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Epithelial cell cycling predicts p53 responsiveness to -irradiation during post-natal mammary gland development

¹ Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA
² Department of Pathology, Baystate Medical Center, Springfield, MA, USA

View larger version (112K): [in a new window]   Fig. 1. Radiation-induced p53 protein expression varies during mammary gland development. Immunohistochemistry using CM5 anti-p53 antisera was performed on BALB/c-Trp53+/+ mammary tissue before (unirradiated, A-D) or 6 hours after (irradiated, E-H) treatment with 5 Gy of whole-body -radiation at various stages of post-natal development. Irradiated age-matched, and developmental stage-matched BALB/c-Trp53–/– mice served as controls (I-L). (Magnification 40x).

View larger version (114K): [in a new window]   Fig. 2. Radiation-induced p21/WAF1 protein expression varies during mammary gland development. Immunohistochemistry using Ab5 anti-p21/WAF1 antisera was performed on BALB/c-Trp53+/+ mammary tissue before (unirradiated, A-D) or 6 hours after (irradiated, E-H) treatment with 5 Gy of whole-body -radiation at various stages of post-natal development. Irradiated age-matched, and developmental stage-matched BALB/c-Trp53–/– mice served as controls (I-L). (Magnification 40x).

View larger version (24K): [in a new window]   Fig. 3. Radiation-induced apoptosis of mammary epithelium varies during post-natal development. The percentage of apoptotic cells in mammary epithelium and intramammary lymph nodes was measured by TUNEL assay on BALB/c-Trp53+/+ mammary tissues before (control) or 6 hours after (irradiated) treatment with 5 Gy of whole-body -radiation at various stages of post-natal development. Asterisks indicate values significantly different from unirradiated controls (P<0.05). Error bars represent s.e.m.

View larger version (65K): [in a new window]   Fig. 4. Apoptosis correlates with PCNA expression in irradiated mammary glands. (A) The percentage of cycling cells was determined immunohistochemically by calculating the percentage of PCNA-positive epithelial cells, while the percentage of apoptotic cells was determined by TUNEL assay on virgin (V), early- (P4) and mid-pregnancy (P15) BALB/c-Trp53+/+ mammary tissues before (control) or 6 hours after (irradiated) treatment with 5 Gy of whole-body -radiation. Asterisks indicate values statistically different from unirradiated controls (P<0.05). Error bars represent s.e.m. (B) Immunohistochemistry was performed using PC10 anti-PCNA antibody (i-iii) while apoptosis was measured by TUNEL staining (iv-vi) on virgin (V), early- (P4) and mid-pregnancy (P15) BALB/c-Trp53+/+ mammary tissues 6 hours after treatment with 5 Gy of whole-body -radiation. (Magnification 40x).

View larger version (87K): [in a new window]   Fig. 5. p53 activity but not ß-casein is abundant in irradiated early-pregnancy (P4) mammary glands. (A) Immunohistochemistry using CM5 anti-p53 (i,iii) and Ab5 anti-p21/WAF1 (ii,iv) antisera was performed on early-pregnancy (P4) BALB/c-Trp53+/+ mammary tissue before (unirradiated) or 6 hours after (irradiated) treatment with 5 Gy of whole-body -radiation. (Magnification 40x). (B) Northern blot analysis of duplicate total RNA samples (10 µg) from fourth inguinal mammary glands of BALB/c-Trp53+/+ mice were hybridized with a cDNA probe of ß-casein (upper panel). Lower panel shows GAPDH expression as loading control.

View larger version (83K): [in a new window]   Fig. 6. p53 and p21/WAF1 expression are induced in proliferating, -irradiated whole organ cultures. Immunohistochemistry using CM5 anti-p53 (A-C) and Ab5 anti-p21/WAF1 (D-F) antisera was performed on whole organ cultures maintained for 96 hours in unsupplemented (untreated) medium (A,D), hormone-supplemented (B,E) or growth factor-supplemented (C,F) medium and 6 hours after treatment with 5 Gy of -radiation. Hormone-supplemented medium contained estrogen (1 ng/ml) plus progesterone (1 µg/ml), while growth factor-supplemented medium contained EGF (20 ng/ml) plus TGF (20 ng/ml). (Magnification 40x.)

View larger version (18K): [in a new window]   Fig. 7. Apoptosis correlates with PCNA expression in -irradiated whole organ cultures. Mammary glands from unprimed virgin BALB/c-Trp53+/+ mice were cultured for 96 hours in unsupplemented (untreated), hormone- or growth factor-supplemented medium. Hormone-supplemented medium contained estrogen (1 ng/ml) plus progesterone (1 µg/ml), while growth factor-supplemented medium contained EGF (20 ng/ml) plus TGF (20 ng/ml). The percentage of cycling cells was determined immunohistochemically by calculating the percentage of PCNA-positive epithelial cells, while the percentage of apoptotic cells was determined by TUNEL assay on whole organ cultures 6 hours after treatment with 5 Gy of -radiation. Asterisks indicate values statistically different from irradiated controls in unsupplemented medium (P<0.05). Error bars represent s.e.m.

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