RESUMO
The polycomb group protein BMI1 has been shown to support normal stem cell proliferation via its putative stem cell factor function, but it is not known if BMI1 may also act as a cancer stem cell factor to promote cancer development. To determine the role of human BMI1 in cancer growth and survival, we performed a loss-of-function analysis of BMI1 by RNA interference (RNAi) in both normal and malignant human cells. Our results indicate that BMI1 is crucial for the short-term survival of cancer cells but not of normal cells. We also demonstrated that loss of BMI1 was more effective in suppressing cancer cell growth than retinoid-treatment, and surviving cancer cells showed significantly reduced tumorigenicity. The cancer-specific growth retardation was mediated by an increased level of apoptosis and a delayed cell cycle progression due to the loss of BMI1. By comparison, BMI1 deficiency caused only a moderate inhibition of the cell cycle progression in normal lung cells. In both normal and cancer cells, the loss of BMI1 led to an upregulation of INK4A-ARF, but with no significant effect on the level of telomerase gene expression, suggesting that other BMI1-cooperative factors in addition to INK4A-ARF activation may be involved in the BMI1-dependent cancer-specific growth retardation. Thus, human BMI1 is critical for the short-term survival of cancer cells, and inhibition of BMI1 has minimal effect on the survival of normal cells. These findings provide a foundation for developing a cancer-specific therapy targeting BMI1.
Assuntos
Apoptose/genética , Neoplasias/genética , Neoplasias/patologia , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Morte Celular , Linhagem Celular Tumoral , Humanos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/fisiologia , Complexo Repressor Polycomb 1 , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/fisiologia , Interferência de RNA , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/fisiologiaRESUMO
Current standard cancer therapies (chemotherapy and radiation) often cause serious adverse off-target effects. Drug design strategies are therefore being developed that will more precisely target cancer cells for destruction while leaving surrounding normal cells relatively unaffected. Telomerase, widely expressed in most human cancers but almost undetectable in normal somatic cells, provides an exciting drug target. This review focuses on recent pharmacogenomic approaches to telomerase inhibition. Antisense oligonucleotides, RNA interference, ribozymes, mutant expression, and the exploitation of differential telomerase expression as a strategy for targeted oncolysis are discussed here in the context of cancer therapeutics. Reports of synergism between telomerase inhibitors and traditional cancer therapeutic agents are also analyzed.
Assuntos
Inibidores Enzimáticos/uso terapêutico , Neoplasias/tratamento farmacológico , Oligonucleotídeos Antissenso/uso terapêutico , RNA Antissenso/uso terapêutico , Telomerase/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Neoplasias/enzimologia , Neoplasias/patologia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , RNA Antissenso/genética , RNA Antissenso/farmacologia , RNA Catalítico/genética , RNA Catalítico/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Telomerase/genética , Telomerase/metabolismoRESUMO
Methylation spreading, which involves a propensity for the mammalian DNA-(cytosine-5)-methyltransferase to de novo methylate cytosine-guanine dinucleotides (CpGs) near pre-existing 5-methylcytosine bases, has been implicated in the control of numerous biological processes. We have assessed methylation spreading by the murine DNA methyltransferase in vitro using synthetic copolymers and oligonucleotides which differ only in their methylation state. Double-stranded oligonucleotides were found to undergo higher levels of de novo methylation overall than otherwise identical single-stranded oligonucleotides. This difference reflects the greater number of de novo methylatable cytosine bases in double-stranded than single-stranded sequences. All tested oligonucleotides containing pre-existing 5-methyl-cytosine(s) were de novo methylated at several fold the rates of non-methylated controls. No mammalian proteins besides the DNA methyltransferase were required for this observed enhancement of de novo methylation. Studies using oligonucleotides differing in patterns of pre-methylation showed that methylation spreading can be initiated by hemimethylated or duplex methylated CpGs indicating that recognition of 5-methylcytosine by the enzyme is sufficient to stimulate methylation spreading. Double and single-stranded oligonucleotides with several bases between CpGs underwent considerably more de novo methylation per CpG than sequences containing sequential uninterrupted methylatable sites. Spacing preferences by the DNA methyltransferase were also observed in hemimethylated oligonucleotides, suggesting that this is a general property of the enzyme. Although methylation spreading outside of CpG dinucleotides was relatively rare, single-stranded DNA incurred higher levels of de novo methylation at sites other than CpG as compared to double-stranded DNA. This indicates less specificity of methylation spreading in single-stranded sequences. Finally, enhanced de novo methylation in the presence of fully methylated CpG sites in double-stranded oligonucleotides was not as high as the rates of methylation of hemimethylated CpGs in otherwise identical oligonucleotides. These studies provide further elucidation of the mechanisms and regulation of the methylation spreading process and its potential role in the biological processes it influences.
Assuntos
Metilação de DNA , DNA/metabolismo , Metiltransferases/metabolismo , 5-Metilcitosina , Animais , Sequência de Bases , Sítios de Ligação , Citosina/análogos & derivados , DNA/síntese química , DNA de Cadeia Simples , Fosfatos de Dinucleosídeos/metabolismo , Cinética , Camundongos , Dados de Sequência Molecular , Oligonucleotídeos/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Recent interest in the regulation of telomerase, the enzyme that maintains chromosomal termini, has lead to the discovery and characterization of the catalytic subunit of telomerase, hTERT. Many studies have suggested that the transcription of hTERT represents the rate-limiting step in telomerase expression and key roles for hTERT have been implied in cellular aging, immortalization, and transformation. Before the characterization of the promoter of hTERT in 1999, regulatory mechanisms suggested for this gene were limited to speculation. The successful cloning and characterization of the hTERT 5' gene regulatory region has enabled its formal investigation and analysis of potential mechanisms controlling hTERT expression. Although these studies have provided important information about hTERT gene regulation, there has been some confusion regarding the nucleotide boundaries of this region, the location, number, and importance of various transcription factor binding motifs, and the results of promoter activity assays. We feel that this uncertainty, combined with the sheer volume of recent publications on hTERT regulation, calls for consolidation and review. In this analysis we examine recent advances in the regulation of the hTERT gene and attempt to resolve discrepancies resulting from the nearly simultaneous nature of publications in this fast-moving area. Additionally, we aim to summarize the extant knowledge of hTERT gene regulation and its role in important biological processes such as cancer and aging.
Assuntos
Regulação Enzimológica da Expressão Gênica , RNA , Telomerase/genética , Animais , Sequência de Bases , Sítios de Ligação , Domínio Catalítico , Sequência Consenso , Metilação de DNA , DNA Complementar , Proteínas de Ligação a DNA , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Telomerase/metabolismo , Telomerase/fisiologia , Fatores de Transcrição/metabolismoRESUMO
Recent studies have indicated that the decline in mitogenesis during the aging process may be related to intracellular defects that become apparent when the cells are subjected to the metabolic stress of cell transformation. We provide the first report of an age-related decline in protein synthesis in human lymphocytes exposed to phytohemagglutinin. This impairment in protein synthetic capacity from aged subjects' transforming cells is apparent from 24 to 72 h of culture. By 72 h of culture the incorporation of radiolabeled leucine in stimulated cells from elderly subjects is about half that for the young. However, cells from the aged have an increased protein content in the face of decreased synthesis suggesting a degradation defect. Since protein synthesis may be necessary for the induction of key effectors which activate glycolysis (which is necessary for transformation), we sought to relate the impaired protein synthesis to the impaired glycolytic enzyme induction with age. Cycloheximide totally inhibited glycolytic enzyme induction as well as cell transformation. A defect in protein synthesis with age may interfere with new enzyme synthesis which is necessary to activate requisite pathways for mitogenesis such as glycolysis.
Assuntos
Envelhecimento , Ativação Linfocitária , Linfócitos/citologia , Mitose , Biossíntese de Proteínas , Adulto , Idoso , Cicloeximida/farmacologia , Glicólise , Humanos , Leucina/metabolismo , Modelos Biológicos , Fito-Hemaglutininas/farmacologia , Fatores de TempoRESUMO
Triosephosphate isomerase was found to have an increased thermolabile component in skin fibroblasts from patients with progeria and Werner's syndrome when compared with normal fibroblasts. Mixtures of cell extracts from progeria or Werner's syndrome with normal fibroblasts gave intermediate levels of the heat-labile triosephosphate isomerase suggesting the absence of cytosolic destabilizing factors. The incorporation of the protease inhibitors 1-tosylamide-2-phenylethyl chloromethyl ketone, N-alpha-p-tosyl-L-lysine chloromethyl ketone, phenylmethyl-sulfonyl fluoride, and pepstatin A in cell extracts failed to affect the level of the labile form of triosephosphate isomerase. The labile component also accumulates in normal fibroblasts in late passage in tissue culture and appears to be identical to the deamidated form of the enzyme which accumulates in other aging cells.
Assuntos
Carboidratos Epimerases/análise , Progéria/enzimologia , Pele/enzimologia , Triose-Fosfato Isomerase/análise , Síndrome de Werner/enzimologia , Adolescente , Adulto , Envelhecimento , Células Cultivadas , Criança , Feminino , Fibroblastos/enzimologia , Temperatura Alta , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
When peripheral lymphocytes from young persons are stimulated in vitro with phytohemagglutinin, an increase in the levels of all the glycolytic enzymes occurs concomitantly with blastogenesis. The specific activities of enzymes increase approximately 200%, with a greater induction of the latter half of the pathway. The increases do not represent a general enzyme induction, since nonglycolytic enzymes such as glucose 6-phosphate dehydrogenase and isocitrate dehydrogenase do not increase during transformation. Human lymphocytes from a geriatric population were also subjected to mitogen stimulation under identical conditions. The initial levels of the enzymes were essentially identical in lymphocytes from young and old subjects as were cultured controls which received no mitogen. However, during mitogen stimulation the cells from the old subjects failed to increase the glycolytic enzymes. This inability to activate glycolysis may be related to the decline in cell-mediated immunity which is known to occur with advancing age.
Assuntos
Envelhecimento , Glicólise , Linfócitos/enzimologia , Adulto , Idoso , Células Cultivadas , Glucosefosfato Desidrogenase/sangue , Humanos , Isocitrato Desidrogenase/sangue , Ativação Linfocitária , Fito-Hemaglutininas/farmacologiaRESUMO
There is a decline in immune capacity with age which is expressed on the organismic level by association with numerous immune-related diseases, on the cellular level by impaired mitogenesis, on the biochemical level by impaired metabolic pathways, and on the molecular level by decreased protein synthesis and degradation. Defects in various cofactors such as calcium and several nucleotides also occur and may be related to the impaired enzyme function during mitogenesis in the aged. The central cause for decreased mitogenesis in the aged could be a decrease in protein synthesis which appears to cause impaired enzyme induction. This impaired enzyme induction accounts in part for the decreased glycolytic flux and DNA synthesis in these cells. Decreased protein synthesis also has been associated with a decreased synthesis of lymphokines which help these cells to proliferate. Numerous other intracellular age-related defects of lymphocytes also occur which may collectively play important interdependent roles in the impaired lymphocyte function of the aged. A potential general underlying mechanism of cellular senescence is proposed based on a genetic "slowing-cycle" effect of transcription, translation, and enzyme induction with immunosenescence presented as an example of an expression of these basic defects.
Assuntos
Envelhecimento/sangue , Linfócitos/metabolismo , HumanosRESUMO
Gene silencing is often mediated by CpG methylation of key protein binding sites within gene regulatory sequences (GRSs). An aging mechanism is proposed based on this gene-silencing phenomenon whereby accumulation over time of methylation within GRSs contributes to cellular senescence. The proposed molecular mechanism for age-related gene silencing is the spreading of methylation through the regulatory sequences of genes resulting in progressive reduction of gene transcription. There is considerable experimental evidence for methylation spreading and its role in gene silencing, but the mechanism responsible for this process has not been elucidated. A four-step mechanism is proposed whereby an original methylation occurs, methyltransferase (MTase) molecules progressively move 5' to 3' from this site, neighboring CpG dinucleotides become methylated, and diminished gene expression ensues. Over time, this process may lead to widespread gene silencing in diverse dividing and nondividing cell types contributing to aging of the organism.
Assuntos
Envelhecimento/genética , Genes Reguladores , Animais , Sítios de Ligação , Senescência Celular/genética , DNA/genética , DNA/metabolismo , Replicação do DNA , Humanos , Metilação , Modelos Genéticos , Ligação ProteicaRESUMO
Although telomerase, which maintains the ends of chromosomes, is down-regulated as cells differentiate leading to attrition of chromosomal termini and ultimate replicative senescence, it is up-regulated in most cancer cells which show no net loss of average telomere length. The mRNA level of the catalytic component of telomerase, hTERT, is the major determinant of telomerase activity but little is known about control of hTERT transcription. We propose mechanisms whereby cytosine methylation may alter the binding of activators such as c-Myc or repressors such as WT1 which interact with the hTERT gene regulatory region to modulate telomerase activity in aging cells and tumorigenesis. Mechanisms are also proposed for control of hTERT expression through changes in the collective binding of its transcription factors in aging and tumorigenic cells. Elucidation of telomerase regulation should facilitate advances in understanding age-related diseases such as cancer and in potential therapeutic modalities.
Assuntos
Transformação Celular Neoplásica/genética , Senescência Celular/genética , Regulação da Expressão Gênica , RNA , Telomerase/genética , Sequência de Bases , DNA , Metilação de DNA , Proteínas de Ligação a DNA , Humanos , Regiões Promotoras GenéticasRESUMO
The pronounced effects of the epigenetic diet (ED) and caloric restriction (CR) have on epigenetic gene regulation have been documented in many pre-clinical and clinical studies. Understanding epigenetics is of high importance because of the concept that external factors such as nutrition and diet may possess the ability to alter gene expression without modifying the DNA sequence. The ED introduces bioactive medicinal chemistry compounds such as sulforaphane (SFN), curcumin (CCM), epigallocatechin gallate (EGCG) and resveratrol (RSV) that are thought to aid in extending the human lifespan. CR, although similar to ED in the target of longevity, mildly reduces the total daily calorie intake while concurrently providing all beneficial nutrients. Both CR and ED may act as epigenetic modifiers to slow the aging process through histone modification, DNA methylation, and by modulating microRNA expression. CR and ED have been proposed as two important mechanisms that modulate and potentially slow the progression of age-related diseases such as cardiovascular disease (CVD), cancer, obesity, Alzheimer's and osteoporosis to name a few. While many investigators have examined CR and ED as separate entities, this review will primarily focus on both as they relate to age-related diseases, their epigenetic effects and their medicinal chemistry.
Assuntos
Restrição Calórica , Química Farmacêutica , Epigênese Genética/fisiologia , Envelhecimento/efeitos dos fármacos , Anticarcinógenos/farmacologia , Catequina/análogos & derivados , Catequina/farmacologia , Curcumina/farmacologia , Epigênese Genética/efeitos dos fármacos , Humanos , Isotiocianatos/farmacologia , Resveratrol , Estilbenos/farmacologia , SulfóxidosRESUMO
It is well established that aberrant gene regulation by epigenetic mechanisms can develop as a result of pathological processes such as cancer. Methylation of CpG islands is an important component of the epigenetic code and a number of genes become abnormally methylated during tumorigenesis. Some bioactive food components have been shown to have cancer inhibition activities by reducing DNA hypermethylation of key cancer-causing genes through their DNA methyltransferase (DNMT) inhibition properties. The dietary polyphenols, (-)-epigallocatechin- 3-gallate (EGCG) from green tea, genistein from soybean and possibly isothiocyanates from plant foods, are some examples of these bioactive food components modulated by epigenetic factors. The activity of cancer inhibition generated from dietary polyphenols is associated with gene reactivation through demethylation in the promoters of methylation-silenced genes such as p16INK4a and retinoic acid receptor beta. The effects of dietary polyphenols such as EGCG on DNMTs appear to have their direct inhibition by interaction with the catalytic site of the DNMT1 molecule, and may also influence methylation status indirectly through metabolic effects associated with energy metabolism. Therefore, reversal of hypermethylation-induced inactivation of key tumor suppression genes by dietary DNMT inhibitors could be an effective approach to cancer prevention and therapy. In this analysis, we focus on advances in understanding the effects of dietary polyphenols on DNA methylation modulation during the process of cancer development, which will offer exciting new opportunities to explore the role of diet in influencing the biology of cancer and to understand the susceptibility of the human epigenome to dietary effects.
Assuntos
Anticarcinógenos/química , Metilação de DNA/efeitos dos fármacos , Neoplasias/prevenção & controle , Anticarcinógenos/uso terapêutico , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/uso terapêutico , Catequina/análogos & derivados , Catequina/uso terapêutico , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/metabolismo , Genisteína/uso terapêutico , Humanos , Isotiocianatos/uso terapêutico , Neoplasias/tratamento farmacológico , Selênio/uso terapêuticoAssuntos
Envelhecimento , Proteínas/metabolismo , Idoso , Aminoácidos/metabolismo , Doenças Cardiovasculares/tratamento farmacológico , Demência/etiologia , Diabetes Mellitus/etiologia , Terapia Enzimática , Humanos , Longevidade , Microssomos Hepáticos/metabolismo , Presbiopia/etiologia , Biossíntese de ProteínasRESUMO
There is a general slowing of protein biosynthesis with increasing age that appears to be universal and involved in the mechanism of aging. One of the apparent expressions of this aging mechanism is an age-related decreased induction of many enzymes including, as the focus of this analysis, those of carbohydrate metabolism. The impaired induction of these enzymes contributes to the slowing of several carbohydrate metabolic pathways, and parallels a marked age-related diminution in the generation of anabolic metabolites and energy compounds from these pathways. Intracellular carbohydrate metabolism contributes to the synthesis of virtually all macromolecules of the cell, and the age-related slowing and impairment in carbohydrate metabolism appears to play a role in the expression of cellular senescence. Moreover, these intracellular impairments in the aged contribute, in part, to the expression of several physiologic decrements and pathologies that accompany the aging process, such as glucose intolerance and diabetes.
Assuntos
Envelhecimento , Metabolismo dos Carboidratos , Enzimas/genética , Genes , Animais , Glicólise , HumanosRESUMO
A simultaneous increase is found in the level of protein synthesis and the major regulatory glycolytic enzyme, phosphofructokinase (PFK), in early phytohemagglutinin exposure of human lymphocytes. The induction of DNA synthesis is demonstrated to be a much later event. This indicates that the increase of glycolysis in mitogen-stimulated cells precedes cell proliferation, but occurs simultaneously with a general increase in protein synthesis. Chemical inhibitors are used to clarify the interrelationship of protein synthesis, glycolytic enzymes levels, and DNA synthesis. Inhibition of protein synthesis with cycloheximide in the mitogen-exposed lymphocytes prevents any increase in PFK levels, implicating protein synthesis as a cause for the increased glycolysis. Cycloheximide also prevents entry into S phase in mitogen-stimulated lymphocytes which may be due to inhibition of the synthesis of enzymes necessary for DNA synthesis, such as DNA polymerase. Aphidicolin, a specific DNA polymerase inhibitor, is found to have no effect on the increase in protein synthesis and PFK levels that precedes DNA synthesis. The increase in glycolysis in mitogen-stimulated lymphocytes occurs simultaneously with, and is dependent upon, increased protein synthesis, and precedes DNA synthesis and lymphocyte proliferation; thus, the high glycolytic rate of mitogen-stimulated cells is not merely a secondary manifestation of rapid cell proliferation as has been previously reported.
Assuntos
Glicólise , Linfócitos/metabolismo , Adulto , Afidicolina , Divisão Celular , Cicloeximida/farmacologia , DNA/biossíntese , Diterpenos/farmacologia , Feminino , Glicólise/efeitos dos fármacos , Humanos , Técnicas In Vitro , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Masculino , Mitógenos/farmacologia , Fosfofrutoquinase-1/metabolismo , Fito-Hemaglutininas/farmacologia , Biossíntese de ProteínasRESUMO
There is an age-related decline in immune capacity which has been linked to a decreased response of lymphocytes to mitogens in vitro. During transformation, lymphocytes require a marked increase in energy production and biosynthesis which is supplied primarily by glycolysis. In the elderly, the glycolytic enzymes increase significantly in transforming lymphocytes at least 24 hr later than in the young and then at significantly reduced levels. Glucose utilization is also impaired in stimulated lymphocytes from the elderly but follows the impairment of glycolysis. In stimulated cells from the young, increases in glycolytic enzyme activity levels accompany sharp increases in blastogenesis while a delayed increase in glycolytic enzyme activity in the elderly is accompanied by a delay in blastogenesis. Maximal glycolytic enzyme activity levels are significantly reduced in transformed lymphocytes from the elderly though the number of transformed cells is also significantly reduced. However, glycolytic enzyme activity levels are significantly lower in the elderly than in the young even on a per transformed cell basis. Thus, this reduction cannot be attributed to the lower number of transformed cells that are present in the elderly. This defect in the increase of glycolysis in stimulated cells from the elderly suggests an intracellular mechanism which could be related to the impaired lymphocyte stimulation in vitro in the aged.
Assuntos
Envelhecimento , Carboidratos/sangue , Ativação Linfocitária , Linfócitos/metabolismo , Adulto , Idoso , Contagem de Células , Sobrevivência Celular , Células Cultivadas , Feminino , Glucose/metabolismo , Glicólise , Humanos , Linfócitos/enzimologia , MasculinoRESUMO
Glycolytic enzyme activity is significantly (P less than 0.05) induced between 24 and 48 hours of incubation in phytohemagglutinin-stimulated human lymphocytes. Nonstimulated cultured cells do not show this induction although these cells have an approximate daily doubling of thymidine incorporation. Maximal glycolytic enzyme activity is reached between 96 and 120 hours of culture in stimulated cells (3.5-fold increase) and maintained until at least 168 hours. There is no significant induction of the hexosemonophosphate shunt or the TCA cycle during seven-day transformation. Induction of glucose utilization becomes significantly (P less than 0.05) greater in stimulated as compared to nonstimulated cultures between 48 and 72 hours of culture and is significantly elevated for at least an additional 96 hours. There is a 17% increase in total protein in the stimulated cells after 24 hours of culture and higher levels of protein content are then maintained over the control. Thymidine incorporation is significantly greater in stimulated cells from 24-144 hours of culture but is not significantly different from the nonstimulated cells at 168 hours (P = 0.98) although glycolytic enzyme activity remains elevated in the stimulated cells. There is a greater enzyme induction of the latter phase of glycolysis during transformation and this phenomenon continues in extended cultures. Increases in glycolytic enzyme activity during mitogenesis appear to be an intrinsic phenomenon independent of cell proliferation and glucose transport. The mitogen-induced increase in the activity of the glycolytic enzymes accompanies blastogenesis and the sustained elevated activity of these enzymes to be related to the high metabolic rate of transformed cells.
Assuntos
Replicação do DNA/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Ativação Linfocitária , Linfócitos/citologia , Mitógenos/farmacologia , Adulto , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Glucose/metabolismo , Glucose-6-Fosfato Isomerase/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Humanos , Isocitrato Desidrogenase/metabolismo , Cinética , L-Lactato Desidrogenase/metabolismo , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Triose-Fosfato Isomerase/metabolismoRESUMO
The incorporation of radiolabeled leucine into phytohemagglutinin-stimulated human lymphocytes increases by 9 hours after mitogen addition in the young whereas this process is delayed by two-fold in the aged (18 hours). Once induced, the leucine incorporation is about 56% less in the aged as compared to the young. The induction of phosphofructokinase (PFK) catalytic activity mimics the induction of protein synthesis in both young (9 hours) and aged (18 hours) subjects also taking twice as long to induce in the aged and attaining much lower levels of induction with increasing subject age. The increase of thymidine incorporation in mitogen-stimulated cells does not occur until 12 hours after the increase in leucine incorporation in both the young (21 hours) and aged (30 hours) which also represents a 9 hour age-related delay in induction. The marked increase in protein synthesis rate occurs in a concerted manner with the induction of glycolysis and the delay and impairment in protein biosynthesis in the aged appears to relate to the similar age-related findings for glycolytic enzyme induction. The mitogen-induced increase in DNA synthesis is a later event and the age-related delay in DNA synthesis induction may be secondary to the delay in the induction of protein synthesis. Other enzyme-dependent processes besides DNA synthesis and glycolysis may also be secondary to a primary slowing of protein synthesis in the aged and related to the delayed cell cycle time frequently observed in aged subjects.
Assuntos
Envelhecimento , Replicação do DNA , Ativação Linfocitária , Linfócitos/metabolismo , Fosfofrutoquinase-1/biossíntese , Biossíntese de Proteínas , Adulto , Idoso , Células Cultivadas , DNA/biossíntese , Indução Enzimática , Humanos , Linfócitos/citologia , Linfócitos/imunologiaRESUMO
Besides modulating specific DNA-protein interactions, methylated cytosine, frequently referred to as the fifth base of the genome, also influences DNA structure, recombination, transposition, repair, transcription, imprinting, and mutagenesis. DNA (cytosine-5-)-methyltransferase catalyzes cytosine methylation in eukaryotes. We have cloned and expressed this enzyme in Escherichia coli, purified it to apparent homogeneity, characterized its properties, and we have shown that it hemimethylates DNA. The cDNA for murine maintenance methyltransferase was reconstructed and cloned for direct expression in native form. Immunoblotting revealed a unique protein (M(r) = 190,000) not present in control cells. The mostly soluble overexpressed protein was purified by DEAE, Sephadex, and DNA cellulose chromatography. Peak methylating activity correlated with methyltransferase immunoblots. The purified enzyme preferentially transferred radioactive methyl moieties to hemimethylated DNA in assays and on autoradiograms. All of the examined properties of the purified recombinant DNA methyltransferase are consistent with the enzyme purified from mammalian cells. Further characterization revealed enhanced in vitro methylation of premethylated oligodeoxynucleotides. The cloning of hemimethyltransferase in E. coli should allow facilitated structure-function mutational analysis of this enzyme, studies of its biological effects in prokaryotes, and potential large scale methyltransferase production for crystallography, and it may have broad applications in maintaining the native methylated state of cloned DNA.
Assuntos
DNA (Citosina-5-)-Metiltransferases/isolamento & purificação , Animais , Sequência de Bases , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Escherichia coli/genética , Vetores Genéticos , Immunoblotting , Mamíferos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Due in part to the complexity of mammalian systems, some of the proposed biological influences of mammalian DNA methylation have not been fully established. Escherichia coli cells, which normally contain negligible CpG methylation, exhibited progressive slowing of replication and lengthened generation times when expressing the murine DNA maintenance methyltransferase. Genomic analysis indicated significant amounts of CpG methylation in expressing cells which was absent from control cells. Expressing cells exposed to the cytosine demethylating agent, 5-azacytidine, rapidly reverted to propagation levels of controls. Substitution of cysteine with alanine in the carboxyl-terminal region proline-cysteine dipeptide of the methyltransferase completely inactivated methylating activity and cells expressing the inactive enzyme replicated as well as controls. These findings strongly implicate a role of epigenetic de novo CpG methylation in modulating cellular propagation, demonstrate that the maintenance methyltransferase can de novo methylate in vivo, and show that the methyltransferase requires an active site cysteine for activity.