Distinct signaling pathways after higher or lower doses of radiation in three closely related human lymphoblast cell lines

Abstract

Purpose: The tumor suppressor p53 plays an essential role in cellular responses to DNA damage caused by ionizing radiation; therefore, this study aims to further explore the role that p53 plays at different doses of radiation.

Materials and methods: The global cellular responses to higher-dose (10 Gy) and lower dose (iso-survival dose, i.e., the respective D0 levels) radiation were analyzed using microarrays in three human lymphoblast cell lines with different p53 status: TK6 (wild-type p53), NH32 (p53-null), and WTK1 (mutant p53). Total RNAs were extracted from cells harvested at 0, 1, 3, 6, 9, and 24 h after higher and lower dose radiation exposures. Template-based clustering, hierarchical clustering, and principle component analysis were applied to examine the transcriptional profiles.

Results: Differential expression profiles between 10 Gy and iso-survival radiation in cells with different p53 status were observed. Moreover, distinct gene expression patterns were exhibited among these three cells after 10 Gy radiation treatment, but similar transcriptional responses were observed in TK6 and NH32 cells treated with iso-survival radiation.

Conclusions: After 10 Gy radiation exposure, the p53 signaling pathway played an important role in TK6, whereas the NFkB signaling pathway appeared to replace the role of p53 in WTK1. In contrast, after iso-survival radiation treatment, E2F4 seemed to play a dominant role independent of p53 status. This study dissected the impacts of p53, NFkB and E2F4 in response to higher or lower doses of gamma-irradiation.

Fig. 1.

(A) Hierarchical clustering of RNA expression profiles for TK6, NH32, and WTK1 cells after 10 Gy and iso-survival doses of radiation. For each cell line, expression levels relative to time 0 are shown at 0, 1, 3, 6, 9, and 24 h after both 10 Gy and iso-survival radiation exposures. The Venn diagrams for each cell line are shown on the right side. (B) Principal component analysis of the genes with twofold changes for each cell line. Red spots indicate 10 Gy treatment; green spots indicate iso-survival treatments. (C) Venn diagrams of genes in response to 10 Gy and iso-survival radiation for each cell line.

Fig. 2.

Downregulation of E2F4-related cell-cycle genes in TK6, WTK1, and NH32 after iso-survival treatments. (A) Hierarchical clustering of 36 E2F4-related cell-cycle genes. (B) Gene network of the E2F4-related cell-cycle genes was revealed by using ingenuity pathway analysis. The solid lines indicate direct evidence of an interaction between the two genes according to published literature reports, whereas the dash lines indicate indirect evidence. Green areas denote genes that were downregulated at 24 h after iso-survival irradiation in TK6. Dose-response curves of MAD2L1, PTTG1, and STMN1 in TK6 (C) and NH32 (D) using quantitative reverse transcription polymerase chain reaction (RT-PCR). Cells were harvested at 24 h after radiation. The expression values were first normalized to ACTB and presented as the relative ratio to control (0 Gy). The data represent an average of three independent quantitative RT-PCR reactions.

Fig. 3.

Activation of p53- and NFkB-signaling pathways in TK6 and WTK1, respectively, after 10 Gy radiation treatment. (A) Hierarchical clustering (left) and p53-centered gene network (right) at 24 h after irradiation in TK6 of the 25 cell death-related genes. (B) Hierarchical clustering (left) and NFkB-centered gene network (right) at 9 h (maximum response) after irradiation in WTK1 of the eight survival-related genes. Gray (white) areas denote nonresponsive genes. Other details of the gene network are the same as in Fig. 2b.

Fig. 4.

The proposed model of differential transcriptional responses to higher and lower doses of ionizing radiation. Solid boxes represent cellular responses. The box with a dotted line indicates evidence inferred from the literature. Question marks denote yet-to-be established connections.