1 Department of Clinical Research, University of Berne, Tiefenaustrasse 120, CH-3004 Bern, Switzerland
2 Institute for Genetics, Karlsruhe Research Center, D-76021 Karlsruhe, Germany*Both authors contributed equally to this work
Author for correspondence (e-mail: andrew.ziemiecki{at}dkf3.unibe.ch)
Accepted October 2, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Mammary gland, Mammary tumour, Apoptosis, Whole mount, Epithelial proliferation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mammary gland consists of two main components, the ectodermal parenchyma and the mesodermal stroma. The parenchyma, which is composed of secretory and ductal epithelial cells, contractile myoepithelial cells and pluripotent stem cells, develops and functions within the stroma, which consists of fibroblasts and adipose cells (Smith and Cepko, 2001). Unlike other organs, the mammary gland develops mainly in the juvenile and adult organism. With the onset of ovarian function at puberty, the rudimentary epithelial anlagen are induced to proliferate and to invade the surrounding fatty tissue, giving rise to a primitive epithelial ductal tree characteristic of the virgin gland. During pregnancy, the mammary epithelium differentiates and expands drastically until the entire gland is filled with secretory epithelium producing milk to nourish the young. After weaning, the mammary epithelium regresses by massive apoptotic cell death (Richert et al., 2000). Although the mediators of the complex interplay involved in mammary gland development and function are not fully characterized, protein tyrosine kinases, either as receptors or intracellular signal transducers, have been implicated (Fox and Harris, 1997; Hynes et al., 1997).
The murine EphB4 receptor protein tyrosine kinase was originally isolated from the mature mouse mammary gland, and tightly controlled expression was observed during mammary gland development and experimental carcinogenesis (Andres et al., 1994). The Eph family of receptor protein tyrosine kinases (RPTKs), which has 14 characterized members, represents the largest family of RPTKs to date (Pasquale, 1997). The ligands of the Eph family RPTKs, the protein ephrins, are also membrane associated either by a glycosyl-phosphatidylinositol tail (ephrin A family) or are bona fide transmembrane proteins (ephrin B family) (Pandey et al., 1995). The cytoplasmic domains of both the receptors, as well as the ligands (ephrin-B family), become phosphorylated on conserved tyrosine residues following interaction, suggesting that signalling cascades can ensue not only from the receptors but also from the ligands. This may provoke bi-directional signalling and mutual cell-cell communication (Holland et al., 1996; Brückner et al., 1997). This contention is supported by the demonstrations that both Eph receptors and ephrin ligands interact with PDZ-domain-containing proteins (Hock et al., 1998; Torres et al., 1998; Brückner et al., 1999; Lin et al., 1999), proteins implicated in the formation of submembranous scaffolds for the assembly of macromolecular signalling complexes (Garner et al., 2000). Possible mechanism(s) for modulating both receptor and ligand activities exist. Both molecule types associate with phosphatases (Dodelet and Pasquale, 2000) and recently an extracellular metalloprotease, Kuzbanian, has been found to associate with the ephrin-A2 ligand. This protease is activated after ligand activation and cleaves the extracellular moiety of the ligand molecule, thereby terminating ligand signalling and the physical association between cells (Hattori et al., 2000).
The observation that Eph receptors and their ligands exhibit reciprocal expression patterns during embryonic development has led to the suggestion that these molecules play a role in the development and patterning of a variety of tissues during embryogenesis. Indeed, Eph family members are involved in gastrulation, cell migration from the neural crest, segmentation of the early embryo and formation of the somites (Holder and Klein, 1999). The best-studied system so far is the development of the nervous system, where the Eph family and its ligands have a pivotal role in axon guidance, fasciculation and, together with NMDA receptors, in synaptogenesis (Klein, 2001). Recently, it has been shown that the Eph family, in particular EphB4 and its ligand ephrin-B2, are intimately involved in the development of the vascular system during embryogenesis (Wang et al., 1998; Gerety et al., 1999). In contrast to embryonic development, little is known about the function of the Eph family in post-natal and adult life, although some members, including EphB4, are expressed in adult organs such as the kidney, lung and mammary gland (Andres et al., 1994).
We have previously investigated the expression of the EphB4 receptor and the ephrin-B2 ligand proteins during mammary gland development. Expression of both was parenchyma specific, developmentally regulated and estrogen dependent, implicating this receptor-ligand pair in the hormone-dependent morphogenesis of the mammary gland (Nikolova et al., 1998). Expression of the ephrin-B2 ligand was confined to the epithelial cells, whereas the EphB4 receptor was expressed in both the myoepithelial and epithelial cells. Interestingly, the epithelial expression of EphB4 was only observed during proliferative phases of mammary gland development, such as puberty and the follicular phase of the cycle (Nikolova et al., 1998). We have now established transgenic mice exhibiting overexpression of the EphB4 receptor in the mammary epithelium to investigate the effects of untimely epithelial expression of this receptor on glandular growth and differentiation. We demonstrate that unscheduled expression of EphB4 interferes with the architecture of the mammary epithelial tree, alters the response of the epithelial cells to proliferative and apoptotic signals and contributes to the invasive phenotype of mouse mammary tumours.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Histological analyses
The fourth inguinal mammary glands were used routinely for histological examination. For whole-mount staining, the mammary glands were spread on coated slides and fixed for four hours in Carnoys solution (ethanol:chloroform:glacial acid, 6:3:1). Tissues were washed for 15 minutes in 70% ethanol, rehydrated and stained overnight in carmine alum (2 g/L carmine, 5 g/L potassium sulfate). The next day, tissues were washed in 70% ethanol, dehydrated, cleared with xylene and mounted in Eukitt. For paraffin embedding, the contralateral fourth inguinal mammary glands were fixed for 24 hours in 4% formaldehyde, dehydrated and embedded in paraffin. 4 µm sections were either stained with hematoxilin and eosin or were subjected to immunohistochemical detection of the EphB4 protein as described previously (Nikolova et al., 1998). Sections were examined using a Leica DMRD microscope and images were recorded digitally using a DC200 camera and the Leica LMS programme (Leica, Glattbrug, Switzerland).
Detection of cell proliferation and apoptosis
Animals were injected with 200 µg per g body weight of bromodeoxyuridine (BdUr, Sigma, Buchs, Switzerland) in PBS three hours before sacrificing. Incorporation of BdUr into DNA was determined immunohistochemically on formaldehyde fixed sections using anti-BdUr-specific antibodies (Roche Diagnostics) and peroxidase-labelled AB Complex (Dako, Glostrup, Denmark). Apoptotic cell death was analysed by the TUNEL assay on formaldehyde fixed sections using the In Situ Cell Death Detection Kit with TMR-red-labelled dUTP, according to the manufacturers instructions (Roche Diagnostics, Rotkreuz, Switzerland).
RNA and protein analyses
For RNA analyses, the third mammary gland was snap frozen in liquid nitrogen and stored at 70°C until required. RNA preparation and northern blot analyses were done as described in Andres et al. (Andres et al., 1994). Equal loading of the gels was verified by ethidium bromide staining. For RT-PCR, the RNA was treated with 10 U of DNase for 60 minutes at room temperature and re-isolated by phenol extraction and ethanol precipitation. RT-PCR was performed using the Titan One Tube RT-PCR Kit according to the manufacturers instructions (Roche Diagnostics, Rotkreuz, Switzerland). The primers used to detect transgene-derived transcripts were directed to sequences corresponding to the 3' untranslated region of the EphB4 cDNA and the SV40 polyadenylation signal of the transgene construct yielding a transgene-specific fragment of 480 bp. The correctness of the PCR product was verified by cloning and sequencing. The absence of contaminating DNA was controlled by conventional PCR. For protein analyses, the snap-frozen contra-lateral third mammary glands were macerated in SDS-PAGE sample buffer, boiled and subjected to immunoprecipitation and/or western blot analyses. Protein concentration was determined visually by amido black staining of 5 µl aliquots of extracts spotted onto nitrocellulose and approximately equal loading confirmed by Coomassie staining of western blot filters. The EphB4 antibodies (Nikolova et al., 1998) and the clone 4G10 phospho-tyrosine antibodies were utilised using conventional immunoprecipitation and western blotting methodology (Küng et al., 1997).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Transgene expression interferes with the development and architecture of the mammary epithelial tree
Whole-mount staining of the entire mammary epithelial tree at different stages of development served to assess the effect(s) of unscheduled EphB4 expression on the gross architecture of the mammary gland. In control animals, at the onset of ovarian function at 3.5 weeks of age, the rudimentary mammary epithelial anlagen are induced to proliferate and invade the fatty tissue by twisting and branching, giving rise to the epithelial tree characteristic of the mature mammary gland (Fig. 2A,C). At 10 weeks of age, the entire fat pad was populated by epithelium and the pubertal growth phase was concluded, as evidenced by the reshaping of terminal end buds to ductal structures (Fig. 2E,G,I,K). In contrast, the mammary anlagen of EphB4 transgenic animals exhibited severe growth retardation. At 5.5 weeks of age, the epithelial network was rudimentary, with reduced side-branching activity and an absence of second and third order branching (Fig. 2B,D). At 10 weeks of age, the epithelial network was expanded and the extent of the epithelialization of the gland and the ductal morphology were similar to that found in 5.5 week-old control females (Fig. 2F,H). In mature transgenic females at 13.5 weeks, the epithelial network was clearly developed; however, epithelial-free areas of adipose tissue and a large number of growing terminal end buds were still visible, indicating that the growth phase had not yet been concluded (Fig. 2J,L).
|
|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The EphB4 transgenic mice exhibited developmentally regulated transgene expression consistent with the properties of the MMTV-LTR promoter the highest expression being detected in lactating mammary glands (Hennighausen, 2000; Dickson et al., 2000). The observed patchy expression pattern of the transgene appears to be a characteristic of the normal lactating mammary gland, as the same phenomenon is also true for milk proteins. It is thought that this phenomenon may be the consequence of local tissue regeneration or local adaptation to variable milk consumption by the young (Bchini et al., 1991; Wilde, 1999) and reflects the remarkable plasticity of this organ. Alternatively, the patchy EphB4 expression could also reflect an attempt of the organ to compensate for possible detrimental effects by silencing transgene expression (Clark, 1998).
Transgenic mammary glands were characterized by disturbed development of the epithelial tree. Beginning at puberty, transgenic epithelial ducts exhibited less branching activity and developed less alveolar buds. This phenomenon was even more evident during pregnancy-induced morphogenesis of the mammary gland. Although not as extensive, this phenotypic consequence is very reminiscent of the defects observed in the mammary epithelium of progesterone receptor (PR) knockout mice (Brisken et al., 1998). These PR knockout animals have confirmed the observations made in vitro that progesterone is responsible for the development of ductal side branches during pubertal and pregnancy-induced development. The local regulators of the frequency of and spacing of the side-branches as well as of bifurcation of the alveolar buds are still unknown. The phenotype observed in the EphB4 transgenic females suggests that this receptor could serve as a negative local control element in these processes.
The low branching activity of the epithelial tree in the EphB4 transgenic mice resulted in a smaller number of individual lobules during pregnancy. The single lobules of the transgenic animals, however, contained more, but smaller, alveoli than control animals. Furthermore, the histological appearance of the single alveoli at lactation of the transgenic animals revealed an irregular, fragile morphology, suggestive of perturbances in cell-cell and cell-matrix interactions. E-and P-cadherin, members of the cell adhesion molecule gene family, are important factors regulating mammary alveolar growth and function. Recent data have described a close relationship between E-cadherin and the Eph receptor family in terms of expression, localization and activation (Zantek et al., 1999; Orsulic and Kemler, 2000). Moreover, ectopic expression of EphA4 in early Xenopus embryos has been shown to disrupt the cadherin-mediated cell adhesion during gastrulation (Winning et al., 1996; Jones et al., 1998). In addition to the cross-talk with the cadherin cell adhesion molecules, the Eph family has also been shown to mediate cell attachment or detachment by regulating integrin function (Huynh-Do et al., 1999; Davy and Parker, 2000). Thus, it is likely that the observed phenotypic consequences of EphB4 overexpression on the mammary epithelial morphology may, at least in part, be due to a disturbed cell-cell and cell-matrix attachment.
The most striking effect observed in the EphB4 transgenic mice concerned the altered proliferative and apoptotic response of the mammary epithelial cells. The analysis of BdUr incorporation revealed that a considerable portion of the transgenic mammary epithelial cells undergo DNA synthesis at day two of involution, a time point at which in control glands apoptosis is initiated and little if any BdUr incorporation was seen. The subsequent, approximately one day delayed, induction of epithelial regression in transgenic glands makes it unlikely that the BdUr-positive cells finished the cell cycle and underwent mitosis. Conceivably, these cells are arrested at the G2/M checkpoint of the cell cycle and subsequently undergo apoptosis. Normally, cells predetermined to proliferate or undergo cell death enter the cell cycle, traverse early G1 phase, whereupon their exact fate is fulfilled (King and Cidlowski, 1998). Recent evidence suggests that, apart from being involved in the control of cell migration, the Eph family is also involved in the regulation of cell death and survival. EphA4 is transiently expressed in motoneurons, which are predestined to undergo cell death during the development of the spinal cord (Ohta et al., 1996). Moreover, several members of the EphA family are responsible for inhibiting cell migration and induction of cell death in ventral spinal cord neurons (Yue et al., 1999). In contrast, overexpression of the ectodomain of the EphB2 receptor in the subventricular zone of the adult brain inhibited cell migration but stimulated proliferation of neuroblasts (Conover et al., 2000).
Our observation of DNA synthesis in the involuting mammary epithelium may reflect an altered response of EphB4 overexpressing cells to death/survival signals in early G1. Furthermore, the high proportion of apoptotic cell death observed in transgenic animals during the proliferative phase at pregnancy may similarly reflect an altered cellular response to proliferative signals, possibly correlated to a disturbed functioning of the E-cadherins. In mammary epithelial cells, E-cadherin can regulate cell survival by activating the retinoblastoma (Rb) gene, and via Rb it can initiate a growth signal conflict in an epithelial cell population induced to undergo apoptosis (Day et al., 1999). It is interesting that in the same time frame that DNA synthesis was observed in the absence of apoptosis, clusterin, a gene involved in protecting cells from apoptosis by unknown mechanisms (Wilson and Easterbrook, 2000), was highly induced in the transgenic mammary glands. This suggests that epithelial survival is, at least transiently, favoured in the transgenic mammary glands predestined for involution.
Although EphB4 overexpression apparently interferes with the growth response of the mammary epithelial cells during involution, tumour formation was not observed in the EphB4 transgenic females. In contrast, in neuT/EphB4 double transgenic females, tumour formation was accelerated and tumour growth was more aggressive than in the single transgenic neuT animals. Similar observations have been made in transgenic animals overexpressing the bcl-2 gene in the mammary epithelium where a delay in post-lactational involution was observed. Similar to the results reported here, bcl-2 overexpression alone did not lead to tumorigenesis; instead, it favoured tumour formation in c-myc/bcl2 double transgenic mice (Jäger et al., 1997). Interestingly, we have observed a considerable increase in the tumour latency time in the single transgenic neuT females crossed into the C57Bl6 genetic background over that in NeuT transgenic females of the pure FeBV strain. This is in agreement with the observations made with other oncogene-bearing mice or with spontaneous tumour formation that the high tumour resistance of the C57Bl6 strain is genetically determined (Macleod and Jacks, 1999). In the EphB4/neuT transgenic females, tumours not only appeared with reduced latency but also metastasised to the lung. Several reports have positively correlated overexpression of Eph family members with carcinogenesis, although direct evidence for the transforming potential is still missing (Dodelet and Pasquale, 2000). Our results indicate that EphB4 overexpression by itself is not tumorigenic but instead favours an invasive phenotype on transformed cells. This notion is further supported by our previous observation that in experimental mouse mammary tumours induced by the Ha-ras oncogene only the anaplastic, invasive tumour cells expressed high levels of EphB4, whereas the majority of the tumour mass was EphB4 negative (Nikolova et al., 1998). Thus, disturbance in the expression of molecules involved in the control of pattern formation may not directly induce transformation but may be instrumental in the acquisition of the malignant, invasive phenotype, possibly by modulating integrin and cadherin functions. It remains to be elucidated if epithelial overexpression of EphB4 can also provoke increased angiogenic potential of mammary cells and thereby facilitate tumour invasiveness.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andres, A.-C., Bchini, O., Schubaur, B., Dolder, B., LeMeur, M. and Gerlinger, P. (1991). Ha-ras induced transformation of mammary epithelium is favoured by increased oncogene expression or inhibition of mammary regression. Oncogene 6, 771-779.[Medline]
Andres, A.-C., Reid, H. H., Zuercher, G., Blaschke, R. J., Albrecht, D. and Ziemiecki, A. (1994). Expression of two novel eph-related receptor tyrosine kinases in mammary gland development and carcinogenesis. Oncogene 9, 1461-1467.[Medline]
Andres, A.-C., Zuercher, G., Djonov, V., Flueck, M. and Ziemiecki, A. (1995). Protein tyrosine kinase expression during the estrous cycle and carcinogenesis of the mammary gland. Int. J. Cancer 63, 288-269.[Medline]
Bchini, O., Andres, A.-C., Schubaur, B., Mehtali, M., LeMeur, M., Lathe, R. and Gerlinger, P. (1991). Precocious mammary gland development and milk protein synthesis in transgenic mice ubiquitously expressing human growth hormone. Endocrinology 128, 539-546[Abstract]
Brisken, C., Park, S., Vass, T., J. P., L., OMalley, B. W. and Weinberg, R. A. (1998). A paracrine role for the epithelial progesterone receptor in mammary development. Proc. Natl. Acad. Sci. USA 95, 5076-5081.
Brueckner, K., Pasquale, E. B. and Klein, R. (1997). Tyrosine phosphorlylation of transmembrane ligands for EphB4 receptors. Science 275, 1640-1643.
Brueckner, K., Labrador, J. P., Scheiffele, P., Herb, A., Seeburg, P. H. and Klein, R. (1999). EphrinB ligands recruit GRIP family PDZ adaptor proteins into raft membrane microdomains. Neuron 22, 511-524.[Medline]
Clark, A. J. (1998). Gene expression in the mammary glands of transgenic animals. Biochem. Soc. Symp. 63, 133-140.[Medline]
Conover, J. C., Doetsch, F., Garcia-Verdugo, J. M., Gale, N. W., Yancopoulos, G. D. and Alvarez-Buylla, A. (2000). Disruption of Eph-ephrin signalling affects migration and proliferation in the adult subventricular zone. Nat. Neurosci. 3, 1091-1097.[Medline]
Davy, A. and Parker, S. M. (2000). Ephrin-A5 modulates cell adhesion and morphology in an integrin-dependent manner. EMBO J. 19, 5396-5404.
Day, M. L., Zhao, X., Vallorosi, C. J., Putzi, M., Powell, C. T., Lin, C. and Day, K. C. (1999). E-caherin mediates aggregation-dependent survival of prostate and mammary epithelial cells through the retinoblastoma cell cycle control pathway. J. Biol. Chem. 274, 9656-9664.
Dickson, C., Creer, A. and Fantl. V. (2000). Mammary gland oncogenes as indicators of pathways important in mammary gland development. Oncogene 19, 1097-1101.[Medline]
Dodelet, V. C. and Pasquale, E. B. (2000). Eph receptors and ephrin ligands: embryogenesis to tumorigenesis. Oncogene 19, 5614-5619.[Medline]
Fox, S. B. and Harris, A. L. (1997). The epidermal growth factor receptor in breast cancer. J. Mammary Gland Biol. Neopl. 2, 131-142.[Medline]
Friedmann, Y., Daniel, C. A., Strickland, P. and Daniel, C. W. (1994). Hox genes in normal and neoplastic mouse mammary gland. Cancer Res. 54, 5981-5985.[Abstract]
Garner, C. C., Nash, J. and Hughanir, R. L. (2000). PDZ domains in synapse assembly and signalling. Trends Cell Biol. 10, 274-280[Medline]
Gavin, B. J. and McMahon, A. P. (1992). Differential regulation of the Wnt gene family during pregnancy and lactation suggests a role in postnatal development of the mammary gland. Mol. Cell Biol. 12, 2418-2423.[Abstract]
Gerety, S. S., Wang, H. U. and Anderson, D. J. (1999). Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development. Mol. Cell 4, 403-414.[Medline]
Hattori, M., Osterfield, M. and Flanagan, J. G. (2000). Regulated cleavage of a contact-mediated axon repellent. Science 289, 1360-1365.
Hennighausen, L. (2000). Mouse models for breast cancer. Oncogene 19, 966-967.[Medline]
Hock, B., Boehme, B., Karn, T., Yamamoto, T., Kaibuchi, K., Holtrich, U., Holland, S., Pawson, T., Ruebsamen-Waigmann, H. and Streibhardt, K. (1998). PDZ-domain mediated interaction of the Eph-related receptor tyrosine kinase EphB3 and the ras-binding protein AF6 depends on the kinase activity of the receptor. Proc. Natl. Acad. Sci. USA 95, 9779-9784.
Holder, N. and Klein, R. (1999). Eph receptors and ephrins: effectors of morphogenesis. Development 126, 2033-2044.
Holland, S. J., Gale, N. W., Mbamalu, G., Yancopoulos, G. D., Henkemeyer, M. and Pawson, T. (1996). Bi-directional signalling through the Eph family receptor Nuk and its transmembrane ligands. Nature 383, 722-725.[Medline]
Huynh-Do, U., Stein, E., Lane, A. A., Liu, H., Cerretti, D. P. and Daniel, T. O. (1999). Surface densities of ephrin-B1 determine EphB1-coupled activation of cell attachment through alphavbeta3 and alpha5beta1 integrins. EMBO J. 18, 2165-2173.
Hynes, N. E., Cella, N. and Wartmann, M. (1997) Prolactin mediated signalling in mammary epithelial cells. J. Mammary Gland Biol. Neopl. 2, 19-28.[Medline]
Jäger, R., Hetzer, U., Schenkel, J. and Weiher, H. (1997). Over expression of bcl-2 inhibits alveolar cell apoptosis during involution and accelerates c-myc induced tumorigenesis of the mammary gland. Oncogene 15, 1787-1795.[Medline]
Jaggi, R., Salmons, B., Muellener, D. and Groner, B. (1986). The v-mos and Ha-ras oncogene expression represses glucocorticoid hormone-dependent trasnscription from the mouse mammary tumor virus LTR. EMBO J. 5, 2609-2616.[Abstract]
Jones, T. L., Chong, L. D., Kim, J., Xu, R. H., Kung, H. F. and Daar, I. O. (1998). Loss of cell adhesion in Xenopus laevis embryos mediated by the cytoplasmic domain of Xlerk, an erythropoietin-producing hepatocellular ligand. Proc. Natl. Acad. Sci. USA 95, 576-581.
King, K. L. and Cidlowski, J. A. (1998). Cell cycle regulation and apoptosis. Annu. Rev. Physiol. 60, 601-617.[Medline]
Klein, R. (2001). Exitatory Eph receptors and adhesive ephrin ligands. Curr. Opin. Cell Biol. 13, 196-203.[Medline]
Kueng, P., Nikolova, Z., Djonov, V., Hemphill, A., Rohrbach, V., Boehlen, D., Zuercher, G., Andres, A.-C. and Ziemiecki, A. (1997). A novel family of serine/threonine kinases participating in spermiogenesis. J. Cell Biol. 139, 1851-1859.
Lin, D., Gish, G. D., Songyang, Z. and Pawson, T. (1999). The carboxyl terminus of class B ephrins constitutes a PDZ domain binding motif. J. Biol. Chem. 272, 3726-3733.
Macleod, K. F. and Jacks, T. (1999). Insights into cancer from transgenic mouse models. J. Pathol. 187, 43-60.[Medline]
Monpetit, M. L., Lawless, K. R. and Tenniswood, M. (1986). Androgen-repressed messages in the rat ventral prostrate. Prostate 8, 25-36.[Medline]
Muller, W. J., Sinn, E., Pattengale, P. K., Wallace, R. and Leder, P. (1998). Single-step induction of mammary adenocarcinoma in transgenic mice bearing the activated neu oncogene. Cell 54, 105-115.
Nikolova, Z., Djonov, V., Zuercher, G., Andres, A.-C. and Ziemiecki, A. (1998). Cell-type specific and estrogen dependent expression of the receptor tyrosine kinase EphB4 and its ligand ephrin-B2 during mammary gland morphogenesis. J. Cell Sci. 111, 2741-2751.
Ohta, K., Nakamura, M., Hirokawa, K., Tanaka, S., Iwama, A., Suda, T., Ando, M. and Tanaka, H. (1996). The receptor tyrosine kinase, Cek8, is transiently expressed on subtypes of motoneuron in the spinal cord during development. Mech. Dev. 54, 59-69.[Medline]
Orsulic, S. and Kemler, R. (2000). Expression of Eph receptors and ephrins is differentially regulated by E-cadherin. J. Cell Sci. 113, 1793-1802.
Pandey, A., Lindberg, R. A. and Dixit, V. M. (1995). Cell signalling. Receptor orphans find a family. Curr. Biol. 5, 986-989.[Medline]
Pasquale, E. B. (1997). The Eph family of receptors. Curr. Opin. Cell Biol. 9, 608-615.[Medline]
Richert, M. M., Schwertfeger, K. L., Ryder, J. W. and Anderson, S. M. (2000). An atlas of mouse mammary gland development. (2000). J. Mamm. Gland Biol. Neopl. 5, 227-242.[Medline]
Schmeichel, K. L., Weaver, V. M. and Bissell, M. (1998). Structural cues from tissue microenvironment are essential determinants of the human mammary epithelial phenotype. J. Mammary Gland Biol. Neopl. 3, 201-214.[Medline]
Smith, G. H. and Cepko, G. (2001). Mammary epithelial stem cells. Micr. Res. Tech. 52, 190-203.
Strange, R., Li, F., Saurer, S., Burkhardt, A. and Friis, R. R. (1992). Apoptotic cell death and tissue remodelling during mouse mammary gland involution. Development 115, 49-58.[Abstract]
Torres, R., Firestein, B. L., Dong, H. L., Staudinger, Y., Olson, E. N., Hughanir, R. L., Bredt, D. S., Gale, N. W. and Yancopoulos, G. D. (1998). PDZ proteins bind, cluster, and synaptically colocalize with Eph receptors and their ephrin ligands. Neuron 21, 1453-1463.[Medline]
Wang, H. U., Chen, Z. F. and Anderson, D. J. (1998). Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrinB2 and its receptor EphB4. Cell 93, 741-753.[Medline]
Wilde, C. J., Knight, C. H. and Flint, D. J. (1999). Control of milk secretion and apoptosis during mammary involution. J. Mamm. Gland Biol. Neoplasia 4, 129-136.[Medline]
Wilson, M. R. and Easterbrook-Smith, S. B. (2000). Clusterin is a secreted mammalian chaperone. Trends Biochem. Sci. 25, 95-98.[Medline]
Winning, R. S., Scales, J. B. and Sargent, T. D. (1996). Disruption of cell adhesion in Xenopus by Pagliaccio, an Eph-class receptor tyrosine kinase. Dev. Biol. 179, 309-319.[Medline]
Yue, Y., Su, J., Cerretti, D. P., Fox, G. M., Jing, S. and Zhou, R. (1999). Selective inhibition of spinal cord neurite outgrowth and cell survival by the Eph family ligand ephrin-A5. J. Neurosci. 15, 10026-10035.
Zantek, N. D., Azimi, M., Fedor-Chaiken, M., Wang, B., Brackenbury, R. and Kinch, M. S. (1999). E-cadherin regulates the function of EphA2 receptor tyrosine kinase. Cell Growth Differ. 10, 629-638.
Zisch, A. H., Stallcup, W. B., Chong, L. D., Dahlin-Huppe, K., Voshol, J., Schachner, M. and Pasquale, E. B. (1997). Tyrosine phosphorylation of L1 family adhesion molecules: implication of the Eph kinase Cek5. J. Neurosci. Res. 47, 655-665.[Medline]