Effect of irradiation on cell transcriptome and proteome of rat submandibular salivary glands

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 α-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.

Figure 1. Effect of 15-Gy head and neck irradiation on saliva secretion.

A. Saliva flow rate after irradiation: normalized, pilocarpine-induced saliva secretion from non-irradiated (control) and 4-, 8- and 12-week post-irradiation rats. The number of rats used in each experiment is indicated inside the bars. ***P<0.001 vs. control. B. H&E staining of rat submandibular gland tissue from control and irradiated rats. C. Representative 2-D electrophoretic analysis of pooled saliva before and after 15-Gy irradiation: saliva protein (45 µg) from non-treated animals, and animals 4 weeks, 8 weeks and 12 weeks post-irradiation were separated by 2-D electrophoresis. Proteins with reduced expression during the 12 weeks following IR are represented by red ellipses, those with higher expression by blue ellipses and those with erratic expression by black rectangles. D. Changes in normalized optical density ratios and MS identification of selected cluster spots after 15-Gy irradiation: identified spot proteins are shown in decreasing order of matched peptides.

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.

Figure 3. Fold change in gene expression following irradiation.

Relative quantification (RQ) values of Muc19, Cdh22, Psp, Prb1, CysS, Mgmt, cyclin d1, p21, p57 and Ywhaq gene expression during the 12 weeks post-irradiation. Validation of microarray values for salivary gland functional gene expression following irradiation by TaqMan real-time PCR. The data are expressed as means of n = 3. *P<0.05, **P<0.005, ***P<0.001 compared to control levels, unless otherwise stated.

Figure 4. Gene expression, SGIE colony forming efficiency and immunofluorescence staining during 12 weeks post-irradiation.

A. In irradiated glands, c-KIT expression decreases progressively (4W, 8W, 12W) compared to control glands. Immunofluorescence staining shows a similar pattern (bar = 50 µm). B. SGIE colony forming efficiency in control and 4, 8 and 12-week post-IR. The number of rats used in each experiment is indicated inside the bars. *Significantly different at P<0.02. C. α-amylase gene expression is increased (4W, 8W, 12W) compared to control glands. Immunofluorescence staining shows a similar pattern (bar = 50 µm). Data are expressed as means of n = 3. *P<0.05, ***P<0.001.