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First published online 2 February 2005
doi: 10.1242/dev.01655

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Developmental stage determines the effects of MYC in the mammary epithelium

Collin M. Blakely^*, Louis Sintasath^*, Celina M. D'Cruz, Kristina T. Hahn, Katherine D. Dugan, George K. Belka and Lewis A. Chodosh

Departments of Cancer Biology, Cell and Developmental Biology, Medicine, and The Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6160, USA

View larger version (26K): [in a new window]   Fig. 1. Mammary epithelial expression of Myc during pregnancy affects pup survival postpartum. (A) Oligonucleotide microarray of analysis of endogenous Myc expression during mouse mammary gland development. Total RNA (30 µg) was isolated from the mammary glands of three separate FVB wild-type mice and hybridized to Affymetrix Mu6500 GeneChips as previously described (Master et al., 2002). Raw expression data are indicated on the y-axis. Black boxes indicate detectable expression, whereas gray boxes indicate samples in which Myc expression was not detected. Gene expression changes determined to be statistically significant by the analysis described in Master et al. (Master et al., 2005) are indicated by vertical arrowheads. x indicates gene expression changes determined not to be statistically significant. Fold-changes in Myc expression, as compared with the baseline (`B') expression of 10-week-old G0P0 animals, are indicated at the top of each column. (B) Effect of MYC transgene expression during pregnancy on postpartum pup survival. MTB/TOM mice were administered 2.0 mg/ml doxycycline in their drinking water to induce MYC transgene expression during the time period of pregnancy indicated at the top. Parturition occurred after 18.5 days of gestation, and pup survival within the first 24 hours was assessed. Shaded bars indicate induction conditions for which all pups died within the first 24 hours of parturition. Non-shaded bars indicate induction conditions in which all pups survived beyond 24 hours of parturition. Hatched bars indicate induction conditions in which a fraction of the litter died within 24 hours of parturition. The effects of induction conditions on pup growth are indicated as follows: +, affected; -, not affected; N/A, not applicable because of pup death. The light-gray region from D12.5-D15.5 of pregnancy indicates the window of highest susceptibility to MYC induction.

View larger version (41K): [in a new window]   Fig. 2. MMTV-myc and MTB/TOM mice exhibit similar mammary gland phenotypes during pregnancy. Hematoxylin and Eosin-stained number 4 mammary gland sections from FVB wild-type, MMTV-myc and MTB/TOM mice at D18.5 of pregnancy. MTB/TOM mice were induced with doxycycline from D0.5-D18.5 of pregnancy. Results are representative of sections from three animals per group.

View larger version (128K): [in a new window]   Fig. 3. Mammary epithelial expression of MYC from D12.5-D15.5 of pregnancy promotes morphological changes consistent with precocious lactation. Hematoxylin and Eosin staining of number four mammary gland histological sections from MTB and MTB/TOM mice induced with doxycycline from D12.5-D15.5 of pregnancy and harvested at 1-day increments from D12.5 of pregnancy through day 1 postpartum (D1PP) (A-P). Uninduced MTB/TOM glands harvested at increasing intervals during lactation are also shown (Q-T). Results are representative of sections from three animals per group.

View larger version (58K): [in a new window]   Fig. 4. Mammary epithelial expression of MYC from D12.5-D15.5 of pregnancy promotes precocious expression of markers of lactation. (A) Northern hybridization analysis of total RNA extracted from MTB and MTB/TOM mouse mammary glands induced with doxycycline from D12.5-D15.5 of pregnancy and harvested at 1-day increments from D12.5 pregnancy through D1PP. Northern blots were hybridized with probes specific to the human MYC transgene (MYC Tg), endogenous mouse Myc, Csnd and prolactin-inducible protein (Pip). Ethidium bromide staining of 28S RNA is shown as a loading control. (B) Western blot analysis of milk protein expression. Mammary protein lysates from uninduced MTB and MTB/TOM mice at D12.5 pregnancy and MTB and MTB/TOM mice induced from D12.5-D15.5 of pregnancy were separated by SDS-PAGE and transferred to nitrocellulose. Mammary lysates from FVB wild-type virgin mice and mice at D18.5 of pregnancy are included as controls. Membranes were probed with polyclonal antiserum specific to mouse milk proteins and ß-tubulin. Expression of -casein and ß-casein are indicated.

View larger version (100K): [in a new window]   Fig. 5. MYC expression during pregnancy induces abnormal mammary epithelial proliferation. (A) BrdU immunohistochemistry of uninduced MTB and MTB/TOM mice at D12.5 of pregnancy; MTB and MTB/TOM mice induced with doxycycline from D12.5-D13.5 sacrificed at D13.5 of pregnancy; and MTB and MTB/TOM mice induced from D12.5-D15.5 and sacrificed at D16.5 of pregnancy. Mice were injected i.p. with 50 mg/kg BrdU 2 hours prior to sacrifice. Histological sections from number four mammary glands were assessed for incorporation of BrdU by IHC analysis. Results are representative of three animals per group. (B) Northern hybridization analysis of total RNA extracted from the mammary glands of MTB and MTB/TOM mice induced with doxycycline as indicated. Total RNA was separated by gel electrophoresis, transferred to nitrocellulose and hybridized with a radiolabeled probe specific to cyclin A mRNA. Ethidium bromide staining of 28S RNA is shown as a loading control.

View larger version (45K): [in a new window]   Fig. 6. Mammary epithelial expression of MYC from D12.5-D15.5 of pregnancy promotes premature Stat5 hyperactivation. MTB and MTB/TOM mice induced with doxycycline from D12.5-D15.5 were sacrificed at 24-hour intervals from D12.5 pregnancy-D1PP. Mammary protein lysates were separated by SDS-PAGE and blotted onto nitrocellulose membranes. Blots were probed with antibodies specific to Y694/Y699 phosphorylated Stat5a/b, total Stat5a/b, and ß-tubulin. Results are representative of experiments performed on two separate sets of animals.

View larger version (98K): [in a new window]   Fig. 7. Expression of MYC from D12.5-D15.5 of pregnancy promotes apoptosis of mammary epithelial cells. Terminal deoxynucleotidyltansferase-mediated dUTP nick end-labeling (TUNEL) analysis was performed using the Apoptag Peroxidase Kit on mammary histological sections from MTB/TOM (A-D) and MTB control mice (E-H) induced with doxycycline from D12.5-D15.5 of pregnancy. TUNEL analysis was also performed on mammary gland histological sections from MTB/TOM (I-L) and MTB mice (M-P) during the post-induction period from day 16.5 of pregnancy to day 1 postpartum (D1PP). Images are representative of three animals per group.

View larger version (61K): [in a new window]   Fig. 8. MYC expression promotes activation of the Stat3 and Tgfb3 involution pathways. (A) The Y705-phosphorylation status of Stat3 was determined by western blot analysis. Mammary protein lysates from MTB and MTB/TOM mice induced with doxycycline from D12.5-D15.5 of pregnancy and sacrificed at 24-hour intervals from D12.5 of pregnancy through D1PP were separated by SDS-PAGE and transferred to nitrocellulose membranes. Lysates from FVB wild-type mice harvested during virgin development, D18.5 of pregnancy and D2 of regression were included as controls. Membranes were probed with antibodies specific to Y705-phosphorylated Stat3 and total Stat3. Results are representative of experiments performed on two separate sets of animals. (B) Total RNA was extracted from mammary glands of MTB and MTB/TOM animals induced from D12.5-D15.5 of pregnancy and sacrificed at D17.5-D1PP. Expression of Tgfb3 mRNA and a direct Tgfb3 target, Serpine1, were assessed by northern hybridization analysis with radiolabeled probes.

View larger version (97K): [in a new window]   Fig. 9. MYC-induced Stat5 activation and precocious lactation are specific to MYC induction from D12.5-D15.5 of pregnancy. (A) Hematoxylin and Eosin staining of mammary histological sections from MTB and MTB/TOM mice induced with doxycycline from D6.5-D9.5 of pregnancy and sacrificed (sac) at D9.5 or D12.5 of pregnancy, as well as from mice induced from D12.5-D15.5 and sacrificed at D15.5 or D18.5 of pregnancy. Results are representative of experiments performed on three separate sets of animals. (B) Expression of the Myc transgene, targets of MYC transcriptional activation Cdk4, Fbl and Shmt1, and the lactation markers Csnd and Pip were determined by northern hybridization analysis. Total mammary RNA was prepared from MTB and MTB/TOM mice induced from D6.5-D9.5 or D12.5-D15.5 of pregnancy. (C) Western blot analysis of Stat5a/b Y694/Y699 phosphorylation. Mammary protein extracts were prepared from MTB and MTB/TOM mice induced from D6.5-D7.5, D6.5-D8.5 and D6.5-D9.5 or D12.5-D13.5, D12.5-D14.5 and D12.5-D15.5 of pregnancy. Proteins were separated by SDS-PAGE and blotted to nitrocellulose membranes. Blots were probed with antibodies specific to Y694/Y699 phosphorylated Stat5a/b, total Stat5a/b, and ß-tubulin. Results are representative of experiments performed on two separate sets of animals.

View larger version (68K): [in a new window]   Fig. 10. MYC downregulates mammary expression of Cav1 in a developmental stage-specific manner. (A) Northern hybridization analysis. Total RNA was extracted from the mammary glands of MTB and MTB/TOM mice induced with doxycycline from D6.5-D9.5 of pregnancy and sacrificed at D9.5 or D12.5 of pregnancy, as well as from MTB and MTB/TOM mice induced from D12.5-D15.5 and sacrificed at D15.5 or D18.5 of pregnancy. Total RNA was subject to gel electrophoresis and blotted to nitrocellulose membranes. Blots were probed with radiolabeled cDNA fragments specific to the human MYC transgene, as well as mouse Cav1, Cish and CK18. Ethidium bromide staining of 28S RNA is shown as a loading control. (B) Western blot analysis of Cav1 expression. Mammary protein lysates from MTB and MTB/TOM mice induced from D6.5-D8.5 or D12.5-D14.5 of pregnancy were separated by SDS-PAGE and blotted to nitrocellulose. FVB wild-type mammary lysates from virgin, D7.5, D12.5 and D18.5 of pregnancy were included as controls. Blots were probed with antibodies specific to Cav1 and ß-tubulin.

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