help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bonnette, S. G.
Right arrow Articles by Hadsell, D. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bonnette, S. G.
Right arrow Articles by Hadsell, D. L.
Endocrinology Vol. 142, No. 11 4937-4945
Copyright © 2001 by The Endocrine Society

ARTICLES

Targeted Disruption of the IGF-I Receptor Gene Decreases Cellular Proliferation in Mammary Terminal End Buds

Sharon G. Bonnette and Darryl L. Hadsell

U.S. Department of Agriculture/Agricultural Research Service Children’s Nutrition Research Center, Departments of Pediatrics (S.G.B., D.L.H.) and Molecular and Cellular Biology (D.L.H.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: Dr. Darryl L. Hadsell, Children’s Nutrition Research Center, 10th floor, 1100 Bates Street, Houston, Texas 77030. E-mail: dhadsell{at}bcm.tmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF-I mediates mammary ductal development through stimulation of terminal end bud (TEB) development; however, no published data exist on the mechanism through which this occurs. The mechanism of IGF-I action on the TEB was studied by determining the requirement for the IGF-I receptor (IGF-IR) in IGF-I-dependent ductal development. We hypothesized that loss of the IGF-IR would disrupt mammary ductal development through a combination of decreased proliferation or increased apoptosis. Because IGF-IR null mice die at birth, embryonic mammary gland transplantation was used to study the effects of a disrupted IGF-IR gene. Analyses of grafts after 4 or 8 wk of development demonstrated a limited growth potential of the null mammary epithelium in virgin hosts. Bromodeoxyuridine labeling and terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick-end labeling showed that cell proliferation was significantly decreased in null TEBs, but apoptosis was not. In addition, both the size and number of TEBs were reduced in null outgrowths. In pregnant hosts, null ductal growth was stimulated beyond the level seen in virgin hosts. These findings directly establish a proliferation-dependent role for the IGF-IR in the cells of the TEB. Additionally, this study indicates that pregnancy-dependent compensatory mechanisms can stimulate mammary development in the absence of an IGF-IR.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE TERMINAL END bud (TEB) is the proliferative compartment within the virgin mouse mammary gland that is responsible for the development of virtually the entire ductal system. These bulb-shaped structures consist predominantly of an outer layer of epithelial cells, termed cap cells, and an inner layer of epithelial cells, termed body cells (1). The extent of proliferation in the TEB has been estimated to be as much as 5-fold more than that observed in mature ducts (2). Within the TEB, the cap cells often display the highest proliferative activity (3, 4). The TEB is also a site of significant apoptosis in the developing gland, and it is this apoptosis that is believed to cause canalization of the developing ducts (5). Regulation of TEB development occurs at several levels involving steroid and peptide hormones as well as local production of growth factors (4, 6).

Numerous studies suggest that IGF-I plays an important role in mammary gland development. Firstly, IGF-I is a potent mitogen for normal mammary epithelial cells in culture, and ductal growth can be induced in mammary gland explant cultures by IGF-I in combination with mammogenic hormones (7). Secondly, in vivo local administration of IGF-I induces mammary TEB development (8) and transgenic mice that overexpress IGF-I specifically in the mammary gland during pregnancy and lactation exhibit an increased incidence of mammary hyperplasia and tumorigenesis (9, 10). Lastly, the mRNAs for both IGF-I and the IGF-I receptor (IGF-IR) are expressed in both the mammary stroma (11) and the developing TEB (7), and studies by Ruan and co-workers (12) demonstrated that targeted deletion of IGF-I inhibits normal TEB development.

In cell culture models, IGF-I inhibits apoptosis through IGF-IR-dependent activation of insulin receptor (IR) substrate-1, followed by downstream activation of PI3K and the serine/threonine kinase Akt (13, 14). Much earlier studies in cell culture models have established that IGF-I stimulates cell cycle progression (15). The mechanism through which this occurs is pleiotropic, involving such things as the Ras/Raf/MAPK pathway (16) and a ß-catenin-regulated pathway (17, 18). Transgenic models that overexpress IGF-I in the mammary gland during lactation exhibit delayed mammary involution in conjunction with inhibited apoptosis (9, 19). Delayed postlactational mammary involution and inhibited apoptosis also occur in response to transgenic overexpression of IGF-II (20). This response has been linked with prolonged phosphorylation of Akt. Unfortunately, there has been limited success at overexpressing IGFs in the virgin mammary gland, and analysis of TEB development in the IGF-I ligand knockout has provided little in terms of the intracellular mechanisms of IGF-I action on mammary cells.

In various tumor cell types, blockage of IGF-IR signaling causes extensive cell death (21). In primary cultures of mouse mammary cells, IGF-I or high concentrations of insulin inhibit apoptosis (22). In contrast, IGF-I treatment of mammary gland organ cultures derived from virgin mice stimulates DNA synthesis (23). Because TEB development involves both cellular proliferation and apoptosis (3, 4), and no published data exist on the cellular mechanism through which TEB development is stimulated by IGF-I, an analysis of the relative involvement of apoptosis and cell cycle progression would be a logical first step in defining the mechanism of IGF-I action on the TEB. In addition, because IGF-I can stimulate biological responses through activation of IR:IGF-IR heterodimers (24, 25), the analysis of TEB development in IGF-IR-null mammary tissue would serve as an important means of formally establishing the importance of the IGF-IR to TEB development.

Independent laboratories have previously described mice that carry a targeted mutation of the IGF-IR and IR genes (26, 27). Homozygous IGF-IR-null mice exhibit decreased prenatal growth and diminished bone, skeletal muscle, and skin development. These mice die within minutes of birth. Through the use of a fetal tissue transplantation technique similar to that used to study other knockout models (28), we have been able to directly examine the in vivo function of the IGF-IR in mammary epithelial cell proliferation and death. Preliminary studies conducted in our laboratory with this model suggested that loss of the IGF-IR inhibited mammary ductal development (29). Hence, the goals of this study were 1) to investigate in further detail how loss of the IGF-IR affects mammary TEB development, 2) to determine if the effects of IGF-IR were mediated through cell proliferation or apoptosis, and 3) to determine whether the hormones of pregnancy can affect the development of mammary epithelium that lacks IGF-IR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
All mice were maintained in a closed conventional animal facility, The Clinical Nutrition Research Facility, at the Children’s Nutrition Research Center (Houston, TX). Mice were fed 5053 PicoLab Rodent Diet 20 (PMI Nutrition International, Inc., Brentwood, MO) ad libitum and maintained at 21-22 C with a 12-h light-dark cycle. Heterozygous IGF-IR mice were obtained from Dr. Argiris Efstratiadis (Columbia University, New York, NY) and bred into an FVB background to allow syngeneic transplantation of mammary tissue. Mice from 9–11 backcrossed generations (F9–F11) were used for this study. Recipient female FVB mice were either born in-house or purchased from Charles River Laboratories, Inc. (Wilmington, MA) or Harlan Sprague Dawley, Inc. (Indianapolis, IN). Animal protocols were approved by the animal care and use committee of Baylor College of Medicine and were conducted in accordance with NIH guidelines (NRC 1996).

Mammary epithelium transplantation
F9–F11 heterozygous males and females were bred to generate wild-type (Igf1r+/+), heterozygous (Igf1r+/-), and null (Igf1r-/-) donor embryos for mammary transplants. On d 16–18 of pregnancy, embryos were harvested by cesarean section and kept on ice in HBSS until dissection.

Three-week-old FVB recipient females were prepared by clearing the inguinal fat pad of endogenous host epithelium as described by DeOme et al. (30). After clearing, embryonic mammary buds were dissected by cutting around the teat of the embryo and transplanting the mammary bud along with the overlying skin into the cleared inguinal gland of the recipient. The transplanted epithelium was allowed to develop for 4 or 8 wk in virgin hosts and then harvested for analysis. To test the effects of pregnancy on the grafted epithelium, hosts were bred 3.5 wk after grafting, and the glands were harvested 10 d postbreeding. Pregnancy was confirmed by observation of embryos in the host uteri.

Embryo genotyping
Embryo genotype was established by Southern analysis as previously described (27). The sex of the embryo was established by PCR on embryo DNA. Amplification of the sex-determining region of the Y chromosome (SRY) gene was used to identify male embryos. The forward SRY primer was 5'-cgccccatgaatgcatttatg-3', and the reverse primer was 5'-cctccgatgaggctgatat-3'. PCR cycling was for 1 min at 94 C, 2 min at 55 C, and 2 min at 72 C for 30 cycles.

Mammary gland whole mounts and morphometric analysis
The transplanted outgrowths were harvested and fixed in 10% neutral buffered formalin overnight and prepared as whole mounts as described by Medina et al. (31). The whole mounts were analyzed for growth by measuring the percentage of the fat pad filled (PFPF) by the ductal system of the outgrowth. PFPF was estimated by measuring the distance the ductal outgrowth extended across the fat pad divided by the total length of the fat pad. Whole mounts were also analyzed for branch point and TEB number and area. These analyses were performed using Adobe Photoshop (Adobe Systems, San Jose, CA) and/or Scion Image (Scion Corp., Frederick, MD) image processing and analysis software. Gray scale tagged-image file format (TIFF) images of whole mounts were captured with a Dage black and white CCD72 video camera (Dage-MTI, Inc., Michigan City, IN) and Scion LG3 frame grabber (Scion Corp.) at a resolution of 72 pixels/inch. To quantitate branch points, images of whole mounts (x10 magnification) were viewed in Adobe Photoshop. Nodes (branch points) in the ductal tree were marked on an overlying layer using the paintbrush tool. The layer containing the marked nodes was saved in a TIFF format, and the marks representing nodes or branches were counted by Scion Image software. To measure TEB number and area, a similar method was used. Briefly, x10 images of whole mounts, captured as described, were displayed in Adobe Photoshop. TEBs were traced or outlined and then filled. The layer containing only the traced images of the TEBs was saved in a TIFF format and quantitated using Scion Image software. The analyze particles function of the Scion Image software was used to quantitate the area in square millimeters and the total number of the traced TEBs. The total number of terminal structures possessing an area 0.03 mm2 or more and with morphology distinct to a TEB structure were recorded following the paradigm of Ball (32). The area of the two largest TEBs was used to determine the mean TEB area, because the minimal average number of TEBs in all groups was 1.8. One Igf1r-/- outgrowth included in the mean TEB area calculation possessed only one TEB.

Cell proliferation and cell death assays
Anti-bromodeoxyuridine (anti-BrdU) immunohistochemistry was used to measure cell proliferation in the mammary outgrowths, whereas the terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick-end labeling (TUNEL) assay was used to measure cell death. These procedures were conducted as previously described (10, 33). Counting at least 1000 cells/section quantitated the percentage of cells proliferating or dying. In some cases, sections of the Igf1r-/- outgrowths did not contain 1000 cells because of the limited growth of the Igf1r-/- epithelium. In these instances, all cells in the entire section of multiple (2, 3, 4, 5, 6) sections were counted.

Statistical analysis
All data were analyzed by one-way ANOVA using Minitab statistical software (State College, PA). Mean separation was accomplished using Fisher’s pairwise comparison. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Embryonic mammary buds can be transplanted into wild-type hosts to study pubertal mammary development
As Igf1r-/- mice die at birth, it is not possible to study organs that develop postnatally, such as the mammary gland. To determine the role that the IGF-IR plays in pubertal mammary development, we transplanted embryonic mammary buds from 16- to 18-d-old Igf1r+/+, Igf1r+/-, and Igf1r-/- mouse embryos into the cleared fat pads of 21-d-old syngeneic recipients. Figure 1Go, A–C, show that transplanted embryonic mammary buds of all three genotypes were able to grow in the host fat pad to form a mammary ductal tree. When transplanting wild-type mammary epithelium, the rates of successful grafts for Igf1r+/+ and Igf1r+/- epithelium were 78% and 71%, respectively, whereas the rate of successful grafts of Igf1r-/- mammary epithelium was only 36% (Fig. 1DGo). Therefore, although mammary epithelium from all three genotypes can be successfully transplanted to study postnatal mammary development, the Igf1r-/- epithelium does not possess the same growth potential as its wild-type or heterozygous counterpart.



View larger version (125K):
[in this window]
[in a new window]
  		Figure 1. Mammary gland whole mounts of ductal outgrowths derived from TEB. Mammary glands were harvested 8 wk posttransplantation and analyzed for ductal growth. Representative whole mounts of Igf1r+/+ (A), Igf1r+/- (B), and Igf1r-/- (C) grafts illustrate the limited and abnormal morphology of the Igf1r-/- ductal system (C). Ductal development of the grafts was expressed as the PFPF by the ductal outgrowth ({blacksquare}; D). The proportion of successful grafts was determined by dividing the number of outgrowths observed by the total number of embryonic mammary buds transplanted ({square}; D). Data are expressed as the mean ± SEM. Numbers in parentheses indicate the number of outgrowths analyzed, and the asterisk indicates a statistical difference (P < 0.05).

 
Igf1r-/- ductal growth is limited but cellular organization is normal
Eight weeks after the embryonic epithelium was transplanted, the glands were harvested and whole-mounted to assess growth. The PFPF by transplanted Igf1r+/+ and Igf1r+/- mammary epithelium averaged 90.2 ± 3.9% vs. 25.0 ± 5.4% for Igf1r-/- epithelium (Fig. 1DGo). The ductal morphology of the Igf1r+/+ (Fig. 1AGo) and Igf1r+/- (Fig. 1BGo) outgrowths appeared normal. In contrast, the Igf1r-/- (Fig. 1CGo) ducts appeared unorganized and displayed irregular branching. Although growth and morphogenesis appeared abnormal in Igf1r-/- outgrowths, studies of hematoxylin- and eosin-stained paraffin sections did not reveal abnormalities at the cellular level (data not shown).

Four-week outgrowths exhibit limited ductal development that is restored by pregnancy
The initial studies of grafts harvested 8 wk posttransplantation established that Igf1r-/- epithelium gave rise to a limited mammary ductal system. To further explore the defect in ductal growth, we performed an analysis of grafts harvested 4 wk posttransplantation. Study of the outgrowths at this time point would allow an analysis of the highly proliferative compartment of the developing ductal system, the TEB, which is absent at the 8-wk point. Representative whole mounts from each genotype are shown (Fig. 2Go, A–C). Igf1r+/+ and Igf1r+/- outgrowths exhibited a greater level of development than Igf1r-/- outgrowths (Fig. 2CGo). Quantitative analysis of the outgrowths showed that after 4 wk, the Igf1r-/- epithelium filled only 17.5 ± 2.4% of the fat pad vs. 66.9 ± 7.4% and 65.9 ± 4.1% for Igf1r+/+ and Igf1r+/- outgrowths, respectively (Fig. 2GGo).



View larger version (42K):
[in this window]
[in a new window]
  		Figure 2. Analyses of ductal outgrowth morphology. Igf1r+/+ (A), Igf1r+/- (B), and Igf1r-/- (C) ductal outgrowths were harvested 4 wk posttransplantation and whole mounted to study growth and morphology. Igf1r+/+ (A) and Igf1r+/- (B) outgrowths show normal ductal and TEB structures. However, study of Igf1r-/- (C) outgrowths reveals a growth-inhibited ductal phenotype. Some hosts were bred 3 wk postgrafting, and the outgrowths were harvested on d 10 of pregnancy. Igf1r+/+ (D), Igf1r+/- (E), and Igf1r-/- (F) grafts from pregnant hosts exhibit increased growth. Ductal development was expressed as the PFPF filled by the ductal outgrowth of Igf1r+/+, Igf1r+/-, and Igf1r-/- grafts in virgin ({blacksquare}) and pregnant ({square}) hosts (G). S, An outgrowth of embryonic skin and hair follicles that was grafted along with the mammary bud. Data are expressed as the mean ± SEM. Numbers in parentheses indicate the number of outgrowths analyzed. Bars with different superscripts differ significantly (P < 0.05).

 
Interestingly, if the host animal was bred 3 wk after grafting and the outgrowths harvested at 10 d of pregnancy (Fig. 2Go, D–F), the extent of ductal elongation in Igf1r-/- grafts (Fig. 2FGo) was restored to levels observed in Igf1r+/+ and Igf1r+/- grafts grown in virgin hosts (Fig. 2Go, A and B). This development was more representative of what would be observed in early pregnant mammary gland development characterized by increased branching and alveolar budding. The extent of growth in Ig1fr+/+ and Igf1r+/- grafts from pregnant hosts was also increased above levels observed in virgin hosts (Fig. 2Go, D and E). Growth in Igf1r-/- grafts from pregnant hosts was 3.5 times greater than growth of Igf1r-/- grafts that were allowed to develop for a similar amount of time in virgin hosts (Fig. 2GGo). Although the extent of growth in Igf1r-/- grafts from pregnant hosts was still significantly less than Igf1r+/+ and Igf1r+/- growth in pregnant hosts, the magnitude of difference was smaller than that observed in grafts from virgin hosts (Fig. 2GGo). Therefore, although Igf1r-/- ductal development was growth inhibited in virgin hosts, the hormonal environment provided by pregnancy was able to partially restore ductal growth.

TEBs in Igf1r-/- outgrowths are smaller
A morphometric study of TEB structures in the 4-wk ductal outgrowths was also performed. Images of whole mounts were used to quantitate TEB number and size. The mean number of TEBs found in Igf1r+/+ and Igf1r+/- outgrowths was 3.6- and 4.0-fold greater, respectively, than that observed in Igf1r-/- outgrowths (Table 1Go). Additionally, the mean TEB area of Igf1r+/+ and Igf1r+/- outgrowths was 31% greater than the mean of Igf1r-/- outgrowths (Table 1Go). These findings indicate that reduced ductal growth in the IGF-IR null tissue is due to reduced TEB development.


View this table:
[in this window]
[in a new window]
  		Table 1. Morphometric analyses of TEBs

 
Igf1r-/- outgrowth histology
Examination of TEB and ductal structures in hematoxylin- and eosin-stained paraffin sections did not reveal any obvious differences in cellular organization of mammary ducts or TEBs among the three genotypes (Fig. 3Go). Higher magnifications of the outgrowths are shown in Fig. 3Go, A–C, to allow closer examination of the TEB structures in gland whole mounts. As described in the TEB morphometric analyses, TEBs in Igf1r-/- outgrowths appeared smaller, but exhibited normal morphology (Fig. 3CGo). Hematoxylin and eosin-stained sections of the whole mounted outgrowths revealed that ductal cell organization was normal in Igf1r-/- outgrowths (Fig. 3FGo). In all three genotypes, a single layer of ductal epithelium lined the lumen (Fig. 3Go, D–F). TEB anatomy also appeared normal (Fig. 3Go, G–I). A layer of cap cells was present in the leading edge of Igf1r-/- end bud structures along with a normal body cell component and lumen (Fig. 3Go, G and I). Therefore, although TEB number and size are reduced in Igf1r-/- outgrowths, the cellular structure of the TEB is normal.



View larger version (130K):
[in this window]
[in a new window]
  		Figure 3. Histology of 4-wk ductal outgrowths. Igf1r+/+ (A), Igf1r+/- (B), and Igf1r-/- (C) whole mounts display TEB structures on the leading edge of the outgrowths. Ductal epithelial organization of Igf1r+/+ (D), Igf1r+/- (E), and Igf1r-/- (F) grafts appears normal in hematoxylin- and eosin-stained sections. All ducts display a single layer of epithelial cells lining the lumen. TEB anatomy also appears normal in Igf1r+/+ (G), Igf1r+/- (H), and Igf1r-/- (I) outgrowths. A cap cell layer at the leading edge of the bud that envelops the body cells can be seen. Additionally, a thickened basal lamina is observed at the neck of the TEB. Classic longitudinal sections of a TEB are illustrated in G and I. The section in H represents a more transverse section, because the thickened basal lamina that is usually present only around the neck of the TEB can be seen surrounding the structure. t, TEB; d, duct; l, lumen; c, cap cells; b, body cells; bl, basal lamina; s, skin and hair follicle grafted along with mammary bud.

 
Differences in TEB cell proliferation account for the limited growth of the Igf1r-/- epithelium
To test whether the limited growth of the Igf1r-/- epithelium was due to decreased proliferation or increased cell death, paraffin sections of outgrowths harvested 4 wk posttransplantation were subjected to anti-BrdU immunohistochemistry or TUNEL analysis (Fig. 4Go). BrdU- or TUNEL-positive cells were counted separately in the TEBs or ductal epithelium of the outgrowths. The number of BrdU-labeled cells was significantly lower in Igf1r-/- TEBs, whereas the ductal epithelium showed no difference in the number of labeled cells among the three genotypes (Fig. 5AGo). The percentage of BrdU-labeled cells in Igf1r-/- TEBs was 40% less than that observed in Igf1r+/+ and Igf1r+/- TEBs (Fig. 5AGo). Closer analysis of the TEBs shown in Fig. 4Go, A and C, suggests that as much as a 2-fold difference in proliferation exists between the cap cells found in Igf1r+/+ TEBs and those found in Igf1r-/- TEBs. The majority (86%) of cells in the cap cell layer of the Igf1r+/+ TEB were BrdU labeled (Fig. 4AGo), in contrast to the minimal number (38%) seen in an Igf1r-/- TEB (Fig. 4CGo). This finding coupled with the observation that Igf1r-/- TEBs are smaller support the conclusion that IGF-IR-dependent proliferation in this very small, but important, population of cells mediates TEB growth and the subsequent development of the mammary ductal system.



View larger version (116K):
[in this window]
[in a new window]
  		Figure 4. BrdU and TUNEL labeling in Igf1r+/+ and Igf1r-/- TEBs. Cell proliferation and apoptosis were detected by anti-BrdU labeling (A and C) and TUNEL assay (B and D), respectively, in grafted mammary epithelial outgrowths. A BrdU-labeled TEB from a Igf1r+/+ outgrowth (A) exhibits extensive labeling (dark nuclei) especially at the leading edge of epithelial cells or cap cells (red arrows), whereas labeling in a TEB from a Igf1r-/- outgrowth shows a significantly less number of labeled cells in the cap cell layer (red arrows; C). TUNEL staining in Igf1r-/- TEBs (D) was similar to that in Igf1r+/+ TEBs (B).

 


View larger version (17K):
[in this window]
[in a new window]
  		Figure 5. Quantitative analysis of cell proliferation and cell death in TEBs and ducts of 4-wk outgrowths. Cell proliferation and apoptosis were detected by anti-BrdU labeling (A) and TUNEL assay (B), respectively, in grafted mammary epithelial outgrowths. Positive cells were counted separately for TEB ({blacksquare}) and ductal structures ({square}). Data are expressed as the mean ± SEM. Numbers in parentheses indicate the number of outgrowths analyzed. a, Significantly less than +/+ and +/- (P < 0.05); b, significantly greater than -/- (P < 0.05); c, significantly greater than +/+ and -/- (P < 0.05).

 
Haploinsufficiency results in increased apoptosis and decreased ductal branching
To determine the importance of apoptosis to IGF-IR-dependent TEB development, cell death rates were determined in paraffin sections of Igf1r+/+, Igf1r+/-, and Igf1r-/- outgrowths using the TUNEL assay (Fig. 4Go, B and D). Surprisingly, Igf1r-/- TEBs or ductal epithelium did not show an increased percentage of TUNEL-positive cells over Igf1r+/+ epithelium (Fig. 5BGo). However, the Igf1r+/- ductal epithelium exhibited a significantly higher number of apoptotic cells than either the Igf1r+/+ or the Igf1r-/- ducts (Fig. 5BGo). Igf1r+/- ductal epithelium exhibited 2.4-fold more apoptotic cells than Igf1r+/+ ducts and 2.6-fold more than Igf1r-/- ducts. Although a statistically significant difference between Igf1r+/- and Igf1r+/+ TEB apoptosis was not observed, Igf1r+/- mammary epithelium exhibited significantly more cell death than either Igf1r+/+ or Igf1r-/- epithelial cells when the percentage of apoptosis among ducts and TEBs was considered as a whole (data not shown). These results suggest that IGF-IR haploinsufficiency results in increased mammary cell apoptosis. Although this increased apoptosis was not large enough to influence the percentage of the fat pad filled by the outgrowth (Fig. 2GGo) or TEB development (Table 1Go), analysis of ductal branching in 4-wk outgrowths revealed that Igf1r+/- ductal systems exhibited a reduction in branches compared with Igf1r+/+ grafts. The ducts in Igf1r+/- grafts had only 66% the number of branches observed in Igf1r+/+ ducts (Fig. 6Go). By contrast, branching in Igf1r-/- outgrowths was only 14% of that observed in Igf1r+/+ and 22% of that observed in Igf1r+/- outgrowths. These data demonstrate links between branching, and proliferation and apoptosis and support the conclusion that IGF-IR haploinsufficiency may inhibit some aspects of mammary gland development. More importantly, these results also clearly show that reduced TEB development in response to the complete loss of IGF-IR signaling is not due to increased apoptosis.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 6. Quantitation of ductal branching in grafted outgrowths. Ductal complexity was quantitated by counting the number of nodes or branch points in the entire gland. Data are expressed as the mean ± SEM. Numbers in parentheses indicate the number of outgrowths analyzed. Bars with different superscripts differ significantly (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These studies demonstrate that targeted disruption of the IGF-IR gene inhibits normal mammary gland ductal development through effects on the development of TEBs. Although this result is not unexpected, it is the first direct in vivo demonstration of the need for the IGF-IR in normal TEB development. Results from the studies of Ruan and co-workers (12) demonstrated that IGF-I regulates mammary ductal development by influencing TEB development. The most striking example of this is in their recent analysis of IGF-I knockout mice (12). Based on these previously published studies, the requirement of IGF-IR for normal TEB development would be predicted. However, the fact that IGF-I can interact with and signal through IR:IGF-IR heterodimers (25, 34) left open the possibility that IGF-IR may not be the sole mediator of IGF-I-dependent TEB development. The results obtained from these grafting studies formally prove that IGF-IR is necessary for TEB development. These results coupled with the recently published observation (28) that targeted mutation of the IR gene does not inhibit mammary ductal development support the conclusion that IGF-IR, not IR, is the dominant mediating receptor in the action of IGF-I on TEB development. Beyond directly proving a requirement for IGF-IR in mammary ductal development, however, these studies contribute several new findings to the understanding of IGF-I action on the mammary gland.

In addition to demonstrating a requirement for IGF-IR within the context of an intact animal, these studies make several novel observations on the mechanism of IGF-I action in the mammary gland. These are as follows. 1) The most dramatic effect of loss of the IGF-IR is decreased proliferation in a very small, but important, population of cells, the TEB cells. 2) Complete loss of the IGF-IR does not affect apoptosis in the TEB. 3) Pregnancy partially restores the development of Igf1r-/- mammary glands. 4) IGF-IR haploinsufficiency increases mammary cell apoptosis and decreases ductal branching. These observations, although limited in the degree to which they define the molecular mechanisms of IGF-I action, serve to identify an important target cell for IGF-I in the mammary gland and provide a basis for more technically challenging studies, which could eventually define IGF-IR-dependent signaling pathways of importance to TEB development.

The cap cell was originally defined by its physical presence in the outer layer of cells on the TEB (1). Ultrastructural and immunohistochemical analyses of TEBs demonstrated that these cells are myoepithelial cell precursors (1, 35). In addition, previously published analysis of cell proliferation within the TEB suggests that proliferation is often highest in the cap cell layer (3, 4). These observations coupled with the finding that proliferation in the Igf1r-/- TEB was inhibited most dramatically in the cap cell layer support the idea that continued study of IGF-I action on this particular cell population within the TEB will yield valuable insights into the role of the IGF-IR in mammary gland development.

The fact that total loss of the IGF-IR reduced proliferation without increasing apoptosis supports the suggestion that stimulation of cell survival pathways by IGF-IR may not be as important to TEB development as stimulation of cell cycle pathways. In this respect, analysis of the phosphorylation state of proteins involved in cell survival, such as Akt (14), or proteins involved in proliferation, such as MAPK (16) or ß-catenin (17, 18), would be valuable. However, because the amount of epithelium in the grafts generated by these studies varies tremendously between Igf1r+/+ and Igf1r-/- tissue, and because the cells of the TEB represent only a fraction of the total cellular mass of the grafts, an immunohistochemical approach would theoretically be the only method by which these signaling events could be detected. Unfortunately, the in situ analysis of the phosphorylation of these proteins appears to only be possible in models in which the proteins are artificially activated above normal levels (36). Current efforts in our laboratory to detect localized phosphorylation of IGF-IR signaling molecules in the TEB have had limited success. However, even if such techniques become possible, the most convincing test of involvement for a specific pathway would rely on genetic crosses of the Igf1r-/- mice with transgenic mice in which specific signaling pathways are constitutively activated. These studies are currently in the planning phase.

The relief of Igf1r-/- mammary cell dependence on IGF-IR signaling during early pregnancy may result from increased levels of E and progesterone that cause secondary changes in local growth factor expression and subsequent activation of alternate growth factor receptors. In other knockout models where mammary development is compromised, multiple cycles of pregnancy and lactation are able to restore mammary gland function (37, 38), indicating that exposure to signals from pregnancy is able to compensate for the loss of otherwise important mammary signaling pathways. The restoration of Igf1r-/- ductal growth observed during pregnancy may also result from changes in mammary cell sensitivity to insulin-like signals mediated by the IR. There is genetic evidence that the IR can mediate the growth-promoting function of IGF-II (39). Additionally, IGF-IR null fibroblasts that overexpress IR are able to grow in serum-free medium supplemented solely with insulin or IGF-II, but not IGF-I (40). Furthermore, changes in insulin responsiveness during the development of rodent mammary epithelium have been documented (41, 42). Carrascosa et al. (41) have shown that increased insulin sensitivity during pregnancy is not due to changes in the level of IR binding, but to up-regulation of IR tyrosine kinase activity. In addition, our own preliminary studies (Lee, A. V., and D. L. Hadsell, unpublished data) suggest that the IGF signaling molecules, IR substrate-1 and –2, are also up-regulated by pregnancy. Again, the ultimate demonstration of these as underlying mechanisms for pregnancy-dependent growth compensation will require genetic crosses of the Igf1r-/- mice with gain of function transgenic mice, as described above.

The most surprising and perplexing result from these studies is the fact that increased apoptosis was observed in the epithelium of Igf1r+/-, but not Igf1r-/-, grafts. The observation that branchpoint number was also reduced in Igf1r+/- supports the surprising conclusion that IGF-IR haploinsufficiency may have biological effects on the mammary gland that would not have been expected based on previously published studies with the Igf1r-/- mice (43). These results also support the suggestion that discreet IGF-IR expression thresholds exist for maintaining cell cycle progression as opposed to cell survival. Studies performed in tumor cells have shown that inhibition of IGF-IR signaling induces apoptosis (21). In contrast, studies performed in normal fibroblast that lack an IGF-IR have shown that these cells are not more susceptible to apoptosis (44). The increased sensitivity of tumor cells to apoptotic signals may be related to the fact that they are rapidly dividing. There is evidence that suggests that cell cycle progression and apoptosis are linked (45). Therefore, the ability to proliferate normally may make the grafted Igf1r+/- epithelium sensitive to a halving of the potential survival signals that would normally come from a full genetic complement of the IGF-IR gene.

In conclusion, we found the IGF-IR to be involved in the processes of cell proliferation and cell death in the developing mouse mammary gland. Moreover, we found the IGF-IR to be required for TEB cell proliferation during virgin morphogenesis and to be less important for pregnancy-dependent growth processes. Future studies involving genetic crosses of the Igf1r-/- mice with transgenic mice that exhibit activation of specific signaling pathways should allow for more definitive analysis of the molecular mechanisms of IGF-IR action on TEB development and mammary tumorigenesis.


   Acknowledgments
 
The authors thank Dr. Argiris Efstratiadis (Columbia University, New York, NY) for the IGF-IR knockout mice, Drs. Peter Young and Gerald Cunha (University of California, San Francisco, CA) for help with the initial transplant studies, Frances Kittrell (Baylor College of Medicine, Houston, TX) for additional help with the embryonic transplant technique, and Jessy George (Children’s Nutrition Research Center, Houston, TX) for technical assistance. The authors also thank Drs. Dan Medina, Jeff Rosen, and Adrian Lee (Baylor College of Medicine, Houston, TX) for critical comments and suggestions on this manuscript, Ms. Leslie Loddeke for editorial assistance, and Ms. Jane Schoppe for secretarial assistance.


   Footnotes
 
This work was supported by federal funds from NIH Grants DK-52197-01 and the U.S. Department of Agriculture/Agricultural Research Service under Cooperative Agreement 58-6250-6001. This work is a publication of the USDA/Agricultural Research Service, Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine and Texas Children’s Hospital (Houston, TX). The contents of this publication do not necessarily reflect the views or policies of the USDA, nor does the mention of trade names, commercial products, or organizations imply endorsement by the U.S. government.

Abbreviations: BrdU, Bromodeoxyuridine; IGF-IR, IGF-I receptor; IR, insulin receptor; PFPF, percentage of the fat pad filled; TEB, terminal end bud; TIFF, tagged-image file format; TUNEL, terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick-end labeling.

Received April 13, 2001.

Accepted for publication July 30, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Williams JM, Daniel CW 1983 Mammary ductal elongation: differentiation of myoepithelium and basal lamina during branching morphogenesis. Dev Biol 97:274–290[CrossRef][Medline]
  2. Russo J, Russo IH 1980 Influence of differentiation and cell kinetics on the susceptibility of the rat mammary gland to carcinogenesis. Cancer Res 40:2677–2687
  3. Dulbecco R, Henahan M, Armstrong B 1982 Cell types and morphogenesis in the mammary gland. Proc Natl Acad Sci USA 79:7346–7350[Abstract/Free Full Text]
  4. Humphreys RC 1999 Programmed cell death in the terminal endbud. J Mammary Gland Biol Neoplasia 4:213–220[CrossRef][Medline]
  5. Humphreys RC, Krajewska M, Krnacik S, Jaeger R, Weiher H, Krajewski S, Reed JC, Rosen JM 1996 Apoptosis in the terminal endbud of the murine mammary gland: a mechanism of ductal morphogenesis. Development 122:4013–4022[Abstract]
  6. Kleinberg DL 1997 Early mammary development: growth hormone and IGF-1. J Mammary Gland Biol Neoplasia 2:49–57[CrossRef][Medline]
  7. Richert M, Wood TL 1999 The insulin-like growth factors (IGF) and IGF type I receptor during postnatal growth of the murine mammary gland: sites of messenger ribonucleic acid expression and potential functions. Endocrinology 140:454–461[Abstract/Free Full Text]
  8. Kleinberg DL 1998 Role of IGF-I in normal mammary development. Breast Cancer Res Treat 47:201–208[CrossRef][Medline]
  9. Hadsell DL, Greenberg NM, Fligger JM, Baumrucker CM, Rosen JM 1996 Targeted expression of des (1–3) human insulin-like growth factor I in transgenic mice influences mammary gland development and IGF-binding protein expression. Endocrinology 137:321–330[Abstract]
  10. Hadsell DL, Murphy KL, Bonnette SG, Reece N, Laucirica R, Rosen JM 2000 Cooperative interaction between mutant p53 and des(1–3)IGF-I accelerates mammary tumorigenesis. Oncogene 19:889–898[CrossRef][Medline]
  11. Walden PD, Ruan W, Feldman M, Kleinberg DL 1998 Evidence that the mammary fat pad mediates the action of growth hormone in mammary gland development. Endocrinology 139:659–662[Abstract/Free Full Text]
  12. Ruan W, Kleinberg DL 2000 Insulin-like growth factor I is essential for terminal end bud formation and ductal morphogenesis during mammary development. Endocrinology 140:5075–5081[Abstract/Free Full Text]
  13. Parrizas M, Saltiel AR, LeRoith D 1997 Insulin-like growth factor 1 inhibits apoptosis using the phosphatidylinositol 3'-kinase and mitogen-activated protein kinase pathways. J Biol Chem 272:154–161[Abstract/Free Full Text]
  14. Kulik G, Klippel A, Weber MJ 1997 Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt. Mol Cell Biol 17:1595–1606[Abstract]
  15. Stiles CD, Capone GT, Scher CD, Antoniades HN, Van Wyk JJ, Pledger WJ 1979 Dual control of cell growth by somatomedins and platelet-derived growth factor. Proc Natl Acad Sci USA 76:1279–1283[Abstract/Free Full Text]
  16. Sasaoka T, Rose DW, Jhun BH, Saltiel AR, Draznin B, Olefsky JM 1994 Evidence for a functional role of Shc proteins in mitogenic signaling induced by insulin, insulin-like growth factor-1, and epidermal growth factor. J Biol Chem 269:13689–13694[Abstract/Free Full Text]
  17. Desbois-Mouthon C, Cadoret A, Blivet-Van Eggelpoel MJ, Bertrand F, Cherqui G, Perret C, Capeau J 2001 Insulin and IGF-1 stimulate the ß-catenin pathway through two signalling cascades involving GSK-3ß inhibition and Ras activation. Oncogene 20:252–259[CrossRef][Medline]
  18. Playford MP, Bicknell D, Bodmer WF, Macaulay VM 2000 Insulin-like growth factor 1 regulates the location, stability, and transcriptional activity of ß-catenin. Proc Natl Acad Sci USA 97:12103–12108[Abstract/Free Full Text]
  19. Neuenschwander S, Schwartz A, Wood TL, Roberts CTJ, Henninghausen L, LeRoith D 1996 Involution of the lactating mammary gland is inhibited by the IGF system in a transgenic mouse model. J Clin Invest 97:2225–2232[Medline]
  20. Moorehead RA, Fata JE, Johnson MB, Khokha R 2001 Inhibition of mammary epithelial apoptosis and sustained phosphorylation of Akt/PKB in MMTV-IGF-II transgenic mice. Cell Death Differ 8:16–29[CrossRef][Medline]
  21. Baserga R, Resnicoff M, D’Ambrosio C, Valentinis B 1997 The role of the IGF-I receptor in apoptosis. Vitam Horm 53:65–98[Medline]
  22. Farrelly N, Lee YJ, Oliver J, Dive C, Streuli CH 1999 Extracellular matrix regulates apoptosis in mammary epithelium through a control on insulin signaling. J Cell Biol 144:1337–1348[Abstract/Free Full Text]
  23. Wood TL, Richert MM, Stull MA, Allar MA 2000 The insulin-like growth factors (IGFs) and IGF binding proteins in postnatal development of murine mammary glands. J Mammary Gland Biol Neoplasia 5:31–42[CrossRef][Medline]
  24. Belfiore A, Frittitta L, Costantino A, Frasca F, Pandini G, Sciacca L, Goldfine ID, Vigneri R 1996 Insulin receptors in breast cancer. Ann NY Acad Sci 784:173–188[Medline]
  25. Kasuya J, Paz IB, Maddux BA, Goldfine ID, Hefta SA, Fujita-Yamaguchi Y 1993 Characterization of human placental insulin-like growth factor-I/insulin hybrid receptors by protein microsequencing and purification. Biochemistry 32:13531–13536[CrossRef][Medline]
  26. Accili D, Drago J, Lee EJ, Johnson MD, Cool MH, Salvatore P, Asico LD, Jose PA, Taylor SI, Westphal H 1996 Early neonatal death in mice homozygous for a null allele of the insulin receptor gene. Nat Genet 12:106–109[CrossRef][Medline]
  27. Liu J-P, Baker J, Perkins AS, Robertson EJ, Efstratiadis A 1993 Mice carrying null mutatuons of the genes encoding insulin-like growth factor I (igf-1) and type 1 IGF receptor (igf1r). Cell 75:59–72[Medline]
  28. Robinson GW, Accili D, Hennighausen L 2000 Rescue of mammary epithelium of early lethal phenotypes by embryonic mammary gland transplantation as exemplified with insulin receptor null mice. In: Ip MM, Asch BB, eds. Methods in mammary gland biology and breast cancer research. New York: Kluwer Academic/Plenum Press; 307–316
  29. Hadsell DL, Bonnette SG 2000 IGF and insulin action in the mammary gland: lessons from transgenic and knockout models. J Mammary Gland Biol Neoplasia 5:19–30[CrossRef][Medline]
  30. DeOme KB, Faulkin Jr LJ, Bern HA, Blair PE 1959 Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H mice. Cancer Res 19:515–520
  31. Medina D, Kittrell FS, Liu YJ, Schwartz M 1993 Morphological and functional properties of TM preneoplastic mammary outgrowths. Cancer Res 53:663–667[Abstract/Free Full Text]
  32. Ball SM 1998 The development of the terminal end bud in the prepubertal-pubertal mouse mammary gland. Anat Rec 250:459–464[CrossRef][Medline]
  33. Hadsell DL, Alexeenko T, Klemintidis Y, Torres D, Lee AV 2001 Inability of overexpressed des(1–3)human insulin-like growth factor I (IGF-I) to inhibit forced mammary gland involution is associated with decreased expression of IGF signaling molecules. Endocrinology 142:1479–1488[Abstract/Free Full Text]
  34. Soos MA, Field CE, Siddle K 1993 Purified hybrid insulin/insulin-like growth factor-I receptors bind insulin-like growth factor-I, but not insulin, with high affinity. Biochem J 290:419–426
  35. Dulbecco R, Unger M, Armstrong B, Bowman M, Syka P 1983 Epithelial cell types and their evolution in the rat mammary gland determined by immunological markers. Proc Natl Acad Sci USA 80:1033–1037[Abstract/Free Full Text]
  36. Li Y, Podsypanina K, Liu X, Crane A, Tan LK, Parsons R, Varmus HE 2001 Deficiency of Pten accelerates mammary oncogenesis in MMTV-Wnt-1 transgenic mice. BMC Mol Biol 2:2[CrossRef][Medline]
  37. Ormandy CJ, Camus A, Barra J, Damotte D, Lucas B, Buteau H, Brousse N, Babinet N, Binart N, Kelly PA 1997 Null mutation of the prolactin receptor gene produces multiple reproductive defects in the mouse. Genes Dev 11:167–178[Abstract/Free Full Text]
  38. Liu X, Gallego MI, Smith GH, Robinson GW, Hennighausen L 1998 Functional release of Stat5a-null mammary tissue through the activation of compensating signals including Stat5b. Cell Growth Differ 9:795–803[Abstract]
  39. Louvi A, Accili D, Efstratiadis A 1997 Growth-promoting interaction of IGF-II with the insulin receptor during mouse embryonic development. Dev Biol 189:33–48[CrossRef][Medline]
  40. Morrione A, Valentinis B, Xu SQ, Yumet G, Louvi A, Efstratiadis A, Baserga R 1997 Insulin-like growth factor II stimulates cell proliferation through the insulin receptor. Proc Natl Acad Sci USA 94:3777–3782[Abstract/Free Full Text]
  41. Carrascosa JM, Ramos P, Molero JC, Herrera E 1998 Changes in the kinase activity of the insulin receptor account for an increased insulin sensitivity of mammary gland in late pregnancy. Endocrinology 139:520–526[Abstract/Free Full Text]
  42. Oka T, Perry JW, Topper YJ 1974 Changes in insulin responsiveness during development of mammary epithelium. J Cell Biol 62:550–556[Free Full Text]
  43. Baker J, Liu J-P, Robertson EJ, Efstratiadis A 1993 Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75:73–82[CrossRef][Medline]
  44. Sell C, Dumenil G, Deveaud C, Miura M, Coppola D, DeAngelis T, Rubin R, Efstratiadis A, Baserga R 1994 Effect of a null mutation of the insulin-like growth factor I receptor gene on growth and transformation of mouse embryo fibroblasts. Mol Cell Biol 14:3604–3612[Abstract/Free Full Text]
  45. Meikrantz W, Schlegel R 1995 Apoptosis and the cell cycle. J Cell Biochem 58:160–174[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Am. J. Pathol.Home page
M. P.V. Shekhar, S. Santner, K. A. Carolin, and L. Tait
Direct Involvement of Breast Tumor Fibroblasts in the Modulation of Tamoxifen Sensitivity
Am. J. Pathol., May 1, 2007; 170(5): 1546 - 1560.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
H.-J. Kim, B. C. Litzenburger, X. Cui, D. A. Delgado, B. C. Grabiner, X. Lin, M. T. Lewis, M. M. Gottardis, T. W. Wong, R. M. Attar, et al.
Constitutively Active Type I Insulin-Like Growth Factor Receptor Causes Transformation and Xenograft Growth of Immortalized Mammary Epithelial Cells and Is Accompanied by an Epithelial-to-Mesenchymal Transition Mediated by NF-{kappa}B and Snail
Mol. Cell. Biol., April 15, 2007; 27(8): 3165 - 3175.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
D.S. Fernandez-Twinn, S. Ekizoglou, B.A. Gusterson, J. Luan, and S.E. Ozanne
Compensatory mammary growth following protein restriction during pregnancy and lactation increases early-onset mammary tumor incidence in rats
Carcinogenesis, March 1, 2007; 28(3): 545 - 552.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
A. V. Loladze, M. A. Stull, A. M. Rowzee, J. DeMarco, J. H. Lantry III, C. J. Rosen, D. LeRoith, K.-U. Wagner, L. Hennighausen, and T. L. Wood
Epithelial-Specific and Stage-Specific Functions of Insulin-Like Growth Factor-I during Postnatal Mammary Development
Endocrinology, November 1, 2006; 147(11): 5412 - 5423.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
M J Meyer, A V Capuco, Y R Boisclair, and M E Van Amburgh
Estrogen-dependent responses of the mammary fat pad in prepubertal dairy heifers.
J. Endocrinol., September 1, 2006; 190(3): 819 - 827.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
T. Vargo-Gogola, B. M. Heckman, E. J. Gunther, L. A. Chodosh, and J. M. Rosen
P190-B Rho GTPase-Activating Protein Overexpression Disrupts Ductal Morphogenesis and Induces Hyperplastic Lesions in the Developing Mammary Gland
Mol. Endocrinol., June 1, 2006; 20(6): 1391 - 1405.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
W. Ruan, F. Fahlbusch, D. R. Clemmons, M. E. Monaco, P. D. Walden, A. P. Silva, H. A. Schmid, and D. L. Kleinberg
SOM230 Inhibits Insulin-Like Growth Factor-I Action in Mammary Gland Development by Pituitary Independent Mechanism: Mediated through Somatostatin Subtype Receptor 3?
Mol. Endocrinol., February 1, 2006; 20(2): 426 - 436.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
S.-Q. Kuang, L. Liao, S. Wang, D. Medina, B. W. O'Malley, and J. Xu
Mice Lacking the Amplified in Breast Cancer 1/Steroid Receptor Coactivator-3 Are Resistant to Chemical Carcinogen-Induced Mammary Tumorigenesis
Cancer Res., September 1, 2005; 65(17): 7993 - 8002.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. M. Carboni, A. V. Lee, D. L. Hadsell, B. R. Rowley, F. Y. Lee, D. K. Bol, A. E. Camuso, M. Gottardis, A. F. Greer, C. P. Ho, et al.
Tumor Development by Transgenic Expression of a Constitutively Active Insulin-Like Growth Factor I Receptor
Cancer Res., May 1, 2005; 65(9): 3781 - 3787.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
M. T. Tilli, R. Reiter, A. S. Oh, R. T. Henke, K. McDonnell, G. I. Gallicano, P. A. Furth, and A. T. Riegel
Overexpression of an N-Terminally Truncated Isoform of the Nuclear Receptor Coactivator Amplified in Breast Cancer 1 Leads to Altered Proliferation of Mammary Epithelial Cells in Transgenic Mice
Mol. Endocrinol., March 1, 2005; 19(3): 644 - 656.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
W. Ruan, M. E. Monaco, and D. L. Kleinberg
Progesterone Stimulates Mammary Gland Ductal Morphogenesis by Synergizing with and Enhancing Insulin-Like Growth Factor-I Action
Endocrinology, March 1, 2005; 146(3): 1170 - 1178.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
R. G. Richards, D. M. Klotz, M. P. Walker, and R. P. DiAugustine
Mammary Gland Branching Morphogenesis Is Diminished in Mice with a Deficiency of Insulin-like Growth Factor-I (IGF-I), But Not in Mice with a Liver-Specific Deletion of IGF-I
Endocrinology, July 1, 2004; 145(7): 3106 - 3110.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. A. Allar and T. L. Wood
Expression of the Insulin-Like Growth Factor Binding Proteins during Postnatal Development of the Murine Mammary Gland
Endocrinology, May 1, 2004; 145(5): 2467 - 2477.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Med.Home page
S. Yeh, Y.-C. Hu, P.-H. Wang, C. Xie, Q. Xu, M.-Y. Tsai, Z. Dong, R.-S. Wang, T.-H. Lee, and C. Chang
Abnormal Mammary Gland Development and Growth Retardation in Female Mice and MCF7 Breast Cancer Cells Lacking Androgen Receptor
J. Exp. Med., December 15, 2003; 198(12): 1899 - 1908.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
A. V. Lee, P. Zhang, M. Ivanova, S. Bonnette, S. Oesterreich, J. M. Rosen, S. Grimm, R. C. Hovey, B. K. Vonderhaar, C. R. Kahn, et al.
Developmental and Hormonal Signals Dramatically Alter the Loca