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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 10  |  Issue : 1  |  Page : 44-57

Early and late changes in radiation-induced gene expression arrays following radioprotection with amifostine


1 Tech Micro Services, 4417 Maple Avenue, Bethesda, MD 20817, USA
2 Department of Pharmacology and Molecular Therapeutics, F. Edward Hébert School of Medicine; Department of Scientific Research, Armed Forces Radiobiology Research Institute, Uniformed Services University of the Health Sciences, Bethesda, MD, USA

Date of Web Publication22-May-2019

Correspondence Address:
Prof. Vijay K Singh
Department of Pharmacology and Molecular Therapeutics, F. Edward Hébert School of Medicine; Department of Scientific Research, Armed Forces Radiobiology Research Institute, Uniformed Services University of the Health Sciences, Bethesda, MD
USA
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jrcr.jrcr_5_19

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  Abstract 


Aims and Objectives: The study objective was to investigate differential gene expression in lymphohematopoietic tissues (spleens) of mice injected with amifostine and exposed to sublethal doses of 60Co γ-radiation. Materials and Methods: Differential cDNA gene expression arrays were used to examine early- (1 day) and late-occurring (63 days) changes in C3H/HeN mice that were administered either amifostine (100 mg/kg) or vehicle 30 min prior to exposure. Results: Sublethal irradiation initiated both early- and late-arising gene responses that were both specific and global in nature, with some significantly modified by amifostine. Of the early changes, ~15% of the genes were upregulated, whereas a comparable fraction was downregulated by irradiation. Notably, amifostine prophylaxis resulted in significant dampening of irradiation-related gene activity. Late-occurring changes were characterized by a reduction in fractional size (~11%) of upregulated genes, along with a corresponding increase of the downregulated fraction (~17%). Again, amifostine prophylaxis resulted in a significant dampening of gene activity, but only for downregulated genes. A cohort of pr oto-oncogenes responded comparably to the entire group of arrayed genes but with several notable exceptions. Differences in gene expression induced by sublethal whole-body radiation exposure were observed here within the splenic tissues of mice, and amifostine prophylaxis significantly altered patterns of gene expression within a sizable fraction of the arrayed genes. Conclusion: This study continues to illustrate the utility of differential cDNA array assays in identifying and dissecting critical gene events (e.g., hematopoietic growth factors and associated proto-oncogenes) altered by irradiation and by the radioprotective pharmacologic amifostine.

Keywords: Amifostine, gene array, gene expression, mice, radiation


How to cite this article:
Seed TM, Singh VK, Hanlon BK. Early and late changes in radiation-induced gene expression arrays following radioprotection with amifostine. J Radiat Cancer Res 2019;10:44-57

How to cite this URL:
Seed TM, Singh VK, Hanlon BK. Early and late changes in radiation-induced gene expression arrays following radioprotection with amifostine. J Radiat Cancer Res [serial online] 2019 [cited 2019 Dec 10];10:44-57. Available from: http://www.journalrcr.org/text.asp?2019/10/1/44/258720




  Introduction Top


It is well-recognized and documented that sufficiently intense exposure to ionizing radiation (IR) produces bodily injuries that can be expressed either shortly following exposure as acute syndrome (s) or can be manifested as late-arising pathologies (i.e., “delayed-type” syndromes) that are the direct result of the time-dependent evolution and progression of initial injuries.[1] “Acute radiation syndrome” is representative of the early-arising syndromes, while radiation-induced tissue fibrosis (e.g., pulmonary or myelofibrosis) or cancer (e.g., solid tumors, leukemias, or related myeloproliferative diseases [MPDs], etc.) is representative of the late-arising diseases induced by prior radiation exposure.[2],[3],[4],[5],[6]

The induction, prevalence, and severity of these induced pathological syndromes are largely governed by a host of both radiological and biological factors. Prominent radiological factors include not only radiation quality, cumulative dose, and the rate of exposure, but also the duration and extent of bodily exposure, whereas prominent biological factors include the animal species and its physiological and general health status.[7],[8],[9],[10]

Many of the radiological and biological details of radiation injury and associated pathological consequences have been worked out through the use of both large and small animal models, for example, the basic nature and processes of radiation-induced MPDs have been determined over many decades of experimental work using disease-prone strains of inbred mice (e.g., CBA, RF, and C3H), with peak incidences of overt disease occurring within several hundred days following exposure to acute, whole-body radiation doses.[11],[12],[13],[14],[15]

Syndrome-precipitating event lesions, whether they be “initiating” or “progressing” in nature, have been identified and characterized to various degrees, ranging from molecular and subcellular events to cellular and whole-tissue alterations to pronounced dysfunctions in various organ systems at risk.[16],[17],[18],[19],[20],[21]

The spleen is an important, occasionally vital (in times of acute lymphohematopoietic stress) organ of all mammals, including various species and strains of rodents.[22],[23],[24] It is well recognized that the mouse's spleen is not only representative of the hematologic system at large, but also shares and contributes many key pathophysiological processes, especially those associated with early- and late-arising injurious, hematopathological responses to IR.[25],[26],[27],[28] Not only have temporal and IR dose-dependent patterns been established, but also qualitative and quantitative shifts in composition and function of splenic tissues of irradiated rodents been previously evaluated and reported.[29],[30],[31],[32],[33],[34]

Elemental processes at the molecular level, specifically at the level of genome and composite genes of radiation-targeted cells, have been evaluated, but more extensively in terms of “early postirradiation” events, while less so for the “delayed,” postirradiation molecular/cellular events associated with evolving or manifesting “late-arising” pathologies.[21],[35],[36] Initial, postirradiation responses are thought to be largely the direct result of the activation of a complex network of signaling pathways that comprise the “DNA damage response” and consequent series of cell protective responses (e.g., DNA repair, cell-cycle arrest, cell senescence, or cell-death responses), with temporally and causal linkages to activation of several prominent transcription factors (e.g., p53, nuclear factor-κB [NF-κB], AP-1, Nrf2, CREB, etc.).[37] A variety of methodologies, including cDNA expression arrays, have been employed to assay and to characterize these initial radiation-induced biomolecular responses under a wide variety of radiobiological conditions.[37],[38],[39] Further, the application of cDNA expression array technology has been found to be useful in other types of gene evaluation studies as well.[40],[41] Amundson et al.[42],[43] were among the first researchers within the radiobiology community to use cDNA expression arrays for the general survey purposes of early radiation-responsive genes. These studies were followed by more extensive investigations, in which specific “signatures” of radiation-induced genomic injuries elicited under a variety of radiation exposure conditions were sought.[44],[45],[46],[47],[48]

Relative to the action(s) of selected radioprotective agents (e.g., amifostine) on IR-targeted genes and the genome itself, these drug-IR-targeted gene inactions are not as well characterized as radiation-responsive genes alone. Nevertheless, new informations on this topic over the last decade or so from the Grdina laboratory at the University of Chicago (Chicago, IL, USA) have relevance to the cDNA expression array work presented here.[49] For example, cultured human glioma cells (U87) exposed to micromolar concentrations of WR-1065 (i.e., the dephosphorylated, bioactive form of amifostine) for ~16 h, responded by markedly increasing and sustaining expression (~2 folds) of both manganese superoxide dismutase and NF-κB genes over a 24-h period. Further, using cDNA expression arrays containing genes (49 genes) characterized as having specific NF-κB-associated DNA-binding motifs, genic expression of interleukin-2 receptor α (IL-2Ra), Regulated on Activation, Normal T-cell Expressed and Secreted (RANTES), and c-myb were markedly elevated, while glutathione S-transferase-3 and c-myc were suppressed.[50] As observed here in this study, comparable changes in the expression of several of these genes occurred over time following amifostine prophylaxis and subsequent sublethal exposures to test C3H/HeN mice.

Amifostine (WR-2721), an aminothiol with rather broad radioprotective properties,[51],[52],[53] has been shown to (i) reduce/limit the extent of acute radiation injury through mainly free radical quenching, although other cytoprotective mechanisms have been implicated (direct protection of DNA itself, enhancing molecular and cellular repair processes, induction of tissue hypoxia, etc.) and (ii) provide a degree of protection against late-arising, radiation-induced cancers through its early countering actions on radiation-induced mutagenesis that is often linked to the carcinogenic processes.[54],[55]

In this study, we have used differential cDNA gene expression arrays to examine early (1 day) and late (63 days) genic changes within blood-forming tissues of C3H/HeN mice that received sublethal, but potentially, MPD-inducing doses of whole-body γ-radiation following either prophylaxis with amifostine (WR-2721) or without prophylaxis (vehicle). Results indicated that amifostine prophylaxis exerted a series of both early- and late-occurring specific changes, most notable of which were the generalized “dampening effects” of radiation-altered gene activities and that were global as well as specific (select number of individual genes) in nature.


  Materials and Methods Top


General experimental design

Differential cDNA gene expression arrays were used to examine early- (1 day) and late-occurring (63 days) changes within lymphohematopoietic tissues (spleens) of acute, sublethal γ-irradiated (3 Gy), or sham-irradiated (0 Gy) C3H/HeN mice. The C3H/HeN strain of mice, along with several other strains (e.g., CBA, RFM), has a documented sensitivity to develop myeloid leukemia and related MPD following sublethal exposure to IR. IR doses in the range of 2–3 Gy total-body irradiation (TBI) represent maximum doses for ML/MPD induction.[11],[12],[13],[14] The sampling time points, 1 day and 63 days, were selected for the following reasons: 1-day sampling allows for early collection of lymphohematopoietic tissues with manifest, fixed IR-injuries, but absent of substantial tissue repair; 63-day sampling, allows for the collection of tissues that have partially recovered and yet still retain latent injuries. These mice received either amifostine or vehicle 30 min prior to total-body 60 Co γ-radiation exposure [Figure 1].
Figure 1: Schematic of the basic elements of the study's experimental approach in the in vivo examination of both early and late gene responses within lymphohematopoietic tissue (spleen) of mice

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Mice

Male C3H/HeN mice, 8–10 weeks of age, were obtained from the National Cancer Institute (Frederick, MD, USA). Newly purchased mice were quarantined for 2 weeks. Following this period, the health status of the mice was confirmed, and the mice were cleared for experimental use. Animal handling protocols and general aspects of animal husbandry have been described in detail previously.[56] This animal study was conducted in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)- International. All animal-based experiments described were executed according to the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council, US National Academy of Sciences.[57] All animal procedures were performed according to a protocol approved by the Institutional Animal Care and Use Committee.

Amifostine prophylaxis

Amifostine was obtained from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, MD, USA. It was dissolved in sterile phosphate-buffered saline and injected subcutaneously (sc) in small volumes (0.2 ml) ~30 min ± 10 prior to irradiation or sham irradiation.

Irradiation

Test animals were exposed to total-body 60 Co γ-radiation (TBI). TBI was carried out acutely at the dose rate of 60 cGy per min to a total dose of 3 Gy. Control/sham-irradiated animals were handled in a similar way to radiation-exposed animals and placed on the irradiation platform for the same amount of time but were not exposed to radiation. Details of these mouse irradiations and dosimetry can be reviewed within the previously reported article.[56]

Tissue sampling

Spleens were harvested from euthanized mice at 1- or 63-day postirradiation/sham irradiation (day of radiation exposure considered day “0”). Tissues from sacrificed animals within each of the experimental groups (i.e., vehicle-treated- or amifostine-treated sham-irradiated controls or comparably treated, irradiated test animals) were processed further for the gene array work reported here. Specifically, in terms of “group size,” 40 animals were utilized in total for these experiments, with 20 intended for the “early” sampling period (1 day) and 20 for the “late” sampling period (63 days). These groups were divided into two major subgroups, with half of the animals (n = 10) irradiated at day 0 (same day), whereas the remaining half (n = 10) were “sham” irradiated (same day). These subgroups were again divided into two minor subgroups, with half of the animals (n = 5) prophylaxed (30 min prior to irradiation) with amifostine, whereas the remaining half of the animals (n = 5) treated with the vehicle alone (saline). As indicated, animals were sacrificed at the indicated times (i.e., 1- or 63-day postirradiation) and the spleens were collected and processed as indicated for the development of the “differential expression array” profiles. At each of the sampling time points (1 and 63 days), three (n = 3) profiles were selected for their completeness and consistency and composited into single profiles for presentation purposes.

mRNA isolation and labeling

Spleens were homogenized on ice, with dense-tissue fragments (capsular/reticular elements) allowed to settle out, while light-density supernatants (enriched in immune and hematopoietic cells) were collected and used in making radio-labeled cDNA probes using the  Atlas More Details Pure Total RNA Labeling System (Clontech Laboratories, Inc., Palo Alto, CA, USA.). After phenol– chloroform extraction, samples were treated with DNase I to remove genomic DNA. Total RNA (10–50 μg) was mixed with biotinylated oligo (dT) to bind to the poly (A +) RNA. Streptavidin-coated magnetic beads were added to selectively capture the combined biotinylated oligo (dT)/mRNA, using a magnetic particle separator.32 P-labeled cDNA probe synthesis was performed with a gene-specific primer mix (protocol supplied by the manufacturer) in the presence of α-32 P-deoxyadenosine triphosphate (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Differential cDNA expression Clontech Atlas Microarray

The microarray consisted of cDNA fragments representing 588 mouse genes immobilized in duplicate dots on a nylon membrane and arranged into six functional groups (Atlas Mouse cDNA Expression Array; Catalog # 7741-1; Clontech Laboratories, Inc.). The assay was performed following the manufacturer's instructions as described previously [Figure 2].[58] Included on this array were several housekeeping gene cDNAs for positive controls as well as plasmid and bacteriophage cDNAs for negative controls. Each cDNA fragment was 200–600 bp long and was present on the membrane in the amount of 10 ng.
Figure 2: Schematic of methodological approach in using Atlas™ cDNA differential gene expression arrays. The 588-gene array is sectored into six major groups. These major groups are listed and briefly characterized

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Hybridization and analyses

Each set of 32 P-labeled cDNA probes consisting of four individual probes were synthesized from amifostine-treated and vehicle-treated splenic tissues derived from both irradiated and sham-irradiated mice. Individual probes were hybridized to four identical Atlas Mouse cDNA Expression Arrays (Clontech Laboratories, Inc.). After a high-stringency wash (according to the manufacturer's protocol described in the Atlas cDNA Expression Arrays User Manual), the arrays were scanned by a Storm 840 Phosphorimager (Molecular Dynamics, Sunnyvale, CA, USA) following a 2–4 day exposure. The array images were then analyzed and compared using the Clontech Atlas Image software. After all of the signals of the genes on the arrays were averaged (global normalization), the aligned arrays were compared with each other. Phosphor imaging was used to assess hybridization signals; 2–3 membranes (arrays) were used for each of the samples (i.e., one tissue processed per array developed) analyzed.

Statistical treatments of data

Differential hybridization values from drug and vehicle-treated, irradiated, or sham-irradiated animals were compared and evaluated using commercially available statistical tools embedded into Microsoft ® Office Suite software (Microsoft Excel ®). For tests of statistical significance, both unpaired and paired Student's t-tests were applied between unmatched and matched test and control groups, respectively. Significant differences between the groups were defined by P < 0.05.


  Results Top


Global responses

Differential responses of all arrayed genes at 1-day and 63-day postirradiation under select experimental conditions of sublethal (3 Gy) irradiation with or without amifostine prophylaxis are illustrated as “heat maps” [Figure 3]a, [Figure 3]b, [Figure 3]c, [Figure 3]d, [Figure 3]e demonstrating IR-response genes at either time point (1 day or 63 days) [Supplementary Figure 1]a, [Supplementary Figure 1]b, [Supplementary Figure 1]c, [Supplementary Figure 1]d, [Supplementary Figure 1]e, [Supplementary Figure 1]f displays the entire array with all 588 genes, whereas [Supplementary Figure 2]a and [Supplementary Figure 2]b Venn diagrams illustrate the quantitative features of the recorded responses of all 588 genes].
Figure 3: (a-e) Heat map of the differential responses (provided as DIF values) of genes at 1–day and 63-day postirradiation. Genes that were not responsive at either time point were excluded from these heat maps. Figures represent composite heat maps with n = 3

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One day following sublethal irradiation (3 Gy) of mice pretreated solely with vehicle (saline), 14.8% of the genes within the array (87/588) appeared “upregulated” (defined by positive differential hybridization/gene expression (DIF) values) within sampled splenic tissues, relative to the genic responses of unirradiated mice. Similarly, 13.9% of the genes within the array (82/588) were “downregulated” (defined by negative DIF values). The average differential gene activity values for genes within the “irradiated-alone” response sector are listed in [Table 1] (top panel) and shown individually as “heat elements” within the heat map [Figure 3]a, [Figure 3]b, [Figure 3]c, [Figure 3]d, [Figure 3]e and [Supplementary Figure 1]a, [Supplementary Figure 1]b, [Supplementary Figure 1]c, [Supplementary Figure 1]d, [Supplementary Figure 1]e, [Supplementary Figure 1]f. The remaining 71.3% of the genes (419/588) within the array were not significantly modified when compared to the arrayed genes of unirradiated tissues. Prophylaxis with 100 mg/kg of amifostine, 30 min prior to irradiation, altered these global genic responses, albeit modestly (in terms of the total number of responsive genes): 15.7% (92/588) of the arrayed genes were upregulated, 13.8% (81/588) downregulated, and 70.6% (415/588) remained unchanged or unresponsive. The average differential gene activity values for genes within these “amifostine-associated” response sectors are as listed [[Table 1], top panel] and as illustrated for individual genes in [Figure 3]a, [Figure 3]b, [Figure 3]c, [Figure 3]d, [Figure 3]e. However, relative to the cohort of genes that were upregulated (87/588 or 14.8%) by irradiation alone [Figure 4]a and [Figure 4]b, left panels], amifostine prophylaxis (as compared to the control and vehicle treatment) continued to upregulate 66.7% of this sector's genes (58/87), whereas 16.1% (14/87) of this sector's genes appeared to be suppressed or downregulated [Figure 4]a, right top panel]. By contrast, examining the sector of genes that was downregulated by irradiation alone, amifostine prophylaxis appeared to promote the upregulation of a substantial number of these genes (10/82 or 12.2%), while sustaining a reduced number of downregulated genes (62/82 or 75.6%) [Figure 4]b, right bottom panel].
Table 1: Differential gene activities (DIF values): Global responses at 1-day and 63-day postirradiation

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Figure 4: (a and b) Comparison of response of specific sectors of upregulated genes ([a] – top panel) or downregulated genes ([b] – bottom panel) 1-day postirradiation either without prior prophylaxis (left panels) or with amifostine prophylaxis (right panels). Composite data shown of differential expression array sets from three animals/three tissues collected and processed on 1 day

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Global dampening effects of amifostine prophylaxis (on radiation-induced gene responses) were noted not only by the number of genes affected but also by the relative activity of those amifostine-responsive genes. Both the average values and the net activity values for these specific sectors of amifostine-response genes diminished relative to those values of comparable gene sectors from the vehicle control samples [[Table 1], top panel]; for example, for the upregulated gene sector, the average value dropped significantly (P = 0.0004) from 15.64 ± 2.31 differential hybridization intensity units (DIF units) to 9.34 ± 1.93 following amifostine prophylaxis, while eliciting a downward change in average net gene activity of −6.25 ± 1.82 [[Table 1] top panel]. By contrast, the downregulated gene sector, the average value rose slightly, approaching a significant value (P = 0.056), from −9.63 ± 2.75 DIF units to −6.32 ± 1.41 following amifostine prophylaxis, while eliciting an upward change in average net gene activity of 3.32 ± 2.05 [[Table 1], top panel].

Differential responses at 63-day postirradiation of arrayed genes following sublethal irradiation, preceded by either saline/vehicle pretreatment or amifostine prophylaxis, are shown as individual “heat elements” within a “heat map” [Figure 3]a, [Figure 3]b, [Figure 3]c, [Figure 3]d, [Figure 3]e and [Supplementary Figure 1]a, [Supplementary Figure 1]b, [Supplementary Figure 1]c, [Supplementary Figure 1]d, [Supplementary Figure 1]e, [Supplementary Figure 1]f. Following acute 3-Gy irradiation of the animals pretreated with the vehicle alone and relative to the earlier sampling period of 1 day, the total number of upregulated genes declined slightly (64/588 or 10.9%), whereas the number of downregulated genes increased slightly (100/588 or 17%). By contrast, in tissues of the irradiated, amifostine-prophylaxed animals, the number of upregulated genes substantially increased (124/588 or 21.1%), whereas the number of downregulated genes declined (55/588 or 9.4%). The major fraction of genes, however, remained unchanged or unresponsive (409/588 or 69.6%). The average differential gene activity values for genes within these “3 Gy irradiation/saline pretreatment controls” at 63-day postirradiation are listed in [Table 1] (bottom panel), with responses of individual genes illustrated as “heat elements” within the “heat map” [Figure 3]a, [Figure 3]b, [Figure 3]c, [Figure 3]d, [Figure 3]e and [Supplementary Figure 1]a, [Supplementary Figure 1]b, [Supplementary Figure 1]c, [Supplementary Figure 1]d, [Supplementary Figure 1]e, [Supplementary Figure 1]f. Comparing the number of responsive genes within specific (and matched) sectors, declines were noted in the sectors of both upregulated and downregulated genes (i.e., 53/64 or 82.8% of the upregulated sector and 46/100 or 46% of the downregulated sector) [Figure 5]a and [Figure 5]b, right panels]. The noted prophylaxis-mediated decline in number of responsive genes within specific sectors was offset by gains in opposing response sectors; for example, with prophylaxis, the 11 fewer upregulated genes were replaced with 9 downregulated genes, along with 2 additional unresponsive genes [Figure 5]a and [Figure 5]b, right panels].
Figure 5: (a and b) Comparison of response of specific sectors of upregulated genes ([a] – top panel) or downregulated genes ([b] – bottom panel) 63-day postirradiation either without prior prophylaxis (left panels) or with amifostine prophylaxis (right panels). Composite data shown of differential expression array sets from three animals/three tissues collected and processed on 63 days

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The overall dampening effect of amifostine prophylaxis on global gene responses was again noted (as per 1-day samples) at the later sampling period (63 days), but less prominent (relative to 1-day samples). The average and net activity values for specific, up-, and downregulated sectors of amifostine-response genes were marginally diminished relative to those values of comparable gene sectors from the vehicle-pretreated 3-Gy samples [Table 1], bottom panel]; for example, the upregulated gene sector, the average value dropped from 5.84 ± 0.94 DIF units to 4.83 ± 1.23 (P = 0.212) following amifostine prophylaxis, whereas the downregulated gene sector also declined, but significantly so, from −6.34 ± 1.57 to −3.72 ± 1.62 (P = 0.003). The marginal downward change in average net gene activity of −1.02 ± 1.28 calculated for the upregulated sector, contrasted to the upward change in net gene activity of 2.52 ± 0.96 noted for the downregulated sector [[Table 1], bottom panel].

Family A subset – “Proto-oncogenes”

One day following acute irradiation, the response of a subset, a constellation, of 13 proto-oncogenes found within the group of Family A genes was evaluated. [Table 2] lists these surveyed proto-oncogenes, while a corresponding “heat map” [Figure 6], left panel and [Supplementary Table 1] illustrates specific gene activities. For this cohort of genes from control tissues collected from vehicle-treated animals, the percentages (numbers) of up- and downregulated genes were 46.2% (6/13) and 38.5% (5/13), respectively. The two remaining proto-oncogenes, or 15.4% (2/13), appeared unresponsive when compared to the unirradiated, vehicle-treated controls but responded following amifostine treatment. Under amifostine prophylaxis, there were comparable numbers of both upregulated and downregulated genes (6/13 or 46.2% for both response sectors), whereas a single remaining proto-oncogene (1/13; 7.7%) continued to be unresponsive.
Table 2: Listing of surveyed family A proto-oncogenes at 1-day and 63-day postirradiation

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Figure 6: Heat maps of the differential responses (indicated by DIF values) of all arrayed “proto-oncogenes” responsive to irradiation alone (A × C series) or in combination with amifostine prophylaxis (A × D series) at 1-day and 63-day postirradiation, relative to baseline gene responses of control tissues. Figures represent composites heat map with n = 3

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Comparing the responses of specific sectors of either upregulated or downregulated proto-oncogenes following control, drug-vehicle pretreatments versus those following amifostine prophylaxis, the responses were mixed: the number of upregulated genes was largely sustained under amifostine prophylaxis (83.3%; 5/6), whereas a single, previously upregulated gene was now downregulated [Figure 7]a. Similarly, within the downregulated sector, the number of downregulated genes was mainly sustained (80%; 4/5), whereas a single, previously downregulated gene was unaltered by prophylaxis [Figure 7]b. Within the response sector that showed no change in the absence of prophylaxis now had two responding genes under amifostine prophylaxis, with one gene upregulated and one gene downregulated (these amifostine prophylaxis-associated responses of the two initially unresponsive under control conditions are not illustrated).
Figure 7: (a and b) Cohort of proto-oncogenes of family A genes with comparison of specific sectors of upregulated genes ([a] – left panels) or downregulated genes ([b] – right panels) 1-day postirradiation either without prior prophylaxis (larger “pie” figures) or with amifostine prophylaxis (smaller “pie” figures). Composite data shown of differential expression array sets from three animals/three tissues collected and processed on 1 day

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In terms of the shift/direction in magnitude of signal strength recorded at 1-day postirradiation (i.e., the relative differential of hybridization intensity between control arrays and test arrays), amifostine prophylaxis uniformly dampened signal strength (DIF values) of the responding, upregulated proto-oncogenes (6/6; 100%). the calculated average DIF value of this sector declined from 14.7 ± 10.1 to 9.3 ± 0.5 with a negative, calculated differential response value of −5.0 ± 1.7 [Table 3], top panel]. By contrast, proto-oncogenes within the downregulated sector responded to amifostine prophylaxis in a mixed fashion: a fraction (40%; 2/5) of the sector exhibited a further dampening of gene activity, whereas the remaining fraction (60%; 3/5) exhibited a lessening of suppression. The calculated average DIF value of this sector declined from −5.20 ± 2.31 to −7.60 ± 3.41. However, the net overall trend elicited by amifostine prophylaxis was a dampening effect, with a negative, calculated differential response value of −2.40 ± 1.72 [[Table 3], top panel].
Table 3: Measured activity (DIF values) of the subset of proto-oncogenes A at 1-day and at 63-day postirradiation

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Focusing on individual proto-oncogenes that were highly responsive 1 day following sublethal irradiation, c-fms (CSF1) and A-Raf were upregulated significantly (with positive DIF values) at 1-day postirradiation, whereas B-Myb and c-myc were significantly downregulated. Amifostine prophylaxis just prior to irradiation served to markedly dampen the radiation-induced responses of both c-fms and A-Raf, as well as lessening the degree of downregulation of c-myc. By contrast, amifostine prophylaxis appeared to promote further downregulation of B-Myb.

Sixty-three days following acute radiation exposure, an expanded, but over-lapping lapping group of responding proto-oncogenes was observed [Table 2]. A corresponding “heat map” [Figure 6], right panel and [Supplementary Table 2] illustrates gene activities. Equal fractions (42.1%; 8/19) of both upregulated and downregulated proto-oncogenes were noted, along with a smaller, unresponsive fraction (15.8%; 3/19). Sixty-three days following earlier amifostine prophylaxis, the fractional size of the upregulated genes significantly increased (68.4%; 13/19), whereas the fractional size of the downregulated sector of genes correspondingly declined (31.6%; 6/19).



Specific cohorts of responsive proto-oncogenes changed not only in terms of relative size, but also in terms of absolute numbers under the influence of earlier amifostine prophylaxis (when compared to those of vehicle-controls). For example, the absolute number of upregulated genes declined from eight to six, as two genes of the sector being replaced by downregulated genes [Figure 8]a. By contrast, the number of downregulated genes sector declined by four genes while being replaced by an equal number of upregulated genes [Figure 8]b.
Figure 8: (a and b) Cohort of proto-oncogenes of Family A genes with comparisons specific sectors of upregulated genes ([a] – left panels) or downregulated genes ([b] – right panels) 63-day postirradiation either without prior prophylaxis (larger “pie” figures) or with amifostine prophylaxis (smaller “pie” figures). Composite data shown of differential expression array sets from three animals/three tissues collected and processed on 63 days

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In terms of the shift/direction in magnitude of signal strength recorded at 63-day postirradiation and in contrast with the induced responses seen earlier at 1-day postirradiation, amifostine prophylaxis failed to dampen the signal strength (DIF value) of the majority fraction of responding proto-oncogenes. Only a minority of proto-oncogenes (21.1%; 4/19) exhibited a prophylaxis-mediated dampening, whereas the majority exhibited either an enhanced signal (52.3%; 10/19) or no change in signal (5/19; 26.3%) [[Table 3], bottom panel].

The calculated average values in DIF for these specific, prophylaxis-mediated responses were as follows: for the upregulated genes, the average DIF value declined from 4.87 ± 1.45 to 2.00 ± 3.53, with a net negative change of − 3.12 ± 3.77 and for the downregulated genes, average values rose from −1.25 ± 0.18 to −0.13 ± 0.91, with a net positive change of 1.13 ±0.99 [[Table 3], bottom panel]. Individual proto-oncogenes that were highly responsive at 1-day postirradiation were not necessarily the same as those responsive genes noted much later at 63-day postirradiation; for example, previously upregulated A-Raf (at 1-day postirradiation) appeared downregulated at 63 days, whereas c-fms, also upregulated at 1 day, was only moderately responsive at 63 days. By contrast, c-Myc and B-Myb, significantly downregulated initially (at 1 day), were now significantly upregulated at 63 days. Amifostine prophylaxis just prior to irradiation induced markedly different gene responses at this late, 63-day sampling: B-Myb was moderately dampened by the early administered prophylaxis, whereas c-Myc appeared enhanced by prophylaxis. The upregulated and moderately active c-fms, as well as H-Ras, were significantly dampened (below control levels) by early amifostine prophylaxis.


  Discussion Top


The Atlas cDNA expression microarrays used in this study have the capacity to examine simultaneously the expression of some 588 active mouse genes.[59],[60],[61] Assuming that the mouse genome contains ~23,000 genes, the small number of arrayed genes would represent only ~2.5% of the mouse genome.[62] However, as these arrayed genes are sampled from some six major gene families that comprise the greater mouse genome itself, it is possible (yet unconfirmed) that the array represents a microcosm of the mouse genome itself. Considering the later possibility, it might be expected that the recorded responses of the array are representative of the genic responses of the entire mouse genome. Accordingly, with this possibility in mind, the study here suggests that there are significant genomic changes induced by acute, sublethal, whole-body irradiation, and that these changes can be modulated by prophylaxis with the well-known radioprotectant, amifostine. Further, these early irradiation- and drug-induced genomic responses, i.e., both global and specific genic responses, appear to be altered over time.

In terms of “global” change, the most impressive general trend noted following amifostine prophylaxis was a generalized “dampening effect” by the drug on induced genic responses; i.e., the magnitude of irradiation-induced gene enhancement appeared to be lessened by prophylaxis, while conversely, the extent of irradiation-induced suppression was lessened. In other words, a major global effect of amifostine prophylaxis appeared to be a “bringing back” to steady state the irradiation-altered genome and its composite, radiation-responsive genes. The broad response observed here might be expected considering the basic nature of molecular binding of amifostine's principal metabolites (WR1065/WR33278) to sequence-dependent “hot spots” within the minor groove of targeted genomic DNA;[39] however, these observations are notable in terms of the rather widespread effect of amifostine prophylaxis in the gene expression of the sublethally irradiated mouse. The basic underlying molecular mechanisms for the noted changes in gene expression over time remain uncertain and without current information on this issue in the open literature. Additional studies will be needed to properly address the basic underlying molecular “mechanisms” by which amifostine affects gene function within the irradiated mouse.

When the gene families were examined individually shortly following radiation exposures (1 day), all of the gene families (six of the six families) responded to amifostine prophylaxis with the above-mentioned “dampening” effect. However, and by contrast, at the late sampling period (63 days), only half of groups of arrayed maintained the full complement of this dampening action by amifostine, whereas the remaining groups of genes exhibited mixed responses that trended in an opposite direction of enhancing (rather than suppressing) initial, irradiation-related genic changes (as noted earlier, at 1-day postirradiation).

Although clearly not definitive, it is quite probable that these overall, time-dependent shifts in the amifostine-induced gene responses of specific gene groupings were directly due to the nature of their composite genes. For example, noting the contrasting overall early response versus late responses of one such select grouping and that the group is heavily weighted (19/98 or ~19%) in genes characterized as “proto-oncogenes” and often associated with carcinogenesis due to their potential mutability and later cell-transforming capacities,[63] it might be reasonable to expect the differential, time-dependent effects of early prophylaxis (i.e., early dampening followed by later genic enhancement). The average measured activity of this gene cohort (Family A proto-oncogenes) was elevated under control conditions but suppressed by amifostine prophylaxis during the early period (1 day) following irradiation. however, during the later sampling period (63 days), this dampening effect by amifostine was muted and clearly less impressive (i.e., than those initial responses noted at 1 day). Moreover, with extended postirradiation time, six additional, previously unresponsive proto-oncogenes were shown (63 days) to be responsive to either prophylaxis or to irradiation or to both in combination. This additional responsiveness might well suggest a type of progression of the proto-oncogenic cohort at large. All of these additional genes have been linked specifically to leukemogenesis, or in general to carcinogenesis, within specific, susceptible strains of mice.[64],[65],[66],[67],[68],[69],[70],[71],[72],[73],[74]

Nevertheless, noted responses of specific proto-oncogenes during the late tissue sampling were consistent with the working hypothesis that early-arising lymphohematopoietic tissue injuries induced by either acute or chronic radiation exposures might be temporally related and perhaps causally related to radiation-induced lymphohematopoietic disorders.[10],[21],[35],[75] Although this study did not directly assess the prevalence of such late-arising blood disorders within the test animals, still heightened responses of selected proto-oncogenes long after the initial radiation exposure event might well be suggestive of an evolving preclinical tissue environment, as reflected by the heightened activity of several major genic players associated with oncogenesis (e.g., c-fms, B-myb, c-myc, h-ras). Further, it is of interest to note that while early prophylaxis with amifostine appeared to significantly dampened the activity of most of these proto-oncogenes, not all responded in this fashion (e.g., amifostine prophylaxis appeared to dampened activity of c-fms, myb-b, and h-ras, while elevating activity of c-myc and AP-1/c-Jun, but with little effect on cKit).

These data, i.e., amifostine-associated/proto-oncogene responses, are interesting to note and perhaps relevant to early processes of late-arising, radiation-induced leukemia and related malignancies of the mouse's lymphohematopoietic system. However, it needs to be pointed out that proto-oncogenes represent only one of the two major classes of genes thought to be key in the “cancer induction process (es).” This second class of genes encompasses the “tumor suppressor genes,”[66] with the Rb tumor suppressor gene being representative. The primary function of both of these groups of unaltered, normal genes is to encode for cell-growth regulating proteins; but with selected spontaneous or induced mutations of these genes, they can function aberrantly and serve to promote cancer development.[66] Interestingly, the Rb gene was relatively inactive in the late (63 days), postirradiated animals, but appeared mildly activated within comparably irradiated and amifostine-prophylaxed animals. Regardless of these noted results, they are still only “observational” by nature and need to be explored and followed up by detailed “mechanistic” studies in order to understand the underlying bases of these genomic changes.

Overall, this study clearly reveals differences in gene expression induced by sublethal whole-body radiation exposure within spleens of mice and that pretreatments with amifostine could indeed alter the expression of these genes, regardless of the location of tissue cells (i.e., spleen vs. marrow) that bear these genes. As noted previously, the spleen-like bone marrow of mice is an actively lymphohematopoietic organ and is highly responsive over a broad range of radiation exposures, both in terms of induction of early-arising tissue hypoplasia, subsequent reparative responses, and consequent pathological conditions.[25],[26],[27],[28],[29],[30],[31],[32],[33],[34] Further and despite the fact that the differential cDNA expression arrays have been used for more than several decades; this study clearly and most notably demonstrates amifostine's capacity to modulate the activity of a large number of genes within the sublethally irradiated mouse. The study continues to illustrate the potential power and utility of the differential display cDNA array approach in identifying and dissecting critical gene events altered by IR over a time course and selectively targeted and modulated by radioprotective pharmacologic agents such as amifostine. It would be interesting to study the long-term effect of amifostine alone in the absence of radiation.


  Conclusion Top


Amifostine is an aminothiol with broad radioprotective properties but its FDA approval is only for limited clinical indications. This study illustrates the use of differential cDNA array in identifying critical gene events altered by amifostine treatment in irradiated mice. Such studies may be helpful to better understand its mechanism of action.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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