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1.
Nucleic Acids Res ; 49(5): 2598-2608, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33591315

RESUMO

Aberrant end joining of DNA double strand breaks leads to chromosomal rearrangements and to insertion of nuclear or mitochondrial DNA into breakpoints, which is commonly observed in cancer cells and constitutes a major threat to genome integrity. However, the mechanisms that are causative for these insertions are largely unknown. By monitoring end joining of different linear DNA substrates introduced into HEK293 cells, as well as by examining end joining of CRISPR/Cas9 induced DNA breaks in HEK293 and HeLa cells, we provide evidence that the dNTPase activity of SAMHD1 impedes aberrant DNA resynthesis at DNA breaks during DNA end joining. Hence, SAMHD1 expression or low intracellular dNTP levels lead to shorter repair joints and impede insertion of distant DNA regions prior end repair. Our results reveal a novel role for SAMHD1 in DNA end joining and provide new insights into how loss of SAMHD1 may contribute to genome instability and cancer development.


Assuntos
Reparo do DNA por Junção de Extremidades , Proteína 1 com Domínio SAM e Domínio HD/fisiologia , Proteína 9 Associada à CRISPR/metabolismo , Quebra Cromossômica , Desoxirribonucleotídeos/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteína 1 com Domínio SAM e Domínio HD/metabolismo
2.
Mol Cancer Res ; 16(3): 428-438, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29222170

RESUMO

Cancer is a genetic disease caused by mutations and chromosomal abnormalities that contribute to uncontrolled cell growth. In addition, cancer cells can rapidly respond to conventional and targeted therapies by accumulating novel and often specific genetic lesions leading to acquired drug resistance and relapsing disease. In chronic lymphocytic leukemia (CLL), however, diverse chromosomal aberrations often occur. In many cases, improper repair of DNA double-strand breaks (DSB) is a major source for genomic abnormalities. Therefore, this study examined the repair of DNA DSBs by nonhomologous end joining (NHEJ) in CLL by performing plasmid-based repair assays in primary CLL cells and normal B cells, isolated from patients, as well as TALEN/Cas9-induced chromosomal deletions in the CLL cell line Mec1. It is demonstrated that DNA repair is aberrant in CLL cells, featuring perturbed DNA break structure preference with efficient joining of noncohesive ends and more deletions at repair junctions. In addition, increased microhomology-mediated end joining (MMEJ) of DNA substrates was observed in CLL together with increased expression of MMEJ-specific repair factors. In summary, these data identify major differences in DNA repair efficiency between CLL cells and normal B cells isolated from patients.Implications: This study suggests inherently aberrant DNA DSB repair in the acquisition of subclonal genomic structural variations important for clonal evolution and treatment resistance in CLL. Mol Cancer Res; 16(3); 428-38. ©2017 AACR.


Assuntos
Reparo do DNA por Junção de Extremidades , Leucemia Linfocítica Crônica de Células B/genética , Linhagem Celular Tumoral , DNA Ligase Dependente de ATP/biossíntese , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Transfecção , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/biossíntese , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/metabolismo
3.
Oncoimmunology ; 7(1): e1371399, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29296521

RESUMO

While research on T cell exhaustion in context of cancer particularly focuses on CD8+ cytotoxic T cells, the role of inhibitory receptors on CD4+ T-helper cells have remained largely unexplored. TIGIT is a recently identified inhibitory receptor on T cells and natural killer (NK) cells. In this study, we examined TIGIT expression on T cell subsets from CLL patients. While we did not observe any differences in TIGIT expression in CD8+ T cells of healthy controls and CLL cells, we found an enrichment of TIGIT+ T cells in the CD4+ T cell compartment in CLL. Intriguingly, CLL patients with an advanced disease stage displayed elevated numbers of CD4+ TIGIT+ T cells compared to low risk patients. Autologous CLL-T cell co-culture assays revealed that depleting CD4+ TIGIT+ expressing T cells from co-cultures significantly decreased CLL viability. Accordingly, a supportive effect of TIGIT+CD4+ T cells on CLL cells in vitro could be recapitulated by blocking the interaction of TIGIT with its ligands using TIGIT-Fc molecules, which also impeded the T cell specific production of CLL-prosurvival cytokines. Our data reveal that TIGIT+CD4+T cells provide a supportive microenvironment for CLL cells, representing a potential therapeutic target for CLL treatment.

4.
Oncotarget ; 7(31): 49459-49469, 2016 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-27385215

RESUMO

Chronic lymphocytic leukemia develops within a complex network driven by genetic mutations and microenvironmental interactions. Among the latter a complex interplay with the immune system is established by the clone. Next to a proposed recruitment of support from T and myeloid cells, potential anti-CLL immune reactions need to be subverted. By using TCL1 mice as a CLL model, we show that TCR-Vß7+ NK1.1+ T cells are overrepresented in this disease model and constitute a main subset of peripheral CD3+ cells with biased TCR usage, showing that these cells account for a major part for T cell skewing in TCL1 mice. Moreover, we show that overrepresentation is dependent on CD1d expression in TCL1 mice, implicating that these cells belong to a NKT-like cell fraction which are restricted to antigen presented by the MHC-like surface marker CD1d. Accordingly, we observed a high fraction of CD161+ cells within overrepresented T cells in CLL patients and we found downregulation of CD1d on the surface of CLL cells, both in TCL1 mice and patients. Finally, we show that in TCL1 mice, CD1d deficiency resulted in shortened overall survival. Our results point to an interaction between CLL and CD161+ T cells that may represent a novel therapeutic target for immune modulation.


Assuntos
Antígenos CD1d/metabolismo , Linfócitos T CD8-Positivos/citologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Células T Matadoras Naturais/citologia , Idoso , Idoso de 80 Anos ou mais , Animais , Complexo CD3/metabolismo , Progressão da Doença , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Mutação , Subfamília B de Receptores Semelhantes a Lectina de Células NK/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo
5.
Eur J Immunol ; 45(8): 2208-11, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26151367

RESUMO

The activation induced deaminase (AID) catalyses the two key events underlying humoral adaptive immunity: class switch recombination and somatic hypermutation of antibody genes in B lymphocytes. AID accomplishes this task by directly deaminating cytosines within the genomic immunoglobulin locus, thereby triggering a complex mutagenic process eventually leading to improved effector function of antibodies. However, it has long been noticed that AID can be aberrantly expressed in cancer and that its activity is not absolutely restricted to antibody genes, as substantial genome-wide off-target mutations have been observed, which contribute to tumorigenesis and clonal evolution of AID-expressing malignancies. In this issue of the European Journal of Immunology, Montamat-Sicotte et al. [Eur. J. Immunol. 2015. 45: 2365-2376] investigate the feasibility and efficacy of in vivo inhibition of AID with HSP90 inhibitors in a mouse model of B-cell leukemia and in vitro with a human breast cancer cell line, thereby demonstrating that cancer patients may benefit from preventing noncanonical AID functions.


Assuntos
Linfócitos B/imunologia , Neoplasias da Mama/imunologia , Citidina Desaminase/imunologia , Proteínas de Choque Térmico HSP90/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias Experimentais/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Animais , Feminino , Humanos
6.
Oncoscience ; 2(4): 320-33, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26097867

RESUMO

Mutations are the basis for evolution and the development of genetic diseases. Especially in cancer, somatic mutations in oncogenes and tumor suppressor genes alongside the occurrence of passenger mutations have been observed by recent deep-sequencing approaches. While mutations have long been considered random events induced by DNA-replication errors or by DNA damaging agents, genome sequencing led to the discovery of non-random mutation signatures in many human cancer. Common non-random mutations comprise DNA strand-biased mutation showers and mutations restricted to certain DNA motifs, which recently have become attributed to the activity of the AID/APOBEC family of DNA deaminases. Hence, APOBEC enzymes, which have evolved as key players in natural and adaptive immunity, have been proposed to contribute to cancer development and clonal evolution of cancer by inducing collateral genomic damage due to their DNA deaminating activity. This review focuses on how mutagenic events through AID/APOBEC deaminases may contribute to cancer development.

7.
Br J Haematol ; 170(4): 515-22, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25940792

RESUMO

Although chronic lymphocytic leukaemia (CLL) is a B cell malignancy, earlier studies have indicated a role of T cells in tumour growth and disease progression. In particular, the functional silencing of antigen-experienced T cells, called T cell exhaustion, has become implicated in immune evasion in CLL. In this study, we tested whether T cell exhaustion is recapitulated in the TCL1(tg) mouse model for CLL. We show that T cells express high levels of the inhibitory exhaustion markers programmed cell death 1 (PDCD1, also termed PD-1) and lymphocyte-activation gene 3 (LAG3), whereas CLL cells express high levels of CD274 (also termed PD-ligand 1). In addition, the fraction of exhausted T cells increases with CLL progression. Finally, we demonstrate that exhausted T cells are reinvigorated towards CLL cytotoxicity by inhibition of PDCD1/CD274 interaction in vivo. These results suggest that T cell exhaustion contributes to CLL pathogenesis and that interference with PDCD1/CD274 signalling holds high potential for therapeutic approaches.


Assuntos
Regulação Leucêmica da Expressão Gênica/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias Experimentais/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Animais , Regulação Leucêmica da Expressão Gênica/genética , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/genética , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Transdução de Sinais/genética , Linfócitos T/patologia
8.
Eur J Immunol ; 44(12): 3747-57, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25179679

RESUMO

The activation-induced cytidine deaminase (AID) mediates somatic hypermutation and class switch recombination of the Ig genes by directly deaminating cytosines to uracils. As AID causes a substantial amount of off-target mutations, its activity has been associated with lymphomagenesis and clonal evolution of B-cell malignancies. Although it has been shown that AID is expressed in B-cell chronic lymphocytic leukemia (CLL), a clear analysis of in vivo AID activity in this B-cell malignancy remained elusive. In this study performed on primary human CLL samples, we report that, despite the presence of a dominant VDJ heavy chain region, a substantial intraclonal diversity was observed at VDJ as well as at IgM switch regions (Sµ), showing ongoing AID activity in vivo during disease progression. This AID-mediated heterogeneity was higher in CLL subclones expressing CD86, which we identified as the proliferative CLL fraction. Finally, CD86 expression correlated with shortened time to first treatment and increased γ-H2AX focus formation. Our data demonstrate that AID is active in CLL in vivo and thus, AID likely contributes to clonal evolution of CLL.


Assuntos
Antígeno B7-2/imunologia , Proliferação de Células , Citidina Desaminase/imunologia , Dano ao DNA/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Proteínas de Neoplasias/imunologia , Feminino , Regulação Leucêmica da Expressão Gênica/imunologia , Genes de Cadeia Pesada de Imunoglobulina/imunologia , Histonas/imunologia , Humanos , Região de Troca de Imunoglobulinas/imunologia , Cadeias mu de Imunoglobulina/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Masculino
9.
Eur J Immunol ; 44(7): 2175-87, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24668151

RESUMO

Activation-induced deaminase (AID) is a DNA-mutating enzyme that mediates class-switch recombination as well as somatic hypermutation of antibody genes in B cells. Due to off-target activity, AID is implicated in lymphoma development by introducing genome-wide DNA damage and initiating chromosomal translocations such as c-myc/IgH. Several alternative splice transcripts of AID have been reported in activated B cells as well as malignant B cells such as chronic lymphocytic leukemia (CLL). As most commercially available antibodies fail to recognize alternative splice variants, their abundance in vivo, and hence their biological significance, has not been determined. In this study, we assessed the protein levels of AID splice isoforms by introducing an AID splice reporter construct into cell lines and primary CLL cells from patients as well as from WT and TCL1(tg) C57BL/6 mice (where TCL1 is T-cell leukemia/lymphoma 1). The splice construct is 5'-fused to a GFP-tag, which is preserved in all splice isoforms and allows detection of translated protein. Summarizing, we show a thorough quantification of alternatively spliced AID transcripts and demonstrate that the corresponding protein abundances, especially those of splice variants AID-ivs3 and AID-ΔE4, are not stoichiometrically equivalent. Our data suggest that enhanced proteasomal degradation of low-abundance proteins might be causative for this discrepancy.


Assuntos
Processamento Alternativo , Citidina Desaminase/genética , Leucemia Linfocítica Crônica de Células B/enzimologia , Animais , Citidina Desaminase/análise , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real
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