Interaction between Tissue and Cellular Stress Responses: Effect of TGF-ß Depletion on Radiation-Induced p53 Response

M.H. Barcellos-Hoff, S.A. Ravani, R.L. Henshall, K.B. Ewan, R.L. Warters*, B. Parvin
Lawrence Berkeley National Laboratory
*University of Utah

One of the most widely studied cellular responses to radiation is the activation of the transcription factor, p53, whose abundance and action dictates individual cellular fate deciscions regarding proliferation, differentiation and death. A cell's response to damage needs to be rapid. Thus it is not surprising that the activation of the p53 stress response primarily involves post-translational changes in the p53 protein.Whereas intracellular radiation-induced mediators of p53 stability have been the subject of intense study, little is known about the extracellular factors that affect the p53 response to ionizing radiation. We have postulated that transforming growth factor 1 (TGF-ß) acts as an extracellular sensor of oxidative stress that orchestrates multicellular responses to cellular damage to facilitate reestablishment of homeostasis. A number of striking similarities exist between p53 and TGF-ß: both regulate complex cellular decisions regarding fate by mediating cell proliferation and apoptosis, both are induced by a variety of damage and specifically ionizing radiation, both exist in latent forms, both exhibit redox modulation of protein activity, both are very rapidly activated (within minutes of exposure), and both are translationally and transcriptionally controlled to moderate later events.

We examined the p53 response in irradiated mouse mammary gland as a function of TGF-ß depletion. Phosphorylation of Ser18 in the amino terminus of the protein results in dissociation of p53 from the mdm2 protein, which otherwise promotes p53 proteolysis. Radiation-induced phosphorylation of Ser18 was significantly reduced when TGF-ß was depleted by administering neutralizing antibodies shortly before irradiation (Figure 1). Nuclear immunofluorescence was elevated in irradiated animals receiving non-specific control antibody (compare Figure 1 A to B). Animals that received TGF- neutralizing antibody showed a significant reduction of the intensity and frequency of nuclear immunoreactivity (compare Figure 1 B to D).

This response was replicated in TGF-ß null heterozygote mammary epithelial cells. TGF-ß null heterozygote exhibit greater than 90% reduction of TGF-ß protein due to it's autoregulation. We used imaging bioinformatics to determine the degree of ser18 phosphorylation in irradiated TGF-ß null heterozygotes compared to wildtypes. Over 2511 epithelial cells were automatically segmented (Figure 2 A) and the ser18 phospo-specific p53 immunoreactivity (Figure 2B) was measured. The response of each nucleus was characterized by a vector that represents the top 10% intensity values on that nucleus. These vectors were then accumulated for all the 2511 nuclei to construct an intensity histogram that was subsequently normalized. The normalized histogram is simply the probability density function for nuclei responses. The difference between between sham and irradiated (5Gy, 1hr) is shown in Figure 2C. The difference sham and irradiated in wild type versus heterozygotes is shown in Figure 2D. The change in intensity distribution between wild type sham and irradiated is almost twice as much as in the heterozygote mammary gland.

Future studies will define the spatial and temporal patterns of p53 response as a function of HZE ion irradiation in cell culture and tissues using antibodies to discriminate between protein abundance and phosphorylation modifications.

Figure 2 A: Representative confocal image segmentation of stromal (red) and lumen (yellow) nuclei. B: Corresponding immunoreactivity of ser18 phospho-specific p53. C: Measurement of peak pixels in sham (red) vs irradiated (blue) wildtype epithelial cells. D: Calculated differential between wildtype (blue) and heterozygote (red) epithelial ser18 phospho-specific p53 immunoreactivity.

Supported by NASA Biomedical Research and Countermeasures Program.