TAT-Mediated Delivery of a DNA Repair Enzyme to Skin Cells Rapidly Initiates Repair of UV-Induced DNA Damage

This month's featured paper is from the Journal of Investigative Dermatology, and is titled “TAT-Mediated Delivery of a DNA Repair Enzyme to Skin Cells Rapidly Initiates Repair of UV-Induced DNA Damage.”

This research was conducted as part of an investigative collaboration, and included the Center for Research on Occupational and Environmental Toxicology’s (CROET) Jodi Johnson, PhD, Brian Lowell, MS2, Olga Ryabinina MD, R. Stephen Lloyd, PhD, and Amanda McCullough, PhD.*

More than one million cases of non-melanoma skin cancers are diagnosed annually in the United States. Of these, the two most common forms of skin cancer, squamous cell carcinomas (SCC) and basal cell carcinomas (BCC), are the result of exposure to ultraviolet light (UV) in sunlight. The DNA damage caused by this exposure, if unrepaired or repaired incorrectly, can lead to mutations in genes.

Studies have shown that although human cells possess a mechanism to repair UV-induced DNA damage, mutagenesis still occurs when DNA is replicated prior to repair of these photoproducts. Furthermore, while human cells have most of the enzymes necessary to complete an alternate repair pathway, base excision repair (BER), they lack a DNA glycosylase that can initiate BER of UV-induced dipyrimidine photoproducts.

This research demonstrates that certain prokaryotes and viruses produce pyrimidine dimer-specific DNA glycosylases (pdgs) that initiate BER of cyclobutane pyrimidine dimers (CPDs), the predominant UV-induced lesions. Such a pdg was identified in the Chlorella virus PBCV-1 and termed Cv-pdg. The Cv-pdg protein was engineered to contain a nuclear localization sequence (NLS) and a membrane permeabilization peptide (transcriptional transactivator, TAT).

The authors’ study shows that the Cv-pdg-NLS-TAT protein could be delivered to repair-proficient keratinocytes and fibroblasts, and to a human skin model, where it rapidly initiated removal of CPDs. “These results are encouraging,” said Amanda McCullough, PhD, Associate Professor, Department of Molecular & Medical Genetics.  “It suggests a potential strategy for prevention of human skin cancer.”

Research into DNA damage, replication and repair is crucial to the health and welfare of society. While non-melanoma skin cancers are seldom lethal, DNA damage in skin cells can lead to significant health issues if left unrepaired, including malignant melanoma.  “Associated health care costs to treat patients with these malignancies make this an economic issue as well.  Therefore, the development of new treatment and preventive modalities is crucial” said Dr. McCullough.

The immediate follow-up studies to this research include large-scale manufacturing and pharmacology and toxicology safety testing of the DNA repair enzyme.  Down the road, scientists at the Lloyd-McCullough laboratory plan to further investigate the role that enhanced DNA repair of sunlight-induced DNA damage plays in UV-induced immune suppression; since dampening of the immune system can lead to expansion of cancerous cells and viral infections.  Maintaining immune surveillance is crucial.  Assisting in this research is a solar simulator that creates the visible and UV spectrum that reaches the earth’s surface.  This technology will allow scientists to precisely mimic human skin sun exposures and provide an accurate assessment of the efficacy of their DNA repair-based therapeutics. 

“The overall objective of our studies is to translate several decades of basic science research to practical, therapeutic applications for the prevention of skin cancers.  Our strategy represents a transition of NIH-sponsored research into company sponsored preclinical studies,” said R. Stephen Lloyd, PhD, Professor, Department of Molecular & Medical Genetics.  The company co-sponsoring this research, Restoration Genetics, Inc, is an OHSU start-up biotechnology company.

Figure 1. Upper left: H&E staining of the skin model; lower left: IIF for expression of K14 (red) and loricrin (green) with DAPI (blue) for nuclei; right: IIF of Cv-pdg (red) after culturing with buffer (upper) or Cv-pdg-NLS-TAT (lower). Solid bar¼50 mm. Dashed bar¼20 mm. Cv, Chlorella virus; DAPI, 40,6-diamidino-2-phenylindole; H&E, hematoxylin–eosin; IIF, indirect immunofluorescence; NLS, nuclear localization sequence; pdg, pyrimidine dimer-specific DNA glycosylase; TAT, transcriptional transactivator peptide.

 
Pictured: (Top left to right) R. Stephen Lloyd, Amanda McCullough, Olga Ryabinina; (Bottom left to right) Brian Lowell, Jodi Johnson

*Jodi Johnson, PhD, Brian Lowell, MS2, Olga Ryabinina MD, R. Stephen Lloyd, PhD, Professor, Department of Molecular & Medical Genetics, and Amanda McCullough, PhD, Associate Professor, Department of Molecular & Medical Genetics

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ABOUT THE PAPER OF THE MONTH

The School of Medicine newsletter spotlights a recently published faculty research paper in each issue. The goals are to highlight the great research happening at OHSU and to share this information across departments, institutes and disciplines. The monthly paper summary is selected by Associate Dean for Basic Science Mary Stenzel-Poore, PhD.

This paper shines a light on critical basic science research that builds towards an important therapeutic goal to minimize the risk of skin cancer. This group’s scientific program illustrates a research spectrum of basic discovery and pre-clinical testing that is directed towards drug development and commercialization—a goal made more feasible through one of OHSU’s new biotechnology startup companies.