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2.
Haematologica ; 98(9): 1397-406, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23349304

RESUMO

Inactivation of the DNA mismatch repair pathway manifests as microsatellite instability, an accumulation of mutations that drives carcinogenesis. Here, we determined whether microsatellite instability in acute myeloid leukemia and myelodysplastic syndrome correlated with chromosomal instability and poly (ADP-ribose) polymerase (PARP) inhibitor sensitivity through disruption of DNA repair function. Acute myeloid leukemia cell lines (n=12) and primary cell samples (n=18), and bone marrow mononuclear cells from high-risk myelodysplastic syndrome patients (n=63) were profiled for microsatellite instability using fluorescent fragment polymerase chain reaction. PARP inhibitor sensitivity was performed using cell survival, annexin V staining and cell cycle analysis. Homologous recombination was studied using immunocytochemical analysis. SNP karyotyping was used to study chromosomal instability. RNA silencing, Western blotting and gene expression analysis was used to study the functional consequences of mutations. Acute myeloid leukemia cell lines (4 of 12, 33%) and primary samples (2 of 18, 11%) exhibited microsatellite instability with mono-allelic mutations in CtIP and MRE11. These changes were associated with reduced expression of mismatch repair pathway components, MSH2, MSH6 and MLH1. Both microsatellite instability positive primary acute myeloid leukemia samples and cell lines demonstrated a downregulation of homologous recombination DNA repair conferring marked sensitivity to PARP inhibitors. Similarly, bone marrow mononuclear cells from 11 of 56 (20%) patients with de novo high-risk myelodysplastic syndrome exhibited microsatellite instability. Significantly, all 11 patients with microsatellite instability had cytogenetic abnormalities with 4 of them (36%) possessing a mono-allelic microsatellite mutation in CtIP. Furthermore, 50% reduction in CtIP expression by RNA silencing also down-regulated homologous recombination DNA repair responses conferring PARP inhibitor sensitivity, whilst CtIP differentially regulated the expression of homologous recombination modulating RecQ helicases, WRN and BLM. In conclusion, microsatellite instability dependent mutations in DNA repair genes, CtIP and MRE11 are detected in myeloid malignancies conferring hypersensitivity to PARP inhibitors. Microsatellite instability is significantly correlated with chromosomal instability in myeloid malignancies.


Assuntos
Proteínas de Transporte/genética , Reparo de Erro de Pareamento de DNA/genética , Proteínas de Ligação a DNA/genética , Instabilidade de Microssatélites , Mutação/genética , Proteínas Nucleares/genética , Poli(ADP-Ribose) Polimerases/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Sobrevivência Celular/genética , Endodesoxirribonucleases , Feminino , Técnicas de Silenciamento de Genes/métodos , Humanos , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Proteína Homóloga a MRE11 , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/enzimologia , Síndromes Mielodisplásicas/genética , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genética , Inibidores de Poli(ADP-Ribose) Polimerases
3.
Haematologica ; 94(5): 638-46, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19407318

RESUMO

UNLABELLED: Background Aberrant or impaired repair of double-strand DNA breaks is a common feature of de novo acute myeloid leukemia and myelodysplastic syndromes. Since poly (ADP-ribose) polymerase (PARP) inhibitors have been recently shown to selectively target cells with defects in double-strand DNA repair, the aim of this study was to explore the possibility of exploiting defects in DNA repair in leukemic cells using PARP inhibitors. DESIGN AND METHODS: Leukemic cell lines were exposed to various PARP inhibitors alone and in combination with non-cytotoxic concentrations of DNA methyltransferase inhibitor, 5' aza-2'-deoxycytidine and/or the histone deacetylase inhibitor, MS275, to test for potentiation of apoptosis with these agents. RESULTS: PARP inhibitors, KU-0058948 and PJ34, induced cell cycle arrest and apoptosis of primary myeloid leukemic cells and myeloid leukemic cell lines in vitro. Immunofluorescence analysis also revealed that PARP inhibitor sensitivity in these leukemic cells was due to a defect in homologous recombination DNA repair. Addition of 5' aza-2'-deoxycytidine failed to increase the cytotoxicity of PARP inhibitors. In contrast, MS275 potentiated the cytotoxic effect of KU-0058948 and PJ34 in all PARP inhibitor-sensitive leukemic cells. Immunofluorescence analysis supported the idea that histone deacetylase inhibitors potentiate cytotoxicity by inhibiting DNA repair processes. Conclusions On the basis of the data presented here, we suggest that PARP inhibitors can potentially exploit defects in double-strand DNA break repair in leukemic cells, paving the way for testing the therapeutic potential of these agents in myelodysplastic syndromes and acute myeloid leukemia.


Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Benzamidas/farmacologia , Butiratos/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Metilases de Modificação do DNA/antagonistas & inibidores , Metilases de Modificação do DNA/metabolismo , Decitabina , Sinergismo Farmacológico , Citometria de Fluxo , Imunofluorescência , Fluorbenzenos/farmacologia , Células HL-60 , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Imuno-Histoquímica , Células K562 , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Fenantrenos/farmacologia , Ftalazinas/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Piridinas/farmacologia , Células U937
4.
Haematologica ; 93(12): 1886-9, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18838476

RESUMO

Poly ADP-ribose polymerase inhibitors have been shown to target cells with homologous recombination DNA repair defects. We report that poly ADP-ribose polymerase inhibitors induces apoptosis in cells deficient in other key DNA repair components. Chromosomal instability disorders, Fanconi Anemia and Bloom's syndrome have dysfunctional DNA repair and an increased likelihood of leukemic transformation. PI addition to Fanconi Anemia and Bloom's syndrome cells resulted in significant apoptosis. Furthermore, poly ADP-ribose polymerase inhibitors induced apoptosis in DNA repair signaling defective ATM(-/-) and NBS(-/-) fibroblasts. Immunocytochemistry showed homologous recombination was abrogated in NBS(-/-) and ATM(-/-) fibroblasts, compromised in Fanconi anemia and normal in Bloom's syndrome cells in response to poly ADP-ribose polymerase inhibitors. Strikingly, poly ADP-ribose polymerase inhibitors increases non-homologous end joining repair activity, whilst non-homologous end joining deficient cells are extremely sensitive to poly ADP-ribose polymerase inhibitors. These data suggest poly ADP-ribose polymerase inhibitors target cells with DNA repair and signaling defects rather than solely defects in homologous recombination improving the potential of poly ADP-ribose polymerase inhibitors therapy in a wider range of cancers.


Assuntos
Distúrbios no Reparo do DNA/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases , Apoptose/efeitos dos fármacos , Síndrome de Bloom/tratamento farmacológico , Síndrome de Bloom/genética , Síndrome de Bloom/patologia , Células Cultivadas , Instabilidade Cromossômica/efeitos dos fármacos , Reparo do DNA , Inibidores Enzimáticos/uso terapêutico , Anemia de Fanconi/tratamento farmacológico , Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Humanos , Síndrome
5.
Methods Mol Biol ; 1608: 343-370, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28695521

RESUMO

This chapter describes some of the techniques in use in our laboratories for the investigation of PARP inhibitors in clinical medicine. More specifically, we are involved in investigating the utility of PARP inhibitors in the treatment of hematopoietic malignancies. We are also actively investigating the properties of the PARP systems in cell biology. We begin the chapter with a very brief history of the invention and use of PARP inhibitors. We then explain the underlying logic of the use of PARP inhibitors either in combination with chemo- or radiotherapy or as single agents used alone. We then provide in full detail the protocols that we use to study PARP inhibitors in cell biology to identify patients that should be susceptible to PARP inhibitor treatment and to manage and investigate these patients throughout their treatment.


Assuntos
Adjuvantes Farmacêuticos/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Humanos , Leucemia/tratamento farmacológico , Leucemia/terapia
6.
Methods Mol Biol ; 780: 239-66, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21870265

RESUMO

This chapter describes some of the techniques in use in our laboratories for the investigation of PARP inhibitors in clinical medicine. More specifically, we are involved in investigating the utility of PARP inhibitors in the treatment of hematopoietic malignancies. We are also actively investigating the properties of the PARP systems in cell biology. We begin the chapter with a very brief history of the invention and use of PARP inhibitors. We then explain the underlying logic of the use of PARP inhibitors either in combination with chemo- or radiotherapy or as single agents used alone. We then provide in full detail the protocols that we use to study PARP inhibitors in cell biology to identify patients that should be susceptible to PARP inhibitor treatment and to manage and investigate these patients throughout their treatment.


Assuntos
Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Neoplasias/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos SCID , Neoplasias/radioterapia , Ensaios Antitumorais Modelo de Xenoenxerto
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