Monitoring of FLT3 phosphorylation status and its response to drugs by flow cytometry in AML blast cells

Grafone, Tiziana; Palmisano, Michela; Nicci, Chiara; Martelli, Maria Paola; Ottaviani, Emanuela; Storti, Sergio; Baccarani, Michele; Martinelli, Giovanni

doi:10.1002/hon.854

Cited by 5 publications

(5 citation statements)

References 22 publications

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“…Various mechanisms of FLT3 activations may be present in different AML patients: FLT3 mutations are associated with a constitutive tyrosine kinase activity, consequently the inhibition of phosphorylated targets is growing in importance. 109 Up to now, different compounds, including lestaurtinib, sunitinib, midostaurin and AC220 have been investigated in vitro and in vivo as FLT3 inhibitors. 59 However, clinical trials have produced only partial and transitory results.…”

Section: Discussionmentioning

confidence: 99%

An overview on the role of FLT3-tyrosine kinase receptor in acute myeloid leukemia: biology and treatment

Grafone¹,

Palmisano²,

Nicci³

et al. 2012

Oncol Rev

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209

203

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Hematopoiesis, the process by which the hematopoietic stem cells and progenitors differentiate into blood cells of various lineages, involves complex interactions of transcription factors that modulate the expression of downstream genes and mediate proliferation and differentiation signals. Despite the many controls that regulate hematopoiesis, mutations in the regulatory genes capable of promoting leukemogenesis may occur. The FLT3 gene encodes a tyrosine kinase receptor that plays a key role in controlling survival, proliferation and differentiation of hematopoietic cells. Mutations in this gene are critical in causing a deregulation of the delicate balance between cell proliferation and differentiation. In this review, we provide an update on the structure, synthesis and activation of the FLT3 receptor and the subsequent activation of multiple downstream signaling pathways. We also review activating FLT3 mutations that are frequently identified in acute myeloid leukemia, cause activation of more complex downstream signaling pathways and promote leukemogenesis. Finally, FLT3 has emerged as an important target for molecular therapy. We, therefore, report on some recent therapies directed against it.

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Section: Discussionmentioning

confidence: 99%

An overview on the role of FLT3-tyrosine kinase receptor in acute myeloid leukemia: biology and treatment

Grafone¹,

Palmisano²,

Nicci³

et al. 2012

Oncol Rev

Self Cite

209

203

View full text Add to dashboard Cite

show abstract

“…Immunostaining of proteins was conducted as described in previous studies with some modifications . In brief, cells were fixed and permeabilized with BD Pharmingen Transcription Factor Buffer Set (BD Bioscience) for 40 minutes at the room temperate according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

“…15,16 In brief, cells were fixed and permeabilized with BD Pharmingen Transcription Factor Buffer Set We analysed the protein expression using ratio of fluorescence intensity (RFI), which was calculate by the mean fluorescence intensity of the stained samples to the isotypic control samples. 15,16 In brief, cells were fixed and permeabilized with BD Pharmingen Transcription Factor Buffer Set We analysed the protein expression using ratio of fluorescence intensity (RFI), which was calculate by the mean fluorescence intensity of the stained samples to the isotypic control samples.…”

Section: Flow Cytometrymentioning

confidence: 99%

“…Immunostaining of proteins was conducted as described in previous studies with some modifications. 15,16 In brief, cells were fixed and permeabilized with BD Pharmingen Transcription Factor Buffer Set (BD Bioscience) for 40 minutes at the room temperate according to the manufacturer's instructions. The cells were then incubated for 40 minutes with a 1/100 dilution of primary antibodies, or polyclonal rabbit immunoglobulins (Cell Signaling Technology) as the isotypic control.…”

Section: Flow Cytometrymentioning

confidence: 99%

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Krüppel‐like factor 4 regulates stemness and mesenchymal properties of colorectal cancer stem cells through the TGF‐β1/Smad/snail pathway

Leng

Zhou

et al. 2019

J Cellular Molecular Medi

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| INTRODUC TI ONCancer metastasis contributes to approximately 90% of cancer-related deaths. 1 Accumulating evidence indicates that cancer stem cells (CSCs) are responsible for the seeding and colonization of cancer metastasis. 2-4 For the past decades, studies have explored and investigated the mechanisms of human carcinogenesis to help develop novel therapeutic strategies. To date, cancer treatment has not been much improved in advanced and chemo-resistant human cancers. Thus, a better understanding of CSCs behaviour in human cancers, including colorectal cancers (CRC), could provide insight into defining the molecular mechanisms of tumorigenesis and cancer progression. Indeed, identification of CSC markers is a very hot topic and interesting data have been generated. 5 For example, CSC markers for breast, prostate, AbstractKrüppel-like factor 4 (KLF4) was closely associated with epithelial-mesenchymal transition and stemness in colorectal cancer stem cells (CSCs)-enriched spheroid cells.Nonetheless, the underlying molecular mechanism is unclear. This study showed that KLF4 overexpression was accompanied with stemness and mesenchymal features in Lgr5 + CD44 + EpCAM + colorectal CSCs. KLF4 knockdown suppressed stemness, mesenchymal features and activation of the TGF-β1 pathway, whereas enforced KLF4 overexpression activated TGF-β1, phosphorylation of Smad 2/3 and Snail expression, and restored stemness and mesenchymal phenotypes. Furthermore, TGF-β1 pathway inhibition invalidated KLF4-facilitated stemness and mesenchymal features without affecting KLF4 expression. The data from the current study are the first to demonstrate that KLF4 maintains stemness and mesenchymal properties through the TGF-β1/Smad/Snail pathway in Lgr5 + CD44 + EpCAM + colorectal CSCs. K E Y W O R D Scancer stem cell, colorectal cancer, Krüppel-like factor 4, snail, TGF-β1

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“…K562 cells were seeded at 5 × 10 3 cells per well in 96-well plates and exposed to increasing concentrations of the NCI compounds for 96 hours. The number of surviving cells was then determined using the XTT colorimetric dye reduction assay (36).…”

Section: Methodsmentioning

confidence: 99%

In Silico Screening Reveals Structurally Diverse, Nanomolar Inhibitors of NQO2 That Are Functionally Active in Cells and Can Modulate NF-κB Signaling

Nolan

Dunstan

Caraher

et al. 2012

Molecular Cancer Therapeutics

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The National Cancer Institute chemical database has been screened using in silico docking to identify novel nanomolar inhibitors of NRH:quinone oxidoreductase 2 (NQO2). The inhibitors identified from the screen exhibit a diverse range of scaffolds and the structure of one of the inhibitors, NSC13000 cocrystalized with NQO2, has been solved. This has been used to aid the generation of a structure-activity relationship between the computationally derived binding affinity and experimentally measured enzyme inhibitory potency. Many of the compounds are functionally active as inhibitors of NQO2 in cells at nontoxic concentrations. To show this, advantage was taken of the NQO2-mediated toxicity of the chemotherapeutic drug CB1954. The toxicity of this drug is substantially reduced when the function of NQO2 is inhibited, and many of the compounds achieve this in cells at nanomolar concentrations. The NQO2 inhibitors also attenuated TNFa-mediated, NF-kB-driven transcriptional activity. The link between NQO2 and the regulation of NF-kB was confirmed by using short interfering RNA to NQO2 and by the observation that NRH, the cofactor for NQO2 enzyme activity, could regulate NF-kB activity in an NQO2-dependent manner. NF-kB is a potential therapeutic target and this study reveals an underlying mechanism that may be usable for developing new anticancer drugs.

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Monitoring of FLT3 phosphorylation status and its response to drugs by flow cytometry in AML blast cells

Cited by 5 publications

References 22 publications

An overview on the role of FLT3-tyrosine kinase receptor in acute myeloid leukemia: biology and treatment

An overview on the role of FLT3-tyrosine kinase receptor in acute myeloid leukemia: biology and treatment

Krüppel‐like factor 4 regulates stemness and mesenchymal properties of colorectal cancer stem cells through the TGF‐β1/Smad/snail pathway

In Silico Screening Reveals Structurally Diverse, Nanomolar Inhibitors of NQO2 That Are Functionally Active in Cells and Can Modulate NF-κB Signaling

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