Deletion of p66Shc in mice increases the frequency of size‐change mutations in the lacZ transgene

Abstract

Summary Upon oxidative challenge the genome accumulates adducts and breaks that activate the DNA damage response to repair, arrest or eliminate the damaged cell. Thus, reactive oxygen species (ROS) generated by endogenous oxygen metabolism are thought to affect mutation frequency. However, few studies determined the mutation frequency when oxidative stress is reduced. To test whether in vivo spontaneous mutation frequency is altered in mice with reduced oxidative stress and cell death rate, we crossed p66Shc knock out (p66KO) mice, characterized by reduced intracellular concentration of ROS and by impaired apoptosis, with a transgenic line harboring multiple copies of the lacZ mutation reporter gene as part of a plasmid that can be recovered from organs into E. coli to measure mutation rate. Liver and small intestine from 2- and 24- month old, lacZ (p66Shc+/+) and lacZp66KO mice, were investigated revealing no difference in overall mutation frequency but a significant increase of the frequency of size-change mutations in the intestine of lacZp66KO mice. This difference was further increased upon irradiation of mice with X-Ray. Additionally, we found that knocking down cyclophilin D, a gene that facilitates mitochondrial apoptosis acting downstream of p66Shc, increased the size-change mutation frequency in small intestine. Size-change mutations also accumulated in death-resistant embryonic fibroblasts from lacZp66KO mice treated with H2O2. These results indicate that p66Shc plays a role in the accumulation of DNA rearrangements and suggest that p66Shc functions to clear damaged cells rather than affect DNA metabolism.

Mitochondrial Apoptosis Reduces Mutagenesis Regardless Oxidative Stress

“…It was demonstrated that both lines give comparable results in mutagenesis studies [52]. Other plasmid transgenic mouse models were generated: rpsL transgenic mouse [53] (and 6-TG selection assay [54].…”

Impaired B lymphopoiesis in old age: a role for inflammatory B cells?

“…These “antigen experienced B cells,” which can include a variety of B cell phenotypes [66], include a newly recognized subset termed “age-associated B cells” or “ABC” [6–8]. While the phenotype and functions of ABC vary somewhat among laboratories, and research into the properties of ABC is only beginning, several features of ABC are notable: (1) ABC are increased in the spleens of old mice of several strains with the CD23 − CD21/35 − mature ABC pool increased by 5–10-fold at 2 years of age [6–8]; (2) ABC are derived, at least in part, from FO B cells, possibly via antigen-driven selection [6–8], (3) in old age, B cells with the ABC phenotype are pro-inflammatory and secrete TNFα while B cells with the ABC phenotype from young mice do not [8], and (4) ABC respond strongly to TLR stimulation, but relatively weakly to typical T cell-dependent signals [6]. …”

“…Repeated depletion of B cells in vivo, followed by autoreconstitution of the bone marrow, “corrected” the poor B lymphopoiesis seen in old mice [67] and mice with genetic defects which caused a lack of mature B cells did not exhibit impaired B lymphopoiesis in old age [68]. ABC are an attractive candidate to negatively regulate B cell development: (1) ABC are increased, both in number and proportion, not only in the spleens of old mice, but also in the bone marrow [6–8]; (2) ABC in old mice produce TNFα and are pro-inflammatory [8, 51]; (3) recently, we have reported that the decline in B cell precursors seen in the bone marrow of aged mice is significantly correlated with increases in bone marrow ABC and, more precisely, with increases in the ABC to FO-like/recirculating B cell ratio within the bone marrow [8]; and 4) adoptive transfer of old ABC into RAG-2 knockout recipients resulted in a significant decline in bone marrow pro-B cells in vivo [8]. …”

“…Both ABC and FO/recirculating B cells gain the capacity to produce TNFα during old age [8, 51], and ABC-derived TNFα induces apoptosis of pro-B cells in vitro [8]. While FO/recirculating B cells also produce TNFα, some B cells with an FO phenotype secrete the anti-inflammatory cytokine IL-10 [8].…”

“…The purpose of this review is to discuss the abnormal B lymphopoiesis seen in old age, primarily in the mouse model, and connect the altered development of new B cells in old age to the dysfunctions seen in B cell immunity. We will focus on recent studies by ourselves and others that show the potential of pro-inflammatory B cells to promote changes within the bone marrow microenvironment that affect both B cell production and function [6–8]. We hypothesize that, as a consequence of increased inflammation locally within the bone marrow, the pathways of B lymphopoiesis are deviated from normal in old age.…”