Molecular analysis of mutations induced in human cells by N-ethyl-N-nitrosourea

✍ Scribed by Kristin A. Eckert; Caroline A. Ingle; Donna K. Klinedinst; Norman R. Drinkwater

• Publisher: John Wiley and Sons
• Year: 1988
• Tongue: English
• Weight: 596 KB
• Volume: 1
• Category: Article
• ISSN: 0899-1987
• DOI: 10.1002/mc.2940010111

✦ Synopsis

Mutational activation of cellular proto-oncogenes is an important event in the pathogenesis of chemically induced tumors. We have used the ori P-tk shuttle vector, pHET, to analyze the types o f DNA sequence changes induced after treating mammalian cells with the carcinogen N-ethyl-N-nitrosourea (ENU). This shuttle vector contains the putative replication origin of the Epstein-Barr virus (EBV) and is stably maintained as a plasmid in EBV-transformed human lymphoblastoid cells. Populations of plasmid-bearing cells were treated with ENU, and plasmid DNA was isolated approximately 7-8 population doublings after treatment for analysis of mutations induced at the herpes simplex virus type 1 thymidine kinase (HSV-tk) target gene. After ENU treatment, frequencies of four of the six possible base substitution mutations significantly increased. Transition mutations were the most common sequence change: 48% of the 46 mutants sequenced were GC-tAT transitions and 17% were AT-rGC transitions. In addition, the number of AT-TA (20%) and AT+CG (9%) transversion mutations significantly increased after ENU treatment. Based on the comparison of mutations induced by ENU in human cells with the types o f base pair changes previously reported for other alkylating agents, we propose that the 02-ethylthymine adduct may be a significant premutagenic lesion in mammalian cells, capable of resulting in AT base pair transversion mutations. Studies from other laboratories have demonstrated the importance of AT+TA transversion mutations in the activation of cellular proto-oncogenes by ENU.