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1.
Br J Cancer ; 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38877108

RESUMO

BACKGROUND: Targeting DNA damage repair factors, such as DNA-dependent protein kinase catalytic subunit (DNA-PKcs), may offer an opportunity for effective treatment of multiple myeloma (MM). In combination with DNA damage-inducing agents, this strategy has been shown to improve chemotherapies partially via activation of cGAS-STING pathway by an elevated level of cytosolic DNA. However, as cGAS is primarily sequestered by chromatin in the nucleus, it remains unclear how cGAS is released from chromatin and translocated into the cytoplasm upon DNA damage, leading to cGAS-STING activation. METHODS: We examined the role of DNA-PKcs inhibition on cGAS-STING-mediated MM chemosensitivity by performing mass spectrometry and mechanism study. RESULTS: Here, we found DNA-PKcs inhibition potentiated DNA damage-inducing agent doxorubicin-induced anti-MM effect by activating cGAS-STING signaling. The cGAS-STING activation in MM cells caused cell death partly via IRF3-NOXA-BAK axis and induced M1 polarization of macrophages. Moreover, this activation was not caused by defective classical non-homologous end joining (c-NHEJ). Instead, upon DNA damage induced by doxorubicin, inhibition of DNA-PKcs promoted cGAS release from cytoplasmic chromatin fragments and increased the amount of cytosolic cGAS and DNA, activating cGAS-STING. CONCLUSIONS: Inhibition of DNA-PKcs could improve the efficacy of doxorubicin in treatment of MM by de-sequestrating cGAS in damaged chromatin.

2.
Genome Med ; 15(1): 80, 2023 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-37803452

RESUMO

BACKGROUND: Primary liver cancer has significant intratumor genetic heterogeneity (IGH), which drives cancer evolution and prevents effective cancer treatment. CRISPR/Cas9-induced mouse liver cancer models can be used to elucidate how IGH is developed. However, as CRISPR/Cas9 could induce chromothripsis and extrachromosomal DNA in cells in addition to targeted mutations, we wondered whether this effect contributes to the development of IGH in CRISPR/Cas9-induced mouse liver cancer. METHODS: CRISPR/Cas9-based targeted somatic multiplex-mutagenesis was used to target 34 tumor suppressor genes (TSGs) for induction of primary liver tumors in mice. Target site mutations in tumor cells were analyzed and compared between single-cell clones and their subclones, between different time points of cell proliferation, and between parental clones and single-cell clones derived from mouse subcutaneous allografts. Genomic instability and generation of extrachromosomal circular DNA (eccDNA) was explored as a potential mechanism underlying the oscillation of target site mutations in these liver tumor cells. RESULTS: After efficiently inducing autochthonous liver tumors in mice within 30-60 days, analyses of CRISPR/Cas9-induced tumors and single-cell clones derived from tumor nodules revealed multiplexed and heterogeneous mutations at target sites. Many target sites frequently displayed more than two types of allelic variations with varying frequencies in single-cell clones, indicating increased copy number of these target sites. The types and frequencies of targeted TSG mutations continued to change at some target sites between single-cell clones and their subclones. Even the proliferation of a subclone in cell culture and in mouse subcutaneous graft altered the types and frequencies of targeted TSG mutations in the absence of continuing CRISPR/Cas9 genome editing, indicating a new source outside primary chromosomes for the development of IGH in these liver tumors. Karyotyping of tumor cells revealed genomic instability in these cells manifested by high levels of micronuclei and chromosomal aberrations including chromosomal fragments and chromosomal breaks. Sequencing analysis further demonstrated the generation of eccDNA harboring targeted TSG mutations in these tumor cells. CONCLUSIONS: Small eccDNAs carrying TSG mutations may serve as an important source supporting intratumor heterogeneity and tumor evolution in mouse liver cancer induced by multiplexed CRISPR/Cas9.


Assuntos
Sistemas CRISPR-Cas , Neoplasias Hepáticas , Camundongos , Animais , Neoplasias Hepáticas/genética , Edição de Genes , Mutação , Genes Supressores de Tumor , DNA , Instabilidade Genômica , DNA Circular
3.
Nat Commun ; 13(1): 4285, 2022 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-35879372

RESUMO

Analysis of human cancer genome sequences has revealed specific mutational signatures associated with BRCA1-deficient tumors, but the underlying mechanisms remain poorly understood. Here, we show that one-ended DNA double strand breaks (DSBs) converted from CRISPR/Cas9-induced nicks by DNA replication, not two-ended DSBs, cause more characteristic chromosomal aberrations and micronuclei in Brca1-deficient cells than in wild-type cells. BRCA1 is required for efficient homologous recombination of these nick-converted DSBs and suppresses bias towards long tract gene conversion and tandem duplication (TD) mediated by two-round strand invasion in a replication strand asymmetry. However, aberrant repair of these nick-converted one-ended DSBs, not that of two-ended DSBs in Brca1-deficient cells, generates mutational signatures such as small indels with microhomology (MH) at the junctions, translocations and small MH-mediated TDs, resembling those in BRCA1-deficient tumors. These results suggest a major contribution of DNA nicks to mutational signatures associated with BRCA1 deficiency in cancer and the underlying mechanisms.


Assuntos
Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Proteína BRCA1/genética , Reparo do DNA , Replicação do DNA/genética , Conversão Gênica , Recombinação Homóloga , Humanos
4.
Nucleic Acids Res ; 45(18): 10614-10633, 2017 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-28977657

RESUMO

Phosphorylated histone H2AX, termed 'γH2AX', mediates the chromatin response to DNA double strand breaks (DSBs) in mammalian cells. H2AX deficiency increases the numbers of unrepaired DSBs and translocations, which are partly associated with defects in non-homologous end joining (NHEJ) and contributing to genomic instability in cancer. However, the role of γH2AX in NHEJ of general DSBs has yet to be clearly defined. Here, we showed that despite little effect on overall NHEJ efficiency, H2AX deficiency causes a surprising bias towards accurate NHEJ and shorter deletions in NHEJ products. By analyzing CRISPR/Cas9-induced NHEJ and by using a new reporter for mutagenic NHEJ, we found that γH2AX, along with its interacting protein MDC1, is required for efficient classical NHEJ (C-NHEJ) but with short deletions and insertions. Epistasis analysis revealed that ataxia telangiectasia mutated (ATM) and the chromatin remodeling complex Tip60/TRRAP/P400 are essential for this H2AX function. Taken together, these data suggest that a subset of DSBs may require γH2AX-mediated short-range nucleosome repositioning around the breaks to facilitate C-NHEJ with loss of a few extra nucleotides at NHEJ junctions. This may prevent outcomes such as non-repair and translocations, which are generally more destabilizing to genomes than short deletions and insertions from local NHEJ.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Histonas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Sequência de Bases , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular , Linhagem Celular , Proteína Quinase Ativada por DNA/fisiologia , Proteínas de Ligação a DNA/fisiologia , Histonas/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Nucleotídeos/análise , Deleção de Sequência
5.
Cell Biosci ; 6: 32, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27182434

RESUMO

Molecular characterization of individual patients' tumor cells is becoming increasingly important in offering effective treatment for patients in clinical practice. Recent advances in the field have indicated that circulating tumor DNA (ctDNA) has huge potential to serve as a biomarker for early detection and precision treatment as well as prognosis of hepatocellular carcinoma (HCC). As ctDNA in HCC patients harbors the molecular characteristics of HCC tumor cells, ctDNA analysis in the blood may be sufficient for convenient, non-invasive and accurate detection, providing information for HCC diagnosis, treatment and prognosis. In this review, we will summarize and discuss current trends and challenges of ctDNA application in HCC.

6.
J Virol ; 76(23): 11809-18, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12414923

RESUMO

When tethered in cis to DNA, the transcriptional corepressor mSin3B inhibits polyomavirus (Py) ori-dependent DNA replication in vivo. Histone deacetylases (HDACs) appear not to be involved, since tethering class I and class II HDACs in cis does not inhibit replication and treating the cells with trichostatin A does not specifically relieve inhibition by mSin3B. However, the mSin3B L59P mutation that impairs mSin3B interaction with N-CoR/SMRT abrogates inhibition of replication, suggesting the involvement of N-CoR/SMRT. Py large T antigen interacts with mSin3B, suggesting an HDAC-independent mechanism by which mSin3B inhibits DNA replication.


Assuntos
Replicação do DNA/genética , DNA Viral/biossíntese , DNA Viral/genética , Polyomavirus/genética , Polyomavirus/metabolismo , Origem de Replicação , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Linhagem Celular , Histona Desacetilases/metabolismo , Camundongos , Modelos Biológicos
7.
Mol Cell Biol ; 22(22): 7907-18, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12391158

RESUMO

The PCAF and GCN5 acetyltransferases, but not p300 or CBP, stimulate DNA replication when tethered near the polyomavirus origin. Replication stimulation by PCAF and GCN5 is blocked by mutational inactivation of their acetyltransferase domains but not by deletion of sequences that bind p300 or CBP. Acetylation of histones near the polyomavirus origin assembled into chromatin in vivo is not detectably altered by expression of these acetyltransferases. PCAF and GCN5 interact with polyomavirus large T antigen in vivo, PCAF acetylates large T antigen in vitro, and large T-antigen acetylation in vivo is dependent upon the integrity of the PCAF acetyltransferase domain. These data suggest replication stimulation occurs through recruitment of large T antigen to the origin and acetylation by PCAF or GCN5.


Assuntos
Acetiltransferases/metabolismo , Antígenos Transformantes de Poliomavirus/metabolismo , Replicação do DNA , Origem de Replicação , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Proteínas Virais/metabolismo , Células 3T3 , Acetilação , Animais , Antígenos Transformantes de Poliomavirus/genética , Células COS , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Histona Acetiltransferases , Histonas/metabolismo , Substâncias Macromoleculares , Camundongos , Polyomavirus/enzimologia , Polyomavirus/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ativação Transcricional , Proteínas Virais/genética
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