RNA synthesis and splicing interferes with DNA in situ end labeling techniques used to detect apoptosis

Area of Science:

• Cell Biology
• Molecular Biology
• Pathology

Background:

• The Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique is widely used to detect apoptosis by identifying DNA fragmentation.
• DNA fragmentation is considered a hallmark of the late, irreversible stages of apoptosis.
• High TUNEL labeling in certain conditions, like atherosclerotic plaques, has raised questions about its specificity.

Purpose of the Study:

• To investigate the specificity of the TUNEL technique in detecting apoptosis.
• To determine if non-apoptotic processes can also lead to TUNEL labeling.
• To propose improvements for accurate apoptosis assessment in tissue sections.

Main Methods:

• Development of a co-localization system to study TUNEL labeling.
• Simultaneous analysis of TUNEL labeling with markers for RNA transcription and splicing.
• Evaluation of TUNEL labeling in conjunction with morphological criteria.

Main Results:

• The TUNEL technique labels not only apoptotic nuclei but also non-apoptotic nuclei exhibiting active gene transcription.
• TUNEL labeling is selective rather than specific for apoptotic nuclei, as non-apoptotic cells can also show DNA fragmentation.
• Observed TUNEL labeling in atherosclerotic plaques varied significantly, suggesting potential confounding factors.

Conclusions:

• The TUNEL technique alone is not entirely specific for the executive phase of apoptosis.
• Combining TUNEL with markers of transcription and morphological analysis is crucial for accurate apoptosis detection.
• Further validation is needed to refine apoptosis assessment in complex biological samples.