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First published online June 28, 2004
doi: 10.1242/10.1242/dev.01213

Development 131, 3469-3479 (2004)
Published by The Company of Biologists 2004
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Metastasis-associated protein 1 deregulation causes inappropriate mammary gland development and tumorigenesis

Rozita Bagheri-Yarmand1, Amjad H. Talukder1, Rui-An Wang1, Ratna K. Vadlamudi1 and Rakesh Kumar1,2,*

1 Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA
2 Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA



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  		Fig. 1. Generation of MTA1-TG mice and expression of the MTA1 transgene. (A) The MMTV-MTA1 transgene. (B) Southern blot detection of the MTA1 transgene in the tail genomic DNA of transgenic (TG) and wild-type littermates of F1 from lines 30, 31, 32 and 33. (C) Representative PCR analysis of genomic DNA from potential founder mice. Lanes marked 10 and 1 show respective copy number equivalents of control MTA1 cDNA plasmid. (D) Time course of transgene expression in line 31 MTA1-TG mice analyzed by RT-PCR followed by Southern blotting. (E) Protein lysates from mammary gland of MTA1-TG and wild-type, virgin 12-week-old mice and pregnancy day 10, were immunoprecipitated with anti-T7 antibodies and western blotted with anti-T7 antibody. MCF7-MTA1 cell lysate was used as a positive control. (F) Immunohistochemical analysis of T7-MTA1 expression using anti-T7 antibodies in the mammary gland from 12-week-old virgin. A negative control without first antibody was shown. Note that the transgene is located in the nucleus of the luminal epithelial cells.

 


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  		Fig. 2. Accelerated ductal extension, excessive side-branching and precocious lobuloalveolar development in wild-type virgin and MTA1-TG females. (A) Whole-mount preparations of wild type (a) and MTA1-TG mammary gland (b) at 6 weeks of age. (Right panel) Quantitative representation of distances from the center of the lymph node to the far end of terminal end-buds (five to seven mice per line). (B) Carmine Red-stained whole mounts of inguinal mammary glands from control (a,b) and MTA1-TG mice (d,e) at 12 weeks of age. Images in b and e are higher magnifications of a and d. Hematoxylin and Eosin-stained sections of mammary glands of 12-week-old wild-type (c) and MTA1-TG mice (f-i). Dilated ducts (f), increased budding (h), a lobuloalveolar-like structure (g) and an indistinct epithelial-stromal boundary (arrowhead, i) can be seen in the mammary glands of MTA1-TG mice. Western blot showing expression of ß-casein (C) and ß-catenin (D) in mammary glands of 12-week-old wild-type and virgin MTA1-TG mice. Cytokeratin 18 used as a control for epithelial cell content. Vinculin was used as a loading control.

 


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  		Fig. 3. Impaired pregnancy-associated morphogenesis in MTA1-TG mice. Whole-mount (A) and histological analysis by Hematoxylin and Eosin stained sections (B) of number 4 abdominal glands on days 10 and 15 of pregnancy, and day 2 of lactation in wild-type and MTA1-TG mice.

 


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  		Fig. 4. Impaired cell proliferation in ductal and alveolar epithelium of MTA1-TG mice. (A) BrdU incorporation into the nuclei of mammary epithelial cells from wild type and MTA1-TG virgin at 12 weeks of age and on day 10 of pregnancy. (B) Quantitation of BrdU incorporation in the nuclei of mammary epithelial cells at 6 and 12 weeks in virgin mice, and at day 10 and day 15 of pregnancy in wild-type and MTA1-TG mice. Five thousand cells per mouse were counted, and six mice per line were examined. The proliferative index was calculated as follows: (number of BrdU-labeled cells/total number of cells)x100. Student's t-test, P<0.01. Error bars indicate s.e.m.

 


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  		Fig. 5. Ductal growth in the mammary glands of ovariectomized MTA1-TG mice and MTA1-TG male mice. (A) Whole mounts of inguinal mammary glands stained with carmine alum from wild-type (a,b) and MTA1-TG (b,d) mice ovariectomized at 3 weeks of age and examined after 5 or 9 weeks are shown. (B) Whole mount of 6-week-old wild-type (a) and MTA1-TG (b) male littermates.

 


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  		Fig. 6. Differential regulation of progesterone receptor isoform by MTA1. (A) RT-PCR analysis of PR isoforms and K18 expression in 12-week-old virgin MTA1-TG and wild-type female mice. (B) Western blot analysis of PR isoforms in 12-week-old virgin MTA1-TG (line 31 and line 33) and wild-type female mice. (C) IHC detection of PR total and PRA isoform in mammary glands of wild-type and MTA1-TG mice. (D) Quantification of PR staining. Western blot analysis of T7-MTA1 expression in MCF-7/MTA1 (E) and HC11/MTA1 (F), and control clones using an anti-T7 monoclonal antibody. Western blot analysis of PR isoforms expression in MCF-7/MTA1 (G) and HC11/MTA1 (H).

 


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  		Fig. 7. Impaired cyclin D1 and Bcl-XL expression in MTA1-TG mice. (A) Western blot analysis of cyclin D1 and cytokeratin 18 in wild-type and MTA1-transgenic from virgin 12 weeks, pregnant 15 days and lactation day 2 mammary glands. (B) Northern blot analysis of cyclin D1 mRNA level in MTA1-MCF7 overexpressing cell line. mRNA was extracted from MCF-7/PCDNA and MCF-7/MTA1 cells. Northern blots were prepared and probed with cDNAs encoding cyclin D1 and K18 as a control to permit normalization for epithelial content of the mRNA samples. (C) Western blot of total proteins from the inguinal mammary glands at various stages of development, virgin 12 weeks, pregnancy day 10, and lactation day 2 with anti-Bcl-XL and anti-vinculin antibody on the same blot. (D) Western blot analysis of Bcl-XL and vinculin in HC11/pcDNA and HC11/MTA1-overexpressing cells.

 


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  		Fig. 8. Involution is severely affected in the mutant mammary glands. (A) Hematoxylin and Eosin staining of mammary glands from wild-type mice (a,c,g) and mammary glands from MTA1-TG mice (b,d,h). Day 1 of involution (a,b), day 3 of involution (c,d), day 21 of involution (g,h). Carmine Red-stained whole mounts of mammary glands at day 21 of involution (e,f). Hyperplastic nodules are present in MTA1-TG mice. (B) TUNEL analysis of wild-type (a,c) and MTA1-TG (b,d) involuting mammary gland 1 day (a,b) and 3 days (c,d) of involution. (C) Quantification of tunnel-positive cells. Ten 200x magnification fields of view were randomly counted. The apoptosis index was calculated as follows: (number of TUNEL positive cells/total number of cells)x100. Statistical analysis was performed using Student's t-test. Error bars represent s.e.m. (D) Western blot analysis of Bcl-XL expression in wild-type and MTA1 transgenic mice at days 1, 3 and 7 of involution (Inv). Vinculin was used as a loading control.

 


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  		Fig. 9. MTA1 transgenic mice develop mammary gland hyperplasia, hyperplastic nodules and mammary tumors. (A) Hematoxylin and Eosin staining showing ductal hyperplasia in mammary glands from 6 weeks virgin MTA1-transgenic mice. Pleomophic nuclei and mitotic figures are present (arrows). (B) MTA1-TG mammary glands after one pregnancy showed intra-luminal focal hyperplasia. Note the ductal epithelial cells contain multilayers and protrude into the lumen (arrows). (C) Founder mice line 30 with single large mammary tumor on the thoracic mammary gland. (D) Founder mice line 31 with a single large tumor on the thoracic mammary gland. (E) Whole-mount staining of number 4 right inguinal mammary glands from 18-month-old virgin MTA1 transgenic glands with several hyperplastic nodules (arrows). (F) Hematoxylin and Eosin-stained section of mammary adenocarcinoma in a 15-month-old multiparous MTA1-TG female. (G) Hematoxylin and Eosin-stained section of malignant lymphomas in a 24-month-old multiparous MTA1 transgenic mammary gland. (H) Western blot analysis of cyclin D1 and Bcl-XL in mammary tumors from MTA1 transgenic mice (T1, T2) and wild-type mouse mammary tissue (WT). The blot was reprobed with T7 antibody to show transgene expression. Vinculin was used as a loading control.

 

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© The Company of Biologists Ltd 2004