Protein Kinase C δ Is Critical for Nucleotide Excision Repair of Cyclobutane Pyrimidine Dimers

Nucleotide excision repair (NER) is the process by which cells identify and repair bulky, helix-distorting DNA lesions such as ultraviolet (UV) radiation-induced cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP). NER in human cells is a complex biochemical process requiring several complementation groups. Defects in complementation group genes result in rare, autosomal recessive cancer syndromes such as xeroderma pigmentosum (XP), cockayne syndrome (CS), and trichothiodystrophy, supporting the tumor suppressive effects of NER. Protein Kinase C delta (PKCδ) expression is lost in squamous cell carcinomas where it functions as a tumor suppressor gene. Additionally, in response to UV radiation, PKCδ is known to function in the maintenance of the G2/M cell cycle checkpoint and apoptosis. Because cell cycle checkpoints are coupled to DNA damage repair pathways, this thesis investigates the role PKCδ plays in DNA damage repair of UV-induced adducts. To examine the role of PKCδ in the repair of DNA damage CPD\u27s, wild type mouse embryonic fibroblasts (MEFs) were exposed to a non-apoptotic UV dose (5 mJ/cm2), allowed time for repair, and then fluorescently stained for CPDs. Through quantitative immunofluorescence microscopy, we found wild type MEFs were competent at repairing CPD\u27s whereas PKCδ null MEFs had significantly (p\u3c0.001) reduced capacity for repair of UV-induced CPD adducts. These results were also supported using southwestern blotting for CPDs in DNA isolated from wild type and PKCδ null cells exposed to high dose UV radiation. We also found that re-expressing PKCδ in PKCδ-/- cells restores DNA damage repair of CPDs. Cells infected with PKCδ expressing vector obtained levels of CPD repair consistent with WT cells. Immunofluorescene staining was also used to show decreased levels of nuclear xeroderma pigmentosum complementation groups A and C (XPA; XPC) protein in PKCδ-/- MEFs. This data was also supported by western blot data which showed low endogenous levels and delayed induction of XPA and XPC in PKCδ-/- cells after UV irradiation. Thus, our results indicate a role for PKCδ in DNA damage repair of CPDs through NER signaling