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1.
Nat Genet ; 15(1): 78-82, 1997 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8988173

RESUMO

The 52-kD Activator Protein (AP2) is a DNA-binding transcription factor implicated in signalling terminal differentiation. Profound developmental abnormalities have been recently observed in AP2-null mice. The molecular events by which AP2 promotes differentiation or development are, however, unknown. Increased expression of the universal cell cycle inhibitor p21WAF1/CIP1 occurs in growth-arrested terminally differentiating cells. In a search for cellular factors that could activate p21 during phorbol ester (TPA)-induced differentiation, we identified AP2 as a regulator of p21 expression. Mutagenesis of an AP2 DNA-binding site within a p21 promoter-luciferase reporter inhibited its activation by either AP2 transfection or TPA stimulation. Endogenous p21 protein levels were elevated and DNA synthesis was inhibited in AP2 versus control vector-transfected cells. Overexpression of AP2 in HepG2 human hepatoblastoma and SW480 human colon adenocarcinoma cells inhibited cell division and stable colony formation. These results link the differentiation-associated factor AP2 to negative cell cycle and growth control, possibly through p21 activation.


Assuntos
Divisão Celular/fisiologia , Ciclinas/genética , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica , Fatores de Transcrição/fisiologia , Cromossomos Humanos Par 6 , Clonagem Molecular , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Humanos , Luciferases/genética , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Fator de Transcrição AP-2 , Células Tumorais Cultivadas
2.
Nat Genet ; 1(1): 45-9, 1992 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-1301998

RESUMO

Recent experiments have suggested that p53 action may be mediated through its interaction with DNA. We have now identified 18 human genomic clones that bind to p53 in vitro. Precise mapping of the binding sequences within these clones revealed a consensus binding site with a striking internal symmetry, consisting of two copies of the 10 base pair motif 5'-PuPuPuC(A/T)(T/A)GPyPyPy-3' separated by 0-13 base pairs. One copy of the motif was insufficient for binding, and subtle alterations of the motif, even when present in multiple copies, resulted in loss of affinity for p53. Mutants of p53, representing each of the four "hot spots" frequently altered in human cancers, failed to bind to the consensus dimer. These results define the DNA sequence elements with which p53 interacts in vitro and which may be important for p53 action in vivo.


Assuntos
DNA/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Sequência Consenso , DNA/genética , Humanos , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase , Ligação Proteica , Proteína Supressora de Tumor p53/genética
3.
Nat Med ; 1(6): 570-7, 1995 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7585125

RESUMO

For several human tumour types, allelic loss data suggest that one or more tumour suppressor genes reside telomeric to the p53 gene at chromosome 17p13.1. In the present study we have used a new strategy, involving molecular analysis of a DNA site hypermethylated in tumour DNA, to identify a candidate gene in this region (17p13.3). Our approach has led to identification of HIC-1 (hypermethylated in cancer), a new zinc-finger transcription factor gene which is ubiquitously expressed in normal tissues, but underexpressed in different tumour cells where it is hypermethylated. Multiple characteristics of this gene, including the presence of a p53 binding site in the 5' flanking region, activation of the gene by expression of a wild-type p53 gene and suppression of G418 selectability of cultured brain, breast and colon cancer cells following insertion of the gene, make HIC-1 gene a strong candidate for a tumour suppressor gene in region 17p13.3.


Assuntos
Cromossomos Humanos Par 17 , Ilhas de CpG , Regulação da Expressão Gênica , Genes Supressores de Tumor , Inibidores do Crescimento/genética , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/fisiologia , Dedos de Zinco/genética , Sequência de Aminoácidos , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Divisão Celular , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Feminino , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Inibidores do Crescimento/biossíntese , Humanos , Fatores de Transcrição Kruppel-Like , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Metilação , Dados de Sequência Molecular , Especificidade de Órgãos , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/biossíntese , Transfecção , Células Tumorais Cultivadas
4.
Science ; 279(5358): 1954-8, 1998 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-9506948

RESUMO

FADD (also known as Mort-1) is a signal transducer downstream of cell death receptor CD95 (also called Fas). CD95, tumor necrosis factor receptor type 1 (TNFR-1), and death receptor 3 (DR3) did not induce apoptosis in FADD-deficient embryonic fibroblasts, whereas DR4, oncogenes E1A and c-myc, and chemotherapeutic agent adriamycin did. Mice with a deletion in the FADD gene did not survive beyond day 11.5 of embryogenesis; these mice showed signs of cardiac failure and abdominal hemorrhage. Chimeric embryos showing a high contribution of FADD null mutant cells to the heart reproduce the phenotype of FADD-deficient mutants. Thus, not only death receptors, but also receptors that couple to developmental programs, may use FADD for signaling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose , Proteínas de Transporte/fisiologia , Desenvolvimento Embrionário e Fetal , Coração/embriologia , Animais , Proteínas de Transporte/genética , Transformação Celular Neoplásica , Células Cultivadas , Doxorrubicina/farmacologia , Endotélio Vascular/embriologia , Proteína de Domínio de Morte Associada a Fas , Feminino , Expressão Gênica , Marcação de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Oncogenes , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/fisiologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia , Receptor fas/genética , Receptor fas/fisiologia
5.
Oncogene ; 26(16): 2353-64, 2007 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-17043653

RESUMO

Epidermal growth factor receptor (EGFR) overexpression and activation is critical in the initiation and progression of cancers, especially those of epithelial origin. EGFR activation is associated with the induction of divergent signal transduction pathways and a gamut of cellular processes; however, the cell-type and tissue-type specificity conferred by certain pathways remains to be elucidated. In the context of the esophageal epithelium, a prototype stratified squamous epithelium, EGFR overexpression is relevant in the earliest events of carcinogenesis as modeled in a three-dimensional organotypic culture system. We demonstrate that the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, and not the MEK/MAPK (mitogen-activated protein kinase) pathway, is preferentially activated in EGFR-mediated esophageal epithelial hyperplasia, a premalignant lesion. The hyperplasia was abolished with direct inhibition of PI3K and of AKT but not with inhibition of the MAPK pathway. With the introduction of an inducible AKT vector in both primary and immortalized esophageal epithelial cells, we find that AKT overexpression and activation is permissive for complete epithelial formation in organotypic culture, but imposes a growth constraint in cells grown in monolayer. In organotypic culture, AKT mediates changes related to cell shape and size with an expansion of the differentiated compartment.


Assuntos
Esôfago/citologia , Proteína Oncogênica v-akt/fisiologia , Diferenciação Celular , Divisão Celular , Células Cultivadas , Senescência Celular , Células Epiteliais/citologia , Receptores ErbB/fisiologia , Vetores Genéticos , Humanos , Imuno-Histoquímica , Técnicas de Cultura de Órgãos , Retroviridae
8.
Mol Cell Biol ; 19(5): 3916-28, 1999 May.
Artigo em Inglês | MEDLINE | ID: mdl-10207115

RESUMO

The tumor suppressor p16(INK4a) inhibits cyclin-dependent kinases 4 and 6. This activates the retinoblastoma protein (pRB) and, through incompletely understood events, arrests the cell division cycle. To permit biochemical analysis of the arrest, we generated U2-OS osteogenic sarcoma cell clones in which p16 transcription could be induced. In these clones, binding of p16 to cdk4 and cdk6 abrogated binding of cyclin D1, p27(KIP1), and p21(WAF1/CIP1). Concomitantly, the total cellular level of p21 increased severalfold via a posttranscriptional mechanism. Most cyclin E-cdk2 complexes associated with p21 and became inactive, expression of cyclin A was curtailed, and DNA synthesis was strongly inhibited. Induction of p21 alone, in a sibling clone, to the level observed during p16 induction substantially reproduced these effects. Overexpression of either cyclin E or A prevented p16 from mediating arrest. We then extended these studies to HCT 116 colorectal carcinoma cells and a p21-null clone derived by homologous recombination. In the parental cells, p16 expression also augmented total cellular and cdk2-bound p21. Moreover, p16 strongly inhibited DNA synthesis in the parental cells but not in the p21-null derivative. These findings indicate that p21-mediated inhibition of cdk2 contributes to the cell cycle arrest imposed by p16 and is a potential point of cooperation between the p16/pRB and p14(ARF)/p53 tumor suppressor pathways.


Assuntos
Apoptose/genética , Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Replicação do DNA , Regulação da Expressão Gênica , Humanos , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/metabolismo , Polirribossomos , Ligação Proteica , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo
9.
Mol Cell Biol ; 18(5): 2462-73, 1998 May.
Artigo em Inglês | MEDLINE | ID: mdl-9566867

RESUMO

Gfi-1 is a cellular proto-oncogene that was identified as a target of provirus integration in T-cell lymphoma lines selected for interleukin-2 (IL-2) independence in culture and in primary retrovirus-induced lymphomas. Gfi-1 encodes a zinc finger protein that functions as a transcriptional repressor. Here we show that Gfi-1B, a Gfi-1 related gene expressed in bone marrow and spleen, also encodes a transcriptional repressor. IL-6-induced G1 arrest and differentiation of the myelomonocytic cell line M1 were linked to the downregulation of Gfi-1B and the parallel induction of the cyclin-dependent kinase inhibitor p21WAF1. Experiments addressing the potential mechanism of the apparent coordinate regulation of these genes revealed that Gfi-1B represses p21WAF1 directly by binding to a high-affinity site at -1518 to -1530 in the p21WAF1 promoter. Forced expression of Gfi-1B, but not of Gfi-1B deletion mutants lacking the repressor domain, blocked the IL-6-mediated induction of p21WAF1 and inhibited G1 arrest and differentiation. We conclude that Gfi-1B is a direct repressor of the p21WAF1 promoter, the first such repressor identified to date, and that sustained expression of Gfi-1B blocks IL-6-induced G1 arrest and differentiation of M1 cells perhaps because it prevents p21WAF1 induction by IL-6.


Assuntos
Ciclinas/biossíntese , Células-Tronco Hematopoéticas/citologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Dedos de Zinco , Células 3T3 , Sequência de Aminoácidos , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Epistasia Genética , Regulação da Expressão Gênica no Desenvolvimento , Interleucina-6/farmacologia , Camundongos , Dados de Sequência Molecular , Monócitos/citologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Ratos , Proteínas Repressoras/genética , Homologia de Sequência de Aminoácidos , Baço , Distribuição Tecidual
10.
Mol Biol Cell ; 12(9): 2629-45, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11553704

RESUMO

Previous studies have argued that enhanced activity of the epidermal growth factor receptor (EGFR) and the mitogen-activated protein kinase (MAPK) pathway can promote tumor cell survival in response to cytotoxic insults. In this study, we examined the impact of MAPK signaling on the survival of primary hepatocytes exposed to low concentrations of deoxycholic acid (DCA, 50 microM). Treatment of hepatocytes with DCA caused MAPK activation, which was dependent upon ligand independent activation of EGFR, and downstream signaling through Ras and PI(3) kinase. Neither inhibition of MAPK signaling alone by MEK1/2 inhibitors, nor exposure to DCA alone, enhanced basal hepatocyte apoptosis, whereas inhibition of DCA-induced MAPK activation caused approximately 25% apoptosis within 6 h. Similar data were also obtained when either dominant negative EGFR-CD533 or dominant negative Ras N17 were used to block MAPK activation. DCA-induced apoptosis correlated with sequential cleavage of procaspase 8, BID, procaspase 9, and procaspase 3. Inhibition of MAPK potentiated bile acid-induced apoptosis in hepatocytes with mutant FAS-ligand, but did not enhance in hepatocytes that were null for FAS receptor expression. These data argues that DCA is causing ligand independent activation of the FAS receptor to stimulate an apoptotic response, which is counteracted by enhanced ligand-independent EGFR/MAPK signaling. In agreement with FAS-mediated cell killing, inhibition of caspase function with the use of dominant negative Fas-associated protein with death domain, a caspase 8 inhibitor (Ile-Glu-Thr-Asp-p-nitroanilide [IETD]) or dominant negative procaspase 8 blocked the potentiation of bile acid-induced apoptosis. Inhibition of bile acid-induced MAPK signaling enhanced the cleavage of BID and release of cytochrome c from mitochondria, which were all blocked by IETD. Despite activation of caspase 8, expression of dominant negative procaspase 9 blocked procaspase 3 cleavage and the potentiation of DCA-induced apoptosis. Treatment of hepatocytes with DCA transiently increased expression of the caspase 8 inhibitor proteins c-FLIP-(S) and c-FLIP-(L) that were reduced by inhibition of MAPK or PI(3) kinase. Constitutive overexpression of c-FLIP-(s) abolished the potentiation of bile acid-induced apoptosis. Collectively, our data argue that loss of DCA-induced EGFR/Ras/MAPK pathway function potentiates DCA-stimulated FAS-induced hepatocyte cell death via a reduction in the expression of c-FLIP isoforms.


Assuntos
Ácido Desoxicólico/farmacologia , Receptores ErbB/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Receptor fas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Ácidos e Sais Biliares/metabolismo , Caspase 9 , Inibidores de Caspase , Caspases/metabolismo , Células Cultivadas , Grupo dos Citocromos c/metabolismo , Precursores Enzimáticos/metabolismo , Proteína Ligante Fas , Hepatócitos/efeitos dos fármacos , Humanos , Membranas Intracelulares/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Permeabilidade , Ratos , Receptor fas/genética , Proteínas ras/metabolismo
13.
Cancer Res ; 57(2): 320-5, 1997 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-9000576

RESUMO

Growth arrest and differentiation of leukemic cells by phorbol 12-myristate 13-acetate (PMA) is accompanied by p53-independent activation of p21WAF1/CIP1 and c-myc down-regulation. We show that despite p21 induction in 7 of 12 human cancer cell lines treated with PMA, growth inhibition was observed only in two cell lines (SKBr3 breast and LNCaP prostate cancer cells). Treatment of SKBr3 and LNCaP cells with PMA was followed by Raf-1 hyperphosphorylation, p21 induction, Rb hypophosphorylation, c-myc down-regulation and growth inhibition. The 10 remaining PMA-resistant cell lines were comprised of 5 that failed to induce p21 and 5 that induced p21 but had defects in steps putatively downstream of this (Rb hypophosphorylation and c-myc down-regulation). Exogenous expression and subsequent failure to down-regulate c-myc protein expression in SKBr3 and LNCaP cells was correlated with acquisition of resistance to the growth inhibitory effect of PMA. Exogenous p21 expression down-regulated c-myc protein in PMA-sensitive cancer cells. Our findings suggest that induction of p21 and down-regulation of c-myc may be necessary steps in a PMA-induced growth-inhibitory pathway in cancer cells.


Assuntos
Neoplasias da Mama/metabolismo , Carcinógenos/farmacologia , Ciclinas/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteína do Retinoblastoma/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21 , Regulação para Baixo , Resistência a Medicamentos , Feminino , Genes myc , Humanos , Masculino , Fosforilação , Neoplasias da Próstata/patologia , Transdução de Sinais , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo
14.
Cancer Res ; 56(10): 2250-5, 1996 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-8625293

RESUMO

p53 induction and cell cycle arrest occur following DNA damage, possibly to allow repair prior to replication. p21WAF1/CIP1, a cyclin-cyclin-dependent kinase inhibitor and proliferating cell nuclear antigen-interacting protein, is induced by p53 and mediates the cell cycle arrest. To investigate a role for p21 in DNA repair in vivo, we studied the expression of in vitro damaged reporter DNA transfected into p21 +/+ or -/- HCT116 human colon cancer cells. Introduction of UV-damaged or cisplatinum-damaged cytomegalovirus-driven beta-galactosidase reporter DNA into tumor cells revealed a significant decrease (2-5-fold) in reporter expression in p21 -/- versus +/+ cells. In the absence of DNA damage, there was a significant increase (2-3-fold) in the number of 6-TG-resistant colonies derived from p21 -/- versus +/+ cells. Reintroduction of wild-type p21, but not a p21 C-terminal truncation mutant which lacks the proliferating cell nuclear antigen interaction domain, stimulated (2-3-fold) the repair capacity of the p21-deficient cells. We conclude that p21 deficiency is associated with a defect in DNA repair, which could lead to an increased sensitivity of tumor cells to DNA damage.


Assuntos
Ciclo Celular/genética , Neoplasias do Colo/patologia , Ciclinas/deficiência , Reparo do DNA/genética , DNA de Neoplasias/metabolismo , Proteínas de Neoplasias/deficiência , Divisão Celular , Cisplatino/farmacologia , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Ciclinas/fisiologia , Dano ao DNA , Replicação do DNA , DNA de Neoplasias/efeitos dos fármacos , DNA de Neoplasias/efeitos da radiação , Genes Reporter/efeitos dos fármacos , Genes Reporter/efeitos da radiação , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Tioguanina/farmacologia , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-Tronco , Proteína Supressora de Tumor p53/fisiologia , Raios Ultravioleta
15.
Cancer Res ; 61(4): 1334-7, 2001 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-11245430

RESUMO

Anchorage-independent growth is a hallmark of tumor cells. We compared gene expression profiles of anchored and nonanchored human mammary carcinoma cells to study this phenomenon. In this study, we show that anchorage had striking effects on cell growth and morphology but altered transcript levels from a limited number of genes. Only about 1% of mRNA transcripts detected in these cells was altered by anchorage. These include genes related to amino acid and polyamine metabolism, apoptosis, ion channels, cytoskeletal and stress proteins, transcription factors, and growth factors. Some of these may be crucial for the survival of transformed cells. For example, clusterin and the tumor necrosis factor-related apoptosis inducing ligand (TRAIL) were suppressed by anchorage, which could help prevent programmed cell death of these tumor cells. In addition to suppressing TRAIL expression, anchorage also decreased the susceptibility of these tumor cells to TRAIL-induced apoptosis as determined by poly(ADP-ribose) phosphorylase cleavage, annexin-V binding (P < 0.01), and cell cycle analysis (P < 0.0001). These data may help explain mechanisms by which anchorage prevents apoptosis of cells that would otherwise experience anoikis. Thus, genes found to be altered by this analysis could serve as potential targets for anticancer therapy. These findings suggest that TRAIL may be used as a means to target circulating epithelial tumor cells before their attachment and colonization at new sites.


Assuntos
Anoikis/fisiologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Glicoproteínas de Membrana/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Proteínas Reguladoras de Apoptose , Neoplasias da Mama/metabolismo , Adesão Celular/genética , Divisão Celular/fisiologia , Perfilação da Expressão Gênica , Humanos , Glicoproteínas de Membrana/biossíntese , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/biossíntese
16.
Cancer Res ; 60(22): 6259-65, 2000 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-11103780

RESUMO

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis of transformed and cancer cells but not of most normal cells. Recent studies have revealed an unforeseen toxicity of TRAIL toward normal human hepatocytes, thereby bringing into question the safety of systemic administration of TRAIL in humans with cancer. We found that SW480 colon adenocarcinoma, or H460 non-small cell lung cancer cell lines, which are sensitive to TRAIL, were not protected by the caspase 9 inhibitor Z-LEHD-FMK from TRAIL-induced apoptosis. However, a human colon cancer cell line HCT116 and a human embryonic kidney cell line 293, which are sensitive to TRAIL, were protected by Z-LEHD-FMK from TRAIL-mediated death. Both HCT116 and SW480 cells were protected from TRAIL by the caspase 8 inhibitor Z-IETD-FMK, dominant-negative FADD and cellular FLIP-s and interestingly both cell lines displayed caspase 9 cleavage to a similar extent after TRAIL exposure. We confirmed that normal human liver cells are sensitive to TRAIL. Moreover, we found that normal human liver cells could be protected from TRAIL-induced apoptosis by simultaneous exposure to Z-LEHD-FMK. A similar brief exposure to TRAIL plus Z-LEHD-FMK inhibited colony growth of SW480 but not HCT116 cells. Because some cancer cell lines are not protected from TRAIL-mediated killing by Z-LEHD-FMK, we believe that a brief period of caspase 9 inhibition during TRAIL administration may widen the therapeutic window and allow cancer cell killing while protecting normal liver cells. This strategy could be further developed in the effort to advance TRAIL into clinical trials.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Apoptose/efeitos dos fármacos , Inibidores de Caspase , Inibidores de Cisteína Proteinase/farmacologia , Hepatócitos/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/farmacologia , Oligopeptídeos/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Proteínas Reguladoras de Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Transporte/fisiologia , Caspase 8 , Caspase 9 , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/enzimologia , Neoplasias do Colo/patologia , Interações Medicamentosas , Proteína de Domínio de Morte Associada a Fas , Feminino , Hepatócitos/citologia , Hepatócitos/enzimologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Glicoproteínas de Membrana/toxicidade , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/toxicidade
17.
Cancer Res ; 59(12): 2770-5, 1999 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-10383128

RESUMO

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors are members of the tumor necrosis factor superfamily. TRAIL selectively kills cancer cells but not normal cells. We report here the cloning of the mouse homologue of the TRAIL receptor KILLER/DR5 (MK). The cDNA of MK is 1146 bp in length and encodes a protein of 381 amino acids. MK contains an extracellular cysteine-rich domain, a transmembrane domain, and a cytoplasmic death-domain characteristic of Fas, tumor necrosis factor, and human TRAIL receptors. MK is highly homologous and binds TRAIL with similar affinity as human DR4 and KILLER/DR5. MK induces apoptosis in mouse and human cells and inhibits colony growth of NIH3T3 cells. Expression of MK is p53-dependent and up-regulated by tumor suppressor p53 and by DNA damaging agents in mouse cells undergoing apoptosis. This is the first report describing a mouse TRAIL receptor gene and also demonstrating that the p53-dependent regulation of KILLER/DR5-mediated apoptosis is conserved between human and mouse.


Assuntos
Receptores do Fator de Necrose Tumoral/genética , Células 3T3 , Sequência de Aminoácidos , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose , Sequência de Bases , Clonagem Molecular , Ensaio de Unidades Formadoras de Colônias , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/fisiologia , Homologia de Sequência de Aminoácidos , Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/metabolismo , Proteína Supressora de Tumor p53/fisiologia
18.
Cancer Res ; 57(1): 130-5, 1997 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-8988053

RESUMO

Recent studies have shown that paclitaxel leads to activation of Raf-1 kinase and have suggested that this activation is essential for bcl-2 phosphorylation and apoptosis. In the present study, we demonstrate that, in addition to paclitaxel, other agents that interact with tubulin and microtubules also induce Raf-1/bcl-2 phosphorylation, whereas DNA-damaging drugs, antimetabolites, and alkylating agents do not. Activation of Raf-1 kinase by paclitaxel is linked to tubulin polymerization; the effect is blunted in paclitaxel-resistant cells, the tubulin of which does not polymerize following the addition of paclitaxel. In contrast, vincristine and vinblastine, drugs to which the paclitaxel-resistant cells retain sensitivity were able to bring about Raf-1 phosphorylation. The requirement for disruption of microtubules in this signaling cascade was strengthened further using paclitaxel analogues by demonstrating a correlation between tubulin polymerization, Raf-1/bcl-2 phosphorylation, and cytotoxicity. Inhibition of RNA or protein synthesis prevents Raf-1 activation and bcl-2 phosphorylation, suggesting that an intermediate protein(s) acts upstream of Raf-1 in this microtubule damage-activating pathway. A model is proposed that envisions a pathway of Raf-1 activation and bcl-2 phosphorylation following disruption of microtubular architecture, serving a role similar to p53 induction following DNA damage.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Microtúbulos/efeitos dos fármacos , Paclitaxel/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Tubulina (Proteína)/efeitos dos fármacos , Antineoplásicos/farmacologia , Resistência a Medicamentos , Ativação Enzimática , Feminino , Humanos , Fosforilação , Polímeros/metabolismo , Inibidores de Proteínas Quinases , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-raf , Tubulina (Proteína)/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo
19.
Cancer Res ; 55(13): 2910-9, 1995 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-7796420

RESUMO

The p53-regulated gene product p21WAF1/CIP1 is the prototype of a family of small proteins that negatively regulate the cell cycle. To learn more about p21WAF1/CIP1 regulation in vivo, monoclonal antibodies were developed for immunohistochemistry. These revealed that p21WAF1/CIP1 expression followed radiation-induced DNA damage in human skin in a pattern consistent with its regulation by p53. A detailed comparison of the human, rat, and mouse p21WAF1/CIP1 promoter sequences revealed that this induction was probably mediated by conserved p53-binding sites upstream of the transcription start site. In unirradiated tissues, p21WAF1/CIP1 expression was apparently independent of p53 and was observed in a variety of cell types. Moreover, there was a striking compartmentalization of p21WAF1/CIP1 expression throughout the gastrointestinal tract that correlated with proliferation rather than differentiation. As epithelial cells migrated up the crypts, the Ki67-expressing proliferating compartment near the crypt base ended abruptly, with the coincident appearance of a nonproliferating compartment expressing p21WAF1/CIP1. In colonic neoplasms, this distinct compartmentalization was largely abrogated. Cell cycle inhibitors are thus subject to precise topological control, and escape from this regulation may be a critical feature of neoplastic transformation.


Assuntos
Ciclinas/metabolismo , Adenoma/genética , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Carcinoma/genética , Neoplasias Colorretais/genética , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Primers do DNA/química , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Ratos , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Pele/metabolismo , Transcrição Gênica , Proteína Supressora de Tumor p53/fisiologia
20.
Cancer Res ; 58(20): 4531-6, 1998 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-9788595

RESUMO

The p53 tumor suppressor gene plays an instrumental role in transcriptional regulation of target genes involved in cellular stress responses. p53-dependent transactivation and transrepression require its interaction with p300/CBP, a coactivator that also interacts with the RelA subunit of nuclear factor-kappaB. We find that p53 inhibits RelA-dependent transactivation without altering RelA expression or inducible kappaB-DNA binding. p53-mediated repression of RelA is relieved by p300 overexpression and the increased RelA activity conferred by p53-deficiency is counteracted by either transactivation domain-deficient p300 fragments that bind RelA or a transdominant mutant of IkappaB alpha. Our results suggest that p53 can regulate diverse kappaB-dependent cellular responses.


Assuntos
Genes p53/fisiologia , Ligases/antagonistas & inibidores , Proteínas Nucleares/fisiologia , Transativadores/fisiologia , Proteína de Ligação a CREB , DNA/metabolismo , Humanos , Ligases/análise , Ligases/química , NF-kappa B/metabolismo , Transcrição Gênica
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