Researchers from Peking University and the University of Chicago have just developed a universal genome-wide assay to detect the specific sites where cisplatin—one of the most widely used agents in cancer chemotherapy—cross-links DNA molecules. The new findings from this study were just published in Angewandte Chemie through an article entitled “Base-Resolution Analysis of Cisplatin–DNA Adducts at the Genome Scale,” and support one of the long-held theories that the mitochondrial genome is one of cisplatin's primary targets.
Since cancer cells are known to be extraordinarily active via DNA replicating at an extremely rapid rate, some anticancer drugs have been designed to target DNA, with cisplatin being one of the most effective DNA-damaging agents. The mechanisms of action for cisplatin are relatively straightforward, as the platinum atom binds to two adjacent DNA guanine bases and cross-links them, thus widening the DNA duplex structure. If the DNA repair machinery cannot excise and refill all the damaged positions, the cell reprograms itself to induce apoptosis and cell death.
While the cisplatin action mechanism is entirely clear, there has been only indirect evidence as to the exact part of the genome preferentially targeted by cisplatin. In the new study, the investigators have developed a universal test system called "cisplatin-seq" to identify specifically the genomic parts preferentially attacked by cisplatin.
The main factor in the researcher’s new assay is the enrichment of cisplatin-affected sites by taking advantage of a particular DNA-binding protein called HMGB1. A segment of this protein (domain A) was used to enhance the fragments containing the cisplatin-modified DNA and subject them to high-throughput sequencing—with the goal of identifying the exact bases where cisplatin was added. "Owing to the ability of cisplatin-DNA adducts to stall DNA synthesis, cisplatin crosslinking sites can be identified at base resolution through the genome," the authors wrote. In other words, the sequencing stops exactly at the site where the cross-linking has occurred.
“We present 'cisplatin-seq' to identify genome-wide cisplatin crosslinking sites at base resolution,” the authors penned. “Cisplatin-seq reveals that mitochondrial DNA is a preferred target of cisplatin. For nuclear genomes, cisplatin–DNA adducts are enriched within promoters and regions harboring transcription termination sites. While the density of GG dinucleotides determines the initial crosslinking of cisplatin, binding of proteins to the genome largely contributes to the accumulative pattern of cisplatin–DNA adducts.”
The big advantage of cisplatin-seq is the detection of cisplatin-DNA adducts genome-wide. Thus the scientists were able to verify the previous assumption that the mitochondrial DNA is especially sensitive to cisplatin action. "Mitochondrial DNA, which is devoid of histone proteins or nucleotide excision repair (one of the DNA repair systems), was found to be a major target of cisplatin," they authors added. “While DNA in the nucleus of cancer cells, binding of proteins to DNA largely contributes to the accumulative pattern of cisplatin-DNA adducts."
The investigators are optimistic that further applications of cisplatin-seq could be utilized to aid in cancer therapy and analytics, such as the profiling of cisplatin-DNA adducts under different dosages of cisplatin treatment.