contact and/or discrimination between favored and non-favored substrates, families of proteins mutant at these two sites were expressed in alkyltransferase-deficient bacteria and analyzed for stability, ability to repair O6-methylguanine (MG)-containing DNA, and ability to differentially repair a preferred (MG-containing DNA) versus a non-preferred (free base MG) substrate. All seven proteins mutant at glutamine 90 (except a proline mutant) were stable in bacteria and repaired MG-containing DNA (> 50% of wild-type levels). A representative glutamine 90 mutant protein was not, however, significantly different from the wild-type protein in the preferential repair of MG-containing DNA versus MG free base. Of eight proteins mutant at asparagine 137, only glutamine and serine mutants repaired MG-containing DNA to any degree (8.5% and 0.8% of wild-type respectively) and only the glutamine mutant protein was detectable in bacterial sonicates by Western blot analysis. Alanine and leucine mutant alkyltransferases, inactive and unstable as non-fusion proteins, could, however, be stably expressed in bacteria as glutathione S-transferase fusion proteins, although the proteins were still inactive in repair. These results suggest that while glutamine 90 has no direct role in MG-DNA methyltransferase-mediated repair or free base/lesioned DNA substrate specificity, asparagine 137 is important in both the stability and activity of the protein and may contribute to the formation or function of the active site of the protein.
Subject(s)
Asparagine/metabolism , Glutamine/metabolism , Methyltransferases/metabolism , Asparagine/chemistry , Asparagine/genetics , Binding Sites , DNA/metabolism , DNA Repair , Enzyme Stability , Glutamine/chemistry , Glutamine/genetics , Guanine/analogs & derivatives , Guanine/metabolism , Methyltransferases/chemistry , Methyltransferases/genetics , Mutation , O(6)-Methylguanine-DNA Methyltransferase , Substrate SpecificityABSTRACT
Expression of the O6-methylguanine DNA methyltransferase (MGMT) gene in human glioma cell lines is strongly associated with resistance to the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea. To examine the possibility that methylation of the body and promoter regions of the MGMT gene is associated with MGMT expression in a graded, rather than a completely on/off fashion, the present study analyzed the methylation status of the MGMT gene in human glioma cell lines exhibiting a wide range of MGMT expression. Methylation in the body of the gene was uniform within each cell line and correlated directly with MGMT expression. The level of MGMT promoter methylation was also graded across the cell lines, at 21 of 25 CpGs tested, but correlated inversely with MGMT expression. Two sites in the MGMT promoter were also much more accessible to restriction enzyme digestion, and thus in a more open chromatin conformation, in nuclei from high MGMT expressors relative to nuclei from cells with little or no MGMT expression. We conclude that the level of methylation, in both the body and promoter of the MGMT gene, is associated with MGMT expression in a graded fashion and may be important in setting the transcriptional state of the MGMT promoter through changes in chromatin structure.
Subject(s)
Gene Expression Regulation, Enzymologic , Methyltransferases/genetics , Promoter Regions, Genetic , Base Sequence , Cell Nucleus/metabolism , DNA Primers/chemistry , Genes , Glioma , Humans , Introns , Methylation , Molecular Sequence Data , O(6)-Methylguanine-DNA Methyltransferase , RNA, Messenger/genetics , Restriction Mapping , Tumor Cells, CulturedABSTRACT
The human O6-methylguanine DNA methyltransferase (MGMT) repairs O6-methylguanine (O6-MG) in DNA at a much lower rate than the Escherichia coli Ada protein, and only MGMT repairs the altered base, O6-benzylguanine (O6-BG). The diversity in DNA repair properties between MGMT and Ada may be a result of divergent amino acid sequences outside their common proline-cysteine-histidine-arginine-valine (PCHRV) acceptor site. One notable sequence difference is an MGMT 28-amino acid carboxyl-terminal tail which is highly conserved among all mammalian alkyltransferases. The role of this tail sequence in substrate specificity was assessed by expressing full-length MGMT and Ada proteins, and mutant MGMT proteins lacking either 10 or 28 amino acids from the carboxyl terminus, as GST fusion proteins in alkyltransferase-deficient E. coli cells, and comparing rates of repair of O6-MG containing DNA and O6-BG by these fusion proteins at 4 degrees C and 37 degrees C. The MGMT carboxyl-terminal tail was not required for repair of O6-MG in DNA at 37 degrees C although the deletion of this tail sequence reversibly inhibited the ability of MGMT to repair O6-MG in DNA at 4 degrees C. Therefore, the absence of this region affects the ability of the protein to repair O6-MG in DNA at lower temperatures. Furthermore, removal of the tail sequence from MGMT decreased the rate of O6-BG repair 5-fold. We conclude that the 28-amino acid carboxyl-terminal MGMT tail, while not required for activity, modulates the rate of MGMT repair at reduced temperatures and plays a role in substrate specificity.