Hooker David J, Gorry Paul R, Ellett Anne M, Wesselingh Steven L, Cherry Catherine L
Centre for Virology, Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Victoria, Australia.
J Cell Mol Med. 2009 May;13(5):948-58. doi: 10.1111/j.1582-4934.2008.00612.x. Epub 2008 Dec 16.
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.
细胞凋亡在正常生理过程中起关键作用，而其失调与某些病理学存在因果联系。细胞凋亡的一个生化特征，即核小体间基因组DNA片段化，可通过连接介导的聚合酶链反应（LM-PCR）检测到。在此，我们通过将微量的600 bp凋亡扩增子与单拷贝管家基因的扩增子相除，将LM-PCR转化为一种新的细胞凋亡定量方法，生成LM-PCR“值”。动态范围约为17倍，与凋亡片段化程度约200倍的差异相关。凝胶间和凝胶内的可靠性均极佳，支持LM-PCR在大量样本集上的实用性。包括细胞随时间暴露于星形孢菌素的验证实验表明，LM-PCR与半胱天冬酶-3/酶联免疫吸附测定（ELISA）一样敏感，且比末端脱氧核苷酸转移酶介导的dUTP缺口末端标记/荧光激活细胞分选（TUNEL/FACS）更敏感，用于区分低程度的细胞凋亡（体内最相关的谱）。LM-PCR谱与半胱天冬酶-3/ELISA的谱相似，但与TUNEL/FACS的不同。然后我们将这种分子工具应用于临床研究。细胞凋亡增加与脂肪萎缩（皮下脂肪消耗）有关，脂肪萎缩是抗HIV高效抗逆转录病毒疗法（HAART）中使用的一些核苷类似物逆转录酶抑制剂（NRTIs）的一种严重、持续的毒性。我们在105份外周血单核细胞样本中证明，在用司他夫定（d4T）治疗期间，LM-PCR值升高，司他夫定是一种毒性特别大的NRTI（与未进行HAART相比，P<0.0001，不成对t检验）。升高的值也与脂肪萎缩的临床证据独立相关（P=0.007，多元逻辑回归模型），但与患者年龄、CD4 T细胞计数或HIV病毒载量无关（每项P>0.8）。这些数据共同表明，LM-PCR是一种强大且可靠的细胞凋亡定量方法，具有基础科学和临床研究的潜力。
