Multiple mechanisms of N -phosphonacetyl- l -aspartate resistance in human cell lines: Carbamyl- P synthetase/aspartate transcarbamylase/dihydro-orotase gene amplification is frequent only when chromosome 2 is rearranged

Abstract

Rodent cells resistant to N-phosphonacetyl-

High rate of CAD gene amplification in human cells deficient in MLH1 or MSH6

“…Eight of eight resistant isolates derived from MLH1 Ϫ/Ϫ HCT116 cells displayed a 2-to 3-fold increase in CAD gene dosage as judged by this method, and seven of eight isolates derived from MSH6 Ϫ/Ϫ MT1 cells behaved in a similar manner ( Table 2). Because a 2-fold increase in CAD gene copy number is sufficient to confer a 5-to 10-fold increase in PALA resistance (18), these observations suggest that CAD gene amplification is the predominant mechanism responsible for development of PALA resistance in MLH1-and MSH6-deficient human cells.…”

“…A fluorescent probe for human chromosome 2 alpha satellite DNA was purchased from Vysis (Downers Grove, IL). A fluorescent CAD DNA probe was prepared by nick translation of cosmid HU-CAD-69 (18) in the presence of fluorophore-labeled dUTP (Vysis). CAD and human chromosome 2 probes were denatured at 75°C for 5 min in hybridization buffer (Vysis) and hybridized at 37°C for 24 h to metaphase spreads according to recommendations of the manufacturer.…”

“…PALA blocks pyrimidine biosynthesis by inhibition of aspartate transcarbamylase, one activity of a trifunctional polypeptide encoded by the mammalian CAD gene (27). Although CAD gene amplification is a common mechanism of PALA resistance in rodent cell lines (28), CAD amplification is typically rare in cultured human cells (18,(29)(30)(31); however, exceptions to this generalization have been noted. Defective p53 function is sufficient to confer a permissive state for amplification of the human CAD locus, but other permissive pathways may also exist (32,33).…”

“…Defective p53 function is sufficient to confer a permissive state for amplification of the human CAD locus, but other permissive pathways may also exist (32,33). PALA resistance due to mechanisms other than CAD amplification have also been observed in human cells (18).…”

Amplification of the human dihydrofolate reductase gene via double minutes is initiated by chromosome breaks

“…5, 6, and 18). In human (19,20) and rat (e.g., ref. 21) cell lines, DHFR amplicons can be manifested either as HSRs or DMs, although in human tumor samples, they most often appear as DMs (22).…”