Tissues irradiated with low or high doses of protons exhibited significant structural abnormalities ( and ). Compared to nonirradiated controls, EPI-200 irradiated with the high dose lacked a continuous basal layer (). Proliferating cells and postmitotic squamous cells also appeared together in the same layer. Moreover, the granular layer had lost integrity and was represented by rare cells underlying a thickened cornified layer. In contrast, tissues irradiated with the low dose showed elevated cell proliferation (), suggesting initiation of tissue recovery processes. Cornification was also milder than that occurring after the high dose (), suggesting a dose-dependent effect for induction of terminal differentiation. Furthermore, all irradiated samples contained numerous nucleated cells in the cornified layer. Such nucleated corneocytes indicate premature cornification in irradiated tissues and suggest failure of nuclear disintegration at the transition from squamous to cornified layers. The cornified layer in irradiated tissues was also sharply delineated and appeared very dense, suggesting dysfunction of the epidermal barrier.
Although there was little overlap of the genes that significantly responded to high and low doses or at the different times, gene ontology analysis can provide insight into common or divergent processes potentially affected by different sets of genes. Gene ontology analysis of the expression profiling data () indicated that processes involved in transcriptional regulation are most affected at early times by exposure to the low dose, while cell cycle and proliferation functions dominate the early response to the high dose. Effects on genes related to cell cycle, proliferation and differentiation continued in tissues exposed to the high dose until at least 16 h after exposure and were also significant in the low-dose-exposed tissues at this time. By 24 h after exposure, signal transduction and developmental processes strongly dominated the tissues exposed to high doses, while significant gene expression changes were no longer detected in the tissues exposed to low doses. These processes are generally consistent with the altered structure ultimately observed in the irradiated tissue and suggest a shift from tissue restoration at the lower dose to greater damage followed by terminal differentiation and tissue restructuring at the higher dose.
In earlier studies of gene expression changes after low-dose irradiation, Yin et al.
) reported the induction in mouse brain of genes involved in protective functions and downregulation of genes involved in specialized functions, such as neural signaling. Although significance of enrichment was not calculated, cell cycle control and signal transduction functions were well represented among genes responding to 10 cG γ rays in that study. In cell culture studies, normal human fibroblasts regulated a large number of genes between 1 and 24 h after exposure to 2 cGy X rays (19
), with functions of signal transduction and development being significantly over-represented among responders. Normal human keratinocytes also responded to 1 cGy X rays by regulating expression of many genes, mostly at 48 and 72 h after exposure (20
). The genes reported as responding only to 1 cGy and not to 1 Gy were significantly enriched for protein metabolism and modification (P
= 1.74 × 10−5
by PANTHER analysis), a category not seen in our study, perhaps due to our focus on earlier times. The category of cell cycle, significant in our low-dose analysis, was the next most significant in the 2D keratinocyte response. Its significance was low (P
= 0.1), however, perhaps due to the exclusion from that analysis of genes responding to both high and low doses. The differences with our results may also be due in part to the measurement of only about 7000 genes in the older studies rather than the whole genome, to the later timeframe of the keratinocyte study, or to differences in radiation response and cellular interactions between cells grown in 2- and 3D cultures.
In a whole genome expression study of full-thickness human skin irradiated in vivo
with low doses during radiotherapy, variation between donors was found to be too great to allow confident identification of individual responding genes, leading to the development of a unique gene set enrichment analysis approach (35
). This approach was used to identify gene groups and pathways responding linearly to exposures of 1 and 10 cGy and 1 Gy. Among the responders, cytokines, MAP kinases and zinc finger proteins were significantly upregulated, while S100 and keratins were downregulated (23
). Although our study did not look for linear dose dependence, these responses are consistent with our significant enrichment of signaling and tissue remodeling functions at high dose and transcriptional regulation at low dose.
Perhaps the strongest theme emerging from the gene ontology analyses of the EPI-200 data was that of functions related to cell cycle and proliferation. These functions dominated the high-dose response 4 h after exposure and both high- and low-dose responses 16 h after exposure (). A more detailed analysis revealed coordinate downregulation of many promitotic genes. Coordinate regulation of such cell cycle genes as CDC2
, cyclins, topoisomerase II and centromere-associated proteins has been described previously (36
), and the coordinate downregulation of these and other mitosis-related genes represents the most consistent response to ionizing radiation observed across a set of 60 diverse cancer cell lines (37
In the 3D tissue model, the cyclins CCNA2, CCNB1
as well as CENPE, CENPF
were downregulated after both high- and low-dose irradiation. In contrast, microtubule motor (KIF2C, KIF4A, KIFC1, KNTC1, KIF11, KIF15, KIF20A
) and non-motor (CKAP5
) binding proteins, centromere proteins (CENPA, CENPJ, CENPM
), and KNTC2
, a gene involved in spindle checkpoint signaling (38
), were all downregulated only in response to the high dose. Similarly, the G1
checkpoint inhibitors CDKN1A
were upregulated only by the high dose. By 24 h after exposure to the high but not the low dose, MYC
expression was strongly elevated (2.6-fold). Overexpression of Myc in keratinocytes has been shown to stimulate their terminal differentiation (39
were also upregulated in these samples. These genes are involved in calcium signaling, which is the dominant signaling pathway regulating terminal differentiation of keratinocytes. Taken together, this suggests a period of strong cell cycle arrest followed by increasing terminal differentiation in response to high-dose radiation exposure, consistent with the decrease in proliferating cells () and increased cornification () observed at later times in these tissues.
In contrast, signals promoting cell cycle arrest after low-dose exposure appear to be less broadly based and shorter lived. In addition, some cell cycle-related genes such as KIF26A
were upregulated only by the low dose. SESN2
is of particular interest because it helps regulate cellular protection against oxidative stress (40
), which may promote cell survival and help reduce the propensity toward terminal differentiation after radiation exposure.
Genes linked to blood circulation and gas exchange were also significantly enriched in tissues exposed to both the low dose (at 16 h) and high dose (at 24 h). These responses may be explained by the need to maintain homeostasis of the oxygen supply as the differentiation state and integrity of the tissue changes after radiation exposure. Being avascular tissue, epidermis depends on oxygen supplies from external sources. In the normal human epidermis, the upper skin layers to a depth of 0.25–0.40 mm are almost exclusively supplied by external oxygen (41
), whereas the bloodstream contributes to oxygen supply at deeper tissue levels. Significant hypercornification observed after irradiation ( and ) suggests impairment of the epidermal barrier function, possibly disrupting normal gas exchange.
Among the genes in the gas exchange functional group, three hemoglobin genes (HBA, HBM
) were upregulated, suggesting a response to an insufficient oxygen supply. Hemoglobins are normally expressed in a variety of tissues including the skin (42
). At least one of the represented genes, CYGB
, is known to be directly regulated by hypoxia (43
). Besides its main function, CYGB
also contributes to scavenging NO and reactive oxygen species, contributing a protective function in response to oxidative stress (44
). Other upregulated genes involved in gas exchange represent cation channels. SCNN1D, SCNN1G
, regulated by the low dose, are involved in regulation of pH in the intracellular milieu (45
). Their upregulation may be a response to a pH change in the tissue. P2RX2
, the only ion channel gene upregulated by the high dose, is also upregulated by hypoxia (47
). Moreover, P2RX2 is an ATP receptor and calcium channel, possibly suggesting another link to calcium signaling, which accelerates differentiation and cornification of keratinocytes, as observed in the tissues exposed to the high dose.
Network analysis of the microarray data indicated TP53 as a major hub involved in the response to high-dose radiation with lesser response after low-dose exposure, consistent with prior studies (15
). In addition, network analysis implicated HNF4A as a transcription factor responsive to ionizing radiation for the first time. The increased levels of the active phosphorylated form of HNF4A protein present in the nuclear protein fraction after low-dose radiation exposure () further supports the involvement of HNF4A in radiation response. A role for HNF4A in stress response is not completely unprecedented, however, because it has been shown to differentially activate specific target genes in response to oxidative stress (50
). The acute-phase gene expression response induced in liver by tunicamycin, which causes endoplasmic reticulum stress, was also shown to be partly dependent on HNF4A (51
Because the transcriptional activity of HNF4A had been shown to be repressed by TP53 (52
), this presented an attractive possible mechanism for the observed shift in the pattern of regulated genes as the radiation dose was increased and levels of TP53 rose. We postulated that disruption of the TP53 gene might allow continued increases in HNF4A levels in cells irradiated by higher doses. This was found not to be the case in HCT116, however, because in contrast to the wild-type cells, the HCT116 p53−/−
cells showed no detectable levels of HNF4A protein and no increase in activation () after either low- or high-dose exposure. Although this finding supports a role for TP53 in the regulation of HNF4A expression, it suggests a more complex relationship, possibly involving interactions with additional regulatory factors. For instance, c-Myc competes with HNF4A for control of the promoters of genes including CDKN1A and APOC3 (53
), and MYC mRNA was regulated by exposure to high but not low doses in the present study. Binding of SMAD3 and SMAD4 to the HNF4A transactivation domain also affects HNF4A transcriptional activity (54
). Expression of different splice variants has also been implicated in shaping the transactivation profile of HNF4A, with at least 6 adult and 3 prenatal isoforms described (55
). In the absence of wild-type p53, it is possible that methylation of the HNF4A promoter may account for the observed lack of HNF4A expression. The overall methylation level of genomic DNA is often higher in cell lines with disrupted or non-functional TP53 (56
), and HCT116 p53−/−
has specifically been shown to have a 6-fold higher expression of the methylation enzyme DNMT1 than its p53 wild-type parent line (57
), suggesting increased methylation and silencing of target genes compared to the parent cell line. HNF4A expression has recently been shown to be regulated by methylation of the promoter (58
), making this a plausible potential explanation. Further studies will be required to unravel the complex regulation of HNF4A, which has been shown here for the first time to respond specifically to low-dose ionizing radiation.