scholarly journals Somatic Mutations in Oncogenes Are in Chronic Myeloid Leukemia Acquired De Novo via Deregulated Base-Excision Repair and Alternative Non-Homologous End Joining

2021 ◽  
Vol 11 ◽  
Author(s):  
Nikola Curik ◽  
Vaclava Polivkova ◽  
Pavel Burda ◽  
Jitka Koblihova ◽  
Adam Laznicka ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Base Excision Repair ◽  
Myeloid Leukemia ◽  
Somatic Mutations ◽  
De Novo ◽  
Excision Repair ◽  
Leukemic Cells ◽  
End Joining ◽  
Base Excision

Somatic mutations are a common molecular mechanism through which chronic myeloid leukemia (CML) cells acquire resistance to tyrosine kinase inhibitors (TKIs) therapy. While most of the mutations in the kinase domain of BCR-ABL1 can be successfully managed, the recurrent somatic mutations in other genes may be therapeutically challenging. Despite the major clinical relevance of mutation-associated resistance in CML, the mechanisms underlying mutation acquisition in TKI-treated leukemic cells are not well understood. This work demonstrated de novo acquisition of mutations on isolated single-cell sorted CML clones growing in the presence of imatinib. The acquisition of mutations was associated with the significantly increased expression of the LIG1 and PARP1 genes involved in the error-prone alternative nonhomologous end-joining pathway, leading to genomic instability, and increased expression of the UNG, FEN and POLD3 genes involved in the base-excision repair (long patch) pathway, allowing point mutagenesis. This work showed in vitro and in vivo that de novo acquisition of resistance-associated mutations in oncogenes is the prevalent method of somatic mutation development in CML under TKIs treatment.

In Vitro Base Excision Repair Assay Using Mammalian Cell Extracts

1999 ◽  
pp. 301-315 ◽  
Author(s):  
Guido Frosina ◽  
Enrico Cappelli ◽  
Paola Fortini ◽  
Eugenia Dogliotti
Keyword(s):  
Mammalian Cell ◽  
Base Excision Repair ◽  
Excision Repair ◽  
Cell Extracts ◽  
Base Excision

Studies on the base excision repair (BER) complex in Pyrococcus furiosus

2009 ◽  
Vol 37 (1) ◽  
pp. 79-82 ◽  
Author(s):  
Shinichi Kiyonari ◽  
Saki Tahara ◽  
Maiko Uchimura ◽  
Tsuyoshi Shirai ◽  
Sonoko Ishino ◽  
...  
Keyword(s):  
Base Excision Repair ◽  
Pyrococcus Furiosus ◽  
Excision Repair ◽  
Repair Process ◽  
Nuclear Antigen ◽  
Physical Interaction ◽  
Ap Endonuclease ◽  
Reading Frame ◽  
Base Excision

We have been studying the functions of PCNA (proliferating-cell nuclear antigen) for the assembly and reassembly of the replisome during replication fork progression. We have identified the functional interactions between PCNA and several proteins involved in DNA replication and repair from Pyrococcus furiosus. We recently reported that the activity of UDG (uracil–DNA glycosylase) in P. furiosus (PfuUDG) is stimulated by PCNA (PfuPCNA) in vitro, and identified an atypical PCNA-binding site, AKTLF, in the PfuUDG protein. To understand further the function of the complex in the BER (base excision repair) process, we investigated the AP (apurinic/apyrimidinic) endonuclease, which can process the BER pathway after uracil removal by UDG. Interestingly, one candidate ORF (open reading frame) for the AP endonuclease was found in the operon containing the gene encoding UDG in the P. furiosus genome. However, this ORF did not exhibit any activity. Instead, we identified the AP endonuclease activity from the other candidate gene products, and designated the protein as PfuAP. We discovered a physical interaction between PfuAP and PfuPCNA, suggesting the formation of a BER complex in one of the repair systems in P. furiosus.

scholarly journals Spontaneous Mutagenesis Is Enhanced in Apex Heterozygous Mice

2004 ◽  
Vol 24 (18) ◽  
pp. 8145-8153 ◽  
Author(s):  
Jessica Huamani ◽  
C. Alex McMahan ◽  
Damon C. Herbert ◽  
Robert Reddick ◽  
John R. McCarrey ◽  
...  
Keyword(s):  
Base Excision Repair ◽  
Germ Line ◽  
Excision Repair ◽  
Spontaneous Mutant ◽  
Spermatogenic Cells ◽  
Ap Endonuclease ◽  
Repair Activity ◽  
Base Excision ◽  
Mutant Frequency

ABSTRACT Germ line DNA directs the development of the next generation and, as such, is profoundly different from somatic cell DNA. Spermatogenic cells obtained from young adult lacI transgenic mice display a lower spontaneous mutant frequency and greater in vitro base excision repair activity than somatic cells and tissues obtained from the same mice. However, spermatogenic cells from old lacI mice display a 10-fold higher mutant frequency. This increased spontaneous mutant frequency occurs coincidentally with decreased in vitro base excision repair activity for germ cell and testicular extracts that in turn corresponds to a decreased abundance of AP endonuclease. To directly test whether a genetic diminution of AP endonuclease results in increased spontaneous mutant frequencies in spermatogenic cell types, AP endonuclease heterozygous (Apex +/−) knockout mice were crossed with lacI transgenic mice. Spontaneous mutant frequencies were significantly elevated (approximately twofold) for liver and spleen obtained from 3-month-old Apex +/− lacI + mice compared to frequencies from Apex +/+ lacI + littermates and were additionally elevated for somatic tissues from 9-month-old mice. Spermatogenic cells from 9-month-old Apex +/− lacI + mice were significantly elevated twofold compared to levels for 9-month-old Apex +/+ lacI + control mice. These data indicate that diminution of AP endonuclease has a significant effect on spontaneous mutagenesis in somatic and germ line cells.

scholarly journals MSH2–MSH6 stimulates DNA polymerase η, suggesting a role for A:T mutations in antibody genes

2005 ◽  
Vol 201 (4) ◽  
pp. 637-645 ◽  
Author(s):  
Teresa M. Wilson ◽  
Alexandra Vaisman ◽  
Stella A. Martomo ◽  
Patsa Sullivan ◽  
Li Lan ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Base Excision Repair ◽  
Somatic Hypermutation ◽  
Excision Repair ◽  
Cytidine Deaminase ◽  
Abasic Site ◽  
Cell Extracts ◽  
Activation Induced Cytidine Deaminase ◽  
Base Excision

Activation-induced cytidine deaminase deaminates cytosine to uracil (dU) in DNA, which leads to mutations at C:G basepairs in immunoglobulin genes during somatic hypermutation. The mechanism that generates mutations at A:T basepairs, however, remains unclear. It appears to require the MSH2–MSH6 mismatch repair heterodimer and DNA polymerase (pol) η, as mutations of A:T are decreased in mice and humans lacking these proteins. Here, we demonstrate that these proteins interact physically and functionally. First, we show that MSH2–MSH6 binds to a U:G mismatch but not to other DNA intermediates produced during base excision repair of dUs, including an abasic site and a deoxyribose phosphate group. Second, MSH2 binds to pol η in solution, and endogenous MSH2 associates with the pol in cell extracts. Third, MSH2–MSH6 stimulates the catalytic activity of pol η in vitro. These observations suggest that the interaction between MSH2–MSH6 and DNA pol η stimulates synthesis of mutations at bases located downstream of the initial dU lesion, including A:T pairs.

scholarly journals Phenotypic heterogeneity of primitive leukemic hematopoietic cells in patients with chronic myeloid leukemia

Blood ◽  
1992 ◽  
Vol 80 (10) ◽  
pp. 2522-2530 ◽  
Author(s):  
C Udomsakdi ◽  
CJ Eaves ◽  
PM Lansdorp ◽  
AC Eaves
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Philadelphia Chromosome ◽  
Significant Proportion ◽  
Hematopoietic Cells ◽  
Chronic Phase ◽  
Leukemic Cells ◽  
Clonogenic Cell ◽  
Clonogenic Cells

Abstract The peripheral blood of chronic myeloid leukemia (CML) patients with chronic-phase disease and elevated white blood cell (WBC) counts typically contains markedly increased numbers of a variety of neoplastic pluripotent and lineage-restricted hematopoietic progenitors. These include cells detected in standard colony assays as well as their more primitive precursors. The latter are referred to as long-term culture-initiating cells (LTC-IC) because of their ability to generate clonogenic cell progeny detectable after a minimum of 5 weeks incubation on competent fibroblast feeder layers. In this study, we have investigated a number of the properties of the LTC-IC and clonogenic cells present in the blood of such CML patients with high WBC counts. This included an analysis of the light scattering properties of these progenitors, as well as their expression of CD34 and HLA-DR, Rhodamine-123 staining, and in vitro sensitivity to 4- hydroperoxycyclophosphamide. In the case of LTC-IC, the production of different types of lineage-restricted and multipotent progeny was also analyzed. Most of the circulating LTC-IC and clonogenic cells in the CML patients studied (on average approximately 70% and approximately 90%, respectively) showed features of proliferating or activated cells. This is in marked contrast to the majority of progenitors in the blood of normal individuals and most of the LTC-IC in normal marrow, all of which exhibit a phenotype expected of quiescent cells. Interestingly, a significant proportion of the circulating clonogenic cells and LTC-IC in the CML samples studied (on average approximately 10% and approximately 30%, respectively) appeared to be phenotypically similar to normal circulating progenitors, although their absolute numbers were indicative of a neoplastic origin. Both phenotypes of circulating CML clonogenic cells and LTC-IC could be obtained at approximately 10% to 20% purity by differential multiparameter sorting. These findings suggest that expansion of the Philadelphia chromosome-positive clone at the level of the earliest types of hematopoietic cells results from the activation of mechanisms that enable some, but not all, signals that block the cycling of normal stem cells to be bypassed or overcome. In addition, they provide strategies for purifying these primitive leukemic cells that should facilitate further analysis of the mechanisms underlying their abnormal proliferative behavior.

DC-Based Vaccines Show in-Vitro Activity against Chronic Myeloid Leukemia Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4276-4276
Author(s):  
Yuen-Fen Tan ◽  
Soon-Keng Cheong ◽  
Chooi-Fun Leong ◽  
SAW Fadhilah
Keyword(s):  
T Cells ◽  
Immune System ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Leukemic Cells ◽  
Gm Csf ◽  
Dc Vaccine ◽  
Hla Dr

Abstract Chronic Myeloid leukemia is a common myeloproliferative disease. Despite recent advances in targeted therapy, only 7–12% of patients achieve molecular remission. Leukemic cells arrange multiple mechanisms to avoid recognition by the immune system. Dendritic cells (DC) are professional antigen presenting cells of the immune system playing a crucial role in the induction of anti-tumor responses. The use of DC is an attractive immunotherapeutic strategy against cancers, especially in minimal residual disease state. In this study, DC vaccine against chronic myeloid leukemia was generated and evaluated in-vitro. Monocytes were isolated and enriched from peripheral blood. These monocytes were subsequently cultured in RPMI medium supplemented with GM-CSF and IL-4 to induce them to become DC. These DC were then co-cultured with tumor lysates obtained from CML cell line in culture medium supplemented with GM-CSF, IL-4 and TNF alpha to become DC-based CML vaccine. The generated DC-based CML vaccines retained their DC morphology, showed strong expression of CD 86 and HLA-DR, and were negative for CD14. Mixed lymphocyte reaction indicated that the generated DC-based CML vaccines were capable of inducing proliferative responses to allogeneic lymphocytes. DC-based CML vaccines were shown to stimulate T cells to express DC-ligands, ie CD28 and CD154, as well as HLA-DR, CD71 and CD 25. In addition, the stimulated T cells were cytotoxic to CML cells used to prepare tumor lysates.

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