A single whole-body low dose X-irradiation does not affect L1, B1 and IAP repeat element DNA methylation longitudinally.

The low dose radioadaptive response has been shown to be protective against high doses of radiation as well as aging-induced genomic instability. We hypothesised that a single whole-body exposure of low dose radiation would induce a radioadaptive response thereby reducing or abrogating aging-related changes in repeat element DNA methylation in mice. Following sham or 10 mGy X-irradiation, serial peripheral blood sampling was performed and differences in Long Interspersed Nucleic Element 1 (L1), B1 and Intracisternal-A-Particle (IAP) repeat element methylation between samples were assessed using high resolution melt analysis of PCR amplicons. By 420 days post-irradiation, neither radiation- or aging-related changes in the methylation of peripheral blood, spleen or liver L1, B1 and IAP elements were observed. Analysis of the spleen and liver tissues of cohorts of untreated aging mice showed that the 17-19 month age group exhibited higher repeat element methylation than younger or older mice, with no overall decline in methylation detected with age. This is the first temporal analysis of the effect of low dose radiation on repeat element methylation in mouse peripheral blood and the first to examine the long term effect of this dose on repeat element methylation in a radiosensitive tissue (spleen) and a tissue fundamental to the aging process (liver). Our data indicate that the methylation of murine DNA repeat elements can fluctuate with age, but unlike human studies, do not demonstrate an overall aging-related decline. Furthermore, our results indicate that a low dose of ionising radiation does not induce detectable changes to murine repeat element DNA methylation in the tissues and at the time-points examined in this study. This radiation dose is relevant to human diagnostic radiation exposures and suggests that a dose of 10 mGy X-rays, unlike high dose radiation, does not cause significant short or long term changes to repeat element or global DNA methylation.

Retrotransposition of limited deletion type of intracisternal A-particle elements in the myeloid leukemia Clls of C3H/He mice.

The murine genome has about 1,000 copies of DNA elements for the intracisternal A-particle (IAP) that resembles a retrovirus. We previously reported that the genomic DNA of the cells from radiation-induced acute myeloid leukemia (AML) lines derived from C3H/He inbred mice was frequently rearranged by the integration of the IAP element. In this study, 8 IAP elements from the characteristic integration sites in 6 cell lines of radiation-induced AML from different mice were characterized and compared in structure with 114 IAP elements isolated from the normal C3H/He genome. One of the 8 elements was a full-length type I IAP, and 7 were of type-I Delta 1 with a common deletion site. Although the type I Delta 1 form is a minor population accounting for about 6% of total genomic IAP elements, it is predominantly retrotransposed in the AML cells from different C3H/He mice. This indicates that limited populations of the IAP elements contribute to the unique retrotransposition in AML cells.

Effect of the irradiated microenvironment on the expression and retrotransposition of intracisternal type A particles in hematopoietic cells.

We have previously demonstrated that the frequency of transformation of the factor-dependent hematopoietic cell line FDCP-1JL26 was dramatically increased when cells were cocultured with the irradiated bone marrow cell line D2XRII. In many of our factor-independent subclonal cell lines that we examined, transformation to factor independence appeared to be due to the retrotransposition of intracisternal type A particles (IAP) into the growth factor genes that are normally required for survival and growth of FDCP-1JL26 cells. To determine the role of the irradiated microenvironment in the evolution of factor-independent cells, we have examined the expression and retrotransposition of IAPs after exposure to the irradiated bone marrow stromal cell line D2XRII. Differential display and Northern blot analysis demonstrated that IAPs were overexpressed in a nonautocrine factor-independent subclonal cell line, FI7CL2. The frequency of retrotransposition was determined by the introduction of the IAP-neo(RT) plasmid into FDCP-1JL26 cells. The IAP-neo(RT) contains a neomycin resistance gene (neo) that only becomes active after retrotransposition, and thus the frequency of retrotransposition in FDCP-1JL26 cells was quantified by determining the frequency of neo-resistant cells.No significant increases in the expression of IAPs were observed after the cells were exposed to the irradiated stromal cells. This observation is in agreement with the observation that no increase in the frequency of retrotransposition could be detected. These results suggest that the irradiated bone marrow may have a passive role in the selection of factor-independent cells. During cocultivation, bone marrow stromal cells may provide a factor(s) to hematopoietic cells that allow it to survive in medium lacking IL-3. At random, a retrotransposition may occur that provides a selective advantage to the hematopoietic cells. In the absence of the irradiated stromal cells, the hematopoietic cells are perhaps more likely to die and therefore are not available for a random retrotransposition event to occur. This model is to be distinguished from an active role in which the irradiated microenvironment would synthesize or activate a factor(s) that promotes retrotransposition.

Intracisternal A-particle (IAP)-mediated leukemogenesis: levels and stability of IAP mRNA in FDC-P1 cells exposed to the conditions of an irradiated environment.

Following injection into sublethally irradiated DBA/2 or BALB/c mice, factor-dependent FDC-P1 cells undergo leukemic transformation due to oncogene activation by insertion of intracisternal A-particle (IAP) genetic elements. Similar events are observed in vitro during coculture of FDC-P1 cells with irradiated bone marrow stroma cells. To elucidate the mechanism of IAP transposition, we studied the level of IAP expression under the growth conditions preceding cell transformation. In vitro experiments showed that the type of growth factor, FDC-P1 cell density, costimulation with steroid hormones or abrupt growth factor withdrawal had no effect on IAP mRNA levels (major transcripts of 7.4, 4.0 and 1.9 kb). By contrast, stimulation with suboptimal concentrations of GM-CSF or IL-3 induced a mean 2. 5-fold increase in the intensity of the 7.4 kb band, and induction of macrophage differentiation with retinoic acid resulted in an increased stability of the 4.0 kb band. Although suboptimal growth stimulation and incipient macrophage differentiation have previously been shown to occur in the process of FDC-P1 cell transformation, an increase in IAP expression could not convincingly be demonstrated in FDC-P1 cell populations isolated from irradiated BALB/c mice or stroma cell cocultures. Further experiments are required to define the role of suboptimal growth stimulation and/or macrophage differentiation in this transformation model.

Induction of transcription from the long terminal repeat of the intracysternal particles type A (IAP) by X-irradiation.

Intracisternal A particles (IAPs) are retrovirus-like entities that are present in many embryonic and transformed cells of Mus musculus. They present long terminal repeats (LTRs) which control the promotion and regulation of their transcription. Using a construction expressing a reporter gene under the control of the entire long terminal repeat (LTR) of IAP in transfected murine fibroblast BALB/c 3T3 cells clone D152, we were able to show that the IAP-LTR is activated by X-irradiation in a time-dependent manner. The relative CAT activity increased with increasing X-irradiation doses, reaching a maximum at 75-150 cGy, followed by a drop in activation. In addition, X-induced D152 mouse cells produced extracellular factor(s), in response to X-irradiation, which activated the IAP-LTR in non-irradiated cells. This factor(s) was detected both when transfected cells were cocultured with inducing cells and when conditioned medium from irradiated cultures was added to the cell cultures. The use of suramin, a strong polyanonic molecule which has been reported to trap growth factors, induces a high reduction of the indirect activation.