expression of its gene and protein, was not supported by the 2-DE analysis of whole saliva. This difference could be either genuine i.e., the net effect on the saliva secreted by all glands is a reduction in a-amylase protein, or it could be of technical origin. In a typical 2-DE gel, about 140 a-amylase protein spots are identified [41], but only a few of them were taken for analysis in this proteomic study, preventing an accurate quantitative interpretation. A further quantitative proteomic approach, such as dimethylation based quantitative activity assay, is suggested to clarify this point. Multiple pathways are involved in the maintenance of genetic integrity after exposure to ionizing radiation, most of which are related to the cell cycle [42]. Cells commonly respond to DNA- damaging agents by activating cell-cycle checkpoints. These checkpoints provide controlled temporary arrest at a specific stage of the cell cycle to allow the cell to correct possible defects [43]. Interestingly, four cell-cycle regulatory genes: cyclin D1 gene, p21, p57 and Ywhaq, were affected by IR. The first three genes shared the same pattern of expression following IR: their major differential up-regulation occurred 4 weeks post-IR, and it was slightly reduced toward 12 weeks post-IR (second pattern). Ywhaq was only down-regulated at 12 weeks post-IR. Damage to the cells’ proliferation ability might also be a result of direct DNA damage, as seen by the increased transcription of Mgmt (second pattern) following IR. Mgmt is a suicide repair enzyme that removes a methyl group from the O6 position in guanine, thus inactivating itself while repairing the DNA [44]. Thus, Mgmt plays an important role in maintaining genomic integrity when DNA damage occurs. The effect of IR on cell- regulation genes as well as on Mgmt and SG-function genes supports the hypothesis that IR results in signaling damage combined with DNA damage [2,12,13]. Because of the complex cell processes determining cell proliferation and cell-cycle arrest, we also examined the outcome of this balance by monitoring in-vivo c-KIT transcription, c-KIT tissue expression and number of grown salivary cell colonies following IR. c-KIT-expressing cells have been suggested to belong to the SG cell-dividing fraction [15] as well as SGIE cells which were reported to be able to grow in vitro for 20 passages [45]. Indeed, IR reduced c-KIT expression as well as number of SGIE colonies, References 1. Vokes EE, Weichselbaum RR, Lippman SM, Hong WK (1993) Head and neck cancer. N Engl J Med 328: 184–194. 2. Konings AW, Coppes RP, Vissink A (2005) On the mechanism of salivary gland radiosensitivity. Int J Radiat Oncol Biol Phys 62: 1187–1194. 3. Fox PC (1998) Acquired salivary dysfunction. Drugs and radiation. Ann N Y Acad Sci 842: 132–137. 4. Aframian DJ, Cukierman E, Nikolovski J, Mooney DJ, Yamada KM, et al. (2000) The growth and morphological behavior of salivary epithelial cells on matrix protein-coated biodegradable substrata. Tissue Eng 6: 209–216. 5. Aframian DJ, David R, Ben-Bassat H, Shai E, Deutsch D, et al. (2004) Characterization of murine autologous salivary gland graft cells: a model for use with an artificial salivary gland. Tissue Eng 10: 914–920. 6. Coppes RP, Vissink A, Konings AW (2002) Comparison of radiosensitivity of rat parotid and submandibular glands after different radiation schedules. Radiother Oncol 63: 321–328. 7. Cotrim AP, Hyodo F, Matsumoto K, Sowers AL, Cook JA, et al. (2007) Differential radiation protection of salivary glands versus tumor by Tempol with accompanying tissue assessment of Tempol by magnetic resonance imaging. Clin Cancer Res 13: 4928–4933. 8. Kashima HK, Kirkham WR, Andrews JR (1965) Postirradiation Sialadenitis – a Study of Clinical Features Histopathologic Changes and Serum Enzyme Variations Following Irradiation of Human Salivary Glands. American Journal of Roentgenology Radium Therapy and Nuclear Medicine 94: 271–291. 9. Valdez IH, Atkinson JC, Ship JA, Fox PC (1993) Major salivary gland function in patients with radiation-induced xerostomia: flow rates and sialochemistry. Int J Radiat Oncol Biol Phys 25: 41–47. 10. Nagler RM (2002) The enigmatic mechanism of irradiation-induced damage to the major salivary glands. Oral Dis 8: 141–146. Proteome and Transcriptome in Irradiated Glands 10 and 75 fold at 12 weeks post-IR respectively (Fig.4A, 4B) exemplifying cell arrest transcriptomic data. Collectively, our data on the expression patterns of cell-cycle genes, c-KIT and colony forming efficiency suggest that a cell-cycle arrest response occurs mainly at 4 to 8 weeks post-IR. In summary, this study emphasizes the complexity manifested by the multifactorial molecular mechanisms involved in SG damage post IR. It demonstrates that IR damages the cells’ abilities to produce and secrete fluids and proteins, and maintain the physiological barrier between serum and saliva. As shown herein, the mechanisms governing this damage involve the cells’ protein synthesis and secretion machineries. This damage is irreversible because tissue-regeneration ability is also damaged. Cells are damaged by the IR and undergo cell-cycle arrest as seen by the up-regulation of CDK inhibitors and down-regulation of Ywhaq. The data presented in this study can provide treatment targets for cell- and gene-therapy approaches, as well as a baseline to follow up on treatment success by comparing cellular, transcriptomic and proteomic characteristics before and after treatment. As rodents salivary glands respond to irradiation in a different way than humans [2], further human studies should be undertaken to support these findings and currently we analyze the protein profile changes in oral fluids of 131I treated thyroid cancer patients, irradiation treated head and neck cancer patients and normal healthy subjects. Supporting Information Table S1 (DOC) MS identification of selected spots. Table S2 Differentially expressed genes 4, 8 and 12 weeks post-irradiation. (DOC) Author Contributions Conceived and designed the experiments: RSC YN GK. Performed the experiments: RSC. Analyzed the data: RSC. Contributed reagents/ materials/analysis tools: RD OD BZ. Wrote the paper: RSC AP DJA. 11. O’Connell AC, Redman RS, Evans RL, Ambudkar IS (1999) Radiation-induced progressive decrease in fluid secretion in rat submandibular glands is related to decreased acinar volume and not impaired calcium signaling. Radiat Res 151: 150–158. 12. Zeilstra LJ, Vissink A, Konings AW, Coppes RP (2000) Radiation induced cell loss in rat submandibular gland and its relation to gland function. Int J Radiat Biol 76: 419–429. 13. Grundmann O, Mitchell GC, Limesand KH (2009) Sensitivity of salivary glands to radiation: from animal models to therapies. J Dent Res 88: 894–903. 14. Takagi K, Yamaguchi K, Sakurai T, Asari T, Hashimoto K, et al. (2003) Secretion of saliva in X-irradiated rat submandibular glands. Radiat Res 159: 351–360. 15. Lombaert IM, Brunsting JF, Wierenga PK, Faber H, Stokman MA, et al. (2008) Rescue of salivary gland function after stem cell transplantation in irradiated glands. PLoS One 3: e2063. 16. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254. 17. Deutsch O, Fleissig Y, Zaks B, Krief G, Aframian DJ, et al. (2008) An approach to remove alpha amylase for proteomic analysis of low abundance biomarkers in human saliva. Electrophoresis 29: 4150–4157. 18. Beer I, Barnea E, Ziv T, Admon A (2004) Improving large-scale proteomics by clustering of mass spectrometry data. Proteomics 4: 950–960. 19. Smyth GK (2004) Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3: Article3. PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e40636