PROTEIN ALTERATIONS ASSOCIATED WITH GENE AMPLIFICATION IN CULTURED HUMAN AND RODENT CELLS

Genomic stability was investigated in Chinese hamster ovary (CHO) and human hepatocellular carcinoma HepG2 cells selected for growth in the presence of cytotoxic concentrations of N-(phosphonacetyl-L-asparate) (PALA). In CHO cells selected with 9 x LD(50) PALA the carbamyl p-synt hetase, aspartate transcarbamylase and dihydroorotase (CAD) gene compl ex was amplified two-fold while in HepG2 cells selected at comparable PALA concentrations a 7- to 10-fold increase in the CAD gene was obser ved. Concomitant with amplification of the CAD gene were increases in CAD mRNA and protein expression in both CHO and HepG2 cells. In long-t erm cultures of HepG2 cells the CAD gene underwent spontaneous amplifi cation (5-fold) in the absence of PALA treatment with increasing passa ge number. In an attempt to define proteins and/or family of proteins that may either directly or indirectly influence DNA amplification pot ential through a mechanism of enhanced genomic instability, immobilize d pH gradient-two-dimensional polyacrylamide gel electrophoresis (IPG 2-D PAGE) analysis of silver-stained nuclear cytoplasmic polypeptides comcomitant with PALA resistance and CAD amplification was performed. Analysis of silver-stained polypeptides from 3 x LD(50) PALA-selected CHO and HepG2 cells revealed no significant alterations in polypeptide expression. In CHO cells selected at 5 x and 7 x PALA LD(50), and Hep G2 cells selected at 5 x and 9 x PALA LD(50), one subset of 4-8 polype ptides (pI: pI 7.2-7.6/36-38 kDa) were increased 2- to 3-fold in both 5 x and 7 x- and 5 x and 9 x LD(50) PALA-selected CHO and HepG2, respe ctively, while five relatively neutral-to-basic, low M(r) polypeptides (p2: 18/7.30; p3: 16/7.00; p4: 14/7.00; p5: 14/7.40; and p6: 13.5/7.0 0) were markedly increased in CHO cells selected at 7 x LD(50) PALA. I n addition to these PALA-associated increases, four polypeptides (p7a: pI 6.50/40 kDa; p7b: 6.55/40; p7c: 6.60/40; and p7d: 6.65/40) were si gnificantly increased in high-passage (p159) HepG2 cells undergoing sp ontaneous CAD gene amplification in the absence of PALA exposure. In C HO cells, polypeptides p7 a, b, d were increased while the expression of p7c (pI 6.60/40 kDa) was unaltered in 7 x LD(50)-treated CHO cells. Although neither the identity nor biological function of polypeptides 1-7 is known, a proposed mechanism involving interaction with certain growth regulatory proteins such as p53 for mediating genomic instabil ity is given.