Quantification of apoptotic DNA fragmentation in a transformed uterine epithelial cell line, HRE-H9, using capillary electrophoresis with laser-induced fluorescence detector (CE-LIF).
Apoptotic cell death of uterine epithelial cells is thought to play an important role in the onset of menstruation and the successful implantation of an embryo during early pregnancy. Abnormal apoptosis in these cells can result in dysmenorrhoea and infertility. In addition, decreased rate of epithelial apoptosis likely contributes to endometriosis. A key step in the onset of apoptosis in these cells is cleavage of the genomic DNA between nucleosomes, resulting in polynucleosomal-sized fragments of DNA. The conventional technique for assessing apoptotic DNA fragmentation uses agarose (slab) gel electrophoresis (i.e. DNA laddering). However, recent technological advances in the use of capillary electrophoresis (CE), particularly the introduction of the laser-induced fluorescence detector (LIF), has made it possible to perform DNA laddering with improved automation and much greater sensitivity. In the present study, we have further developed the CE-LIF technique by using a DNA standard curve to quantify accurately the amount of DNA in the apoptotic DNA fragments and have applied this new quantitative technique to study apoptosis in a transformed uterine epithelial cell line, the HRE-H9 cells. Apoptosis was induced in the HRE-H9 cells by serum deprivation for 5, 7 and 24 h, resulting in increased DNA fragmentation of 2.2-, 3.1- and 6.2-fold, respectively, above the 0 h or plus-serum controls. This ultrasensitive CE-LIF technique provides a novel method for accurately measuring the actions of pro- or anti-apoptotic agents or conditions on uterine epithelial cell lines.
Fiscus
R R
RR
The Epithelial Cell Biology Research Center, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong. ronfiscus@cuhk.edu.hk
Leung
C P
CP
Yuen
J P
JP
Chan
H C
HC
D000818:Animals; D017209:Apoptosis; D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000648:ultrastructure; D004247:DNA / Q000032:analysis; D053938:DNA Fragmentation; D019075:Electrophoresis, Capillary / Q000379:methods*; D004847:Epithelial Cells / Q000166:cytology / Q000648:ultrastructure; D005260:Female; D005453:Fluorescence; D007700:Kinetics; D007834:Lasers; D011817:Rabbits; D012015:Reference Standards; D014599:Uterus / Q000166:cytology*
D004247:DNA