Long-lived inflammatory signaling in irradiated bone marrow is genome dependent.

Ionizing radiation is carcinogenic, but genotype is a key determinant of susceptibility. Mutational DNA damage is generally attributed to cause disease, but irradiation also affects multicellular interactions as a result of poorly understood bystander effects that may influence carcinogenic susceptibility. In this study, we show that the bone marrow of irradiated mice will retain the ability to kill hemopoietic clonogenic stem cells and to induce chromosomal instability for up to 3 months after irradiation. Chromosomal instability was induced in bone marrow cells derived from CBA/Ca mice, a strain that is susceptible to radiation-induced acute myeloid leukemia (r-AML), but not in C57BL6 mice that are resistant to r-AML. Similarly, clonogenic cell lethality was exhibited in C57BL/6 mice but not CBA/Ca mice. Mechanistic investigations revealed that these genotype-dependent effects involved cytokine-mediated signaling and were mediated by a cyclooxygenase-2-dependent mechanism. Thus, our results suggested that inflammatory processes were responsible for mediating and sustaining the durable effects of ionizing radiation observed on bone marrow cells. Because most exposures to ionizing radiation are directed to only part of the body, our findings imply that genotype-directed tissue responses may be important determinants of understanding the specific consequence of radiation exposure in different individuals.

Chromosomal instability in unirradiated hemaopoietic cells induced by macrophages exposed in vivo to ionizing radiation.

The tumorigenic potential of ionizing radiation has conventionally been attributed to DNA damage in irradiated cells induced at the time of exposure. Recently, there have been an increasing number of reports of damage in unirradiated cells that are either neighbors or descendants of irradiated cells, respectively, regarded as bystander effects and genomic instability and collectively termed nontargeted effects. In this study, we show that descendants of normal murine hemaopoietic clonogenic stem cells exposed to bone marrow-conditioned medium derived from gamma-irradiated mice exhibit chromosomal instability unlike the descendants of directly gamma-irradiated cells. The instability is expressed in bone marrow cells of the radiation-induced acute myeloid leukemia (r-AML) susceptible strain (CBA/Ca) but not in mice resistant to r-AML (C57BL/6). Furthermore, crossgenetic experiments show the induction of the instability phenotype requires both the producer and responder cells to be of the susceptible CBA/Ca genotype. Macrophages are the source of the bystander signals, and the signaling mechanism involves tumor necrosis factor-alpha, nitric oxide, and superoxide. The findings show a genotype-dependent chromosomal instability phenotype induced by radiation-induced macrophage-mediated bystander signaling. As the majority of accidental, occupational, and therapeutic exposures to ionizing radiation are partial body exposures, the findings have implications for understanding the consequences of such exposure.

The MDM2 ubiquitination signal in the DNA-binding domain of p53 forms a docking site for calcium calmodulin kinase superfamily members.

Genetic and biochemical studies have shown that Ser(20) phosphorylation in the transactivation domain of p53 mediates p300-catalyzed DNA-dependent p53 acetylation and B-cell tumor suppression. However, the protein kinases that mediate this modification are not well defined. A cell-free Ser(20) phosphorylation site assay was used to identify a broad range of calcium calmodulin kinase superfamily members, including CHK2, CHK1, DAPK-1, DAPK-3, DRAK-1, and AMPK, as Ser(20) kinases. Phosphorylation of a p53 transactivation domain fragment at Ser(20) by these enzymes in vitro can be mediated in trans by a docking site peptide derived from the BOX-V domain of p53, which also harbors the ubiquitin signal for MDM2. Evaluation of these calcium calmodulin kinase superfamily members as candidate Ser(20) kinases in vivo has shown that only CHK1 or DAPK-1 can stimulate p53 transactivation and induce Ser(20) phosphorylation of p53. Using CHK1 as a prototypical in vivo Ser(20) kinase, we demonstrate that (i) CHK1 protein depletion using small interfering RNA can attenuate p53 phosphorylation at Ser(20), (ii) an enhanced green fluorescent protein (EGFP)-BOX-V fusion peptide can attenuate Ser(20) phosphorylation of p53 in vivo, (iii) the EGFP-BOX-V fusion peptide can selectively bind to CHK1 in vivo, and (iv) the Deltap53 spliced variant lacking the BOX-V motif is refractory to Ser(20) phosphorylation by CHK1. These data indicate that the BOX-V motif of p53 has evolved the capacity to bind to enzymes that mediate either p53 phosphorylation or ubiquitination, thus controlling the specific activity of p53 as a transcription factor.