RESUMO
Cathepsin D (CTSD) is a major lysosomal protease harboring an N-terminal signal peptide (amino acids 1-20) to enable vesicular transport from endoplasmic reticulum to lysosomes. Here, we report the possibility of a mitochondrial targeting sequence and mitochondrial localization of CTSD in cells. Live-cell imaging analysis with C-terminal enhanced green fluorescent protein-tagged CTSD (EGFP-CTSD) indicated that CTSD localizes to mitochondria. CTSD amino acids 21-35 are responsible for its mitochondrial localization, which exhibit typical features of mitochondrial targeting sequences, and are evolutionarily conserved. A proteinase K protection assay and sucrose gradient analysis showed that a small population of endogenous CTSD molecules exists in mitochondria. These results suggest that CTSD is a dual-targeted protein that may localize in both lysosomes and mitochondria.
Assuntos
Catepsina D , Mitocôndrias , Catepsina D/genética , Catepsina D/metabolismo , Mitocôndrias/metabolismo , Peptídeo Hidrolases/metabolismoRESUMO
Mitochondria-eating protein (Mieap) plays a critical role in mitochondrial quality control (MQC) and functions as a p53-inducible tumor suppressor. This study aimed to examine its role in gastric cancer (GC) and esophageal cancer (EC). GC cells were infected with Mieap-overexpressing adenovirus (Ad-Mieap) and subjected to fluorescence-activated cell sorting (FACS), western blotting, and caspase assays. Thereafter, we evaluated the potential disruption of the p53/Mieap-regulated MQC pathway in vivo. Methylation-specific PCR (MSP) for Mieap, NIX, and BNIP3 promoters was performed and p53 mutations were detected using cryopreserved surgical specimens. Exogenous Mieap in GC cells induced the formation of vacuole-like structures (called MIVs, Mieap-induced vacuoles) and caspase-dependent cell death, with the activation of both caspase-3 and caspase-9. Of the 47 GC patients, promoter methylation in Mieap, BNIP3, and NIX was identified in two (4.3%), 29 (61.7%), and zero (0%) specimens, respectively. In total, 33 GC patients (70.2%) inactivated this MQC pathway. Amazingly, BNIP3 promoter in the normal epithelium was highly methylated in 18 of the 47 GC patients (38.3%). In EC patients, this MQC pathway was also inactivated in ten of 12 patients (83.3%). These results indicate that p53/Mieap-regulated MQC plays an important role in upper gastrointestinal (GI) tumor suppression, possibly, in part, through the mitochondrial apoptotic pathway.
Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Neoplasias Gástricas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Idoso , Apoptose/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Metilação de DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Mutação , Regiões Promotoras Genéticas/genética , Transdução de Sinais/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Oncogenic mutations of RAS genes, found in about 30% of human cancers, are considered to play important roles in cancer development. However, oncogenic RAS can also induce senescence in mouse and human normal fibroblasts. In some cell lines, oncogenic RAS has been reported to induce non-apoptotic programed cell death (PCD). Here, we investigated effects of oncogenic RAS expression in several types of normal human epithelial cells. Oncogenic RAS but not wild-type RAS stimulated macropinocytosis with accumulation of large-phase lucent vacuoles in the cytoplasm, subsequently leading to cell death which was indistinguishable from a recently proposed new type of PCD, methuosis. A RAC1 inhibitor suppressed accumulation of macropinosomes and overexpression of MYC attenuated oncogenic RAS-induced such accumulation, cell cycle arrest and cell death. MYC suppression or rapamycin treatment in some cancer cell lines harbouring oncogenic mutations in RAS genes induced cell death with accumulation of macropinosomes. These results suggest that this type of non-apoptotic PCD is a tumour-suppressing mechanism acting against oncogenic RAS mutations in normal human epithelial cells, which can be overcome by MYC overexpression, raising the possibility that its induction might be a novel approach to treatment of RAS-mutated human cancers.
Assuntos
Morte Celular/genética , Células Epiteliais/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas ras/genética , Linhagem Celular Tumoral , Células Cultivadas , Humanos , Neoplasias/genética , Neoplasias/patologia , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Mitochondria-eating protein (Mieap), encoded by a p53-target gene, plays an important role in mitochondrial quality control (MQC). Mieap has been reported to have a critical role in tumor suppression in colorectal cancer. Here, we investigated its role as a tumor suppressor in breast cancer. The enforced expression of exogenous Mieap in breast cancer cells induced caspase-dependent apoptosis, with activation of both caspase-3/7 and caspase-9. Immunohistochemistry revealed endogenous Mieap in the cytoplasm in 24/75 (32%) invasive ductal carcinomas (IDC), 15/27 (55.6%) cases of ductal carcinoma in situ (DCIS) and 16/18 (88.9%) fibroadenomas (FA) (IDC vs DCIS; P = 0.0389, DCIS vs FA; P = 0.0234, IDC vs FA; P < 0.0001). In IDC, the Mieap promoter was methylated in 6/46 (13%) cases, whereas p53 was mutated in 6/46 (13%) cases. Therefore, the p53/Mieap-regulated MQC pathway was inactivated in 12/46 IDC (26.1%). Interestingly, all tumors derived from the 12 patients with Mieap promoter methylation or p53 mutations pathologically exhibited more aggressive and malignant breast cancer phenotypes. Impairment of p53/Mieap-regulated MQC pathway resulted in significantly shorter disease-free survival (DFS) (P = 0.021), although p53 status is more prognostic in DFS than Mieap promoter methylation. These results indicate that p53/Mieap-regulated MQC has a critical role in tumor suppression in breast cancer, possibly in part through mitochondrial apoptotic pathway.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Metilação de DNA , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Mutação , Regiões Promotoras GenéticasRESUMO
The tumor suppressor p53 gene is frequently mutated in human cancers, and the p53 protein suppresses cancer. However, the mechanism behind the p53-mediated tumor suppression is still unclear. Recently, the mitochondria-eating protein (Mieap) was identified as a p53-inducible protein. Mieap induces the accumulation of lysosomal proteins within mitochondria (Mieap-induced accumulation of lysosome-like organelles within mitochondria, or MALM) in response to mitochondrial damage, and eliminates the oxidized mitochondrial proteins to repair unhealthy mitochondria. Furthermore, Mieap also induces vacuole-like structures (Mieap-induced vacuole, or MIV) to eat and degrade unhealthy mitochondria. Therefore, Mieap controls mitochondrial quality by repairing or eliminating unhealthy mitochondria by MALM or MIV, respectively. This mechanism is not mediated by canonical autophagy. Mieap-deficient ApcMin/+ mice show strikingly high rates of intestinal tumor development as well as advanced-grade adenomas and adenocarcinomas. The p53/Mieap/BCL2 interacting protein 3 mitochondrial quality control pathway is frequently inactivated in human colorectal cancers. Defects in Mieap-regulated mitochondrial quality control lead to accumulation of unhealthy mitochondria in cancer cells. Cancer-specific unhealthy mitochondria could contribute to cancer development and aggressiveness through mitochondrial reactive oxygen species and altered metabolism. Mieap-regulated mitochondrial quality control is a newly discovered function of p53 that plays a critical role in tumor suppression.
Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Neoplasias/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Autofagia/fisiologia , Humanos , Lisossomos/metabolismo , OxirreduçãoRESUMO
Human angiosarcoma is a rare malignant vascular tumor associated with extremely poor clinical outcome and generally arising in skin of the head and neck region. However, little is known about the molecular pathogeneses and useful immunohistochemical markers of angiosarcoma. To investigate the mechanisms of angiosarcoma progression, we collected 85 cases of human angiosarcoma specimens with clinical records and analyzed ISO-HAS-B patient-derived angiosarcoma cells. As control subjects, 54 cases of hemangioma and 34 of pyogenic granuloma were collected. Remarkably, consistent with our recent observations regarding the involvement of survivin expression following Hippo pathway inactivation in the neoplastic proliferation of murine hemangioendothelioma cells and human infantile hemangioma, nuclear survivin expression was observed in all cases of angiosarcoma but not in hemangiomas and pyogenic granulomas, and the Hippo pathway was inactivated in 90.3% of yes-associated protein (YAP) -positive angiosarcoma cases. However, survivin expression modes and YAP localization (Hippo pathway activation modes) were not correlated with survival. In addition, we confirmed that survivin small interference RNA (siRNA) transfection and YM155, an anti-survivin drug, elicited decreased nuclear survivin expression and cell proliferation in ISO-HAS-B cells which expressed survivin consistently. Conclusively, these findings support the importance of survivin as a good marker and critical regulator of cellular proliferation for human angiosarcoma and YM155 as a potential therapeutic agent.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Biomarcadores Tumorais/genética , Hemangiossarcoma/genética , Proteínas Inibidoras de Apoptose/genética , Fosfoproteínas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hemangiossarcoma/patologia , Via de Sinalização Hippo , Humanos , Imidazóis , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Masculino , Pessoa de Meia-Idade , Naftoquinonas , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Survivina , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
Cholangiocarcinoma is a relatively rare cancer, but its incidence is increasing worldwide. Although several risk factors have been suggested, the etiology and pathogenesis of the majority of cholangiocarcinomas remain unclear. Recently, a high incidence of early-onset cholangiocarcinoma was reported among the workers of a printing company in Osaka, Japan. These workers underwent high exposure to organic solvents, mainly haloalkanes such as 1,2-dichloropropane (1,2-DCP) and/or dichloromethane. We performed whole-exome analysis on four cases of cholangiocarcinoma among the printing workers. An average of 44.8 somatic mutations was detected per Mb in the genome of the printing workers' cholangiocarcinoma tissues, approximately 30-fold higher than that found in control common cholangiocarcinoma tissues. Furthermore, C:G-to-T:A transitions with substantial strand bias as well as unique trinucleotide mutational changes of GpCpY to GpTpY and NpCpY to NpTpY or NpApY were predominant in all of the printing workers' cholangiocarcinoma genomes. These results were consistent with the epidemiological observation that they had been exposed to high concentrations of chemical compounds. Whole-genome analysis of Salmonella typhimurium strain TA100 exposed to 1,2-DCP revealed a partial recapitulation of the mutational signature in the printing workers' cholangiocarcinoma. Although our results provide mutational signatures unique to occupational cholangiocarcinoma, the underlying mechanisms of the disease should be further investigated by using appropriate model systems and by comparison with genomic data from other cancers.
Assuntos
Colangiocarcinoma/epidemiologia , Colangiocarcinoma/genética , Exoma/genética , Exposição Ocupacional , Adulto , Colangiocarcinoma/induzido quimicamente , Colangiocarcinoma/patologia , Exoma/efeitos dos fármacos , Humanos , Japão/epidemiologia , Masculino , Cloreto de Metileno/toxicidade , Mutação/efeitos dos fármacos , Impressão , Propano/análogos & derivados , Propano/toxicidade , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genéticaRESUMO
Mitochondrial DNA (mtDNA) depletion syndrome (MDS; MIM 251880) is a prevalent cause of oxidative phosphorylation disorders characterized by a reduction in mtDNA copy number. The hitherto recognized disease mechanisms alter either mtDNA replication (POLG (ref. 1)) or the salvage pathway of mitochondrial deoxyribonucleosides 5'-triphosphates (dNTPs) for mtDNA synthesis (DGUOK (ref. 2), TK2 (ref. 3) and SUCLA2 (ref. 4)). A last gene, MPV17 (ref. 5), has no known function. Yet the majority of cases remain unexplained. Studying seven cases of profound mtDNA depletion (1-2% residual mtDNA in muscle) in four unrelated families, we have found nonsense, missense and splice-site mutations and in-frame deletions of the RRM2B gene, encoding the cytosolic p53-inducible ribonucleotide reductase small subunit. Accordingly, severe mtDNA depletion was found in various tissues of the Rrm2b-/- mouse. The mtDNA depletion triggered by p53R2 alterations in both human and mouse implies that p53R2 has a crucial role in dNTP supply for mtDNA synthesis.
Assuntos
Proteínas de Ciclo Celular/genética , DNA Mitocondrial/genética , Deleção de Genes , Doenças Mitocondriais/etiologia , Mutação/genética , Ribonucleotídeo Redutases/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteínas de Ciclo Celular/fisiologia , Células Cultivadas , Análise Mutacional de DNA , Feminino , Fibroblastos , Homozigoto , Humanos , Recém-Nascido , Escore Lod , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias Musculares , Doenças Mitocondriais/patologia , Dados de Sequência Molecular , Linhagem , Ribonucleotídeo Redutases/fisiologia , Proteína Supressora de Tumor p53/genéticaRESUMO
Biomolecular condensates (BCs) are formed by proteins with intrinsically disordered regions (IDRs) via liquid-liquid phase separation. Mieap/Spata18, a p53-inducible protein, participates in suppression of colorectal tumors by promoting mitochondrial quality control. However, the regulatory mechanism involved remains unclear. Here, we report that Mieap is an IDR-containing protein that drives formation of BCs involved in cardiolipin metabolism. Mieap BCs specifically phase separate the mitochondrial phospholipid, cardiolipin. Mieap directly binds to cardiolipin in vitro. Lipidomic analysis of cardiolipin suggests that Mieap promotes enzymatic reactions in cardiolipin biosynthesis and remodeling. Accordingly, four cardiolipin biosynthetic enzymes, TAMM41, PGS1, PTPMT1, and CRLS1 and two remodeling enzymes, PLA2G6 and TAZ, are phase-separated by Mieap BCs. Mieap-deficient cells exhibit altered crista structure, leading to decreased respiration activity and ATP production in mitochondria. These results suggest that Mieap may form membrane-less organelles to compartmentalize and facilitate cardiolipin metabolism, thus potentially contributing to mitochondrial quality control.
RESUMO
Netrin-1 and its receptors DCC (deleted in colorectal cancer) and the UNC5 orthologues (human UNC5A-D and rodent UNC5H1-4) define a new mechanism for both the positive (induction) and negative (suppression) regulation of apoptosis. Accumulating evidence implies that for human cancers, this positive signalling pathway is frequently inactivated. Surprisingly, binding of netrin-1 to its receptors inhibits tumour suppressor p53-dependent apoptosis, and p53 is directly involved in transcriptional regulation of netrin-1 and its receptors. So, the netrin-1 receptor pathways probably play an important part in tumorigenesis.
Assuntos
Moléculas de Adesão Celular/genética , Neoplasias Colorretais/genética , Fatores de Crescimento Neural/genética , Receptores de Superfície Celular/genética , Proteínas Supressoras de Tumor/genética , Axônios/fisiologia , Receptor DCC , Genes DCC , Humanos , Perda de Heterozigosidade , Neoplasias/genética , Neoplasias/patologia , Receptores de Netrina , Netrina-1RESUMO
p53R2, which is regulated by tumor suppressor p53, is a small subunit of ribonucleotide reductase. To determine whether it is involved in DNA repair by supplying deoxyribonucleotides (dNTPs) for resting cells in vivo, we generated a strain of mice lacking Rrm2b (encoding p53R2). These mice developed normally until they were weaned but from then on had growth retardation and early mortality. Pathological examination indicated that multiple organs had failed, and all Rrm2b-null mice died from severe renal failure by the age of 14 weeks. TUNEL staining showed a greater number of apoptotic cells in kidneys of 8-week-old Rrm2b-/- mice relative to wild-type mice. p53 was activated in kidney tissues of Rrm2b-/- mice, leading to transcriptional induction of p53 target genes. Rrm2b-/- mouse embryonic fibroblasts (MEFs) became immortal much earlier than Rrm2b+/+ MEFs. dNTP pools were severely attenuated in Rrm2b-/- MEFs under oxidative stress. Rrm2b deficiency caused higher rates of spontaneous mutation in the kidneys of Rrm2b-/- mice. Our results suggest that p53R2 has a pivotal role in maintaining dNTP levels for repair of DNA in resting cells. Impairment of this pathway may enhance spontaneous mutation frequency and activate p53-dependent apoptotic pathway(s) in vivo, causing severe renal failure, growth retardation and early mortality.
Assuntos
Proteínas de Ciclo Celular , Desoxirribonucleotídeos/metabolismo , Falência Renal Crônica/genética , Falência Renal Crônica/metabolismo , Ribonucleotídeo Redutases/deficiência , Ribonucleotídeo Redutases/genética , Animais , Apoptose , Reparo do DNA , Feminino , Genes p53 , Rim/metabolismo , Rim/patologia , Falência Renal Crônica/etiologia , Falência Renal Crônica/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Insuficiência de Múltiplos Órgãos/etiologia , Insuficiência de Múltiplos Órgãos/genética , Insuficiência de Múltiplos Órgãos/metabolismo , Insuficiência de Múltiplos Órgãos/patologia , Mutação , Estresse OxidativoRESUMO
Mitochondria-eating protein (MIEAP) is a molecule important for non-canonical mitophagy and thought to be a tumor suppressor. Our previous study found that MIEAP expression is defective in thyroid oncocytomas, irrespective of being benign or malignant, and also in non-oncocytic thyroid cancers. Thyroid oncocytomas are composed of large polygonal cells with eosinophilic cytoplasm that is rich in abnormal mitochondria. Thus, our data indicate that, together with increased mitochondrial biogenesis that compensates for the dysfunction of the mitochondria, MIEAP plays a critical role in the accumulation of mitochondria in thyroid oncocytic tumors, whereas a defective MIEAP expression alone is not sufficient for mitochondrial accumulation in non-oncocytic cancers with normal mitochondria. To clarify whether MIEAP is a tumor suppressor in the thyroids and whether MIEAP knockout (KO) alone is sufficient for the oncocytic phenotype and also to extend our effort toward canonical mitophagy (a selective autophagy), we here conducted mouse studies using genetically engineered mice. BrafCA/wt mice developed thyroid cancers 1 year after intrathyroidal injection of adenovirus expressing Cre, while cancer development was observed at 6 months in adenovirus-Cre-injected BrafCA/wt;MieapKO/KO and BrafCA/wt;Atg5flox/flox mice [where autophagy-related 5 (ATG5) is a component of autophagic machinery], although KO of either molecule alone was not sufficient for cancer development. These data demonstrate that MIEAP or ATG5 KO accelerated thyroid cancer development. However, cancers in adenovirus-Cre-injected BrafCA/wt ;MieapKO/KO and BrafCA/wt ;Atg5flox/flox mice were not oncocytic. In conclusion, we here show that MIEAP and ATG5 are both tumor suppressors in thyroid carcinogenesis, but as we have anticipated from our previous data, KO of either molecule does not confer the oncocytic phenotype to BRAFV600E-positive thyroid cancers. The combination of disruptive mitochondrial function and impaired mitochondrial quality control may be necessary to establish a mouse model of thyroid oncocytoma.
Assuntos
Adenoma Oxífilo , Proteínas Mitocondriais , Neoplasias da Glândula Tireoide , Animais , Camundongos , Adenoma Oxífilo/patologia , Autofagia/genética , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mitofagia/genética , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias da Glândula Tireoide/patologiaRESUMO
Although a number of targets for p53 have been reported, the mechanism of p53-dependent apoptosis still remains to be elucidated. Here we report a new p53 target-gene, designated p53RDL1 (p53-regulated receptor for death and life; also termed UNC5B). The p53RDL1 gene product contains a cytoplasmic carboxy-terminal death domain that is highly homologous to rat Unc5H2, a dependence receptor involved in the regulation of apoptosis, as well as in axon guidance and migration of neural cells. We found that p53RDL1 mediated p53-dependent apoptosis. Conversely, when p53RDL1 interacted with its ligand, Netrin-1, p53-dependent apoptosis was blocked. Therefore, p53RDL1 seems to be a previously un-recognized target of p53 that may define a new pathway for p53-dependent apoptosis. We suggest that p53 might regulate the survival of damaged cells by balancing the regulation of Netrin-p53RDL1 signalling, and cell death through cleavage of p53RDL1 for apoptosis.
Assuntos
Apoptose/fisiologia , Receptores de Superfície Celular/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA , DNA Complementar , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Fatores de Crescimento Neural/fisiologia , Receptores de Netrina , Netrina-1 , Ratos , Receptores de Superfície Celular/genética , Transdução de Sinais , Células Tumorais Cultivadas , Proteínas Supressoras de TumorRESUMO
Mitochondria-eating protein (MIEAP; also known as SPATA18), a p53-downstream gene, is involved in mitochondrial quality control (MQC). Enforced MIEAP expression induces caspase-dependent cell death in vitro, and impairment of the p53/MIEAP-regulated MQC pathway is frequently observed in breast cancer (BC), resulting in poor disease-free survival (DFS). To investigate the clinical significance of MIEAP in BC, we identified 2,980 patients from two global, large-scale primary BC cohorts: the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC; n=1,904) and the Cancer Genome Atlas (TCGA; n=1,076). We divided patients in each cohort into high and low groups based on median gene expression levels and analyzed the association between MIEAP expression and clinical outcomes. Compared with normal tumors, MIEAP expression was significantly downregulated in all patients with p53-mutant BC regardless of subtype. MIEAP expression was negatively correlated with KI67 expression. Gene set enrichment analysis demonstrated that cell cycle- and proliferation-associated gene sets were significantly enriched in MIEAP-low tumors compared to MIEAP-high tumors. Patients with MIEAP-high luminal subtype were associated with significantly longer DFS than those with MIEAP-low luminal tumors in both cohorts, whereas significantly longer overall survival was observed only in the METABRIC cohort, which has roughly double the number of samples. These results indicated that the mechanistic role of MIEAP is clinically relevant in the two independent cohorts. This is the first study to use large cohorts to demonstrate the association between MIEAP expression and survival in patients with luminal subtype BC.
RESUMO
Although the regulation of tumor angiogenesis is believed to be one of the core functions of p53, the mechanism still remains to be elucidated. Here, we report that semaphorin 3F (SEMA3F), an axon guidance molecule, is involved in p53-regulated antiangiogenesis. The expression level of SEMA3F mRNA was increased by both exogenous and endogenous p53. Chromatin immunoprecipitation assay indicated that a potent p53-binding sequence in intron 1 of SEMA3F interacts with p53 and that it has a p53-responsive transcriptional activity. Overexpression of SEMA3F inhibited in vitro cell growth of the lung cancer cell line H1299. In nude mice assay, the size of the H1299 tumors expressing SEMA3F was much smaller, and they showed lesser number of blood vessels as compared with the control tumors. Moreover, tumors derived from the p53-knockdown colorectal cancer cell line LS174T displayed a remarkable enhancement of tumor vessel formation as compared with control tumors containing normal levels of p53. The expression levels of SEMA3F and neuropilin-2 (NRP2), the functional receptor for SEMA3F, in p53-knockdown LS174T tumors were lower than those in the control tumors. Adenovirus-mediated SEMA3F gene transfer induced the remarkable in vitro growth suppression of the stable transformant of H1299 cells, which express high levels of NRP2. These results suggest that p53 negatively regulates tumor vessel formation and cell growth via the SEMA3F-NRP2 pathway.
Assuntos
Genes Supressores de Tumor , Proteínas de Membrana/genética , Neoplasias/irrigação sanguínea , Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Cromossomos Humanos Par 3 , Regulação Neoplásica da Expressão Gênica , Genes p53 , Células HCT116 , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/metabolismo , Neuropilina-2/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transdução de Sinais , Transcrição GênicaRESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV) causes both AIDS-related Kaposi's sarcoma (KS) and classic KS, but their clinical presentations are different, and respective mechanisms remain to be elucidated. The KSHV K1 gene is reportedly involved in tumorigenesis through the immunoreceptor tyrosine-based activation motif (ITAM). Since we found the sequence variations in the K1 gene of KSHV isolated from AIDS-related KS and classic KS, we hypothesized that the transformation activity of the K1 gene contributes to the different clinical presentations. To evaluate our hypothesis, we compared the transformation activities of the K1 gene between AIDS-related KS and classic KS. We also analyzed ITAM activities and the downstream AKT and NF-κB. We found that the transformation activity of AIDS-related K1 was greater than that of classic K1, and that AIDS-related K1 induced higher ITAM activity than classic K1, causing more potent Akt and NF-κB activities. K1 downregulation by siRNA in AIDS-related K1 expressing cells induced a loss of transformation properties and decreased both Akt and NF-κB activities, suggesting a correlation between the transformation activity of K1 and ITAM signaling. Our study indicates that the increased transformation activity of AIDS-related K1 is associated with its clinical aggressiveness, whereas the weak transformation activity of classic type K1 is associated with a mild clinical presentation and spontaneous regression. The mechanism of spontaneous regression of classic KS may provide new therapeutic strategy to cancer.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/genética , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Herpesvirus Humano 8/genética , Interações Hospedeiro-Patógeno/genética , Sarcoma de Kaposi/genética , Neoplasias Cutâneas/genética , Proteínas Virais/genética , Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/patologia , Infecções Oportunistas Relacionadas com a AIDS/virologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Fibroblastos/metabolismo , Fibroblastos/virologia , Células HeLa , Herpesvirus Humano 8/crescimento & desenvolvimento , Herpesvirus Humano 8/patogenicidade , Humanos , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Remissão Espontânea , Sarcoma de Kaposi/diagnóstico , Sarcoma de Kaposi/patologia , Sarcoma de Kaposi/virologia , Índice de Gravidade de Doença , Transdução de Sinais , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/virologia , Transformação Genética , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismoRESUMO
Aneuploidy is a hallmark of human cancers. Although the maintenance of genomic integrity by p53 is important in preventing aneuploidy, its mechanism remains to be elucidated. Here we report evidence that B-cell linker protein (BLNK) mediates the inhibition of cytokinesis, which generates tetraploidy but prevents aneuploidy. We identified BLNK as a transcriptional target of p53. Surprisingly, ectopic expression of exogenous BLNK inhibited cytokinesis, resulting in the formation of tetraploid cells. Indeed, BLNK was involved in the generation of spontaneously arising binucleate tetraploid cells. Interestingly, cytokinesis after DNA damage was inhibited in p21(-/-) and p53+/+ cells, but not in p53(-/-) cells. BLNK knockdown in p53+/+ and p21(-/-) cells enhanced cytokinesis after DNA damage, leading to the generation of aneuploid cells. In addition, a BLNK-downregulated human pre-B leukemia cell line showed increased cytokinesis and aneuploidy after DNA damage compared with two other pre-B leukemia cell lines expressing higher levels of BLNK. These results suggest that BLNK acts as a mediator of p53 in the inhibition of cytokinesis, which prevents aneuploidy. We propose that the inhibition of cytokinesis is crucial for the maintenance of genomic integrity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Aneuploidia , Citocinese/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Células HCT116 , Humanos , Hibridização in Situ Fluorescente , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Modelos Genéticos , Proteína Supressora de Tumor p53/genéticaRESUMO
We identified recently the p53AIP1 gene, a novel p53 target that mediates p53-dependent apoptosis. In the experiments reported here, ectopic expression of p53AIP1 induced down-regulation of mitochondrial DeltaPsim and release of cytochrome c from mitochondria in human cells. Immunoprecipitation and immunostaining experiments indicated interaction between p53AIP1 and bcl-2 proteins at mitochondria. Overexpression of bcl-2 blocked the down-regulation of mitochondrial DeltaPsim and the proapoptotic activity of p53AIP1. Our results implicate p53AIP1 as a pivotal mediator of the p53-dependent mitochondrial apoptotic pathway.
Assuntos
Apoptose/fisiologia , Mitocôndrias/fisiologia , Proteínas/fisiologia , Animais , Proteínas Reguladoras de Apoptose , Células COS , Grupo dos Citocromos c/metabolismo , Grupo dos Citocromos c/fisiologia , Regulação para Baixo , Humanos , Membranas Intracelulares/fisiologia , Potenciais da Membrana/fisiologia , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Células Tumorais CultivadasRESUMO
To search for p53 target genes throughout the human genome, we applied a cDNA microarray system using adenovirus-mediated transfer of p53 into p53-deficient U373MG (glioblastoma) cells. In this manner, we detected dozens of genes that appeared to be regulated by wild-type p53. We describe here characterization of one such gene, termed CABC1 [chaperone-activity of bc1 complex in Schizosaccharomyces pombe (ABC1)-like], which encodes a 647-amino acid peptide with significant sequence similarity to activity of bc1 complex (ABC1) in Arabidopsis thaliana and S. pombe. The CABC1 product was located in mitochondria, and colony-formation assays with cancer cell lines indicated its ability to suppress cell growth. Inhibition of CABC1 expression by transfection with antisense oligonucleotide significantly reduced the apoptotic response induced by wild-type p53. These results suggest that CABC1 may play an important role in mediating p53-inducible apoptosis through the mitochondrial pathway.