RESUMEN
The human papillomavirus type 8 (HPV8) is associated with skin cancer development. The goal of this study was to investigate the effects of HPV8 oncoproteins on cellular gene expression and the identification of key regulators. We performed affymetrix microarray analyses to identify differentially expressed genes and common sequence motifs and identified Sp1/3 binding sites as being crucial. In transient transfection assays, we confirmed that HPV8-E7 stimulates the activity of Sp1/3 promoters. Interestingly, the HPV8-E7L23A mutant, which cannot trigger keratinocyte invasion was unable to activate fibronectin gene expression. In skin models or HPV8 positive skin cancers we found a peculiar deposition of fibronectin in the dermal compartment, and a correlation of Sp3 and fibronectin in the nucleus of HPV8-positive keratinocytes. Taken together, we identified that HPV8-E7 exerts control over cellular gene expression through Sp1/3 binding motifs, which may contribute to HPV8-mediated keratinocyte transformation and subsequent fibronectin-dependent invasion.
Asunto(s)
Regulación de la Expresión Génica , Interacciones Huésped-Patógeno , Papillomaviridae/crecimiento & desarrollo , Proteínas E7 de Papillomavirus/metabolismo , Factor de Transcripción Sp1/biosíntesis , Factor de Transcripción Sp3/biosíntesis , Sitios de Unión , Carcinogénesis , Línea Celular , Fibronectinas/metabolismo , Perfilación de la Expresión Génica , Humanos , Queratinocitos/virología , Análisis por MicromatricesRESUMEN
BACKGROUND: The transcription factors Sp3/Sp1 are expressed in a various types of cancers and BNIP3 is overexpressed in prostate cancer. Although it has been demonstrated that BNIP3 is transcriptionally regulated by HIF-1α and is post-transcriptionally regulated by miR145, our previous data indicated that there might be some other transcription factors regulating BNIP3 in prostate cancer. This study is conducted to investigate whether BNIP3 expression is directly regulated by Sp3/Sp1 or not. MATERIALS AND METHODS: Bioinformatics analysis shows that BNIP3 promoter contains several potential Sp3/Sp1 binding sites. And then it is demonstrated that SP3 could regulate the BNIP3 transcriptionally by binding to the predicted sites by dual reporter gene assays, ChIP, and EMSA. The biological effects of SP3 regulating BNIP3 on prostate cancer cells proliferation are measured by MTT, TUNEL, and flow cytometry. RESULTS: Our data show that Sp3 but not Sp1, is positively related to BNIP3 overexpression in prostate cancer. Sp3 can directly regulate BNIP3 transcription by mainly binding to the Sp3 binding sites (-624~-615 and -350~-343) of BNIP3 promoter. Knockdown of Sp3 by RNA interference could reduce cells growth and lead to cells apoptosis in PC-3 and DU145. Sp3-dependent BNIP3 overexpression might be an important mechanism to promote prostate cancer cells proliferation. CONCLUSION: This is the first study to provide direct evidence of Sp3-dependent BNIP3 expression. Sp3 might be the major transcriptional regulator of BNIP3 in prostate cancer and it is worthy to further study. The regulation of BNIP3 by Sp3 may be a new cancer-specific therapeutic target in prostate cancer.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Proteínas de la Membrana/biosíntesis , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas/biosíntesis , Factor de Transcripción Sp3/biosíntesis , Factores de Transcripción/fisiología , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen/métodos , Humanos , Masculino , Neoplasias de la Próstata/patologíaRESUMEN
Reactive oxygen species (ROS)-inducing anticancer agents such as phenethylisothiocyanate (PEITC) activate stress pathways for killing cancer cells. Here we demonstrate that PEITC-induced ROS decreased expression of microRNA 27a (miR-27a)/miR-20a:miR-17-5p and induced miR-regulated ZBTB10/ZBTB4 and ZBTB34 transcriptional repressors, which, in turn, downregulate specificity protein (Sp) transcription factors (TFs) Sp1, Sp3, and Sp4 in pancreatic cancer cells. Decreased expression of miR-27a/miR-20a:miR-17-5p by PEITC-induced ROS is a key step in triggering the miR-ZBTB Sp cascade leading to downregulation of Sp TFs, and this is due to ROS-dependent epigenetic effects associated with genome-wide shifts in repressor complexes, resulting in decreased expression of Myc and the Myc-regulated miRs. Knockdown of Sp1 alone by RNA interference also induced apoptosis and decreased pancreatic cancer cell growth and invasion, indicating that downregulation of Sp transcription factors is an important common mechanism of action for PEITC and other ROS-inducing anticancer agents.
Asunto(s)
Anticarcinógenos/farmacología , Inhibidores Enzimáticos/farmacología , Isotiocianatos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Animales , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Desnudos , MicroARNs/biosíntesis , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas c-myc/genética , Interferencia de ARN , ARN Interferente Pequeño , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Factor de Transcripción Sp1/biosíntesis , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp3/biosíntesis , Factor de Transcripción Sp4/biosíntesis , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Quercetin and hyperoside (QH) in combination (1:1 ratio) have previously been shown to inhibit the growth of human leukemia cells. Here, we investigated the anticancer activity of the same mixture in 786-O renal cancer cells. QH decreased the generation of reactive oxygen species (ROS) by up to 2.25-fold and increased the antioxidant capacity by up to 3-fold in 786-O cells (3.8-60 µg/ml), whereas IC50 values for viability were 18.2, 18.7 and 11.8 µg/ml, respectively. QH also induced caspase-3 cleavage (2-fold) and increased PARP cleavage. Specificity protein (Sp) transcription factors are overexpressed in cancer cells and regulate genes required for cell proliferation, survival and angiogenesis. QH treatment decreased the expression of Sp1, Sp3 and Sp4 mRNA and this was accompanied by decreased protein expression. Moreover, expression of the Sp-dependent anti-apoptotic survival gene survivin was also significantly reduced, both at the mRNA and protein levels. QH decreased microRNA-27a (miR-27a) and induced the zinc finger protein ZBTB10, an Sp-repressor, suggesting that interactions between QH and the miR-27a-ZBTB10 axis play a role in Sp downregulation. This was confirmed by transfection of cells with a specific mimic for miR-27a, which partially reversed the effects of QH. These findings are consistent with previous studies on botanical anticancer agents in colon cancer cells.
Asunto(s)
Antioxidantes/farmacología , Carcinoma de Células Renales/tratamiento farmacológico , MicroARNs/genética , Quercetina/análogos & derivados , Quercetina/farmacología , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/biosíntesis , Especies Reactivas de Oxígeno/metabolismo , Proteínas Represoras , Factor de Transcripción Sp1/biosíntesis , Factor de Transcripción Sp3/biosíntesis , Factor de Transcripción Sp4/biosíntesisRESUMEN
The dormancy-inducing factors of bacteria inhibit tumor cell growth. In the present study, we evaluated the antitumor effects of the dormancy-inducing factor 4-hexylresorcinol (4-HR) using real-time cell electronic sensing (RT-CES) in SCC-9 cells (tongue squamous cell carcinoma cells). Treatment with 4-HR suppressed the growth of SCC-9 cells in a dose-dependent manner. We used a DNA microarray to identify genes that showed a significant change in expression upon 4-HR administration in SCC-9 cells. Among the differentially expressed genes, the protein expression of several cell proliferation related factors, including E2F1, E2F2, E2F3, E2F4, E2F5, E2F6, Sp1 and Sp3, were determined by western blot analyses. Treatment with 4-HR strongly suppressed E2F2 and slightly suppressed E2F3 but did not change the expression of E2F1, E2F4, E2F5 and E2F6 relative to no treatment. Furthermore, 4-HR increased Sp1 expression in a dose-dependent manner and decreased Sp3 expression. Therefore, the ratio of Sp1 to Sp3, an important driving force of epithelial cell differentiation, was drastically increased. Consistent with this observation, 4-HR increased the expression of the epithelial cell differentiation markers involucrin and keratin 10. Together, our results indicate that 4-HR induces the differentiation of SCC-9 via the modulation of the E2F-mediated signaling pathway.
Asunto(s)
Carcinoma de Células Escamosas/patología , Factor de Transcripción E2F2/biosíntesis , Factor de Transcripción E2F3/biosíntesis , Hexilresorcinol/farmacología , Factor de Transcripción Sp1/biosíntesis , Factor de Transcripción Sp3/biosíntesis , Neoplasias de la Lengua/patología , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Diferenciación Celular/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Factor de Transcripción E2F2/genética , Factor de Transcripción E2F3/genética , Expresión Génica/efectos de los fármacos , Humanos , Análisis por Micromatrices , Transducción de Señal/efectos de los fármacos , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp3/genética , Neoplasias de la Lengua/genética , Neoplasias de la Lengua/metabolismoRESUMEN
Non-metastatic cells 5 (NME5), a recently found gene belonging to the NDPK-like molecules gene family, is highly expressed in testis and some types of human cancer. Current studies have revealed diverse potential functions of NME5 and we have reported that NME5 is associated with innate resistance to gemcitabine in human pancreatic cancer cells in previous study. However, the mechanism underlying the transcriptional regulation of NME5 has not been elucidated yet. In this study, we analyzed the 5'-flanking region of the human NME5 gene and revealed its transcription start site (TSS) at -35 bp relative to its translation start codon ATG. Using 5' unidirectional deletion analysis, we demonstrated that the proximal promoter of NME5 is located within -1051 bp to +35 bp. Two functional GC-boxes (-300 bp and -323 bp) were identified within the promoter region. Mutation of either GC-box led to significant reduction in NME5 promoter activity, whereas overexpression of Sp1 activated NME5 promoter activity in MIA PaCa-2 and 293T cells. In silico analysis predicted that transcription factor Sp1 binds to both GC-boxes, which were confirmed by EMSA and ChIP. In addition, we found that compared with MIA PaCa-2, Sp1 was highly expressed in PAXC002, a well characterized human pancreatic cancer cell line with innate gemcitabine resistance where NME5 was reported to be highly expressed, indicating that Sp1 induces NEM5 expression in PAXC002 cells. In conclusion, our study characterized for the first time the human NME5 promoter which is controlled by Sp1 transcription factor in pancreatic cancer.
Asunto(s)
Nucleósido Difosfato Quinasas NM23/genética , Neoplasias Pancreáticas/genética , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , Activación Transcripcional , Secuencia de Bases , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Secuencias Reguladoras de Ácidos Nucleicos , Análisis de Secuencia de ADN , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp3/biosíntesis , Factor de Transcripción Sp3/genética , Factor de Transcripción Sp3/metabolismo , Sitio de Iniciación de la Transcripción , Transcripción Genética , GemcitabinaRESUMEN
Increased expression of the calcium-sensing receptor (CASR), which controls blood calcium homeostasis, leads to a decrease in the extracellular calcium set-point, thereby reducing parathyroid hormone secretion and renal calcium reabsorption and increasing calcitonin secretion resulting in reduced circulating calcium levels. Critically ill patients with elevated proinflammatory cytokine levels commonly have hypocalcemia, although the mechanism is not known. After intraperitoneal injection of interleukin (IL)-6 in the rat, circulating levels of parathyroid hormone, 1,25-dihydroxyvitamin D, and calcium fell within hours and remained low at 24 h. Expression of CASR (mRNA and protein) increased within hours in parathyroid, thyroid, and kidney and remained elevated at 24 h. The CASR gene has two promoters (P1 and P2) yielding transcripts having alternative 5'-untranslated regions but encoding the same receptor protein. Activities of P1 and P2 promoter/luciferase reporter constructs were stimulated approximately 2-3-fold by IL-6 in proximal tubule HKC cells and TT thyroid C-cells. Studies with P1 deleted and mutated promoter-reporter and Stat1 and/or Stat3 dominant-negative constructs showed that a Stat1/3 element downstream of the P1 start site accounted for the IL-6 induction. There are no Stat elements in the P2 promoter, but Sp1/3 elements are clustered at the transcription start site. A series of transfection P2 promoter-reporter analyses showed that Sp1 together with Stat1/3 was critical for IL-6 responsiveness of P2. By oligonucleotide precipitation assay, IL-6 rapidly promoted a complex containing both Sp1/3 and Stat1/3 on the Sp1/3 elements. In conclusion, Stat1/3 directly controls promoter P1, and the Stats indirectly regulate promoter P2 via Sp1/3 in response to IL-6. By this mechanism, the cytokine likely contributes to altered extracellular calcium homeostasis.
Asunto(s)
Regulación de la Expresión Génica , Interleucina-6/fisiología , Receptores Sensibles al Calcio/biosíntesis , Receptores Sensibles al Calcio/genética , Factor de Transcripción STAT1/biosíntesis , Factor de Transcripción STAT3/biosíntesis , Factor de Transcripción Sp1/biosíntesis , Factor de Transcripción Sp3/biosíntesis , Transcripción Genética , Regulación hacia Arriba , Animales , Secuencia de Bases , Humanos , Masculino , Datos de Secuencia Molecular , Ratas , Ratas Sprague-DawleyRESUMEN
A375-6 human melanoma cells are sensitive to the antiproliferative effect of IL-1. After a long period of culturing, we have obtained cells resistant to IL-1. The resistant clone A375-R8 constitutively produced IL-1 alpha. In this study, we identified a sequence, CGCC, located at -48 to -45 upstream of the transcription start site, to be essential for the constitutive IL-1 alpha gene activation. Specificity protein 1 (Sp1) and Sp3 bound to the nucleotide containing the sequence. Although the binding level to the nucleotide and expression level of Sp1 and Sp3 are comparable in A375-R8 and A375-6 cells, transactivation activity of Sp1 is higher in A375-R8 cells as compared with A375-6 cells. Sp3 could not transactivate the IL-1 alpha promoter. These results suggest that Sp1 but not Sp3 is important for IL-1 alpha gene activation. Trichostatin A (TSA), an inhibitor of histone deacetylase (HDAC), greatly augmented the IL-1 alpha promoter activity in A375-6 cells to the level comparable with that in A375-R8 cells. TSA also induced IL-1 alpha mRNA expression in A375-6 cells. Sp1 and Sp3 bound to HDAC1 in A375-R8 and A375-6 cells. The chromatin immunoprecipitation assay revealed the binding of Sp1 and HDAC1 to the promoter region of the IL-1 alpha gene. The activities of HDAC bound to Sp1 and Sp3, and that of HDAC1 was lower in A375-R8 cells as compared with A375-6 cells. These results indicate that the reduction in the activity and interaction of HDAC1 with Sp1 are critical for the constitutive IL-1 alpha gene expression.
Asunto(s)
Perfilación de la Expresión Génica , Histona Desacetilasas/metabolismo , Interleucina-1alfa/biosíntesis , Interleucina-1alfa/genética , Melanoma/inmunología , Factor de Transcripción Sp1/metabolismo , Línea Celular Tumoral , ADN/inmunología , Activación Enzimática/inmunología , Histona Desacetilasa 1 , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción Sp1/biosíntesis , Factor de Transcripción Sp3/biosíntesis , Factor de Transcripción Sp3/metabolismo , Células Tumorales CultivadasRESUMEN
Combinatorial interactions between cis elements and trans-acting factors are required for regulation of cardiac gene expression during normal cardiac development and pathological cardiac hypertrophy. Sp factors bind GC boxes and are implicated in recruitment and assembly of the basal transcriptional complex. In this study, we show that the cardiac troponin T (cTnT) promoter contains a GC box that is necessary for basal and cAMP-mediated activity of cTnT promoter constructs transfected in embryonic cardiomyocytes. Cardiac nuclear proteins bind the cTnT GC box in a sequence-specific fashion and consist of Sp1, Sp2, and Sp3 protein factors. By chromatin immunoprecipitation, Sp1 binds the cTnT promoter "in vivo." Cotransfected Sp1 trans-activates the cTnT promoter in cardiomyocytes in culture. Sp3 represses Sp1-mediated transcriptional activation of the cTnT gene in embryonic cardiomyocytes. Sp3 repression of Sp1-mediated cTnT promoter activation is dose dependent, inferring a mechanism of competitive binding/inhibition. To evaluate the role of Sp factors in cardiac gene expression in vivo, we have established a clinically relevant animal model of pathological cardiac hypertrophy where the fetal cardiac program is activated. In this animal model, cardiac hypertrophy results from increased left-right shunting, volume loading of the left ventricle, and pressure loading of the right ventricle. Sp1 expression is increased in all four hypertrophied cardiac chambers, whereas Sp3 expression is diminished. This observation is consistent with the in vitro activating function of Sp1 and inhibitory effects of Sp3 on activity of cTnT promoter constructs. Sp factor levels are modulated during the hypertrophic cardiac program in vivo.
Asunto(s)
Cardiomegalia/metabolismo , Corazón/fisiología , Regiones Promotoras Genéticas/fisiología , Factor de Transcripción Sp2/antagonistas & inhibidores , Factor de Transcripción Sp3/biosíntesis , Factor de Transcripción Sp3/farmacología , Troponina T/genética , Animales , Western Blotting , Cardiomegalia/genética , Núcleo Celular/metabolismo , Células Cultivadas , Embrión de Pollo , Cromatina/metabolismo , ADN/biosíntesis , ADN/genética , Regulación hacia Abajo/fisiología , Drosophila/metabolismo , Elementos E-Box/genética , Ensayo de Cambio de Movilidad Electroforética , Femenino , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Corazón/efectos de los fármacos , Inmunohistoquímica , Inmunoprecipitación , Plásmidos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sarcómeros/metabolismo , Ovinos , Factor de Transcripción Sp2/farmacología , Factor de Transcripción Sp3/fisiología , Técnicas de Cultivo de Tejidos , TransfecciónRESUMEN
In the present investigation, we have examined the transcriptional regulation of adenomatous polyposis coli (APC) gene expression in the spontaneously immortalized human normal breast epithelial cell line, MCF10A, in response to carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) treatment. The APC mRNA levels and the APC gene's promoter (pAPCP) activity were increased in MCF10A cells after treatment with DMBA. A sequential deletion analysis and site-directed mutagenesis of the pAPCP promoter revealed that the DMBA response is mediated through a GC-box element. Also, the GC-box binding agent mithramycin A, which prevents binding of proteins to the GC-box region, abolished DMBA-mediated increase of the pAPCP promoter activity. The specificity of the proteins binding to the GC-box region was characterized by gel-shift analysis. An increased binding of the GC-box binding proteins was observed in the gel-shift analysis with nuclear extracts from DMBA-treated MCF10A cells, which corresponded to the increased levels of Sp1 and Sp3 proteins. However, a super-shift of the DNA-protein complexes was observed with only anti-Sp3 antibody. Based on the chromatin-immunoprecipitation assay results, the Sp3 appeared to be a genuine protein binding to the GC-box site of the pAPCP promoter. In RNA interference experiments, in which the Sp3 expression was knocked down, the DMBA response on the pAPCP promoter activity was reduced, suggesting that the binding of Sp3 to the GC-box site is critical for DMBA-induced pAPCP promoter activity. From these results we conclude that the increased pAPCP promoter activity in the MCF10A cell line in response to DMBA treatment is mediated by Sp3.