RESUMO
Genetic studies in mice and human cancers established BCL11B as a haploinsufficient tumor suppressor gene. Paradoxically, BCL11B is overexpressed in some human cancers where its knockdown is synthetic lethal. We identified the BCL11B protein in a proximity-dependent biotinylation screen performed with the DNA glycosylase NTHL1. In vitro DNA repair assays demonstrated that both BCL11B and a small recombinant BCL11B213-560 protein lacking transcription regulation potential can stimulate the enzymatic activities of two base excision repair (BER) enzymes: NTHL1 and Pol ß. In cells, BCL11B is rapidly recruited to sites of DNA damage caused by laser microirradiation. BCL11B knockdown delays, whereas ectopic expression of BCL11B213-560 accelerates, the repair of oxidative DNA damage. Inactivation of one BCL11B allele in TK6 lymphoblastoid cells causes an increase in spontaneous and radiation-induced mutation rates. In turn, ectopic expression of BCL11B213-560 cooperates with the RAS oncogene in cell transformation by reducing DNA damage and cellular senescence. These findings indicate that BCL11B functions as a BER accessory factor, safeguarding normal cells from acquiring mutations. Paradoxically, it also enables the survival of cancer cells that would otherwise undergo senescence or apoptosis due to oxidative DNA damage resulting from the elevated production of reactive oxygen species.
Assuntos
Reparo por Excisão , Proteínas Repressoras , Animais , Humanos , Camundongos , Dano ao DNA , Reparo do DNA/genética , Genes Supressores de Tumor , Oncogenes , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Base excision repair is initiated by DNA glycosylases that recognize specific altered bases. DNA glycosylases for oxidized bases carry both a glycosylase activity that removes the faulty base and an apyrimidinic/apurinic lyase activity that introduces a single-strand DNA incision. In particular, the CUT domains within the CUX1 and CUX2 proteins were recently shown to interact with the 8-oxoguanine (8-oxoG) DNA glycosylase and stimulate its enzymatic activities. SATB1, which contains two CUT domains, was originally characterized as a T cell-specific genome organizer whose aberrant overexpression in breast cancer can promote tumor progression. Here we investigated the involvement of SATB1 in DNA repair. SATB1 knockdown caused a delay in DNA repair following exposure to H2O2, an increase in OGG1-sensitive oxidized bases within genomic DNA, and a decrease in 8-oxoG cleavage activity in cell extracts. In parallel, we observed an increase in phospho-CHK1 and γ-H2AX levels and a decrease in DNA synthesis. Conversely, ectopic expression of SATB1 accelerated DNA repair and reduced the levels of oxidized bases in genomic DNA. Moreover, an enhanced GFP-SATB1 fusion protein was rapidly recruited to laser microirradiation-induced DNA damage. Using purified proteins, we showed that SATB1 interacts directly with OGG1, increases its binding to 8-oxoG-containing DNA, promotes Schiff base formation, and stimulates its glycosylase and apyrimidinic/apurinic lyase enzymatic activities. Structure/function analysis demonstrated that CUT domains, but not the homeodomain, are responsible for the stimulation of OGG1. Together, these results identify another CUT domain protein that functions both as a transcription factor and an accessory factor in base excision repair.
Assuntos
Dano ao DNA , DNA Glicosilases/metabolismo , Reparo do DNA , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , DNA Glicosilases/química , DNA Glicosilases/genética , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Células Jurkat , Proteínas de Ligação à Região de Interação com a Matriz/química , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Relação Estrutura-Atividade , Fatores de TranscriçãoRESUMO
The Cut homeobox 1 (CUX1) gene is a target of loss-of-heterozygosity in many cancers, yet elevated CUX1 expression is frequently observed and is associated with shorter disease-free survival. The dual role of CUX1 in cancer is illustrated by the fact that most cell lines with CUX1 LOH display amplification of the remaining allele, suggesting that decreased CUX1 expression facilitates tumor development while increased CUX1 expression is needed in tumorigenic cells. Indeed, CUX1 was found in a genome-wide RNAi screen to identify synthetic lethal interactions with oncogenic RAS. Here we show that CUX1 functions in base excision repair as an ancillary factor for the 8-oxoG-DNA glycosylase, OGG1. Single cell gel electrophoresis (comet assay) reveals that Cux1âº/â» MEFs are haploinsufficient for the repair of oxidative DNA damage, whereas elevated CUX1 levels accelerate DNA repair. In vitro base excision repair assays with purified components demonstrate that CUX1 directly stimulates OGG1's enzymatic activity. Elevated reactive oxygen species (ROS) levels in cells with sustained RAS pathway activation can cause cellular senescence. We show that elevated expression of either CUX1 or OGG1 prevents RAS-induced senescence in primary cells, and that CUX1 knockdown is synthetic lethal with oncogenic RAS in human cancer cells. Elevated CUX1 expression in a transgenic mouse model enables the emergence of mammary tumors with spontaneous activating Kras mutations. We confirmed cooperation between Kras(G12V) and CUX1 in a lung tumor model. Cancer cells can overcome the antiproliferative effects of excessive DNA damage by inactivating a DNA damage response pathway such as ATM or p53 signaling. Our findings reveal an alternate mechanism to allow sustained proliferation in RAS-transformed cells through increased DNA base excision repair capability. The heightened dependency of RAS-transformed cells on base excision repair may provide a therapeutic window that could be exploited with drugs that specifically target this pathway.
Assuntos
Dano ao DNA , Reparo do DNA/fisiologia , Genes ras/fisiologia , Proteínas de Homeodomínio/fisiologia , Proteínas Nucleares/fisiologia , Proteínas Repressoras/fisiologia , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Células Cultivadas , Senescência Celular/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Neoplasias Mamárias Experimentais/genética , Camundongos Transgênicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estresse Oxidativo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de TranscriçãoRESUMO
CUX1 and CUX2 proteins are characterized by the presence of three highly similar regions called Cut repeats 1, 2, and 3. Although CUX1 is ubiquitously expressed, CUX2 plays an important role in the specification of neuronal cells and continues to be expressed in postmitotic neurons. Cut repeats from the CUX1 protein were recently shown to stimulate 8-oxoguanine DNA glycosylase 1 (OGG1), an enzyme that removes oxidized purines from DNA and introduces a single strand break through its apurinic/apyrimidinic lyase activity to initiate base excision repair. Here, we investigated whether CUX2 plays a similar role in the repair of oxidative DNA damage. Cux2 knockdown in embryonic cortical neurons increased levels of oxidative DNA damage. In vitro, Cut repeats from CUX2 increased the binding of OGG1 to 7,8-dihydro-8-oxoguanine-containing DNA and stimulated both the glycosylase and apurinic/apyrimidinic lyase activities of OGG1. Genetic inactivation in mouse embryo fibroblasts or CUX2 knockdown in HCC38 cells delayed DNA repair and increased DNA damage. Conversely, ectopic expression of Cut repeats from CUX2 accelerated DNA repair and reduced levels of oxidative DNA damage. These results demonstrate that CUX2 functions as an accessory factor that stimulates the repair of oxidative DNA damage. Neurons produce a high level of reactive oxygen species because of their dependence on aerobic oxidation of glucose as their source of energy. Our results suggest that the persistent expression of CUX2 in postmitotic neurons contributes to the maintenance of genome integrity through its stimulation of oxidative DNA damage repair.
Assuntos
Córtex Cerebral/metabolismo , Dano ao DNA , Reparo do DNA , Proteínas de Homeodomínio/metabolismo , Neurônios/metabolismo , Animais , Linhagem Celular , Córtex Cerebral/citologia , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Neurônios/citologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oxirredução , Ratos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Cell populations able to generate a large repertoire of genetic variants have increased potential to generate tumor cells that survive through the multiple selection steps involved in tumor progression. A mechanism for the generation of aneuploid cancer cells involves passage through a tetraploid stage. Supernumerary centrosomes, however, can lead to multipolar mitosis and cell death. Using tissue culture and transgenic mouse models of breast cancer, we report that Cut homeobox 1 (CUX1) causes chromosomal instability by activating a transcriptional program that prevents multipolar divisions and enables the survival of tetraploid cells that evolve to become genetically unstable and tumorigenic. Transcriptional targets of CUX1 involved in DNA replication and bipolar mitosis defined a gene expression signature that, across 12 breast cancer gene expression datasets, was associated with poor clinical outcome. The signature not only was higher in breast tumor subtypes of worse prognosis, like the basal-like and HER2(+) subtypes, but also identified poor outcome among estrogen receptor-positive/node-negative tumors, a subgroup considered to be at lower risk. The CUX1 signature therefore represents a unique criterion to stratify patients and provides insight into the molecular determinants of poor clinical outcome.
Assuntos
Ciclo Celular , Instabilidade Cromossômica/fisiologia , Proteínas de Homeodomínio/fisiologia , Mitose/fisiologia , Proteínas Nucleares/fisiologia , Proteínas Repressoras/fisiologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Linhagem Celular , Replicação do DNA , Feminino , Perfilação da Expressão Gênica , Humanos , Regiões Promotoras Genéticas , Fatores de TranscriçãoRESUMO
BACKGROUND: Overexpression of the Cut homeobox 1 gene, CUX1, inversely correlates with patient survival in breast cancers. Cell-based assays and molecular studies have revealed that transcriptional regulation by CUX1 involves mostly the proteolytically processed p110 isoform. As there is no antibody specific to p110 CUX1 only, an alternate strategy must be employed to identify its targets. RESULTS: We expressed physiological levels of a tagged-p110 CUX1 protein and performed chromatin affinity purification followed by hybridization on ENCODE and promoter arrays. Targets were validated by chromatin immunoprecipitation and transcriptional regulation by CUX1 was analyzed in expression profiling and RT-qPCR assays following CUX1 knockdown or p110 CUX1 overexpression. Approximately 47% and 14% of CUX1 binding sites were respectively mapped less than 4 Kbp, or more than 40 Kbp, away from a transcription start site. More genes exhibited changes in expression following CUX1 knockdown than p110 CUX1 overexpression. CUX1 directly activated or repressed 7.4% and 8.4% of putative targets identified on the ENCODE and promoter arrays respectively. This proportion increased to 11.2% for targets with 2 binding sites or more. Transcriptional repression was observed in a slightly higher proportion of target genes. The CUX1 consensus binding motif, ATCRAT, was found at 47.2% of the CUX1 binding sites, yet only 8.3% of the CUX1 consensus motifs present on the array were bound in vivo. The presence of a consensus binding motif did not have an impact on whether a target gene was repressed or activated. Interestingly, the distance between a binding site and a transcription start site did not significantly reduced the ability of CUX1 to regulate a target gene. Moreover, CUX1 not only was able to regulate the next adjacent gene, but also regulated the gene located beyond this one as well as the gene located further away in the opposite direction. CONCLUSION: Our results demonstrate that p110 CUX1 can activate or repress transcription when bound at a distance and can regulate more than one gene on certain genomic loci.
Assuntos
Proteínas de Homeodomínio/genética , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Sítios de Ligação/genética , Imunoprecipitação da Cromatina , Feminino , Técnicas de Silenciamento de Genes , Humanos , Isoformas de Proteínas/metabolismo , Análise Serial de Tecidos , Fatores de Transcrição , Sítio de Iniciação de Transcrição , Ativação Transcricional/fisiologiaRESUMO
We identified the BCL11A protein in a proximity-dependent biotinylation screen performed with the DNA glycosylase NTHL1. In vitro, DNA repair assays demonstrate that both BCL11A and a small recombinant BCL11A160-520 protein that is devoid of DNA binding and transcription regulatory domains can stimulate the enzymatic activities of two base excision repair enzymes: NTHL1 and DNA Pol ß. Increased DNA repair efficiency, in particular of the base excision repair pathway, is essential for many cancer cells to proliferate in the presence of elevated reactive oxygen species (ROS) produced by cancer-associated metabolic changes. BCL11A is highly expressed in triple-negative breast cancers (TNBC) where its knockdown was reported to reduce clonogenicity and cause tumour regression. We show that BCL11A knockdown in TNBC cells delays repair of oxidative DNA damage, increases the number of oxidized bases and abasic sites in genomic DNA, slows down proliferation and induces cellular senescence. These phenotypes are rescued by ectopic expression of the short BCL11A160-520 protein. We further show that the BCL11A160-520 protein accelerates the repair of oxidative DNA damage and cooperates with RAS in cell transformation assays, thereby enabling cells to avoid senescence and continue to proliferate in the presence of high ROS levels.
RESUMO
The p110 CUX1 homeodomain protein participates in the activation of DNA replication genes in part by increasing the affinity of E2F factors for the promoters of these genes. CUX1 expression is very weak in quiescent cells and increases during G(1). Biochemical activities associated with transcriptional activation by CUX1 are potentiated by post-translational modifications in late G(1), notably a proteolytic processing event that generates p110 CUX1. Constitutive expression of p110 CUX1, as observed in some transformed cells, leads to accelerated entry into the S phase. In this study, we investigated the post-translation regulation of CUX1 during mitosis and the early G(1) phases of proliferating cells. We observed a major electrophoretic mobility shift and a complete inhibition of DNA binding during mitosis. We show that cyclin B/CDK1 interacts with CUX1 and phosphorylates it at multiple sites. Serine to alanine replacement mutations at 10 SP dipeptide sites were required to restore DNA binding in mitosis. Passage into G(1) was associated with the degradation of some p110 CUX1 proteins, and the remaining proteins were gradually dephosphorylated. Indirect immunofluorescence and subfractionation assays using a phospho-specific antibody showed that most of the phosphorylated protein remained in the cytoplasm, whereas the dephosphorylated protein was preferentially located in the nucleus. Globally, our results indicate that the hyperphosphorylation of CUX1 by cyclin B/CDK1 inhibits its DNA binding activity in mitosis and interferes with its nuclear localization following cell division and formation of the nuclear membrane, whereas dephosphorylation and de novo synthesis contribute to gradually restore CUX1 expression and activity in G(1).
Assuntos
Proteína Quinase CDC2/metabolismo , Núcleo Celular/metabolismo , Ciclina B/metabolismo , Fase G1/fisiologia , Proteínas de Homeodomínio/metabolismo , Mitose/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Proteína Quinase CDC2/genética , Linhagem Celular , Núcleo Celular/genética , Ciclina B/genética , DNA/genética , DNA/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Proteínas Repressoras/genéticaRESUMO
The full-length CUX1 protein isoform was previously shown to function as an auxiliary factor in base excision repair (BER). Specifically, CUT domains within CUX1 stimulate the enzymatic activities of the OGG1 DNA glycosylase and APE1 endonuclease. Moreover, ectopic expression of CUX1 or CUT domains increased the resistance of cancer cells to treatments that cause oxidative DNA damage and mono-alkylation of bases. Stimulation of OGG1 AP/lyase and APE1 endonuclease activities, however, cannot explain how CUT domains confer resistance to these treatments since these enzymes produce DNA single-strand breaks that are highly toxic to cells. In the present study, we show that CUT domains stimulate the polymerase and deoxyribose phosphate (dRP)-lyase activities of DNA polymerase ß to promote BER completion. In agreement with these results, CUX1 knockdown decreases BER completion in cell extracts and causes an increase in the number of abasic sites in genomic DNA following temozolomide treatment. We also show that CUT domains stimulate bypass of intrastrand G-crosslinks by Pol ß in vitro, while the resistance of cancer cells to cisplatin treatment is reduced by CUX1 knockdown but restored by ectopic expression of CUT domains. Altogether our results establish CUX1 as an important auxiliary factor that stimulates multiple steps of base excision repair, from the recognition and removal of altered bases to the addition of new nucleotides and removal of 5'-deoxyribose phosphate required for ligation and BER completion. These findings provide a mechanistic explanation for the observed correlation between CUX1 expression and the resistance of cancer cells to genotoxic treatments.
Assuntos
DNA Polimerase beta/química , DNA Polimerase beta/metabolismo , Reparo do DNA , Domínios e Motivos de Interação entre Proteínas , Sítios de Ligação , Linhagem Celular , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Ativação Enzimática , Técnicas de Inativação de Genes , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Ligação Proteica , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Proteolytic processing of the CUX1 transcription factor generates an isoform, p110 that accelerates entry into S phase. To identify targets of p110 CUX1 that are involved in cell cycle progression, we performed genome-wide location analysis using a promoter microarray. Since there are no antibodies that specifically recognize p110, but not the full-length protein, we expressed physiological levels of a p110 isoform with two tags and purified chromatin by tandem affinity purification (ChAP). Conventional ChIP performed on synchronized populations of cells confirmed that p110 CUX1 is recruited to the promoter of cell cycle-related targets preferentially during S phase. Multiple approaches including silencing RNA (siRNA), transient infection with retroviral vectors, constitutive expression and reporter assays demonstrated that most cell cycle targets are activated whereas a few are repressed or not affected by p110 CUX1. Functional classes that were over-represented among targets included DNA replication initiation. Consistent with this finding, constitutive expression of p110 CUX1 led to a premature and more robust induction of replication genes during cell cycle progression, and stimulated the long-term replication of a plasmid bearing the oriP replicator of Epstein Barr virus (EBV).
Assuntos
Replicação do DNA/genética , Proteínas de Homeodomínio/fisiologia , Proteínas Nucleares/fisiologia , Proteínas Repressoras/fisiologia , Ativação Transcricional , Animais , Linhagem Celular , Cromatina/isolamento & purificação , Cromatografia de Afinidade , Genes cdc , Genômica , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Fase S/genética , Análise de Sequência de DNA , Fatores de TranscriçãoRESUMO
Proteolytic processing by cathepsin L generates p110 Cut homeobox 1 (CUX1) at the end of the G(1) phase, whereas an alternative transcript encodes p75 CUX1. These short CUX1 isoforms were reported to be overexpressed in cancer cells, and transgenic mice overexpressing the p75 isoform were found to develop myeloproliferative disease-like myeloid leukemias. In the present study, we report that the neutrophil elastase can also generate a short CUX1 isoform in the MV4;11 acute myeloid leukemia cell line. Proteolytic processing was so efficient that the full-length CUX1 protein was detected only when cells were maintained in the presence of the specific elastase inhibitor III. In agreement with these findings, higher levels of the processed cyclin E isoforms were also detected in MV4;11 cells. Reappearance of full-length cyclin E and CUX1 could be induced upon the treatment of MV4;11 cells with the differentiation inducer phorbol 12-myristate 13-acetate or, unexpectedly, following overexpression of a short recombinant CUX1 protein. In both cases, the mechanism involved transcriptional repression of the neutrophil elastase gene. This result revealed a negative feedback loop whereby CUX1 shuts down the expression of the protease that cleaves it. Overall, the findings in MV4;11 and other cancer cells suggest that various mechanisms are used in cancer to favor the expression of short CUX1 isoforms.
Assuntos
Proteínas de Homeodomínio/metabolismo , Leucemia Mieloide/enzimologia , Elastase de Leucócito/metabolismo , Processamento de Proteína Pós-Traducional , Extratos Celulares , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Ciclina E/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/química , Humanos , Leucemia Mieloide/patologia , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Inibidores de Serina Proteinase/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The CDP/Cux transcription factor was previously found to acquire distinct DNA binding and transcriptional properties following a proteolytic processing event that takes place at the G1/S transition of the cell cycle. In the present study, we have investigated the role of the CDP/Cux processed isoform, p110, in cell cycle progression. Populations of cells stably expressing p110 CDP/Cux displayed a faster division rate and reached higher saturation density than control cells carrying the empty vector. p110 CDP/Cux cells reached the next S phase faster than control cells under various experimental conditions: following cell synchronization in G0 by growth factor deprivation, synchronization in S phase by double thymidine block treatment, or enrichment in G2 by centrifugal elutriation. In each case, duration of the G1 phase was shortened by 2 to 4 h. Gene inactivation confirmed the role of CDP/Cux as an accelerator of cell cycle progression, since mouse embryo fibroblasts obtained from Cutl1z/z mutant mice displayed a longer G1 phase and proliferated more slowly than their wild-type counterparts. The delay to enter S phase persisted following immortalization by the 3T3 protocol and transformation with H-RasV12. Moreover, CDP/Cux inactivation hindered both the formation of foci on a monolayer and tumor growth in mice. At the molecular level, expression of both cyclin E2 and A2 was increased in the presence of p110 CDP/Cux and decreased in its absence. Overall, these results establish that p110 CDP/Cux functions as a cell cycle regulator that accelerates entry into S phase.
Assuntos
Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Fase S/fisiologia , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Testes de Carcinogenicidade , Proliferação de Células , Transformação Celular Neoplásica , Células Cultivadas , Centrifugação , Ciclina A/genética , Ciclina A/metabolismo , Ciclina A2 , Ciclinas/genética , Ciclinas/metabolismo , Feminino , Fibroblastos/citologia , Genes ras , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Mutantes , Camundongos Nus , Proteínas Nucleares/genética , Isoformas de Proteínas , Proteínas Repressoras/genética , Timidina/metabolismo , Fatores de Transcrição/genéticaRESUMO
It is generally accepted that the role of cathepsin L in cancer involves its activities outside the cells once it has been secreted. However, cathepsin L isoforms that are devoid of a signal peptide were recently shown to be present in the nucleus where they proteolytically process the CCAAT-displacement protein/cut homeobox (CDP/Cux) transcription factor. A role for nuclear cathepsin L in cell proliferation could be inferred from the observation that the CDP/Cux processed isoform can accelerate entry into S phase. Here, we report that in many transformed cells the proteolytic processing of CDP/Cux is augmented and correlates with increased cysteine protease expression and activity in the nucleus. Taking advantage of an antibody that recognizes the prodomain of human cathepsin L, we showed that human cells express short cathepsin L species that do not contain a signal peptide, do not transit through the endoplasmic reticulum, are not glycosylated, and localize to the nucleus. We also showed that transformation by the ras oncogene causes rapid increases both in the production of short nuclear cathepsin L isoforms and in the processing of CDP/Cux. Using a cell-based assay, we showed that a cell-permeable inhibitor of cysteine proteases is able to delay the progression into S phase and the proliferation in soft agar of ras-transformed cells, whereas the non-cell-permeable inhibitor had no effect. Taken together, these results suggest that the role of cathepsin L in cancer might not be limited to its extracellular activities but may also involve its processing function in the nucleus.
Assuntos
Catepsinas/genética , Núcleo Celular/enzimologia , Transformação Celular Neoplásica , Cisteína Endopeptidases/genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células 3T3 , Animais , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Catepsina L , Divisão Celular , Linhagem Celular Tumoral , Precursores Enzimáticos/genética , Feminino , Humanos , CamundongosRESUMO
Background: Cut Like homeobox 1 (CUX1), which encodes an auxiliary factor in base excision repair, resides on 7q22.1, the most frequently and highly amplified chromosomal region in glioblastomas. The resistance of glioblastoma cells to the mono-alkylating agent temozolomide is determined to some extent by the activity of apurinic/apyrimidinic endonuclease 1 (APE1). Methods: To monitor the effect of CUX1 and its CUT domains on APE1 activity, DNA repair assays were performed with purified proteins and cell extracts. CUX1 protein expression was analyzed by immunohistochemistry using a tumor microarray of 150 glioblastoma samples. The effect of CUX1 knockdown and overexpression on the resistance of glioblastoma cell lines to temozolomide was investigated. Results: We show that CUT domains stimulate APE1 activity. In agreement with these findings, CUX1 knockdown causes an increase in the number of abasic sites in genomic DNA and a decrease in APE1 activity as measured in cell extracts. Conversely, ectopic CUX1 expression increases APE1 activity and lowers the number of abasic sites. Having established that CUX1 is expressed at high levels in most glioblastomas, we next show that the resistance of glioblastoma cells to temozolomide and to a combined treatment of temozolomide and ionizing radiation is reduced following CUX1 knockdown, but increased by overexpression of CUX1 or a short protein containing only 2 CUT domains, which is active in DNA repair but devoid of transcriptional activity. Conclusion: These findings indicate that CUX1 expression level impacts on the response of glioblastoma cells to treatment and identifies the CUT domains as potential therapeutic targets.
Assuntos
Biomarcadores Tumorais/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/tratamento farmacológico , Glioblastoma/enzimologia , Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Temozolomida/farmacologia , Antineoplásicos Alquilantes/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Dano ao DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Glioblastoma/genética , Glioblastoma/patologia , Proteínas de Homeodomínio/genética , Humanos , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Fatores de Transcrição , Células Tumorais CultivadasRESUMO
CDP/Cux (CCAAT-displacement protein/cut homeobox) contains four DNA binding domains, namely, three Cut repeats (CR1, CR2, and CR3) and a Cut homeodomain. CCAAT-displacement activity involves rapid but transient interaction with DNA. More stable DNA binding activity is up-regulated at the G(1)/S transition and was previously shown to involve an N-terminally truncated isoform, CDP/Cux p110, that is generated by proteolytic processing. CDP/Cux has been previously characterized as a transcriptional repressor. However, here we show that expression of reporter plasmids containing promoter sequences from the human DNA polymerase alpha (pol alpha), CAD, and cyclin A genes is stimulated in cotransfections with N-terminally truncated CDP/Cux proteins but not with full-length CDP/Cux. Moreover, expression of the endogenous DNA pol alpha gene was stimulated following the infection of cells with a retrovirus expressing a truncated CDP/Cux protein. Chromatin immunoprecipitation (ChIP) assays revealed that CDP/Cux was associated with the DNA pol alpha gene promoter specifically in the S phase. Using linker scanning analyses, in vitro DNA binding, and ChIP assays, we established a correlation between binding of CDP/Cux to the DNA pol alpha promoter and the stimulation of gene expression. Although we cannot exclude the possibility that stimulation of gene expression by CDP/Cux involved the repression of a repressor, our data support the notion that CDP/Cux participates in transcriptional activation. Notwithstanding its mechanism of action, these results establish CDP/Cux as an important transcriptional regulator in the S phase.
Assuntos
DNA Polimerase I/genética , Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Células 3T3 , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , DNA/genética , DNA/metabolismo , Genes Reporter , Células HeLa , Humanos , Técnicas In Vitro , Camundongos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Fase S , Deleção de Sequência , Fatores de Transcrição , Ativação TranscricionalRESUMO
Ionizing radiation generates a broad spectrum of oxidative DNA lesions, including oxidized base products, abasic sites, single-strand breaks and double-strand breaks. The CUX1 protein was recently shown to function as an auxiliary factor that stimulates enzymatic activities of OGG1 through its CUT domains. In the present study, we investigated the requirement for CUX1 and OGG1 in the resistance to radiation. Cancer cell survival following ionizing radiation is reduced by CUX1 knockdown and increased by higher CUX1 expression. However, CUX1 knockdown is sufficient by itself to reduce viability in many cancer cell lines that exhibit high levels of reactive oxygen species (ROS). Consequently, clonogenic results expressed relative to that of non-irradiated cells indicate that CUX1 knockdown confers no or modest radiosensitivity to cancer cells with high ROS. A recombinant protein containing only two CUT domains is sufficient for rapid recruitment to DNA damage, acceleration of DNA repair and increased survival following radiation. In agreement with these findings, OGG1 knockdown and treatment of cells with OGG1 inhibitors sensitize cancer cells to radiation. Together, these results validate CUX1 and more specifically the CUT domains as therapeutic targets.
Assuntos
Reparo do DNA/genética , Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares/metabolismo , Tolerância a Radiação/genética , Proteínas Repressoras/metabolismo , Linhagem Celular Tumoral , DNA Glicosilases/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Humanos , Immunoblotting , Microscopia Confocal , Proteínas Nucleares/genética , Reação em Cadeia da Polimerase , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/genética , Fatores de TranscriçãoRESUMO
Two isoforms of the CCAAT-displacement protein/cut homeobox (CDP/Cux) transcription factor have been characterized thus far. The full length protein, p200, which contains four DNA binding domains, transiently binds to DNA and carries the CCAAT-displacement activity. The p110 isoform is generated by proteolytic processing at the G1-S transition and is capable of stable interaction with DNA. Here we demonstrate the existence of a shorter CDP/Cux isoform, p75, which contains only two DNA binding domains, Cut repeat 3 and the Cut homeodomain, and binds more stably to DNA. CDP/Cux p75 was able to repress a reporter carrying the promoter for the cyclin-dependent kinase inhibitor p21 gene and to activate a DNA polymerase alpha gene reporter. Expression of CDP/Cux p75 involved a novel mechanism: transcription initiation within intron 20. The intron 20-initiated mRNA (I20-mRNA) was expressed at higher level in the thymus and in CD4+/CD8+ and CD4+ T cells. I20-mRNA was expressed only weakly or not at all in normal human mammary epithelial cells and normal breast tissues but was detected in many breast tumor cells lines and breast tumors. In invasive tumors a significant association was established between higher I20-mRNA expression and a diffuse infiltrative growth pattern (n = 41, P = 0.0137). In agreement with these findings, T47D breast cancer cells stably expressing p75 could not form tubule structures in collagen but rather developed as solid undifferentiated aggregates of cells. Taken together, these results suggest that aberrant expression of the CDP/Cux p75 isoform in mammary epithelial cells may be associated with the process of tumorigenesis in breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Células 3T3 , Animais , Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Núcleo Celular/metabolismo , Colágeno/metabolismo , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Proteínas de Homeodomínio , Humanos , Íntrons , Camundongos , Peso Molecular , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Isoformas de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Fatores de Transcrição , Células Tumorais CultivadasRESUMO
Despite having long telomeres, mouse embryo fibroblasts (MEFs) senesce more rapidly than human diploid fibroblasts because of the accumulation of oxidative DNA damage. The CUX1 homeodomain protein was recently found to prevent senescence in RAS-driven cancer cells that produce elevated levels of reactive-oxygen species. Here we show that Cux1-/- MEFs are unable to proliferate in atmospheric (20%) oxygen although they can proliferate normally in physiological (3%) oxygen levels. CUX1 contains three domains called Cut repeats. Structure/function analysis established that a single Cut repeat domain can stimulate the DNA binding, Schiff-base formation, glycosylase and AP-lyase activities of 8-oxoguanine DNA glycosylase 1, OGG1. Strikingly and in contrast to previous reports, OGG1 exhibits efficient AP-lyase activity in the presence of a Cut repeat. Repair of oxidative DNA damage and proliferation in 20% oxygen were both rescued in Cux1-/- MEFs by ectopic expression of CUX1 or of a recombinant Cut repeat protein that stimulates OGG1 but is devoid of transcription activation potential. These findings reinforce the causal link between oxidative DNA damage and cellular senescence and suggest that the role of CUX1 as an accessory factor in DNA repair will be critical in physiological situations that generate higher levels of reactive oxygen species.
Assuntos
Senescência Celular/genética , Reparo do DNA , Fibroblastos/citologia , Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares/metabolismo , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/metabolismo , Animais , Dano ao DNA , Fibroblastos/metabolismo , Células HCT116 , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Proteínas Nucleares/genética , Oxirredução , Estresse Oxidativo/genética , Proteínas Repressoras/genética , Fatores de TranscriçãoRESUMO
Proteinase-activated receptors (PARs) are G-protein-coupled receptors that have been linked to an array of cellular processes, including inflammation, migration, and proliferation. Although signal transduction downstream of PARs has been actively investigated, little is known about the mechanisms that lead to changes in transcriptional programs. Here we show that the CUX1 homeodomain protein is a downstream effector of PAR2. Treatment of epithelial and fibroblastic cells with trypsin or the PAR2-activating peptide (PAR2-AP) caused a rapid increase in CUX1 DNA binding activity. The stimulation of CUX1 was specific to PAR2 because no effect was observed with thrombin or the PAR1-AP. Using a panel of recombinant CUX1 proteins, the regulation was found to involve the cut repeat 3 (CR3) and the cut homeodomain, two DNA binding domains that are present in all CUX1 isoforms. Expression analysis in cux1(-/-) mouse embryo fibroblasts led to the identification of three genes that are regulated downstream of both PAR2 and CUX1 as follows: interleukin-1alpha, matrix metalloproteinase-10, and cyclo-oxygenase-2. p110 CUX1 was able to activate each of these genes, both in reporter assays and following the infection of cells. Moreover, the treatment of Hs578T breast tumor cells with trypsin led to a rapid recruitment of p110 CUX1 to the promoter of these genes and to a concomitant increase in their mRNA steady-state levels. Altogether, these results suggest a model whereby activation of PAR2 triggers a signaling cascade that culminates with the stimulation of p110 CUX1 DNA binding and the transcriptional activation of target genes.
Assuntos
Fibroblastos/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/metabolismo , Modelos Biológicos , Proteínas Nucleares/metabolismo , Receptor PAR-2/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/genética , Embrião de Mamíferos , Fibroblastos/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Inflamação/genética , Inflamação/metabolismo , Interleucina-1alfa/biossíntese , Interleucina-1alfa/genética , Metaloproteinase 10 da Matriz/biossíntese , Metaloproteinase 10 da Matriz/genética , Camundongos , Camundongos Knockout , Mutação , Células NIH 3T3 , Proteínas Nucleares/genética , Peptídeos/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Receptor PAR-2/agonistas , Receptor PAR-2/genética , Proteínas Repressoras/genética , Transdução de Sinais/efeitos dos fármacos , Tripsina/farmacologiaRESUMO
Proteolytic processing at the end of the G(1) phase generates a CUX1 isoform, p110, which functions either as a transcriptional activator or repressor and can accelerate entry into S phase. Here we describe a second proteolytic event that generates an isoform lacking two active repression domains in the COOH terminus. This processing event was inhibited by treatment of cells with synthetic and natural caspase inhibitors. In vitro, several caspases generated a processed isoform that co-migrated with the in vivo generated product. In cells, recombinant CUX1 proteins in which the region of cleavage was deleted or in which Asp residues were mutated to Ala, were not proteolytically processed. Importantly, this processing event was not associated with apoptosis, as assessed by terminal dUTP nick end labeling assay, cytochrome c localization, poly(ADP-ribose) polymerase cleavage, and fluorescence-activated cell sorting. Moreover, processing was observed in S phase but not in early G(1), suggesting that it is regulated through the cell cycle. The functional importance of this processing event was revealed in reporter and cell cycle assays. A recombinant, processed, CUX1 protein was a more potent transcriptional activator of several cell cycle-related genes and was able to accelerate entry into S phase, whereas mutants that could not be processed were inactive in either assay. Conversely, cells treated with the quinoline-Val Asp-2,6-difluorophenoxymethylketone caspase inhibitor proliferated more slowly and exhibited delayed S phase entry following exit from quiescence. Together, our results identify a substrate of caspases in proliferating cells and suggest a mechanism by which caspases can accelerate cell cycle progression.