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Recognition of platinum-induced DNA damage by nuclear proteins: screening for mechanisms
B Marples1, H Adomat, P C Billings
1Department of Medical Biophysics, British Columbia Cancer Research Centre, Vancouver, Canada.
Abstract:
The association of high mobility group (HMG) proteins with cisplatin-damaged DNA has attracted considerable interest because of a possible relationship with drug-acquired resistance and the repair of DNA damage caused by this important chemotherapeutic agent. We have further characterized the binding of HMG proteins to cisplatin-damaged DNA using a modification of the damaged DNA affinity precipitation assay (DDAP) and proteins isolated from the nuclei of V79 cells. HMG proteins recognized cisplatin adducts only in double-stranded DNA sequences. Pre-treatment of cells with cisplatin (1 microM) prior to protein extraction decreased the apparent yield of HMG proteins. However, the pre-treatment of isolated protein did not prevent recognition of the DNA adducts. To investigate the possible role of HMG proteins in toxicity and resistance, we have extended the DDAP method to study other platinum agents, some of which are active in cisplatin-resistant cells. A comparison of trans- and cis-[PtCl2(NH3)quinoline] is presented as an example. HMG proteins recognized DNA damage caused by the cis, but not the trans isomer. However, the trans isomer is known to be significantly more toxic and is highly active in cisplatin-resistant cells, suggesting a mechanism of action different from cisplatin. Therefore, the toxicity of trans-[PtCl2(NH3)quinoline] appears to be unrelated to the recognition of damage by HMG proteins. The DDAP assay may provide an additional screen for new mechanisms of cytotoxic platinum agents.
Insights
High mobility group (HMG) proteins bind to cisplatin-damaged DNA, but not to DNA damaged by the toxic trans isomer. This suggests HMG proteins are not involved in the toxicity of trans platinum agents.
Area of Science:
- Molecular Biology
- Cancer Research
- Biochemistry
Background:
- High mobility group (HMG) proteins are implicated in DNA repair and drug resistance.
- Cisplatin is a widely used chemotherapeutic agent that damages DNA.
- Understanding HMG protein interactions with damaged DNA is crucial for cancer therapy.
Purpose of the Study:
- To characterize the binding of HMG proteins to cisplatin-damaged DNA.
- To investigate the role of HMG proteins in cellular resistance and toxicity to platinum-based drugs.
- To explore the utility of the damaged DNA affinity precipitation assay (DDAP) for screening platinum agents.
Main Methods:
- Modified damaged DNA affinity precipitation assay (DDAP) using V79 cell nuclear proteins.
- Analysis of HMG protein binding to DNA adducts formed by cisplatin and other platinum agents.
- Comparison of binding specificity for cis and trans isomers of platinum compounds.
Main Results:
- HMG proteins specifically recognize cisplatin adducts in double-stranded DNA.
- Pre-treating cells with cisplatin reduced HMG protein yield, but not protein recognition of adducts.
- HMG proteins bind to DNA damaged by cis-[PtCl2(NH3)quinoline] but not by the trans isomer.
- The high toxicity of the trans isomer appears independent of HMG protein recognition.
Conclusions:
- HMG protein binding to platinum-damaged DNA is specific to the DNA adducts and isomer type.
- The DDAP assay is a valuable tool for studying platinum agent-DNA interactions.
- HMG proteins may not play a significant role in the toxicity mechanism of certain platinum agents like the trans isomer.