Article Figures & Data
Figures
- Fig. 1.
Irradiated TGF-β1 +/− mammary gland shows reduced levels of active TGF-β1 and Smad 2/3. TGF-β1 +/− and +/+ mice were irradiated whole body to a dose of 5 Gy and killed 1 h later. A, false color digital micrographs of dual immunofluorescence of antigen-purified TGF-β1 antibody (red) and LAP antibody (green) visualized simultaneously with DAPI-stained nuclei (blue). Comparison of mammary gland tissue from irradiated TGF-β1 +/+ and +/− mice indicates that TGF-β1 immunoreactivity (yellow orange) is greater in TGF-β1 +/+ mice, whereas TGF-β1+/− mice show predominant LAP immunoreactivity (green). The prominent localization of TGF-β1 in the irradiated wild-type mice reflects radiation-induced activation (5) . Note that all cells stain with antibodies to LAP before radiation exposure (7) . B, false color digital micrographs of Smad 2/3 antibody (green) localized simultaneously with DAPI-stained nuclei (blue). Comparison of mammary gland cryosections from sham (a and c) or irradiated (b and d) TGF-β1+/+ (a and b) and +/− (c and d) mice indicates that IR induced significant Smad2/3 immunoreactivity. Immunofluorescence intensity was markedly reduced in irradiated TGF-β1 +/− mice.
- Fig. 2.
TGF-β1 gene dosage correlates with reduced apoptosis and cell cycle block in response to radiation. A, the frequency of apoptotic nuclei detected using terminal deoxynucleotidyltransferase-mediated nick end labeling reaction was determined in the mammary epithelium of TGF-β1 +/− and +/+ mice (means; bars, SE; n = 3 animals). Sham-irradiated (▪) and whole-body irradiated (
) wild-type mice were significantly different (t test; P = 0.02). The irradiated TGF-β1 heterozygote mice were not significantly different from sham-irradiated heterozygote mice but were significantly different from irradiated wild-type mice (t test; P = 0.006). Pregnant NIH/OlaHsd TGF-β1 +/− dams were irradiated whole body (5 Gy) on day 12.5 of gestation. Embryos irradiated in utero were collected 6 h after irradiation. Apoptotic nuclei were detected using the terminal deoxynucleotidyltransferase-mediated nick end labeling reaction in liver (B) and epidermis (C) from TGF-β1 +/+, +/−, and −/− embryos. Apoptosis was decreased in control TGF-β1 +/− and −/− embryo tissues. Significantly increased apoptosis was absent from both liver and epidermis of irradiated TGF-β1 +/− and −/− embryos. The frequency of cycling cells was detected using PCNA antibodies in sham-irradiated (▪) and irradiated (
) embryos. Radiation-induced cell cycle block was evidenced by a 2–3-fold reduction of PCNA-positive cells after irradiation in utero in the liver (D) and epidermis (E) from TGF-β +/+ and +/− embryos. The frequency of PCNA-positive cells was not significantly different between sham and IR embryos of −/− genotype, indicating abrogation of radiation-induced cell cycle block. - Fig. 3.
p53 Ser-18P is induced in irradiated mammary epithelial cells. A, antibodies to Ser-18P p53 were used in Western blotting of total tissue protein extracts of irradiated BALB/c mammary tissue. No signal was evident in sham-irradiated tissue. A single band was detected at 1 h and was present up to 24 h after radiation exposure by Western blotting. B, false color images of immunofluorescence localization of p53 Ser-18 phosphorylation detected using secondary antibodies labeled with Alexa 488 (appears green/turquoise). Nuclei were counterstained with DAPI (blue). Immunofluorescence was absent from controls in which the primary antibody was deleted (a) and discernable in only a few epithelial cells in sham-irradiated tissue (b). Prominent nuclear immunoreactivity was evident throughout the epithelium from 1 h (c), 4 h (d), 15 h (e), and 24 h (g) after radiation exposure.
- Fig. 4.
Decreased radiation-induced p53 Ser-18 P after irradiation and chronic or transient TGF-β1 depletion. A, Western blot of tissue extracts from wild-type (wt) or Tgfβ1 heterozygote (ht) mice sham and 1–6 h after IR. p53 Ser-18 phosphorylation was significantly reduced in Tgfβ1 +/− mice 1–6 h after IR. B, p53 Ser-18P was localized as indicated in Fig. 3 <$REFLINK> using cryosections of C57BL/6/129Sv TGF-β1 +/+ mice (a–c) or TGF-β1 +/− mice (d–f) subjected to sham exposure (a and d) or irradiated with 5 Gy, 1 h (b and e) or 6 h (c and f) before sacrifice. Nuclear localization of p53 Ser-18P 1 h after IR was significantly reduced in TGF-β1 +/− animals compared with wild-type animals. By 6 h, p53 Ser-18P was decreased in both genotypes. C, Western blot of tissue extracts from BALB/c adult female mice injected i.p. with an irrelevant IgG antibody as a control (C) or TGF-β1 neutralizing (N) antibody before irradiation. p53 Ser-18P was significantly reduced 1 h after IR when TGF-β1 neutralizing antibodies were administered before irradiation. D, nuclear immunolocalization of p53 Ser-18P was significantly reduced in animals treated with TGF-β neutralizing antibody. Mice received control (a and b) or TGF-β pan-isoform neutralizing monoclonal antibody (c and d) 3 h before sham exposure (a and c) or whole-body irradiation with 5 Gy (b and d). p53 Ser-18P was localized in cryosections as indicated in Fig. 3 <$REFLINK> .
Volume 62, Issue 20
