23rd Annual NASA Space Radiation Investigators' Workshop (2012)
8020.pdf
NEW VIEWS: DNA Demethylation May Play a Role in Space Radiation-Induced
Cell Polyploidization
1
Elizabeth A. Kosmacek , Michael A. Mackey2,1, and Fiorenza Ianzini1,2*
Departments of 1Pathology and 2Biomedical Engineering, University of Iowa, Iowa City,
IA. *Presenting Author
We have demonstrated that one of the effects of charged particle radiation on normal
cells is to induce changes in DNA ploidy, in that the affected cells, similarly to what is
usually measured in X- or γ-irradiated cancer cells, become large polyploid cells. These
cells present condensed and non-condensed chromatin accompanied by cytologically
observed changes in chromatin texture, where the latter is tightly packed or highly fluffy.
These traits are common in cancer cells and are thought to be due to disrupted and
deregulated DNA methylation. To determine the extent of epigenetic changes in these
cells that have assumed a cancer phenotype by becoming polyploid, we looked for
changes in specific gene products that are known to be involved in the regulation of
DNA methylation, in chromatin condensation, and in changes in ploidy. The lymphoidspecific helicase (Lsh) gene fits these requisites. Lsh is a member of the SNF2 helicase
family of chromatin-remodeling ATPases and is required for the proper establishment
and maintenance of DNA methylation. Loss of function mutations in this gene leads to
hypomethylation of CpG and reactivation of transposable elements; its overexpression
leads to re-establishment of methylation sites through the protein's ability to regulate
DNA methyltransferases 3a and 3b which are involved in the establishment of de novo
DNA methylation patterns. Lsh also plays a role during meiosis; lack of Lsh is
associated with incomplete homologous chromosome synapsis and persistent H2AX
foci at the asynapsed chromosomes and at the axial gaps at the synaptonemal complex
in oocytes and spermatogonia. Lsh is preferentially associated with pericentric
heterochromatin; accordingly, CpG demethylation due to suppression of Lsh is most
substantial around pericentromeric sequences. Disruption of normal organization of
pericentric heterochromatin is associated with changes in cell ploidy, aberrant chromatin
organization, aberrant mitoses, and skewed cell proliferation. Our preliminary results
show that doses of 2 Gy of 1 GeV/n iron ion radiation cause a dramatic decrease in Lsh
mRNA and protein levels during the first 3-5 days post-irradiation with a reversion to
quasi-normal levels at later times post-irradiation in MRC-5 cells. RT-PCR data reveal a
decrease in Lsh mRNA of about 7 fold relative to control at day 3 post-irradiation.
Likewise, immunofluorescence co-staining measurements of Lsh and γ-H2AX
expression show a dramatic drop in Lsh protein by day 3-5 post-irradiation with
persistent expression of γ-H2AX. Moreover, cells with large and fragmented nuclei
appear by day 3 post-irradiation and these nuclei stain negative for Lsh, suggesting that
radiation induces Lsh loss of function. Lsh loss of function interferes with proper CpG
methylation, these are indeed hypomethylated as demonstrated by our preliminary
results on DNA extracts incubated with McrBC that indicate that iron ion radiation
induces genome wide loss of CpG methylation within 72 h post-exposure.
These findings are the first to link radiation-induced morphologic and phenotypic cell
changes to genetic and epigenetic modifications. These modifications affect the overall
cellular genetic stability of the exposed populations and their progeny and may
contribute to augmented cancer risk from space radiation.
Support: NIH/NCI 2P30CA086862; NASA NRA NNJ06HH68G; NASA NNX10AJ31G