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Gene and human tumour cell line specific differencesin nitrogen mustard induced DNA alkylation and interstrand
A Sunters1, K A Grimaldi, R L Souhami
1CRC Drug-DNA Interactions Research Group, Department of Oncology, University College London Medical School, 91 Riding House Street, London W1P 8BT, UK.
Abstract:
The levels of N-alkyl purine and DNA interstrand crosslink formation, produced by the clinically used nitrogen mustard antitumour drug mechlorethamine (HN2), were quantitated at the level of specific genes in a panel of human tumour cell lines using modified Southern blotting methods. When purified genomic DNA was treated with HN2 in vitro, no significant difference in the extent of N-alkyl purine or interstrand crosslink formation in the N-ras, c-myc or CD3delta genes was observed. When the cell lines LS174T, Colo320HSR, J6 and U937 were treated with HN2, however, there was significant heterogeneity in the levels of N-alkyl purine formation in the three genes. The rank order of the extent of damage in the three genes was also different in the cell lines. The level of alkylation did not correlate with either the transcriptional activity of a gene or drug sensitivity. Crosslinks were not detectable in the N-ras or c-myc genes of LS174T, J6 or U937 cells treated with HN2, and only detectable in the amplified c-myc gene of the Colo320HSR cell line. In the related cell line Colo320DM, which has both native and translocated c-myc alleles which are both amplified and episomal, crosslinks were detected in the amplified native and rearranged c-myc alleles, and also in the N-ras gene which is also amplified in this cell line. For bifunctional alkylating agents such as HN2, therefore, heterogeneity of DNA damage can occur between different genes in human cells and can also vary for different lesions produced by the same agent. In addition, this heterogeneity can differ between human tumour cell lines.
Insights
Mechlorethamine (HN2) causes varied DNA damage across genes in human tumor cells, unlike in purified DNA. This gene-specific damage heterogeneity differs between cell lines and lesion types.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- Mechlorethamine (HN2) is a nitrogen mustard antitumour drug.
- DNA damage by HN2 includes N-alkyl purine and interstrand crosslinks.
Purpose of the Study:
- Quantitate DNA damage by HN2 at specific genes in human tumor cell lines.
- Investigate heterogeneity of DNA damage between genes and cell types.
Main Methods:
- Modified Southern blotting techniques.
- Treatment of human tumor cell lines (LS174T, Colo320HSR, J6, U937, Colo320DM) with HN2.
- In vitro treatment of purified genomic DNA with HN2.
Main Results:
- In vitro DNA treatment showed no significant gene-specific damage differences.
- In vivo cell line treatment revealed heterogeneous N-alkyl purine formation across genes.
- Interstrand crosslinks were gene- and cell line-specific, often undetectable.
- Damage levels did not correlate with gene transcriptional activity or drug sensitivity.
Conclusions:
- Bifunctional alkylating agents like HN2 cause heterogeneous DNA damage between genes in human cells.
- This heterogeneity in DNA damage can vary for different lesions produced by the same agent.
- Heterogeneity of DNA damage differs significantly between human tumor cell lines.