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 CultivadasRESUMO
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/genéticaRESUMO
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/fisiologiaAssuntos
Dano ao DNA , Genes p53 , Receptores do Fator de Necrose Tumoral/genética , Sequência de Aminoácidos , Sequência de Bases , Morte Celular/genética , Cromossomos Humanos Par 8/genética , DNA/genética , Humanos , Dados de Sequência Molecular , RNA Mensageiro/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Homologia de Sequência de AminoácidosRESUMO
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/metabolismoRESUMO
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 TecidualAssuntos
Ciclo Celular/fisiologia , Ciclinas/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Feminino , Humanos , Modelos Biológicos , Proteínas Proto-Oncogênicas c-akt , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Transdução de Sinais/fisiologiaAssuntos
Proteína BRCA1/metabolismo , Neoplasias da Mama/genética , Neoplasias Ovarianas/genética , Dedos de Zinco , Animais , Feminino , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Ligação Proteica , Proteínas/genética , Proteínas GADD45RESUMO
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/metabolismoRESUMO
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 UltravioletaRESUMO
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/toxicidadeRESUMO
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/biossínteseRESUMO
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/fisiologiaRESUMO
The present study assessed the role of the p53 tumor suppressor gene in cell cycle arrest and apoptosis following treatment of Burkitt's lymphoma and lymphoblastoid cell lines with gamma-rays, etoposide, nitrogen mustard, and cisplatin. Cell cycle arrest was measured by flow cytometry; p53 and p21Waf1/Cip1 protein levels were measured by Western blotting; cell survival was measured in 72-96-h growth inhibition assays and by trypan blue staining, and apoptotic DNA fragmentation was assessed by either agarose gel electrophoresis or a modified filter elution method. We found that gamma-rays and etoposide induced a strong G1 arrest in the wild-type p53 lines while nitrogen mustard and cisplatin induced relatively little G1 arrest. All agents failed to induce G1 arrest in cells containing mutant p53 genes. The degree of G1 arrest observed with these agents correlated with the rate of p53 and p21Waf1/Cip1 protein accumulation: gamma-rays and etoposide induced rapid accumulation of both p53 and p21Waf1/Cip1; nitrogen mustard and cisplatin induced slow accumulation of p53 and no major accumulation of the p21Waf1/Cip1 protein. Despite differences in G1 arrest and kinetics of p53 or p21Waf1/Cip1 protein accumulation, all agents tended to decrease survival to a greater extent in the wild-type p53 lines compared to the mutant p53 lines. Cell death in the wild-type p53 lines was associated with intracellular DNA degradation into oligonucleosomal sized DNA fragments, indicative of apoptosis. We also observed an inverse sensitivity relationship between nitrogen mustard/cisplatin and etoposide in the mutant p53 lines and this was found to correlate with topoisomerase II mRNA levels in the cells. Our results suggest that p53 gene status is an important determinant of both radio- and chemosensitivity in lymphoid cell lines and that p53 mutations are often associated with decreased sensitivity to DNA damaging agents.
Assuntos
Apoptose/genética , Linfoma de Burkitt/genética , Cisplatino/farmacologia , Ciclinas/metabolismo , Etoposídeo/farmacologia , Fase G1/genética , Raios gama , Genes p53/genética , Mecloretamina/farmacologia , Mutação/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Linfoma de Burkitt/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , DNA Topoisomerases Tipo II/análise , Resistência a Medicamentos/genética , Citometria de Fluxo , Fase G1/efeitos dos fármacos , Fase G1/efeitos da radiação , Genes p53/fisiologia , Humanos , Linfócitos , Mutação/genética , Nocodazol/farmacologia , RNA Mensageiro/análise , Tolerância a Radiação/genética , Células Tumorais CultivadasRESUMO
The c-Myc oncoprotein is a transcription factor involved in cellular transformation as well as apoptotic cell death. We show here that over-expression of c-Myc delivered by an adenovirus vector up-regulates endogenous proapoptotic bax mRNA and protein expression in human cells. In contrast, the cytotoxic tumor necrosis factor-related apoptosis-inducing ligand induces cell death without up-regulating bax expression. c-Myc/Max heterodimers bind to canonical E-box elements located in the bax promoter region as demonstrated by electrophoretic mobility shift analysis and DNaseI foot-printing assays. Analysis of bax regulatory region mutants suggests a model involving myc-dependent activation as well as relief of repression through distinct E-box elements. c-Myc-null cells are deficient in bax-promoter activation as compared with wild-type c-Myc-expressing cells. Overexpression of c-Myc in serum-starved human or mouse embryonic cells leads to apoptosis which is significantly reduced in the presence of growth factor-containing serum. c-Myc-induced apoptosis appears to be deficient in bax-null as compared with bax-wild-type mouse embryonic fibroblasts. The results suggest that the cell death-promoting gene bax is directly downstream of c-Myc in a pathway leading to apoptosis.
Assuntos
Apoptose/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas c-myc/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Adenoviridae/genética , Animais , Apoptose/genética , Regulação da Expressão Gênica , Genes myc , Vetores Genéticos/genética , Humanos , Camundongos , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Transdução de Sinais/fisiologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas , Regulação para Cima , Proteína X Associada a bcl-2RESUMO
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/metabolismoRESUMO
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/fisiologiaRESUMO
The chromosomal region 8p21 contains a number of putative tumor suppressor genes and is a frequent site of translocations in head and neck cancers. Recently, a novel tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor gene, KILLER/DR5, a member of the tumor necrosis factor receptor family, was identified as a potential mediator in p53-dependent apoptosis and mapped to 8p21 by fluorescence in situ hybridization. We have determined the genomic structure of KILLER/DR5 and performed sequence analysis of all 10 coding exons in 20 primary head and neck cancers with allelic loss of chromosome 8p. To screen for a subset of mutations localized to the functional cytoplasmic death domain, we sequenced this region in an additional 40 primary head and neck cancers. We found two alterations in this domain, including a 2-bp insertion at a minimal repeat site, introducing a premature stop codon and resulting in a truncated protein. This KILLER/DR5 mutation was also present in the germ line of the affected patient, and the tumor did not have a p53 mutation by sequence analysis. Transfection studies in head and neck squamous cell carcinoma and colon and ovarian carcinoma cell lines revealed loss of growth-suppressive function associated with the tumor-derived KILLER/DR5 truncation mutant. These observations provide the first evidence for mutation of a TRAIL death receptor gene in a human cancer, leading to loss of its apoptotic function.
Assuntos
Cromossomos Humanos Par 8/genética , Deleção de Genes , Genes Supressores de Tumor/genética , Neoplasias de Cabeça e Pescoço/genética , Receptores do Fator de Necrose Tumoral/genética , Sequência de Bases , Mapeamento Cromossômico , Éxons/genética , Humanos , Íntrons/genética , Perda de Heterozigosidade , Dados de Sequência Molecular , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Ensaio Tumoral de Célula-TroncoRESUMO
CD437 is a novel retinoid that can induce apoptosis in a variety of tumor cell types by an unknown mechanism. We found that CD437 up-regulated the expression of p21(WAF1/CIP1), Bax, and Killer/DR5 and induced G1 arrest and rapid apoptosis in three human non-small cell lung carcinoma cell lines with wild-type p53 but not in five cell lines with mutant p53, suggesting a role for p53 in the effects of CD437. Using H460 cells in which wild-type p53 protein was degraded by transfection of the human papillomavirus 16 E6 (HPV-16 E6) gene and H460 cells transfected with a control plasmid only, we found that CD437 increased p53, p21(WAF1/CIP1), Bax, and Killer/DR5 in the control transfectants. In contrast, the constitutive p53 protein level was suppressed, and the ability of CD437 to increase p53 and its downstream genes was compromised in E6 transfectants. In addition, CD437 induced G1 arrest and apoptosis in the control transfectants but not in the E6-transfected cells. These results indicate that p53 plays a role in CD437-induced growth inhibition and apoptosis in human non-small cell lung carcinoma cells.
Assuntos
Antineoplásicos/farmacologia , Apoptose , Carcinoma Pulmonar de Células não Pequenas/patologia , Fase G1/efeitos dos fármacos , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Repressoras , Retinoides/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Caspase 3 , Caspases/metabolismo , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/genética , Grupo dos Citocromos c/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/genética , Transdução de Sinais , Transfecção , Células Tumorais Cultivadas , Proteína X Associada a bcl-2RESUMO
The death receptor (DR) KILLER/DR5 gene has recently been identified as a doxorubicin-regulated transcript that was also induced by exogenous wild-type p53 in p53-negative cells. KILLER/DR5 gene encodes a DR containing cell surface protein that is highly homologous to DR4, another DR of the tumor necrosis factor (TNF) receptor family. Both DR4 and KILLER/DR5 independently bind to their specific ligand TRAIL and engage the caspase cascade to induce apoptosis. TRID (also known as TRAIL-R3) is an antiapoptotic decoy receptor that lacks the cytoplasmic death domain and competes with KILLER/DR5 and DR4 for binding to TRAIL. In this study, we demonstrate that the DR KILLER/DR5 gene is regulated in a p53-dependent and -independent manner during genotoxic and nongenotoxic stress-induced apoptosis. Just like other p53-regulated genes, ionizing radiation induction of KILLER/DR5 occurs in p53 wild-type cells, whereas methyl methanesulfonate regulation of KILLER/DR5 occurs in a p53-dependent and -independent manner. However, unlike other p53-regulated genes, KILLER/DR5 is not regulated following UV irradiation. TNF-alpha, a nongenotoxic cytokine, also induced the expression of KILLER/DR5 in a number of cancer cell lines, irrespective of p53 status. TNF-alpha did not alter the KILLER/DR5 mRNA stability, suggesting that the TNF-alpha regulation of KILLER/DRS expression appears transcriptional. We also provide evidence that KILLER/DR5 is regulated in a trigger and cell type-specific manner and that its induction by TNF-alpha, p53, or DNA damage is not the consequence of apoptosis induced by these agents. Unlike KILLER/DR5, none of the other KILLER/DR5 family members, including DR4, TRID, or the ligand TRAIL, displayed genotoxic stress or TNF-alpha regulation in a p53 transcription-dependent manner. Thus, KILLER/DR5 appears a bona fide downstream target of p53 that is also regulated in a cell type-specific, trigger-dependent, and p53-independent manner.