Measuring and monitoring apoptosis and drug toxicity in HIV patients by ligation-mediated polymerase chain reaction.
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.
Hooker
David J
DJ
Centre for Virology, Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Victoria, Australia. dahoo@burnet.edu.au
Gorry
Paul R
PR
Ellett
Anne M
AM
Wesselingh
Steven L
SL
Cherry
Catherine L
CL
D023241:Antiretroviral Therapy, Highly Active / Q000009:adverse effects; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D015658:HIV Infections / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects; D008060:Lipodystrophy / Q000139:chemically induced / Q000473:pathology; D016133:Polymerase Chain Reaction / Q000379:methods; D018894:Reverse Transcriptase Inhibitors / Q000009:adverse effects; D019311:Staurosporine / Q000494:pharmacology; D018119:Stavudine / Q000009:adverse effects
D018894:Reverse Transcriptase Inhibitors; D018119:Stavudine; D053148:Caspase 3; D019311:Staurosporine