Usefulness of PKH fluorescent labelling to study leukemic cell proliferation with various cytostatic drugs or acetyl tetrapeptide – AcSDKP

Boutonnât, Jean; Faussat, Anne-Marie; Marie, Jean‐Pierre; Bignon, Jérôme; Wdzieczak‐Bakala, Joanna; Barbier, Magali; Thierry, Josiane; Ronot, Xavier; Colle, Pierre-Emmanuel

doi:10.1186/1471-2407-5-120

Cited by 14 publications

(8 citation statements)

References 23 publications

(20 reference statements)

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“…The PkH26 and the PkH67 fluorescent cell linkers (Sigma-Aldrich) were used to stably incorporate a dye with long aliphatic tails into the lipid receptors to labeling membrane of rapidly growing cells. These fluorochomes have been used for labeling epithelial cells in the gut [113] , leukemia cells [114] , lymphocytes [115] , epithelial and mesenchymal stem cells [116] , and cord blood T lymphocytes [117] for ex vivo and in vivo cell proliferation analysis. The chickens in these groups received alternate injections of red (PkH26) or green (PkH67) fluorochromes intravenously every two weeks (see Methods ).…”

Section: Resultsmentioning

confidence: 99%

Inhibition of Autoimmune Chagas-Like Heart Disease by Bone Marrow Transplantation

et al. 2014

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BackgroundInfection with the protozoan Trypanosoma cruzi manifests in mammals as Chagas heart disease. The treatment available for chagasic cardiomyopathy is unsatisfactory.Methods/Principal FindingsTo study the disease pathology and its inhibition, we employed a syngeneic chicken model refractory to T. cruzi in which chickens hatched from T. cruzi inoculated eggs retained parasite kDNA (1.4 kb) minicircles. Southern blotting with EcoRI genomic DNA digests revealed main 18 and 20 kb bands by hybridization with a radiolabeled minicircle sequence. Breeding these chickens generated kDNA-mutated F1, F2, and F3 progeny. A targeted-primer TAIL-PCR (tpTAIL-PCR) technique was employed to detect the kDNA integrations. Histocompatible reporter heart grafts were used to detect ongoing inflammatory cardiomyopathy in kDNA-mutated chickens. Fluorochromes were used to label bone marrow CD3+, CD28+, and CD45+ precursors of the thymus-dependent CD8α+ and CD8β+ effector cells that expressed TCRγδ, vβ1 and vβ2 receptors, which infiltrated the adult hearts and the reporter heart grafts.Conclusions/SignificanceGenome modifications in kDNA-mutated chickens can be associated with disruption of immune tolerance to compatible heart grafts and with rejection of the adult host's heart and reporter graft, as well as tissue destruction by effector lymphocytes. Autoimmune heart rejection was largely observed in chickens with kDNA mutations in retrotransposons and in coding genes with roles in cell structure, metabolism, growth, and differentiation. Moreover, killing the sick kDNA-mutated bone marrow cells with cytostatic and anti-folate drugs and transplanting healthy marrow cells inhibited heart rejection. We report here for the first time that healthy bone marrow cells inhibited heart pathology in kDNA+ chickens and thus prevented the genetically driven clinical manifestations of the disease.

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Section: Resultsmentioning

confidence: 99%

Inhibition of Autoimmune Chagas-Like Heart Disease by Bone Marrow Transplantation

et al. 2014

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“…AcSDKP, a negative regulator of hematopoiesis, had the effect of radioprotection on hematopoietic progenitors (Charrier et al, 2004). AcSDKP has direct and reversible inhibitory effect on the growth of human CD34 þ subpopulations in response to growth factors and inhibits the proliferation of leukemic cells Boutonnat et al, 2005). In vivo, its administration into mice increases their rate of survival after lethal dose of cytosine arabinoside chemotherapy or irradiation and inhibits the CFU-S entry into cycle (Watanabe et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

Thymosin β4 and AcSDKP inhibit the proliferation of HL‐60 cells and induce their differentiation and apoptosis

Huang

Wang

2006

Cell Biology International

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Our previous works have shown that bone marrow stromal cells secrete thymosin beta4 (Tbeta4) and AcSDKP. Tbeta4 and AcSDKP are existed in the conditioned medium of bone marrow endothelial cells. They exerted inhibitory effects on hematopoietic cells and then had protective effect on the early hematopoietic cells, which were cultured in the presence of hematopoietic stimulators. Thymosin beta4 consists of 43 peptides with a molecular weight of 4963. It contains at its N-terminal end the sequence of the acetylated tetrapeptide Ac-N-Ser-Asp-Lys-Pro (AcSDKP). This study was performed to evaluate the effect of Tbeta4 and AcSDKP on the growth of HL-60 cells. It was showed that Tbeta4 (10(-11)-10(-7)mol/L) and AcSDKP (10(-11)-10(-7)mol/L) had the dose-dependent inhibitory effect on the proliferation of HL-60 cells. Based on cell morphology and NBT reduction, Tbeta4 and AcSDKP induced differentiation of HL-60 cells. Morphologic and DNA fragment analysis proved that Tbeta4 and AcSDKP induced apoptosis of HL-60 cells. In order to analyze the mechanism of the effects of Tbeta4 and AcSDKP, intracellular free Ca(2+) concentration ([Ca(2+)](i)) of HL-60 leukemic cells was tested and Atlas cDNA Expression Array was performed. The results showed that Tbeta4 and AcSDKP could increased [Ca(2+)](i) by stimulating the release of Ca(2+) from intracellular Ca(2+) pool. Moreover, AcSDKP could also elicit a potent extracelluar calcium influx in HL-60 cells. Tbeta4 could also change apoptotic-related gene expression in leukemic cells, and resulted in the inhibition of proliferation and induction of differentiation and apoptosis of leukemic cells.

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“…Although bromodeoxyuridine labeling of DNA is frequently used in LRC assays, recent studies show its cytotoxic nature (18). The use of membrane-labeling dyes such as PKH67/ PKH26 that have a good correlation with bromodeoxyuridine incorporation and slow-cycling cells is suggested (19)(20)(21). These vital dyes consist of a fluorophore attached to an aliphatic carbon backbone that irreversibly binds to the lipid bilayer on cell membranes.…”

Section: Introductionmentioning

confidence: 99%

Cancer Stem Cells and Aneuploid Populations within Developing Tumors Are the Major Determinants of Tumor Dormancy

2009

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Tumor formation involves substantial cell division and genetic instability, but the relationship between quiescent cancer stem cells (CSC) and dividing progenitors in these events is poorly understood. Likewise, the implication of aneuploid cells in solid tumors is uncertain. CSCs are postulated to contribute to tumor dormancy and present a formidable obstacle in limiting treatment outcomes for a majority of cancers, whereas the genetic heterogeneity conjured by aneuploid cells may influence tumor drug resistance. However, direct confirmation of these events remains forthcoming. In the present study, we addressed the identification of tumor dormancy in terms of isolation of therapy-refractory residual tumor cells from tumors that persist in a state of quiescence as labelretaining cells. The choices of label were PKH67/PKH26 dyes that irreversibly bind to the lipid bilayer on cell membranes and get equally partitioned among daughter cells subsequent to each cell division. Consequent characterization revealed that label-retaining cells encompass two different populations capable of remaining in a state of quiescence, i.e., stem-like cells and aneuploid cells. The former express a reversibility of quiescence through retention of functionality and also exhibit therapeutic refractoriness; the latter seem to be either quiescent or proliferation-arrested at steady-state. Subsequent to exposure to selective pressure of chemotherapy, a fraction of these cells may acquire the potential to proliferate in a drug-refractory manner and acquire stem-like characteristics. Collectively, the findings of the present study reveal that tumor-derived CSCs and aneuploid populations contribute to drug resistance and tumor dormancy in cancer progression. [Cancer Res 2009;69(24):9245-53]

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Usefulness of PKH fluorescent labelling to study leukemic cell proliferation with various cytostatic drugs or acetyl tetrapeptide – AcSDKP

Cited by 14 publications

References 23 publications

Inhibition of Autoimmune Chagas-Like Heart Disease by Bone Marrow Transplantation

Inhibition of Autoimmune Chagas-Like Heart Disease by Bone Marrow Transplantation

Thymosin β4 and AcSDKP inhibit the proliferation of HL‐60 cells and induce their differentiation and apoptosis

Cancer Stem Cells and Aneuploid Populations within Developing Tumors Are the Major Determinants of Tumor Dormancy

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