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Role of inositol polyphosphates in programmed cell death

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Abstract

The role of inositol polyphosphates (InsPs) in the mediation of cellular apoptosis was investigated in mouse MC3T3 osteoblastic cell line. Extracellular administration of InsP4, InsP5, and InsP6 increased apoptosis in a dose-dependent manner. InsP6 was more potent than InsP5 and InsP4 in promoting apoptosis. Inositol hexasulfate (InsS6), a structural analog of InsP6, was used to determine specificity of InsP6-induced apoptosis as measured by acridine orange/ethidium bromide, flow cytometry, and DNA degradation. In order to study the effects of endogenous InsPs on apoptosis, we used NaF and antimycin A as treatment agents to manipulate intracellular levels of InsPs. NaF is known to increase levels of higher InsPs by inhibiting InsPs phosphatases, a process that is reversed by antimycin A because InsPs kinases are inhibited as a result of depletion of cellular ATP pools. Apoptosis was induced in MC3T3 cells in a NaF dose- and time-dependent manner. Approximately 50% apoptosis was observed at 1 mM NaF in 8 h. Prior treatment with 10 μM antimycin A for 30 min significantly reduced the NaF-induced apoptosis as compared with its control. Additionally, we measured changes in AKT phosphorylation, cleavage of caspase-3 and caspase-9, and release of cytochrome C from mitochondria into cytosol. These changes coincided with total cellular InsPs under similar conditions. The data indicated that NaF-induced changes in apoptotic markers could be due to an increased endogenous InsPs that were partially reversed by antimycin A treatment.

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Abbreviations

MEM:

Minimum essential medium

FBS:

Fetal bovine serum

PH:

Plekstrin homology domain

InsPs:

Inositol polyphosphates

PIP:

Phosphatidlylinositol phosphates

AKT/PKB:

Protein kinase B

PI-3K:

Phosphatidylinositol 3-Kinase

DAB:

Diaminobenzidine

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Acknowledgment

This work was supported in part by funds from the Graduate Institute of Technology, Arkansas Space Grant Consortium, Thomsen Hall Foundation, Arkansas NSF-EPSCoR, and a seed grant from the Office of Research and Sponsored Programs, UALR. Authors thank Ms. Diane Haynes for critically reading the manuscript. Rakhee Agarwal thanks Graduate Institute of Technology for a Graduate Assistantship.

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  1. Graduate Institute of Technology, University of Arkansas at Little Rock, ETAS-329, 2801 South University Avenue, Little Rock, AR, 72204, USA

    Rakhee Agarwal, Hamid Mumtaz & Nawab Ali

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  1. Rakhee Agarwal
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  2. Hamid Mumtaz
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  3. Nawab Ali
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Correspondence to Nawab Ali.

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Agarwal, R., Mumtaz, H. & Ali, N. Role of inositol polyphosphates in programmed cell death. Mol Cell Biochem 328, 155–165 (2009). https://doi.org/10.1007/s11010-009-0085-6

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