Transforming growth factor alpha and mouse models of human breast cancer.

Transforming growth factor alpha (TGFalpha) is a principal molecule in the normal and neoplastic development of the mammary gland. Binding of TGFalpha to the epidermal growth factor receptor (EGFR), activates the EGFRs' endogenous tyrosine kinase activity and stimulates growth of the epithelium in the virgin and pregnant mouse mammary gland. TGFalpha expression can be detected in breast cancer cells in vivo and in vitro and overexpression can elicit partial transformation or immortalized human and rodent mammary epithelial cells. Despite evidence implicating TGFalpha in the development of mammary neoplasia, the actual mechanism of TGFalpha-induced transformation is unclear. Transgenic mouse models targeting heterologus TGFalpha to the mammary gland have established TGFalpha overexpression can induce hyperproliferation, hyperplasia and occasional carcinoma. These transgenic studies demonstrated a facilitating, proliferative role for TGFalpha in the development of neoplasia and implicated several oncogenes that can cooperate with TGFalpha to transform the mammary epithelium. From studies of EGFR signaling pathways, inhibitory and modulating agents such as anti-EGFR antibodies and specific kinases inhibitors have been used to block the action of this pathway and prevent the development of TGFalpha-induced neoplasia and tumor formation. Studies in Stat5a knockout mice have established that the JAK2/Stat5a pathway can facilitate the survival of the mammary epithelium and can impact the progression of TGFalpha-mandated mammary tumorigenesis. Together these experiments indicate that TGFalpha and the EGFR signaling pathway are potentially amenable to therapies for treatment of human breast disease.

Signal transducer and activator of transcription 5a influences mammary epithelial cell survival and tumorigenesis.

The mammary gland undergoes extensive tissue remodeling and cell death at the end of lactation in a process known as involution. We present evidence that the prolactin-activated transcription factor signal transducer and activator of transcription 5a (Stat5a) has a crucial role in the regulation of cell death during mammary gland involution. In a transforming growth factor-alpha transgenic mouse model that exhibited delayed mammary gland involution, the absence of Stat5a facilitated involution-associated changes in morphology of the gland and the extent and timing of programmed cell death. These Stat5a-dependent changes also affected epidermal growth factor receptor-initiated mammary gland tumorigenesis. Overexpression of the transforming growth factor alpha transgene in the mammary epithelium reproducibly generated mammary hyperplasia and tumors. In the presence of the activated epidermal growth factor receptor, deletion of Stat5a delayed initial hyperplasia and mammary tumor development by 6 weeks. These observations demonstrate that Stat5a is a survival factor, and its presence is required for the epithelium of the mammary gland to resist regression and involution-mediated apoptosis. We also suggest that Stat5a is one of the antecedent, locally acting molecules that initiate the process of epithelial regression and reorganization during involution.

Programmed cell death in the terminal endbud.

Ductal development in the pubertal mouse mammary gland is characterized by dramatic morphological changes in the epithelium driven by proliferation of cap and body cells in the terminal endbuds. Recent experiments revealed a coincident and abundant apoptosis in the body cells of these structures. The cells undergoing apoptosis are occasionally restricted to defined regions within the terminal endbud. Localization adjacent to the presumptive lumina suggests that this process functions to sculpt the lumina of the subtending duct. Members of the Bcl-2 family of apoptosis regulatory molecules; Bcl-2 and Bcl-x, appear to have some role in regulating apoptosis in the terminal endbud. Other possible signals which could regulate this developmental process and a model are presented.

Stably transfected HC11 cells provide an in vitro and in vivo model system for studying Wnt gene function.

The in vitro and in vivo effects of several Wnt family members have been studied using stably transfected HC11 cells, a clonal mammary epithelial cell line derived from a midpregnant mouse mammary gland capable of hormone-dependent differentiation in vitro. Differential effects of Wnt-1, Wnt-2, and Wnt-7B expression were observed both on the morphology of confluent HC11 cells and on the pattern of E-cadherin expression. Wnt-7B had no apparent effect on HC11 cell morphology or E-cadherin expression, as compared to mock-transfected HC11 cells. Injection of stably transfected pools of Wnt-1, Wnt-2, Wnt-7B, and mock-transfected cells into the cleared fat pad of syngeneic BALB/c mice generated reproducible outgrowths after 8 or 12 weeks. Mock-transfected cells produced outgrowths that exhibited some morphologically normal ductal and alveolar-like structures. However, no morphologically normal structures were observed in the fat pads containing Wnt-transfected cells. Instead, these outgrowths were characterized by significant fibrosis, epithelial hyperplasia, and multiple sites of growth. In contrast to the lack of an observed effect in vitro, palpable adenocarcinomas were observed 12 weeks after injection of the Wnt-7B-transfected HC11 cells. These tumors contained significant regions of hyperplastic and transformed epithelium and lacked the fibrotic phenotype observed in the Wnt-1 and -2 outgrowths. These results support the hypothesis that different Wnt family members may elicit distinct functional effects and reinforce the need to perform simultaneous comparisons of Wnt function both in vitro and in vivo. Stably transfected HC11 cells provide a useful model system in which to elucidate the function of different Wnt family members.

Mammary gland development is mediated by both stromal and epithelial progesterone receptors.

A combination of a knockout mouse model, tissue transplantation, and gene expression analysis has been used to investigate the role of steroid hormones in mammary gland development. Mouse mammary gland development was examined in progesterone receptor knockout (PRKO) mice using reciprocal transplantation experiments to investigate the effects of the stromal and epithelial PRs on ductal and lobuloalveolar development. The absence of PR in transplanted donor epithelium, but not in recipient stroma, prevented normal lobuloalveolar development in response to estrogen (E) and progesterone (P) treatment. Conversely, the presence of PR in the transplanted donor epithelium, but not in the recipient stroma, revealed that PR in the stroma may be necessary for ductal development. Members of the Wnt growth factor family, Wnt-2 and Wnt-5B, were employed as molecular markers of steroid hormone action in the mammary gland stroma and epithelium, respectively, to investigate the systemic effects of E and P. Hormonal treatment of intact, ovariectomized, and PR-/- mice and mice after transplantation of PR-/- epithelium into wild type (PR+/+) stroma demonstrated that these two locally acting growth factors are regulated by independent mechanisms. Wnt-2 is acutely repressed by E alone, while Wnt-5B gene expression is induced only after chronic treatment with both E and P. Wnt 5B appears to be one of the few molecular markers of P action in the mammary epithelium. This study suggests that the regulation of mammary gland development by steroid hormones is mediated by distinct effects of the stromal and epithelial PR and differential growth factor expression.

Use of PRKO mice to study the role of progesterone in mammary gland development.

To better understand the distinct physiological roles played by progesterone and estrogen receptors (PR and ER) as well as to study directly PR function in an in vivo context, a novel mutant mouse strain, the PR knockout (PRKO) mouse, was generated carrying a germline loss of function mutation at the PR locus. Mouse mammary gland development has been examined in PRKO mice using reciprocal transplantation experiments to investigate the effects of the stromal and epithelial PRs on ductal and lobuloalveolar development. The absence of PR in transplanted donor epithelium, but not in recipient stroma, prevented normal lobuloalveolar development in response to estrogen and progesterone treatment. Conversely, the presence of PR in the transplanted donor epithelium, but not in the recipient stroma, revealed that PR in the stroma may be necessary for ductal development. Stimulation of ductal development by the PR may, therefore, be mediated by an unknown secondary signaling molecule, possibly a growth factor. The continued stimulation of the stromal PR appears to be dependent on reciprocal signal(s) from the epithelium. Thus, the combination of gene knockout and reciprocal transplantation technologies has provided some new insights into the role of stromal-epithelial interactions and steroid hormones in mammary gland development.

Apoptosis in the terminal endbud of the murine mammary gland: a mechanism of ductal morphogenesis.

Ductal morphogenesis in the rodent mammary gland is characterized by the rapid penetration of the stromal fat pad by the highly proliferative terminal endbud and subsequent formation of an arborized pattern of ducts. The role of apoptosis in ductal morphogenesis of the murine mammary gland and its potential regulatory mechanisms was investigated in this study. Significant apoptosis was observed in the body cells of the terminal endbud during the early stage of mammary ductal development. Apoptosis occurred predominately in defined zones of the terminal endbud; 14.5% of the cells within three cell layers of the lumen were undergoing apoptosis compared to 7.9% outside this boundary. Interestingly, DNA synthesis in the terminal endbud demonstrated a reciprocal pattern; 21.1% outside three cell layers and 13.8% within. Apoptosis was very low in the highly proliferative cap cell laver and in regions of active proliferation within the terminal endbud. In comparison to other stages of murine mammary gland development, the terminal endbud possesses the highest level of programmed cell death observed to date. These data suggest that apoptosis is an important mechanism in ductal morphogenesis. In p53-deficient mice, the level of apoptosis was reduced, but did not manifest a detectable change in ductal morphology, suggesting that p53-dependent apoptosis is not primarily involved in formation of the duct. Immunohistochemical examination of the expression of the apoptotic checkpoint proteins, Bcl-x, Bax and Bcl-2, demonstrated that they are expressed in the terminal endbud. Bcl-x and Bcl-2 expression is highest in the body cells and lowest in the nonapoptotic cap cells, implying that their expression is associated with increased apoptotic potential. Bax expression was distributed throughout the terminal endbud independent of the observed pattern of apoptosis. A functional role for Bcl-2 family members in regulating endbud apoptosis was demonstrated by the significantly reduced level of apoptosis observed in WAP-Bcl-2 transgenic mice. The pattern of apoptosis and ductal structure of endbuds in these mice was also disrupted. These data demonstrate that p53-independent apoptosis may play a critical role in the early development of the mammary gland.

Localization and quantification of Wnt-2 gene expression in mouse mammary development.

The Wnt gene family encodes a group of proteins probably involved in cell-cell communication during several stages of vertebrate development. More than 10 members of this family have been identified and shown to be expressed mainly in developing neural tissue. Using a reverse transcription-polymerase chain reaction (RT-PCR)-based approach with degenerate oligonucleotides directed against conserved sequences in the Wnt genes, Wnt-2 transcripts were detected in RNA isolated from mammary glands of 4- to 6-week-old virgin C3H mice, a period characterized by extensive end bud and ductal proliferation. The spatial and temporal expression of Wnt-2 in the developing mouse mammary gland was studied by in situ hybridization, quantitative RT-PCR, and Northern analysis. Wnt-2 is expressed during the ductal phase of mammary gland development, primarily in the basal layer of mammary ducts and in the body cells of end buds. Wnt-2 RNA transcripts were readily detected in poly(A) RNA isolated from 5-week-old C3H and Balb/c mice. RNA transcript levels measured as molecules per nanogram of total RNA by RT-PCR decreased 10- to 40-fold within 2 days after the onset of pregnancy and remained low during pregnancy and lactation. This is in contrast to the patterns of expression of other Wnt family members, Wnt-5a and -5b, whose expression was either barely or not detectable in the 4- to 6-week-old mammary gland, but increased markedly during pregnancy. These results confirm the differential expression of Wnt gene family members during mammary gland development. Furthermore, they suggest that Wnt-2, as well as several other family members, may play a role in pattern formation during early mammary gland development.

An experimental system for determining the influence of microgravity on B lymphocyte activation and cell fusion.

The influence of microgravity on lymphocyte activation is central to the understanding of immunological function in space. Moreover, the adaptation of groundbased technologies to microgravity conditions presents opportunities for biotechnological applications including high efficiency production of antibody forming hybridomas. Because the emerging technology of microgravity hybridoma generation is dependent upon activation and cultivation of B lymphocytes during flight, we have adapted mitogen-driven B lymphocyte stimulation and culture that allows for the in vitro generation of large numbers of antibody forming cells suitable for cell fusion over a period of 1-2 weeks. We believe that this activation and cultivation system can be flown on near-term space flights to test fundamental hypotheses about mammalian cell activation, cell fusion, metabolism, secretion, growth, and bio-separation.

Identification of isolate-specific sporozoite proteins of Cryptosporidium parvum by two-dimensional gel electrophoresis.

Five isolates of Cryptosporidium parvum collected from human, horse, and calf sources were compared for differences in sporozoite protein patterns by using two-dimensional gel electrophoresis. Silver-stained two-dimensional gels contained over 300 protein spots from detergent-solubilized sporozoites. A distinguishing 106-kilodalton peptide that shifted in isoelectric point was detected in four of the five isolates. Computerized two-dimensional gel analysis was performed to obtain objective quantitation of the pI shift. Three of these four isolates could be differentiated from one other by the pI shift in this peptide. The fifth isolate was distinguished by the absence of the 106-kilodalton peptide and the presence of a 40-kilodalton peptide that was not observed in any other isolate.

A computer-enhanced comparative study of brain region polypeptides and proteins by two-dimensional gel electrophoresis.

A reproducible and quantitative strategy for identifying tissue-specific proteins of the central nervous system is described. The methods include a simple extraction procedure, two-dimensional polyacrylamide gel electrophoresis (2-DGE), silver staining, and computerized analysis. Acetic acid protein extractions of brain regions from three groups of male Sprague-Dawley rats were compared by computer analysis using 2-DGE with GELCODE silver staining. Protein spot mapping and characterizations of molecular weight and pI were compiled for the pineal gland, retina, hypothalamus and cerebral cortex. Regionally specific protein spots were identified using the Visage System (BioImage) for data acquisition and a new set of algorithms (University of Arizona) for assigning isoelectric point (pI) and molecular weight determinations, spot matching and selection of unique spots. Seventeen newly identified acidic proteins are unique to the pineal gland. Some others are also common to the retina but not in other regions examined. Further study of these and other regionally specific proteins are of particular interest under conditions which alter biological or disease mechanisms.

Isolation by preparative isoelectric focusing of a direct acting fibrinolytic enzyme from the venom of Agkistrodon contortrix contortrix (southern copperhead).

Fibrolase, a blood clot-lysing enzyme, was isolated from the venom of the snake Agkistrodon contortrix contortrix using preparative scale isoelectric focusing in the recycling isoelectric focusing (RIEF) apparatus. Two sequential purifications, beginning with 1.0 g of whole, dried venom, were employed. A pH 6-8 range gradient effected the first separation. While 100% of the enzyme was recovered in three fractions, 43% (one fraction) had 70% purity. The second run was a refractionation of three, pooled fractions from the first run, in a 0.7 pH range gradient. Of the fibrolase in the venom, 63% was recovered in four fractions. One of these represented 29% of venom fibrolase, with 97% purity. Gel filtration chromatography removed most of the remaining, higher molecular weight contaminants of the RIEF-purified enzyme.