Publications & Reports

Measuring and monitoring apoptosis and drug toxicity in HIV patients by ligation-mediated polymerase chain reaction.

Hooker DJ, Gorry PR, Ellett AM, Wesselingh SL, Cherry CL
Centre for Virology, Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Victoria, Australia.


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 approximately 17-fold correlating with a approximately 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.


  • Journal: Journal of Cellular and Molecular Medicine
  • Published: 01/05/2009
  • Volume: 13
  • Issue: 5
  • Pagination: 948-958