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1.
PLoS One ; 7(5): e37759, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22662213

RESUMO

It has been previously described that p21 functions not only as a CDK inhibitor but also as a transcriptional co-repressor in some systems. To investigate the roles of p21 in transcriptional control, we studied the gene expression changes in two human cell systems. Using a human leukemia cell line (K562) with inducible p21 expression and human primary keratinocytes with adenoviral-mediated p21 expression, we carried out microarray-based gene expression profiling. We found that p21 rapidly and strongly repressed the mRNA levels of a number of genes involved in cell cycle and mitosis. One of the most strongly down-regulated genes was CCNE2 (cyclin E2 gene). Mutational analysis in K562 cells showed that the N-terminal region of p21 is required for repression of gene expression of CCNE2 and other genes. Chromatin immunoprecipitation assays indicated that p21 was bound to human CCNE2 and other p21-repressed genes gene in the vicinity of the transcription start site. Moreover, p21 repressed human CCNE2 promoter-luciferase constructs in K562 cells. Bioinformatic analysis revealed that the CDE motif is present in most of the promoters of the p21-regulated genes. Altogether, the results suggest that p21 exerts a repressive effect on a relevant number of genes controlling S phase and mitosis. Thus, p21 activity as inhibitor of cell cycle progression would be mediated not only by the inhibition of CDKs but also by the transcriptional down-regulation of key genes.


Assuntos
Proteínas Correpressoras/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Mitose/genética , Fase S/genética , Transcrição Gênica , Linhagem Celular , Análise por Conglomerados , Biologia Computacional/métodos , Quinase 2 Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/química , Ciclinas/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Células K562 , Queratinócitos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica
2.
Genes Dev ; 21(5): 562-77, 2007 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-17344417

RESUMO

Little is known about the regulation and function of the Notch1 gene in negative control of human tumors. Here we show that Notch1 gene expression and activity are substantially down-modulated in keratinocyte cancer cell lines and tumors, with expression of this gene being under p53 control in these cells. Genetic suppression of Notch signaling in primary human keratinocytes is sufficient, together with activated ras, to cause aggressive squamous cell carcinoma formation. Similar tumor-promoting effects are also caused by in vivo treatment of mice, grafted with keratinocytes expressing oncogenic ras alone, with a pharmacological inhibitor of endogenous Notch signaling. These effects are linked with a lesser commitment of keratinocytes to differentiation, an expansion of stem cell populations, and a mechanism involving up-regulation of ROCK1/2 and MRCKalpha kinases, two key effectors of small Rho GTPases previously implicated in neoplastic progression. Thus, the Notch1 gene is a p53 target with a role in human tumor suppression through negative regulation of Rho effectors.


Assuntos
Carcinoma de Células Escamosas/genética , Genes Supressores de Tumor , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Queratinócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptor Notch1/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Queratinócitos/citologia , Camundongos , Miotonina Proteína Quinase , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , RNA Interferente Pequeno , Receptor Notch1/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Células-Tronco/citologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Quinases Associadas a rho
3.
J Biol Chem ; 281(41): 30463-70, 2006 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-16912042

RESUMO

p21 plays a dual role in keratinocyte growth and differentiation control. It restricts the number of keratinocyte stem cell populations while inhibiting the later stages of differentiation independently of the cell cycle. The molecular/biochemical mechanism for the differentiation suppressive function of p21 is unknown. Here we show that elevated p21 expression leads to activation of MAPK family members in a keratinocyte-specific and cell cycle-independent manner, and up-regulation of MAPK activity can explain the inhibitory effects of p21 on differentiation. p21 induces transcription of several genes with MAPK activation potential. Although several of these genes are induced by p21 in a MAPK-dependent manner, expression of IGF-I is induced upstream of MAPK activation. IGF-I stimulation is by itself sufficient to cause MAPK activation and inhibit differentiation and suppression of IGF-I signaling by knock down of the cognate receptor (IGF-R1), diminishing the ability of p21 to activate MAPK and suppress differentiation. Thus, in keratinocytes, the ability of p21 to suppress differentiation can be explained by cell type-specific activation of the MAPK cascade by transcriptional up-regulation of the IGF-I gene.


Assuntos
Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Regulação da Expressão Gênica , Fator de Crescimento Insulin-Like I/biossíntese , Queratinócitos/citologia , Animais , Ciclo Celular , Diferenciação Celular , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Ativação Enzimática , Epiderme/metabolismo , Fator de Crescimento Insulin-Like I/genética , Queratinócitos/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Regiões Promotoras Genéticas , Transcrição Gênica
4.
Genes Dev ; 20(8): 1028-42, 2006 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-16618808

RESUMO

Notch signaling promotes commitment of keratinocytes to differentiation and suppresses tumorigenesis. p63, a p53 family member, has been implicated in establishment of the keratinocyte cell fate and/or maintenance of epithelial self-renewal. Here we show that p63 expression is suppressed by Notch1 activation in both mouse and human keratinocytes through a mechanism independent of cell cycle withdrawal and requiring down-modulation of selected interferon-responsive genes, including IRF7 and/or IRF3. In turn, elevated p63 expression counteracts the ability of Notch1 to restrict growth and promote differentiation. p63 functions as a selective modulator of Notch1-dependent transcription and function, with the Hes-1 gene as one of its direct negative targets. Thus, a complex cross-talk between Notch and p63 is involved in the balance between keratinocyte self-renewal and differentiation.


Assuntos
Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Queratinócitos/citologia , Receptor Notch1/fisiologia , Transativadores/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Sequência de Bases , Primers do DNA , Humanos , Camundongos , Regiões Promotoras Genéticas , RNA Interferente Pequeno , Fatores de Transcrição
5.
Genes Dev ; 19(12): 1485-95, 2005 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-15964998

RESUMO

In keratinocytes, the cyclin/CDK inhibitor p21(WAF1/Cip1) is a direct transcriptional target of Notch1 activation; loss of either the p21 or Notch1 genes expands stem cell populations and facilitates tumor development. The Notch1 tumor-suppressor function was associated with down-regulation of Wnt signaling. Here, we show that suppression of Wnt signaling by Notch1 activation is mediated, at least in part, by down-modulation of Wnts gene expression. p21 is a negative regulator of Wnts transcription downstream of Notch1 activation, independently of effects on the cell cycle. More specifically, expression of the Wnt4 gene is under negative control of endogenous p21 both in vitro and in vivo. p21 associates with the E2F-1 transcription factor at the Wnt4 promoter and causes curtailed recruitment of c-Myc and p300, and histone hypoacetylation at this promoter. Thus, p21 acts as a selective negative regulator of transcription and links the Notch and Wnt signaling pathways in keratinocyte growth control.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores de Superfície Celular/metabolismo , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Ciclo Celular , Proteínas de Ciclo Celular/genética , Diferenciação Celular , Divisão Celular , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21 , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Proteína p300 Associada a E1A , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , Camundongos , Camundongos Knockout , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Notch1 , Receptores de Superfície Celular/genética , Transdução de Sinais , Transativadores/metabolismo , Fatores de Transcrição HES-1 , Fatores de Transcrição/genética , Transcrição Gênica , Proteínas Wnt , Proteína Wnt4
6.
J Biol Chem ; 280(45): 37725-31, 2005 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-16155000

RESUMO

Glucocorticoid-induced tumor necrosis factor receptor (GITR) is a member of the tumor necrosis factor receptor superfamily, is expressed in T lymphocytes, and exerts an anti-apoptotic function in these cells. We reported that GITR is also highly expressed in the skin, specifically in keratinocytes, and that it is under negative transcriptional control of p21(Cip1/WAF1), independently from the cell cycle. Although GITR expression is higher in p21-deficient keratinocytes and skin, it is down-modulated with differentiation and in response to UVB. The combined analysis of keratinocytes with increased GITR expression versus normal keratinocytes and skin of mice with a disruption of the GITR gene indicates that this protein protects keratinocytes from UVB-induced apoptosis both in vitro and in vivo.


Assuntos
Apoptose/efeitos da radiação , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Glucocorticoides/farmacologia , Queratinócitos/efeitos da radiação , Receptores de Fator de Crescimento Neural/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Transcrição Gênica , Raios Ultravioleta , Animais , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Células Epidérmicas , Epiderme/metabolismo , Epiderme/efeitos da radiação , Feminino , Deleção de Genes , Proteína Relacionada a TNFR Induzida por Glucocorticoide , Queratinócitos/citologia , Camundongos
7.
Mol Reprod Dev ; 65(3): 269-77, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12784248

RESUMO

To improve efficiency of transgenesis, we compared M16 and CZB embryo culture media, supporting development to blastocysts of FVB/N mouse pronuclear-eggs, microinjected with enhanced green fluorescent protein (EGFP) transgene. When EGFP-injected-eggs were cultured (120 hr), blastocyst development was significantly (P < 0.03) higher in M16 medium (72.5 +/- 2.4%) than that in CZB (13.2 +/- 4.3%) or CZBG (CZB with 5.6 mM glucose at 48 hr culture) (62.1 +/- 3.7%) media. Blastocyst development of noninjected embryos was higher in M16 (92.0 +/- 2.6%) and CZBG (83.9 +/- 3.9%) media than in CZB (31.9 +/- 2.8%) medium (P < 0.0001). However, percentages of morulae at 72 hr were comparable in all treatments. Developed blastocysts were better in M16 than in CZB or CZBG media. Consistent with this, mean cell number per blastocyst, developed from injected embryos, was significantly (P < 0.002) higher in M16 medium (79.6), than those in CZB (31.3) or CZBG media (60.7); similar with noninjected embryos. Cell allocation to trophectoderm (TE) and inner cell mass (ICM), i.e., TE:ICM ratio, for injected blastocysts in M16 (3.0) was less than (P < 0.05) those in CZB (4.2) and CZBG (4.4) media; similar with noninjected blastocysts. Moreover, blastocysts, developed in M16 and CZBG media, hatched, attached, and exhibited trophoblast outgrowth; 18% of them showed EGFP-expression. Importantly, blastocysts from M16 medium produced live transgenic "green" pups (11%) following embryo transfer. Taken together, our results indicate that supplementation of glucose, at 48 hr of culture (CZBG), is required for morula to blastocyst transition; M16 medium, containing glucose from the beginning of culture, is superior to CZB or CZBG for supporting development of biologically viable blastocysts from EGFP-transgene-injected mouse embryos.


Assuntos
Blastocisto/fisiologia , Proteínas Luminescentes/genética , Animais , Animais Recém-Nascidos , Blastocisto/citologia , Sobrevivência Celular , Meios de Cultura , Transferência Embrionária , Desenvolvimento Embrionário e Fetal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Transgênicos , Microinjeções , Mórula/citologia , Mórula/fisiologia , Transfecção/métodos
8.
Biochem Biophys Res Commun ; 313(4): 1030-6, 2004 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-14706646

RESUMO

The impact of embryonic enhanced green fluorescent protein (EGFP)-expression on development is not clear. In this study, we comprehensively assessed EGFP-expression pattern and its effect on early mouse development, following pronuclear-microinjection of the EGFP-transgene, containing chicken-beta-actin promoter and cytomegalovirus enhancer. Preimplantation embryos exhibited differential EGFP-expression patterns. While blastocyst development of non-expressing embryos was 77.3+/-1.8%, that of expressing embryos was only 43.9+/-1.6% (P<0.0001). Developmental competence of embryos negatively correlated (r=-0.99) with the levels of EGFP-expression. Faint-, moderate-, and intense-expressing embryos developed to 83.1+/-5.3%, 50+/-5%, and 9.5+/-3.9% blastocysts, respectively (P<0.002). Interestingly, blastocysts expressing faint-moderate levels of EGFP were developmentally competent through the post-implantation period and delivered viable transgenic 'green' mice, following embryo transfer. These results indicate that hyper-expression of EGFP affects preimplantation development and faint-moderate level of its expression is compatible with normal embryogenesis in the mouse.


Assuntos
Desenvolvimento Embrionário e Fetal/genética , Proteínas Luminescentes/genética , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Transferência Embrionária , Feminino , Fluorescência , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Transferência de Genes , Marcadores Genéticos , Proteínas de Fluorescência Verde , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microinjeções , Gravidez , Proteínas Recombinantes/genética
9.
Biochem Biophys Res Commun ; 303(4): 994-1001, 2003 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-12684032

RESUMO

One of the limitations of transgenesis is low efficiency. In this study, we generated transgenic mice harboring the enhanced green fluorescent protein (EGFP) gene, under the control of chicken-beta-actin promoter and cytomegalovirus enhancer, using two approaches and compared their efficiencies. One involved culture of EGFP-injected embryos developing through EGFP-expressing "green" blastocysts, followed by their transfer to uterus. The second was oviductal-transfer of EGFP-injected-eggs. Embryo culture-based-transgenesis (ECBT) produced 100% transgenic mice, unlike the second approach. Moreover, ECBT required reduced number of recipients and markedly increased pregnancy rates. Of the nine founders, seven exhibited ubiquitous EGFP-expression, one (GU1) was a mosaic and the other (G18) was non-expressing. The molecular basis for this was attributed to repeat-induced gene silencing, since the G18 had a high copy number (approximately 99/genome) of the non-mutated and non-rearranged EGFP-transgene integrated at a single site. Our results show the superiority of ECBT over the conventional oviductal approach for generating transgenic "green" mice.


Assuntos
Transferência Embrionária , Proteínas Luminescentes/genética , Camundongos Transgênicos , Animais , Animais Recém-Nascidos , Blastocisto/citologia , Técnicas de Cultura de Células , Núcleo Celular/genética , DNA/administração & dosagem , Embrião de Mamíferos/citologia , Feminino , Dosagem de Genes , Inativação Gênica , Genótipo , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Camundongos , Microinjeções , Microscopia de Fluorescência , Fenótipo , Gravidez
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