Apoptosis has a critical role in normal physiology while its dysregulation has causal links with certain pathologies. A biochemical hallmark of apoptosis, internucleosomal genomic DNA fragmentation, is detectable by ligation-mediated polymerase chain reaction (LM-PCR). Here we converted LM-PCR into a new apoptosis quantifier by dividing trace quantities of 600 bp apoptotic amplicons into those of a single copy house-keeping gene, generating the LM-PCR 'value'. Dynamic range was ∼17-fold correlating with a ∼200-fold difference in degree of apoptotic fragmentation. Inter- and intra-gel reliability were both excellent, supporting LM-PCR's utility with large sample sets. Validation experiments comprising cell exposure to staurosporine over time revealed LM-PCR is as sensitive as caspase-3-ELISA and more sensitive than terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling-flourescence- activated cell sorting (TUNEL-FACS) for distinguishing low degrees of apoptosis (the spectrum most relevant in vivo). The LM-PCR profile mirrored that of caspase-3-ELISA but not TUNEL-FACS. We then applied this molecular tool to clinical investigation. Increased apoptosis is implicated in lipoatrophy (subcutaneous fat wasting), a serious, persistent toxicity of some nucleoside analogue reverse transcriptase inhibitors (NRTIs) used in anti-HIV highly active antiretroviral therapy (HAART). We demonstrated in 105 peripheral blood mononuclear cell samples that elevated LM-PCR values are seen during therapy with stavudine (d4T), a particularly toxic NRTI (P < 0.0001 versus no HAART, unpaired t-test). Elevated values were also independently associated with clinical evidence of lipoatrophy (P = 0.007, multiple logistic regression modelling) but not with patient age, CD4 T-cell count nor HIV viral load (P > 0.8 for each). Together these data demonstrate that LM-PCR is a robust and reliable quantifier of apoptosis with potential for basic science and clinical investigation.
- Antiretroviral drugs
- Ligation-mediated polymerase chain reaction
ASJC Scopus subject areas
- Molecular Medicine
- Cell Biology