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1.
J Hum Nutr Diet ; 28(5): 486-92, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24976290

RESUMO

BACKGROUND: Body image distortion/discrepancy leads to psychological stress, disordered eating and mental and physical disease. To begin to assess body image distortion/discrepancy, we compared perceived, desired and measured percentage body fat in male versus female and college-aged versus non-college aged individuals. In addition, we assessed the acute stress response to body composition measurement. METHODS: Body fat percentage of 15 college aged ('College Students'; CS) (mean = 19 years) and 16 non-college aged ('Non-College Aged Students'; NCS) (mean = 39 years) males and females was assessed with the BodPod Body Composition Tracking System (Life Measurement Instruments, Concord, CA, USA). Participants indicated their perception of body fat and their desired body fat using a somatomorphic matrix. Salivary cortisol, heart rate and blood pressure were also measured. Data were analysed by analysis of variance and alpha was set at 0.05. RESULTS: Mean (SD) percentage body fat of males [15.2% (6.1%)] was significantly lower than that of females [28.4% (6.4%)] (P < 0.0001). Both CS and NCS females perceived their body fat to be lower (5%) than measured body fat and desired their body fat to be lower (12%) than measured (P < 0.05). CS and NCS male participants demonstrated the opposite result; both CS and NCS male populations perceived their body fat to be higher (5%) than measured body fat and desired their body fat to be higher (4%) than measured (P < 0.05). No differences between any groups were observed in heart rate, blood pressure or cortisol response to body fat measurement. CONCLUSIONS: Sex-related but not age-related differences in perceived, desired and measured percentage body fat were observed.


Assuntos
Tecido Adiposo , Composição Corporal , Imagem Corporal , Fatores Sexuais , Adulto , Fatores Etários , Emoções , Feminino , Humanos , Masculino , Percepção , Estresse Psicológico , Adulto Jovem
2.
J Intern Med ; 273(5): 429-36, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23600398

RESUMO

Exposure to environmental toxins is associated with a variety of age-related diseases including cancer and neurodegeneration. For example, in Parkinson's disease (PD), chronic environmental exposure to certain toxins has been linked to the age-related development of neuropathology. Neuronal damage is believed to involve the induction of neuroinflammatory events as a consequence of glial cell activation. Cellular senescence is a potent anti-cancer mechanism that occurs in a number of proliferative cell types and causes the arrest of proliferation of cells at risk of malignant transformation following exposure to potentially oncogenic stimuli. With age, senescent cells accumulate and express a senescence-associated secretory phenotype (SASP; that is the robust secretion of many inflammatory cytokines, growth factors and proteases). Whereas cell senescence in peripheral tissues has been causally linked to a number of age-related pathologies, little is known about the induction of cellular senescence and the SASP in the brain. On the basis of recently reported findings, we propose that environmental stressors associated with PD may act in part by eliciting senescence and the SASP within non neuronal glial cells in the ageing brain, thus contributing to the characteristic decline in neuronal integrity that occurs in this disorder.


Assuntos
Envelhecimento , Senescência Celular , Exposição Ambiental/efeitos adversos , Poluentes Ambientais/efeitos adversos , Neuroglia , Doença de Parkinson/etiologia , Doença de Parkinson/patologia , Humanos , Mediadores da Inflamação/metabolismo , Neuroglia/patologia , Doença de Parkinson/metabolismo , Fenótipo , Transdução de Sinais , Fatores de Tempo
3.
Nat Genet ; 23(4): 405-12, 1999 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-10581025

RESUMO

Telomeres are DNA-protein structures that cap linear chromosomes and are essential for maintaining genomic stability and cell phenotype. We identified a novel human telomere-associated protein, TIN2, by interaction cloning using the telomeric DNA-binding-protein TRF1 as a bait. TIN2 interacted with TRF1 in vitro and in cells, and co-localized with TRF1 in nuclei and metaphase chromosomes. A mutant TIN2 that lacks amino-terminal sequences effects elongated human telomeres in a telomerase-dependent manner. Our findings suggest that TRF1 is insufficient for control of telomere length in human cells, and that TIN2 is an essential mediator of TRF1 function.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a Telômeros , Telômero/genética , Telômero/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , Proteínas de Ligação a DNA/química , Expressão Gênica , Humanos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Proteína 1 de Ligação a Repetições Teloméricas , Distribuição Tecidual
4.
Curr Opin Cell Biol ; 3(2): 230-4, 1991 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-1883615

RESUMO

Differentiated eukaryotic cells have only a finite capacity for cell division. This limitation is thought to be a cellular manifestation of organismal aging, and a restraint to tumor progression. The molecular basis for cellular senescence is not known, but a molecular framework for understanding this phenomenon has recently been established.


Assuntos
Sobrevivência Celular/genética , Animais , Divisão Celular , Humanos
5.
Nat Protoc ; 16(5): 2471-2498, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33911261

RESUMO

The development of genetic tools allowed for the validation of the pro-aging and pro-disease functions of senescent cells in vivo. These discoveries prompted the development of senotherapies-pharmaceutical interventions aimed at interfering with the detrimental effect of senescent cells-that are now entering the clinical stage. However, unequivocal identification and examination of cellular senescence remains highly difficult because of the lack of universal and specific markers. Here, to overcome the limitation of measuring individual markers, we describe a detailed two-phase algorithmic assessment to quantify various senescence-associated parameters in the same specimen. In the first phase, we combine the measurement of lysosomal and proliferative features with the expression of general senescence-associated genes to validate the presence of senescent cells. In the second phase we measure the levels of pro-inflammatory markers for specification of the type of senescence. The protocol can help graduate-level basic scientists to improve the characterization of senescence-associated phenotypes and the identification of specific senescent subtypes. Moreover, it can serve as an important tool for the clinical validation of the role of senescent cells and the effectiveness of anti-senescence therapies.


Assuntos
Algoritmos , Senescência Celular , Técnicas Citológicas/métodos , Biomarcadores/metabolismo , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo
6.
Mater Sci Eng C Mater Biol Appl ; 106: 110261, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31753330

RESUMO

Limitations in effectiveness and the invasive nature of current cancer treatment options emphasize the need for further clinical advancements. Among other approaches, targeted hyperthermia is as a new strategy aimed at targeting cancerous cells to improve the efficacy of radiotherapy or cytotoxic drugs. However, the testing of magnetic vehicles has mainly focused on the use of nanoparticles. In this work, Fe77B10Si10C3 glass-coated amorphous magnetic microwires were assessed for the first time as magnetic vehicles with high potential for the localized heating of osteosarcoma cells by means of an AC magnetic field. The results from the in vitro assays performed inside a microfluidic device demonstrated the ability of these magnetic microwires to induce malignant cell death. Exposing the system to different magnetic fields for less than 1 h provoked a reduction up to 89% of the osteosarcoma cell population, whereas healthy myoblastoma cells remained nearly unaffected. The proposed technology demonstrates in vitro the effectiveness of these microwires as vehicles for targeted magnetic hyperthermia.


Assuntos
Compostos Férricos/química , Vidro/química , Hipertermia Induzida/métodos , Campos Magnéticos , Animais , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Camundongos , Osteossarcoma/metabolismo , Osteossarcoma/patologia
7.
Trends Cell Biol ; 11(11): S27-31, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11684439

RESUMO

Organisms with renewable tissues had to evolve mechanisms to prevent the development of cancer. One such mechanism is cellular senescence, which irreversibly arrests the growth of cells at risk for neoplastic transformation. Recent findings have revealed the complexities of the senescence phenotype and unexpected possible consequences for the organism.


Assuntos
Transformação Celular Neoplásica , Senescência Celular/fisiologia , Genes Supressores de Tumor/fisiologia , Neoplasias/etiologia , Animais , Divisão Celular , Senescência Celular/genética , Genes p53/genética , Humanos , Neoplasias/fisiopatologia , Fenótipo , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Telômero/metabolismo
8.
J Cell Biol ; 153(2): 367-80, 2001 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-11309417

RESUMO

Bloom syndrome (BS) is an autosomal recessive disorder characterized by a high incidence of cancer and genomic instability. BLM, the protein defective in BS, is a RecQ-like helicase, presumed to function in DNA replication, recombination, or repair. BLM localizes to promyelocytic leukemia protein (PML) nuclear bodies and is expressed during late S and G2. We show, in normal human cells, that the recombination/repair proteins hRAD51 and replication protein (RP)-A assembled with BLM into a fraction of PML bodies during late S/G2. Biochemical experiments suggested that BLM resides in a nuclear matrix-bound complex in which association with hRAD51 may be direct. DNA-damaging agents that cause double strand breaks and a G2 delay induced BLM by a p53- and ataxia-telangiectasia mutated independent mechanism. This induction depended on the G2 delay, because it failed to occur when G2 was prevented or bypassed. It coincided with the appearance of foci containing BLM, PML, hRAD51 and RP-A, which resembled ionizing radiation-induced foci. After radiation, foci containing BLM and PML formed at sites of single-stranded DNA and presumptive repair in normal cells, but not in cells with defective PML. Our findings suggest that BLM is part of a dynamic nuclear matrix-based complex that requires PML and functions during G2 in undamaged cells and recombinational repair after DNA damage.


Assuntos
Adenosina Trifosfatases/metabolismo , Ciclo Celular/fisiologia , Núcleo Celular/metabolismo , Dano ao DNA , DNA Helicases/metabolismo , Proteínas Nucleares/metabolismo , Adenosina Trifosfatases/genética , Síndrome de Bloom/genética , Western Blotting , Fracionamento Celular , Células Cultivadas , DNA Helicases/genética , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Citometria de Fluxo , Humanos , Microscopia de Fluorescência , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteína da Leucemia Promielocítica , Proteínas/metabolismo , Rad51 Recombinase , RecQ Helicases , Proteína de Replicação A , Fatores de Transcrição/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas Supressoras de Tumor , Raios X
9.
Science ; 289(5487): 2062-3, 2000 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-11032557

RESUMO

Model organisms such as yeast have proved exceptionally valuable for revealing new information about the molecular pathways involved in the aging of cells. In her Perspective, Campisi comments on new work showing that caloric restriction increases longevity in yeast by activating the SIR2 gene, which alters the compactness of chromatin and thus regulates gene expression (Lin et al.).


Assuntos
Envelhecimento/fisiologia , Cromatina/fisiologia , Ingestão de Energia , Inativação Gênica , Histona Desacetilases/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae , Transativadores/metabolismo , Animais , Divisão Celular , Reparo do DNA , Replicação do DNA , DNA Circular/metabolismo , DNA Fúngico/metabolismo , DNA Ribossômico/metabolismo , Glucose/metabolismo , Histona Desacetilases/genética , Histonas/metabolismo , Longevidade , Mutação , NAD/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Recombinação Genética , Saccharomyces cerevisiae/genética , Sirtuína 2 , Sirtuínas , Transativadores/genética
10.
Science ; 247(4939): 205-9, 1990 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-2104680

RESUMO

Normal cells in culture invariably undergo senescence, whereby they cease proliferation after a finite number of doublings. Irreversible changes in gene expression occurred in senescent human fetal lung fibroblasts: a non-cell cycle-regulated mRNA was partially repressed; an unusual polyadenylated histone mRNA was expressed; although serum induced c-H-ras, c-myc, and ornithine decarboxylase mRNA normally, ornithine decarboxylase activity was deficient; and serum did not induce mRNA for a replication-dependent histone and for the c-fos proto-oncogene. The loss of c-fos inducibility was the result of a specific, transcriptional block. The results suggest that senescent fibroblasts were unable to proliferate because of, at least in part, selective repression of c-fos; moreover, the multiple changes in gene expression support the view that cellular senescence is a process of terminal differentiation.


Assuntos
Fibroblastos/citologia , Expressão Gênica/fisiologia , Proteínas Proto-Oncogênicas/genética , Supressão Genética , Transcrição Gênica , Sangue , Divisão Celular , Linhagem Celular , Sobrevivência Celular/fisiologia , Embrião de Mamíferos , Fibroblastos/metabolismo , Humanos , Ornitina Descarboxilase/genética , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-fos , Proteínas Proto-Oncogênicas c-myc , Proteínas Proto-Oncogênicas p21(ras) , RNA Mensageiro/biossíntese , RNA Mensageiro/genética
11.
Science ; 199(4335): 1336-7, 1978 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-564549

RESUMO

By use of a spin label fatty acid, 5-doxylstearate, an increase in bulk membrane fluidity was observed after fertilization of two species of sea urchin eggs. Eggs partially activated by ammonia showed a similar effect. The data suggest that a structural change involving membrane lipids accompanies activation.


Assuntos
Fertilização , Lipídeos de Membrana/fisiologia , Óvulo/fisiologia , Zigoto/fisiologia , Amônia/farmacologia , Animais , Feminino , Membranas/fisiologia , Óvulo/efeitos dos fármacos , Ouriços-do-Mar , Marcadores de Spin , Viscosidade , Zigoto/ultraestrutura
13.
Curr Biol ; 11(21): 1706-10, 2001 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-11696330

RESUMO

An important quest in modern biology is to identify genes involved in aging. Model organisms such as the nematode Caenorhabditis elegans are particularly useful in this regard. The C. elegans genome has been sequenced [1], and single gene mutations that extend adult life span have been identified [2]. Among these longevity-controlling loci are four apparently unrelated genes that belong to the clk family. In mammals, telomere length and structure can influence cellular, and possibly organismal, aging. Here, we show that clk-2 encodes a regulator of telomere length in C. elegans.


Assuntos
Envelhecimento/genética , Proteínas de Caenorhabditis elegans/genética , Genes de Helmintos , Proteínas de Saccharomyces cerevisiae , Proteínas de Ligação a Telômeros , Telômero/genética , Sequência de Aminoácidos , Animais , Proteínas de Ligação a DNA/genética , Dados de Sequência Molecular , Mutação , RNA Antissenso , RNA Interferente Pequeno , Tolerância a Radiação , Homologia de Sequência de Aminoácidos , Raios X
14.
Mol Cell Biol ; 6(2): 518-24, 1986 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-3785153

RESUMO

We have shown that c-myc mRNA levels decrease more than 20-fold when F9 teratocarcinoma stem cells are induced to arrest growth and terminally differentiate to parietal endoderm after exposure to retinoic acid and cyclic AMP (Campisi et al., Cell 36:241-247, 1984). Here, we demonstrate that although growth arrest and full expression of the differentiated phenotype required about 3 days, c-myc mRNA declined abruptly between 8 and 16 h after the addition of retinoic acid and cyclic AMP. The decline was independent of cyclic AMP. We found little or no change in the level of c-myc transcription during differentiation, although two other genes showed marked transcriptional regulation. Thus, decreased c-myc mRNA is a consequence of very early posttranscriptional regulation directed by retinoic acid. Differentiation was not fundamental to this regulation. We have shown that sodium butyrate blocks expression of the differentiated phenotype if added within 8 h of retinoic acid and cyclic AMP (Levine et al., Dev. Biol. 105:443-450, 1984). However, butyrate did not inhibit the decrease in c-myc mRNA. Furthermore, F9 cells partially arrested growth without differentiating when grown in isoleucine-deficient medium. Under these conditions, c-myc mRNA levels also declined. Our results suggest that induction of differentiation-specific genes may be under retinoic acid-mediated control dissimilar from that responsible for the decay of c-myc mRNA. In addition, they raise the possibility that growth arrest may be initiated by reduced c-myc expression.


Assuntos
Genes Reguladores , Oncogenes , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cinética , Camundongos , RNA Mensageiro/genética , Teratoma/patologia
15.
Mol Cell Biol ; 4(9): 1807-14, 1984 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-6387447

RESUMO

The control of eucaryotic cell proliferation is governed largely by a series of regulatory events which occur in the G1 phase of the cell cycle. When stimulated to proliferate, quiescent (G0) 3T3 fibroblasts require transcription, rapid translation, and three growth factors for the growth state transition. We examined exponentially growing 3T3 cells to relate the requirements for G1 transit to those necessary for the transition from the G0 to the S phase. Cycling cells in the G1 phase required transcription, rapid translation, and a single growth factor (insulin-like growth factor [IGF] I) to initiate DNA synthesis. IGF I acted post-transcriptionally at a late G1 step. All cells in the G1 phase entered the S phase on schedule if either insulin (hyperphysiological concentration) or IGF I (subnanomolar concentration) was provided as the sole growth factor. In medium lacking all growth factors, only cells within 2 to 3 h of the S phase were able to initiate DNA synthesis. Similarly, cells within 2 to 3 h of the S phase were less dependent on transcription and translation for entry into the S phase. Cells responded very differently to inhibited translation than to growth factor deprivation. Cells in the early and mid-G1 phases did not progress toward the S phase during transcriptional or translational inhibition, and during translational inhibition they actually regressed from the S phase. In the absence of growth factors, however, these cells continued progressing toward the S phase, but still required IGF at a terminal step before initiating DNA synthesis. We conclude that a suboptimal condition causes cells to either progress or regress in the cell cycle rather than freezing them at their initial position. By using synchronized cultures, we also show that in contrast to earlier events, this final, IGF-dependent step did not require new transcription. This result is in contrast to findings that other growth factors induce new transcription. We examined the requirements for G1 transit by using a chemically transformed 3T3 cell line (BPA31 cells) which has lost some but not all ability to regulate its growth. Early- and mid-G1-phase BPA31 cells required transcription and translation to initiate DNA synthesis, although they did not regress from the S phase during translational inhibition. However, these cells did not need IGF for entry into the S phase.


Assuntos
Replicação do DNA/efeitos dos fármacos , Insulina/farmacologia , Peptídeos/farmacologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Somatomedinas/farmacologia , Animais , Benzo(a)pireno/toxicidade , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Células Clonais , Fibroblastos/citologia , Citometria de Fluxo , Cinética , Camundongos
16.
Mol Cell Biol ; 9(8): 3411-7, 1989 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-2677672

RESUMO

Although much is known about the structure of ras-encoded proteins, little is known about how expression is regulated. In serum-stimulated murine fibroblasts, c-ras-Ha mRNA levels fluctuated with the growth state but not with the position in the cell cycle. Two types of growth factors regulated c-ras-Ha expression: insulin (IN) or insulinlike growth factor I, each apparently acting through its cognate receptor, and epidermal growth factor (EGF). In quiescent cells, IN or insulinlike growth factor I induced c-ras-Ha mRNA three- to fivefold within 4 h, but thereafter the mRNA declined. By contrast, EGF had little effect in 4 h but induced the mRNA after 4 to 6 h. When quiescent cells were given serum or IN and EGF simultaneously, c-ras-Ha mRNA rose steadily, beginning 1 to 2 h after stimulation, and reached a stable five- to sevenfold elevation in 16 h. Thus, c-ras-Ha gene expression was sequentially regulated by two growth factors, one of which (IN) does not induce expression of other growth-regulated protooncogenes. A transformed derivative cell line that does not require IN for G1 progression has lost early IN-dependent but not late serum-dependent regulation. The results support the possibility that c-ras-Ha and IN action are functionally linked.


Assuntos
Fator de Crescimento Epidérmico/fisiologia , Regulação da Expressão Gênica , Fator de Crescimento Insulin-Like I/fisiologia , Insulina/fisiologia , Proteínas Proto-Oncogênicas/genética , Somatomedinas/fisiologia , Animais , Northern Blotting , Ciclo Celular , Linhagem Celular Transformada , Células Cultivadas , Fibroblastos/metabolismo , Camundongos , Proteínas Proto-Oncogênicas p21(ras) , RNA Mensageiro/análise , RNA Mensageiro/biossíntese
17.
Mol Cell Biol ; 20(1): 273-85, 2000 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10594030

RESUMO

Normal cells do not divide indefinitely due to a process known as replicative senescence. Human cells arrest growth with a senescent phenotype when they acquire one or more critically short telomeres as a consequence of cell division. Recent evidence suggests that certain types of DNA damage, chromatin remodeling, and oncogenic forms of Ras or Raf can also elicit a senescence response. We show here that E2F1, a multifunctional transcription factor that binds the retinoblastoma (pRb) tumor suppressor and that can either promote or suppress tumorigenesis, induces a senescent phenotype when overexpressed in normal human fibroblasts. Normal human cells stably arrested proliferation and expressed several markers of replicative senescence in response to E2F1. This activity of E2F1 was independent of its pRb binding activity but dependent on its ability to stimulate gene expression. The E2F1 target gene critical for the senescence response appeared to be the p14(ARF) tumor suppressor. Replicatively senescent human fibroblasts overexpressed p14(ARF), and ectopic expression of p14(ARF) in presenescent cells induced a phenotype similar to that induced by E2F1. Consistent with a critical role for p14(ARF), cells with compromised p53 function were immune to senescence induction by E2F1, as were cells deficient in p14(ARF). Our findings support the idea that the senescence response is a critical tumor-suppressive mechanism, provide an explanation for the apparently paradoxical roles of E2F1 in oncogenesis, and identify p14(ARF) as a potentially important mediator of the senescent phenotype.


Assuntos
Proteínas de Transporte , Senescência Celular/genética , Proteínas de Ligação a DNA , Proteínas/genética , Fatores de Transcrição/genética , Proteínas de Ciclo Celular/genética , Diferenciação Celular/genética , Divisão Celular/genética , Linhagem Celular , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Regulação da Expressão Gênica , Genes Supressores de Tumor , Humanos , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Proteína Supressora de Tumor p14ARF
18.
Mol Cell Biol ; 16(6): 2987-97, 1996 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8649410

RESUMO

p21Sdi1/WAF1/Cip1 inhibits cyclin-dependent protein kinases and cell proliferation. p21 is presumed to inhibit growth by preventing the phosphorylation of growth-regulatory proteins, including the retinoblastoma tumor suppressor protein (pRb). The ultimate effector(s) of p21 growth inhibition, however, is largely a matter of conjecture. We show that p21 inhibits the activity of E2F, an essential growth-stimulatory transcription factor that is negatively regulated by unphosphorylated pRb. p21 suppressed the activity of E2F-responsive promoters (dihydrofolate reductase and cdc2), but E2F-unresponsive promoters (c-fos and simian virus 40 early) were unaffected. Moreover, the simian virus 40 early promoter was rendered p21 suppressible by introducing wild-type, but not mutant, E2F binding sites; p21 deletion mutants showed good agreement in their abilities to inhibit E2F transactivation and DNA synthesis; and E2F-1 (which binds pRb), but not E2F-4 (which does not), reversed both inhibitory effects of p21. Despite the central role for pRb in regulating E2F, p21 suppressed growth and E2F activity in cells lacking a functional pRb. Moreover, p21 protein (wild type but not mutant) specifically disrupted an E2F-cyclin-dependent protein kinase 2-p107 DNA binding complex in nuclear extracts of proliferating cells, whether or not they expressed normal pRb. Thus, E2F is a critical target and ultimate effector of p21 action, and pRb is not essential for the inhibition of growth or E2F-dependent transcription.


Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Quinases Ciclina-Dependentes/antagonistas & inibidores , Ciclinas/farmacologia , Proteínas de Ligação a DNA , Inibidores Enzimáticos/farmacologia , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Células 3T3 , Animais , Sequência de Bases , Sítios de Ligação/genética , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21 , DNA/biossíntese , DNA/genética , DNA/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fator de Transcrição E2F4 , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Proteína do Retinoblastoma/genética , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Transcrição Gênica
19.
Mol Cell Biol ; 15(6): 3398-404, 1995 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7760836

RESUMO

Cell proliferation and differentiation are precisely coordinated during the development and maturation of the mammary gland, and this balance invariably is disrupted during carcinogenesis. Little is known about the cell-specific transcription factors that regulate these processes in the mammary gland. The mouse mammary epithelial cell line SCp2 grows well under standard culture conditions but arrests growth, forms alveolus-like structures, and expresses beta-casein, a differentiation marker, 4 to 5 days after exposure to basement membrane and lactogenic hormones (differentiation signals). We show that this differentiation entails a marked decline in the expression of Id-1, a helix-loop-helix (HLH) protein that inactivates basic HLH transcription factors in other cell types. SCp2 cells stably transfected with an Id-1 expression vector grew more rapidly than control cells under standard conditions, but in response to differentiation signals, they arrested growth and formed three-dimensional structures similar to those of control cells. Id-1-expressing cells did not, however, express beta-casein. Moreover, 8 to 10 days after receiving differentiation signals, they lost three-dimensional organization, invaded the basement membrane, and then resumed growth. SCp2 cells expressing an Id-1 antisense vector grew more slowly than controls; in response to differentiation signals, they remained stably growth arrested and fully differentiated, as did control cells. We suggest that Id-1 renders cells refractory to differentiation signals and receptive to growth signals by inactivating one or more basic HLH proteins that coordinate growth and differentiation in the mammary epithelium.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/farmacologia , Sequências Hélice-Alça-Hélice , Glândulas Mamárias Animais/efeitos dos fármacos , Proteínas Repressoras , Fatores de Transcrição , Animais , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais , Epitélio/efeitos dos fármacos , Proteína 1 Inibidora de Diferenciação , Camundongos , Transdução de Sinais
20.
Mol Cell Biol ; 18(8): 4577-88, 1998 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9671467

RESUMO

Mammary epithelial cells undergo changes in growth, invasion, and differentiation throughout much of adulthood, and most strikingly during pregnancy, lactation, and involution. Although the pathways of milk protein expression are being elucidated, little is known, at a molecular level, about control of mammary epithelial cell phenotypes during normal tissue morphogenesis and evolution of aggressive breast cancer. We developed a murine mammary epithelial cell line, SCp2, that arrests growth and functionally differentiates in response to a basement membrane and lactogenic hormones. In these cells, expression of Id-1, an inhibitor of basic helix-loop-helix transcription factors, declines prior to differentiation, and constitutive Id-1 expression blocks differentiation. Here, we show that SCp2 cells that constitutively express Id-1 slowly invade the basement membrane but remain anchorage dependent for growth and do not form tumors in nude mice. Cells expressing Id-1 secreted a approximately 120-kDa gelatinase. From inhibitor studies, this gelatinase appeared to be a metalloproteinase, and it was the only metalloproteinase detectable in conditioned medium from these cells. A nontoxic inhibitor diminished the activity of this metalloproteinase in vitro and repressed the invasive phenotype of Id-1-expressing cells in culture. The implications of these findings for normal mammary-gland development and human breast cancer were investigated. A gelatinase of approximately 120 kDa was expressed by the mammary gland during involution, a time when Id-1 expression is high and there is extensive tissue remodeling. Moreover, high levels of Id-1 expression and the activity of a approximately 120-kDa gelatinase correlated with a less-differentiated and more-aggressive phenotype in human breast cancer cells. We suggest that Id-1 controls invasion by normal and neoplastic mammary epithelial cells, primarily through induction of a approximately 120-kDa gelatinase. This Id-1-regulated invasive phenotype could contribute to involution of the mammary gland and possibly to the development of invasive breast cancer.


Assuntos
Células Epiteliais/fisiologia , Sequências Hélice-Alça-Hélice , Proteínas Repressoras , Fatores de Transcrição/fisiologia , Animais , Testes de Carcinogenicidade , Divisão Celular , Linhagem Celular , Movimento Celular , Células Epiteliais/metabolismo , Gelatinases/metabolismo , Humanos , Proteína 1 Inibidora de Diferenciação , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/fisiologia , Metaloendopeptidases/antagonistas & inibidores , Camundongos , Camundongos Nus , Invasividade Neoplásica , Fenótipo , Fatores de Transcrição/genética , Células Tumorais Cultivadas
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