quantifying-level-8-oxo-dg-using-elisa-assay-to-evaluate-oxidative

Quantifying the Level of 8-oxo-dG Using ELISA Assay to Evaluate Oxidative DNA Damage in MCF-7 Cells

8-Oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dG) base is the predominant form of commonly observed DNA oxidative damage. DNA impairment profoundly ...

Suzhou Vocational Health College

Objective To construct the eukaryotic expression vectors of human fms related tyrosine kinase 3 (FLT3) gene and FLT3-internal tandem duplication (FLT3-ITD) mutants and purify the native proteins through immunoprecipitation from HEK293T cell lysates. Methods The cDNA fragments of FLT3wt and FLT3-ITD were amplified from bone marrow cells of healthy individuals and FLT3-ITD-mutated acute myeloid leukemia (AML) patients with specific primers, and the PCR products were cloned in CD530A-T2A-GFP expression vectors. FLT3wt and FLT3-ITD plasmids were transfected in HEK293T cells by Polyjet reagent, and the recombinant proteins were purified by immunoprecipitation and competing elution methods. Results FLT3wt and FLT3-ITD-mutated DNA sequences were successfully cloned in CD530A-T2A-GFP expression vectors. FLT3wt and FLT3-ITD mutated proteins were successfully expressed and purified in HEK293T cells as verified by Western blotting and sliver staining. Conclusion FLT3wt and FLT3-ITD expression vectors were successfully constructed, and purified proteins were successfully obtained from HEK293T cells.

[Expression and purification of FLT3 and FLT3-ITD mutated proteins in HEK293T cells].

To detect and analyze the mutation status of FANCJ gene in adult AML patients, so as to provide the basis for studying the mechanism of FANCJ driven AML and guiding the preventim and treatment of deseese.

[Mutation Spectrum of FANCJ Gene in Adult Acute Myeloid Leukemia].

Flap endonuclease-1 (FEN1) plays a central role in DNA replication and DNA damage repair process. In mammals, FEN1 functional sites variation is related to cancer and chronic inflammation, and supports the role of FEN1 as a tumor suppressor. However, FEN1 is overexpressed in multiple types of cancer cells and is associated with drug resistance, supporting its role as an oncogene. Hence, it is vital to explore the multi-functions of FEN1 in normal cell metabolic process. This study was undertaken to examine how the gene expression profile changes when FEN1 is downregulated in 293T cells.

Transcriptome Analysis of FEN1 Knockdown HEK293T Cell Strain Reveals Alteration in Nucleic Acid Metabolism, Virus Infection, Cell Morphogenesis and Cancer Development.

Lung cancer (especially, non-small cell lung cancer [NSCLC]) is one of the most malignant cancers in the world. Hinesol is the major component of the essential oil of Atractylodes lancea (Thunb.) DC and possesses the most promising anticancer function. However, the effects and molecular mechanism of hinesol on antiproliferation in NSCLC cells has not been well understood. In this study, we found that hinesol effectively inhibited the A549 and NCI-H1299 cells in a dose- and time-dependent manner by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay. In addition, hinesol induced cell cycle arrest at G0/G1 phase and apoptosis assessed by flow cytometry in A549 cells. Furthermore, Western blot analysis showed that hinesol decreased phosphorylation of mitogen-activated protein kinase, extracellular signal-regulated kinase, IκBα, and p65 inhibited the expressions of Bcl-2, cyclin D1 and upregulated the expression of Bax. Based on these results, hinesol might be a potential drug candidate of anti-NSCLC for therapy.

The antitumor effect of hinesol, extract from Atractylodes lancea (Thunb.) DC. by proliferation, inhibition, and apoptosis induction via MEK/ERK and NF-κB pathway in non-small cell lung cancer cell lines A549 and NCI-H1299.

Cervical cancer is one of the most common causes of cancer-associated mortality among affected women in the world. At present, treatment with weekly cisplatin plus ionizing radiation (IR) therapy is the standard regimen for cervical cancer, especially for locally advanced cervical cancer. The purpose of this study is to determine whether FEN1 inhibitors could enhance the therapeutic effect of IR therapy.

FEN1 inhibitor increases sensitivity of radiotherapy in cervical cancer cells.

Ursolic acid (UA), a primary bioactive triterpenoid, was reported as an anti-cancer agent. However, the current knowledge of UA and its potential anti-cancer mechanisms and targets in breast cancer cells are limited. In this study, we aimed to illustrate the potential mechanisms and targets of UA in breast cancer cells MCF-7.

The Analysis of the Anti-Tumor Mechanism of Ursolic Acid Using Connectively Map Approach in Breast Cancer Cells Line MCF-7.

Acute myeloid leukemia (AML) with t(8;21) is categorized as favorable-risk AML, but KIT mutations show a significantly poor prognostic impact in such patients. Persistent vulnerability to relapse is a major challenge in the treatment of this subtype of patients. Venetoclax is a BCL-2 selective inhibitor. The venetoclax+HMA strategy is also a notable salvage regimen that achieves good clinical outcomes in the treatment of relapsed or refractory (R/R) AML. However, in our clinical practice, we found that disease progressed rapidly even after venetoclax+azacitidine (AZA) therapy in two relapsed t(8;21) AML patients with KIT mutations. We report for the first time the therapeutic potential of venetoclax+midostaurin as a new combination therapy for relapsed t(8;21) AMLs with KIT mutations showing resistance to venetoclax+AZA therapy. Our ex vivo study also showed that midostaurin alone could inhibit proliferation and induce apoptosis of Kasumi-1 cells (e.g. Midostaurin induced G2 phase cell arrest, down-regulated p-KIT and BCL-2, while Bax protein levels were up-regulated) and observed a synergistic anti effect when the two drugs were combined. Our study shows that the venetoclax+midostaurin regimen may be a promising treatment option for R/R t(8;21) AML with KIT mutations.

Combination of Venetoclax and Midostaurin Efficiently Suppressed Relapsed t(8;21)Acute Myeloid Leukemia With Mutant KIT After Failure of Venetoclax Plus Azacitidine Treatment.

To uncover the role of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)/runt-related transcription factor 2 (RUNX2)/interleukin-11 (IL-11) pathway in the activation of Müller glial cells (MGCs) and the breakdown of blood-retina barrier (BRB) during diabetic retinopathy (DR).

MEK/ERK/RUNX2 Pathway-Mediated IL-11 Autocrine Promotes the Activation of Müller Glial Cells during Diabetic Retinopathy.

Acute myeloid leukemia (AML) is a hematological malignancy with high molecular and clinical heterogeneity, and is the most common type of acute leukemia in adults. Due to limited treatment options, AML is prone to relapse and has a poor prognosis. Excision repair cross-complementing 3 (ERCC3) is an important member of nucleotide excision repair (NER) that is overexpressed in types of solid cancers and potentially regarded as a prognostic factor. However, its role in AML remains unclear. The purpose of this study was to explore ERCC3 expression and functions in AML.

Comprehensive analysis of ERCC3 prognosis value and ceRNA network in AML.

Fms-like tyrosine kinase 3 (FLT3) is frequently mutated in haematological malignancies. Although canonical FLT3 mutations including internal tandem duplications (ITDs) and tyrosine kinase domains (TKDs) have been extensively studied, little is known about the clinical significance of non-canonical FLT3 mutations. Here, we first profiled the spectrum of FLT3 mutations in 869 consecutively newly diagnosed acute myeloid leukaemia (AML), myelodysplastic syndrome and acute lymphoblastic leukaemia patients. Our results showed four types of non-canonical FLT3 mutations depending on the affected protein structure: namely non-canonical point mutations (NCPMs) (19.2%), deletion (0.7%), frameshift (0.8%) and ITD outside the juxtamembrane domain (JMD) and TKD1 regions (0.5%). Furthermore, we found that the survival of patients with high-frequency (>1%) FLT3-NCPM in AML was comparable to those with canonical TKD. In vitro studies using seven representative FLT3-deletion or frameshift mutant constructs showed that the deletion mutants of TKD1 and the FLT3-ITD mutant of TKD2 had significantly higher kinase activity than wild-type FLT3, whereas the deletion mutants of JMD had phosphorylation levels comparable with wild-type FLT3. All tested deletion mutations and ITD were sensitive to AC220 and sorafenib. Collectively, these data enrich our understanding of FLT3 non-canonical mutations in haematological malignancies. Our results may also facilitate prognostic stratification and targeted therapy of AML with FLT3 non-canonical mutations.

Mutation spectrum of FLT3 and significance of non-canonical FLT3 mutations in haematological malignancy.

As a core member of the FA complex, in the Fanconi anemia pathway, FAAP24 plays an important role in DNA damage repair. However, the association between FAAP24 and patient prognosis in AML and immune infiltration remains unclear. The purpose of this study was to explore its expression characteristics, immune infiltration pattern, prognostic value and biological function using TCGA-AML and to verify it in the Beat AML cohort.

Jiahui Du

Suzhou Key Laboratory of Medical Biotechnology

Suzhou Vocational Health College

Quantifying the Level of 8-oxo-dG Using ELISA Assay to Evaluate Oxidative DNA Damage in MCF-7 Cells

Jiahui Du

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Suzhou Vocational Health College

Quantifying the Level of 8-oxo-dG Using ELISA Assay to Evaluate Oxidative DNA Damage in MCF-7 Cells

Suzhou Key Laboratory of Medical Biotechnology