RESUMO
Natural killer (NK)-cells have potent anti-tumor effects, yet it remains unclear if they are effective for patients with relapsed acute myeloid leukemia (AML). In a phase I clinical trial, we treated 12 patients (median age 60 years) with refractory AML (median 5 lines of prior therapy, median bone marrow blast count of 47%) with fludarabine/cytarabine followed by 6 infusions of NK-cells expanded from haploidentical donors using K562 feeder cells expressing membrane-bound IL21 and 4-1BBL. Patients received 106-107/kg/dose. No toxicity or graft-versus-host disease (GVHD) was observed and MTD was not reached. Seven patients (58.3%) responded and achieved a complete remission (CR) with/without count recovery. Median time to best response was 48 days. Five responding patients proceeded to a haploidentical transplant from the same donor. After a median follow-up of 52 months, 1-year overall survival (OS) for the entire group was 41.7%, better for patients who responded with CR/CRi (57.14%), and for patients who responded and underwent transplantation (60%). Persistence and expansion of donor-derived NK-cells were identified in patients' blood, and serum IFNγ levels rose concurrently with NK cell infusions. A higher count-functional inhibitory KIR was associated with higher likelihood of achieving CR/CRi. In conclusion, we observed a significant response to ex vivo expanded NK-cell administration in refractory AML patients without adverse effects.
Assuntos
Doença Enxerto-Hospedeiro , Leucemia Mieloide Aguda , Humanos , Pessoa de Meia-Idade , Células Matadoras Naturais/patologia , Doença Enxerto-Hospedeiro/etiologia , Citarabina , HaplótiposRESUMO
Antitumor activity of immune cells such as T cells and NK cells has made them auspicious therapeutic regimens for adaptive cancer immunotherapy. Enhancing their cytotoxic effects against malignancies and overcoming their suppression in tumor microenvironment (TME) may improve their efficacy to treat cancers. Clustered, regularly interspaced short palindromic repeats (CRISPR) genome editing has become one of the most popular tools to enhance immune cell antitumor activity. In this review we highlight applications and practicability of CRISPR/Cas9 gene editing and engineering strategies for cancer immunotherapy. In addition, we have reviewed several approaches to study CRISPR off-target effects.
RESUMO
BACKGROUND: The purpose of this review is to summarize our own experimental studies carried out over a 13-year period of time using the F98 rat glioma as model for high grade gliomas. We evaluated a binary chemo-radiotherapeutic modality that combines either cisplatin (CDDP) or carboplatin, administered intracerebrally (i.c.) by means of convection-enhanced delivery (CED) or osmotic pumps, in combination with either synchrotron or conventional X-irradiation. METHODS: F98 glioma cells were implanted stereotactically into the brains of syngeneic Fischer rats. Approximately 14 days later, either CDDP or carboplatin was administered i.c. by CED, followed 24 h later by radiotherapy using either a synchrotron or, subsequently, megavoltage linear accelerators (LINAC). RESULTS: CDDP was administered at a dose of 3 µg in 5 µL, followed 24 h later with an irradiation dose of 15 Gy or carboplatin at a dose of 20 µg in 10 µL, followed 24 h later with 3 fractions of 8 Gy each, at the source at the European Synchrotron Radiation Facility (ESRF). This resulted in a median survival time (MeST) > 180 days with 33% long term survivors (LTS) for CDDP and a MeST > 60 days with 8 to 22% LTS, for carboplatin. Subsequently it became apparent that comparable survival data could be obtained with megavoltage X-irradiation using a LINAC source. The best survival data were obtained with a dose of 72 µg of carboplatin administered by means of Alzet® osmotic pumps over 7 days. This resulted in a MeST of > 180 days, with 55% LTS. Histopathologic examination of all the brains of the surviving rats revealed no residual tumor cells or evidence of significant radiation related effects. CONCLUSIONS: The results obtained using this combination therapy has, to the best of our knowledge, yielded the most promising survival data ever reported using the F98 glioma model.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Encefálicas/terapia , Quimiorradioterapia/métodos , Sistemas de Liberação de Medicamentos , Glioma/terapia , Animais , Neoplasias Encefálicas/patologia , Carboplatina/administração & dosagem , Cisplatino/administração & dosagem , Convecção , Glioma/patologia , Infusões Intralesionais , RatosRESUMO
BACKGROUND: Recurrent pediatric medulloblastoma and ependymoma have a grim prognosis. We report a first-in-human, phase I study of intraventricular infusions of ex vivo expanded autologous natural killer (NK) cells in these tumors, with correlative studies. METHODS: Twelve patients were enrolled, 9 received protocol therapy up to 3 infusions weekly, in escalating doses from 3 × 106 to 3 × 108 NK cells/m2/infusion, for up to 3 cycles. Cerebrospinal fluid (CSF) was obtained for cellular profile, persistence, and phenotypic analysis of NK cells. Radiomic characterization on pretreatment MRI scans was performed in 7 patients, to develop a non-invasive imaging-based signature. RESULTS: Primary objectives of NK cell harvest, expansion, release, and safety of 112 intraventricular infusions of NK cells were achieved in all 9 patients. There were no dose-limiting toxicities. All patients showed progressive disease (PD), except 1 patient showed stable disease for one month at end of study follow-up. Another patient had transient radiographic response of the intraventricular tumor after 5 infusions of NK cell before progressing to PD. At higher dose levels, NK cells increased in the CSF during treatment with repetitive infusions (mean 11.6-fold). Frequent infusions of NK cells resulted in CSF pleocytosis. Radiomic signatures were profiled in 7 patients, evaluating ability to predict upfront radiographic changes, although they did not attain statistical significance. CONCLUSIONS: This study demonstrated feasibility of production and safety of intraventricular infusions of autologous NK cells. These findings support further investigation of locoregional NK cell infusions in children with brain malignancies.
Assuntos
Neoplasias Encefálicas , Neoplasias Cerebelares , Ependimoma , Células Matadoras Naturais/transplante , Meduloblastoma , Adolescente , Neoplasias Encefálicas/líquido cefalorraquidiano , Neoplasias Encefálicas/terapia , Neoplasias Cerebelares/líquido cefalorraquidiano , Neoplasias Cerebelares/terapia , Criança , Ependimoma/líquido cefalorraquidiano , Ependimoma/tratamento farmacológico , Feminino , Humanos , Infusões Intraventriculares , Células Matadoras Naturais/imunologia , Masculino , Meduloblastoma/líquido cefalorraquidiano , Meduloblastoma/terapia , Recidiva Local de NeoplasiaAssuntos
Infecções por Adenoviridae , Transferência Adotiva , Doenças do Recém-Nascido , Linfócitos T , Infecções por Adenoviridae/imunologia , Infecções por Adenoviridae/patologia , Infecções por Adenoviridae/terapia , Feminino , Humanos , Recém-Nascido , Doenças do Recém-Nascido/imunologia , Doenças do Recém-Nascido/patologia , Doenças do Recém-Nascido/terapia , Recém-Nascido Prematuro , Linfócitos T/imunologia , Linfócitos T/transplanteRESUMO
CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGFß. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.
Assuntos
Sistemas CRISPR-Cas/genética , Engenharia Genética/métodos , Terapia Genética/métodos , Imunoterapia/métodos , Células Matadoras Naturais/metabolismo , Ribonucleoproteínas/metabolismo , HumanosRESUMO
The aim of this study was to evaluate four different platinated bioconjugates containing a cisplatin (cis-diamminedichloroplatinum [cis-DDP]) fragment and epidermal growth factor receptor (EGFR)-targeting moieties as potential therapeutic agents for the treatment of brain tumors using a human EGFR-expressing transfectant of the F98 rat glioma (F98EGFR) to assess their efficacy. The first two bioconjugates employed the monoclonal antibody cetuximab (C225 or Erbitux(®)) as the targeting moiety, and the second two used genetically engineered EGF peptides. C225-G5-Pt was produced by reacting cis-DDP with a fifth-generation polyamidoamine dendrimer (G5) and then linking it to C225 by means of two heterobifunctional reagents. The second bioconjugate (C225-PG-Pt) employed the same methodology except that polyglutamic acid was used as the carrier. The third and fourth bioconjugates used two different EGF peptides, PEP382 and PEP455, with direct coordination to the Pt center of the cis-DDP fragment. In vivo studies with C225-G5-Pt failed to demonstrate therapeutic activity following intracerebral (ic) convection-enhanced delivery (CED) to F98EGFR glioma-bearing rats. The second bioconjugate, C225-PG-Pt, failed to show in vitro cytotoxicity. Furthermore, because of its high molecular weight, we decided that lower molecular weight peptides might provide better targeting and microdistribution within the tumor. Both PEP382-Pt and PEP455-Pt bioconjugates were cytotoxic in vitro and, based on this, a pilot study was initiated using PEP455-Pt. The end point for this study was tumor size at 6 weeks following tumor cell implantation and 4 weeks following ic CED of PEP455-Pt to F98 glioma-bearing rats. Neuropathologic examination revealed that five of seven rats were either tumor-free or only had microscopic tumors at 42 days following tumor implantation compared to a mean survival time of 20.5 and 26.3 days for untreated controls. In conclusion, we have succeeded in reformatting the toxicity profile of cis-DDP and demonstrated the therapeutic efficacy of the PEP455-Pt bioconjugate in F98 glioma-bearing rats.
RESUMO
In this report we describe studies with N5-2OH, a carboranyl thymidine analog (CTA), which is a substrate for thymidine kinase 1 (TK1), using the F98 rat glioma model. In vivo BNCT studies have demonstrated that intracerebral (i.c.) osmotic pump infusion of N5-2OH yielded survival data equivalent to those obtained with i.v. administration of boronophenylalanine (BPA). The combination of N5-2OH and BPA resulted in a modest increase in MST of F98 glioma bearing rats compared to a statistically significant increase with the RG2 glioma model, as has been previously reported by us (Barth et al., 2008). This had lead us to synthesize a second generation of CTAs that have improved in vitro enzyme kinetics and in vivo tumor uptake (Agarwal et al., 2015).
Assuntos
Terapia por Captura de Nêutron de Boro , Neoplasias Encefálicas/radioterapia , Timidina Quinase/efeitos dos fármacos , Timidina/administração & dosagem , Animais , Ratos , Timidina/análogos & derivadosRESUMO
A library of sixteen 2nd generation amino- and amido-substituted carboranyl pyrimidine nucleoside analogs, designed as substrates and inhibitors of thymidine kinase 1 (TK1) for potential use in boron neutron capture therapy (BNCT) of cancer, was synthesized and evaluated in enzyme kinetic-, enzyme inhibition-, metabolomic-, and biodistribution studies. One of these 2nd generation carboranyl pyrimidine nucleoside analogs (YB18A [3]), having an amino group directly attached to a meta-carborane cage tethered via ethylene spacer to the 3-position of thymidine, was approximately 3-4 times superior as a substrate and inhibitor of hTK1 than N5-2OH (2), a 1st generation carboranyl pyrimidine nucleoside analog. Both 2 and 3 appeared to be 5'-monophosphorylated in TK1(+) RG2 cells, both in vitro and in vivo. Biodistribution studies in rats bearing intracerebral RG2 glioma resulted in selective tumor uptake of 3 with an intratumoral concentration that was approximately 4 times higher than that of 2. The obtained results significantly advance the understanding of the binding interactions between TK1 and carboranyl pyrimidine nucleoside analogs and will profoundly impact future design strategies for these agents.
Assuntos
Compostos de Boro/uso terapêutico , Terapia por Captura de Nêutron de Boro , Glioma/radioterapia , Inibidores de Proteínas Quinases/farmacologia , Nucleosídeos de Pirimidina/farmacologia , Timidina Quinase/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Glioma/metabolismo , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Nucleosídeos de Pirimidina/síntese química , Nucleosídeos de Pirimidina/química , Ratos , Relação Estrutura-Atividade , Timidina Quinase/metabolismoRESUMO
The purposes of this study were (i) to investigate the differences in effects between 160-kV low-energy and 6-MV high-energy X-rays, both by computational analysis and in vitro studies; (ii) to determine the effects of each on platinum-sensitized F98 rat glioma and murine B16 melanoma cells; and (iii) to describe the in vitro cytotoxicity and in vivo toxicity of a Pt(II) terpyridine platinum (Typ-Pt) complex. Simulations were performed using the Monte Carlo code Geant4 to determine enhancement in absorption of low- versus high-energy X-rays by Pt and to determine dose enhancement factors (DEFs) for a Pt-sensitized tumor phantom. In vitro studies were carried out using Typ-Pt and again with carboplatin due to the unexpected in vivo toxicity of Typ-Pt. Cell survival was determined using clonogenic assays. In agreement with computations and simulations, in vitro data showed up to one log unit reduction in surviving fractions (SFs) of cells treated with 1-4 µg/ml of Typ-Pt and irradiated with 160-kV versus 6-MV X-rays. DEFs showed radiosensitization in the 50-200 keV range, which fell to approximate unity at higher energies, suggesting marginal interactions at MeV energies. Cells sensitized with 1-5 or 7 µg/ml of carboplatin and then irradiated also showed a significant decrease (P < 0.05) in SFs. However, it was unlikely this was due to increased interactions. Theoretical and in vitro studies presented here demonstrated that the tumoricidal activity of low-energy X-rays was greater than that of high-energy X-rays against Pt-sensitized tumor cells. Determining whether radiosensitization is a function of increased interactions will require additional studies.
Assuntos
Apoptose/efeitos da radiação , Carboplatina/administração & dosagem , Modelos Biológicos , Neoplasias Experimentais/patologia , Neoplasias Experimentais/radioterapia , Radioterapia de Alta Energia/métodos , Absorção de Radiação , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Simulação por Computador , Relação Dose-Resposta à Radiação , Camundongos , Modelos Estatísticos , Aceleradores de Partículas , Doses de Radiação , Radiossensibilizantes/administração & dosagem , Ratos , Resultado do TratamentoRESUMO
Unnatural cyclic amino acids (UNAAs) are a new class of boron delivery agents that are in a pre-clinical stage of evaluation. In the present study, the biodistribution of racemic forms of the cis- and trans-isomers of the boronated UNAA 1-amino-3-boronocyclopentanecarboxylic acid (ABCPC) and 1-amino-3-boronocycloheptanecarboxylic acid (ABCHC) were evaluted in B16 melanoma bearing mice and this was compared to l-p-boronophenylalanine (BPA). Boron concentrations were determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES) at 2.5h following intraperitoneal (i.p.) injection of the test agents at a concentration equivalent to 24mg/B/kg. While all compounds attained comparable tumor boron concentrations, the tumor/blood (T/Bl) boron concentration ratios were far superior for both cis-ABCPC and cis-ABCHC compared to BPA (T/Bl=16.4, and 15.1 vs. 5.4). Secondary ion mass spectrometry (SIMS) imaging revealed that the cis-ABCPC delivered boron to the nuclei, as well as the cytoplasm of B16 cells. Next, a biodistribution study of cis-ABCPC and BPA was carried out in F98 glioma bearing rats following i.p. administration. Both compounds attained comparable tumor boron concentrations but the tumor/brain (T/Br) boron ratio was superior for cis-ABCPC compared to BPA (6 vs. 3.3). Since UNAAs are water soluble and cannot be metabolized by tumor cells, they could be potentially more effective boron delivery agents than BPA. Our data suggest that further studies are warranted to evaluate these compounds prior to the initiation of clinical studies.
Assuntos
Aminoácidos Cíclicos/farmacocinética , Aminoácidos Cíclicos/uso terapêutico , Compostos de Boro/farmacocinética , Compostos de Boro/uso terapêutico , Terapia por Captura de Nêutron de Boro/métodos , Glioma/metabolismo , Melanoma/metabolismo , Aminoácidos Cíclicos/química , Animais , Compostos de Boro/química , Linhagem Celular Tumoral , Portadores de Fármacos , Feminino , Glioma/radioterapia , Melanoma/radioterapia , Taxa de Depuração Metabólica , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Ratos , Ratos Endogâmicos F344 , Distribuição Tecidual , Resultado do TratamentoRESUMO
BACKGROUND: In this study we determined if treatment combining radiation therapy (RT) with intracerebral (i.c.) administration of carboplatin to F98 glioma bearing rats could improve survival over that previously reported by us with a 15 Gy dose (5 Gy × 3) of 6 MV photons. METHODS: First, in order to reduce tumor interstitial pressure, a biodistribution study was carried out to determine if pretreatment with dexamethasone alone or in combination with mannitol and furosemide (DMF) would increase carboplatin uptake following convection enhanced delivery (CED). Next, therapy studies were carried out in rats that had received carboplatin either by CED over 30 min (20 µg) or by Alzet pumps over 7 d (84 µg), followed by RT using a LINAC to deliver either 20 Gy (5 Gy × 4) or 15 Gy (7.5 Gy × 2) dose at 6 or 24 hrs after drug administration. Finally, a study was carried out to determine if efficacy could be improved by decreasing the time interval between drug administration and RT. RESULTS: Tumor carboplatin values for D and DMF-treated rats were 9.4 ± 4.4 and 12.4 ± 3.2 µg/g, respectively, which were not significantly different (P = 0.14). The best survival data were obtained by combining pump delivery with 5 Gy × 4 of X-irradiation with a mean survival time (MST) of 107.7 d and a 43% cure rate vs. 83.6 d with CED vs. 30-35 d for RT alone and 24.6 d for untreated controls. Treatment-related mortality was observed when RT was initiated 6 h after CED of carboplatin and RT was started 7 d after tumor implantation. Dividing carboplatin into two 10 µg doses and RT into two 7.5 Gy fractions, administered 24 hrs later, yielded survival data (MST 82.1 d with a 25% cure rate) equivalent to that previously reported with 5 Gy × 3 and 20 µg of carboplatin. CONCLUSIONS: Although the best survival data were obtained by pump delivery, CED was highly effective in combination with 20 Gy, or as previously reported, 15 Gy, and the latter would be preferable since it would produce less late tissue effects.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/radioterapia , Carboplatina/administração & dosagem , Glioma/tratamento farmacológico , Glioma/radioterapia , Animais , Neoplasias Encefálicas/mortalidade , Terapia Combinada , Glioma/mortalidade , Infusões Intraventriculares , Masculino , Dosagem Radioterapêutica , Ratos , Ratos Endogâmicos F344 , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
3-[5-{2-(2,3-Dihydroxyprop-1-yl)-o-carboran-1-yl}pentan-1-yl]thymidine (N5-2OH) is a first generation 3-carboranyl thymidine analog (3CTA) that has been intensively studied as a boron-10 ((10)B) delivery agent for neutron capture therapy (NCT). N5-2OH is an excellent substrate of thymidine kinase 1 and its favorable biodistribution profile in rodents led to successful preclinical NCT of rats bearing intracerebral RG2 glioma. The present study explored cellular influx and efflux mechanisms of N5-2OH, as well as its intracellular anabolism beyond the monophosphate level. N5-2OH entered cultured human CCRF-CEM cells via passive diffusion, whereas the multidrug resistance-associated protein 4 appeared to be a major mediator of N5-2OH monophosphate efflux. N5-2OH was effectively monophosphorylated in cultured murine L929 [thymidine kinase 1 (TK1(+))] cells whereas formation of N5-2OH monophosphate was markedly lower in L929 (TK1(-)) cell variants. Further metabolism to the di- and triphosphate forms was not observed in any of the cell lines. Regardless of monophosphorylation, parental N5-2OH was the major intracellular component in both TK1(+) and TK1(-) cells. Phosphate transfer experiments with enzyme preparations showed that N5-2OH monophosphate, as well as the monophosphate of a second 3-carboranyl thymidine analog [3-[5-(o-carboran-1-yl)pentan-1-yl]thymidine (N5)], were not substrates of thymidine monophosphate kinase. Surprisingly, N5-diphosphate was phosphorylated by nucleoside diphosphate kinase although N5-triphosphate apparently was not a substrate of DNA polymerase. Our results provide valuable information on the cellular metabolism and pharmacokinetic profile of 3-carboranyl thymidine analogs.
Assuntos
Compostos de Boro/administração & dosagem , Compostos de Boro/metabolismo , Terapia por Captura de Nêutron de Boro , Proteínas de Transporte de Nucleosídeos/metabolismo , Timidina Quinase/metabolismo , Timidina/análogos & derivados , Animais , Transporte Biológico , Compostos de Boro/química , Compostos de Boro/farmacologia , Terapia por Captura de Nêutron de Boro/métodos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Camundongos , Estrutura Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Transporte de Nucleosídeos/genética , Fosforilação , Saccharomyces cerevisiae/genética , Especificidade por Substrato , Timidina/administração & dosagem , Timidina/química , Timidina/metabolismo , Timidina/farmacologia , TransfecçãoRESUMO
The purpose of this study was to evaluate two novel liposomal formulations of cisplatin as potential therapeutic agents for treatment of the F98 rat glioma. The first was a commercially produced agent, Lipoplatin™, which currently is in a Phase III clinical trial for treatment of non-small cell lung cancer (NSCLC). The second, produced in our laboratory, was based on the ability of cisplatin to form coordination complexes with lipid cholesteryl hemisuccinate (CHEMS). The in vitro tumoricidal activity of the former previously has been described in detail by other investigators. The CHEMS liposomal formulation had a Pt loading efficiency of 25% and showed more potent in vitro cytotoxicity against F98 glioma cells than free cisplatin at 24 h. In vivo CHEMS liposomes showed high retention at 24 h after intracerebral (i.c.) convection enhanced delivery (CED) to F98 glioma bearing rats. Neurotoxicologic studies were carried out in non-tumor bearing Fischer rats following i.c. CED of Lipoplatin™ or CHEMS liposomes or their "hollow" counterparts. Unexpectedly, Lipoplatin™ was highly neurotoxic when given i.c. by CED and resulted in death immediately following or within a few days after administration. Similarly "hollow" Lipoplatin™ liposomes showed similar neurotoxicity indicating that this was due to the liposomes themselves rather than the cisplatin. This was particularly surprising since Lipoplatin™ has been well tolerated when administered intravenously. In contrast, CHEMS liposomes and their "hollow" counterparts were clinically well tolerated. However, a variety of dose dependent neuropathologic changes from none to severe were seen at either 10 or 14 d following their administration. These findings suggest that further refinements in the design and formulation of cisplatin containing liposomes will be required before they can be administered i.c. by CED for the treatment of brain tumors and that a formulation that may be safe when given systemically may be highly neurotoxic when administered directly into the brain.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Cisplatino/administração & dosagem , Glioma/tratamento farmacológico , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidade , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/química , Cisplatino/farmacocinética , Cisplatino/farmacologia , Cisplatino/toxicidade , Relação Dose-Resposta a Droga , Glioma/patologia , Lipossomos , Masculino , Ratos , Transplante HomólogoRESUMO
Nucleosomes, the fundamental units of chromatin structure, are regulators and barriers to transcription, replication and repair. Post-translational modifications (PTMs) of the histone proteins within nucleosomes regulate these DNA processes. Histone H3(T118) is a site of phosphorylation [H3(T118ph)] and is implicated in regulation of transcription and DNA repair. We prepared H3(T118ph) by expressed protein ligation and determined its influence on nucleosome dynamics. We find H3(T118ph) reduces DNA-histone binding by 2 kcal/mol, increases nucleosome mobility by 28-fold and increases DNA accessibility near the dyad region by 6-fold. Moreover, H3(T118ph) increases the rate of hMSH2-hMSH6 nucleosome disassembly and enables nucleosome disassembly by the SWI/SNF chromatin remodeler. These studies suggest that H3(T118ph) directly enhances and may reprogram chromatin remodeling reactions.
Assuntos
Montagem e Desmontagem da Cromatina , Histonas/metabolismo , Nucleossomos/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histonas/química , Humanos , Proteína 2 Homóloga a MutS/metabolismo , Nucleossomos/química , Fosforilação , Ligação ProteicaRESUMO
Histone post-translational modifications are essential for regulating and facilitating biological processes such as RNA transcription and DNA repair. Fifteen modifications are located in the DNA-histone dyad interface and include the acetylation of H3-K115 (H3-K115Ac) and H3-K122 (H3-K122Ac), but the functional consequences of these modifications are unknown. We have prepared semisynthetic histone H3 acetylated at Lys-115 and/or Lys-122 by expressed protein ligation and incorporated them into single nucleosomes. Competitive reconstitution analysis demonstrated that the acetylation of H3-K115 and H3-K122 reduces the free energy of histone octamer binding. Restriction enzyme kinetic analysis suggests that these histone modifications do not alter DNA accessibility near the sites of modification. However, acetylation of H3-K122 increases the rate of thermal repositioning. Remarkably, Lys --> Gln substitution mutations, which are used to mimic Lys acetylation, do not fully duplicate the effects of the H3-K115Ac or H3-K122Ac modifications. Our results are consistent with the conclusion that acetylation in the dyad interface reduces DNA-histone interaction(s), which may facilitate nucleosome repositioning and/or assembly/disassembly.
Assuntos
DNA/metabolismo , Histonas/química , Histonas/metabolismo , Nucleossomos/química , Proteínas de Xenopus/metabolismo , Acetilação , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Histonas/síntese química , Nucleossomos/genética , Nucleossomos/metabolismo , Ligação Proteica , Proteínas de Xenopus/química , Xenopus laevisRESUMO
Acetylcholine plays an important role in regulation of nervous system development and function. We are developing zebrafish (Danio rerio) as a model system to study the role of specific neuronal nicotinic acetylcholine receptor (nAChR) subtypes in development and the effects of nicotine on the developing vertebrate nervous system. We previously characterized the expression of several zebrafish nAChR subunits. To further develop the zebrafish model, here we report a study on the molecular characterization of two additional nAChR subunit genes, designated chrna6 and chrna4. Both zebrafish nAChRs have a high degree of sequence identity to nAChRs expressed in a variety of mammalian species. Reverse transcription polymerase chain reaction was used to show that both nAChR subunit RNAs were expressed early in zebrafish development, with the chrna4 transcript present at 3 hours postfertilization (hpf) and the chrna6 RNA present at 10 hpf. In situ hybridization was used to localize chrna6 and chrna4 RNA expression in 24, 48, 72, and 96 hpf zebrafish. The chrna6 and chrna4 RNAs were each expressed in a unique pattern, which changed during development. At various ages, chrna6 was expressed in Rohon-Beard sensory neurons, trigeminal ganglion, retina, and the pineal gland. Most notably, chrna6 was expressed in catecholaminergic neurons in the midbrain, but was also present in noncatecholaminergic cells in both midbrain and hindbrain. The expression of chrna6 RNA in catecholaminergic cells supports the use of zebrafish as a valid model system to better understand the molecular basis of cholinergic regulation of dopaminergic signaling and the role of alpha6-containing nAChRs in Parkinson's disease. The most notable chrna4 expression was in neural crest cells at 24 hpf and reticulospinal neurons in hindbrain at 48 hpf. chrna4 RNA exhibited a widespread and robust expression pattern in the midbrain in 72 hpf and 96 hpf zebrafish.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Receptores Nicotínicos/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Sequência Conservada , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Filogenia , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Alinhamento de Sequência , Peixe-Zebra/genéticaRESUMO
At infection sites, polymorphonuclear leukocyte (PMN) function is enhanced ("primed") by granulocyte-macrophage colony-stimulating factor (GM-CSF) or lipopolysaccharide (LPS) and activated by formyl peptides. In this study, GM-CSF or LPS alone had no significant effects on PMN ciprofloxacin transport. Through a mechanism involving protein kinase C, activation by formyl-Met-Leu-Phe (fMLP) significantly decreased the K(m) of ciprofloxacin transport and enhanced ciprofloxacin accumulation. This effect was dramatically enhanced when PMNs were primed with GM-CSF or LPS prior to activation by fMLP.
Assuntos
Fatores Quimiotáticos/farmacologia , Ciprofloxacina/farmacocinética , Neutrófilos/efeitos dos fármacos , Proteína Quinase C/metabolismo , Transporte Biológico/efeitos dos fármacos , Cálcio/química , Quelantes/farmacologia , Fatores Quimiotáticos/metabolismo , Diacilglicerol Quinase/antagonistas & inibidores , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Proteína Quinase C/antagonistas & inibidoresRESUMO
BACKGROUND: Fluoroquinolones and tetracyclines can penetrate epithelial cells, but the mechanism by which they cross the plasma membrane is unclear. In this study, a cell line derived from oral epithelium was used as a model to demonstrate a role for active transport. METHODS: Transport of ciprofloxacin and minocycline by confluent cell monolayers was assayed by measuring the increase in cell-associated fluorescence. RESULTS: Uptake of both agents was saturable and was inhibited at low temperatures. At 37 degrees C, the cells transported ciprofloxacin and minocycline with Km values of 351 and 133 microg/ml, respectively, and maximum velocities of 5.11 and 13.4 ng/min/microg cell protein, respectively. When ciprofloxacin and minocycline were removed from the extracellular medium, the intracellular levels of both agents decreased. Ciprofloxacin efflux from loaded cells occurred more rapidly than with minocycline. Cells accumulated intracellular drug levels that were at least 8-fold higher than extracellular levels for ciprofloxacin and at least 40-fold higher for minocycline. Transport of ciprofloxacin and minocycline was significantly influenced by pH and was most favorable at pH 7.7 and 7.2, respectively. While ciprofloxacin transport was Na+ independent, minocycline transport was strongly inhibited when sodium in the medium was replaced with choline. Transport of both agents was inhibited by a variety of organic cations, but the pattern of inhibition was different. Papaverine, phenylephrine, and doxycycline competitively inhibited minocycline transport, but inhibited ciprofloxacin transport by a non-competitive mechanism. CONCLUSIONS: Epithelial cells take up ciprofloxacin and minocycline via different active transport systems. These transporters may play an important role in enhancing the effectiveness of these agents against invasive pathogens.