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1.
Nat Chem Biol ; 13(11): 1164-1171, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28892090

RESUMO

Small ubiquitin-like modifier (SUMO) family proteins regulate target-protein functions by post-translational modification. However, a potent and selective inhibitor targeting the SUMO pathway has been lacking. Here we describe ML-792, a mechanism-based SUMO-activating enzyme (SAE) inhibitor with nanomolar potency in cellular assays. ML-792 selectively blocks SAE enzyme activity and total SUMOylation, thus decreasing cancer cell proliferation. Moreover, we found that induction of the MYC oncogene increased the ML-792-mediated viability effect in cancer cells, thus indicating a potential application of SAE inhibitors in treating MYC-amplified tumors. Using ML-792, we further explored the critical roles of SUMOylation in mitotic progression and chromosome segregation. Furthermore, expression of an SAE catalytic-subunit (UBA2) S95N M97T mutant rescued SUMOylation loss and the mitotic defect induced by ML-792, thus confirming the selectivity of ML-792. As a potent and selective SAE inhibitor, ML-792 provides rapid loss of endogenously SUMOylated proteins, thereby facilitating novel insights into SUMO biology.


Assuntos
Inibidores Enzimáticos/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/antagonistas & inibidores , Sumoilação , Proliferação de Células/efeitos dos fármacos , Segregação de Cromossomos/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes myc , Humanos , Mitose/efeitos dos fármacos , Neoplasias/genética , Neoplasias/patologia , Processamento de Proteína Pós-Traducional , Células Tumorais Cultivadas
2.
Mol Cell Proteomics ; 10(11): M111.009183, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21873567

RESUMO

Cullin-RING ubiquitin ligases (CRLs) are responsible for the ubiquitination of many cellular proteins, thereby targeting them for proteasomal degradation. In most cases the substrates of the CRLs have not been identified, although many of those that are known have cancer relevance. MLN4924, an investigational small molecule that is a potent and selective inhibitor of the Nedd8-activating enzyme (NAE), is currently being explored in Phase I clinical trials. Inhibition of Nedd8-activating enzyme by MLN4924 prevents the conjugation of cullin proteins with NEDD8, resulting in inactivation of the entire family of CRLs. We have performed stable isotope labeling with amino acids in cell culture analysis of A375 melanoma cells treated with MLN4924 to identify new CRL substrates, confidently identifying and quantitating 5122-6012 proteins per time point. Proteins such as MLX, EID1, KLF5, ORC6L, MAGEA6, MORF4L2, MRFAP1, MORF4L1, and TAX1BP1 are rapidly stabilized by MLN4924, suggesting that they are novel CRL substrates. Proteins up-regulated at later times were also identified and siRNA against their corresponding genes were used to evaluate their influence on MLN4924-induced cell death. Thirty-eight proteins were identified as being particularly important for the cytotoxicity of MLN4924. Strikingly, these proteins had roles in cell cycle, DNA damage repair, and ubiquitin transfer. Therefore, the combination of RNAi with stable isotope labeling with amino acids in cell culture provides a paradigm for understanding the mechanism of action of novel agents affecting the ubiquitin proteasome system and a path to identifying mechanistic biomarkers.


Assuntos
Ciclopentanos/farmacologia , Proteoma/metabolismo , Pirimidinas/farmacologia , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Cinética , Fenótipo , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Proteólise , Proteoma/genética , Proteômica , Interferência de RNA , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitinação
3.
Proc Natl Acad Sci U S A ; 107(8): 3698-703, 2010 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-20133671

RESUMO

Gene expression signatures are used in the clinic as prognostic tools to determine the risk of individual patients with localized breast tumors developing distant metastasis. We lack a clear understanding, however, of whether these correlative biomarkers link to a common biological network that regulates metastasis. We find that the c-MYC oncoprotein coordinately regulates the expression of 13 different "poor-outcome" cancer signatures. In addition, functional inactivation of MYC in human breast cancer cells specifically inhibits distant metastasis in vivo and invasive behavior in vitro of these cells. These results suggest that MYC oncogene activity (as marked by "poor-prognosis" signature expression) may be necessary for the translocation of poor-outcome human breast tumors to distant sites.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Neoplasias da Mama/genética , Movimento Celular/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Metástase Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico
4.
Clin Cancer Res ; 29(23): 4870-4882, 2023 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-37733811

RESUMO

PURPOSE: Tumors activate protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK, also called EIF2AK3) in response to hypoxia and nutrient deprivation as a stress-mitigation strategy. Here, we tested the hypothesis that inhibiting PERK with HC-5404 enhances the antitumor efficacy of standard-of-care VEGF receptor tyrosine kinase inhibitors (VEGFR-TKI). EXPERIMENTAL DESIGN: HC-5404 was characterized as a potent and selective PERK inhibitor, with favorable in vivo properties. Multiple renal cell carcinoma (RCC) tumor models were then cotreated with both HC-5404 and VEGFR-TKI in vivo, measuring tumor volume across time and evaluating tumor response by protein analysis and IHC. RESULTS: VEGFR-TKI including axitinib, cabozantinib, lenvatinib, and sunitinib induce PERK activation in 786-O RCC xenografts. Cotreatment with HC-5404 inhibited PERK in tumors and significantly increased antitumor effects of VEGFR-TKI across multiple RCC models, resulting in tumor stasis or regression. Analysis of tumor sections revealed that HC-5404 enhanced the antiangiogenic effects of axitinib and lenvatinib by inhibiting both new vasculature and mature tumor blood vessels. Xenografts that progress on axitinib monotherapy remain sensitive to the combination treatment, resulting in ∼20% tumor regression in the combination group. When tested across a panel of 18 RCC patient-derived xenograft (PDX) models, the combination induced greater antitumor effects relative to monotherapies. In this single animal study, nine out of 18 models responded with ≥50% tumor regression from baseline in the combination group. CONCLUSIONS: By disrupting an adaptive stress response evoked by VEGFR-TKI, HC-5404 presents a clinical opportunity to improve the antitumor effects of well-established standard-of-care therapies in RCC.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Animais , Humanos , Carcinoma de Células Renais/patologia , Axitinibe/farmacologia , Axitinibe/uso terapêutico , Neoplasias Renais/patologia , Inibidores de Proteínas Quinases/uso terapêutico
5.
Sci Transl Med ; 13(611): eaba7791, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34524860

RESUMO

SUMOylation, the covalent conjugation of small ubiquitin-like modifier (SUMO) proteins to protein substrates, has been reported to suppress type I interferon (IFN1) responses. TAK-981, a selective small-molecule inhibitor of SUMOylation, pharmacologically reactivates IFN1 signaling and immune responses against cancers. In vivo treatment of wild-type mice with TAK-981 up-regulated IFN1 gene expression in blood cells and splenocytes. Ex vivo treatment of mouse and human dendritic cells promoted their IFN1-dependent activation, and vaccination studies in mice demonstrated stimulation of antigen cross-presentation and T cell priming in vivo. TAK-981 also directly stimulated T cell activation, driving enhanced T cell sensitivity and response to antigen ex vivo. Consistent with these observations, TAK-981 inhibited growth of syngeneic A20 and MC38 tumors in mice, dependent upon IFN1 signaling and CD8+ T cells, and associated with increased intratumoral T and natural killer cell number and activation. Combination of TAK-981 with anti-PD1 or anti-CTLA4 antibodies improved the survival of mice bearing syngeneic CT26 and MC38 tumors. In conclusion, TAK-981 is a first-in-class SUMOylation inhibitor that promotes antitumor immune responses through activation of IFN1 signaling. TAK-981 is currently being studied in phase 1 clinical trials (NCT03648372, NCT04074330, NCT04776018, and NCT04381650) for the treatment of patients with solid tumors and lymphomas.


Assuntos
Imunidade , Sumoilação
6.
J Biomol Screen ; 20(8): 957-64, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25924619

RESUMO

Gene knockdown by small interfering RNA (siRNA) has been used extensively to investigate the function of genes in targeted and genome-wide studies. One of the primary challenges of siRNA studies of any scale is to achieve sufficient gene knockdown to produce the biological changes that lead to measurable phenotypes. Reverse, lipid-based transfection efficiency minimally requires the optimization of the following parameters: cell number, knockdown duration, siRNA oligonucleotide concentration, type/brand of transfection lipid, and transfection lipid concentration. In this study, we describe a methodology to utilize the flexibility and low-volume range of the Echo acoustic liquid handler to rapidly screen a matrix of transfection conditions. The matrix includes six different transfection lipids from three separate vendors across a broad range of concentrations. Our results validate acoustic liquid transfer for the delivery of siRNAs and transfection reagents. Finally, this methodology is applied to rapidly optimize transfection conditions across many tissue culture cell lines derived from various originating tissues.


Assuntos
Técnicas de Transferência de Genes , RNA Interferente Pequeno/genética , Transfecção/métodos , Linhagem Celular Tumoral , Sobrevivência Celular , Técnicas de Silenciamento de Genes , Humanos , Reprodutibilidade dos Testes
7.
Int J Biochem Cell Biol ; 35(7): 1085-97, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12672479

RESUMO

3-Hydroxyanthranilic acid 3,4-dioxygenase (EC 1.13.11.6; HADO) was purified to homogeneity from beef liver with the use of two dye columns (Cibacron Blue and Reactive Green 19) and hydroxyapatite. Two active peaks of enzyme were isolated from the hydroxyapatite column or by nondenaturing chromatofocusing of the enzyme prior to hydroxyapatite. The two active forms moved with different electrophoretic mobilities when they were subjected to nondenaturing polyacrylamide gel electrophoresis, regardless of the method of isolation. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), however, these species had apparently identical mobilities and have, therefore, close molecular mass. Analysis by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry gave them a molecular mass of 32566 and 32515 Da, respectively, for the species with apparent pI values of 5.60 and 4.98, respectively, suggesting that they differ only in the presence or absence of the iron cofactor. The N-terminal group appears to be blocked as no amino-terminal sequence was possible from direct Edman degradation. A new inactivator of the enzyme, 6-chloro-3-hydroxyanthranilic acid, was synthesized and was shown to exhibit time-dependent inactivation. A possible mechanism for inactivation is proposed.


Assuntos
Dioxigenases , Fígado/enzimologia , Oxigenases/isolamento & purificação , 3-Hidroxiantranilato 3,4-Dioxigenase , Animais , Bovinos , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Cinética , Oxigenases/antagonistas & inibidores , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
8.
Mol Cancer Ther ; 13(6): 1625-35, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24672057

RESUMO

MLN4924 is an investigational small-molecule inhibitor of the Nedd8-activating enzyme currently in phase I clinical trials. MLN4924 induces DNA damage via rereplication in most cell lines. This distinct mechanism of DNA damage may affect its ability to combine with standard-of-care agents and may affect the clinical development of MLN4924. As such, we studied its interaction with other DNA-damaging agents. Mitomycin C, cisplatin, cytarabine, UV radiation, SN-38, and gemcitabine demonstrated synergy in combination with MLN4924 in vitro. The combination of mitomycin C and MLN4924 was shown to be synergistic in a mouse xenograft model. Importantly, depletion of genes within the ataxia telangiectasia and Rad3 related (ATR) and BRCA1/BRCA2 pathways, chromatin modification, and transcription-coupled repair reduced the synergy between mitomycin C and MLN4924. In addition, comet assay demonstrated increased DNA strand breaks with the combination of MLN4924 and mitomycin C. Our data suggest that mitomycin C causes stalled replication forks, which when combined with rereplication induced by MLN4924 results in frequent replication fork collisions, leading to cell death. This study provides a straightforward approach to understand the mechanism of synergy, which may provide useful information for the clinical development of these combinations.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Ciclopentanos/administração & dosagem , Sinergismo Farmacológico , Mitomicina/administração & dosagem , Pirimidinas/administração & dosagem , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Linhagem Celular Tumoral , Cromatina/efeitos dos fármacos , Cromatina/genética , Dano ao DNA/efeitos dos fármacos , Humanos , Camundongos , Enzimas Ativadoras de Ubiquitina/genética , Raios Ultravioleta , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Cancer Res ; 73(1): 225-34, 2013 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23100467

RESUMO

MLN4924 is an investigational small-molecule inhibitor of the NEDD8-activating enzyme (NAE) in phase I clinical trials. NAE inhibition prevents the ubiquitination and proteasomal degradation of substrates for cullin-RING ubiquitin E3 ligases that support cancer pathophysiology, but the genetic determinants conferring sensitivity to NAE inhibition are unknown. To address this gap in knowledge, we conducted a genome-wide siRNA screen to identify genes and pathways that affect the lethality of MLN4924 in melanoma cells. Of the 154 genes identified, approximately one-half interfered with components of the cell cycle, apoptotic machinery, ubiquitin system, and DNA damage response pathways. In particular, genes involved in DNA replication, p53, BRCA1/BRCA2, transcription-coupled repair, and base excision repair seemed to be important for MLN4924 lethality. In contrast, genes within the G(2)-M checkpoint affected sensitivity to MLN4924 in colon cancer cells. Cell-cycle analysis in melanoma cells by flow cytometry following RNAi-mediated silencing showed that MLN4924 prevented the transition of cells from S-G(2) phase after induction of rereplication stress. Our analysis suggested an important role for the p21-dependent intra-S-phase checkpoint and extensive rereplication, whereas the ATR-dependent intra-S-phase checkpoint seemed to play a less dominant role. Unexpectedly, induction of the p21-dependent intra-S-phase checkpoint seemed to be independent of both Cdt1 stabilization and ATR signaling. Collectively, these data enhance our understanding of the mechanisms by which inhibition of NEDD8-dependent ubiquitination causes cell death, informing clinical development of MLN4924.


Assuntos
Antineoplásicos/farmacologia , Ciclopentanos/farmacologia , Dano ao DNA/efeitos dos fármacos , Melanoma/metabolismo , Pirimidinas/farmacologia , Ubiquitinas/metabolismo , Western Blotting , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Proteína NEDD8 , Reação em Cadeia da Polimerase
10.
Sci Signal ; 5(214): ra19, 2012 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-22394561

RESUMO

Epithelial cells respond to growth factors including epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), and insulin. Using high-content immunofluorescence microscopy, we quantitated differences in signaling networks downstream of EGF, which stimulated proliferation of mammary epithelial cells, and insulin or IGF-1, which enhanced the proliferative response to EGF but did not stimulate proliferation independently. We found that the abundance of the cyclin-dependent kinase inhibitors p21Cip1 and p57Kip2 increased in response to IGF-1 or insulin but decreased in response to EGF. Depletion of p57Kip2, but not p21Cip1, rendered IGF-1 or insulin sufficient to induce cellular proliferation in the absence of EGF. Signaling through the PI3K (phosphatidylinositol 3-kinase)-Akt-mTOR (mammalian target of rapamycin) pathway was necessary and sufficient for the increase in p57Kip2, whereas MEK [mitogen-activated or extracellular signal-regulated protein kinase (ERK) kinase]-ERK activity suppressed this increase, forming a regulatory circuit that limited proliferation in response to unaccompanied Akt activity. Knockdown of p57Kip2 enhanced the proliferative phenotype induced by tumor-associated PI3K mutant variants and released mammary epithelial acini from growth arrest during morphogenesis in three-dimensional culture. These results provide a potential explanation for the context-dependent proliferative activities of insulin and IGF-1 and for the finding that the CDKN1C locus encoding p57Kip2 is silenced in many breast cancers, which frequently show hyperactivation of the PI3K pathway. The status of p57Kip2 may thus be an important factor to assess when considering targeted therapy against the ERK or PI3K pathways.


Assuntos
Proliferação de Células , Fator de Crescimento Epidérmico/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Glândulas Mamárias Humanas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p57 , Humanos , Glândulas Mamárias Humanas/química
11.
Cancer Res ; 70(11): 4318-26, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20460535

RESUMO

Multiple pathways have been proposed to explain how proteasome inhibition induces cell death, but mechanisms remain unclear. To approach this issue, we performed a genome-wide siRNA screen to evaluate the genetic determinants that confer sensitivity to bortezomib (Velcade (R); PS-341). This screen identified 100 genes whose knockdown affected lethality to bortezomib and to a structurally diverse set of other proteasome inhibitors. A comparison of three cell lines revealed that 39 of 100 genes were commonly linked to cell death. We causally linked bortezomib-induced cell death to the accumulation of ASF1B, Myc, ODC1, Noxa, BNIP3, Gadd45alpha, p-SMC1A, SREBF1, and p53. Our results suggest that proteasome inhibition promotes cell death primarily by dysregulating Myc and polyamines, interfering with protein translation, and disrupting essential DNA damage repair pathways, leading to programmed cell death.


Assuntos
Antineoplásicos/farmacologia , Ácidos Borônicos/farmacologia , Morte Celular/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Inibidores de Proteassoma , Pirazinas/farmacologia , RNA Interferente Pequeno/genética , Bortezomib , Morte Celular/genética , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Dano ao DNA , Técnicas de Silenciamento de Genes , Células HCT116 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-myc/genética , Ribossomos/efeitos dos fármacos , Serina-Treonina Quinases TOR , Transfecção
13.
Proc Natl Acad Sci U S A ; 104(10): 3787-92, 2007 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-17360431

RESUMO

The formation of a lumen in three-dimensional mammary epithelial acinar structures in vitro involves selective apoptosis of centrally localized cells that lack matrix attachment. Similarly, apoptosis is induced by forced detachment of mammary epithelial cells from matrix, a process referred to as anoikis. Through microarray analysis, we found that mRNA levels of the proapoptotic BH3-only protein Bmf are up-regulated during both anoikis and acinar morphogenesis. Importantly, down-regulation of Bmf expression by small interfering RNAs is sufficient to prevent anoikis and acinar cell death and promote anchorage-independent growth to a similar extent as down-regulation of another BH3-only protein, Bim, which was previously shown to be required for these processes. Knockdown of the BH3-only proteins Bad or Bid does not suppress anoikis or luminal apoptosis or promote anchorage-independent growth, but protects from other defined apoptotic stimuli, indicating specificity of BH3-only function. Bmf mRNA is significantly up-regulated upon loss of matrix attachment or disruption of the actin cytoskeleton, but not in response to several other stresses. Interestingly, constitutive activation of the Mek/Erk or phosphatidylinositol 3-kinase/Akt pathways suppresses the transcriptional up-regulation of Bmf during anoikis. Thus, Bmf is a central mediator of anoikis in mammary cells and a target of oncogenes that contribute to the progression of glandular epithelial tumors. Finally, Bmf is expressed during involution of the mouse mammary gland, suggesting that Bmf may also critically contribute to developmental processes in vivo.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Anoikis , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Animais/metabolismo , Morfogênese , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Citoesqueleto/metabolismo , Progressão da Doença , Humanos , Camundongos , Proteína de Morte Celular Associada a bcl/metabolismo
14.
Proc Natl Acad Sci U S A ; 99(3): 1461-6, 2002 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-11830665

RESUMO

Although the biochemical targets of most drugs are known, the biological consequences of their actions are typically less well understood. In this study, we have used two whole-genome technologies in Saccharomyces cerevisiae to determine the cellular impact of the proteasome inhibitor PS-341. By combining population genomics, the screening of a comprehensive panel of bar-coded mutant strains, and transcript profiling, we have identified the genes and pathways most affected by proteasome inhibition. Many of these function in regulated protein degradation or a subset of mitotic activities. In addition, we identified Rpn4p as the transcription factor most responsible for the cell's ability to compensate for proteasome inhibition. Used together, these complementary technologies provide a general and powerful means to elucidate the cellular ramifications of drug treatment.


Assuntos
Ácidos Borônicos/farmacologia , Cisteína Endopeptidases/metabolismo , Genoma Fúngico , Genômica/métodos , Complexos Multienzimáticos/metabolismo , Inibidores de Proteases/farmacologia , Pirazinas/farmacologia , Saccharomyces cerevisiae/genética , Bortezomib , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Reparo do DNA , DNA Fúngico/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Perfilação da Expressão Gênica , Testes de Sensibilidade Microbiana , Análise de Sequência com Séries de Oligonucleotídeos , Complexo de Endopeptidases do Proteassoma , RNA Fúngico/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Transcrição Gênica
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