RESUMO
We show that femtosecond laser irradiation of polydimethylsiloxane (PDMS) enables selective and patterned cell growth by altering the wetting properties of the surface associated with chemical and/or topographical changes. In the low pulse energy regime, the surface becomes less hydrophobic and exhibits a low water contact angle compared to the pristine material. X-ray photoelectron spectroscopy (XPS) also reveals an increased oxygen content in the irradiated regions, to which the C2C12 cells and rabbit anti-mouse protein were found to attach preferentially. In the high pulse energy regime, the laser-modified regions exhibit superhydrophobicity and were found to inhibit cell adhesion, whereas cells were found to attach to the surrounding regions due to the presence of nanoscale debris generated by the ablation process.
Assuntos
Adesão Celular/fisiologia , Dimetilpolisiloxanos/química , Lasers , Impressão Molecular/métodos , Mioblastos/citologia , Mioblastos/fisiologia , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/efeitos da radiação , Linhagem Celular , Dimetilpolisiloxanos/efeitos da radiação , Teste de Materiais , Camundongos , Propriedades de Superfície/efeitos da radiaçãoRESUMO
OBJECTIVE: To investigate the influence of menses on the vaginal microbiota and determine whether tampons that differ in material composition influence these bacterial communities in different ways. DESIGN: A single-centre trial with randomised, complete block design. SETTING: Procter & Gamble facility. SAMPLE: Seven self-declared healthy, female volunteers of reproductive age. METHODS: Volunteers used a pad and two types of tampons during the study, one product exclusively each month for three sequential menstrual cycles. During menses and once each mid-cycle, vaginal bacterial community composition was characterised by cultivation-independent methods based on pyrosequencing of V1-V2 variable regions of 16S ribosomal RNA genes. MAIN OUTCOME MEASURES: Changes in the species composition, abundance and diversity in vaginal bacterial communities over time and between treatments. RESULTS: The vaginal microbiotas of all seven women were dominated by Lactobacillus spp. at mid-cycle, and the compositions of those communities were largely consistent between cycles. Community dynamic patterns during menses varied considerably and were more or less individualised. In three of the seven women the community diversity during pad use was significantly different from at least one tampon cycle. CONCLUSIONS: Changes in the composition of the vaginal microbiota during menses were common, but the magnitude of change varied between women. Despite these changes, most communities were capable of resuming a composition similar to previous mid-cycle sampling times following menstruation. Overall we conclude that the two tampons tested do not significantly impact the vaginal microbiota in different ways; however, larger studies should be performed to confirm these findings.
Assuntos
Bactérias/classificação , Produtos de Higiene Menstrual , Menstruação , Metagenoma , Vagina/microbiologia , Adolescente , Adulto , Bactérias/genética , Bactérias/isolamento & purificação , Feminino , Humanos , Pessoa de Meia-Idade , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Adulto JovemRESUMO
Many tumors exhibit elevated chromosome mis-segregation termed chromosome instability (CIN), which is likely to be a potent driver of tumor progression and drug resistance. Causes of CIN are poorly understood but probably include prior genome tetraploidization, centrosome amplification and mitotic checkpoint defects. This study identifies epigenetic alteration of the centromere as a potential contributor to the CIN phenotype. The centromere controls chromosome segregation and consists of higher-order repeat (HOR) alpha-satellite DNA packaged into two chromatin domains: the kinetochore, harboring the centromere-specific H3 variant centromere protein A (CENP-A), and the pericentromeric heterochromatin, considered important for cohesion. Perturbation of centromeric chromatin in model systems causes CIN. As cancer cells exhibit widespread chromatin changes, we hypothesized that pericentromeric chromatin structure could also be affected, contributing to CIN. Cytological and chromatin immunoprecipitation and PCR (ChIP-PCR)-based analyses of HT1080 cancer cells showed that only one of the two HORs on chromosomes 5 and 7 incorporate CENP-A, an organization conserved in all normal and cancer-derived cells examined. Contrastingly, the heterochromatin marker H3K9me3 (trimethylation of H3 lysine 9) mapped to all four HORs and ChIP-PCR showed an altered pattern of H3K9me3 in cancer cell lines and breast tumors, consistent with a reduction on the kinetochore-forming HORs. The JMJD2B demethylase is overexpressed in breast tumors with a CIN phenotype, and overexpression of exogenous JMJD2B in cultured breast epithelial cells caused loss of centromere-associated H3K9me3 and increased CIN. These findings suggest that impaired maintenance of pericentromeric heterochromatin may contribute to CIN in cancer and be a novel therapeutic target.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Centrômero/genética , Centrômero/metabolismo , Instabilidade Cromossômica , Heterocromatina/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Linhagem Celular Tumoral , Cromossomos Humanos Par 5/genética , Feminino , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Cinetocoros/metabolismo , Invasividade NeoplásicaRESUMO
Repair of double-stranded breaks (DSBs) is vital to maintaining genomic stability. In mammalian cells, DSBs are resolved in one of the following complex repair pathways: nonhomologous end-joining (NHEJ), homologous recombination (HR), or the inclusive DNA damage response (DDR). These repair pathways rely on factors that utilize reversible phosphorylation of proteins as molecular switches to regulate DNA repair. Many of these molecular switches overlap and play key roles in multiple pathways. For example, the NHEJ pathway and the DDR both utilize DNA-PK phosphorylation, whereas the HR pathway mediates repair with phosphorylation of RPA2, BRCA1, and BRCA2. Also, the DDR pathway utilizes the kinases ATM and ATR, as well as the phosphorylation of H2AX and MDC1. Together, these molecular switches regulate repair of DSBs by aiding in DSB recognition, pathway initiation, recruitment of repair factors, and the maintenance of repair mechanisms.
RESUMO
Although Smad 3 is known to serve as a signaling intermediate for the transforming growth factor beta (TGFbeta) family in nonreproductive tissues, its role in the ovary is unknown. Thus, we used a recently generated Smad 3-deficient (Smad 3-/-) mouse model to test the hypothesis that Smad 3 alters female fertility and regulates the growth of ovarian follicles from the primordial stage to the antral stage. In addition, we tested whether Smad 3 affects the levels of proteins that control apoptosis, survival, and proliferation in the ovarian follicle. To test this hypothesis, breeding studies were conducted using Smad 3-/- and wild-type mice. In addition, ovaries were collected from Smad 3-/- and wild-type mice on Postnatal Days 2-90. One ovary from each animal was used to estimate the total number of primordial, primary, and antral follicles. The other ovary was used for immunohistochemical analysis of selected members of the B-cell lymphoma/leukemia-2 family of protooncogenes (Bax, Bcl-2, Bcl-x), proliferating cell nuclear antigen (PCNA), and cyclin-dependent kinase 2 (Cdk-2). The results indicate that Smad 3-/- mice have reduced fertility compared with wild type mice. The results also indicate that Smad 3 may not affect the size of the primordial follicle pool at birth, but it may regulate growth of primordial follicles to the antral stage. Further, the results indicate that Smad 3 may regulate the expression of Bax and Bcl-2, but not Bcl-x, Cdk-2, and PCNA. Collectively, these data suggest that Smad 3 may play an important role in the regulation of ovarian follicle growth and female fertility.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ligação a DNA/fisiologia , Folículo Ovariano/crescimento & desenvolvimento , Transativadores/fisiologia , Animais , Apoptose , Peso Corporal , Divisão Celular , Sobrevivência Celular , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/análise , Proteínas de Ligação a DNA/deficiência , Feminino , Fertilidade , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Folículo Ovariano/citologia , Ovário/anatomia & histologia , Ovário/química , Ovário/fisiologia , Antígeno Nuclear de Célula em Proliferação/análise , Proteínas Serina-Treonina Quinases/análise , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas c-bcl-2/análise , Proteína Smad3 , Transativadores/deficiência , Proteína X Associada a bcl-2 , Proteína bcl-XRESUMO
The antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C) has been used as a highly effective agent for the treatment of leukemia. The active metabolite 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP) is a potent inhibitor of DNA polymerases alpha, delta, and epsilon, and is responsible for inhibiting intact cell DNA synthesis. We have shown that a multiprotein complex, exhibiting many of the properties expected of the human cell DNA replication apparatus, can be readily isolated from human cells and tissues and is capable of supporting origin-dependent DNA synthesis in vitro. DNA polymerases alpha, delta, and epsilon are components of this multiprotein complex, termed the DNA synthesome, and we report here that the activities of these DNA synthesome-associated DNA polymerases are inhibited differentially by ara-CTP. Inhibition of the DNA synthesome-associated DNA polymerase alpha increased in a concentration-dependent manner, and was correlated closely with the inhibition of simian virus 40 (SV40) origin-dependent in vitro DNA replication, whereas DNA synthesome-associated DNA polymerase delta activity was not inhibited significantly by ara-CTP at 100 microM. Recent work has shown that the synthesome-associated DNA polymerase epsilon does not function in in vitro SV40 DNA replication, suggesting that only polymerases alpha and delta drive the DNA replication fork. Therefore, our results suggest that inhibition of the activity of the mammalian cell DNA synthesome by ara-CTP is due primarily to the inhibition of the DNA synthesome-associated DNA polymerase alpha. This observation implies that the drug may target specific phases of the DNA synthetic process in human cells.
Assuntos
Arabinofuranosilcitosina Trifosfato/farmacologia , DNA Polimerase III/antagonistas & inibidores , DNA Polimerase I/antagonistas & inibidores , Replicação do DNA/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Linhagem Celular , DNA/biossíntese , DNA/efeitos dos fármacos , DNA Polimerase I/metabolismo , DNA Polimerase III/metabolismo , Células HeLa , Humanos , Vírus 40 dos Símios/fisiologiaRESUMO
PURPOSE: An intact and fully functional multiprotein DNA replication complex (DNA synthesome) from human as well as from murine mammary carcinoma cells was first isolated and characterized in our laboratory. The human cell synthesome supports the in vitro origin-specific simian virus 40 (SV40) DNA replication reaction in the presence of the viral large T-antigen using a semiconservative mechanism and has been shown to contain all the proteins and enzymes required to support DNA synthesis. We are currently using the DNA synthesome as a unique model for analyzing the mechanism of action of anticancer drugs affecting DNA replication. The purpose of this study was to further investigate the mechanism of action of ara-C using the DNA synthesome isolated from the human breast cancer cell line MDA MB-468. METHODS: Synthesome-mediated SV40 DNA replication was performed in the presence of various concentrations of ara-CTP (the active metabolite of ara-C) and the types of daughter DNA molecules produced were analyzed lusing neutral and alkaline gel electrophoresis. We also examined the effect of ara-C on intact MDA MB-468 cell DNA synthesis and on cell proliferation. In addition, we studied the effect of ara-CTP on the activity of some of the synthesome target proteins (the DNA polymerases alpha and delta). RESULTS: Full-length daughter DNA molecules were obtained in the presence of low concentrations of ara-CTP while at higher concentrations, there was an inhibition of full-length daughter DNA synthesis. The findings suggest that specifically the initiation phase of DNA synthesis was inhibited by ara-CTP since the production of the short Okazaki fragments was suppressed at all concentrations of the drug above 10 microM. In addition, it was found that the IC50 of ara-CTP for inhibition of synthesome-mediated in vitro DNA replication was comparable to that required to inhibit intact cell DNA synthesis. Further experimentation has shown that ara-CTP preferentially inhibits the activity of the synthesome-associated DNA polymerase alpha enzyme while the DNA polymerase delta seems to be resistant to the inhibitory effect of that drug. CONCLUSIONS: Our results indicate that ara-C's action on DNA replication is mediated primarily through DNA polymerase alpha and suggest that this enzyme plays a key role in DNA synthetic initiation events. The results also provide definitive support for the use of the DNA synthesome as a unique and powerful model for analyzing the mechanism of action of anticancer drugs which directly affect DNA replication.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Citarabina/farmacologia , Replicação do DNA/efeitos dos fármacos , DNA de Neoplasias/biossíntese , Antígenos Transformantes de Poliomavirus/metabolismo , Arabinofuranosilcitosina Trifosfato/farmacologia , Neoplasias da Mama/metabolismo , Divisão Celular/efeitos dos fármacos , DNA Polimerase I/biossíntese , DNA Polimerase III/biossíntese , Humanos , Replicon/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
PURPOSE: Gemcitabine (dFdC) and cytarabine (araC) are both analogs of deoxycytidine. Gemcitabine is a relatively new drug that has been shown in both clinical trials and in vitro systems to have more potent antitumor activity than araC. We have previously isolated a fully functional multiprotein DNA replication complex from human cells and termed it the DNA synthesome. Using the DNA synthesome, we have successfully examined the mechanism of action of several anticancer drugs that directly affect DNA synthesis. In this study, we compared the effects of dFdC and araC on in vitro DNA synthesis mediated by the DNA synthesome with the effects of these drugs on intact MCF7 cell DNA synthesis. METHODS: We examined the effects of dFdC and araC on intact MCF7 cell DNA synthesis and clonogenicity. We also performed in vitro SV40 replication assays mediated by the MCF7 cell-derived DNA synthesome in presence of dFdCTP and araCTP. The types of daughter molecules produced in the assay were analyzed by neutral and alkaline agarose gel electrophoresis. Finally, we examined the effects ofdFdCTP and araCTP on the synthesome-associated DNA polymerase alpha and delta activities. RESULTS: Our results showed that dFdC was more potent than araC at inhibiting intact MCF7 cell DNA synthesis and clonogenicity. [3H]Thymidine incorporation was inhibited by 50% at a dFdC concentration of 10 microM, which was about tenfold lower than the concentration of araC required to inhibit intact cell DNA synthesis by the same amount. As examined by clonogenicity assay, dFdC was also significantly more cytotoxic than araC after a 24-h incubation. In vitro SV40 replication assays using the DNA synthesome derived from MCF7 cells demonstrated that the formation of full-length DNA along with replication intermediates were inhibited by dFdCTP in a concentration-dependent manner. Full-length DNA was produced in the in vitro DNA replication assay even when the dFdCTP was incubated in the assay at concentrations of up to 1 mM. We observed that in the presence of 10 microM dCTP, 3 microM dFdCTP and 60 microM araCTP were required to inhibit in vitro SV40 DNA synthesis by 50%. Although dFdCTP is more potent than araCTP at inhibiting in vitro SV40 DNA synthesis, there was no significant difference between the inhibitory effect of these two drugs on the activity of the MCF7 synthesome-associated DNA polymerases alpha and delta. It was found that the drug concentrations required to inhibit 50% of the synthesome-associated DNA polymerase delta activity were much higher than those required to inhibit 50% of DNA polymerase alpha activity for both dFdCTP and araCTP. CONCLUSION: Taken together, our results demonstrated that: (1) dFdC is a more potent inhibitor of intact cell DNA synthesis and in vitro SV40 DNA replication than araC; (2) the decrease in the synthetic activity of synthesome-mediated in vitro SV40 origin-dependent DNA synthesis by dFdCTP and araCTP correlates with the inhibition of DNA polymerase alpha activity; and (3) the MCF7 cell DNA synthesome can serve as a unique and relevant model to study the mechanism of action of anticancer drugs that directly affect DNA synthesis.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Citarabina/farmacologia , DNA de Neoplasias/biossíntese , Desoxicitidina/análogos & derivados , Antígenos Transformantes de Poliomavirus/biossíntese , Antígenos Transformantes de Poliomavirus/genética , Arabinofuranosilcitosina Trifosfato/farmacologia , Neoplasias da Mama/genética , Clonagem Molecular , DNA Polimerase I/metabolismo , DNA Polimerase III/metabolismo , Desoxicitidina/farmacologia , Humanos , Replicon/efeitos dos fármacos , Replicon/genética , Células Tumorais Cultivadas , GencitabinaRESUMO
The mechanisms responsible for creating genetic errors and genomic instability in cancer cells have not been fully defined. Recently, it has been shown that human cells contain a highly organized complex of proteins, termed the DNA synthesome, that is fully competent to carry out all phases of SV40 in vitro DNA replication (J. M. Coll et al, Oncol. Res., 8: 435-447, 1996; L. H. Malkas et al., Biochemistry, 29: 6362-6374, 1990; Y. Wu et al., J. Cell. Biochem., 54: 32-46, 1994; N. Applegren et al., J. Cell. Biochem., 54: 32-46, 1994). DNA replication fidelity analyses of the DNA synthesome derived from malignant and nonmalignant human breast cells demonstrate that the malignant cell synthesome is mutagenic. The decrease in tumor cell replication fidelity was not due to an increased proliferative capacity of the tumor cells or an increase in the synthetic activity of their DNA synthesome. The ratios of insertions, deletions, and mismatches created by the synthesome from malignant and nonmalignant breast cells were essentially identical, despite the greater overall number of mutations made by the breast cancer cell synthesome. These data define, for the first time, a mechanism unique to cancer cells that contributes to the observed increase in genetic mutation in cancer cells.
Assuntos
Neoplasias da Mama/metabolismo , Replicação do DNA , DNA de Neoplasias/biossíntese , Adulto , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/patologia , Feminino , Humanos , Hiperplasia/metabolismo , Hiperplasia/patologia , Reprodutibilidade dos Testes , Células Tumorais CultivadasRESUMO
Despite extensive research efforts to identify unique molecular alterations in breast cancer, to date, no characteristic has emerged that correlates exclusively with malignancy. Recently, it has been shown that the multiprotein DNA replication complex (synthesome) from breast cancer cells has a significantly decreased replication fidelity compared to that of nonmalignant breast cells. Proliferating cell nuclear antigen (PCNA) functions in DNA replication and DNA repair and is a component of the synthesome. Using two-dimensional PAGE analysis, we have identified a novel form of PCNA in malignant breast cells. This unique form is not the result of a genetic alteration, as demonstrated by DNA sequence analysis of the PCNA gene from malignant and nonmalignant breast cells. This novel form is most likely the result of an alteration in the post-translational modification of PCNA in malignant breast cells. These findings are significant in that it is now possible to link changes in the fidelity of DNA replication with a specific alteration of a component of the DNA synthetic apparatus of breast cancer cells. The novel form of PCNA may prove to be a new signature for malignant breast cells.
Assuntos
Neoplasias da Mama/metabolismo , Antígeno Nuclear de Célula em Proliferação/biossíntese , Animais , Sequência de Bases , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Divisão Celular/fisiologia , DNA de Neoplasias/biossíntese , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , Antígeno Nuclear de Célula em Proliferação/genética , Células Tumorais CultivadasRESUMO
Poly(ADP-ribose) polymerase (PARP) is a component of the multiprotein DNA replication complex (MRC, DNA synthesome) that catalyzes replication of viral DNA in vitro. PARP poly(ADP-ribosyl)ates 15 of the approximately 40 proteins of the MRC, including DNA polymerase alpha (DNA pol alpha), DNA topoisomerase I (topo I), and proliferating-cell nuclear antigen (PCNA). Although about equal amounts of MRC-complexed and free forms of PCNA were detected by immunoblot analysis of HeLa cell extracts, only the complexed form was poly(ADP-ribosyl)ated, suggesting that poly(ADP-ribosyl)ation of PCNA may regulate its function within the MRC. NAD inhibited the activity of DNA pol delta in the MRC in a dose-dependent manner, whereas the PARP inhibitor, 3-AB, reversed this inhibitory effect. The roles of PARP in modulating the composition and enzyme activities of the DNA synthesome were further investigated by characterizing the complex purified from 3T3-L1 cells before and 24 h after induction of a round of DNA replication required for differentiation of these cells; at the latter time point, approximately 95% of the cells are in S phase and exhibit a transient peak of PARP expression. The MRC was also purified from similarly treated 3T3-L1 cells depleted of PARP by antisense RNA expression; these cells do not undergo DNA replication nor terminal differentiation. Both PARP protein and activity and essentially all of the DNA pol alpha and delta activities exclusively cosedimented with the MRC fractions from S phase control cells, and were not detected in the MRC fractions from PARP-antisense or uninduced control cells. Immunoblot analysis further revealed that, although PCNA and topo I were present in total extracts from both control and PARP-antisense cells, they were present in the MRC fraction only from induced control cells, indicating that PARP may play a role in their assembly into an active DNA synthesome. In contrast, expression of DNA pol alpha, DNA primase, and RPA was down-regulated in PARP-antisense cells, suggesting that PARP may be involved in the expression of these proteins. Depletion of PARP also prevented induction of the expression of the transcription factor E2F-1, which positively regulates transcription of the DNA pol alpha and PCNA genes; thus, PARP may be necessary for expression of these genes when quiescent cells are stimulated to proliferate.
Assuntos
Proteínas de Transporte , Proteínas de Ciclo Celular , Proteínas de Ligação a DNA , DNA Polimerase Dirigida por DNA/biossíntese , Complexos Multienzimáticos/biossíntese , Poli(ADP-Ribose) Polimerases/metabolismo , Células 3T3 , Animais , DNA Polimerase I/genética , DNA Polimerase I/metabolismo , DNA Polimerase III/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Camundongos , Complexos Multienzimáticos/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Antissenso/biossíntese , Proteína 1 de Ligação ao Retinoblastoma , Fator de Transcrição DP1 , Fatores de Transcrição/biossíntese , Fatores de Transcrição/fisiologia , TransfecçãoRESUMO
Mercuric ion is cytotoxic and mutagenic to cells; however, the mechanisms of mercuric ion-induced cytotoxicity are not well understood. Numerous studies have suggested that these effects may be due in part to the alteration and inhibition of a variety of cellular processes including DNA replication, DNA repair, RNA transcription, and protein synthesis. Studies utilizing whole cells to examine these activities are not able to specifically identify the precise mechanism or site of the effect. Other studies carried out using whole cell extracts and variously purified DNA polymerases are not able to adequately represent the highly ordered environment in which DNA replication occurs in the intact cell. We report here, for the first time, the use of an intact human cell multiprotein complex (which we have termed the DNA synthesome) to carry out full-length DNA replication and DNA synthesis in the presence of Hg2+ ion in vitro. In this study we report that DNA replication and DNA polymerase activity, as well as DNA replication fidelity of the human cell DNA synthesome, are specifically inhibited by physiologically attainable concentrations of mercuric ion.
Assuntos
Replicação do DNA/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/toxicidade , Inibidores da Síntese de Proteínas/toxicidade , Antígenos Virais de Tumores/efeitos dos fármacos , Antígenos Virais de Tumores/genética , Células HeLa , Humanos , Substâncias Macromoleculares , Modelos Biológicos , Complexos Multiproteicos , Testes de Mutagenicidade , Vírus 40 dos Símios/genética , Vírus 40 dos Símios/imunologiaRESUMO
In this report we describe, for the first time, the purification and characterization of a replication-competent multiprotein form of DNA polymerase (designated the DNA synthesome) from the human leukemia cell line (HL-60) using a series of centrifugation, ion-exchange chromatography and velocity sedimentation steps. The proteins and enzymatic activities thus far identified to co-purify with the leukemia cell DNA synthesome include the DNA polymerases alpha and delta, DNA primase, proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), replication protein A (RP-A), and DNA topoisomerases I and II. We have demonstrated that the DNA synthesome is fully competent to replicate simian virus 40 (SV40) replication origin containing DNA in vitro in the presence of the viral large T-antigen. This result implies that all of the cellular activities required for large T-antigen-dependent in vitro SV40 DNA synthesis are present in the isolated human leukemia cell DNA synthesome. Since SV40 is extensively dependent on the host cell's DNA synthetic machinery for its own DNA replication, our results indicate that the isolated leukemia cell DNA synthesome may play a role not only in viral DNA synthesis but also in human leukemia cell DNA replication. We recently proposed a model to represent the DNA synthesome that was isolated from HeLa and murine cells. Our data indicate that the organization of the DNA synthesome from HL-60 cells also fits this proposed model. The purified DNA synthesome will not only allow the further study of the molecular mechanisms required to carry out human leukemia cell DNA replication, but may also provide a tool for eventually dissecting some of the regulatory controls of the cell's DNA synthetic machinery.
Assuntos
Replicação do DNA , DNA Viral/biossíntese , DNA Polimerase Dirigida por DNA/isolamento & purificação , Complexos Multienzimáticos/isolamento & purificação , Cromatografia por Troca Iônica , DNA Polimerase Dirigida por DNA/metabolismo , Células HL-60 , Humanos , Substâncias Macromoleculares , Complexos Multienzimáticos/metabolismoRESUMO
The precise mechanisms involved in the regulation of the mammalian cell DNA-synthesizing machinery are poorly understood. In vitro DNA replication systems, in particular the employment of the simian virus 40 (SV40)-based cell-free DNA replication system, has identified several mammalian enzymes and proteins required for DNA synthesis. Although these proteins have been identified as playing a role in DNA replication, their functional organization allowing for the efficient replication of DNA has not been well defined. This review describes the proteins that have currently been defined as having a role in mammalian DNA replication and their proposed mechanisms of action. How these proteins may organize themselves to form multiprotein complexes, or larger DNA replication factories, allowing for efficient chromosomal DNA synthesis is discussed. In addition, the cell cycle regulation of mammalian DNA synthesis and the current status concerning mammalian DNA replication origins is described.
Assuntos
Replicação do DNA , Animais , Cromossomos/genética , Cromossomos/metabolismo , Ciclinas/farmacologia , DNA Viral/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Origem de Replicação/genética , Vírus 40 dos Símios/metabolismoRESUMO
We have previously described the isolation and characterization of an intact multiprotein complex for DNA replication, designated the DNA synthesome, from human breast cancer cells and biopsied human breast tumor tissue. The purified DNA synthesome was observed to fully support DNA replication in vitro. We had also proposed a model for the breast cell DNA synthesome, in which DNA polymerases alpha, delta, and epsilon, DNA primase, and replication factor C (RF-C) represent members of the core component, or tightly associated, proteins of the complex. This model was based on the observed fractionation, chromatographic, and sedimentation profiles for these proteins. We report here that poly(ADP-ribose)polymerase (PARP) and DNA ligase 1 are also members of the breast cell DNA synthesome core component. More importantly, in this report we present the results of coimmunoprecipitation studies that were designed to map the protein-protein interactions between several members of the core component of the DNA synthesome. Consistent with our proposed model for the breast cell DNA synthesome, our data indicate that DNA polymerases alpha and delta, DNA primase, RF-C, as well as proliferating cell nuclear antigen (PCNA), tightly associate with each other in the complex, whereas DNA polymerase epsilon, PARP, and several other components were found to interact with the synthesome via a direct contact with only PCNA or DNA polymerase alpha. The association of PARP with the synthesome core suggests that this protein may serve a regulatory function in the complex. Also, the coimmunoprecipitation studies suggest that the three DNA polymerases alpha, delta, and epsilon all participate in the replication of breast cell DNA. To our knowledge this is the first report ever to describe the close physical association of polypeptides constituting the intact human breast cell DNA replication apparatus.
Assuntos
Mama/enzimologia , DNA Polimerase Dirigida por DNA/metabolismo , Complexos Multienzimáticos/metabolismo , Células Cultivadas , DNA Ligase Dependente de ATP , DNA Ligases/metabolismo , DNA Polimerase I/metabolismo , DNA Primase/metabolismo , Replicação do DNA , Feminino , Humanos , Mapeamento de Peptídeos , Poli(ADP-Ribose) Polimerases/metabolismo , Antígeno Nuclear de Célula em Proliferação/farmacologia , Ligação ProteicaRESUMO
Increasing evidence has supported the concept that many of the enzymes and factors involved in the replication of mammalian DNA function together as a multiprotein complex. We have previously reported on the partial purification of a multiprotein form of DNA polymerase from human HeLa cells shown to be fully competent to support origin-specific large T-antigen-dependent simian virus 40 (SV40) DNA replication in vitro. In an attempt to more definitively identify the complex or complexes responsible for DNA replication in vitro, partially purified human HeLa cell protein preparations competent to replicate DNA in vitro were subjected to native polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose. The Native Western blots were probed with a panel of antibodies directed against proteins believed to be required for DNA replication in vitro. Apparent complexes of 620 kDa and 500 kDa were identified by monoclonal antibodies directed against DNA polymerase alpha and DNA polymerase delta, respectively. To detect epitopes possibly unexposed within the native multiprotein complexes, blots were also analyzed following denaturation in situ following treatment with detergent and reducing agent. The epitope or access to the epitope recognized by the monoclonal antibody against DNA polymerase alpha was destroyed by exposure of the blots to denaturing conditions. In contrast, an epitope present on a very large complex of approximately 1000 kDa was recognized by a monoclonal antibody against proliferating cell nuclear antigen only following treatment of the native immunoblots with denaturing agents. Identification of these complexes will allow their further purification, characterization, and elucidation of their role in the replication of DNA.
Assuntos
Replicação do DNA , DNA Viral/genética , Proteínas/análise , Receptores de Antígenos de Linfócitos T , Vírus 40 dos Símios , Células HeLa , Humanos , Immunoblotting , Substâncias Macromoleculares , Complexos Multiproteicos , Proteínas/genéticaRESUMO
In recent years, work from a large number of laboratories has greatly expanded our knowledge of the biochemical characteristics and the genetic structure of the DNA polymerases used during papovavirus DNA replication. The development of in vitro DNA replication systems for both SV40 and polyoma virus has been paramount in facilitating the development of the current models describing how DNA polymerase alpha and delta function to replicate the genomes of these two viruses. Our studies have demonstrated that the proteins recognized to be essential for both in vitro SV40 and polyoma viral origin-dependent DNA synthesis can be isolated from cells as an intact complex. We have shown that the human cell MRC closely resembles the murine cell MRC, in both its protein composition and its fractionation and chromatographic profile. In addition, our data regarding both the human and the murine MRC support the dipolymerase model proposed from in vitro DNA replication studies using reconstituted assay systems. In addition, analysis of the nucleotide sequence of the genes encoding DNA polymerase alpha and delta has revealed that the amino acids encoded by several regions of these two genes have been rigorously maintained across evolutionary lines. This information has permitted the identification of protein domains which mediate the complex series of protein-protein interactions that direct the DNA polymerases to the cell nucleus, specify complete or partial exonuclease active sites, and participate in the interaction of each DNA polymerase with the DNA template. Expression studies examining each of the genes encoding DNA polymerase alpha and delta clearly indicate that both DNA polymerases are cell cycle regulated and undergo a dramatic induction in their expression when quiescent cells are stimulated to enter the cell cycle. This is in contrast to the two- to three-fold upregulation in the level of expression of these two genes when cycling cells cross the G1/S boundary. In addition, both proteins are phosphorylated in a cell cycle-dependent manner, and phosphorylation appears to be mediated through the action of a cdc2-dependent protein kinase. Despite all of this new information, much remains to be learned about how papovavirus DNA replication is regulated and how these two DNA polymerases act in vivo to faithfully copy the viral genomes. Studies have yet to be performed which identify all of the cellular factors which potentially mediate papovavirus DNA replication. The reconstituted replication systems have yielded a minimum number of proteins which are required to replicate SV40 and polyoma viral genomes in vitro. However, further studies are needed to identify additional factors which may participate in each step of the initiation, elongation, and termination phases of viral genome replication. As an example, models describing the potential role of cellular helicases, which are components of the MRC isolated from murine and human cells, have yet to be described. It is also conceivable that there are a number of other proteins which serve to attach the MRC to the nuclear matrix, stimulate viral DNA replication, and potentially regulate various aspects of the activity of the MRC throughout viral DNA replication. We are currently working toward characterizing the biochemical composition of the MRC from both murine and human cells. Our goals are to identify all of the structural components of the MRC and to define the role of these components in regulating papovavirus and cellular DNA replication. We have also begun studies to visualize the spatial organization of these protein components within the MRC, examine the regulatory processes controlling the activity of the various components of the MRC, and then develop this information into a coherent picture of the higher order structure of the MRC within the cell nucleus. We believe that this information will enable us to develop an accurate view of the detailed processes mediating both pa
Assuntos
DNA Polimerase II/isolamento & purificação , Replicação do DNA , DNA Polimerase Dirigida por DNA/isolamento & purificação , DNA Polimerase Dirigida por DNA/metabolismo , Papillomaviridae/fisiologia , Polyomaviridae , RNA Nucleotidiltransferases/isolamento & purificação , Vírus 40 dos Símios/fisiologia , Replicação Viral , Animais , Linhagem Celular , Cromatografia de Afinidade/métodos , Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , Clonagem Molecular/métodos , DNA Polimerase II/metabolismo , DNA Polimerase III , DNA Primase , DNA Polimerase Dirigida por DNA/biossíntese , Células HeLa , Humanos , Indicadores e Reagentes , Camundongos , RNA Nucleotidiltransferases/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Vírus 40 dos Símios/enzimologia , Moldes GenéticosRESUMO
In this report, we describe for the first time the isolation and purification of a multiprotein complex for DNA replication from MDA MB-468 human breast cancer cells. This complex, which we designate the DNA synthesome, fully supports the in vitro replication of simian virus 40 (SV40) origin-containing DNA in the presence of the viral large T-antigen. Since the SV40 virus utilizes the host's cellular proteins for its own DNA replication, our results indicate that the DNA synthesome may play a role not only in viral DNA synthesis but in human breast cell DNA replication as well. Our studies demonstrate that the following DNA replication proteins constitute the DNA synthesome: DNA polymerase alpha, DNA primase, DNA polymerase delta, proliferating cell nuclear antigen, replication protein A, replication factor C, DNA topoisomerases I, II, and DNA polymerase epsilon. In addition, we successfully isolated the DNA synthesome from human breast tumor tissue as well as from xenografts from nude mice injected with the human breast cancer cell line MCF-7. The DNA synthesome purified from the breast cancer tissues fully supports SV40 DNA replication in vitro. Furthermore, our results obtained from a novel forward mutagenesis assay suggest that the DNA synthesome isolated from a nonmalignant breast cell line mediates SV40 DNA replication by an error-resistant mechanism. In contrast, the DNA synthesome derived from malignant breast cells and tissue exhibited a lower fidelity for DNA synthesis in vitro. Overall, our data support the role of the DNA synthesome as mediating breast cell DNA replication in vitro and in vivo.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Replicação do DNA/fisiologia , DNA de Neoplasias/biossíntese , Animais , Antígenos Transformantes de Poliomavirus/isolamento & purificação , Neoplasias da Mama/química , Carcinoma Ductal de Mama/química , DNA Primase , DNA Polimerase Dirigida por DNA/análise , Feminino , Humanos , Imuno-Histoquímica , Substâncias Macromoleculares , Camundongos , Camundongos Nus , Complexos Multiproteicos , Transplante de Neoplasias , Antígeno Nuclear de Célula em Proliferação/análise , RNA Nucleotidiltransferases/análise , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
PURPOSE: We have previously reported on the isolation and characterization of a multiprotein DNA replication complex (MRC) from HeLa cells that fully supports in vitro DNA replication. Based upon its ability to replicate DNA in a cell-free environment (devoid of other cellular processes) the MRC may serve as a unique model system for investigating the mechanisms of action of anticancer drugs that directly affect DNA synthesis. The experiments described in this report were performed to establish whether the MRC could serve as a model system to examine in detail the mechanism of action of camptothecin, a DNA topoisomerase I inhibitor. METHODS: We examined the effects of increasing concentrations of camptothecin on HeLa cell survival, intact HeLa cell DNA synthesis and MRC-mediated in vitro DNA replication. We also performed topoisomerase I assays in the presence of increasing concentrations of camptothecin to study the direct effects of the agent on MRC-associated topoisomerase I activity. Furthermore, we employed an SDS precipitation assay to measure the formation of MRC-associated topoisomerase I-cleavable complexes in the presence of increasing concentrations of camptothecin. RESULTS: We found a close correlation between the IC50 values for intact HeLa cell DNA synthesis (0.15 microM) and MRC-mediated in vitro DNA synthesis (0.05 microM). Similarly, we found that 0.05 microM camptothecin inhibited MRC-associated topoisomerase I activity by approximately 50%. In addition, we found that the formation of MRC-associated topoisomerase I-cleavable complexes increased linearly with increasing concentrations of camptothecin. CONCLUSIONS: The data presented in this report support the use of the MRC as a model system to study the mechanism of action of camptothecin. We anticipate that future studies with the MRC will help elucidate the cellular consequences of camptothecin-cleavable complex formation.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacologia , Replicação do DNA/efeitos dos fármacos , DNA/biossíntese , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Inibidores da Topoisomerase IRESUMO
Evidence for multiprotein complexes playing a role in DNA replication has been growing over the years. We have previously reported on a replication-competent multiprotein form of DNA polymerase isolated from human (HeLa) cell extracts. The proteins that were found at that time to co-purify with the human cell multiprotein form of DNA polymerase included: DNA polymerase alpha, DNA primase, topoisomerase I, RNase H, PCNA, and a DNA-dependent ATPase. The multiprotein form of the human cell DNA polymerase was further purified by Q-Sepharose chromatography followed by glycerol gradient sedimentation and was shown to be fully competent to support origin-specific and large T-antigen dependent simian virus 40 (SV40) DNA replication in vitro [Malkas et al. (1990b): Biochemistry 29:6362-6374]. In this report we describe the further characterization of the human cell replication-competent multiprotein form of DNA polymerase designated MRC. Several additional DNA replication proteins that co-purify with the MRC have been identified. These proteins include: DNA polymerase delta, RF-C, topoisomerase II, DNA ligase I, DNA helicase, and RP-A. The replication requirements, replication initiation kinetics, and the ability of the MRC to utilize minichromosome structures for DNA synthesis have been determined. We also report on the results of experiments to determine whether nucleotide metabolism enzymes co-purify with the human cell MRC. We recently proposed a model to represent the MRC that was isolated from murine cells [Wu et al. (1994): J Cell Biochem 54:32-46]. We can now extend this model to include the human cell MRC based on the fractionation, chromatographic and sedimentation behavior of the human cell DNA replication proteins. A full description of the model is discussed. Our experimental results provide further evidence to suggest that DNA synthesis is mediated by a multiprotein complex in mammalian cells.