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1.
Clin Cancer Res ; 29(20): 4128-4138, 2023 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-37566240

RESUMO

PURPOSE: Non-inflamed (cold) tumors such as leiomyosarcoma do not benefit from immune checkpoint blockade (ICB) monotherapy. Combining ICB with angiogenesis or PARP inhibitors may increase tumor immunogenicity by altering the immune cell composition of the tumor microenvironment (TME). The DAPPER phase II study evaluated the safety, immunologic, and clinical activity of ICB-based combinations in pretreated patients with leiomyosarcoma. PATIENTS AND METHODS: Patients were randomized to receive durvalumab 1,500 mg IV every 4 weeks with either olaparib 300 mg twice a day orally (Arm A) or cediranib 20 mg every day orally 5 days/week (Arm B) until unacceptable toxicity or disease progression. Paired tumor biopsies, serial radiologic assessments and stool collections were performed. Primary endpoints were safety and immune cell changes in the TME. Objective responses and survival were correlated with transcriptomic, radiomic, and microbiome parameters. RESULTS: Among 30 heavily pretreated patients (15 on each arm), grade ≥ 3 toxicity occurred in 3 (20%) and 2 (13%) on Arms A and B, respectively. On Arm A, 1 patient achieved partial response (PR) with increase in CD8 T cells and macrophages in the TME during treatment, while 4 had stable disease (SD) ≥ 6 months. No patients on Arm B achieved PR or SD ≥ 6 months. Transcriptome analysis showed that baseline M1-macrophage and B-cell activity were associated with overall survival. CONCLUSIONS: Durvalumab plus olaparib increased immune cell infiltration of TME with clinical benefit in some patients with leiomyosarcoma. Baseline M1-macrophage and B-cell activity may identify patients with leiomyosarcoma with favorable outcomes on immunotherapy and should be further evaluated.

2.
PLoS One ; 13(6): e0199361, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29928062

RESUMO

The presence of cancer stem cells (CSCs) and the induction of epithelial-to-mesenchymal transition (EMT) in tumors are associated with tumor aggressiveness, metastasis, drug resistance, and poor prognosis, necessitating the development of reagents for unambiguous detection of CSC- and EMT-associated proteins in tumor specimens. To this end, we generated novel antibodies to EMT- and CSC-associated proteins, including Goosecoid, Sox9, Slug, Snail, and CD133. Importantly, unlike several widely used antibodies to CD133, the anti-CD133 antibodies we generated recognize epitopes distal to known glycosylation sites, enabling analyses that are not confounded by differences in CD133 glycosylation. For all target proteins, we selected antibodies that yielded the expected target protein molecular weights by Western analysis and the correct subcellular localization patterns by immunofluorescence microscopy assay (IFA); binding selectivity was verified by immunoprecipitation-mass spectrometry and by immunohistochemistry and IFA peptide blocking experiments. Finally, we applied these reagents to assess modulation of the respective markers of EMT and CSCs in xenograft tumor models by IFA. We observed that the constitutive presence of human hepatocyte growth factor (hHGF) in the tumor microenvironment of H596 non-small cell lung cancer tumors implanted in homozygous hHGF knock-in transgenic mice induced a more mesenchymal-like tumor state (relative to the epithelial-like state when implanted in control SCID mice), as evidenced by the elevated expression of EMT-associated transcription factors detected by our novel antibodies. Similarly, our new anti-CD133 antibody enabled detection and quantitation of drug-induced reductions in CD133-positive tumor cells following treatment of SUM149PT triple-negative breast cancer xenograft models with the CSC/focal adhesion kinase (FAK) inhibitor VS-6063. Thus, our novel antibodies to CSC- and EMT-associated factors exhibit sufficient sensitivity and selectivity for immunofluorescence microscopy studies of these processes in preclinical xenograft tumor specimens and the potential for application with clinical samples.


Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Microambiente Tumoral/efeitos dos fármacos , Antígeno AC133/metabolismo , Animais , Anticorpos Monoclonais/biossíntese , Antineoplásicos/uso terapêutico , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Feminino , Técnicas de Introdução de Genes , Fator de Crescimento de Hepatócito/genética , Humanos , Indicadores e Reagentes , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos Transgênicos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Pirazinas/farmacologia , Pirazinas/uso terapêutico , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Semin Oncol ; 43(4): 484-91, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27663480

RESUMO

Robust pharmacodynamic assay results are valuable for informing go/no-go decisions about continued development of new anti-cancer agents and for identifying combinations of targeted agents, but often pharmacodynamic results are too incomplete or variable to fulfill this role. Our experience suggests that variable reagent and specimen quality are two major contributors to this problem. Minimizing all potential sources of variability in procedures for specimen collection, processing, and assay measurements is essential for meaningful comparison of pharmacodynamic biomarkers across sample time points. This is especially true in the evaluation of pre- and post-dose tumor biopsies, which suffer from high levels of tumor insufficiency due to variations in biopsy collection techniques and significant specimen heterogeneity within and across patients. Developing methods to assess heterogeneous biopsies is necessary in order to evaluate a majority of tumor biopsies collected for pharmacodynamic biomarker studies. Improved collection devices and standardization of methods are being sought in order to improve the tumor content and quality of tumor biopsies. In terms of reagent variability, we have found that stringent initial reagent qualification and quality control of R&D-grade reagents is critical to minimize lot-to-lot variability and prevent assay failures, especially for clinical pharmacodynamic questions, which often demand assay performance that meets or exceeds clinical diagnostic assay standards. Rigorous reagent specifications and use of appropriate assay quality control methodologies help to ensure consistency between assay runs, laboratories and trials to provide much needed pharmacodynamic insights into the activity of investigational agents.


Assuntos
Antineoplásicos/farmacocinética , Biomarcadores Tumorais/análise , Manejo de Espécimes/métodos , Biópsia , Humanos , Indicadores e Reagentes , Neoplasias/patologia , Reprodutibilidade dos Testes , Manejo de Espécimes/normas
4.
Cancer Chemother Pharmacol ; 78(1): 73-81, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27169793

RESUMO

PURPOSE: Indenoisoquinolines are non-camptothecin topoisomerase I (TopI) inhibitors that overcome the limitations of camptothecins: chemical instability and camptothecin resistance. Two dosing schedules of the novel indenoisoquinoline, indotecan (LMP400), were evaluated in patients with advanced solid tumors. METHODS: The maximum tolerated dose (MTD), toxicities, and pharmacokinetics of two indotecan drug administration schedules (daily for 5 days or weekly) were investigated. Modulation of TopI and the phosphorylation of histone H2AX (γH2AX) were assayed in tumor biopsies; γH2AX levels were also evaluated in circulating tumor cells (CTCs) and hair follicles to assess DNA damage response. RESULTS: An MTD of 60 mg/m(2)/day was established for the daily regimen, compared to 90 mg/m(2) for the weekly regimen. The TopI response to drug showed target engagement in a subset of tumor biopsies. Pharmacokinetics profiles demonstrated a prolonged terminal half-life and tissue accumulation compared to topotecan. Dose-dependent decreases in total CTCs were measured in seven patients. Formation of γH2AX-positive foci in CTCs (day 3) and hair follicles (4-6 h) was observed following treatment. CONCLUSIONS: We established the MTD of two dosing schedules for a novel TopI inhibitor, indotecan. Target engagement was demonstrated as Top1 downregulation and γH2AX response. No objective responses were observed on either schedule in this small patient cohort. The principal toxicity of both schedules was myelosuppression; no significant gastrointestinal problems were observed. Increased DNA damage response was observed in CTCs, hair follicles, and a subset of tumor biopsies.


Assuntos
Benzodioxóis/administração & dosagem , DNA Topoisomerases Tipo I/metabolismo , Histonas/metabolismo , Isoquinolinas/administração & dosagem , Neoplasias/tratamento farmacológico , Inibidores da Topoisomerase I/administração & dosagem , Adulto , Idoso , Benzodioxóis/efeitos adversos , Benzodioxóis/farmacocinética , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Esquema de Medicação , Feminino , Folículo Piloso/metabolismo , Meia-Vida , Humanos , Isoquinolinas/efeitos adversos , Isoquinolinas/farmacocinética , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Neoplasias/patologia , Fatores de Tempo , Distribuição Tecidual , Inibidores da Topoisomerase I/efeitos adversos , Inibidores da Topoisomerase I/farmacocinética , Topotecan/farmacocinética , Adulto Jovem
5.
J Exp Clin Cancer Res ; 35: 56, 2016 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-27029323

RESUMO

BACKGROUND: DNA topoisomerase I (Top1) is a DNA unwinding protein and the specific target of the camptothecin class of chemotherapeutic drugs. One of these, irinotecan, acting through its active metabolite SN-38, is used in the treatment of metastatic colorectal cancer. However, resistance to irinotecan represents a major clinical problem. Since molecular alterations in Top1 may result in resistance to irinotecan, we characterized Top1 in three human colon cancer cell lines with acquired resistance to SN-38. METHODS: Three SN-38 resistant (20-67 fold increased resistance) cell lines were generated and compared to wild-type parental cells with regards to: TOP1 gene copy number and gene sequence, Top1 expression (mRNA and protein), Top1 enzymatic activity in the absence and presence of drug, and Top1-DNA cleavage complexes in drug treated cells. TOP1 mutations were validated by PCR using mutant specific primers. Furthermore, cross-resistance to two indenoisoquinoline Top1-targeting drugs (NSC 725776 and NSC 743400) and two Top2-targeting drugs (epirubicin and etoposide) was investigated. RESULTS: Two of three SN-38 resistant cell lines carried TOP1 gene copy number aberrations: A TOP1 gene copy gain and a loss of chromosome 20, respectively. One resistant cell line harbored a pair of yet unreported TOP1 mutations (R364K and G717R) in close proximity to the drug binding site. Mutant TOP1 was expressed at a markedly higher level than wild-type TOP1. None or very small reductions were observed in Top1 expression or Top1 activity in the absence of drug. In all three SN-38 resistant cell lines Top1 activity was maintained in the presence of high concentrations of SN-38. None or only partial cross-resistance were observed for etoposide and epirubicin, respectively. SN-38 resistant cells with wild-type TOP1 remained sensitive to NSC 743400, while cells with mutant TOP1 was fully cross-resistant to both indenoisoquinolines. Top1-DNA cleavage complex formation following drug treatment supported the other findings. CONCLUSIONS: This study adds to the growing knowledge about resistance mechanisms for Top1-targeting chemotherapeutic drugs. Importantly, two yet unreported TOP1 mutations were identified, and it was underlined that cross-resistance to the new indenoisoquinoline drugs depends on the specific underlying molecular mechanism of resistance to SN-38.


Assuntos
Camptotecina/análogos & derivados , Neoplasias do Colo/genética , DNA Topoisomerases Tipo I/genética , Resistencia a Medicamentos Antineoplásicos , Mutação , Benzodioxóis/farmacologia , Sítios de Ligação , Camptotecina/farmacologia , Linhagem Celular Tumoral , Deleção Cromossômica , Neoplasias do Colo/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Epirubicina/farmacologia , Etoposídeo/farmacologia , Dosagem de Genes , Guanidinas/farmacologia , Células HCT116 , Células HT29 , Humanos , Hidrazonas/farmacologia , Irinotecano , Isoquinolinas/farmacologia
6.
Clin Cancer Res ; 22(14): 3683-94, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27001313

RESUMO

PURPOSE: Rational development of targeted MET inhibitors for cancer treatment requires a quantitative understanding of target pharmacodynamics, including molecular target engagement, mechanism of action, and duration of effect. EXPERIMENTAL DESIGN: Sandwich immunoassays and specimen handling procedures were developed and validated for quantifying full-length MET and its key phosphospecies (pMET) in core tumor biopsies. MET was captured using an antibody to the extracellular domain and then probed using antibodies to its C-terminus (full-length) and epitopes containing pY1234/1235, pY1235, and pY1356. Using pMET:MET ratios as assay endpoints, MET inhibitor pharmacodynamics were characterized in MET-amplified and -compensated (VEGFR blockade) models. RESULTS: By limiting cold ischemia time to less than two minutes, the pharmacodynamic effects of the MET inhibitors PHA665752 and PF02341066 (crizotinib) were quantifiable using core needle biopsies of human gastric carcinoma xenografts (GTL-16 and SNU5). One dose decreased pY1234/1235 MET:MET, pY1235-MET:MET, and pY1356-MET:MET ratios by 60% to 80% within 4 hours, but this effect was not fully sustained despite continued daily dosing. VEGFR blockade by pazopanib increased pY1235-MET:MET and pY1356-MET:MET ratios, which was reversed by tivantinib. Full-length MET was quantifiable in 5 of 5 core needle samples obtained from a resected hereditary papillary renal carcinoma, but the levels of pMET species were near the assay lower limit of quantitation. CONCLUSIONS: These validated immunoassays for pharmacodynamic biomarkers of MET signaling are suitable for studying MET responses in amplified cancers as well as compensatory responses to VEGFR blockade. Incorporating pharmacodynamic biomarker studies into clinical trials of MET inhibitors could provide critical proof of mechanism and proof of concept for the field. Clin Cancer Res; 22(14); 3683-94. ©2016 AACR.


Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Células A549 , Animais , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Crizotinibe , Células HEK293 , Células HT29 , Humanos , Imunoensaio/métodos , Indazóis , Indóis/farmacologia , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Camundongos , Camundongos Nus , Pirazóis/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Sulfonamidas/farmacologia , Sulfonas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
7.
Biochemistry ; 55(10): 1494-502, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26885726

RESUMO

Noncovalent second-shell interactions are important in controlling metal-binding affinity and activity in metalloenzymes, but fine-tuning these interactions in designed metalloenzymes has not been fully explored. As a result, most designed metalloenzymes have low metal-binding affinity and activity. Here we identified three mutations in the second coordination shell of an engineered Mn(II)-binding site in cytochrome c peroxidase (called MnCcP.1, containing Glu45, Glu37, and Glu181 ligands) that mimics the native manganese peroxidase (MnP), and explored their effects on both Mn(II)-binding affinity and MnP activity. First, removing a hydrogen bond to Glu45 through Tyr36Phe mutation enhanced Mn(II)-binding affinity, as evidenced by a 2.8-fold decrease in the KM of Mn(II) oxidation. Second, introducing a salt bridge through Lys179Arg mutation improved Glu35 and Glu181 coordination to Mn(II), decreasing KM 2.6-fold. Third, eliminating a steric clash that prevented Glu37 from orienting toward Mn(II) resulted in an 8.6-fold increase in kcat/KM, arising primarily from a 3.6-fold decrease in KM, with a KM value comparable to that of the native enzyme (0.28 mM vs 0.19 mM for Pleurotus eryngii MnP PS3). We further demonstrated that while the effects of Tyr36Phe and Lys179Arg mutations are additive, because involved in secondary-shell interactions to different ligands, other combinations of mutations were antagonistic because they act on different aspects of the Mn(II) coordination at the same residues. Finally, we showed that these MnCcP variants are functional models of MnP that mimic its activity in both Mn(II) oxidation and degradation of a phenolic lignin model compound and kraft lignin. In addition to achieving KM in a designed protein that is similar to the that of native enzyme, our results offer molecular insight into the role of noncovalent interactions around metal-binding sites for improving metal binding and overall activity; such insight can be applied to rationally enhance these properties in other metalloenzymes and their models.


Assuntos
Citocromo-c Peroxidase/metabolismo , Manganês/metabolismo , Peroxidases/metabolismo , Sítios de Ligação/fisiologia , Cristalização , Citocromo-c Peroxidase/química , Ativação Enzimática/fisiologia , Manganês/química , Peroxidases/química , Estrutura Secundária de Proteína
8.
Cancer Res ; 74(23): 6968-79, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25269479

RESUMO

Camptothecin and its derivatives, topotecan and irinotecan, are specific topoisomerase I (Top1) inhibitors and potent anticancer drugs killing cancer cells by producing replication-associated DNA double-strand breaks, and the indenoisoquinoline LMP-400 (indotecan) is a novel Top1 inhibitor in clinical trial. To develop novel drug combinations, we conducted a synthetic lethal siRNA screen using a library that targets nearly 7,000 human genes. Depletion of ATR, the main transducer of replication stress, came as a top candidate gene for camptothecin synthetic lethality. Validation studies using ATR siRNA and the ATR inhibitor VE-821 confirmed marked antiproliferative synergy with camptothecin and even greater synergy with LMP-400. Single-cell analyses and DNA fiber combing assays showed that VE-821 abrogates the S-phase replication elongation checkpoint and the replication origin-firing checkpoint induced by camptothecin and LMP-400. As expected, the combination of Top1 inhibitors with VE-821 inhibited the phosphorylation of ATR and Chk1; however, it strongly induced γH2AX. In cells treated with the combination, the γH2AX pattern changed over time from the well-defined Top1-induced damage foci to an intense peripheral and diffuse nuclear staining, which could be used as response biomarker. Finally, the clinical derivative of VE-821, VX-970, enhanced the in vivo tumor response to irinotecan without additional toxicity. A key implication of our work is the mechanistic rationale and proof of principle it provides to evaluate the combination of Top1 inhibitors with ATR inhibitors in clinical trials.


Assuntos
Replicação do DNA/efeitos dos fármacos , DNA Topoisomerases Tipo I/metabolismo , Compostos Organotiofosforados/farmacologia , Pirazinas/farmacologia , Origem de Replicação/efeitos dos fármacos , Sulfonas/farmacologia , Inibidores da Topoisomerase I/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Dano ao DNA , Células HT29 , Histonas/genética , Histonas/metabolismo , Humanos , Irinotecano , Fosforilação/efeitos dos fármacos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Análise de Célula Única/métodos , Topotecan/farmacologia
9.
Biochemistry ; 53(23): 3781-9, 2014 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-24901481

RESUMO

The location of the Trp radical and the catalytic function of the [Fe(IV)═O Trp191(•+)] intermediate in cytochrome c peroxidase (CcP) are well-established; however, the unambiguous identification of the site(s) for the formation of tyrosyl radical(s) and their possible biological roles remain elusive. We have now performed a systematic investigation of the location and reactivity of the Tyr radical(s) using multifrequency Electron Paramagnetic Resonance (EPR) spectroscopy combined with multiple-site Trp/Tyr mutations in CcP. Two tyrosines, Tyr71 and Tyr236, were identified as those contributing primarily to the EPR spectrum of the tyrosyl radical, recorded at 9 and 285 GHz. The EPR characterization also showed that the heme distal-side Trp51 is involved in the intramolecular electron transfer between Tyr71 and the heme and that formation of Tyr71(•) and Tyr236(•) is independent of the [Fe(IV)═O Trp191(•+)] intermediate. Tyr71 is located in an optimal position to mediate the oxidation of substrates binding at a site, more than 20 Šfrom the heme, which has been reported recently in the crystal structures of CcP with bound guaicol and phenol [Murphy, E. J., et al. (2012) FEBS J. 279, 1632-1639]. The possibility of discriminating the radical intermediates by their EPR spectra allowed us to identify Tyr71(•) as the reactive species with the guaiacol substrate. Our assignment of the surface-exposed Tyr236 as the other radical site agrees well with previous studies based on MNP labeling and protein cross-linking [Tsaprailis, G., and English, A. M. (2003) JBIC, J. Biol. Inorg. Chem. 8, 248-255] and on its covalent modification upon reaction of W191G CcP with 2-aminotriazole [Musah, R. A., and Goodin, D. B. (1997) Biochemistry 36, 11665-11674]. Accordingly, while Tyr71 acts as a true reactive intermediate for the oxidation of certain small substrates that bind at a site remote from the heme, the surface-exposed Tyr236 would be more likely related to oxidative stress signaling, as previously proposed. Our findings reinforce the view that CcP is the monofunctional peroxidase that most closely resembles its ancestor enzymes, the catalase-peroxidases, in terms of the higher complexity of the peroxidase reaction [Colin, J., et al. (2009) J. Am. Chem. Soc. 131, 8557-8563]. The strategy used to identify the elusive Tyr radical sites in CcP may be applied to other heme enzymes containing a large number of Tyr and Trp residues and for which Tyr (or Trp) radicals have been proposed to be involved in their peroxidase or peroxidase-like reaction.


Assuntos
Citocromo-c Peroxidase/metabolismo , Expectorantes/metabolismo , Guaiacol/metabolismo , Heme/metabolismo , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/metabolismo , Tirosina/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Biocatálise , Citocromo-c Peroxidase/química , Citocromo-c Peroxidase/genética , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Expectorantes/química , Guaiacol/química , Heme/química , Cinética , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oxirredução , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Propriedades de Superfície , Triptofano/química , Triptofano/metabolismo , Tirosina/química
10.
PLoS One ; 7(12): e50494, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23284638

RESUMO

BACKGROUND: Topoisomerase I (Top1) is a proven target for cancer therapeutics. Recent data from the Fluorouracil, Oxaliplatin, CPT-11: Use and Sequencing (FOCUS) trial demonstrated that nuclear staining of Top1 correlates with chemotherapeutic efficacy. Such a correlation may help identify patients likely to respond to Top1 inhibitors and illuminate their mechanism of action. Cellular response to Top1 inhibitors is complex, but Top1 target engagement is a necessary first step in this process. This paper reports the development and validation of a quantitative immunoassay for Top1 in tumors. METHODOLOGY/PRINCIPAL FINDINGS: We have developed and validated a two-site enzyme chemiluminescent immunoassay for quantifying Top1 levels in tumor biopsies. Analytical validation of the assay established the inter-day coefficient of variation at 9.3%±3.4% and a 96.5%±7.3% assay accuracy. Preclinical fit-for-purpose modeling of topotecan time- and dose-effects was performed using topotecan-responsive and -nonresponsive xenografts in athymic nude mice. Higher baseline levels of Top1 were observed in topotecan-responsive than -nonresponsive tumors. Top1 levels reached a maximal decrease 4 to 7 hours following treatment of engrafted mice with topotecan and the indenoisoquinoline NSC 724998. CONCLUSIONS/SIGNIFICANCE: Our analysis of Top1 levels in control and treated tumors supports the previously proposed mechanism of action for Top1 inhibitor efficacy, wherein higher baseline Top1 levels lead to formation of more covalent-complex-dependent double-strand break damage and, ultimately, cell death. In contrast, xenografts with lower baseline Top1 levels accumulate fewer double-stand breaks, and may be more resistant to Top1 inhibitors. Our results support further investigation into the use of Top1 levels in tumors as a potential predictive biomarker. The Top1 immunoassay described in this paper has been incorporated into a Phase I clinical trial at the National Cancer Institute to assess pharmacodynamic response in tumor biopsies and determine whether baseline Top1 levels are predictive of response to indenoisoquinoline Top1 inhibitors.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , Imunoensaio/métodos , Neoplasias/enzimologia , Neoplasias/patologia , Animais , Biópsia , Linhagem Celular Tumoral , Ensaios Clínicos como Assunto , DNA Topoisomerases Tipo I/sangue , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Isoquinolinas/química , Isoquinolinas/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/enzimologia , Camundongos , Neoplasias/tratamento farmacológico , Poli(ADP-Ribose) Polimerases/metabolismo , Fatores de Tempo , Inibidores da Topoisomerase I/farmacologia , Topotecan/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Cancer Res ; 70(6): 2191-203, 2010 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-20215517

RESUMO

Because topoisomerase 1 (TOP1) is critical for the relaxation of DNA supercoils and because it is the target for the anticancer activity of camptothecins, we assessed TOP1 transcript levels in the 60 cell line panel (the NCI-60) of the National Cancer Institute's anticancer drug screen. TOP1 expression levels varied over a 5.7-fold range across the NCI-60. HCT116 colon and MCF-7 breast cancer cells were the highest expressers; SK-MEL-28 melanoma and HS578T breast carcinoma cells were the lowest. TOP1 mRNA expression was highly correlated with Top1 protein levels, indicating that TOP1 transcripts could be conveniently used to monitor Top1 protein levels and activity in tissues. Assessment of the TOP1 locus by array comparative genomic hybridization across the NCI-60 showed copy numbers ranging from 1.71 to 4.13 and a statistically significant correlation with TOP1 transcript levels (P < 0.01). Further analyses of TOP1 expression on an exon-specific basis revealed that exon 1 expression was generally higher and less variable than expression of the other exons, suggesting some form of transcriptional pausing regulation between exons 1 and 2. Accordingly, we found the presence of multiple evolutionarily conserved potential G-quadruplex-forming sequences in the first TOP1 intron. Physicochemical tests for actual quadruplex formation by several of those sequences yielded quadruplex formation for two of them and duplex formation for one. The observations reported here suggest the hypothesis that there is a conserved negative transcription regulator within intron 1 of the TOP1 gene associated with a quadruplex-prone region.


Assuntos
DNA Topoisomerases Tipo I/genética , DNA de Neoplasias/genética , Éxons , Íntrons , Neoplasias/genética , Linhagem Celular Tumoral , Dicroísmo Circular , DNA Topoisomerases Tipo I/biossíntese , DNA de Neoplasias/metabolismo , Quadruplex G , Dosagem de Genes , Guanosina/genética , Guanosina/metabolismo , Humanos , Neoplasias/enzimologia , Análise de Sequência com Séries de Oligonucleotídeos , Transcrição Gênica
12.
Clin Cancer Res ; 16(3): 1073-84, 2010 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-20103672

RESUMO

PURPOSE: Circulating tumor cells (CTC) in peripheral blood of patients potentially represent a fraction of solid tumor cells available for more frequent pharmacodynamic assessment of drug action than is possible using tumor biopsy. However, currently available CTC assays are limited to cell membrane antigens. Here, we describe an assay that directly examines changes in levels of the nuclear DNA damage marker gammaH2AX in individual CTCs of patients treated with chemotherapeutic agents. EXPERIMENTAL DESIGN: An Alexa Fluor 488-conjugated monoclonal gammaH2AX antibody and epithelial cancer cell lines treated with topotecan and spiked into whole blood were used to measure DNA damage-dependent nuclear gammaH2AX signals in individual CTCs. Time-course changes in both CTC number and gammaH2AX levels in CTCs were also evaluated in blood samples from patients undergoing treatment. RESULTS: The percentage of gammaH2AX-positive CTCs increased in a concentration-dependent manner in cells treated with therapeutically relevant concentrations of topotecan ex vivo. In samples from five patients, percent gammaH2AX-positive cells increased post-treatment from a mean of 2% at baseline (range, 0-6%) to a mean of 38% (range, 22-64%) after a single day of drug administration; this increase was irrespective of increases or decreases in the total CTC count. CONCLUSIONS: These data show promise for monitoring dynamic changes in nuclear biomarkers in CTCs (in addition to CTC count) for rapidly assessing drug activity in clinical trials of molecularly targeted anticancer therapeutics as well as for translational research.


Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/sangue , Monitoramento de Medicamentos/métodos , Histonas/sangue , Neoplasias/sangue , Células Neoplásicas Circulantes/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Dano ao DNA , Humanos , Neoplasias/tratamento farmacológico , Variações Dependentes do Observador
13.
Mol Cancer Ther ; 8(7): 1878-84, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19584232

RESUMO

Topoisomerase I (Top1) is a proven target for cancer therapeutics, and the level of Top1 in tumors has been used as a biomarker for chemotherapeutic efficacy. In this study, we report the development and validation of a two-site enzyme chemiluminescent immunoassay for Top1, which we used to measure Top1 levels in the NCI-60 cancer cell line panel. Top1 levels ranged from 0.9 to 9.8 ng/mL/microg protein extract in these cell lines. Levels varied both within and between cancer types but were generally highest in colon cancer and leukemia cell lines and lowest in central nervous system and renal cancer cell lines. Top1 mRNA levels in the NCI-60 cell lines were also measured by microarray; mRNA values generally showed a good correlation with protein levels (Pearson correlation = 0.8). When these expression levels were compared with the activity of the indenoisoquinoline class of Top1 inhibitors across the NCI-60 cell panel, low levels of Top1 were associated with increased resistance to these drugs. The results of our studies indicate that our Top1 assay can be used to quantify Top1 levels in untreated cells as well as cells treated with Top1 inhibitors and that the assay has the potential to be adapted for use in predicting clinical response to Top1-active antineoplastic agents.


Assuntos
Antineoplásicos/farmacologia , DNA Topoisomerases Tipo I/análise , Isoquinolinas/química , Western Blotting , Linhagem Celular Tumoral/química , Proliferação de Células/efeitos dos fármacos , DNA Topoisomerases Tipo I/genética , Ensaio de Imunoadsorção Enzimática , Humanos , Indenos/química , Medições Luminescentes , Análise em Microsséries , National Cancer Institute (U.S.) , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estados Unidos
14.
J Biol Inorg Chem ; 12(1): 126-37, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17021923

RESUMO

Manganese peroxidase (MnP) from the white rot fungus Phanerochaete chrysosporium contains a manganese-binding site that plays a critical role in its function. Previously, a Mn(II)-binding site was designed into cytochrome c peroxidase (CcP) based on sequence homology (Yeung et al. in Chem. Biol. 4:215-222, 1997; Gengenbach et al. in Biochemistry 38:11425-11432, 1999). Here, we report a redesign of this site based on X-ray structural comparison of MnP and CcP. The variant, CcP(D37E, V45E, H181E), displays 2.5-fold higher catalytic efficiency (k (cat)/K (M)) than the variant in the original design, mostly due to a stronger K (M) of 1.9 mM (vs. 4.1 mM). High-resolution X-ray crystal structures of a metal-free form and a form with Co(II) at the designed Mn(II) site were also obtained. The metal ion in the engineered metal-binding site overlays well with Mn(II) bound in MnP, suggesting that this variant is the closest structural model of the Mn(II)-binding site in MnP for which a crystal structure exists. A major difference arises in the distances of the ligands to the metal; the metal-ligand interactions in the CcP variant are much weaker than the corresponding interactions in MnP, probably owing to partial occupancy of metal ion at the designed site, difference in the identity of metal ions (Co(II) rather than Mn(II)) and other interactions in the second coordination sphere. These results indicate that the metal ion, the ligands, and the environment around the metal-binding site play important roles in tuning the structure and function of metalloenzymes.


Assuntos
Citocromo-c Peroxidase/química , Manganês/química , Sítios de Ligação , Cristalografia por Raios X , Citocromo-c Peroxidase/genética , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oxirredução , Sensibilidade e Especificidade , Homologia de Sequência de Aminoácidos , Espectrofotometria Ultravioleta/métodos , Relação Estrutura-Atividade
15.
Arch Insect Biochem Physiol ; 62(1): 43-54, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16612809

RESUMO

Larvae of the goldenrod gall moth, Epiblema scudderiana, use the freeze avoidance strategy of winter cold hardiness and show multiple metabolic adaptations for subzero survival including accumulation of large amounts of glycerol as a colligative antifreeze. Induction and regulation of cold hardiness adaptations requires the intermediary action of signal transduction enzymes. Changes in the activities of several signaling enzymes including cAMP-dependent protein kinase (PKA), protein phosphatases 1 (PP1), 2A, 2C, and protein tyrosine phosphatases (PTPs) were monitored over the winter and during experimental exposures of larvae to subzero temperatures (-4 degrees C, a temperature that triggers rapid glycerol synthesis, or -20 degrees C, a common midwinter ambient temperature) or anoxia. A strong increase in the amount of active PP1 in the latter part of the winter may be responsible for shutting off glycogenolysis once glycerol levels are maximized. There appears to be a limited role for PKA in overwintering but PP2A and PP2C activities rose when larvae were exposed to -20 degrees C and PTP activities rose significantly over the winter months and also in response to laboratory subzero (-20 degrees C) and anoxia exposures. The strong responses by PTPs suggest that these may be involved in cell cycle and growth arrest during winter diapause.


Assuntos
Temperatura Baixa , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Mariposas/enzimologia , Mariposas/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Animais , Congelamento , Regulação Enzimológica da Expressão Gênica , Hipóxia/metabolismo , Larva , Estações do Ano , Fatores de Tempo
16.
Insect Biochem Mol Biol ; 36(1): 18-24, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16360946

RESUMO

Freeze-tolerant larvae of the goldenrod gall fly, Eurosta solidaginis Fitch, show multiple metabolic adaptations for subzero survival including the autumn synthesis of high concentrations of polyols. The induction and regulation of cold hardiness adaptations requires the intermediary action of signal transduction enzymes. The present study evaluates changes in the activities of cAMP-dependent protein kinase (PKA), protein phosphatases 1 (PP1), 2A, 2C, and protein tyrosine phosphatases (PTPs) over the course of the winter season and also in insects exposed to -4, -20 degrees C, or anoxic conditions in the laboratory. The increased PKA and decreased PP1 over the winter season and/or at subzero temperature support a regulatory role for these enzymes in cryoprotectant polyol synthesis. PTP activities were also strongly increased under these conditions and may act to antagonize tyrosine kinase mediated cell growth and proliferation responses and, thereby, contribute to hypometabolism and diapause over the winter.


Assuntos
Adaptação Fisiológica , Temperatura Baixa/efeitos adversos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dípteros/enzimologia , Dípteros/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Animais , Congelamento , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Hipóxia , Larva/enzimologia , Larva/fisiologia , Estações do Ano , Fatores de Tempo
17.
J Am Chem Soc ; 127(16): 5750-1, 2005 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-15839648

RESUMO

The physiological electron-transfer (ET) partners, cytochrome c peroxidase (CcP) and cytochrome c (Cc)1, can be modified to exhibit photoinitiated ET through substitution of Zn (or Mg) for Fe in either partner. Laser excitation of the Zn-porphyrin (ZnP) to its triplet excited state (3ZnP) initiates direct heme-heme ET to the ferriheme center of its partner across the protein-protein interface. This photoinitiated ET produces the charge-separated intermediate, I = [ZnP+CcP, Fe2+Cc], with a metalloporphyrin pi-cation radical (ZnP+) in the donor protein and a ferroheme acceptor protein. I, in general, is thought to return to the ground state by a thermal ET process that involves direct heme-heme back-ET to complete a simple photocycle. We here contrast intracomplex ET between yeast iso-1 Cc and ZnCcP(WT) (wild-type) with that for two ZnCcP(X) variants: X = W191F, with redox-active W191 replaced by Phe; WYM4, a W191F mutant with further replacement of four other potentially redox-active sites (W51F, Y187F, Y229F, and Y236F). The results show that W191 acts as an ET mediator, which "short-circuits" the direct heme-heme back-ET through a two-step, hopping process in which the ZnP+ cation radical formed by photoinitiated ET rapidly oxidizes W191, and the resultant W191+, in turn, rapidly oxidizes Fe2+Cc.


Assuntos
Citocromo-c Peroxidase/química , Citocromos c/química , Zinco/química , Citocromo-c Peroxidase/metabolismo , Citocromos c/metabolismo , Heme/química , Heme/metabolismo , Cinética , Oxirredução , Fotoquímica , Zinco/metabolismo
18.
J Biol Chem ; 280(13): 12858-66, 2005 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15664991

RESUMO

Myoglobin (Mb) is used as a model system for other heme proteins and the reactions they catalyze. The latest novel function to be proposed for myoglobin is a P450 type hydroxylation activity of aromatic carbons (Watanabe, Y., and Ueno, T. (2003) Bull. Chem. Soc. Jpn. 76, 1309-1322). Because Mb does not contain a specific substrate binding site for aromatic compounds near the heme, an engineered tryptophan in the heme pocket was used to model P450 hydroxylation of aromatic compounds. The monooxygenation product was not previously isolated because of rapid subsequent oxidation steps (Hara, I., Ueno, T., Ozaki, S., Itoh, S., Lee, K., Ueyama, N., and Watanabe, Y. (2001) J. Biol. Chem. 276, 36067-36070). In this work, a Mb variant (F43W/H64D/V68I) is used to characterize the monooxygenated intermediate. A modified (+16 Da) species forms upon the addition of 1 eq of H2O2. This product was digested with chymotrypsin, and the modified peptide fragments were isolated and characterized as 6-hydroxytryptophan using matrix-assisted laser desorption ionization time-of-flight tandem mass spectroscopy and 1H NMR. This engineered Mb variant represents the first enzyme to preferentially hydroxylate the indole side chain of Trp at the C6 position. Finally, heme extraction was used to demonstrate that both the formation of the 6-hydroxytryptophan intermediate (+16 Da) and subsequent oxidation to form the +30 Da final product are catalyzed by the heme cofactor, most probably via the compound I intermediate. These results provide insight into the mechanism of hydroxylation of aromatic carbons by heme proteins, demonstrating that non-thiolate-ligated heme enzymes can perform this function. This establishes Mb compound I as a model for P450 type aromatic hydroxylation chemistry.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Peróxido de Hidrogênio/farmacologia , Mioglobina/genética , 5-Hidroxitriptofano/química , Animais , Sítios de Ligação , Catalase/química , Catálise , Cromatografia Líquida de Alta Pressão , Quimotripsina/química , Escherichia coli/metabolismo , Heme/química , Hemeproteínas , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Mutação , Mioglobina/química , Oxigênio/química , Oxigênio/metabolismo , Peptídeos/química , Fosfatos/química , Compostos de Potássio/química , Estrutura Terciária de Proteína , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria , Triptofano/química , Raios Ultravioleta , Baleias
19.
J Am Chem Soc ; 126(35): 10812-3, 2004 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-15339144

RESUMO

Introducing nonnative metal ions or metal-containing prosthetic groups into a protein can dramatically expand the repertoire of its functionalities and thus its range of applications. Particularly challenging is the control of substrate-binding and thus reaction selectivity such as enantioselectivity. To meet this challenge, both non-covalent and single-point attachments of metal complexes have been demonstrated previously. Since the protein template did not evolve to bind artificial metal complexes tightly in a single conformation, efforts to restrict conformational freedom by modifying the metal complexes and/or the protein are required to achieve high enantioselectivity using the above two strategies. Here we report a novel site-selective dual anchoring (two-point covalent attachment) strategy to introduce an achiral manganese salen complex (Mn(salen)), into apo sperm whale myoglobin (Mb) with bioconjugation yield close to 100%. The enantioselective excess increases from 0.3% for non-covalent, to 12.3% for single point, and to 51.3% for dual anchoring attachments. The dual anchoring method has the advantage of restricting the conformational freedom of the metal complex in the protein and can be generally applied to protein incorporation of other metal complexes with minimal structural modification to either the metal complex or the protein.


Assuntos
Heme/química , Metaloproteínas/química , Animais , Apoproteínas/química , Apoproteínas/metabolismo , Sítios de Ligação , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Cromo/química , Citocromo-c Peroxidase/química , Citocromo-c Peroxidase/metabolismo , Etilenodiaminas/química , Etilenodiaminas/metabolismo , Heme/metabolismo , Heme/fisiologia , Metaloproteínas/síntese química , Metaloproteínas/metabolismo , Modelos Moleculares , Mioglobina/química , Mioglobina/metabolismo , Compostos Organometálicos/química , Compostos Organometálicos/metabolismo , Espectrofotometria Ultravioleta , Estereoisomerismo
20.
J Biol Inorg Chem ; 8(7): 699-706, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-14505074

RESUMO

Cytochrome c peroxidase (C cP) variants with an engineered Mn(II) binding site, including MnC cP [C cP(MI, G41E, V45E, H181D)], MnC cP(W191F), and MnC cP(W191F, W51F), that mimic manganese peroxidase (MnP), have been characterized by resonance Raman (RR) spectroscopy. Analysis of the Raman bands in the 200-700 cm(-1) and 1300-1650 cm(-1) regions indicates that both the coordination and spin state of the heme iron in the variants differ from that of C cP(MI), the recombinant yeast C cP containing additional Met-Ile residues at the N-terminus. At neutral pH the frequencies of the nu(3) mode indicate that a pure five-coordinate heme iron exists in C cP(MI) whereas a six-coordinate low-spin iron is the dominant species in the C cP variants with the engineered Mn(II) binding site. The H181D mutation, which weakens the proximal linkage to the heme iron, may be responsible for these spectral and structural changes. Raman spectra of the variants C cP(MI, W191F) and C cP(MI, W191F, W51F) were also obtained to clarify the structural and functional roles of mutations at two tryptophan sites. The W51F mutation was found to disrupt H-bonding to the distal water molecules and the resulting variants tended to form transitional or mixed coordination states that possess spectral and structural features similar to that of MnP. Such structural features, with a loosened distal water, may facilitate the binding of H(2)O(2) and increase the rate constant for compound I formation. This effect, in addition to the elimination of an H-bond to ferryl oxygen by the same mutation, accounts for the increased MnP specific activity of MnC cP(W191F, W51F).


Assuntos
Citocromo-c Peroxidase/química , Citocromo-c Peroxidase/genética , Peroxidases/metabolismo , Análise Espectral Raman , Substituição de Aminoácidos , Sítios de Ligação , Citocromo-c Peroxidase/metabolismo , Proteínas Fúngicas , Heme/química , Peróxido de Hidrogênio/metabolismo , Cinética , Mimetismo Molecular , Mutagênese Sítio-Dirigida , Engenharia de Proteínas
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