Gene Expression Profiles of Fibroblasts Irradiated with Two Protocols in vitro

Abstract

Irradiation of exponentially growing fibroblasts induces premature differentiation to postmitotic functional cells depositing increased amounts of extracellular matrix (ECM) proteins. On the other hand, a predictive test for patients’ risk of developing fibrosis was based on a gene expression signature after fractionated irradiation of confluent cultures (Alsner et al., Radiother. Oncol. 83:261-6, 2007). The purpose was to characterize the radiation-induced gene expression profiles for the two protocols. Established skin fibroblast cultures were irradiated with 6 MV X-rays in vitro. Fibroblast differentiation was determined by cytomorphological scoring in clonal culture. Gene expression was determined using Affymetrix HgU133+ microarrays. After batch normalization, differentially expressed genes were identified by ANOVA using JMP Genomics software. The expression of selected genes was validated by real-time PCR. Pathway analysis was performed by Gene Set Enrichment Analysis using the R-package ReactomePA, filtering pathways with normalized enrichment scores (NES) >1.5 or < -1.5 and adjusted p-values <0.05. On day 5 after irradiation with 4 Gy, exponentially growing cultures showed the terminally differentiated phenotype. Microarray analysis showed >4-fold upregulation of 157 genes, including several collagen genes and >4-fold downregulation of 244 genes, including many cell cycle-related genes. Irradiation of confluent cultures with 3×4 Gy yielded 91 up- and 195 down-regulated genes (> 4-fold). Differential gene expression for the two protocols correlated better for down- than for up-regulated genes but GDF15 was strongly upregulated in both. Pathway analysis showed 37 common pathways, 51% of which were related to ECM or glycosaminoglycans (GAG), 14% to inflammation, and 11% to cholesterol and bile acid/salts. 37 pathways were more specific for 1×4 Gy (exponential cultures) with 41% related to GAG or cell-cell/cell-matrix interactions, and 16% to inflammation. 27 pathways were more specific to 3×4 Gy (confluent cultures) with 22% related to cholesterol and bile acid/salts, 15% to each of translation and metabolism, and 11% to inflammation. The expression profile of exponentially growing cells reflected premature terminal differentiation to a postmitotic phenotype. However, both protocols upregulated ECM and, notably, inflammatory pathways. Additional upregulated pathways represented GAG and cell-cell/cell-matrix interactions relating to differentiation after 1×4 Gy (exponential cultures). By contrast, upregulated cholesterol and bile acid/salts, translation, and metabolism, pathways after 3×4 Gy (confluent cultures) might indicate a role of lipid metabolism. Thus, different aspects of the fibrogenic process appear to be detected with the two protocols which may complement each other as model systems for mechanistic studies of radiation-induced fibrosis.