Mammary whole mount images (late pregnancy) from mice expressing the SV40 T-antigen (left) and wild-type litter mates (right)
Summary
Disruption of cell cycle regulation is associated with developmental abnormalities and tumorigenesis. The simian virus 40 large T antigen (Tag) interferes with cell cycle control by interacting with the pRb family and p53. Mice carrying a transgene composed of the whey acidic protein (WAP) gene promoter and the Tag coding sequence express Tag during pregnancy and are unable to nurse their young. Tag expression induced apoptosis in mammary epithelial cells during late pregnancy. At least 5% of mammary epithelial cells were undergoing apoptosis at any one time. In contrast, less than 0.2% of mammary epithelial cells in non-transgenic littermates were undergoing apoptosis. Apoptosis in Tag mice was associated with increased steady state RNA levels of bax and bcl-xL+S, with a relative increase in bcl-xS expression. Since p53 was sequestered by Tag, it is likely that p53 independent mechanisms precipitated apoptosis. The Tag expressing mammary alveolar cells which did not undergo apoptosis continued to differentiate through late pregnancy as measured by the sequential activation of milk protein gene expression. However, milk protein production, processing and secretion was impaired resulting in lactation failure.
Histological evaluation of tumor progression in WAP-TAg mice
Citations
Other ReferencesTzeng, Y.-J., Guhl, M., Graessmann, M. and Graessmann, A. (1993) Breast cancer formation in transgenic animals induced by the whey acidic protein SV40T antigen (WAP-SV-T) hybrid gene. Oncogene 8, 1965-1971. Medline Abstract
Li, M., Hu, J., Heermeier, K., Hennighausen, L. and Furth, P.A. (1996a) Expression of a viral oncoprotein during mammary gland development altyers cel fate and function: induction of p53 independent apoptosis is followed by impaired milk production in surviving cells. Cell Growth & Differentiation 7, 3-11. Medline Abstract
Li, M., Hu, J., Heermeier, K., Hennighausen, L. and Furth, P.A. (1996b) Apoptosis and remodeling of mammary gland tissue during involution proceeds through p53 independent pathways. Cell Growth & Differentiation 7, 13-20 (Medline Abstract).
Li, M., Liu, X., Robinson, G., Bar-Paled, U., Wagner, K-U, Young, W.S, Hennighausen, L. and Furth, P.A. (1997) Mammary derived signals activate programmed cell during the involuting mammary gland. Proc. Natl. Acad. Sci. U.S.A., 94, 3425-3430 (Medline Abstract). (PDF reprint)
Tzeng et al. (1998) Medline Abstract
Tzeng et al. (1996) Medline Abstract
Santarelli et al. (1996) Medline Abstract
Huesler et al. (1998) Medline Abstract
Background
The retinoblastoma protein (pRb) family and p53 have critical functions in regulating the cell cycle. In transgenic mice expression of the simian virus 40 large T antigen (Tag) and the papillomavirus E6 and E7 proteins can lead to developmental defects and cancer by disrupting cell cycle control through interactions with pRb and p53. We used mice carrying a transgene composed of the whey acidic protein (WAP) promoter and the Tag coding sequence to identify cellular pathways disrupted through the expression of this viral oncoprotein in the developing mammary gland.
Female mice carrying a WAP-Tag transgene specifically express Tag in mammary tissue beginning around day 13 of pregnancy but tumor development does not occur until after 3-5 pregnancies. These mice are unable to nurse their young starting with the first pregnancy. This suggests that expression of a viral oncoprotein leads to functional defects in mammary epithelial cells prior to malignant transformation.
Mammary phenotype
ducts
Overall ductal outgrowth during puberty and pregnancy appears to be normal.
alveoli
Alveolar development during pregnancy is impaired and the fat pad is not completely filled
(slide 2). There is some variation in alveolar outgrowth; while in some of the mice
at parturition, the alveolar structures are scarce, in others they mimic an 18 day
pregnant gland.
Epithelial cells expressing Tag differentiate through late pregnancy as measured by the sequential activation of milk protein genes, such as WAP, beta-casein, WDNM1 and alpha-lactalbumin, but milk protein production was impaired (slide 3).
The presence of milk proteins was evaluated by immunohistochemistry using antibodies directed against either mouse WAP or total mouse milk proteins. Milk proteins were abundant in both mammary epithelial cells and the alveolar lumina of control mice (slide 3). In contrast, only little milk protein was detected in either epithelial cells or alveolar lumens in Tag mice (slide 3).
Gene expression
Tag induced activation of bax
A several-fold induction of bax protein was observed in late pregnant WAP-Tag mice as
compared to non-transgenic littermates (slide 4).
Tag induced activation of bcl-x(short)
A relative increase of bcl-x(short) over bcl-x(long) occurs in WAP-Tag as comapred to
non-transgenic controls (slide 5).
Mechanistic implications
Tag induced apoptosis in mammary epithelial cells during pregnancy
Since both pRb and p53 binding domains of Tag were expressed in mammary tissue coincident
with PCD, we suggest that p53 independent apoptosis was activated by expression of Tag in
the mammary gland (model: slide 6). This is consistent with the concept that p53
independent apoptosis is important in normal mammary gland development and involution. In
support of this hypothesis, we performed a related study which demonstrated that mammary
gland involution does not require functional p53 (Abstract).
Expression of Tag during pregnancy led to impaired milk protein production and failure of lactation. Tag synthesis in mammary epithelial cells did not destroy alveolar structures but resulted in impaired milk protein production and secretion despite sufficient steady state amounts of milk protein mRNAs. These results further demonstrate that expression of a viral oncoprotein impaired cellular function prior to evidence of malignant transformation.
Milk protein production in Tag mice may be inhibited by either direct or indirect mechanisms. It does not appear that direct apoptotic destruction of the gland is responsible since alveolar structures and milk protein mRNAs are present throughout late pregnancy and parturition. In Tag mice, mammary gland involution and lobulo-alveolar collapse with reduced levels of milk protein mRNAs does not occur until after parturition and lactation failure. There are several mechanisms through which Tag could act to inhibit milk production. First, Tag synthesis might affect milk protein production by association with pRb family members and dysregulation of cell cycle control. Tag could inhibit milk protein production by interfering with the ability of mammary epithelial cells to exit from the cell cycle and undergo terminal differentiation. In normal mammary tissue, epithelial cell proliferation falls during late pregnancy coincident with the onset of milk protein synthesis. pRb or a related family member could be required for terminal differentiation and exit from the cell cycle of mammary epithelial cells. Exit from the cell cycle could be required for proper and abundant milk protein synthesis. It appears less likely that interruption of p53 function by Tag was responsible for the inability to produce milk proteins since p53 -/- mice lactate normally (Absrtact).
Other information
The BglII - Asp718 fragment spanning the mouse WAP gene promoter from -1573 to +24 was linked to the SV40 T-antigen gene.
The transgene was introduced into the NMRI background.
Keywords
T-antigen, transgenic, apoptosis, p53, WAP, lactation deficiency, milchlos, mammary development, tumors
Related Research Results local signals mediate apoptosis
Contributed By
Priscilla A. Furth
Institute of Human Virology
University of Maryland Medical School
Phone: 410-706-4606
Fax: 410-706-4619
e-mail: furth@umbi.umd.edu
More info about the Furth lab ("http://mammary.nih.gov/Groups/Furth/Furth_group.html")
last update: January 1999