Proteome analysis of proliferative response of bystander cells adjacent to cells exposed to ionizing radiation - PubMed (original) (raw)

Proteome analysis of proliferative response of bystander cells adjacent to cells exposed to ionizing radiation

Bogdan I Gerashchenko et al. Proteomics. 2007 Jun.

Abstract

Recently (Cytometry 2003, 56A, 71-80), we reported that direct cell-to-cell contact is required for stimulating proliferation of bystander rat liver cells (WB-F344) cocultured with irradiated cells, and neither functional gap junction intercellular communication nor long-range extracellular factors appear to be involved in this proliferative bystander response (PBR). The molecular basis for this response is unknown. Confluent monolayers of WB-F344 cells were exposed to 5-Gray (Gy) of gamma-rays. Irradiated cells were mixed with unirradiated cells and co-cultured for 24 h. Cells were harvested and protein expression was examined using 2-DE. Protein expression was also determined in cultures of unirradiated and 5-Gy irradiated cells. Proteins were identified by MS. Nucleophosmin (NPM)-1, a multifunctional nucleolar protein, was more highly expressed in bystander cells than in either unirradiated or 5-Gy irradiated cells. Enolase-alpha, a glycolytic enzyme, was present in acidic and basic variants in unirradiated cells. In bystander and 5-Gy irradiated cells, the basic variant was weakly expressed, whereas the acidic variant was overwhelmingly present. These data indicate that the presence of irradiated cells can affect NPM-1 and enolase-alpha in adjacent bystander cells. These proteins appear to participate in molecular events related to the PBR and suggest that this response may involve cellular defense, proliferation, and metabolism.

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Figures

Figure 1

Figure 1

Representative patterns of protein expression after 2-DE separation of protein samples obtained from a 24-h culture of: (A) unirradiated cells (95% of the total cell population) with irradiated cells (5% of the total cell population), (B) unirradiated (100% population), and (C) 5-Gy irradiated cells (100% population). The horizontal axis represents the range of p_I_ values of proteins between 3 and 10. The vertical axis represents the molecular weigh of proteins in kDa. The arrows indicate spots, the proteins of which were identified by MS/MS (the names of identified proteins are presented in Table 1). The decision to select these protein spots for MS/MS analysis was based on intersample comparison of 2-D gel images obtained from three independent experiments. The regions enclosed within rectangles are shown enlarged in (D).

Figure 2

Figure 2

Results of densitometric analysis of changes in expression of protein spot 1 (NPM 1) shown in Fig. 1. For intergel comparisons, the spot volume was normalized to give a fraction value of the total spot volume per gel. Data presented are the mean ± SEM of three independent experiments.

Figure 3

Figure 3

Western blot analysis of changes in NPM 1 content of protein samples obtained from 24-h cultures of unirradiated cells (100% population), 5-Gy irradiated cells (100% population), and unirradiated cells (95% of the total cell population) with irradiated cells (5% of the total cell population). Two of three independent experiments (marked as Exp. #2 and Exp. #3) showed similar results.

Figure 4

Figure 4

Results of densitometric analysis of changes in expression of NPM 1 shown in Fig. 3. These results are expressed as a “fold increase” of densitometric values of samples 0 Gy (95%) 5 Gy (5%) and 5 Gy (100%) compared to the densitometric value of sample 0 Gy (100%) considered as a control sample. Data presented are the mean ± SEM of three independent experiments. The photographic films were analyzed by scanning densitometry with ImageJ software.

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