Elucidation of changes in molecular signalling leading to increased cellular transformation in oncogenically progressed human bronchial epithelial cells exposed to radiations of increasing LET

Abstract

The early transcriptional response and subsequent induction of anchorage-independent growth after exposure to particles of high Z and energy (HZE) as well as γ-rays were examined in human bronchial epithelial cells (HBEC3KT) immortalised without viral oncogenes and an isogenic variant cell line whose p53 expression was suppressed but that expressed an active mutant K-RAS(V12) (HBEC3KT-P53KRAS). Cell survival following irradiation showed that HBEC3KT-P53KRAS cells were more radioresistant than HBEC3KT cells irrespective of the radiation species. In addition, radiation enhanced the ability of the surviving HBEC3KT-P53RAS cells but not the surviving HBEC3KT cells to grow in anchorage-independent fashion (soft agar colony formation). HZE particle irradiation was far more efficient than γ-rays at rendering HBEC3KT-P53RAS cells permissive for soft agar growth. Gene expression profiles after radiation showed that the molecular response to radiation for HBEC3KT-P53RAS, similar to that for HBEC3KT cells, varies with radiation quality. Several pathways associated with anchorage independent growth, including the HIF-1α, mTOR, IGF-1, RhoA and ERK/MAPK pathways, were over-represented in the irradiated HBEC3KT-P53RAS cells compared to parental HBEC3KT cells. These results suggest that oncogenically progressed human lung epithelial cells are at greater risk for cellular transformation and carcinogenic risk after ionising radiation, but particularly so after HZE radiations. These results have implication for: (i) terrestrial radiation and suggests the possibility of enhanced carcinogenic risk from diagnostic CT screens used for early lung cancer detection; (ii) enhanced carcinogenic risk from heavy particles used in radiotherapy; and (iii) for space radiation, raising the possibility that astronauts harbouring epithelial regions of dysplasia or hyperplasia within the lung that contain oncogenic changes, may have a greater risk for lung cancers based upon their exposure to heavy particles present in the deep space environment.

Figure 1.

Clonogenic survival and anchorage-independent growth of HBEC3KT and HBEC3KT-P53KRAS cells that were irradiated by ⁵⁶Fe, ²⁸Si or γ-rays. (A) Survival curves were plotted using a two-component survival fit. Error bars: standard error. For all radiation types, HBEC3KT-P53KRAS cells were more radioresistant than parental HBEC3KT cells. (B) Plot of transformation frequencies that compares colonies grown in soft agar between irradiated and sham-treated HBEC3KT and HBEC3KT-P53KRAS cells, indicated enhanced anchorage independent growth of bronchial epithelial cells with p53 knock-down and mutant K-RAS after γ-ray and HZE radiations. (C) transformation frequencies corrected with survival fractions after radiation. Error bar: standard error. Lines indicate P < 0.05 between groups.

Figure 2.

Comparison of gene expression profiles between HBEC3KT and HBEC-P53KRAS cells. (A) Principal component analysis of whole transcriptome profiles showed no clear segregation of the two cell lines. (B) PCA of 382 differentially expressed genes from SAM analysis separate two cell populations. (C) Gene functional categories that significantly changed in HBEC3KT-P53KRAS cells. (D) Signalling pathways that significantly changed in HBEC3KT-P53KRAS cells.

Figure 3.

Altered p53, KRAS and HIF-1α signalling pathways in HBEC-P53KRAS cells. (A) Upstream analysis based on gene expression profiles indicated activation of KRAS and suppression of TP53. The function of AKT, NFκB, JUN and MAP2K1 was also activated and was predicated based on the literature and consistent to the result of activated KRAS and suppressed p53. The predicted activation of p53 by KRAS was inconsistent to the finding that p53 was suppressed. (B) Scheme demonstrating the activation of HIF-1α pathway. Red: down-regulation. Green: up-regulation. (C) Western blot showed higher protein level of HIF-1α in HBEC3KT-P53KRAS. (D), Gene expression of HIF-1α showed no significant changes of transcripts between HBEC3KT and HBEC3KT-P53KRAS cells from microarray data. (E) Comparison of gene expression indicating up-regulation of HIF-1α-activating genes (top row) in HBEC3KT-P53KRAS and down-regulation of HIF-1α-degrading genes (bottom row). The boxplot shows median value, 75 percentile and the whisker represents 90 percentile. Dots indicate outliers.

Figure 4.

Gene expression profiling of HBEC3KT-P53KRAS cells that were irradiated by ⁵⁶Fe, ²⁸Si or γ-rays. (A) Unsupervised hierarchical clustering indicated distinct response of cells to different radiation qualities. (B) Temporal patterns of genes associated with p53-signalling were consistent and up-regulated in HBEC3KT cell lines after radiation by different radiation types. The up-regulations were weakened with smaller fold changes or delayed peaks and were radiation type dependent in HBEC3KT-P53KRAS cells.

Fig. 5.

Signaling pathways associated with anchorage independent growth increased significantly in response to all radiation types in HBEC3KT-P53KRAS cells when compared with the syngeneic HBEC3KT cells. The ratio represents the significance of each pathways that changed in response to radiations.