Chronic irradiation of human cells reduces histone levels and deregulates gene expression

Abstract

Over the past decades, there have been huge advances in understanding cellular responses to ionising radiation (IR) and DNA damage. These studies, however, were mostly executed with cell lines and mice using single or multiple acute doses of radiation. Hence, relatively little is known about how continuous exposure to low dose ionising radiation affects normal cells and organisms, even though our cells are constantly exposed to low levels of radiation. We addressed this issue by examining the consequences of exposing human primary cells to continuous ionising γ-radiation delivered at 6-20 mGy/h. Although these dose rates are estimated to inflict fewer than a single DNA double-strand break (DSB) per hour per cell, they still caused dose-dependent reductions in cell proliferation and increased cellular senescence. We concomitantly observed histone protein levels to reduce by up to 40%, which in contrast to previous observations, was not mainly due to protein degradation but instead correlated with reduced histone gene expression. Histone reductions were accompanied by enlarged nuclear size paralleled by an increase in global transcription, including that of pro-inflammatory genes. Thus, chronic irradiation, even at low dose-rates, can induce cell senescence and alter gene expression via a hitherto uncharacterised epigenetic route. These features of chronic radiation represent a new aspect of radiation biology.

Figure 1

Chronic low dose γ-radiation retards cell growth and induces cell responses to DNA damage. (a) Dose rates used for chronic γ-radiation exposures, corresponding cumulative doses over a 7-day period and DSB estimates based on 1 Gy generating 40 DSBs in a human cell (Olive, 1998). (b) Quantification of DSBs by immunofluorescence staining of 53BP1 foci in primary fibroblasts exposed to chronic radiation for 7 days (>200 cells measured per condition, displayed as blue circles). Mean numbers of foci per cell are shown by crosses, and Tukey analyses are shown by boxes. (c) Proliferation rates of fibroblasts exposed to various dose-rates of chronic γ-radiation, data shown as triplicate measurements of one donor. (d) Chronic radiation-induced apoptosis in fibroblasts measured luminescence generated by caspase 3 cleavage in a population of cells as a positive marker of apoptosis. Staurosprorine treated cells were used as positive control. (e) Induction of senescence by chronic radiation in primary fibroblasts as measured by GalactoLight assay for senescence-associated beta-galactosidase activity after 7 days of chronic irradiation at the stated dose rates. The graph shows the total activity in a population of cells of the same number, measured in arbitrary units of luminescence (a.u.).

Figure 2

Chronic γ-radiation reduces histone levels. (a) Western blot analyses of histone H2AX and γH2AX in primary fibroblasts exposed to various dose-rates of chronic γ-radiation for 7 days. Cells irradiated with a single acute dose of 4 Gy X-ray were included as control. (b) Effect of chronic γ-irradiation on H2AX levels in three different isogenic primary cell types from a different donor to that used in (a) at the same dose rates as in (a). (c) Immunoblots of other histones in chronically irradiated primary fibroblasts. (d) All significant histone level changes detected by SILAC LC-MS/MS protein analyses of samples from primary fibroblasts exposed or mock-exposed to chronic γ-radiation for 7 days.

Figure 3

Reduced histone levels in senescent cells is induced in vitro by different means, and in vivo from aged donors. (a) Phase-contrast images of primary fibroblasts induced into senescence by chronic γ-radiation, oncogene over-expression or exhaustive replication (replicative senescence), and DNA damage from a single acute 4 Gy X-ray dose at an early time-point (1 hour) as a control for DNA damage without senescence. Scale bars 200 µm. (b) Western immunoblot analyses of histones in fibroblasts described in (a). (c) Histone levels in dermal fibroblasts isolated from human neonatal (age 0, donors a and b) and adult donors.

Figure 4

p53 but not ATM is required for chronic γ-radiation-induced histone reductions. (a) Western blots of histone proteins in wild-type, TP53^−/− and ATM^−/− RPE-1 cells following exposures to 20 mGy/h chronic radiation for 7 days. (b) ATM activation, as shown by auto-phosphorylation on S1981, in response to chronic radiation in primary fibroblasts.

Figure 5

Reduction of histone levels in chronically irradiated cells occurs mainly though reduced transcription, not protein degradation. (a) Effect of inhibiting the protein degradation capability in fibroblasts exposed to chronic radiation for 7 days. Proteasome inhibition (5 µM MG132 for 24 h) or lysosomal autophagy inhibition (10 µM chloroquine for 24 h) of fibroblasts exposed to chronic γ-radiation for 7 days. (b) Differential expression of histone gene transcripts from RNA-seq analyses of primary fibroblasts after 7 days exposure to 20 mGy/h chronic γ-radiation with changes greater than 1.5-fold and p < 0.01. (c) Changes in core histone levels in fibroblasts as measured by qRT-PCR after 7 days exposure to the same radiation conditions as in (b).

Figure 6

Chronic γ-radiation increases nuclear size and global RNA synthesis. (a) Nuclei of fibroblasts exposed to chronic γ-radiation for 7 days, stained with DAPI. All images were acquired by fluorescent microscopy with 10X objective under similar conditions and quantified by automated software detection and nuclear area calculated for >1000 cells. (b) Global RNA synthesis in primary keratinocytes and fibroblasts; detected by pulse labelling with Alexa 488-labelled EU and ensuing measurement of average fluorescence intensity per cell. *Is p < 0.1, *** is p < 0.001 relative to un-irradiated samples.