Increased gene amplification in immortal rodent cells deficient for the DNA-dependent protein kinase catalytic subunit.
Gene amplification is one of the most frequent genome anomalies observed in tumor cells, whereas it has never been detected in cells of normal origin. A large body of evidence indicates that DNA double-strand breaks (DSBs) play a key role in initiating gene amplification. In mammals, DSBs are mainly repaired through the nonhomologous end-joining pathway (NHEJ) that requires a functional DNA-dependent protein kinase catalytic subunit (DNA-PKcs). In rodent cell lines, N-(phosphonacetyl)-L-aspartate (PALA) resistance is considered a measure of gene amplification because it is mainly attributable to amplification of the carbamyl-P-synthetase aspartate transcarbamylase dihydro-orotase (CAD) gene. In this paper we show that the radiosensitive hamster cell line V3, which is defective in DSB repair because of a mutation in the DNA-PKcs gene, displays also an increased frequency of gene amplification. In these cells, we found that the amplification of the CAD gene occurs with a frequency and a rate more than one order of magnitude higher than in control cell lines, although it relies on the same mechanisms. When the same analysis was performed in mouse embryo fibroblasts (MEFs) obtained from animals in which the DNA-PKcs gene was ablated by homologous recombination, a higher frequency of amplification compared with the controls was found only after cellular immortalization. In primary DNA-PKcs(-/-) MEFs, PALA treatment induced a block in the cell cycle, and no PALA-resistant clones were found. Our results indicate that the lack of DNA-PKcs increases the probability that gene amplification occurs in a genetic background already permissive, like that of immortalized cells, although it is not sufficient to make normal cells able to amplify.
Mondello
C
C
Istitituto di Genetica Biochimica ed Evoluzionistica, Consiglio Nazionale delle Ricerche, Via Abbiategrasso 207, 27100 Pavia, Italy. mondello@igbe.pv.cnr.it
Rebuzzini
P
P
Dolzan
M
M
Edmonson
S
S
Taccioli
G E
GE
Giulotto
E
E
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001221:Aspartate Carbamoyltransferase / Q000235:genetics; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D004080:Dihydroorotase / Q000235:genetics; D019008:Drug Resistance, Neoplasm; D005347:Fibroblasts / Q000201:enzymology / Q000502:physiology; D005784:Gene Amplification; D051379:Mice; D009097:Multienzyme Complexes / Q000235:genetics; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency* / Q000235:genetics*
D000964:Antimetabolites, Antineoplastic; C033626:CAD trifunctional enzyme; D004268:DNA-Binding Proteins; D009097:Multienzyme Complexes; D001224:Aspartic Acid; C013195:sparfosic acid; D001221:Aspartate Carbamoyltransferase; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D010746:Phosphonoacetic Acid