Somatic cell cloned transgenic bovine neurons for transplantation in parkinsonian rats.

Parkinson's disease symptoms can be improved by transplanting fetal dopamine cells into the putamen of parkinsonian patients. Because the supply of human donor tissue is limited and variable, an alternative and genetically modifiable non-human source of tissue would be valuable. We have generated cloned transgenic bovine embryos, 42% of which developed beyond 40 days. Dopamine cells collected from the ventral mesencephalon of the cloned fetuses 42 to 50 days post-conception survived transplantation into immunosuppressed parkinsonian rats and cells from cloned and wild-type embryos improved motor performance. Somatic cell cloning can efficiently produce transgenic animal tissue for treating parkinsonism.

An ubiquitously expressed gene 3.5 kilobases upstream of the glycerol-3-phosphate dehydrogenase gene in mice.

While studying the organization of the mouse glycerol-phosphate dehydrogenase gene (Gdc-1 on chromosome 15), we identified a novel transcriptional unit located only 3.4 kilobases (kb) upstream of the 5' end of the Gdc-1 gene. This gene has been provisionally named D15Kz1. The unusual proximity of these two genes led us to investigate the pattern of expression and sequence characteristics of the new gene for comparison with those of Gdc-1. D15Kz1 was found to have transcripts of 3.2 and 3.4 kb in length. The 3.4-kb transcript was expressed at low levels in all tissues examined, whereas the 3.2-kb transcript was detected only in the cerebral cortex and the brown fat. D15Kz1 and Gdc-1 are not coordinately regulated, as evidenced by the characteristics of their expression in several tissues and in differentiating 3T3-F442A adipocyte cultures. A cDNA sequence of 3,105 bases isolated from an embryonal carcinoma lambda gt10 cDNA library had a large open reading frame of 461 amino acids at one end followed by 1.6 kb of sequence with multiple stop codons. Algorithms used to search the protein and nucleic acid data bases detected no significant sequence similarity to any other protein or gene. Southern blot analysis of genomic DNA using the D15Kz1 cDNA as a probe indicated that D15Kz1 is a single-copy gene in the mouse genome and that it is conserved in humans, rats, and chickens. This conservation of gene sequences suggests that D15Kz1 encodes a protein with an important cellular function.

Production of calves from G1 fibroblasts.

Since the landmark study of Wilmut et al. describing the birth of a cloned lamb derived from a somatic cell nucleus, there has been debate about the donor nucleus cell cycle stage required for somatic cell nuclear transfer (NT). Wilmut et al. suggested that induction of quiescence by serum starvation was critical in allowing donor somatic cells to support development of cloned embryos. In a subsequent report, Cibelli et al. proposed that G0 was unnecessary and that calves could be produced from actively dividing fibroblasts. Neither study conclusively documented the importance of donor cell cycle stage for development to term. Other laboratories have had success with NT in several species, and most have used a serum starvation treatment. Here we evaluate methods for producing G0 and G1 cell populations and compare development following NT. High confluence was more effective than serum starvation for arresting cells in G0. Pure G1 cell populations could be obtained using a "shake-off" procedure. No differences in in vitro development were observed between cells derived from the high-confluence treatment and from the "shake-off" treatment. However, when embryos from each treatment were transferred to 50 recipients, five calves were obtained from embryos derived from "shake-off" cells, whereas no embryos from confluent cells survived beyond 180 days of gestation. These results indicate that donor cell cycle stage is important for NT, particularly during late fetal development, and that actively dividing G1 cells support higher development rates than cells in G0.

Cytoplasmic sequestration and functional repression of p53 in the mammary epithelium is reversed by hormonal treatment.

Proper function of the p53 tumor suppressor gene is critical for inhibiting tumor development in a broad spectrum of tissues. Although the mammary gland is highly susceptible to tumor formation, the functional status of p53 in the normal tissue had not been investigated. Therefore, expression, localization, and activity of p53 were examined in normal mammary tissues. High levels of p53 protein were found expressed in the cytoplasm of the ductal epithelium of the quiescent mammary gland. Ionizing radiation failed to recruit p53 to the nucleus, and p53-dependent responses were minimal. However, transient hormonal stimulation resulted in nuclear accumulation of p53, an induction of p21/WAF1, and a 5-fold increase in apoptosis after ionizing radiation. Therefore, the functional state of wild-type p53 in the mammary epithelium can be regulated by hormonal stimuli.

Mutations in p53 are frequent in the preneoplastic stage of mouse mammary tumor development.

Preneoplastic lesions in the mammary gland represent a population of cells at increased risk of progression to tumors. Because p53 is the most commonly mutated gene in human breast cancer, we sought to determine whether mutations in p53 were present in preneoplastic lesions or were acquired during progression to overt tumors. In the mouse mammary gland, hyperplastic alveolar nodules (HAN) are the most common preneoplastic lesion. Analysis of the TM series of transplantable murine HAN outgrowths and tumors allowed the status of p53 to be determined at distinct stages of mammary tumorigenesis. Alterations in the p53 gene or in the pattern of p53 protein expression were observed in all five HAN outgrowth lines examined. Altered expression of p53 protein was detected in 3 of 5 TM HAN outgrowth lines as determined by immunohistochemistry. Overexpression of nuclear p53 was detected in only a fraction of the cells (10-50%) in TM3 and TM4 HAN outgrowths, whereas in tumors that arose from TM4 HAN outgrowths, the proportion of cells overexpressing p53 protein approached approximately 100%. Despite overexpression of p53 in TM3 HAN outgrowths, no tumors have developed in this line. The TM9 outgrowth line exhibited a different pattern of p53 expression by immunohistochemistry: p53 protein was overexpressed in the cytoplasm of virtually all cells in the HAN outgrowths but was localized to the nuclei of TM9 tumor cells. Direct sequencing of p53 transcripts from tumors and cell lines revealed various genetic changes: point mutations in exons 4 and 5 (TM2H, nonsense; TM4, missense); a deletion in exon 5 (TM4); and an insertion in exon 7 (TM3). Although p53 protein was overexpressed in TM9 tumors, it was shown to be wild-type both by immunoprecipitation and direct sequencing of the entire coding region of the cDNA. TM4 cells were homogeneous with respect to mutant p53 genotype and uniformly expressed p53 by immunohistochemical staining in vitro, but transplantation of TM4 cells to fat pads of BALB/c hosts resulted in HAN outgrowths in situ in which < 50% of the cells expressed the mutant p53 at detectable levels. In summary, mutation of the p53 gene and overexpression of p53 protein can occur in preneoplastic mammary epithelial cells, and those mutations are maintained in tumors that arise from the HAN. Conversely, expression of mutant p53 was decreased when cells were grown in situ, implicating the presence of cellular factors that can suppress p53 expression in vivo. These observations demonstrate that the p53 pathway may be a common target for mutation in murine mammary tumorigenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

p53 mutations selected in vivo when mouse mammary epithelial cells form hyperplastic outgrowths are not necessary for establishment of mammary cell lines in vitro.

Breast cancer is a consequence of multiple alterations occurring over a long period of time. Genetic changes in early stages of tumorigenesis have not been defined. A recently developed murine system permits the study of mammary preneoplastic cells in vivo and in vitro (F. S. Kittrell et al., Cancer Res., 52: 1924-1932, 1992). To assess the potential role of p53 mutations in early stages of breast cancer, the status of p53 was determined in a series of mouse mammary epithelial cell lines which give rise to preneoplastic outgrowths (hyperplastic alveolar nodules) when transplanted into cleared mammary fat pads of syngeneic mice. Protein stability and conformation were analyzed using immunoprecipitations and immunochemical assays; p53 transcripts were sequenced using a polymerase chain reaction approach. The parental cell lines (FSK lines) showed no evidence of p53 alterations at either the protein or the nucleic acid level, indicating that p53 mutations are not essential for the establishment of mammary epithelial cell lines in vitro. In contrast, cell lines (TM lines) derived from hyperplastic alveolar nodule outgrowths induced by FSK cells expressed only mutant p53 genes. The mutation in one outgrowth cell line (TM-2H) resulted in the loss of p53 protein synthesis, whereas two other outgrowth lines (TM-3, TM-4) overexpressed mutant p53 protein. Mutation of p53 appears to correlate with preneoplastic growth in vivo. Although it is not clear if the mutations occur before or after transplantation of cells in vivo, there appears to be a pronounced growth advantage in the mammary gland for cells expressing mutant p53.

Delayed involution of the mammary epithelium in BALB/c-p53null mice.

In mammals, weaning of neonates and subsequent milk stasis initiates removal of the secretory epithelium of the mammary gland by apoptosis. The p53 tumor suppressor gene is induced rapidly following weaning of neonates, but its role in the process of involution has not been defined. Therefore, experiments were performed to identify the cell types in which the p53 gene is expressed during involution and determine the consequences of its absence in BALB/c-p53null mice. Both p53 mRNA and protein were detected in the mammary epithelium within 48 h following weaning and resulted in an eightfold increase in levels of p21WAF1 mRNA. Induction of p21WAF1 mRNA was absent in BALB/c-p53null mice, and therefore, was shown to be p53-dependent. The BALB/c-p53null mice exhibited delayed involution of the mammary epithelium, as measured by 60% greater epithelial area compared to BALB/c-p53(wt) mice through 5 days post-weaning. The delay was transient with no differences being apparent at 7 days post-weaning. Expression of the stromal protease stromelysin-1 was unaffected by the absence of p53 suggesting that stromal responses were intact. These data demonstrate that p53 participates in the first stage of involution initiated by the epithelium itself, but does not affect the second phase during which stromal proteases are induced.

A mammary-specific model demonstrates the role of the p53 tumor suppressor gene in tumor development.

Although alterations in the p53 tumor suppressor gene are detected frequently in human breast cancers, mammary tumors are observed infrequently in p53(null) mice. This has led to the suggestion that absence of p53 alone is not sufficient for induction of mammary tumors. However, early death of p53(null) mice from thymic lymphomas may obscure tumor phenotypes that would develop later. Therefore, p53(null) mammary epithelium was transplanted into cleared mammary fat pads of wild type p53 BALB/c hosts to allow long-term analysis of mammary tumor phenotypes. Five treatments were compared for their effects on tumor incidence in hosts bearing transplants of p53(null) and p53wt mammary epithelium. The treatment groups were: (1) untreated; (2) continuous hormone stimulation with pituitary isografts; (3) multiple pregnancies; (4) DMBA alone; and (5) DMBA+pituitary isografts. The tumor incidences in p53(null) vs p53wt mammary transplants for each treatment group were 62% vs 0%, 100% vs 0%, 68% vs 0%, 60% vs 4% and 91% vs 14%, respectively. The mammary tumors that developed in the p53(null) mammary epithelium were all adenocarcinomas and were frequently aneuploid. These data demonstrate that the absence of p53 is sufficient to cause development of mammary tumors and that hormonal stimulation enhances the tumorigenicity of p53(null) mammary epithelium to a greater extent than DMBA exposure alone. This model provides an in situ approach to examine the molecular basis for the role of p53 in the regulation of mammary tumorigenesis.

Monitoring coupling of peptides to carrier proteins using biotinylated peptide.

The amount of synthetic peptide coupled to a carrier protein is a critical parameter in production of antipeptide antibodies. In the method described, biotinylated peptide was included as a tracer to monitor the coupling of peptide to keyhole limpet hemocyanin or BSA. The extent of coupling was assessed by first fractionating free and coupled peptide by gel filtration, then slot blotting an aliquot of each fraction onto nitrocellulose. The biotinylated peptide was detected using an avidin-horseradish peroxidase conjugate. To estimate the mass of peptide incorporated, the fractions were analyzed using a competitive ELISA. The limit of detection of this assay was < 0.1 pmol and was linear to 2 pmol. The mass of biotinylated peptide in each column fraction was determined using this assay and the total mass of peptide coupled to the carrier was calculated. Using these techniques, biotin-labeling provided a sensitive and versatile means to assess the quality of peptide-carrier protein conjugates.

Binding and bioactivity of ovine and porcine prolactins in porcine mammary tissue.

Differential binding of homologous and heterologous prolactin was investigated in porcine mammary tissue. Specific binding of ovine prolactin to porcine mammary membranes or tissue slices was significantly greater than specific binding of the homologous porcine prolactin. Ovine prolactin was also more potent than porcine prolactin in stimulating proliferation of Nb2 cells. In contrast, stimulation of glucose metabolism in porcine mammary explants by porcine prolactin was greater than that by ovine prolactin. Differences in specific binding were probably not due to damage during iodination, as low concentrations of iodinated prolactins were similar to unlabelled prolactins in their abilities to stimulate proliferation of Nb2 cells. Furthermore, electrophoretic analysis of medium from binding reactions suggested that differences in specific binding were not due to proteolytic cleavage of the homologous prolactin into large (greater than 10 kDa) fragments. These studies suggest that ovine prolactin either binds to sites in addition to the authentic lactogenic receptor in porcine mammary tissue or that a significantly higher affinity of ovine prolactin for the porcine lactogenic receptor has little effect on its biological activity.

Quantitation of prolactin-dependent responses in porcine mammary explants.

A system was developed to quantitate prolactin-dependent responses in porcine mammary tissue obtained from pregnant gilts. Metabolic responses to prolactin (Prl) and cortisol (C) in the presence of varying doses of insulin (I) were examined in mammary explants cultured on the surface of the medium or submerged in medium, under an atmosphere of humidified air. Explants suspended on grids at the surface of medium oxidized 45% more glucose (P less than .05) and incorporated 67% more glucose into lipids (P less than .05) than explants submerged in culture medium. In explants cultured on grids, both 100 and 1,000 ng I/ml increased glucose oxidation (by 50%) and glucose incorporation into lipids (by 150%) compared with 10 ng/ml (P less than .05), but responses to 100 and 1,000 ng I/ml were not different. Therefore, in subsequent studies, explants were cultured on grids with 100 ng I/ml. Rates of glucose metabolism for mammary explants cultured with I + C for 48 or 72 h were not different from those in fresh tissue. However, addition of Prl (200 or 1,000 ng/ml) increased oxidation rate 130% and fat synthesis 400% compared with I + C (P less than .05). Addition of triiodothyronine to I + C + Prl further increased rate of fat synthesis by 87%. Dose-dependent responses to Prl were demonstrated and were within the concentrations of Prl found in blood of gestating gilts. These studies demonstrate that the lactogenic complex of I, C and Prl induces metabolic activity in porcine mammary tissue from late pregnancy.

MiR203 mediates subversion of stem cell properties during mammary epithelial differentiation via repression of ΔNP63α and promotes mesenchymal-to-epithelial transition.

During reproductive life, the mammary epithelium undergoes consecutive cycles of proliferation, differentiation and apoptosis. Doing so relies on the retained proliferative capacity, prolonged lifespan and developmental potency of mammary stem cells (MaSCs). ΔNp63α, the predominant TP63 isoform in mammary epithelia, is robustly expressed in MaSCs and is required for preservation of self-renewing capacity in diverse epithelial structures. However, the mechanism(s) underlying subversion of this activity during forfeiture of self-renewing capacity are poorly understood. MicroRNAs (miRNAs) govern critical cellular functions including stem cell maintenance, development, cell cycle regulation and differentiation by disrupting translation of target mRNAs. Data presented here indicate that expression of miR203, a miRNA that targets ΔNp63α and ΔNp63ß is activated during luminal epithelial differentiation and that this pattern is observed in the murine mammary hierarchy. In addition, we present evidence that the transcription factor Zeb1 represses miR203 expression, thus enhancing ΔNp63α protein levels. Furthermore, ectopic miR203 suppresses ΔNp63α expression, proliferation and colony formation. The anti-clonogenic effects mediated by miR203 require suppression of ΔNp63α. In addition, ectopic miR203 promotes mesenchymal-to-epithelial transition and disrupts activities associated with epithelial stem cells. These studies support a model in which induction of miR203 mediates forfeiture of self-renewing capacity via suppression of ΔNp63α and may also have anti-tumorigenic activity through its reduction of EMT and cancer stem cell populations.

siRNA screening identifies differences in the Fanconi anemia pathway in BALB/c-Trp53+/- with susceptibility versus C57BL/6-Trp53+/- mice with resistance to mammary tumors.

BALB/c mice heterozygous for Trp53 develop a high proportion of spontaneous mammary tumors, a phenotype distinct from other mouse strains. BALB/c-Trp53+/- female mice, thus, resemble the hereditary Li-Fraumeni syndrome (LFS) characterized by early-onset of breast cancer, even though LFS involves TP53 mutations, which may involve not only loss- but also gain-of-function. Previous analysis of tumors in BALB/c-Trp53+/- females showed frequent loss of heterozygosity involving the wild-type allele of Trp53 and displayed characteristics indicative of mitotic recombination. Critical involvement of DNA double-strand break (DSB) repair dysfunction, particularly of homologous recombination (HR), was also noticed in the etiology of human breast cancer. To better define functional alterations in BALB/c-Trp53+/- mice, we applied a fluorescence-based DSB repair assay on mouse embryonic fibroblasts (MEFs) from BALB/c-Trp53+/- versus C57BL/6J-Trp53+/- mice. This approach revealed deregulation of HR but not non-homologous end-joining (NHEJ) in BALB/c-Trp53+/-, which was further confirmed for mammary epithelial cells. Screening of a small interfering RNA-library targeting DSB repair, recombination, replication and signaling genes, identified 25 genes causing differences between homologous DSB repair in the two strains upon silencing. Interactome analysis of the hits revealed clustering of replication-related and fanconi anemia (FA)/breast cancer susceptibility (BRCA) genes. Further dissection of the functional change in BALB/c-Trp53+/- by immunofluorescence microscopy of nuclear 53BP1, Replication protein A (RPA) and Rad51 foci uncovered differences in crosslink and replication-associated repair. Chromosome breakage, G2 arrest and biochemical analyses indicated a FA pathway defect downstream of FancD2 associated with reduced levels of BRCA2. Consistent with polygenic models for BRCA, mammary carcinogenesis in BALB/c-Trp53+/- mice may, therefore, be promoted by a BRCA modifier allele in the FA pathway in the context of partial p53 loss-of-function.

Regulation of apoptosis during mammary involution by the p53 tumor suppressor gene.

Regulation and functions of the p53 tumor suppressor gene have been studied extensively with respect to its critical role in maintaining the stability of genomic DNA following genotoxic insults. However, p53 is also induced by physiologic stimuli resulting in cell cycle arrest and apoptosis. In other situations, the activity of p53 must be repressed to prevent inappropriate removal of cells. The mammary gland provides a valuable system in which to study the mechanisms by which the expression and biological responses to p53 can be regulated under a variety of physiological circumstances. The pro-apoptotic role of p53 in the secretory mammary epithelium may be especially relevant to lactation in livestock. We have utilized p53-deficient mice to establish the molecular targets of p53 in the mammary gland and biological consequences when it is absent. The p21/WAF1 gene (Cdkn1a) is a transcriptional target gene of the p53 protein that responds to elevated levels of p53 during milk stasis providing an endogenous reporter of p53 activity. Abrogation of p53 resulted in delayed involution of the mammary epithelium, demonstrating the physiological role of p53 in regulating involution. Though delayed, stromal proteases were induced in the mammary gland by 5 d postweaning, providing a p53-independent mechanism that resulted in removal of the residual secretory epithelium. These processes can be interrupted by treatment with hydrocortisone. These data establish p53 as a physiological regulator of involution that acts to rapidly initiate apoptosis in the secretory epithelium in response to stress signals, but also indicate the presence of compensatory pathways to effect involution. Additional mechanisms involving intracellular stress signaling pathways (e.g., Stat3) and stromal-mediated pathways have been identified and, together with p53 pathways, may be used to identify animals with greater persistency of lactation.

Regulation of p53 and its targets during involution of the mammary gland.

Post-lactational involution of the mammary gland provides a system in which to study the expression and function of genes that regulate apoptosis in the context of a normal tissue. The functions of the p53 tumor suppressor gene have been extensively studied as a mediator of apoptosis in response to DNA damage, but its regulation in normal physiologic processes has been poorly characterized. Expression of p53 mRNA was shown to be among the first genes to be induced in mammary tissue following weaning of neonates. Although involution proceeds in the absence of a functional p53 gene, it is delayed compared to normal individuals. Therefore, involution can be viewed as biphasic with initial responses being sensitive to p53, whereas secondary responses being p53-independent. These observations can be exploited to determine the subset of genes that are p53-responsive and that mediate the effects of p53 in normal mammary tissue.

Expression of MDM2 during mammary tumorigenesis.

The MDM2 oncoprotein encodes a 90 kDa nuclear phosphoprotein capable of abrogating the growth suppressive functions of p53 and pRb tumor suppressor proteins by direct interaction. Alternative splicing of MDM2 protein coding sequences has been documented during tumor progression in human ovarian and bladder carcinomas. The aim of this study was to determine whether alternative splicing of MDM2 occurs during breast tumorigenesis in mice and humans and whether protein coding sequences were affected. Specimens representing normal and malignant breast tissues from the murine D2 mammary tumor model system and human breast carcinomas were examined. Three distinct mdm2 mRNA transcripts of 3.3, 1.6 and 1.5 kb were detected in normal and malignant murine mammary tissues by Northern blot analysis using a full-length mdm2 cDNA probe. Additional Northern blot analysis using a probe derived from exon 12 of murine mdm2 demonstrated that the 1.5 and 1.6 kb transcripts lack sequences encoding the C-terminus of the protein. No evidence of internal deletions of protein coding sequences of mdm2 was detected in any of the normal mammary tissues or D2 murine mammary tumors examined by reverse transcription PCR (RT-PCR). Three distinct MDM2 transcripts of 6.7, 4.7 and 1.9 kb were detected in malignant human breast tissue by Northern blot analysis using a cDNA probe specific for the complete open reading frame of human MDM2. However, a cDNA probe specific for the last exon of human MDM2 hybridized only to the 6.7 and 4.7 kb transcripts, demonstrating that the 1.9 kb transcript lacked protein coding sequences contained in exon 12. Similarly, no internal deletions were detected in a panel of malignant human breast tissues using RT-PCR and analogous primers within human MDM2. Therefore, breast tumors differ from other solid tumors reported previously in that no internal deletions of MDM2 protein coding sequences were observed. However, the data document the presence of multiple MDM2 mRNA transcripts in both normal and malignant breast tissues. A subset of MDM2 transcripts were shown to lack the last exon which contains sequences coding for the RING and zinc fingers and domains which are targets for caspase-3 mediated proteolytic degradation and are required to target p53 for proteosomal degradation.

Effect of fibroblast donor cell age and cell cycle on development of bovine nuclear transfer embryos in vitro.

The effects of cell cycle stage and the age of the cell donor animal on in vitro development of bovine nuclear transfer embryos were investigated. Cultures of primary bovine fibroblasts were established from animals of various ages, and the in vitro life span of these cell lines was analyzed. Fibroblasts from both fetuses and calves had similar in vitro life spans of approximately 30 population doublings (PDs) compared with 20 PDs in fibroblasts obtained from adult animals. When fibroblasts from both fetuses and adult animals were cultured as a population, the percentage of cells in G1 increased linearly with time, whereas the percentage of S-phase cells decreased proportionately. Furthermore, the percentage of cells in G1 at a given time was higher in adult fibroblasts than in fetal fibroblasts. To study the individual cells from a population, a shake-off method was developed to isolate cells in G1 stage of the cell cycle and evaluate the cell cycle characteristics of both fetal and adult fibroblasts from either 25% or 100% confluent cultures. Irrespective of the age, the mean cell cycle length in isolated cells was shorter (9.6-15.5 h) than that observed for cells cultured as a population. Likewise, the length of the G1 stage in these isolated cells, as indicated by 5-bromo-deoxyuridine labeling, lasted only about 2-3 h. There were no differences in either the number of cells in blastocysts or the percentage of blastocysts between the embryos reconstructed with G1 cells from 25% or 100% confluent cultures of fetal or adult cell lines. This study suggests that there are substantial differences in cell cycle characteristics in cells derived from animals of different ages or cultured at different levels of confluence. However, these factors had no effect on in vitro development of nuclear transfer embryos.

Heregulin induces in vivo proliferation and differentiation of mammary epithelium into secretory lobuloalveoli.

Mammary gland development and differentiation is mediated through the combined activities of systemic hormones and locally synthesized growth factors. To determine the in vivo response of mammary epithelium to heregulin (HRG), we implanted Elvax pellets containing HRG alpha or HRG beta within the mammary glands of prepubescent female mice in the presence or absence of exogenous estradiol and progesterone (E/ P). Mice treated in the same way with transforming growth factor alpha (TGF-alpha) were included as a positive control. Each growth factor treatment induced epithelial ductal branching in the presence or absence of E/P. In the absence of E/P, HRG beta did not effect terminal end bud formation, mammary epitheilum branching, or ductal migration. In contrast, TGF-alpha and HRG alpha induced ductal branching and HRG alpha induced ductal migration in the absence of E/P. The overall mammary response to growth factors was potentiated by the concomitant presence of E/P. In every case, the in vivo mammary epithelial responses to HRG alpha were more robust than TGF-alpha. Limited lobuloalveolar development was also observed in growth factor-treated mammary glands when E/P was present. Histological examination of growth factor-induced lobuloalveoli revealed secretory products within the lumen of HRG alpha and HRG beta lobuloalveoli. TGF-alpha-induced lobuloalveoli lacked similar secretory products.

Development of spontaneous mammary tumors in BALB/c p53 heterozygous mice. A model for Li-Fraumeni syndrome.

Breast cancer is the most frequent tumor type among women in the United States and in individuals with Li-Fraumeni syndrome. The p53 tumor suppressor gene is altered in a large proportion of both spontaneous breast malignancies and Li-Fraumeni breast cancers. This suggests that loss of p53 can accelerate breast tumorigenesis, yet p53-deficient mice rarely develop mammary tumors. To evaluate the effect of p53 loss on mammary tumor formation, the p53(null) allele was back-crossed onto the BALB/c genetic background. Median survival was 15.4 weeks for BALB/c-p53(-/-) mice compared to 54 weeks for BALB/c-p53(+/-) mice. Sarcomas and lymphomas were the most frequent tumor types in BALB/c-p53(-/-) mice, whereas 55% of the female BALB/c-p53(+/-) mice developed mammary carcinomas. The mammary tumors were highly aneuploid, frequently lost the remaining wild-type p53 allele, but rarely lost BRCA1. Although mammary tumors were rarely detected in BALB/c-p53(-/-) female mice, when glands from BALB/c-p53(-/-) mice were transplanted into wild-type BALB/c hosts, 75% developed mammary tumors. The high rate of mammary tumor development in the BALB/c background, but not C57Bl/6 or 129/Sv, suggests a genetic predisposition toward mammary tumorigenesis. Therefore, the BALB/c-p53(+/-) mice provide a unique model for the study of breast cancer in Li-Fraumeni syndrome. These results demonstrate the critical role that the p53 tumor suppressor gene plays in preventing tumorigenesis in the mammary gland.