The Cyclin-Dependent Kinase Inhibitor p27 (Kip1) Regulates Both DNA Synthesis and Apoptosis in Mammary Epithelium But Is Not Required for Its Functional Development during Pregnancy

Elizabeth A. Davison, Christine S. L. Lee, Matthew J. Naylor, Samantha R. Oakes, Robert L. Sutherland, Lothar Hennighausen, Christopher J. Ormandy and Elizabeth A. Musgrove

Cancer Research Program (E.A.D., C.S.L.L., M.J.N., S.R.O., R.L.S., C.J.O., E.A.M.), Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, New South Wales 2010, Australia; and Laboratory of Genetics and Physiology (L.H.), National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0822

Address all correspondence and requests for reprints to: Elizabeth A. Musgrove, Cancer Research Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, New South Wales 2010, Australia. E-mail: e.musgrove{at}garvan.org.au.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Decreased expression of the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) is common in breast cancer and is associated with poor prognosis. p27 is also an important mediator of steroidal regulation of cell cycle progression. We have therefore investigated the role of p27 in mammary epithelial cell proliferation. Examination of the two major functions of p27, assembly of cyclin D1-Cdk4 complexes and inhibition of Cdk2 activity, revealed that cyclin D1-Cdk4 complex formation was not impaired in p27-/- mammary epithelial cells in primary culture. However, cyclin E-Cdk2 activity was increased approximately 3-fold, indicating that the CDK inhibitory function of p27 is important in mammary epithelial cells. Increased epithelial DNA synthesis was observed during pregnancy in p27-/- mammary gland transplants, but this was paralleled by increased apoptosis. During pregnancy and at parturition, development and differentiation of p27+/+ and p27-/- mammary tissue were indistinguishable. These results demonstrate a role for p27 in both the proliferation and survival of mammary epithelial cells. However, the absence of morphological and cellular defects in p27-/- mammary tissue during pregnancy raises the possibility that loss of p27 in breast cancer may not confer an overall growth advantage unless apoptosis is also impaired.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DECREASED EXPRESSION OF the cyclin-dependent kinase inhibitor (CKI), p27 (Kip1), resulting from reduced stability of the protein rather than gene deletion or mutation, is common in many epithelial cancers, including breast cancer (1). Loss of p27 expression in breast cancer is associated with poor prognosis in most studies, although some studies have not found a significant association between p27 expression and outcome (2). Furthermore, p27 expression is decreased in hyperplasias, suggesting that deregulation of p27 expression occurs early in the development of breast cancer (3). These data suggest a role for p27 as a tumor suppressor in breast cancer, consistent with its function as a negative regulator of cell cycle progression and the demonstration that mice with a homozygous or heterozygous disruption of the p27 gene are more susceptible to tumorigenesis in response to radiation and diverse chemical carcinogens (4, 5, 6). In vitro studies have demonstrated that p27 is an important regulator of cellular proliferation in breast cancer cells, where its induction by progestins and antiestrogens is critical to inhibition of proliferation (7, 8, 9, 10). In addition, altered posttranslational modification of p27 has been implicated in resistance to TGFß-mediated growth inhibition of normal human mammary epithelial cells in culture (11). However, relatively little is known about the role of this cell cycle-regulatory molecule in the mammary gland in vivo.

Cell cycle progression is driven by interactions between cyclins, cyclin-dependent kinases (CDKs), and CKIs. The D-type cyclins associated with Cdk4 or Cdk6 are critical during G1 phase, as is cyclin E-Cdk2. Once activated by cyclin binding, these CDKs phosphorylate members of the family of pocket proteins including pRb to allow progress into S phase (12). An additional level of CDK regulation is provided by CKIs including p27 and the related p21 (Waf1, Cip1) and p57 (Kip2). Overexpression of the Cip/Kip CKIs leads to cell cycle arrest in G1 phase, because of their potent inhibition of Cdk2 (12). These inhibitors have a dual role: they stabilize complexes between D-type cyclins and their CDK partners, and so can also function as positive regulators of cell cycle progression (13, 14). A significant fraction of p27 is associated with cyclin D1, leading to the hypothesis that a major function of cyclin D1 is to sequester p27 and thus prevent p27 from inactivating cyclin E-Cdk2. The ability of deletion of p27 to restore normal development in tissues, including the mammary gland, which display defects in mice lacking cyclin D1, illustrates the importance of this role in vivo (15, 16). Mice lacking p27 are larger than their wild-type siblings because of an overall increase in cell number, most obvious in tissues that normally express p27 at the highest levels (17, 18, 19), demonstrating that p27 is a critical negative regulator of cell proliferation in vivo.

Mammary gland development is initiated in the embryo and occurs in defined stages associated with sexual development and reproduction, under the influence of ovarian, placental, and pituitary hormones (20). In the prepubertal mouse, the mammary epithelium forms a rudimentary ductal structure extending into the mammary fat pad from the nipple. Initiation of ovarian hormone secretion at the onset of puberty accelerates ductal growth and branching (21, 22). The final stages of development do not occur until pregnancy, when the epithelial cells within the ducts undergo extensive proliferation and differentiation, resulting in expansion of the lobuloalveolar compartment and differentiation of epithelial cells necessary for milk secretion (21, 22). Recent studies using knockout mice have documented critical roles for a number of hormones (estrogen, progesterone, and prolactin) as well as transcription factors (signal transducer and activator of transcription 5A and CCAAT enhancer binding protein-ß) in mammary development during puberty and pregnancy (21, 22). However, despite the necessity for precise temporal and spatial control of proliferation during mammary development, relatively few studies have examined the role of cell cycle-regulatory molecules in this process. A notable exception is the demonstration that cyclin D1 is necessary for normal alveolar development and lactation (23, 24). In addition, although pRb is not required for mammary development (25), expression of phosphorylation-resistant pRb alleles impairs mammary development and leads to precocious differentiation (26). In this study we have investigated the role of p27 in proliferation of mammary epithelial cells and development of the mammary gland, using mice lacking p27. The function of p27 as a CKI suggested that, in the absence of p27, mammary epithelial cell proliferation might be increased, consistent with a role for decreased p27 expression in loss of growth control during tumorigenesis. Our data confirm this hypothesis but also demonstrate increased apoptosis. As a result the overall morphology and histology of mammary tissue are the same in the presence or absence of p27. This is in contrast with data published during the course of this investigation (27), which assigned an essential role to p27 in normal mammary development.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Decreased Expression of p27 during Pregnancy
To establish whether p27 expression was altered during physiological regulation of proliferation in normal mammary tissue, we first determined the pattern of p27 expression during pregnancy. Immunohistochemistry using sections of paraffin-embedded mammary glands from mature female mice at estrus and at various stages though pregnancy revealed intense nuclear p27 staining in the epithelial cells, as shown in Fig. 1AGo. At estrus approximately 90% of the ductal epithelium expressed p27, but by 2.5–4.5 dpc (days postcoitus) this was reduced to approximately 60% (Fig. 1BGo; 2.5 dpc, P = 0.0008; 3.5 dpc, P = 0.002; 4.5 dpc, P = 0.0008 compared with estrus). The proportion of p27-positive cells rose at 5.5 dpc (no significant difference at 5.5 dpc compared with estrus, P = 0.1) but subsequently decreased again and remained below that present at estrus until at least 15.5 dpc (Fig. 1Go; 7.5 dpc, P = 0.05; 11.5 dpc, P = 0.001; 15.5 dpc P = 0.04 compared with estrus). In the same mammary glands, bromodeoxyuridine (BrdU) staining was low at estrus, but increased significantly by 2.5 dpc (2.5 dpc, P = 0.002; 3.5 dpc, P = 0.04; 4.5 dpc, P = 0.01 compared with estrus), returned toward control at 5.5 dpc (P = 0.06), but then remained elevated thereafter (Fig. 1AGo; 7.5 dpc, P = 0.009; 11.5 dpc, P = 0.0001; 15.5 dpc, P = 0.002 compared with estrus). When the proportions of BrdU-positive and p27-positive cells were quantitated, it was clear that p27 expression during pregnancy mirrored the amount of DNA synthesis (Fig. 1BGo). The inverse relationship between p27 and BrdU positivity was statistically significant: P = 0.005; r2 = 0.32. The close inverse relationship between p27 expression and DNA synthesis is consistent with the hypothesis that p27 might play an important role in control of epithelial cell proliferation as the mammary gland develops during pregnancy.



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Fig. 1. Relationship between Expression of p27 and DNA Synthesis in Murine Mammary Tissue during Pregnancy

A, Immunohistochemical analysis of p27 protein expression and BrdU incorporation (as a measure of DNA synthesis) using paraffin sections of mammary tissue from 16-wk-old C57BL/6;129 x 1/SvJ female mice at estrus (E) and at 2.5 and 11.5 dpc. Bar, 50 µm. B, Quantitation of p27 protein expression (diamonds) and BrdU incorporation (squares) within the luminal epithelium at estrus (E) and during pregnancy. Data are presented as mean ± SEM or range of two to four animals for each timepoint, except BrdU incorporation at 3.5 dpc (n = 7) and 7.5 dpc (n = 11). In some cases the SEM or range is smaller than the size of the symbol used.

 
Formation of Cyclin D1-Cdk4 Complexes in Mammary Epithelial Cells in Culture Does Not Require p27
To examine the biochemical consequences of loss of p27 in the absence of interference from stromal and other contaminating cell types that would be present in lysates of whole mammary glands, we used primary mammary epithelial cell (MEC) cultures from mice lacking functional p27 due to deletion of the cyclin-CDK interaction domain (18). The phenotype of these mice is apparently identical to that of mice lacking the entire coding region of p27 (17, 19), and so they are referred to here as p27-/-. The inguinal mammary glands from p27-/- and p27+/+ mice were dissected, the epithelial cells were purified by several rounds of collagenase digestion, and the resulting MECs were harvested after several days culture. Initially, we examined whether the absence of functional p27 led to alterations in the level of other cell cycle-regulatory proteins. Western blot analysis of MECs derived from p27+/+ and p27-/- mammary tissue demonstrated no difference in the expression of cyclin D1, cyclin D3, cyclin E, Cdk4, Cdk2, or p21 in samples normalized for expression of cytokeratin 18, an epithelial cell marker (Fig. 2AGo and Table 1Go). To investigate whether the absence of functional p27 affected complex formation between cyclin D1 and Cdk4, cyclin D1 immunoprecipitates from MEC lysates were Western blotted using an anti-Cdk4 antibody. This revealed that neither cyclin D1 nor Cdk4 levels were significantly different in immunoprecipitates of MEC lysates from p27+/+ and p27-/- glands (Fig. 2BGo and Table 1Go), clearly demonstrating that there was no impairment of complex formation in MECs lacking p27. In the absence of functional p27, however, there was a significant increase in the association between cyclin D1 and p21 (Fig. 2BGo and Table 1Go). No corresponding increase in overall p21 abundance was observed (Fig. 2AGo and Table 1Go), suggesting altered distribution of p21 among its binding partners in the absence of p27 and indicating a role for p21 in the stabilization of cyclin D1-Cdk4 complexes in this context.



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Fig. 2. Cyclin, CDK, and CKI Expression and Complex Formation in Cultured MECs

A, MECs were derived from the inguinal mammary glands of 16-wk-old mice and harvested after 4 d primary culture. Representative Western blots of MEC lysates from two independent cultures (each containing MECs derived from the pooled mammary glands of two or three animals) examining cyclin D1, cyclin D3, cyclin E, Cdk4, Cdk2, p27, and p21 expression are shown. The same membranes were also blotted for the epithelial cell marker cytokeratin 18 as a loading control. B, MEC lysates prepared as in panel A were immunoprecipitated using cyclin D1 antibodies and then Western blotted using antibodies to cyclin D1, Cdk4, and p21 as indicated. Immunoprecipitates of two independent cultures (each containing MECs from two or three animals) for each genotype are shown.

 

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Table 1. Cyclin, CDK, and CKI Levels in MEC Lysates and Immunoprecipitates

 
Cyclin E-Cdk2 Activity Is Increased in the Absence of p27
The data in Fig. 2Go indicate that the assembly factor function of p27 is not essential in MECs. To examine the other major function of p27, CDK inhibition, cyclin E-Cdk2 activity was measured in MECs in culture. This kinase is active during G1 phase and is an important target for p27. However, it is tightly regulated in concert with changes in proliferation rate, and we therefore initially examined regulation of cyclin E-Cdk2 activity during culture of p27+/+ MECs. Freshly purified MECs displayed low proliferation rates (measured by BrdU incorporation) consistent with the low proliferation rates of mammary epithelium in vivo in virgin animals. However, the proportion of BrdU-positive cells increased to 10–15% by 3 d (data not shown). The abundance of p27 was initially high (Fig. 3AGo), consistent with the widespread expression of p27 detected by immunohistochemistry in virgin mice (Fig. 1Go), but decreased with increasing time in culture (Fig. 3AGo). Over the same timeframe, cyclin E-Cdk2 activity increased (Fig. 3AGo). These data suggested that p27 inhibition of cyclin E-Cdk2 was likely to be of most significance in the initial stage of culture, before BrdU incorporation had reached its peak, and we therefore compared cyclin E-Cdk2 activity in p27-/- and p27+/+ MEC cultures harvested after 1–2 d of culture. Cyclin E-Cdk2 activity was increased 3-fold in p27-/- MECs compared with p27+/+ MECs (P = 0.013, Fig. 3BGo). In longer-term cultures there was no apparent difference in the cyclin E-Cdk2 activity of MECs derived from p27-/- and p27+/+ glands (data not shown), consistent with the low levels of p27 in p27+/+ MECs at these time points.



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Fig. 3. Increased Cyclin E-Cdk2 Activity in p27-/- MECs

A, MECs were derived from the inguinal mammary glands of 16-wk-old p27+/+ mice. Lysates were prepared after 2 or 3 d primary culture and Western blotted using antibodies to p27. The kinase activity of cyclin E immunoprecipitates was measured using histone H1 substrate. B, Lysates were prepared from MECs derived from p27+/+ or p27-/- mice after 1–2 d primary culture. The kinase activity of cyclin E immunoprecipitates was measured using histone H1 substrate and quantitated. Data are the mean ± SEM of five independent cultures of p27+/+ MECs (representing 10 animals) and three independent cultures of p27-/- MECs (five animals).

 
Loss of p27 Has No Effect on the Development, Architecture, and Differentiation of Mammary Epithelium
Data presented in Figs. 2Go and 3Go indicated that the principal role of p27 in mammary epithelial cell proliferation in vitro was likely to be inhibition of CDKs including cyclin E-Cdk2, rather than acting as an assembly factor for cyclin D1-Cdk4. This implied that loss of p27 in the mammary gland in vivo might lead to increased cell cycle progression and consequent mammary hyperplasia. Since female p27-/- mice are infertile, it was necessary to perform mammary transplants to investigate the effect of lack of p27 on mammary development during pregnancy. Mammary fragments from p27-/- females were transplanted into host mammary fat pads that had been cleared of endogenous epithelium, while the contralateral cleared mammary fat pads received mammary fragments from p27+/+ females. The host animals were prepubertal Rag 1-/- animals, which lack T and B cells (28) and will thus accept allografts, but are endocrinologically normal. After a 10-wk interval, the host animals were mated and the transplanted mammary glands analyzed at intervals during pregnancy.

Under the influence of pubertal hormones from the host, mammary epithelium from the transplanted fragments formed a mammary ductal structure that was morphologically similar to the endogenous mammary glands, but lacked connection to the nipple. Mature virgin animals displayed a branched ductal tree, which often extended to the edges of the host fat pad (Fig. 4AGo). The transplanted epithelium underwent further development during pregnancy to form numerous alveolar buds by 13.5 dpc (Fig. 4AGo), but no differences in gross morphology between p27+/+ and p27-/- glands were apparent. Quantitation of side branching and alveolar bud development in whole mounts of transplanted mammary glands showed, as expected in mature animals, little increase in the number of ductal branches but a progressive increase in the number of alveolar buds as the gland developed through pregnancy (Fig. 4BGo). There was, however, no significant difference between p27+/+ and p27-/- transplants for either of these parameters at any time point examined. Microscopic examination of hematoxylin and eosin-stained sections of these mammary glands did not reveal any differences in tissue architecture between p27-/- and p27+/+ glands either in virgin glands or throughout pregnancy, i.e. there were no apparent increases in the number of cells constituting the epithelium of individual ducts (data not shown).



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Fig. 4. Morphology of p27+/+ and p27-/- Mammary Transplants

A, Whole mounts of mammary tissue derived from transplanted p27+/+ or p27-/- epithelium at estrus and at 5.5 and 13.5 dpc were carmine stained. Bar, 2.5 mm. B, The number of ductal branches and lobuloalveolar buds per 1 mm2 area of transplanted p27+/+ (open boxes) or p27-/- (closed boxes) mammary tissue were quantitated at estrus and at 5.5 and 13.5 dpc. Data were obtained from three to four animals for each time point.

 
The lack of any alteration in morphology in p27-/- transplants was unexpected given the increased cyclin E-Cdk2 activity documented in Fig. 3Go. To establish whether the lack of aberrant development was due to the specific gene knockout mouse strain, one of the authors (L.H.) investigated mammary development in p27-/- mice, which lack the entire coding region (17) rather than lacking the cyclin-CDK-interacting region alone. These mice are on a 129 genetic background and were purchased from the Jackson Laboratory (Bar Harbor, ME). Mammary transplantation experiments were again performed. Tissue fragments from p27-/- females and p27+/+ littermates were transplanted into cleared contralateral fat pads of nude mice, which were mated after 8 wk. At parturition, p27-/- and p27+/+ mammary tissue were histologically indistinguishable (Fig. 5Go), confirming the results of the mammary transplants in Fig. 4Go.



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Fig. 5. Histology of p27+/+ and p27-/- Mammary Tissue at Parturition

Transplanted p27+/+ or p27-/- mammary tissue was collected at parturition, paraffin embedded, and stained using hematoxylin and eosin. Bar in upper panels, 300 µm; bar in lower panels, 50 µm.

 
Increased DNA Synthesis in the Absence of p27
To determine whether the absence of p27 led to alterations in cell proliferation despite the lack of effect on overall morphology, markers of proliferation were measured in p27-/- and p27+/+ mammary transplants. BrdU immunohistochemistry was performed to determine the number of epithelial cells undergoing active DNA synthesis. Serial sections were stained for expression of the proliferating cell nuclear antigen (PCNA), which is expressed in early G1 and S phase and thus has been used as a marker of actively proliferating cells. BrdU-positive and PCNA-positive epithelial cells were present in the epithelium of transplanted mammary glands from both p27-/- and p27+/+ mice, but were more frequent in p27-/- transplants (Fig. 6AGo). Comparison of data from 12 glands with 0–40% PCNA-positive epithelium demonstrated that the number of BrdU-positive cells was proportional to the number of PCNA-positive cells (r2 = 0.6; P = 0.006), and thus only the results from PCNA-stained sections are shown in Fig. 6BGo. Quantitation of the proportion of epithelial cells displaying nuclear staining confirmed consistent elevation of both BrdU and PCNA positivity in p27-/- epithelium (Fig. 6BGo and data not shown). The approximately 2.5-fold increase in PCNA-positive cells was statistically significant at both 5.5 dpc (P = 0.05) and 13.5 dpc (P = 0.03). Thus, although loss of p27 did not affect the overall morphology of the gland, it led to an increase in proliferation, consistent with the increased cyclin E-Cdk2 activity observed in p27-/- MECs. One possible explanation for the apparently normal development of p27-/- mammary transplants despite altered CDK activity in p27-/- MECs in culture is that, because MEC proliferation during pregnancy is strongly steroid-dependent but MEC proliferation in vitro is not, p27 may not perform the same function in both experimental models. However, the increase in proliferation of p27-/- epithelium during pregnancy suggests that p27 is playing a similar role in both contexts, principally as an inhibitor of cyclin E-Cdk2, i.e. that its function in mammary epithelium is not dependent on the factor(s) stimulating proliferation, consistent with the established role of p27 as a target for multiple mitogenic stimuli.



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Fig. 6. Increased Proliferation in p27-/- Mammary Transplants

A, Immunohistochemical detection of BrdU incorporation and PCNA expression in p27+/+ and p27-/- mammary transplants at 13.5 dpc. Bar, 50 µm. B, Quantitation of PCNA expression in p27+/+ (open boxes) and p27-/- (shaded boxes) mammary transplants. Data were obtained from three to five transplants per genotype at each time point, except p27+/+ at 5.5 dpc (n = 2).

 
Increased Apoptosis in the Absence of p27
The increased proliferation but normal morphology of the p27-/- mammary transplants raised the possibility that increased apoptosis was also occurring in the absence of p27. We therefore quantitated apoptosis in the transplanted mammary glands. Both in situ end labeling of DNA strand breaks [terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL)] and immunohistochemical staining for active caspase 3, an effector caspase involved in the DNA fragmentation that characterizes apoptosis, were increased in p27-/- transplants (Fig. 7AGo). The number of caspase 3-positive cells (Fig. 7BGo) increased approximately 3-fold, an increase that was statistically significant at all time points (virgin, P = 0.025; 5.5 dpc, P = 0.01; 13.5–15.5 dpc, P = 0.05). Similarly, the increase in TUNEL-positive cells (Fig. 7BGo) was statistically significant in both virgin animals (P = 0.05) and during pregnancy (13.5–15.5 dpc, P = 0.02). The proportion of nuclei displaying the morphological features of apoptosis (i.e. condensed nuclei) was also increased from 0.2% in p27+/+ transplants to 0.8% in p27-/- transplants (P = 0.003 overall). All three measures indicate increased apoptosis in p27-/- mammary tissue and suggest that, in the absence of p27, increased apoptosis balances increased rates of epithelial cell proliferation to result in a mammary ductal structure that is apparently indistinguishable from wild type in terms of numbers of epithelial cells, overall tissue architecture, and macroscopic morphology.



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Fig. 7. Increased Apoptosis in p27-/- Mammary Transplants

A, Representative active caspase 3 staining in p27+/+ and p27-/- epithelium at 5.5 dpc and TUNEL staining at 13.5 dpc. Bar, 50 µm. B, Quantitation of TUNEL positivity (open boxes) and active caspase 3 staining (hatched boxes) in p27+/+ (open boxes) and p27-/- (shaded boxes) mammary transplants. Data were obtained from four to seven transplanted glands per genotype at each time point except at 5.5 dpc (n = 2 for each genotype) and TUNEL-stained p27+/+ virgin transplants (n = 2). Transplants examined at 13.5 and 15.5 dpc gave similar results and consequently data have been pooled.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have examined the consequences of loss of the CKI, p27, in mammary epithelial cells as a means of gaining insight into its role in normal mammary development and the potential consequences of its decreased expression in breast cancer. Biochemical analysis of p27-/- MECs in culture revealed that loss of p27 resulted in increased cyclin E-Cdk2 activity, but had no effect on cyclin D1-Cdk4 assembly. DNA synthesis during pregnancy was increased approximately 2.5-fold in transplanted p27-/- mouse mammary epithelium compared with transplanted wild-type epithelium. However, the increased mammary epithelial DNA synthesis during pregnancy was accompanied by an increase in apoptosis, indicating that p27 functions in regulation of both proliferation and survival in this tissue. These observations are consistent with the increased Cdk2 activity and hyperplasia observed in other organs in p27-/- mice (17, 18, 19) but contrast with results from another laboratory reported while this study was in progress (27). Although they reported increased proliferation resulting from loss of a single p27 allele, Muraoka and colleagues (27) observed impaired development of transplanted p27-/- mammary epithelium during pregnancy, accompanied by decreased rates of proliferation and delayed differentiation. In whole mammary gland extracts from p27-/- mice, they found significantly decreased levels of cyclin D1 expression (relative to an epithelial cell marker, cytokeratin 14) and failure to form cyclin D1-Cdk4 complexes with consequent marked decreases in Cdk4 activity (27). In contrast, Cdk2 activity was comparable in p27-/- and p27+/+ mammary gland extracts (27). Thus in their hands the absence of p27 principally results in lack of cyclin D1 function, such that the phenotype of p27-/- mammary glands mirrors that of cyclin D1-/- glands (23, 24, 29), while in our hands the major consequences were increases in cyclin E-Cdk2 activity, proliferation, and apoptosis.

Crosses between p27-/- and cyclin D1-/- mice have been generated in two laboratories (15, 16). Analysis of these mice reveals that ablation of p27 restores normal development to cyclin D1-dependent tissues, including the retina and mammary gland. These data are in contrast with what would be predicted if both cyclin D1-/- and p27-/- mammary epithelia exhibit impaired mammary development. In that case an additional, mammary-specific, pathway would need to be activated to allow mammary epithelial cell proliferation and the development of the mammary gland in the absence of both cyclin D1 and p27 (22). However, if loss of p27 activates Cdk2 in mammary epithelium, as demonstrated here, and can do so in the absence of cyclin D1, proliferation and consequent mammary development in mice lacking both cyclin D1 and p27 could be driven by cyclin E-Cdk2, analogous to the ability of cyclin E to replace cyclin D1 in the mammary gland (30) and consistent with our findings of increased proliferation in p27-/- mammary epithelium.

The basis of the differences between our studies and those of Muraoka et al. (27) remains to be determined. The differences do not appear to arise because of differences in animal strains, because we have used the same strain of p27-/- mice as Muraoka et al. and present additional data from an independent strain of p27-/- mice that also displayed normal mammary morphology when transplanted onto immunocompromised recipients. Our data are also in accord with the results of experiments performed in a third laboratory as controls in an investigation of the phenotype of mice lacking both cyclin D1 and p27 (15). Thus, data from independent experiments in three different laboratories and using different p27-/- mouse strains indicate that p27-/- mammary epithelium forms a gland of normal morphology during pregnancy. It is possible that differences in the details of the experimental protocols used in each laboratory contribute to differences in the morphology of transplanted mammary glands, although this would not also account for differences in expression of cell cycle-regulatory proteins in MECs from p27-/- females. One possible factor investigated was the age of the animals used, since Muraoka et al. (27) used 6-wk-old animals as transplant donors and prepared whole mammary lysates from 3-wk-old animals, whereas our initial studies used older animals, typically approximately 16 wk old. However, when we repeated our studies using 6- to 8-wk-old animals, the development of transplanted p27-/- epithelium during pregnancy was not impaired, and there were no differences in cyclin D1 levels in MEC cultures from 5- to 6-wk-old animals. Further experimentation, e.g. direct comparison of p27-/- epithelium from different laboratories within a single experiment, will be required to resolve this issue.

The striking inverse relationship between p27 expression and DNA synthesis during pregnancy is consistent with the established role for p27 as a CKI with a fundamental role in maintenance of quiescence and inhibition of transition from G1 into S phase (12). The change in p27 expression during pregnancy may simply be a consequence of altered proliferation. However, because MECs derived from the mammary glands of p27-/- mice have increased levels of cyclin E-Cdk2 activity, and p27-/- mammary epithelium displays increased DNA synthesis during pregnancy, decreased p27 expression during pregnancy may be a cause of changes in the DNA synthetic rate. Given the steroid hormone dependence of proliferation during pregnancy, this points to p27 as a potential mediator of steroid hormone regulation of proliferation in mammary epithelium in vivo, consistent with data from breast cancer cells in vitro (7, 8, 9, 10). In another steroid-responsive tissue, uterine epithelium, estrogen-mediated cell cycle progression is accompanied by decreased p27 expression, and both cyclin E-Cdk2 and cyclin A-Cdk2 activities are increased in the absence of p27 (31). Although the absence of p27 did not impair the ability of progesterone to inhibit estrogen stimulation of these cells, these data again suggest p27 as a steroid-regulated CDK inhibitor.

The cyclin D1-Cdk4 complexes that formed in the absence of p27 contained increased levels of the related CKI, p21, indicating that p21 can substitute for p27 as a cyclin D1-Cdk4 assembly and stabilization factor in MECs. This was not the case for p27 inhibition of MEC cyclin E-Cdk2 activity, perhaps because the relatively low levels of p21 expressed in the mammary gland are insufficient to perform both functions. The pRb-family member p130 can bind to cyclin E-Cdk2, compensating for absent CKIs and allowing normal CDK regulation in fibroblasts lacking both p27 and p21 (32). However, there was no evidence for p130 binding cyclin E-Cdk2 in MEC lacking p27, perhaps also because p130 is expressed at low levels in these cells (our unpublished data).

Increased apoptosis in the absence of p27 is not observed in all tissues, although increasing evidence suggests that p27 may provide protection from apoptosis under conditions of cellular or physiological stress (33, 34, 35), and decreased rates of apoptosis have been described in some cancer cell lines after enforced overexpression of p27 (36, 35). One possible mechanism for increased apoptosis in the absence of p27 is that it is triggered by deregulated Cdk2 activity. However, because increased apoptosis has been observed in some (33, 37), but not all (17, 18, 19), p27-/- cell types exhibiting increased Cdk2 activity and proliferation, this does not seem to be a general mechanism. A further possibility is that the increased rates of apoptosis observed in p27-/- mammary epithelium may be a consequence of increased proliferation. Significant apoptosis occurs in the lumen of the terminal end buds of the developing mammary gland, and this is thought to be important for ductal morphogenesis (38). A recent study has shown that enhanced proliferation after expression of cyclin D1 or the HPV16 E7 oncoprotein is not sufficient to impair lumen formation in mammary acini in vitro, but rather is balanced by increased apoptosis to maintain a hollow glandular architecture (39). The authors suggested that the apoptosis might be triggered by the inability of some cells to maintain basement membrane contact, with associated loss of polarity (39). Similar mechanisms could possibly lead to apoptotic cell death of the excess ductal cells produced by increased proliferation in p27-/- mammary epithelium.

In summary, our studies of MEC proliferation and mammary gland development in the absence of p27-/- provide evidence for a role for p27 in controlling both the proliferation and survival of normal mammary epithelial cells and raise questions over the implications of decreased p27 expression in breast cancer. If reduced p27 expression increases both proliferation and apoptosis in breast cancers it may not lead to increased tumor burden unless the apoptotic machinery is disrupted. It would thus be of interest to investigate the relationships between p27 levels, apoptotic rates, and outcome in clinical breast cancer.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mice
A colony of p27-/- and p27+/+ mice was established at the Garvan Institute from founder animals kindly provided by Dr. Andrew Koff (Memorial Sloan-Kettering Cancer Center, New York, NY). The p27 coding sequence in these mice is disrupted so that it produces an amino-truncated version of the p27 protein ({Delta}51) that lacks the cyclin-CDK interaction domain and is nonfunctional (18). Wild-type and knockout animals were maintained on a mixed C57Bl/6–129/SvJ background. Littermates were used when possible. All animal experimentation was conducted in accordance with accepted standards of humane animal care. Mice were genotyped by PCR analysis of genomic DNA using the following primers: ACGTGAGAGTGTCTAACGG, AGTGCTTCTCCAAGTCCC and GCGAGGATCTCGTCGTGAC (40). These yielded products of 120 bp (wild-type p27) and 400 bp (neomycin). Rag1-/- mice on a C57/BL6 genetic background (28) were purchased from the Animal Resource Centre, Perth, WA, Australia. All animals were housed with food and water ad libitum with a 12-h light, 12-h dark cycle at 22 C.

Cell Culture
Primary cultures of MECs were prepared from p27+/+ and p27-/- animals by limited collagenase digestion essentially as described previously (41). Briefly, both inguinal mammary glands were surgically removed from mature (16 wk old) female mice. Each gland was finely chopped and washed in HEPES-buffered RPMI 1640 medium supplemented with 2.5% fetal calf serum. The tissue was then subjected to approximately four 1-h rounds of digestion at 37 C with 1–2 mg/ml collagenase type L (Sigma-Aldrich Corp., Castle Hill, New South Wales, Australia) in HEPES-buffered RPMI 1640. The resulting mammary organoids were passed through sterile 400-µm polyester mesh (Small Parts, Inc., Miami Lakes, FL). Organoids derived from two to three animals (i.e. four to six mammary glands) were pooled and then plated into fibronectin-coated 25-cm2 tissue culture flasks in primary culture medium (1:1 DMEM/Hams F-12 catalog no. 11330, Life Technologies, Inc., Melbourne, Victoria, Australia) containing 5 µg/ml insulin, 10 ng/ml epidermal growth factor (Promega Corp., Annandale, New South Wales, Australia), 5 µg/ml hydrocortisone (Sigma), 5 ng/ml cholera toxin (Sigma), 0.01 U/ml penicillin (Life Technologies, Inc.), 10 ng/ml streptomycin (Life Technologies, Inc.), 20 µg/ml gentamicin), and 10% fetal calf serum.

Western Blot Analysis, Immunoprecipitation, and Kinase Assays
MEC cultures were harvested by trypsinization and pelleted by centrifugation. The cell pellets were lysed by incubation on ice (5 min) in lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 10% (vol/vol) glycerol, 0.1% Tween-20, 1 mM EDTA, 2.5 mM EGTA, 10 mM ß-glycerophosphate, 10 µg aprotinin/ml, 10 µg leupeptin/ml, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM sodium orthovanadate, 1 mM NaF]. Cellular debris was cleared by centrifugation, and the lysates stored at -80 C.

For immunoprecipitation, 100 µg of MEC lysate were precleared by incubation for 1 h at 4 C with protein G-Sepharose beads (Zymed Laboratories, Inc., South San Francisco, CA) and then immunoprecipitated by incubation for 2 h at 4 C with cross-linked anticyclin D1 antibody (72–13G, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Antibody cross-linking was performed as described previously (42). The immunoprecipitates were washed with lysis buffer and resuspended in SDS-PAGE sample buffer [63 mM Tris-HCl (pH 6.8), 10% (vol/vol) glycerol, 2% SDS, 5% ß-mercaptoethanol].

Samples of immunoprecipitated or total protein in SDS-PAGE sample buffer were denatured for 3 min at 95 C and then separated by SDS-PAGE and transferred to Immuno-Blot polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc., Hercules, CA). Transfer was confirmed by staining with 0.5% Ponceau S in 10% acetic acid. The membranes were incubated for 2 h at room temperature with the following primary antibodies: cyclin D1 (Ab-3; Neomarkers, Fremont, CA); cyclin D3 (C-16), cyclin E (M-20), Cdk2 (M2), Cdk4 (C-22), and p21 (M-19) from Santa Cruz Biotechnology, Inc.; p27 (K25020; Transduction Laboratories, Inc., Lexington, KY); and cytokeratin 18 (RDI-PRO 61028; Research Diagnostics, Inc., Cleveland, OH).

The histone H1 kinase activity of cyclin E immunoprecipitates (100 µg MEC lysate) was measured as previously described (43) using 10 µg histone H1 as substrate. Little or no background phosphorylation was detected in samples immunoprecipitated by using beads without antibody.

Mammary Gland Transplants
Mammary epithelial transplants were performed as follows. Pieces (1 mm3) of mammary gland excised from mature (>=16 wk old) donors were transplanted into a cleared mammary fat pad in a 3-wk-old Rag 1-/- recipient, i.e. after removal of the endogenous mammary rudiment. Mammary gland portions from one p27-/- donor were transplanted into the cleared inguinal (no. 4) fat pads of several recipients. The contralateral inguinal fat pads were also cleared and transplanted with p27+/+ mammary gland sections. In all, 96.4% of transplants were successful. At least 8 wk after transplant, mammary glands were analyzed in virgin animals, at 5.5 and 13.5–16.5 dpc. The morning of plug detection was designated 0.5 dpc. In one set of transplant experiments, mammary transplants from 6- to 8-wk-old p27-/- donor animals were compared with transplants from age-matched p27+/+ animals or 17-wk-old p27-/- animals by implantation into contralateral glands. Analysis of these transplants revealed that the age of the donor animal did not affect the morphology, proliferation, or apoptotic rate of the transplanted glands; therefore, data from donor animals of different ages have been pooled.

One set of transplant experiments used p27-/- animals obtained from The Jackson Laboratory. These mice lack the entire coding region of p27 (17) and were in a 129 background. Mammary tissue from p27-/- and p27+/+ littermates was transplanted into cleared fat pads of 3-wk-old nude mice, which were then mated after 8 wk. Mammary tissue was isolated at parturition for histological analysis.

Morphological examination of the transplanted glands was used to determine that the observed ductal outgrowth was the result of transplanted epithelium and not the result of endogenous epithelium. Ductal outgrowth resulting from transplanted epithelium originates from the transplant site at the center of the gland, whereas a ductal structure originating from the edge of the gland indicates outgrowth that is derived from endogenous epithelium (44).

Whole-Mount Analysis
For morphological evaluation of transplanted mammary glands, the endogenous third mammary gland and the fourth glands (containing the mammary transplants) were dissected from the skin, spread onto a SuperFrost Plus glass slide (Menzel-Glaser, Braunscheig, Germany), and fixed in 10% neutral buffered formalin. For examination of p27 expression during pregnancy, the fourth mammary glands were dissected and fixed in the same way. Mammary gland whole mounts were defatted in acetone before carmine alum (0.2% carmine, 0.5% aluminium sulfate) staining overnight. The whole mount was dehydrated using a graded ethanol series followed by xylene treatment for 60 min and storage in methyl salicylate (45). Ductal development was examined by low-power microscopy using a Leica M212 microscope and photographed with a Leica DC200 Camera and Leica DC viewer (Leica Microsystems, Wetzlar, Germany).

Histological and Immunohistochemical Analysis
After morphological evaluation, mammary gland whole mounts were removed from the glass slide, embedded in paraffin, and sectioned for histological and immunohistochemical analysis. Histology was examined in paraffin sections that had been dewaxed, rehydrated, and stained with hematoxylin and eosin. To measure DNA synthesis, animals were injected ip with BrdU dissolved in PBS (100 µg BrdU/g body weight), 2 h before euthanasia by cervical dislocation. Immunohistochemical detection of p27, BrdU, PCNA, and activated caspase 3 was performed using a DAKO autostainer (DAKO A/S, Glostrup, Denmark). Paraffin sections (4 µm) were dewaxed and rehydrated. Antigen retrieval was performed using citrate EDTA buffer (DAKO) boiled under pressure for p27, low pH target retrieval solution (DAKO) at 100 C for 30 min for BrdU and PCNA detection, and 10 mM trisodium citrate boiled under pressure for 2 min for active caspase 3. Endogenous peroxidase activity was inhibited with 3% H2O2, and blocked in 10% normal horse serum. The ARK peroxidase kit (DAKO) was used for biotinylation of mouse monoclonal antibodies. Slides were incubated with the following biotin-conjugated mouse monoclonal antibodies: anti-p27 at 1:100 (Kip 1/p27; Transduction Laboratories, Inc.), anti-BrdU at 1:50 (Bu20a; DAKO), anti-PCNA at 1:1250 (PC10; DAKO). The active caspase 3 rabbit polyclonal antibody (R&D Systems, Inc., Minneapolis, MN) was used at 1:6000. The sections were counterstained with Witlock’s hematoxylin and Scott’s blue. Detection of apoptosis in paraffin sections by TUNEL analysis was performed using the DeadEnd Colorimetric TUNEL System (Promega Corp., Annandale, New South Wales, Australia), according to the manufacturer’s instructions. At least 1000 epithelial cells per gland were counted for each animal at x400 magnification using a Leica DMRB microscope and photographed using a Leica DC200 Camera and Leica DC viewer (Leica Microsystems).

Statistical Analysis
ANOVAs were performed using StatView statistical software (Abacus Concepts, Inc., Berkeley, CA), with P values determined using Fisher’s projected least significant difference test.


    ACKNOWLEDGMENTS
 
We thank Dr. Andrew Koff for providing a breeding pair of p27+/- mice, Dr. Danielle Lynch for advice on the preparation of MEC cultures, and Melanie Trivett for advice on caspase 3 staining. We are grateful to Drs. Darren Saunders and Keiko Miyoshi for assistance with some experiments, and thank Dr. Julie Ferguson, Tony Chaplin, Eric Schmied, and other staff of the Garvan Institute animal care facility for advice and expert animal care.


    FOOTNOTES
 
This research was supported by the US Army Medical Research and Materiel Command (under Grants DAMD17-00-1-0252 and BC022015), the National Health and Medical Research Council of Australia, and The Cancer Council, New South Wales. M.J.N. was the recipient of a University of New South Wales Faculty of Medicine Dean’s Research Scholarship.

Abbreviations: BrdU, Bromodeoxyuridine; CDK, cyclin-dependent kinase; CKI, cyclin-dependent kinase inhibitor; dpc, days post coitum; MEC, mammary epithelial cell; PCNA, proliferating cell nuclear antigen; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling.

Received for publication May 30, 2003. Accepted for publication August 15, 2003.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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