Objective: Frequent alterations of microsatellite sequence of cancer cells were found recently in a substantial fraction of human cancers including hereditary non-polyposis colon cancer. This paper aimed to investigate the genetic instability of tumor cells by using microsatellite instability(MI) as the marker in vitro.
Methods: Two RER+ cell lines (replication error phenotype), RKO and HCT116 and one RER- cell line, sw480, were used as the hosts for transfection with an episomal plasmid, pCMV-CAR, containing an exogenous (CA)14 repeat which was inserted within the coding sequence of lacZ reporter gene and thus made lacZ misreading. The transfectant clones were selected and established by hygromicin. Expression and production of lacZ reporter gene of restored reading frame were detected with X-gal staining assay.
Results: After hygromicin selection, stable pCMV-CAR transfectant clones were established. It was shown that mutation of deletion of insertion within(CA)14 occurred in the transfectant RER+ cells but not in the RER-cells. The mutation restored normal reading frame of lacZ gene, and resulted in expression and production of bio-active beta galactosidase which was detected with X-gal staining. This feature of the transfectant clones was maintained during culture passages.
Conclusion: The alterations of the exogenous(CA)14 repeat in the transfectant RER+ clones revealed genetic instability and complicated mutation status of cancer cells. It is suggested that the exogenous (CA)14 in transfectant clones could be a useful target sequence for monitoring the effects of environmental agents on MI of human cancer cells.