RESUMO
Suppression of tumor formation and restoration of normal growth of cells has been an insignia that the retinoblastoma gene (RB1) functions as a tumor suppressor gene. The tumor suppressive functions of RB are suggested to associate with regulation of cell cycle events or gene transcription. We have analysed here the interactions of RB and c-Ha-ras oncogene by gene transfection studies. Mouse fibroblasts stably expressing high levels human wild type (wt) pRB or mutant pRB were transfected with genomic or LTR promoter driven c-Ha-ras(Val-12) expression vectors. We find that expression of normal, but not mutant RB protein in the cells prevents c-Ha-ras oncogene mediated cellular transformation and colony formation in soft agar. Analysis of stable RB and genomic c-Ha-ras cell transfectants for expression of pRB and p21ras by immunoblotting indicates a strong correlation with the presence of high levels of RB protein and inhibition of ras-transformation. Moreover, during culturing the RB and genomic c-Ha-ras expressing clones a progressive transformation of phenotypically normal clones was observed which paralleled loss or decrease of RB expression and concomitant increase in p21ras production. These findings suggest a functional cross-talk between RB protein and p21ras, which balances the cell phenotype between normal and transformed states.
Assuntos
Transformação Celular Neoplásica/genética , Genes ras/fisiologia , Proteína do Retinoblastoma/fisiologia , Células 3T3 , Animais , Comunicação Celular , Transformação Celular Neoplásica/patologia , Ensaio de Unidades Formadoras de Colônias , Humanos , Higromicina B , Camundongos , Mutação , Fenótipo , Proteínas Proto-Oncogênicas p21(ras)/análise , Proteínas Proto-Oncogênicas p21(ras)/efeitos dos fármacos , Proteína do Retinoblastoma/análise , Proteína do Retinoblastoma/genética , TransfecçãoRESUMO
Control of fate of cells encountered with DNA damaging agents is pivotal for normal cellular homeostasis. DNA damage leads in many cases to growth arrest of the cells ensuring sufficient time for damage repair. Growth arrest can be mediated by p53 tumor suppressor protein and loss of its function leads to inability of the cells to both growth arrest and undergo apoptosis. We show here that followed by genotoxic stress, the retinoblastoma gene product, pRB, is associated with growth arrest of cells in a p53 independent manner. In u.v.-treated human and mouse fibroblasts, pRB is rapidly dephosphorylated. pRB dephosphorylation occurs concomitant with growth arrest of cells including cells with p53 mutations (SW 480 colon carcinoma cells), cells expressing SV40 T antigen and rat-transformed cells (T-24 bladder carcinoma cells) unresponsive in regard to p53 stimulation. Furthermore, flow cytometry analysis of u.v.-radiated synchronized G1 cells indicates that the cells transiently arrest in G1 for 10-12 h with pRB dominating in its underphosphorylated form, whereas p53 accumulation occurs only after the cells have entered into S-phase. In addition, u.v.-radiation of late S- and G2/M-phase cells leads to p53 accumulation and cell cycle arrest. The results indicate that p53 accumulation upon u.v.-radiation occurs during DNA replication and is thus not involved in G1 arrest. We suggest that the events that lead to pRB dephosphorylation upon u.v.-radiation provide the cell an efficient G1 arrest which occurs prior and independently of p53.
Assuntos
Ciclo Celular/fisiologia , Proteína do Retinoblastoma/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta , Células 3T3 , Animais , Linhagem Celular , Replicação do DNA/efeitos da radiação , Humanos , Camundongos , Fosforilação , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/efeitos da radiação , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/efeitos da radiaçãoRESUMO
p21Cip1/Waf1 cyclin-dependent kinase inhibitor (p21) is inducible by Raf and mitogen-activated protein kinase kinase (MAPKK), but the level of regulation is unknown. We show here by conditional and transient Ras-expression models that Ras induces p21. Induction of p21 in conditionally Ras-expressing cells is posttranscriptional utilizing mitogen-activated protein kinase (MAPK) pathway. Transient, high-level Ras-expression induces transcriptional activation of p21 mediated by a GC-rich region in p21 promoter -83-54 bp relative to the transcription initiation site containing binding sites for Sp1-family transcription factors. Mutation of either Sp1-binding site 2 or 4 in this region decreases the magnitude of induction of promoter activity by Ras, but only the simultaneous mutation of both sites abolishes fully the induction. Electrophoretic mobility shift assays using an oligonucleotide corresponding to Sp1-binding site 2 indicate that both Sp1 and Sp3 transcription factors bind to this region. The results demonstrate that the central cytosolic growth regulator Ras is a potent transcriptional and posttranscriptional inducer of the nuclear growth inhibitor p21.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Ciclinas/biossíntese , DNA/genética , Regulação da Expressão Gênica/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica , Células 3T3 , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/genética , DNA/metabolismo , Flavonoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Mutagênese Sítio-Dirigida , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , Sequências Reguladoras de Ácido Nucleico , Deleção de SequênciaRESUMO
The vascular endothelial growth factor (VEGF) family has recently been expanded by the isolation of two additional growth factors, VEGF-B and VEGF-C. Here we compare the regulation of steady-state levels of VEGF, VEGF-B and VEGF-C mRNAs in cultured cells by a variety of stimuli implicated in angiogenesis and endothelial cell physiology. Hypoxia, Ras oncoprotein and mutant p53 tumor suppressor, which are potent inducers of VEGF mRNA did not increase VEGF-B or VEGF-C mRNA levels. Serum and its component growth factors, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) as well as transforming growth factor-beta (TGF-beta) and the tumor promoter phorbol myristate 12,13-acetate (PMA) stimulated VEGF-C, but not VEGF-B mRNA expression. Interestingly, these growth factors and hypoxia simultaneously downregulated the mRNA of another endothelial cell specific ligand, angiopoietin-1. Serum induction of VEGF-C mRNA occurred independently of protein synthesis; with an increase of the mRNA half-life from 3.5 h to 5.5-6 h, whereas VEGF-B mRNA was very stable (T 1/2>8 h). Our results reveal that the three VEGF genes are regulated in a strikingly different manner, suggesting that they serve distinct, although perhaps overlapping functions in vivo.
Assuntos
Hipóxia Celular , Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , RNA Mensageiro/metabolismo , Células 3T3 , Animais , Humanos , Camundongos , Fator A de Crescimento do Endotélio Vascular , Fator B de Crescimento do Endotélio Vascular , Fator C de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
p21(Waf1/Cip1) (hereafter referred to as p21) is up-regulated in differentiating and DNA-damaged cells, but it is also up-regulated by serum and growth factors. We show here that fibroblast growth factor-2 (FGF-2), platelet-derived growth factor (PDGF), and transforming growth factor-beta1 (TGF-beta1) all induce p21 expression in mouse fibroblasts, but with markedly different kinetics. We link their effect on p21 to Ras and mitogen-activated protein kinase kinase-1(/2) [MEK1(/2)]-regulated pathways using either a specific MEK1(/2) inhibitor (PD 098059) or cells expressing conditionally activated Ras or dominant negative Ras. We demonstrate that p21 induction by PDGF and TGF-beta1 requires MEK1(/2) and, additionally, that the TGF-beta1 effect on p21 depends on Ras, whereas the PDGF effect does not. In contrast, FGF-2 regulation of p21 is largely independent of MEK and Ras. However, PD 098059 efficiently inhibited S-phase entry of quiescent cells induced by either FGF-2 or PDGF, suggesting separate signaling pathways for FGF-2 in induction of p21 and in S-phase entry. The results suggest different but partly overlapping signaling pathways in growth factor regulation of p21.
Assuntos
Ciclinas/genética , Substâncias de Crescimento/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas ras/metabolismo , Células 3T3 , Animais , Inibidor de Quinase Dependente de Ciclina p21 , Inibidores Enzimáticos/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Flavonoides/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Substâncias de Crescimento/metabolismo , Cinética , MAP Quinase Quinase 1 , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , RNA Mensageiro/análise , Fase S/fisiologia , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Expression of the human retinoblastoma gene (RB1) in tumor cells defective of the gene in many instances abrogates the growth of the cells. Here we have evaluated the characteristics and tumor-suppressive functions of the human retinoblastoma gene product in mouse fibroblasts. Human full-length wild-type or mutant RB cDNAs were transfected into NIH 3T3 cells and cell clones expressing high levels of RB protein were isolated and characterized. Stable expression of RB protein was obtained, and cell growth experiments indicated that the human RB expressing clones maintained unaltered growth rates under normal culture conditions. The growth rates of wild-type RB-expressing clones but not those expressing mutant RB were reduced in lower serum concentrations. This indicates that serum withdrawal may bring out some growth suppressive properties of RB. Analysis of the human RB protein produced by mouse fibroblasts by cell synchronization and immunoblotting indicated that pRB was phosphorylated and dephosphorylated in a cell cycle-dependent manner. This suggests a functional role for the human pRB also in mouse cells. Moreover, cells expressing wild-type pRB were less susceptible to the transforming effects of SV40 large T antigen than cells expressing mutant pRB as shown by cell transfection studies. The results indicate that human pRB produced by mouse fibroblasts is functionally active and show that pRB can suppress the transforming activity of T antigen.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteína do Retinoblastoma/fisiologia , Células 3T3/efeitos dos fármacos , Animais , Antígenos Virais de Tumores , Proteínas Sanguíneas/farmacologia , Ciclo Celular , Análise Mutacional de DNA , Genes Supressores , Humanos , Camundongos , Fosforilação , TransfecçãoRESUMO
Stable coexpression of the human retinoblastoma protein (pRB) cDNA and EJ c-Ha-ras oncogene in murine fibroblasts leads to loss of pRB expression with concomitant transformation of the cells (1). We show here that conditional expression of p21ras in mouse fibroblasts expressing human pRB leads to a rapid decrease of pRB expression at both protein and mRNA levels. The decrease of pRB mRNA is blocked by cycloheximide, suggesting the requirement of ongoing protein synthesis. p21ras expression leads also to decreases of c-myc and tissue metalloproteinase inhibitor-2 mRNAs, whereas cyclin-dependent kinase 4, cyclin D1, E2F-1, and ornithine decarboxylase are unaffected. The decrease in pRB is accompanied by progressive morphological transformation of the cells. The effect of p21ras on pRB expression was serum and growth factor dependent. A shift of the cells to low serum (0.2% FCS) abolished the effects of p21ras on pRB, but this effect was reconstituted by the addition of growth factors epidermal growth factor, fibroblast growth factor-2, transforming growth factor beta 1, and platelet-derived growth factor to the cells. The results suggest a complex interaction between p21ras, pRB, and growth factors in the control of cell growth. p21ras appears to drive the cell cycle by deregulation of key cell cycle regulators, the functions of which in low serum become redundant or require the presence of growth factors positively driving the cell cycle.
Assuntos
Substâncias de Crescimento/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína do Retinoblastoma/genética , Células 3T3/efeitos dos fármacos , Células 3T3/metabolismo , Animais , Antineoplásicos/metabolismo , Proteínas Sanguíneas/farmacologia , Ciclo Celular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Immunoblotting , Camundongos , Mutagênese/fisiologia , Proteínas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RNA Mensageiro/metabolismo , Proteína do Retinoblastoma/metabolismo , Inibidor Tecidual de Metaloproteinase-2 , Transformação GenéticaRESUMO
p53 target genes p21(Cip1/Waf1) cyclin-kinase inhibitor (p21 CKI), GADD45, bax, and cyclin G and genes affecting the redox state of the cells are implicated in p53 damage control responses. In order to attribute their functions and dependency of p53 in UV-damaged cells we undertook an analysis of UVC responses of fibroblasts derived from p53 knock-out mice. UVC radiation efficiently and rapidly inhibited DNA replication in both p53 -/- and +/+ cells. The arrest was persistent in p53 -/- fibroblasts and cells underwent apoptosis, whereas p53 +/+ cells recovered and reentered the cycle. Protein and mRNA analyses of p21 expression showed that it was induced up to sixfold with similar kinetics both in the presence and in the absence of p53. However, high doses of UV abrogated the p21 response in p53 -/- cells, whereas it was maintained in cells with normal p53. UVC radiation transcriptionally activated p21 expression as demonstrated by luciferase reporter assays using deletion constructs of the p21 promoter. The promoter assays further confirmed the independency of p53-binding sites in the activation and linked UV-responsive transcriptional regulation of p21 to two Sp1 consensus binding sites within -61 bp of the transcription initiation site. A weaker regulation was mediated by elements between -1300 to -500 bp relative to the transcription initiation site. The results suggest that in fibroblasts UVC radiation is a rapid and efficient inducer of p21 expression also in a p53-independent manner.