Figure 2. Transcriptomic expression patterns following 15-Gy irradiation. A. Expression profiles of differentially expressed genes (n = 3) 4, 8 and 12 weeks post-irradiation depicted by Heatmap and Expender analysis and grouped into two clusters based on temporal expression profiles: genes with up-regulated expression are shown in red, and those with down-regulated expression are shown in green. B. 81 genes were down-regulated at least twofold 4 weeks post- irradiation, and recovered to around control levels 8 and 12 weeks post-irradiation. *** P,0.0001 for 4-week values vs. control, 8- and 12- week post-irradiation values. C. 14 genes were twofold up-regulated 4 weeks post-irradiation, and further increased threefold 8 and 12 weeks post-irradiation. **P,0.001 for control vs. all 8- and 12-week post- irradiation values. doi:10.1371/journal.pone.0040636.g002 salivary secretory proteins and were up-regulated from week 4 post-IR. The CysS gene was up-regulated following IR to a maximum 1108-fold increase at 8 weeks post-IR and subsided to a 21-fold higher level than controls at 12 weeks post-IR (Fig. 3). Among the cell-cycle pathway genes, four were found to be affected by IR during the 12-week timeframe: cyclin D1 gene, p21 (encoding cyclin-dependent kinase [CDK] inhibitor 1A), p57 (encoding CDK inhibitor 1C) and Ywhaq (encoding tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide). Since changes in these genes are associated with cell-cycle arrest, their expressions were validated by real-time PCR. As shown in Fig. 3, cyclin D1 gene was up-regulated fourfold 4 weeks post-IR and remained ,2.5-fold up-regulated 8 and 12 weeks post-IR. p21 was up-regulated 68-fold at 4 weeks post-IR and remained high at 8 weeks (35-fold) and 12 weeks (30- fold) post-IR. p57 was up-regulated 2.8- to 1.9-fold following IR. Ywhaq, encoding a 14-3-3 family member, was only down- regulated twofold at 12 weeks post-IR. Proteome and Transcriptome in Irradiated Glands Interestingly, the large decrease in the transcription of salivary proteins in the first pattern and the increased expression of cell- cycle-regulating genes in the second pattern were accompanied by increased expression of the gene encoding Mgmt, a DNA repair enzyme, following IR (see below in pattern 2). This gene was analyzed by real-time PCR and exhibited a 7.5-fold increase at 4 weeks, a decrease to 4.5-fold at 8 weeks post-IR, and then remained at a 3.7-fold higher level than controls at 12 weeks post- IR (Fig. 3). The genes encoding CysS and Psp showed the same expression pattern as in the proteomic analysis, i.e. CysS transcription was found to be up-regulated in the mRNA analysis by Taqman (Fig. 3) and in spots 14 and 15 by proteomic analysis. Psp transcription was slightly down-regulated in the mRNA analysis by Taqman (Fig. 3) 4 weeks post-IR, and in the proteomic analysis it was found in several spots: 1, 3, 5, 6 and 8; although the behavior differed slightly for each spot, the prominent trend was down-regulation (Table S1). In the mRNA analysis, expression level was up- regulated at 8 and 12 weeks, but in the proteomic analysis, the damage continued and no return to normal levels was observed. We also measured the effect of IR on the c-KIT salivary cell proliferating fraction by real-time PCR (Fig. 4A). c-KIT expres- sion showed a 10-fold reduction 12 weeks post-IR. We validated these results using confocal microscopy, observing the same decrease in expression pattern (Fig. 4A). To further validate cell proliferation/cell arrest data in another independent approach we counted the number of SGIE cell colonies obtained from the submandibular salivary glands before and 4, 8 and 12 weeks after irradiation (Fig. 4B). The average number of grown colonies per animal without irradiation was 32617, while 4 weeks after head and neck irradiation, SGIE colony growth was 10 times lower, and about 75 lower (compared to control) 8 and 12 weeks post irradiation (0.560.5). As normally, a-amylase is not produced by the SSG [6], we validated these results by Taqman real-time PCR. We found a gradual time-dependent increase in a-amylase transcription from control levels in non-irradiated animals to 13-fold and 25-fold at 8 and 12 weeks post-IR, respectively (Fig. 4C). In contrast, a- amylase protein expression exhibited up-regulation following IR in protein spots 15 and 18 (Table S1), in agreement with the mRNA analysis (Fig. 4C), but in the other a-amylase protein spots it was down-regulated (spots 1, 11). We further validated these results using confocal microscopy which showed the same increase in expression pattern (Fig. 4C). Discussion IR therapy for head and neck cancer patients results in irreversible damage to the salivary parenchymal tissue. In the last decade, attempts have been made to decrease IR damage to the SG using a variety of approaches that can be grouped into three categories: (i) physical approaches such as intensity-modulated radiation therapy and 3-D radiotherapy [25]; (ii) pharmacological approaches such as exploring the usefulness of sialogogues or radical scavengers to protect glands from IR damage; (iii) biological approaches such as gene transfer [26], and tissue- and cell-regeneration methodologies [4,15,27,28]. However to date, the pathogenesis underlying the irreversible damage has not been adequately explored and many questions remain to be addressed [2]. The aim of this study was to analyze proteomic and transcriptomic changes in saliva following IR of SSG in a rodent model. We hypothesized that understanding the global mechanis- tic changes that occur following IR will eventually allow us to promote biological treatment modalities for these patients. The IR PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e40636