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Repair Mechanism of UV-damaged DNA in Xeroderma Pigmentosum

Readthrough of premature termination codon (PTC) in XP-C cells. Confocal microscopic images of fluorescent immunostained XP-C cells with a PTC. The XP-C cells were exposed to UV radiation through a Millipore filter resulting in small areas of UV damage to the nuclei. The cells were then stained for presence of XPC protein. The untreated cells have no detectable XPC protein while the Geneticin treated cells show XPC protein localized to the sites of DNA damage (arrows).
Readthrough of premature termination codon (PTC) in XP-C cells. Confocal microscopic images of fluorescent immunostained XP-C cells with a PTC. The XP-C cells were exposed to UV radiation through a Millipore filter resulting in small areas of UV damage to the nuclei. The cells were then stained for presence of XPC protein. The untreated cells have no detectable XPC protein while the Geneticin treated cells show XPC protein localized to the sites of DNA damage (arrows).

Xeroderma pigmentosum (XP) is a rare, inherited disorder characterized by extreme skin sensitivity to ultraviolet (UV) rays from sunlight. XP is caused by mutations in genes involved in nucleotide excision repair (NER) of damaged DNA. Normal cells are usually able to fix this damage before it leads to problems; however, the DNA damage is not repaired normally in patients with XP. As more abnormalities form in DNA, cells malfunction and eventually become cancerous or die. XP patients have more than a 10,000-fold increased risk of developing skin cancer. Kenneth Kraemer, M.D., in CCR’s Dermatology Branch, has been studying XP patients at the Clinical Center for more than 40 years.

Many human genetic disorders may be the result of premature termination codon (PTC) mutations in different genes. There are three types of PTC mutations—TGA, TAG, and TAA. A PTC can reduce the level of mRNA and protein via nonsense-mediated mRNA decay (NMD), a mechanism that detects and degrades PTC-bearing transcripts. NMD prevents the expression of short proteins that might be nonfunctional or deleterious. NER defective cells from XP patients fall into seven complementation groups (XP-A through XP-G). PTC mutations, which may produce abnormal, short proteins (or no protein at all by NMD), have been identified in 15 percent of XP-C patients.

Agents to induce readthrough—the transcription of a nucleic acid sequence beyond its normal termination—of PTCs are useful research tools and potential therapeutics. Some antibiotics such as aminoglycosides and other compounds have been used to restore full-length proteins by reading through PTC; however, readthrough of XP PTC has not been previously described. In a proof-of-principle study, Kraemer, Christiane Kuschal, Ph.D., a postdoctoral fellow in the Kraemer lab, and colleagues set out to determine the efficiency of readthrough by studying a panel of eight DNA-repair deficient skin cells from XP patients with different PTCs in the XPC DNA repair gene.

The researchers used seven different assays to assess the various steps of the post-UV NER pathway. They used a combination of quantitative real-time PCR, immunoblotting, and immunofluorescence techniques. They found that in six of the eight PTC-containing XP-C cells, treatment with two aminoglycosides—Geneticin and gentamicin—led to induced levels of XPC protein that function in the normal NER pathway and remove DNA damage.

The researchers also reported that the ability of aminoglycosides and nonaminoglycoside small molecules to read through different types of PTC mutations of XP-C depends on type, copy number, and genetic location of the PTC, the downstream nucleotide base, and the readthrough compound used. Of clinical significance, nonaminoglycoside small molecules are less toxic than aminoglycosides and are able to pass through the blood brain barrier, which would enable treatment of neurodegenerative forms of XP.      

Unlike previous studies, this study employed a cell-based system that uses skin cells from XP patients and is probably more likely to be representative of physiological effects in human skin. Taken together, this study demonstrates that readthrough of XPC PTC can occur with certain compounds despite their clinical toxicity. Topical application with antibiotics such as gentamicin might be useful for the prevention of sunlight-induced skin cancers in XP-C patients and would eliminate a lot of the toxicity caused by parenteral delivery.

The XP-C cell assay system is a promising model for assessing readthrough of PTC and for testing new drugs. For example, the assay can be used to identify which XP-C patients are likely to respond to a specific agent. The selection of study patients and agents according to their in vitro efficacy will likely expedite efforts to demonstrate efficacy and minimize toxicity in clinical trials.

Reviewed by Miranda L Hanson

Reference
Kuschal C, DiGiovanna JJ, Khan SG, Gatti RA, Kraemer KH. Repair of UV photolesions in xeroderma pigmentosum group C cells induced by translational readthrough of premature termination codons. Proc Natl Acad Sci USA. November 11, 2013. PubMed Link