Metalloproteinase dependent reduction of cell surface cluster determinants upon the induction of apoptosis

Albert Magro,Sirish Shrestha,Gary Rankin,Kathy Brundage,Alice Magro

doi:10.3892/ijo.2014.2344

Abstract

LN18 glioblastoma cells were used as a model to examine changes in surface cluster determinants (CDs) as the cells undergo apoptosis. LN18 cells proceeding through apoptosis manifested a decrease in cell adhesion molecules, growth factor receptors and other surface proteins. Apoptosis was induced by MK886, a known FLAP and PPAR-α inhibitor, or staurosporine, a known inhibitor of protein kinases including protein kinase C (PKC). The detection and decrease of surface CDs were observed by flow cytometry using CD-specific primary antibodies followed by secondary antibodies conjugated to phycoerythrin. It was determined that there was an apoptotic induced decrease of α and β integrin determinants and the growth factor receptors EGFR and IGF1R. The MHC-1 cell surface marker HLA-ABC was also reduced in the apoptotic cells. The level of EGFR, IGF1R and detected α and β integrin determinants dropped dramatically. The degradation takes place in mid to late apoptosis. It was determined by real-time RT-PCR that the decrease in integrins, EGFR, IGF1R and MHC-1 determinants were not due to a reduction in transcription. Inhibitors of metalloproteinases blocked the apoptotic decrease in cell surface determinants indicating that metalloproteinases mediated the reduction in these CDs in a manner that can reduce growth and survival signals while stimulating the NK surveillance system. Overall, the data indicate that the final stages of the pharmacological induction of apoptosis, while proceeding to a full commitment to non-necrotic cell death, involves the degradation of integrin, insulin and epidermal growth factor receptors caused by a programmed dysregulation of the cell’s metalloproteinases.

Highlights

Programmed cell death was first recognized as a process of tissue restructuring [1] which led to the understanding that it is a gene-guided progression that occurs naturally in the embryonic development of tissues, organs and limbs during the growth and development of an organism [2,3,4,5]
Using the LN18 glioblastoma cell line as a model, we investigated whether integrins, growth factor receptors and MHC-1 determinants are modified as cells proceed throughout the process of apoptosis
There has been little emphasis on determining the functionally opposite process of modulating the cell's surface receptors as the cells progress through apoptosis

Summary

Introduction

Programmed cell death was first recognized as a process of tissue restructuring [1] which led to the understanding that it is a gene-guided progression that occurs naturally in the embryonic development of tissues, organs and limbs during the growth and development of an organism [2,3,4,5] This type of cell death is known as apoptosis and has been recognized as an active regulatory mechanism complementary to, but functionally opposite of, proliferation. Cell death progressions can be initiated by a variety of stimuli including free radicals, hypoxia, hyperthermia, radiation, viral infections, toxins and cytotoxic pharmacologic agents These direct stimuli initiate programmed cell death by what is referred to as intrinsic signaling pathways. The transduction mechanisms of the integrin family of receptors [11] along with insulin type-1 (IGF1R) and epidermal growth factor (EGFR) receptors have been well studied [12,13] and there is an emerging picture of interrelationships that exist between their intracellular signaling systems [14,15]

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Results

Conclusion