Test analyzing cells’ ability to fix different kinds of broken DNA could help doctors predict cancer risk.
Our DNA is under constant attack from many sources, including environmental pollutants, ultraviolet light, and radiation. Fortunately, cells have several major DNA repair systems that can fix this damage, which may lead to cancer and other diseases if not mended.
The effectiveness of these repair systems varies greatly from person to person; scientists believe that this variability may explain why some people get cancer while others exposed to similar DNA-damaging agents do not. A team of MIT researchers has now developed a test that can rapidly assess several of these repair systems, which could help determine individuals’ risk of developing cancer and help doctors predict how a given patient will respond to chemotherapy drugs.
The new test, described in the Proceedings of the National Academy of Sciences the week of April 21, can analyze four types of DNA repair capacity simultaneously, in less than 24 hours. Previous tests have been able to evaluate only one system at a time.
“All of the repair pathways work differently, and the existing technology to measure each of those pathways is very different for each one. It takes expertise, it’s time-consuming, and it’s labor-intensive,” says Zachary Nagel, an MIT postdoc and lead author of the PNAS paper. “What we wanted to do was come up with one way of measuring all DNA repair pathways at the same time so you have a single readout that’s easy to measure.”
The research team, led by professor Leona Samson, used this approach to measure DNA repair in a type of immortalized human blood cells called lymphoblastoid cells, taken from 24 healthy people. They found a huge range of variability, especially in one repair system where some people’s cells were more than 10 times more efficient than others.
“None of the cells came out looking the same. They each have their own spectrum of what they can repair well and what they don’t repair well. It’s like a fingerprint for each person,” says Samson, who is the Uncas and Helen Whitaker Professor, an American Cancer Society Professor, and a member of MIT’s departments of biological engineering and of biology, Center for Environmental Health Sciences, and Koch Institute for Integrative Cancer Research.
With the new test, the MIT team can measure how well cells repair the most common DNA lesions, including single-strand breaks, double-strand breaks, mismatches, and the introduction of alkyl groups caused by pollutants such as fuel exhaust and tobacco smoke.
To achieve this, the researchers created five different circular pieces of DNA, four of which carry a specific type of DNA damage, also called DNA lesions. Each of these circular DNA strands, or plasmids, also carries a gene for a different colored fluorescent protein. In some cases, the DNA lesions prevent those genes from being expressed, so when the DNA is successfully repaired, the cell begins to produce the fluorescent protein. In others, repairing the DNA lesion turns the fluorescent gene off.
By introducing these plasmids into cells and reading the fluorescent output, scientists can determine how efficiently each kind of lesion has been repaired. In theory, more than five plasmids could go into each cell, but the researchers limited each experiment to five reporter plasmids to avoid potential overlap among colors. To overcome that limitation, the researchers are also developing an alternative tactic that involves sequencing the messenger RNA produced by cells when they copy the plasmid genes, instead of measuring fluorescence.