Abstract
Activation-induced deaminase (AID) converts DNA cytosines to uracils in immunoglobulin genes, creating antibody diversification. It also causes mutations and translocations that promote cancer. We examined the interplay between uracil creation by AID and its removal by UNG2 glycosylase in splenocytes undergoing maturation and in B cell cancers. The genomic uracil levels remain unchanged in normal stimulated B cells, demonstrating a balance between uracil generation and removal. In stimulated UNG−/− cells, uracil levels increase by 11- to 60-fold during the first 3 days. In wild-type B cells, UNG2 gene expression and enzymatic activity rise and fall with AID levels, suggesting that UNG2 expression is coordinated with uracil creation by AID. Remarkably, a murine lymphoma cell line, several human B cell cancer lines, and human B cell tumors expressing AID at high levels have genomic uracils comparable to those seen with stimulated UNG−/−splenocytes. However, cancer cells express UNG2 gene at levels similar to or higher than those seen with peripheral B cells and have nuclear uracil excision activity comparable to that seen with stimulated wild-type B cells. We propose that more uracils are created during B cell cancer development than are removed from the genome but that the uracil creation/excision balance is restored during establishment of cell lines, fixing the genomic uracil load at high levels.
SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00589-14.
ACKNOWLEDGMENTS
The uracil quantification assay used in this work was developed largely by Rachel Parisien with assistance from Diane Cabelof (Wayne State University). We thank Anat Kapelnikov and Shaliny Ramachandran (University of Toronto) for preparing some of the murine samples and Diane Cabelof for help with preparation of cell extracts. We thank Vimukthi Senevirathne and Liam Holley (Wayne State University) for assistance in mouse genotyping. We thank Kenneth Honn (Wayne State University) for use of RT-PCR equipment and are grateful to Kang Chen (Wayne State University) for help in obtaining tonsils and assistance in processing tonsillar tissue.
The work described here was supported by NIH grants GM572000 and CA153936 to A.S.B., a Wayne State University graduate fellowship to S.S., and Canadian Cancer Society grant 16080 to A.M.