Expression of a Viral Oncoprotein during Mammary Gland Development Alters Cell Fate and Function: Induction of p53-independent Apoptosis Is Followed by Impaired Milk Protein Production in Surviving Cells1

The disruption of cell cycle regulation is associated with developmental abnormalities and tumorigenesis. The SV4O 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 nontransgenic littermates was 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 bcI-x expression. Since p53 was sequestered by Tag, it is likely that p53-independent mechanisms precipitated apoptosis. The Tag-expressing mammary alveolar cells that 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. Received 6/19/95; revised 10/3/95; accepted 10/5/95. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 1 8 U.S.C. Section 1 734 solely to mdicate this fact. I This work was supported in part by a research contract from Galagen (Arden Hills, MN; to P. A. F.) and Grant 01-067482532 from the Veterans Administration Research Advisory Group (to P. A. F.). 2 To whom requests for reprints should be addressed, at Department of Medicine, Room 5D-136, Baltimore VA Medical Center, University of Maryland Medical School, 10 North Greene Street, Baltimore, MD 21201. Phone: (410) 605-7181; Fax: (410) 605-7914; E-mail: [email protected]. Introduction The pRb3 family and p53 have critical functions in regulating the cell cycle. In transgenic mice, expression of the SV4O large 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 (1-4). We used mice carrying a transgene composed of the WAP promoter and the Tag coding sequence (5) to identify cellular pathways disrupted through the expression of this viral oncoprotein in the developing mammary gland. Mammary gland development consists of well characterized steps that culminate in lactation (6-9). Developmental abnormalities at specific stages can be recognized through the examination of mammary gland structure and the evaluation of cellular differentiation. The function of the gland can be assessed by the presence or absence of a successful lactation. In virgin mice, the mammary parenchyma is composed of an organized system of ducts within the mammary fat pad. The major sites of growth are at the terminal end buds. With each estrous cycle, the lateral end buds differentiate and subdivide progressively. Extensive development mediated by lactogenic hormones begins with pregnancy and is completed at the onset of lactation. A rapid increase in the number and size of alveoli occurring during the second half of pregnancy results in development of fully differentiated and functional secretory lobules. Differentiation of mammary epithelial cells and formation of alveolar structures during pregnancy leads to the sequential expression of milk protein genes (7), followed by milk protein secretion and lactation. Lactation is maintained as long as the dams are suckled. After weaning, the mammaty gland involutes, and the entire lobulo-alveolar compartment collapses through PCD. This results in a ductal network resembling that of a mature virgin. The mammary gland can serve as a paradigm to investigate how loss of cell cycle regulation, mediated by a viral oncoprotein, affects both organ development and function. Female mice carrying a WAP-Tag transgene specifically express Tag in mammary tissue beginning around day 13 of pregnancy (5), but tumor development does not occur until after three to five 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. 3 The abbreviations used are: pRb, retinoblastoma protein; Tag, SV4O large T antigen; WAP, whey acidic protein; PCD, programmed cell death; nt, nucleotide; TGF 31 , transforming growth factor 31 ; MDGI, mammaryderived growth inhibitor; RT-PCR, reverse transcriptase PCR. 4 5V40 Tag Alters Mammary Cell Fate and Function Fig. 1. Mammary glands from Tag mice exhibit decreased alveolar density as compared to control mice. Whole-mount analysis of mammary glands from Tag (A and C) and control (B and D) mice. The inguinal glands of day 16 pregnant mice were mounted on a glass slide, fixed, and stained. Similar results were seen in glands from late pregnancy and on the day of parturition. A and B show the entire gland; C and D show the same glands at increased magnification. LN, lymph node. In this study, we demonstrate that the presence of Tag in differentiating mammary epithelial cells altered both cell fate and function. Tag synthesis induced p53-independent apoptosis in mammary epithelial cells during late pregnancy. This was probably mediated by changes in bax and bcl-xL#{247}S expression. However, only a subset of Tag-expressing cells underwent apoptosis, and alveolar structures were not destroyed. Tag-expressing cells that did not undergo apoptosis continued to differentiate throughout late pregnancy and expressed major milk protein RNAs. However, milk protein production and secretion were impaired in these cells, resuiting in lactation failure. Results Tag Mice Cannot Nurse Their Offspring. Tag was detected in alveolar cells at approximately day 1 3 of the first pregnancy. Although nontransgenic females nursed all of their litters, Tag mice were unable to feed their offspring, beginning with the first litter. Pups delivered to transgenic females were healthy initially and attempted to suckle. However, no milk was observed in their stomachs, and 100% of the offspring died within 48 h. In cross-fostering experiments, 76% of offspring born to Tag mice could be rescued by fostering them to a nontransgenic female. Conversely, 1 00% of offspring born to nontransgenic mice died after being fostered to Tag mice (data not shown). To investigate why Tag mice cannot lactate at the time of delivery, studies were focused on the developmental state of the gland just prior to delivery. In normal mice, milk production and secretion into the alveoli begins a few days prior to delivery. Gestation in the Tag mice and controls was 20 days. Mice were evaluated during the first pregnancy to identify events that occur shortly after Tag expression. Tag-induced, p53-independent Apoptosis Was Associated with Increased Steady-State Levels of bax and bcl-xL+s RNA. Whole-mount analyses of mammary tissue from Tag mice revealed decreased alveolar density, starting at day 16 of pregnancy through parturition (Fig. 1). Histologcal analyses demonstrated that the average alveolar circumference of Tag mice was smaller than that of control mice (Fig. 2). In situ detection of apoptotic cells demonstrated that approximately 5% of mammary epithelial cells were actively undergoing apoptosis in Tag mice at day 18 to day 19 of pregnancy (Table 1 ; Fig. 2, G and H). In contrast, less than 0.2% of cells underwent apoptosis in mammary glands of nontransgenic pregnant mice. Tag expression was detected in the majority of mammary epithelial cells (Fig. 2, D and E). Immunohistochemical staining localized p53 to the nucleus of epithelial cells of transgenic mice but was undetected in nontransgenic mice (data not shown; Ref. 5). These results suggest that the apoptosis induced by Tag expression during pregnancy was mediated by p53-independent mechanisms. To identify which apoptosis pathway genes were transcriptionally activated following Tag expression, we performed Northern blot analyses of steady-state levels of bax, bcl-xL±s, p53, and bcl-2 on RNA extracted from mammary glands of late pregnant mice. Steady-state levels of bax (Fig. 3C) and bcl-xL#{247}s (Fig. 3A) RNA were increased approximately 5and 2-fold, respectively, as compared to nontransgenic controls. To distinguish between bcl-xL and bcl-x RNA, a RT-PCR analysis was performed with primers which simultaneously amplified both forms, followed by a hybridization with oligonucleotides that distinguished between the two forms. Both bcl-xL and bcl-x RNA levels were increased in Tag transgenic mice, but a relatively greater increase in bcl-x RNA levels was observed (Fig. 3B). Bcl-xL RNA reFig. 2. Tag-induced apoptosis in mammary epithelial cells during pregnancy. Histological analysis of mammary tissue from day 18 pregnant Tag (A, B, D, E, G, and H) and control (C, F, and I) mice. Tissue was sectioned and stained with H&E (A, B, and C). Fat pad, alveoli, lumen, and secretions are indicated. A: arrow, an apoptotic cell. lmmunohistochemical analysis for the presence of Tag was performed (0, E, and F). D, solid arrow, a Tag-expressing cell. Note: the majority of nuclei in the Tag mice are positive for Tag. In situ detection of apoptosis was performed (G, H, and I). G: arrows, apoptotic cells. Cells undergoing apoptosis appear brown. Cell Growth & Differentiation 5 Table 1 Percentage of cells undergoing apoptosis in sections of mammary gland tissue from control (C) and Tag (1) mice Each mouse no. represents ma mmary tissue taken from a different mouse. A poptotic cells we re identified usin g the Apotag kit (0 ncor, Gaithersburg, MD).