OVEREXPRESSION OF ASPARAGINE SYNTHETASE IN ALBIZZIIN-RESISTANT MURINEDIPLOID EMBRYONIC STEM-CELLS

Gene amplification is commonly observed in primary tumors and establis hed drug-resistant cell lines, both of which are generally aneuploid. However, this process is undetectable (frequency < 10(-9)) in normal d iploid mammalian cell lines. To investigate whether gene amplification can occur in pluripotent diploid cells, we have selected drug-resista nt mutants of mouse embryonic stem (ES) cells. We had previously found that Chinese hamster ovary (CHO) and human cell lines selected in alb izziin (Alb), an amino acid analog of L-glutamine, overexpress asparag ine synthetase (AS) due to gene amplification. The same drug selection system was applied to ES cells to isolate single-step and multistep d rug-resistant mutants. Albizziin-resistant ES cells exhibited elevated levels of AS; however, drug resistance in ES cells was associated wit h mRNA overexpression without gene amplification. AS gene amplificatio n was observed in only one drug-resistant cell line and was preceded b y AS mRNA overexpression. Gene amplification in the latter coincided w ith the loss of the pluripotent nature of the ES cells.