Effect of Irradiation on Cell Transcriptome and Proteome of Rat Submandibular Salivary Glands Raluca Stiubea-Cohen1, Ran David1, Yoav Neumann 1 , Guy Krief1, Omer Deutsch1, Batia Zacks1, Doron J. Aframian2, Aaron Palmon 1 * 1 Faculty of Dental Medicine, Institute of Dental Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel, 2 Salivary Gland Clinic and Saliva Diagnostic Laboratory, Department of Oral Medicine, The Hebrew University of Jerusalem, Hadassah Medical Center, Jerusalem, Israel Abstract Salivary glands (SGs) are irreversibly damaged by irradiation (IR) treatment in head and neck cancer patients. Here, we used an animal irradiation model to investigate and define the molecular mechanisms affecting SGs following IR, focusing on saliva proteome and global transcription profile of submandibular salivary gland (SSG) tissue. We show that saliva secretion was gradually reduced to 50% of its initial level 12 weeks post-IR. Saliva protein composition was further examined by proteomic analysis following mass spectrometry (MS) analysis that revealed proteins with reduced expression originating from SSGs and proteins with increased expression derived from the serum, both indicating salivary tissue damage. To examine alterations in mRNA expression levels microarray analysis was performed. We found significant alterations in 95 genes, including cell-cycle arrest genes, SG functional genes and a DNA repair gene. Tissue damage was seen by confocal immunofluorescence of a-amylase and c-Kit that showed an increase and decrease, respectively, in protein expression. This was coherent with real-time PCR results. This data indicates that IR damages the SSG cells’ ability to produce and secrete saliva and proteins, and maintain the physiological barrier between serum and saliva. The damage does not heal due to cell- cycle arrest, which prevents tissue regeneration. Taken together, our results reveal a new insight into IR pathobiology. Citation: Stiubea-Cohen R, David R, Neumann Y, Krief G, Deutsch O, et al. (2012) Effect of Irradiation on Cell Transcriptome and Proteome of Rat Submandibular Salivary Glands. PLoS ONE 7(7): e40636. doi:10.1371/journal.pone.0040636 Editor: Sue Cotterill, St. Georges University of London, United Kingdom Received February 6, 2012; Accepted June 11, 2012; Published July 6, 2012 Copyright: 2012 Stiubea-Cohen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by the Tashtiot Foundation, Israeli Ministry of Science and by Israel Science Foundation grant no. 1083/11. No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: aharonp@ekmd.huji.ac.il Introduction Each year, 500,000 patients are diagnosed with head and neck cancer worldwide, and most of them receive irradiation (IR) treatment [1]. This mode of treatment has a pronounced effect on the salivary glands (SGs), resulting in irreversible damage to the parenchymal tissue [2]. Irradiated patients suffer from diminished ability to secrete saliva, with consequent eating and vocal disturbances, as well as frequent mucosal infections, rampant dental caries, periodontal disease and episodes of considerable pain and coughing, all of which significantly decrease their quality of life [3]. In recent years, we and others have concentrated on methods to restore normal SG function in these patients [4,5]. The notion of SG regeneration is based on the assumption that autologous SG graft cells can be isolated before initializing IR therapy, cultivated, and preserved during the IR period. Upon treatment completion, cells can then be implanted into the irradiated gland, replacing the functionally damaged ones. One pivotal point that still needs to be addressed toward this novel strategy is the detailed effect of IR on the salivary transcriptome and proteome, to facilitate our ability to monitor treatment outcome at the molecular level, and not only by measuring saliva output. Several animal models have been established to examine SG radiosensitivity, a common one being the single 15-Gy dose rat model implemented in this study. This model has been used to describe time kinetics of saliva output, limited protein composition (mainly amylase as a marker of acinar function) and tissue structure responses following IR [2,6,7,8,9,10,11,12,13,14,15]. Those studies have demonstrated that in the post-IR stages, function is damaged, as reflected by a 50% loss of salivary flow, reduction in a-amylase activity and subsequent glandular shrink- age. This has been attributed to reduced acinar cell activity, and loss of acinar cells as a result of apoptosis and acinar stem cell death [2,11,12]. Unlike other radiosensitive tissues, SG cells proliferate slowly and are highly differentiated. Thus, the effect of IR cannot be attributed solely to rate of tissue proliferation. To define the molecular mechanisms affecting SGs following IR, we studied the effect of IR on saliva output, global transcription profile of submandibular SG (SSG) tissue and whole saliva proteome before and up to 12 weeks after IR. Materials and Methods Animals Female Sprague-Dawley rats (200–225 g) were used as the animal model. All experiments were approved by the Ethical Committee on animal testing of the Hebrew University (NIH approval number: OPRR-A01-5011, Research number MD-07- 10472-4). All animals were treated according to procedures approved by the Animal Care and Use Committee at our institute PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e40636