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The Effects and the Mechanisms of LukS-PV on Human Acute Myeloid Leukemia Cell Lines, THP-1

Author: XieQiang
Tutor: MaZuoLing
School: Anhui Medical University,
Course: Clinical Laboratory Science
Keywords: leukemia cell cycle proliferation apoptosis Bcl-2
CLC: R733.71
Type: Master's thesis
Year: 2013
Downloads: 2
Quote: 0
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Abstract


Objective: Panton-Valentine leukocidin (PVL) is a cytotoxin secreted fromstaphylococcus aureus. PVL consists of two subunits, LukS-PV and LukF-PV. In aprevious study, we reported that PVL, LukS-PV and LukF-PV could induce apoptosis inpolymorphonuclear neutrophils (PMNs). The aims of this project：（1）To investigatealterations in cell growth, apoptosis, and cell cycle induced by rLukS on the acutemyeloid leukemia THP-1cell lines;(2). To investigate the mechanisms of rLukSinduced cell apoptosis in AML cell lines, THP-1.Methods: we chose optimal concentration by using different concentrations (0，0.05，0.25,0.05,0.75and1μM/L) of rLukS on THP-1.After3,6,9,12h, the cell vitality wasdetected by MTS method. The THP-1cells were incubated with different concentrations(0,0.05,0.5and1μM/L) of rLukS for12h. Cell-cycle arrest was measured by flowcytometric (FCM); Cell cycle regulatory proteins were analyzed by western blotting.Apoptosis was observed by annexin V-FITC/propidium iodide (PI) FCM and TUNELstaining. To characterize changes in the gene expression profile of THP-1cells inresponse to rLukS, we performed cDNA microarray analysis using the real-time PCRarray（SABioscience Human Apoptosis PCR Array PAHS-012A）, which contains84genes including the TNF ligands and their receptors, members of the bcl-2, caspase,IAP and and other related genes. Westernblot method was used to investigate the resultsof cDNA microarray analysis. Results: THP-1cells treated with rLukS, resulted in a significant proliferationinhibition in a dose-and time-dependent manner. Treatment of THP-1cells with rLukSinduced G0/G1phase cell cycle arrest, while down-regulated cyclin D1. RLukS inducedthe apoptosis of THP-1cells in a dose-dependent manner. The cDNA microarray resultsshowed the majority of genes examined showed only small differences in expression.Serveral genes which changed over2-fold were related with Bcl-2family, caspasefamily,the TNF ligands and their receptors. The Westernblot results showed (1)rLukS-treated THP-1cells also exhibited down-regulated expressions of anti-apoptoticproteins Bcl-2, up-regulated expressions of apoptotic proteins Bax.(2) The expressionsof caspase-3,caspase-8,caspase-9were up-regulated after rLukS treatment.Conclusions:(1). RLukS could inhibit the proliferation of THP-1cells and induceapoptosis.(2). Activation of caspase-8and-9, increase level of Bax protein suggestingthat rLukS-induced apoptosis is preferentially mediated by the mitochondrial pathway.These changes lead to caspase-3activation and the ultimate induction of apoptosis inTHP-1cells.

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CLC: > Medicine, health > Oncology > Hematopoietic and lymphoid neoplasms > Leukemia > Acute leukemia
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