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1.
Sci Signal ; 13(645)2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32817374

RESUMO

Synthetic lethality between poly(ADP-ribose) polymerase (PARP) inhibition and BRCA deficiency is exploited to treat breast and ovarian tumors. However, resistance to PARP inhibitors (PARPis) is common. To identify potential resistance mechanisms, we performed a genome-wide RNAi screen in BRCA2-deficient mouse embryonic stem cells and validation in KB2P1.21 mouse mammary tumor cells. We found that resistance to multiple PARPi emerged with reduced expression of TET2 (ten-eleven translocation), which promotes DNA demethylation by oxidizing 5-methylcytosine (5mC) to 5-hydroxymethycytosine (5hmC) and other products. TET2 knockdown in BRCA2-deficient cells protected stalled replication forks (RFs). Increasing 5hmC abundance induced the degradation of stalled RFs in KB2P1.21 and human cancer cells by recruiting the base excision repair-associated apurinic/apyrimidinic endonuclease APE1, independent of the BRCA2 status. TET2 loss did not affect the recruitment of the repair protein RAD51 to sites of double-strand breaks (DSBs) or the abundance of proteins associated with RF integrity. The loss of TET2, of its product 5hmC, and of APE1 recruitment to stalled RFs promoted resistance to the chemotherapeutic cisplatin. Our findings reveal a previously unknown role for the epigenetic mark 5hmC in maintaining the integrity of stalled RFs and a potential resistance mechanism to PARPi and cisplatin.


Assuntos
Neoplasias da Mama/genética , Replicação do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Desoxicitidina/análogos & derivados , Instabilidade Genômica/genética , Neoplasias Ovarianas/genética , 5-Metilcitosina/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Desoxicitidina/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia
2.
J Nat Prod ; 80(7): 1981-1991, 2017 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-28617598

RESUMO

Investigation of aeroponically grown Physalis peruviana resulted in the isolation of 11 new withanolides, including perulactones I-L (1-4), 17-deoxy-23ß-hydroxywithanolide E (5), 23ß-hydroxywithanolide E (6), 4-deoxyphyperunolide A (7), 7ß-hydroxywithanolide F (8), 7ß-hydroxy-17-epi-withanolide K (9), 24,25-dihydro-23ß,28-dihydroxywithanolide G (10), and 24,25-dihydrowithanolide E (11), together with 14 known withanolides (12-25). The structures of 1-11 were elucidated by the analysis of their spectroscopic data, and 12-25 were identified by comparison of their spectroscopic data with those reported. All withanolides were evaluated for their cytotoxic activity against a panel of tumor cell lines including LNCaP (androgen-sensitive human prostate adenocarcinoma), 22Rv1 (androgen-resistant human prostate adenocarcinoma), ACHN (human renal adenocarcinoma), M14 (human melanoma), SK-MEL-28 (human melanoma), and normal human foreskin fibroblast cells. Of these, the 17ß-hydroxywithanolides (17-BHWs) 6, 8, 9, 11-13, 15, and 19-22 showed selective cytotoxic activity against the two prostate cancer cell lines LNCaP and 22Rv1, whereas 13 and 20 exhibited selective toxicity for the ACHN renal carcinoma cell line. These cytotoxicity data provide additional structure-activity relationship information for the 17-BHWs.


Assuntos
Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Neoplasias Renais/tratamento farmacológico , Physalis/química , Neoplasias da Próstata/tratamento farmacológico , Vitanolídeos/isolamento & purificação , Vitanolídeos/farmacologia , Antineoplásicos Fitogênicos/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Masculino , Melanoma/tratamento farmacológico , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Physalis/crescimento & desenvolvimento , Relação Estrutura-Atividade , Vitanolídeos/química
3.
Nat Commun ; 7: 12425, 2016 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-27498558

RESUMO

Poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) olaparib has been approved for treatment of advanced ovarian cancer associated with BRCA1 and BRCA2 mutations. BRCA1- and BRCA2-mutated cells, which are homologous recombination (HR) deficient, are hypersensitive to PARPi through the mechanism of synthetic lethality. Here we examine the effect of PARPi on HR-proficient cells. Olaparib pretreatment, PARP1 knockdown or Parp1 heterozygosity of Brca2(cko/ko) mouse embryonic stem cells (mESCs), carrying a null (ko) and a conditional (cko) allele of Brca2, results in viable Brca2(ko/ko) cells. PARP1 deficiency does not restore HR in Brca2(ko/ko) cells, but protects stalled replication forks from MRE11-mediated degradation through its impaired recruitment. The functional consequence of Parp1 heterozygosity on BRCA2 loss is demonstrated by a significant increase in tumorigenesis in Brca2(cko/cko) mice. Thus, while olaparib efficiently kills BRCA2-deficient cells, we demonstrate that it can also contribute to the synthetic viability if PARP is inhibited before BRCA2 loss.


Assuntos
Proteína BRCA2/deficiência , Poli(ADP-Ribose) Polimerase-1/deficiência , Animais , Proteína BRCA2/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Recombinação Homóloga/efeitos dos fármacos , Humanos , Integrases/metabolismo , Proteína Homóloga a MRE11/metabolismo , Camundongos , Modelos Biológicos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia
4.
Biochem J ; 473(14): 2165-77, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27208174

RESUMO

The presence of latent activities in enzymes is posited to underlie the natural evolution of new catalytic functions. However, the prevalence and extent of such substrate and catalytic ambiguity in evolved enzymes is difficult to address experimentally given the order-of-magnitude difference in the activities for native and, sometimes, promiscuous substrate/s. Further, such latent functions are of special interest when the activities concerned do not fall into the domain of substrate promiscuity. In the present study, we show a special case of such latent enzyme activity by demonstrating the presence of two mechanistically distinct reactions catalysed by the catalytic domain of receptor protein tyrosine phosphatase isoform δ (PTPRδ). The primary catalytic activity involves the hydrolysis of a phosphomonoester bond (C─O─P) with high catalytic efficiency, whereas the secondary activity is the hydrolysis of a glycosidic bond (C─O─C) with poorer catalytic efficiency. This enzyme also displays substrate promiscuity by hydrolysing diester bonds while being highly discriminative for its monoester substrates. To confirm these activities, we also demonstrated their presence on the catalytic domain of protein tyrosine phosphatase Ω (PTPRΩ), a homologue of PTPRδ. Studies on the rate, metal-ion dependence, pH dependence and inhibition of the respective activities showed that they are markedly different. This is the first study that demonstrates a novel sugar hydrolase and diesterase activity for the phosphatase domain (PD) of PTPRδ and PTPRΩ. This work has significant implications for both understanding the evolution of enzymatic activity and the possible physiological role of this new chemistry. Our findings suggest that the genome might harbour a wealth of such alternative latent enzyme activities in the same protein domain that renders our knowledge of metabolic networks incomplete.


Assuntos
Proteínas Tirosina Fosfatases Semelhantes a Receptores/química , Proteínas Tirosina Fosfatases Semelhantes a Receptores/metabolismo , Catálise , Domínio Catalítico , Biologia Computacional , Proteínas Tirosina Fosfatases Semelhantes a Receptores/genética , Eletricidade Estática , Especificidade por Substrato
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