Genomic Aberrations and Cellular Heterogeneity in SV40-Immortalized Human Corneal Epithelial Cells
Kenta Yamasaki 1*,
Satoshi Kawasaki 2,
Robert D Young 3,
Hideki Fukuoka 2,
Hidetoshi Tanioka 2,
Mina Nakatsukasa 2,
Andrew J Quantock 3,
and
Shigeru Kinoshita 2
1 ophthalmology, Kyoto prefectural university of medicine, kyoto, Japan
2 ophthalmology, Kyoto prefectural university of medicine, Kyoto, Japan
3 Optometry and Vision Science, Cardiff University, Cardiff, United Kingdom
* To whom correspondence should be addressed. E-mail: kyamasak{at}ophth.kpu-m.ac.jp.
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Abstract |
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Purpose. Simian virus 40 (SV40)-immortalized human corneal epithelial (HCE-T) cells have been widely used as an in vitro model of human corneal epithelial cells. To better understand the nature of this cell line, we assessed it for genomic aberrations and cellular heterogeneity.
Methods. For the quantitative measurement of genomic aberrations, array-based comparative genomic hybridization (CGH) analysis was performed. For identification of cellular heterogeneity, we analyzed cell morphology, growth kinetics, trans-epithelial electrical resistance, and transfection/transcriptional efficiency. We also carried out real-time PCR and chromosomal fluorescent in situ hybridization (cFISH) against some gained or lost loci in order to assess genomic heterogeneity. To assess differences in the gene expression profiles between HCE-T cells and normal corneal epithelial cells, we collected expressed sequence tags (ESTs) for this cell line. Southern blotting and inverse PCR analyses were used to determine the genomic integration site of the SV40 large T antigen gene (LTAG).
Results. Array CGH analysis demonstrated that the genomic content of HCE-T cells is different from the normal healthy genome. Our results from cellular functional assays, real-time PCR, and cFISH strongly indicated that HCE-T cells consist of a significant number of heterogeneous cell populations. The genomic integration site of the SV40 large T antigen was at p22.1 of chromosome 9.
Conclusions. Our results indicate that HCE-T cells have an altered genomic content and that they are composed of heterogeneous cell populations. This should be considered when conducting experiments or interpreting the results of studies which use this cell line.
Key Words:
corneal epithelium, gene expression, molecular biology
