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Mutational activation of H-ras oncogene transformability by alkylnitrosourea-induced DNA damage
E F Yamasaki1, D P Salamon, A A Wani
1Department of Radiology, Ohio State University, Columbus 43210.
Abstract:
To assess the role of DNA alkylation damage in oncogene activation, plasmid DNA containing H-ras proto-oncogene (p220-EC) and oncogene (p220-EJ) were treated with increasing concentrations of carcinogenic methylnitrosourea (MNU) and ethylnitrosourea (ENU). The modified plasmid DNA were analyzed by transfection-transformation of the NIH/3T3-recipient cells. Treatment with varying doses of MNU (0.1-5 mM) and ENU (1-15 mM) did not result in the inactivation of the plasmid containing target genes. A transformation efficiency of greater than 40% was observed upon treatment of H-ras oncogene with the highest doses of the alkylating agents. The morphologically transformed foci obtained with alkylated p220-EC ranged from 2.8 to 0.3/microgram MNU alkylated and 1.6 to 0.6/microgram ENU alkylated plasmid DNA. A significant proportion of the morphological transformants exhibited growth in soft agar. The HpaII/MspI restriction length polymorphism (RFLP) at codon 12 of H-ras exon-1 was detected with 4 independently isolated clones obtained from MNU-alkylated p220-EC transfections. Allele-specific in situ gel hybridization with a battery of codon 12 and codon 61 oligonucleotide probes confirmed these RFLPs to be due to sequence changes at codon 12. No clone with sequence changes in the H-ras codon 61 could be detected. The data indicate that a high degree of in vitro alkylation damage of the target gene is necessary to elicit mutational activation of H-ras in transfection-transformation assay. Low frequency notwithstanding, the data demonstrate that DNA alkylation damage at critical target sites can initiate neoplastic cellular transformation.
Insights
DNA alkylation damage from methylnitrosourea (MNU) and ethylnitrosourea (ENU) can activate the H-ras oncogene. This study shows that significant DNA damage is required for oncogene activation and neoplastic cell transformation.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- DNA alkylation is a known genotoxic event.
- Oncogene activation is a key step in cancer development.
- The H-ras proto-oncogene is frequently mutated in human cancers.
Purpose of the Study:
- To investigate the role of DNA alkylation damage in activating the H-ras oncogene.
- To determine the relationship between the extent of DNA alkylation and oncogene activation.
- To assess the potential of DNA alkylation to initiate neoplastic cellular transformation.
Main Methods:
- Plasmid DNA containing H-ras proto-oncogene (p220-EC) and oncogene (p220-EJ) were treated with methylnitrosourea (MNU) and ethylnitrosourea (ENU).
- Modified plasmid DNA was analyzed via transfection-transformation of NIH/3T3 cells.
- Morphological transformation, soft agar growth, and restriction fragment length polymorphism (RFLP) analysis were performed.
Main Results:
- High concentrations of MNU and ENU did not inactivate the target genes but induced transformation.
- Transformation efficiency reached over 40% with H-ras oncogene treatment.
- Sequence analysis revealed mutations at codon 12 of H-ras exon-1 in MNU-alkylated transfectants.
Conclusions:
- Significant in vitro DNA alkylation damage is necessary for H-ras mutational activation.
- DNA alkylation at critical sites can initiate neoplastic cellular transformation.
- This study highlights the oncogenic potential of DNA alkylation damage.