Tissue transglutaminase mRNA expression in apoptotic cell death.
BACKGROUND
Tissue transglutaminase (t.TG) is an enzyme that catalyzes the cross-linking of intracellular proteins, thus assembling a protein scaffold that prevents leakage of intracellular components. t.TG is activated during the apoptotic cell death cascade and plays a key role in the formation of apoptotic bodies. The aim of this study was to determine to what amount t.TG-mRNA becomes expressed during apoptosis and whether the t.TG-mRNA expression level could be used as trace marker of recent apoptosis and in individual cases for quantification of apoptosis.


METHODS
Expression of t.TG-mRNA was determined using TaqMan based, real-time RT-PCR, a semi-quantitative RT-PCR technique. The t.TG-mRNA expression was measured in cultured cells (MCF-7, human endothelial cells) and in peripheral blood mononuclear cells (PBMCs) before and after induction of apoptosis in vitro.


RESULTS
The TaqMan RT-PCR of t.TG proved to be reliable, reproducible (CV's inter and intraassay precisions of 0.8-2.8%, measured at two levels), and specific for apoptotic cell death. t.TG-mRNA expression increases in response to apoptosis induction and is not expressed during the process of necrotic cell death. The expression during apoptotic cell death changes in the dose dependent manner in cultured cells as well as in the PBMCs, treated in vitro. The increase t.TG-mRNA expression level was up to 20 times, depending on the intensity of the apoptosis induction treatment and incubation time afterwards. PBMCs of patients with myelodysplasia showed spontaneous expression of t.TG-mRNA in agreement with their increased apoptotic cell death in vivo.


CONCLUSIONS
t.TG-mRNA expression increases significantly in response to apoptosis inducing treatment. The observed changes are dose and time dependent. This leads to the conclusion that t.TG expression can be used as a trace marker for detection and quantification of apoptosis.
Volokhina
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EB
Department of Clinical Chemistry, Medisch Spectrum Twente, Hospital Group, P.O. Box 50000, 7500 KA Enschede, The Netherlands.
Hulshof
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Haanen
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C
Vermes
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I
D017209:Apoptosis / Q000502:physiology*; D005260:Female; D006801:Humans; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D011503:Transglutaminases / Q000096:biosynthesis / Q000235:genetics*; D014407:Tumor Cells, Cultured
D012333:RNA, Messenger; D011503:Transglutaminases