CG&D
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cell Growth & Differentiation

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
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
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Said, T. K.
Right arrow Articles by Medina, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Said, T. K.
Right arrow Articles by Medina, D.
Cell Growth & Differentiation Vol. 12, 285-295, June 2001
© 2001 American Association for Cancer Research

Cyclin-dependent Kinase (cdk) Inhibitors/cdk4/cdk2 Complexes in Early Stages of Mouse Mammary Preneoplasia1

Thenaa K. Said2, Ricardo C. B. Moraes, Uma Singh, Francis S. Kittrell and Daniel Medina

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The level of circulating ovarian hormones (estrogen and progesterone) alone or in combination with pituitary hormones have a potent mitogenic impact in the normal mammary gland, and they also play a pivotal role in the development and progression of mammary carcinoma. The differential effects of hormones on the molecular components of cyclin-dependent kinase (cdk) complexes in mammary epithelium of the hormone-dependent ductal outgrowth line, EL11, and the hormone-independent alveolar outgrowth line, TM2L, were the focus of this study. The two outgrowth lines, which represent early stages in mammary hyperplasia, were compared with normal mammary gland at different hormonal conditions: control, hormone stimulated by pituitary isograft, and hormone depleted by ovariectomy. Hormonal stimulation by a pituitary isograft resulted in DNA synthesis and lobuloalveolar development of normal mammary ducts, DNA synthesis but no lobuloalveolar development in the EL11 ductal outgrowth, and no changes either in DNA synthesis or in lobuloalveolar morphology in the TM2L outgrowth. The levels of cdk4- and cyclin D1-associated kinase activities were correlated with cell proliferation in only the alveolar phenotypes (i.e., in only hormonally stimulated normal virgin gland and in alveolar mammary outgrowth), whereas cyclin D2-dependent kinase activity was correlated with cell proliferation in only the alveolar preneoplasia. p16INK4a and p21Cip1 protein levels were decreased at the earliest stages of preneoplasia, i.e., at immortalization, and were independent from changes in cyclin D1, which occurred later in preneoplasia. Although all cdk inhibitors changed in concordance with hormonal status reflected by proliferation levels, p27Kip1 was the only cdk inhibitor that was up-regulated at the earliest stages of preneoplasia and may have a unique role in blocking alveolar differentiation in response to the loss of one or more of the cell cycle-negative regulators. We hypothesize that up-regulation of p27Kip1 prevents immortalized ductal outgrowths (EL11) from progressing to the neoplastic state, even under hormonal stimulation.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell growth is directed by complex molecular cascades that involve a number of genes, all acting in concert to achieve the biochemical and structural changes required for correct cell duplication. In particular, cell cycle progression is controlled by the products of timely expressed genes (e.g., cyclins and cdks3 ; Refs. 1 and 2 ). The cyclins, the concentration of which in the cell fluctuates characteristically during the different phases of the cell cycle, act predominantly by association with, and thereby regulating, the catalytic subunits of various cdks (2) . In mammalian cells, D-type cyclins (D1–3) and their cdk partners are involved in G1 regulation (1) . The three D-type cyclins show considerable structural and functional homologies with each other, and in certain instances, they may complement each other functionally. On the other hand, some studies have demonstrated that specific isoforms of D-cyclins, particularly cyclins D2 and D3, act to maintain nonproliferative signals and promote differentiation or cell growth arrest (3 , 4) . The differential expression and biological/functional relevance of a specific D-cyclin appear to depend on the cell lineage (5) , intracellular concentration (4 , 6) , influence of other genes (3) , and the stage at which a cell undergoes differentiation or proliferation (7 , 8) .

In mouse mammary tumor models, overexpression of cyclin D1 in virgin mammary gland results in mammary hyperplasia (9) and a very low frequency of carcinoma. Sufficient cyclin D1 expression is obligatory for lobuloalveolar differentiation (10 , 11) of the mammary gland but not ductal elongation or maturation. Estrogen and progesterone hormones stimulate cyclin D1 expression, whereas antiestrogen and antiprogesterone block cyclin D1 expression (12) . On the other hand, it has been reported that cyclin D2 is less efficient than cyclin D1 for G1 progression in the human breast carcinoma cell line T47D (13) . The overexpression of cyclin D1 protein occurs in 80% of invasive lobular breast carcinoma (14) ; however, neither DNA amplification nor mRNA overexpression of cyclin D2 was detected in 1171 and 132 breast cancers, respectively (15) .

A growing family of cdkIs (reviewed in Refs. 5 and 16, 17, 18, 19, 20 ) negatively regulates activities of the G1 cyclin-cdk complexes. Members of the INK4 proteins [p16/INK4A (21) , p15/INK4B (22 , 23) , p18/INK4C (22 , 24) , and p19/INK4D (24 , 25) ] and the Cip/Kip families [p21/Cip1 (26, 27, 28, 29) , p27/Kip1 (30, 31, 32) , and p57/kip2 (33 , 34) ] act by inhibiting cdk4/6 and cdk2 (35) . These kinases are responsible for the coordinate phosphorylation and inactivation of the growth-suppressive functions of Rb (36, 37, 38) . pRb and the related proteins, p107 and p130, act by redirecting or sequestering transcription factors regulating genes required for S-phase (39 , 40) .

Mammary luminal epithelial cells operate within a complex epithelial-stromal environment, where there are numerous opportunities and examples of paracrine interactions (41 , 42) . Because hormones regulate mammary epithelial cell growth, this study focused on the growth regulation of mammary epithelial cells during early stages of neoplastic transformation in vivo and at different hormonal states. In this study, we selected early lesions in mammary preneoplasia represented by well-characterized in vivo mammary outgrowth lines: the immortalized, nontumorigenic, hormone-dependent ductal outgrowth, EL11; and the immortalized, hormone-independent alveolar hyperplasia, TM2L, and compared them with normal mammary gland. We examined morphological development, DNA synthesis, cdkI complexes, and cyclin D1- and cyclin D2-dependent kinase activities in these three groups in control, ovariectomized, and hormonally stimulated conditions.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Effect of Hormones on Mammary Gland Development at Early Stages of Tumorigenesis.
The three groups, normal virgin gland (i.e., duct), immortal hormone-dependent duct (EL11), and hormone-independent alveolar outgrowths (TM2L) were examined in control, hormone-depleted (ovariectomized), and hormone-stimulated conditions by pituitary isograft. Analysis of the morphology and organization of the mammary epithelium by whole mounts and histological sections was correlated with BrdUrd immunohistological staining, which determined the location and number of mammary gland cells in S-phase. In the control condition, the virgin gland is ductal, the EL11 outgrowths are ductal with sparse lateral alveolar buds, and the TM2L outgrowths possess uniform alveolar development equivalent to late pregnancy of normal mammary glands (Fig. 1)Citation . After ovariectomy, inhibition of ductal branching and reduction of duct thickness were observed in the normal virgin gland and to a lesser extent in the EL11 outgrowth (Fig. 1, A and B)Citation , and ducts did not completely fill the fat pads in both groups. This observation was supported by a 95 and 61% decrease in the BrdUrd indexes in normal virgin and EL11 outgrowths (Fig. 2)Citation . In the ovariectomized TM2L hyperplasia, no change in morphology was observed; however, a 36% decrease in BrdUrd index was observed versus the TM2L control, indicating that the TM2L cells were still marginally responsive to steroid hormones. In the groups hormonally stimulated by pituitary isografts, the virgin gland exhibited a significant increase in ductal branching and alveolar development (Fig. 1A)Citation . Extensive growth was confirmed by the 104% increase in BrdUrd index (Fig. 2)Citation , mainly in the alveolar structures. Lobuloalveolar development failed to occur when normal virgin gland in ovariectomized mice was stimulated by a pituitary isograft (Fig. 1A)Citation ; the effect of pituitary stimulation was limited to thicker ducts. The growth stimulation in EL11 ductal outgrowths was limited to an increase in ductal side branching, but no alveolar structures were observed (Fig. 1B)Citation with 59% increase in BrdUrd-labeled cells compared with control (Fig. 2)Citation . The TM2L cells revealed no change morphologically (Fig. 1C)Citation and only a 4% increase in BrdUrd index (Fig. 2)Citation . These results showed one major exception from the extensive literature data on hormonal regulation of normal mammary development. Whereas both normal duct and immortalized duct responded to ovarian depletion by a decrease in proliferation and responded to hormone supplementation by an increase in proliferation, the immortal duct population failed to differentiate in response to hormones.



View larger version (83K):
[in this window]
[in a new window]
 
Fig. 1. Whole-mount analysis of inguinal mammary glands from normal and early stages in tumorigenesis at different hormonal conditions. Whole mount of virgin normal mammary gland (A), EL11 ductal outgrowth (B), and TM2L alveolar outgrowth (C) were analyzed at control, ovariectomized (OVX), and pituitary isograft-stimulated (+Pit) states. Hormone deprivation by ovariectomy in A–C exhibited differences in branching morphogenesis only in virgin and EL11 ductal outgrowths but not in TM2L alveolar outgrowths. Stimulation by pituitary isograft resulted in a striking difference in alveolar development between virgin gland (A) and EL11 outgrowth (B). Arrows, the most prominent features in each group at a specific hormonal status. Bar, 5 mm. The image on the left of each whole mount is an equal enlarged portion of the mammary gland on the right.

 


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 2. BrdUrd incorporation into mammary epithelial cells. Epithelial cells positive for BrdUrd incorporation were counted from a total 1500 cells/section, and the means were calculated from six fat pads/group; bars, SE. The numbers at the top of the columns represent the percentage of BrdUrd-labeled cells that increased or decreased compared with their control counterpart.

 
Total Protein Content in Mammary Epithelial Cells in Early Tumorigenesis and at Different Hormonal Conditions.
The mammary gland, particularly in the virgin gland, is heterogeneous because it is composed of mammary epithelial, adipose, and fibroblast stromal cells. To compare changes in only the mammary epithelial cells at different stages of development and hormone treatment, the epithelial cells were isolated as highly purified populations, as described in "Materials and Methods." As can be surmised from the protein yields (Fig. 3)Citation , a large number of inguinal glands were pooled to do the analyses and were repeated in a separate pool of glands at least once. Protein content per 105 mammary epithelial cells increased by 2.4- and 6.5-fold in control EL11 and TM2L outgrowths compared with virgin (Fig. 3)Citation . After ovariectomy, protein content decreased by 2–2.7-fold in virgin and EL11, respectively, and was marginally lower in TM2L outgrowths compared with their control counterparts. The protein content in the hormonally stimulated groups increased by 2.7-fold in the normal virgin gland, whereas EL11 and TM2L outgrowths revealed marginal increases (1.3-fold) compared with their controls (Fig. 3)Citation . These data suggest that changes in the hormone status affect total protein content in the mammary epithelium in a similar pattern in all of the three groups. All data discussed in this study were normalized to the µg total protein/105 epithelial cells in each group and at each specific hormonal status (Fig. 3)Citation .



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 3. Total protein contents in mammary epithelial cells. Epithelial cells from four to eight fat pads were isolated, and protein was extracted as described in "Materials and Methods." By using the bicinchoninic acid protein assay kit, protein contents per 105 mammary epithelial cells were determined in each group and at each hormonal status. Numbers on the top of the columns represent the fold increase or decrease in total protein content compared with normal mammary virgin gland.

 
Effect of Hormone Status on cdk4 and Cyclin D1- and Cyclin D2-associated Kinase Activities in Early Mammary Preneoplasia.
Our previous study (43) and others (44 , 45) have reported that cyclin D1 expression was increased by estrogen and/or progesterone in normal mammary gland. There are fewer data on the effect of hormones on cyclin D2 expression and its associated kinase activities in mammary tumorigenesis. After quantitation and normalization of the data, the total protein levels of cdk4 and cyclins D1 and D2 in the immortal mammary outgrowths (EL11 and TM2L) showed no significant changes with hormonal status (Fig. 4, A, Ba, Bc, and Bd)Citation . However, upon hormonal stimulation, the cdk4 protein level increased 2.5-fold in the normal mammary gland (Fig. 4, A and Ba)Citation . Although the total protein levels of cyclin D1 were detectable only in the TM2L alveolar outgrowth at all three hormone conditions, cyclin D2 was detectable in all of the three groups and at all three hormonal conditions (Fig. 4, A, Bc, and Bd)Citation .



View larger version (46K):
[in this window]
[in a new window]
 
Fig. 4. Protein levels of cell cycle regulatory molecules in mammary tissues at different hormone conditions. A, total protein levels of cdk4, cdk2, cyclin D1, cyclin D2, p21Cip1, p27Kip1, and actin as control for equal sample loading. +Pit, pituitary isograft stimulated. B, histograms for the same proteins in A after normalization, the results of the protein content in 105 mammary epithelial cells of each group, and the results at each hormonal status. All proteins were analyzed by Western blot in 500 µg of tissue protein extract/sample. Antibodies were tested for specificity prior to use. The virgin/ovariectomized (OVX) sample is missing in the cdk4 panel. All protein bands were visualized by ECL detection reagent system after different exposure times against BioMax Kodak films. The intensity of the protein bands was scanned densitometrically using the Phosphorimager SF analyzer. Numbers on top of columns in B represent the number of fold increase or decrease compared with the internal control of each group. +pit, pituitary isograft stimulated.

 
Of more interest are the effects of hormonal status of the mammary glands with cyclin D1- and cyclin D2-associated kinase activities. Cyclin D1- and cyclin D2-associated kinase activities were measured using full-length RB protein as a substrate. pRB is a preferential substrate to measure cdk4 activities, whereas histone H1 is a preferential substrate to measure cdk2 kinase activity. cdk4 kinase activity reflected the total activity of cdk4 associated with all three cyclin Ds. At control status, a 6-fold increase in cdk4 activity in the alveolar mammary preneoplasia (i.e., TM2L) was observed compared with normal and immortal ductal mammary epithelial cells (Fig. 5A)Citation . The cdk4 kinase activity was elevated (4.6-, 2-, and 2.3-fold) upon hormone stimulation in normal virgin gland, ductal EL11, and alveolar TM2L outgrowths, respectively, compared with their control counterparts (Fig. 5A)Citation . After ovariectomy, no change was observed in all of the three groups. These results suggest that the increase in cdk4 activity correlated with the mammary alveolar phenotype.



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 5. cdk4-, cyclin D1-, and cyclin D2-associated kinase in early mammary preneoplasia at different hormonal conditions. Tissue protein extracts (500 µg) per sample were precleared with normal rabbit serum and then immunoprecipitated with either 4 µg of anti-cdk4 polyclonal antibody, 3 µg of anti-cyclin D1 polyclonal antibody, or anti-cyclin D2 monoclonal antibody. Full-length pRb (p110RB) was used as substrate in all of the assays, as described in "Materials and Methods." Histograms represent relative levels of phosphorylated pRB (substrate), scanned and quantified densitometrically by the Phosphorimager SF analyzer, and results were normalized as described earlier. Numbers on the top of the columns are fold increase or decrease compared with the internal control in each group.

 
Cyclin D1- and cyclin D2-associated kinase activities represent their activities in association with both cdk4 and cdk6. In the control condition, cyclin D1-associated kinase activity increased moderately in TM2L outgrowths, whereas the EL11 outgrowths showed no change compared with normal mammary gland (Fig. 5B)Citation . After ovariectomy, the level of cyclin D1-associated kinase activity was 2-fold lower in only the normal mammary gland, with no change in EL11 and TM2L outgrowths (Fig. 5B)Citation . If we put the results of cdk4 and cyclin D1-associated kinase activities together with cyclin D2-associated kinase activity (Fig. 5C)Citation , we observe that upon hormone stimulation, cdk4, and cyclin D1-associated kinase activity increased 4.6- and 3.2-fold, 2- and 2.4-fold, and 2.3- and 3.5-fold in normal gland, EL11 outgrowths, and TM2L outgrowths, respectively, whereas cyclin D2-associated kinase activity was increased (2.3-fold) in only the alveolar preneoplastic TM2L outgrowths at the same hormone condition compared with control (Fig. 5)Citation . Depletion of hormones did not alter cdk4 and cyclin D1- and cyclin D2-associated kinase activities in all three groups compared with their control status (Fig. 5)Citation . These data suggest that marked elevations in cdk4 and cyclin D1-associated kinase activities are necessary to the alveolar phenotype in both hormonedependent (i.e., virgin) and hormone-independent (i.e., TM2L) outgrowths but not to the immortal ductal (i.e., EL11) outgrowths. These data suggest that cyclin D2-associated kinase activity increased substantially only after transformation of the mammary epithelium from immortal ductal to alveolar preneoplasia.

Hormonal Effect on cdkI Interactions to cdk4/2.
cdk2 protein levels increased by 5- and 6-fold in the immortal ductal (EL11) and alveolar (TM2L) outgrowths compared with normal virgin mammary gland, respectively (Fig. 4, A and Bb)Citation . Upon hormone stimulation, the cdk2 level increased by 2-fold only in the normal virgin mammary gland, whereas no change was observed in EL11 and TM2L outgrowths and only marginal reduction after ovariectomy (Fig. 4Bb)Citation .

The p16INK4a protein inhibits the association between cdk4/6 and D-type cyclins and thereby blocks cyclin D-directed cdk4/6 phosphorylation of RB (21 , 46) . The p16INK4a protein levels in 100 µg of protein samples were undetectable in all three groups, whereas, p21Cip1 and p27Kip1 were detected easily (Fig. 4A)Citation . Using 500 µg of protein, samples were sufficient to detect p16INK4a, p21Cip1, and p27Kip1 associated with cdk4 or cdk2 (Fig. 6)Citation . After quantification and normalization of the data at control status, it appeared that p16INK4a-cdk4 protein levels were at a detectable levels in virgin mammary gland but decreased by 1.6- and 4.2-fold in EL11 and TM2L hyperplasia, respectively (Fig. 6ACitation and Table 1Citation ). Interestingly, p16INK4a associated with cdk4 increased by 2.5- and 1.5-fold after ovariectomy in EL11 and TM2L outgrowths, respectively, compared with their control levels. In contrast, upon hormonal stimulation, p16INK4a-cdk4 levels decreased by 5-, 3-, and 4.2-fold in the three groups, respectively (Fig. 6ACitation and Table 1Citation ).



View larger version (52K):
[in this window]
[in a new window]
 
Fig. 6. Interaction of cdkIs to cdk4 and cdk2 in early mammary preneoplasia at different hormonal conditions. Tissue protein extracts (500 µg) per sample were precleared with normal rabbit serum and then immunoprecipitated with either 4 µg of anti-cdk4 or 3 µg of anti-cdk2 polyclonal antibodies, followed by Western blot analysis using anti-p16INK4a rabbit polyclonal antibody (A), antimouse p21Cip1 antibody (B), or p27Kip1 mouse monoclonal antibody (C and D). Protein bands were revealed after ECL detection reaction and exposure to BioMax Kodak films for 3–10 min. Proteins of interest were scanned and quantified densitometrically by the Phosphorimager SF analyzer. Histograms present arbitrary units of cdkI-cdk4 or cdk2 after data were normalized to the total protein level in 105 epithelial cells in each sample. Numbers on top of columns represent the number of fold increase or decrease compared with the internal control of each group.

 

View this table:
[in this window]
[in a new window]
 
Table 1 Effect of hormonal status on cdkI association with cdk4 and cdk2 at early stages in mammary preneoplasia

 
In the control condition, the p21Cip1 protein level was detectable in normal virgin gland, 2-fold higher in the immortal EL11 cells, and decreased by 7-fold in TM2L outgrowths (Fig. 4, A and Be)Citation . After ovariectomy, p21Cip1 protein levels increased in all three groups but decreased with hormone stimulation (Fig. 4Be)Citation . The pattern of p21Cip1-cdk2 was consistent in all hormonal manipulations. For example, in the control condition, p21Cip1-cdk2 levels decreased 3.7-fold in EL11 and 4.2-fold in TM2L outgrowths. After ovariectomy, the level increased 1.7-, 2.8-, and 1.5-fold in the three groups, respectively (Fig. 6BCitation and Table 1Citation ). With hormone stimulation, the p21Cip1-cdk2 level decreased 12.6-, 1.7-, and 4.9-fold in virgin gland, EL11, and TM2L outgrowths, respectively, compared with their controls (Fig. 6BCitation and Table 1Citation ). These data suggest that hormone manipulation regulates p21Cip1-cdk2 interaction in a reproducible manner but has the strongest effects on the hormone-dependent mammary gland (i.e., virgin and EL11).

Because p27Kip1 is a universal inhibitor of all cdks, it can inhibit the kinase activities of cyclin Ds complexed to cdk4, cdk6, and cyclins D1, E, and A complexed with cdk2, and cyclin B complexed to cdc2 (reviewed in Ref. 47 ). After normalization of the Western blot results for total p27Kip1 protein level, we observed 6- and 2-fold increases in total p27Kip1 protein levels in EL11 and TM2L mammary outgrowths, respectively, compared with normal virgin mammary gland in control hormone conditions (Fig. 4Bf)Citation . After ovariectomy, the total protein level of p27Kip1 decreased marginally in all of the three groups as compared with their control counterparts. However, in these same samples, the level of p27Kip1-cdk4/cdk2 protein increased primarily in EL11 mammary outgrowths (4.7- and 2.2-fold), respectively (Fig. 6, C and DCitation ; Table 1Citation ). In contrast, upon hormone stimulation, we observed profound decreases (2.0–10-fold) in both total (Fig. 4Bf)Citation and complexed p27Kip1 protein to cdk4 and cdk2 in virgin gland and TM2L mammary outgrowths (Fig. 6, C and DCitation ; Table 1Citation ). The exception was in the EL11 ductal outgrowths, where levels of p27Kip1 complexed to cdk4/cdk2 increased with hormone stimulation compared with their respective controls. These data are intriguing and suggest that overexpression of p27Kip1 in the immortalized ductal outgrowths may be a compromise to the decreases in p16INK4a and p21Cip1 in cdk4/cdk2 complexes and may have a critical role in blocking alveolar development.

Mammary Tumor Development at Different Hormonal States.
The tumorigenicity of these mammary phenotypes was followed for a 1-year period. Virgin gland and EL11 immortalized ductal outgrowths did not form tumors in any of the three treatment groups. The absence of tumors in EL11 was in the presence of significant levels of DNA synthesis. The TM2L hormone-independent, immortal alveolar hyperplastic outgrowth produced a tumor incidence of 61, 53, and 74% in control, ovariectomized, and hormonally stimulated animals, respectively (Table 1)Citation . The differences in tumor incidence are not statistically significant (P > 0.05). The latent period was increased by 12 weeks in the hormonally depleted mice; however, tumors developed rapidly thereafter. These results may reflect the decrease in DNA synthesis by 36% after ovariectomy (Fig. 2)Citation .


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Mammary gland development is regulated by the interplay of systemic hormones, local growth factors, and the reciprocal relay of cell-cell interactions between the epithelium and surrounding stroma (48) . A number of previous studies have indicated that the normal proliferation of mammary gland epithelium, as well as the initiation and progression of mammary tumorigenesis, was dependent on the ovarian steroid, estrogen (44 , 49) . Our previous study has provided in vivo support for a significant proliferative role of progesterone, in addition to estrogen, in the murine mammary gland (43) . In addition, progesterone exerts proliferative effects on cultured human breast cancer cells (12) .

The cdkI inhibitory mechanisms are based on their expression level and on their competence to bind and deactivate cdk complexes. The balance between cyclin, cdk, and cdkI expression and phosphorylation status predict their interactions and activity. We found that these interactions are altered by hormone manipulation in mammary epithelial cells.

The progressive decreases in the level of p16INK4a-cdk4 from normal mammary duct, to immortalized duct, to immortalized alveoli suggest a permissive environment for cell proliferation but not the cause of immortalization, because p16INK4a was detectable in all three groups. The cause of immortalization in the ductal and alveolar mammary cells might be attributed to the loss of p15INK4b (50) or to p19ARF (51) . The p19ARF gene product was found to be lost at the protein and RNA levels in EL11 and was very low in TM2L outgrowths.4 The reduced levels of p16INK4a complexes to cdk4 occur in a predictable fashion for permissive proliferation (48) but do not correlate with tumorigenic potential, because the immortal EL11 cell population does not produce tumor, even after hormone stimulation (52) . Hormone manipulation affected the level of p16INK4a-cdk4 complexes because the hormone-sensitive immortal EL11 cells were most responsive to ovariectomy, which suggests that ovariectomy may be effective at the earliest stages in mammary ductal lesions. Upon hormone stimulation, the profound decrease in p16INK4a-cdk4 was predicted in the three groups.

In the control condition, the pattern of gradual decreases in p21Cip1-cdk2 from normal to immortal ductal to alveolar phenotype was similar to that of p16INK4a and was in parallel with increases in BrdUrd indexes. The decrease in p21Cip1-cdk2 may indicate transformation of the normal mammary epithelial cells to preneoplasia. However, hormone deprivation or stimulation induced or reduced the level of free p21Cip1 and p21Cip1-cdk2, respectively, in mammary cells with a difference in their hormone responsiveness. For instance, the normal mammary gland was most responsive to ovariectomy and hormone stimulation, then EL11 was next, followed by the least hormone-independent TM2L preneoplastic cells. Thus, this mouse mammary model represents a gradual loss of hormone responsiveness as mammary epithelial cells progress from normal, to immortal ductal, to alveolar.

Most striking is the effect of hormone status on p27Kip1 expression and interaction with cdk4/2 in the early stages of mammary preneoplasia. It has been reported that p27Kip1 is an inhibitor of the cyclin D1-cdk4 and cyclin E-cdk2 complexes (20 , 30) . In agreement with the notion that cdk2 needs to be active for DNA replication to occur (53 , 54) , in the control condition, the alveolar TM2L cells exhibited the highest BrdUrd index and were able to develop tumor. The ductal EL11 cells were unable to develop tumor (52) and exhibited a BrdUrd index similar to that in the normal gland (Table 1)Citation . These results suggest that p27Kip1 is the only cdkI that was profoundly up-regulated as free and in complex with cdk2 in the immortal ductal EL11 outgrowths and moderately up-regulated in the alveolar TM2L preneoplasia. It is well known that increases in p27Kip1 have been associated with cell growth arrest (30, 31, 32) , cell differentiation (55, 56, 57) , or an increase in apoptotic activities (58, 59, 60) , whereas decreases in p27Kip1 have been correlated with several types of malignancies, including breast cancer (61 , 62) . Because the two mammary outgrowths (i.e., EL11 and TM2L) are immortal, one may predict that up-regulation of the p27Kip1 protein level may have occurred after the cells had lost one of more of their negative regulators. It is conceivable that concomitantly or after the loss or attenuation of p19ARF in EL11 and TM2L cell populations, p27Kip1 was up-regulated and associated with cdk2 as a compensatory mechanism to control cell growth. In EL11 cells, overexpression of p27Kip1 may result in blocked differentiation, whereas in alveolar TM2L cells, a stage after immortalization, cells may have differentiated prior to loss of p27Kip1 expression.

Of interest is that upon hormone stimulation, total p27Kip1 protein levels remained high only in the EL11 outgrowths, whereas levels were decreased in the alveolar mammary cells (normal and TM2L cells). These data raise the possibility that accumulation of p27Kip1 in the EL11 cell population may impede mammary cell differentiation. Unlike virgin ductal gland or TM2L outgrowths, it is important to note that upon hormonal stimulation, increases in p27Kip1-cdk4/cdk2 were found only in EL11 immortal ductal outgrowths (Table 1)Citation . The EL11 cells were unable to differentiate but only were able to proliferate, as indicated by BrdUrd indexes and the mild increases in cdk4 and D1-associated kinase activities after hormone stimulation. It has been reported that at stoichiometric levels, p27Kip1 (and p21Cip1) allows D cyclins to bind cdks and thus serves as an "assembly" factor for cyclin D-cdk complexes (20 , 63 , 64) , suggesting that D-cyclins cause redistribution of p27Kip1 from cyclin E-cdk to cyclin D-cdk complexes (20) , a role that is kinase independent in cell progression by controlling the activity of cyclin E via titration of p27Kip1 (20) . Because all kinase activities examined in the present study were D-cyclin-related activities, studies of cyclin E/cdk2 activity on the same mammary tissues and at the same hormonal status are warranted to prove whether this mechanism exists in this model. Both normal and immortal ductal mammary glands exhibited similar proliferation rates but differences in their differentiation potentials. It is plausible that a difference in differentiation potential may control the activity of cyclin E-cdk2 via p27Kip1 interaction in only a subset of cells that possess the potential to differentiate (65) .

Cyclin D1 overexpression and associated kinase activity and cdk4 activity correlated with the alveolar preneoplastic cells at all hormone conditions and with the differentiated normal mammary gland only after hormone stimulation. These data were in agreement with other reports that demonstrated a correlation between increases in cyclin D1 and progression in human breast cancer, because cyclin D1 gene is frequently amplified and/or overexpressed in both primary human breast carcinoma and breast carcinoma cell lines (10 , 62 , 66, 67, 68, 69) . Although cyclin D2 was detectable in all three groups, its associated kinase activity correlated only with alveolar preneoplasia (TM2L) but not with alveologenesis. It has been reported that cyclin D2 preferentially associates with cdk2 in breast cancer epithelium (70) ; however, in this study, we have used only pRb as a substrate to measure cyclin D1- and cyclin D2-associated kinase activities, which reflected predominantly cyclin D1-cdk4/6 and cyclin D2-cdk4/6 but not cdk2 activities. It is conceivable that the level of cyclin D2 concentration in the mammary epithelial cells is critical in forming either active or inactive D2/cdk complexes at a defined hormonal state. Thus, cyclin D2/cdk2 complexes may foster cell cycle progression in the TM2L alveolar hormone-independent hyperplasia. To understand the possible different activities of cyclin D2/cdk complexes, it is essential to characterize cyclin D2 cellular compartmentalization, interactions, and activities of complexes with cdk6 and cdk2 at early stages of transformation of the normal mammary gland.

Collectively, the changes in cyclin D1 and cdkI results were in accordance with the extensive data on hormonal regulation of normal mammary development with one major exception. Whereas both normal duct and immortalized duct responded to ovarian depletion by a decrease in proliferation and responded to hormone supplementation by an increase in proliferation, the immortal duct population failed to differentiate in response to hormone stimulation by pituitary isograft. We have examined recently estrogen and progesterone receptor content in the normal and immortalized duct populations, and they are equivalent (data not shown). We have also shown that proliferation and alveolar differentiation induced by a pituitary isograft requires the progesterone receptor in the mammary cell (45) . Therefore, the block in mammary epithelial cell differentiation in EL11 is downstream of the progesterone receptor. We suggest that the overexpression of p27Kip1 is a major factor of why EL11 cells do not produce tumors under prolonged hormone stimulation. The link between alveolar differentiation and murine mammary tumorigenesis is well established in the literature (71) . The alveolar cell is one preferred pathway for tumorigenesis, as demonstrated by the occurrence of hyperplastic alveolar nodules in both mouse mammary tumor virusinduced and chemical carcinogen-induced tumorigenesis. Additionally, there are well-documented cases of spontaneous transformation using serially transplanted ductal outgrowth lines, where tumorigenesis only occurred when the ductal lines gained the ability to undergo alveolar differentiation (71 , 72) . These data support the hypothesis that the differentiation block in EL11 may be the cause for the lack of tumorigenesis. We speculate that p27Kip1 up-regulation is the basis for the block in differentiation; thus, additional mechanism-based studies to determine the basis for p27Kip1 up-regulation are warranted. Future experiments will include a second immortal, hormone-dependent mammary outgrowth, EL12, that has a phenotype of being able to differentiate upon hormone stimulation, in contrast to EL11 outgrowth, which differentiates neither at control nor upon hormone stimulation (52) . In this experiment, the difference in the levels of p27Kip1-E/cdk2 and E/cdk2 activity in the ductal versus alveolar phenotypes should confirm the functional role of p27Kip1 in blocking mammary alveologenesis. Another future experiment is planned to determine whether p27Kip1 overexpression would block mammary epithelial differentiation in the EL12 cell line. Additionally, it would be of interest to examine mammary gland development in conditional p27Kip1 transgenic mice, where p27Kip1 is targeted to the mammary gland.

In conclusion, our studies suggest that although all cdkIs changed in concordance with hormonal status reflected by their proliferation rates, p27Kip1 seems to have a central role in alveolar cell differentiation. We hypothesize that up-regulation of p27Kip1 prevents immortalized ductal outgrowths (EL11) from progressing to the neoplastic state, even under hormonal stimulation.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Generation of the Mammary Outgrowth Lines.
The BALB/c mouse mammary outgrowth lines in vivo used in this study have been established and studied extensively in the past 10 years (35 , 71) . Briefly, the EL11 outgrowth line is an immortalized, hormone-dependent, alveolar incompetent ductal outgrowth. This outgrowth line is refractory to 7,12-dimethylbenz(a)anthracene-induced tumorigenesis (35 , 52 , 71) and is now in transplant generation 42. This represents a life span of 11 years, during which time it has had a spontaneous tumor incidence of <0.2%. The TM2L is a stage II hyperplasia that has recently spontaneously progressed from stage I to stage II. It is hormone responsive but not dependent and is now in transplant generation 36 and has had a spontaneous tumor incidence of ~50% by 10 months after transplantation. The progression from stage I to stage II hyperplasia was detected in transplant generation 29, or ~7 years after isolation of the in vivo line.

Hormone Manipulation in Vivo and Whole Mount Preparation.
The experiment was designed to manipulate in vivo hormone conditions affecting mammary epithelial cell regulation. The virgin gland is ductal, the EL11 outgrowths are ductal with sparse lateral alveolar buds, and the TM2L outgrowths possess uniform alveolar development equivalent to late pregnancy of normal glands. Each group was examined at three different hormonal states: (a) control; (b) hormonal stimulation of growth and differentiation achieved via a single pituitary isograft [a pituitary gland was grafted underneath the kidney capsule of 5-week-old mice, as described earlier (45 , 73) ]; and (c) hormone depletion (ovariectomy) at 10 weeks of age. All groups were terminated at 13 weeks of age. The inguinal (no. 4) mammary glands were removed and evaluated for morphological stage of development as whole mounts. Fat pads were fixed in Tellysniczky’s fixative, defatted in acetone, stained in iron hematoxylin, stored in methyl salicylate in glass scintillation vials, and examined under x4–20 power with a dissecting microscope. For histological evaluation, four to six fat pads of each group and at each hormone condition were imbedded in paraffin, sectioned at 5 µm, and stained with H&E.

BrdUrd Immunohistological Staining.
Two h before sacrifice, each animal received an i.p. injection of BrdUrd [70 µg of BrdUrd (Sigma Chemical Co.)/g body weight]. After BrdUrd injection, the animals were killed, and both inguinal glands were dissected, carefully flattened, and fixed in Methacarn (methanol:acetic acid:chloroform, 60:30:10) overnight at room temperature and then postfixed in acetone. Fixed tissues were embedded in paraffin and sectioned (5 µm) for either standard H&E staining or BrdUrd immunostaining. Following the manufacturer’s protocol using the Cell Proliferation kit from Amersham Life Science, Inc. (Arlington Heights, IL), localization of the positive BrdUrd-stained mammary epithelial cells was examined for each tissue section, and a random field of 1500 cells was counted/section.

Quantification of Protein in Mammary Epithelial Cells in Vivo.
The inguinal (no. 4) mammary glands (four to eight fat pads) were removed for each group at a specific hormonal condition. Lymph nodes were discarded. Using sterile technique, tissues were minced into very small pieces (<1 mm) with a scalpel or razor blade, and 1 g of minced tissue/10 ml digestion medium was placed in 50-ml conical tubes. The digestion medium contained DMEM:F12 buffered with HEPES (pH 7.6), 100 units/ml penicillin-streptomycin, 100 µg/ml gentamicin, 60 units/ml nystatin (all from Sigma Chemical Co.), 2 mg/ml collagenase A from Boehringer-Mannheim, and 100 units/ml hyaluronidase (Sigma Chemical Co.). The tubes were incubated at 37°C for ~3 h at 125 rpm in a water bath with gentle shaking. When the tissue was thoroughly digested so that it visually appeared as a cloudy homogenous solution, the mixture was centrifuged at 1000 rpm for 5 min. The cell pellets were rinsed five times in PBS. After the last wash, the cell pellet was a purified epithelial cell population, based on our previous experience (74) . As a standard procedure, we observed complete removal of the adipocytes and 90% of the fibroblasts, based on the growth of cells in primary culture and visualized under the microscope. Hence, the cell pellets were resuspended in 1 ml of PBS and counted by staining 100 µl of the cell suspension with trypan blue. The cells were pelleted by centrifugation for 5 min and lysed in 300 µl of chilled lysis buffer containing 50 mM HEPES (pH 7.8), 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, and 0.1% Tween 20 supplemented with 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 20 units/ml aprotinin, 10 mM ß-glycerophosphate, 1 mM NaF, and 0.1 mM sodium orthovanadate (all protease and phosphatase inhibitors were obtained from Sigma Chemical Co.). Lysed cells were sonicated on ice three times for 10 s using an Ultrasonicator processor. All samples were examined microscopically for complete lysis of the nuclei. Cellular debris was removed after ultracentrifugation at 100,000 x g at 4°C for 30 min, and the protein content of the supernatants was determined by the bicinchoninic acid protein assay (Pierce, Rockford, IL) and calculated as µg protein/105 mammary epithelial cells. Tissue protein extract preparation was as described earlier (73) . All data obtained from tissue extracts were normalized to protein levels in their counterpart epithelial cells alone, as described above.

Immunoprecipitation and Western Blot Analysis.
Protein extracts (100 µg) were analyzed for cdk4, cdk2, cyclin D1, cyclin D2, and cdkIs (p16INK4a, p21Cip1, and p27Kip1) protein levels following the protocol for Western blot analysis as described earlier (75) . The antibodies used were cdk4 (C-22), p16INK4a (M-156) rabbit polyclonal antibodies, and actin (I-19) gout polyclonal antibody (Santa Cruz Biotechnology Inc.); antimouse p21Cip1 serum (PharMingen); rabbit cdk2 and cyclin D1 polyclonal antibodies (Upstate Biotechnology); and mouse cyclin D2 (DCS-3.1) and mouse p27Kip1 monoclonal antibodies from NewMarker and Transduction Laboratories, respectively. Protein bands were revealed using SuperSignal chemiluminescence detection reagents (Pierce), followed by different exposure times to BioMax Kodak films. Protein bands were scanned and quantified by the Phosphorimager SF analyzer (Molecular Dynamics), and data were normalized to the protein level in 105 mammary epithelial cells in each group at a defined hormonal status. Immunoprecipitation assays of 500 µg of protein extract/sample were basically followed as described by Said et al. (73) . Immunoprecipitations of cdk4 and cdk2 were followed by Western blot analysis for cdkI (p16INK4a and p27Kip1) associated with cdk4 and for p21Cip1 and p27Kip1 associated with cdk4 and cdk2, respectively.

Kinase Assays for cdk4, Cyclin D1, and Cyclin D2 Immune Complexes.
The protocol followed was described by Said et al. (73) with minor modification. Briefly, 500 µg of protein extract/sample were mixed with 3 µg of cdk4 polyclonal antibody (Santa Cruz Biotechnology), cyclin D1 polyclonal antibody (Upstate Biotechnology Inc.), cyclin D2 (DCS-5.2) monoclonal antibody (New Marker), or 10 µl of 10% normal rabbit serum, as negative control. Kinase activities of all of the immune complexes were measured using 2 µg/sample of full-length pRb (p110RB; QED Bioscience, Inc.) as a substrate. Phosphorylated pRB visualized by autoradiography was scanned and quantified using a Phosphorimager SF analyzer. The preimmune serum and background values were subtracted to obtain the actual levels of pRB phosphorylation. All values then were normalized as described earlier to obtain the actual kinase activities in mammary epithelial cells in each group and at a specific hormonal condition.

Tumorigenesis Studies at Different Hormonal Conditions.
To study mammary tumorigenesis at different hormonal conditions, 20–28 mice in each group and at each hormonal status (control, ovariectomized, and with a pituitary isograft) were set up as described above. Mice were maintained under observation and palpated weekly for tumors for 1 year. The number of mice that developed tumors and the time for tumor development were calculated.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Grant CA-11944 from the NIH. Back

2 To whom requests for reprints should be addressed, at Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030. Phone: (713) 798-4834; Fax: (713) 790-0545; E-mail: tsaid{at}bcm.tmc.edu Back

3 The abbreviations used are: cdk, cyclin-dependent kinase; cdkI, cdk inhibitor; TM2L, transformed mammary-2 of low tumorigenicity; EL11, elongated life 11; BrdUrd, 5-bromo-2'-deoxyuridine; Rb, retinoblastoma; pRb, Rb protein. Back

4 T. K. Said, F. S. Kittrell, and D. Medina, unpublished data. Back

Received for publication 12/14/00. Revision received 2/19/01. Accepted for publication 4/23/01.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

  1. Sherr C. J. Mammalian G1 cyclins. Cell, 73: 1059-1065, 1993.[Medline]
  2. Sherr C. J. Mammalian G1 cyclins. Cell, 79: 551-555, 1994.[Medline]
  3. Fan J., Bertino J. K-ras modulates the cell cycle via both positive and negative regulatory pathways. Oncogene, 14: 2595-2607, 1997.[Medline]
  4. Meyyappan M., Wong H., Hull C., Riabowol K. T. Increased expression of cyclin D2 during multiple states of growth arrest in primary and established cells. Mol. Cell. Biol., 18: 3163-3172, 1998.[Abstract/Free Full Text]
  5. Sherr C. J. D-type cyclins. Trends Biochem. Sci., 20: 187-190, 1995.[Medline]
  6. Atadja P., Wong H., Veillette C., Riabowol K. Overexpression of cyclin D1 blocks proliferation of normal diploid fibroblasts. Exp. Cell Res., 217: 205-216, 1995.[Medline]
  7. Gao C. Y., Zelenka P. S. Cyclins, cyclin-dependent kinases and differentiation. Bioassays, 19: 307-315, 1997.[Medline]
  8. Li Z., Hromchak R., Bloch A. Differential expression of proteins regulating cell cycle progression in growth vs. differentiation. Biochem. Biophys. Acta, 1356: 149-159, 1997.[Medline]
  9. Wang T. C., Cardiff R. D., Zukerberg L., Lees E., Arnold A., Schmidt E. V. Mammary hyperplasia and carcinoma in MMTV-cyclin D1 transgenic mice. Nature (Lond.), 369: 669-671, 1994.[Medline]
  10. Fantl V., Stamp G., Andrews A., Rosewell I., Dickson D. Mice lacking cyclin D1 are small and show defects in eye and mammary gland development. Genes Dev., 9: 2364-2372, 1995.[Abstract/Free Full Text]
  11. Sicinski P., Donaher J. L., Parker S. B., Li T., Fazeli A., Gardner H., Haslam S. Z., Bronson R. T., Elledge S. J., Weinberg R. A. Cyclin D1 provides a link between development and oncogenesis in the retina and breast. Cell, 82: 621-630, 1995.[Medline]
  12. Musgrove E. A., Hamilton J. A., Lee C. S., Sweeney K. J. E., Watts C. K. W., Sutherland R. L. Growth factor, steroid, and steroid antagonists regulation of cyclin gene expression associated with changes in T-47D human breast cancer cell cycle progression. Mol. Cell. Biol., 13: 3577-3587, 1993.[Abstract/Free Full Text]
  13. Buckley M. F., Sweeney K. J. E., Hamilton J. A., Sini R. L., Manning D. L., Nicholson R. I., deFazio A., Watts C. K. W., Musgrove E. A., Sutherland R. L. Expression and amplification of cyclin genes in human breast cancer. Oncogene, 8: 2127-2133, 1993.[Medline]
  14. Oyama T., Kashiwabara K., Yoshimoto K., Arnold A., Koerner F. Frequent overexpression of the cyclin D1 oncogene in invasive lobular carcinoma of the breast. Cancer Res., 58: 2876-2880, 1998.[Abstract/Free Full Text]
  15. Courjal F., Louason G., Speisser P., Katsaros D., Zeillinger R., Theillet C. Cyclin gene amplification and overexpression in breast and ovarian cancers: evidence for the selection of cyclin D1 in breast and cyclin E in ovarian tumors. Int. J. Cancer, 69: 247-253, 1996.[Medline]
  16. Hirama T., Koeffler H. P. Role of the cyclin-dependent kinase inhibitors in the development of cancer. Blood, 86: 841-854, 1995.[Free Full Text]
  17. Hunter T., Pines J. Cyclin and cancer. Cyclin D and CDK inhibitors come of age. Cell, 79: 573-582, 1994.[Medline]
  18. Kamb A. Cell-cycle regulators and cancer. Trend Genet., 11: 126-140, 1995.
  19. Peter M., Herskowitz I. Joining the complex: cyclin dependent kinase inhibitory proteins and the cell cycle. Cell, 79: 181-184, 1994.[Medline]
  20. Sherr C. J., Roberts J. M. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev., 9: 1149-1163, 1995.[Free Full Text]
  21. Serrano M., Hannon G. J., Beach D. A new regulatory motif in cell cycle control causing specific inhibition of cyclin D/cdk4. Nature (Lond.), 366: 704-707, 1993.[Medline]
  22. Guan K. L., Jenkins C. W., Li Y., Nichols M. A., Wu X., O’Keefe C. L., Matera A. G., Xiong Y. Growth suppression by p18, a p16INK4/MTS1- and p14INK4B/MTS2-related CDK6 inhibitor, correlates with wild-type pRb function. Genes Dev., 8: 2939-2952, 1994.[Abstract/Free Full Text]
  23. Hannon G. J., Beach D. p15/INK4B is a potential effector of TGF-ß-induced cell cycle arrest. Nature (Lond.), 371: 257-261, 1994.[Medline]
  24. Hirai H., Roussel M. F., Kato J. Y., Ashmun R. A., Sherr C. J. Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6. Mol. Cell. Biol., 15: 2672-2681, 1995.[Abstract/Free Full Text]
  25. Chan F., Zang K. J., Cheng L., Shapiro D. N., Winoto A. Identification of human and mouse p19, a novel CDK4 and CDK6 inhibitor with homology to p16INK4. Mol. Cell. Biol., 15: 2682-2688, 1995.[Abstract/Free Full Text]
  26. El-Deiry W., Tokino T., Velculescu V. E., Levy D. B., Parsons R., Trent J. M., Lin D., Mercer W. E., Kinzler K. W., Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell, 75: 817-825, 1993.[Medline]
  27. Harper J. W., Adami G. R., Wei N., Keyomarsi K., Elledge S. J. The p21 cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell, 75: 805-816, 1993.[Medline]
  28. Noda A., Ning Y., Venable S. F., Pereira-Smith O. M., Smith J. R. Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp. Cell Res., 211: 90-98, 1994.[Medline]
  29. Xiong Y., Hannon G. J., Zhang H., Casso D., Kobayashi R., Beach D. p21 is a universal inhibitor of cyclin kinases. Nature (Lond.), 366: 701-704, 1993.[Medline]
  30. Polak K., Kato J. Y., Solomon M. J., Sherr C. J., Massague J., Roberts J. M., Koff A. p27kip1, a cyclin-Cdk inhibitor, links transforming growth factor-ß and contact inhibition to cell cycle arrest. Genes Dev., 8: 9-22, 1994.[Abstract/Free Full Text]
  31. Polak K., Lee M. H., Erdjument-Bromage H., Koff A., Roberts J. M., Tempst P., Massagué J. Cloning of p27kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell, 78: 59-66, 1994.[Medline]
  32. Toyoshima H., Hunter T. p27, a novel inhibitor of G1 cyclin-cdk protein kinase activity, is related to p21. Cell, 78: 67-74, 1994.[Medline]
  33. Lee M. H., Reynisdottir I., Massagué J. Cloning of p57/kip2, a cyclin-dependent kinase inhibitor with unique domain structure and tissue distraction. Genes Dev., 9: 639-649, 1995.[Abstract/Free Full Text]
  34. Matsuoka S., Edwards M. C., Bai C., Parker S., Zhang P., Baldini A., Harper J. W., Elledge S. J. p57/Kip2, a structurally distinct member of the p21/CIP1 Cdk inhibitor family, is a candidate tumor suppressor gene. Genes Dev., 15: 650-662, 1995.
  35. Sheaff R. J., Roberts J. M. Cell cycle control Pagano M. eds. 1-34, Springer New York 1998.
  36. Kitagawa M., Higashi H., Jung H. K., Takahasi K., Suzuki I., Ikeda M., Tamai K., Kato J., Segawa K., Yoshida E., Nishimura S., Taya Y. The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2. EMBO J., 15: 7060-7069, 1996.[Medline]
  37. Lundberg A., Weinberg R. A. Functional interaction of the retinoblastoma protein requires sequential modification by at least two distinct cyclin-cdk complexes. Mol. Cell. Biol., 18: 753-761, 1998.[Abstract/Free Full Text]
  38. Zarkowska T., Mittnacht S. Differential phosphorylation of the retinoblastoma protein by G1/S cyclin-dependent kinases. J. Biol. Chem., 272: 12738-12746, 1997.[Abstract/Free Full Text]
  39. Dyson N. The regulation of E2F by pRb-family proteins. Genes Dev., 12: 2245-2262, 1998.[Free Full Text]
  40. Nevins J. R. Toward an understanding of the functional complexity of the E2F and retinoblastoma families. Cell Growth Differ., 9: 585-593, 1998.[Medline]
  41. Daniel C. W., Smith G. H. The mammary gland: a model for development. J. Mammary Gland Biol. Neoplasia, 4: 3-8, 1999.[Medline]
  42. Robinson G. W., Karpf A. B. C., Kratochwil K. Regulation of mammary gland development by tissue interaction. J. Mammary Gland Biol. Neoplasia, 4: 9-20, 1999.[Medline]
  43. Said T. S., Conneely O. M., Medina D., O’Malley B. W., Lydon J. Progesterone, in addition to estrogen, induces cyclin D1 expression in the murine mammary epithelial cell, in vivo. Endocrinology, 138: 3933-3939, 1997.[Medline]
  44. Laidlaw I. J., Clarke R. B., Howell A., Owen A., Potten C. S., Anderson E. The proliferation of normal human breast tissue implanted into athymic nude mice is stimulated by estrogen but nor progesterone. Endocrinology, 136: 164-171, 1995.[Medline]
  45. Lydon P. J., Ge G., Kittrell F. S., Medina D., O’Malley B. W. Murine mammary gland carcinogenesis is critically dependent on progesterone receptor function. Cancer Res., 59: 4279-4284, 1999.
  46. Quelle D. E., Ashmun R. A., Hannon G. J., Rehberger P. A., Trono D., Richter H., Walker C., Beach D., Sherr C. J., Serrano M. Cloning and characterization of murine p16INK4a and p15INK4b genes. Oncogene, 11: 635-645, 1995.[Medline]
  47. Lloyd R. V., Erickson L. A., Jin L., Kulig E., Qian X., Cheville J. C., Scheithauer B. W. P27Kip1: A multifunctional cyclin-dependent kinase inhibitor with prognostic significance in human cancers. Am. J. Pathol., 154: 313-323, 1999.[Medline]
  48. Imagawa W., Yang J., Guzman R., Nandi S. Control of mammary gland development Knobil E. Neill J. D. eds. . The Physiology of Reproduction, : 1033-1063, Raven Press New York 1994.
  49. Maricarmen D. P-S., Weinberg R. A. Estrogen-dependent cyclin E-cdk2 activation through p21 redistribution. Mol. Cell. Biol., 17: 4059-4069, 1997.[Abstract/Free Full Text]
  50. Malumbres M., Perez de Castro I., Santos J., Melendez B., Mangues R., Serrano M., Pellicer A., Fernandez-Piqueras J. Inactivation of the cyclin-dependent kinase inhibitor p15INK4b by deletion and de novo methylation with independence of p16 INK4a alteration in murine primary T-cell lymphomas. Oncogene, 14: 1361-1370, 1997.[Medline]
  51. Kamijo T., Bodner S., Van de Kamp E., Randle D. H., Sherr C. J. Tumor spectrum in ARF-deficient mice. Cancer Res., 59: 2217-2222, 1999.[Abstract/Free Full Text]
  52. Medina D., Kittrell F. S. Immortalization phenotype dissociated from the preneoplastic phenotype in mouse mammary epithelial outgrowths in vivo. Carcinogenesis (Lond.), 14: 25-28, 1993.[Abstract/Free Full Text]
  53. Pagano M., Pepperkok R., Lukas I., Baldin V., Ansorge W., Bartek I., Draetta G. Regulation of the human cell cycle by the cdk2 protein kinase. J. Cell Biol., 121: 101-111, 1993.[Abstract/Free Full Text]
  54. Van den Heuvel S., Harlow E. Distinct roles for cyclin-dependent kinases in cell cycle control. Science (Wash. DC), 262: 2050-2054, 1993.[Abstract/Free Full Text]
  55. Drissi H., Hushka D., Aslam F., Nguyen Q., Buffone E., Koff A., Wijnen A. J., Lian J. B., Stein J. L., Stein G. S. The cell cycle regulator p27Kip1 contributes to growth and differentiation of osteoblasts. Cancer Res., 59: 3705-3711, 1999.[Abstract/Free Full Text]
  56. Harvat B. L., Wang A., Seth P., Jetten A. M. Up-regulation of p27kip1, p21WAF1/Cip1 and p16Ink4a is associated with, but not sufficient for, induction of squamous differentiation. J. Cell Sci., 111: 1185-1196, 1998.[Abstract/Free Full Text]
  57. Zhan-Rong L., Hromchak R., Mudipalli A., Bloch A. Tumor suppressor proteins as regulators of cell differentiation. Cancer Res., 58: 4282-4287, 1998.[Abstract/Free Full Text]
  58. Hiromura K., Pippin J. W., Fero M. L., Roberts J. M., Shankland S. J. Modulation of apoptosis by the cyclin-dependent kinase inhibitor p27kip1. J. Clin. Investig., 103: 597-604, 1999.[Medline]
  59. Katayose Y., Kim M., Rakkar A. N., Li Z., Cowan K. H., Seth P. Promoting apoptosis: a novel activity associated with the cyclindependent kinase inhibitor p27Kip1. Cancer Res., 57: 5441-5445, 1997.[Abstract/Free Full Text]
  60. Wu J., Shen Z-Z., Lu J-S., Jiang M., Han Q-X., Fontana J. A., Barsky S. H., Shao Z-M. Prognostic role of p27kip1 and apoptosis in human breast cancer. Br. J. Cancer, 79: 1572-1578, 1999.[Medline]
  61. Catzavelos C., Bhattacharya N., Ung Y. C., Wilson J. A., Roncari L., Sandhu C., Shaw P., Yeger H., Morava-Protzner I., Kapusta L., Franssen E., Pritchard K. I., Slingerland J. M. Decreased levels of the cell cycle inhibitor p27Kip1 protein: prognostic implications in primary breast cancer. Nat. Med., 3: 227-230, 1997.[Medline]
  62. Cox L. A., Chen G., Lee E. Y. Tumor suppressor genes and their roles in breast cancer. Breast Cancer Res. Treat., 32: 19-38, 1994.[Medline]
  63. Zhang H., Hannon G. J., Beach D. p21-containing cyclin kinases exit in both active and inactive states. Genes Dev., 8: 1750-1758, 1994.[Abstract/Free Full Text]
  64. LaBaer J., Garrett M. D., Stevenson L. F., Slingerland J. M., Sandhu C., Chou H. S., Fattaey A., Harlow E. New functional activities for the p21 family of CDK inhibitors. Genes Dev., 11: 847-862, 1997.[Abstract/Free Full Text]
  65. Clark R. B., Howell A., Potten C. S., Anderson E. p27KIP1 expression indicates that steroid receptor-positive cells are a non-proliferating, differentiated subpopulation of the normal human breast epithelium. Eur. J. Cancer, 36: S27-S36, 2000.
  66. Bartkova J., Lukas J., Strauss M., Bartek J. Cyclin D1 oncoprotein aberrantly accumulates in malignancies of diverse histogenesis. Oncogene, 10: 775-778, 1995.[Medline]
  67. Devilee P., Schuuring E., Van de Vijver M. J., Cornelisse C. J. Recent developments in the molecular genetic understanding of breast cancer. Crit. Rev. Oncol., 5: 247-270, 1994.
  68. Musgrove E. A., Lee C. S. L., Buckley M. F., Sutherland R. L. Cyclin D1 induction in breast cancer cells shortens G1 and is sufficient for cells arrested in G1 to complete the cell cycle. Proc. Natl. Acad. Sci. USA, 91: 8022-8026, 1994.[Abstract/Free Full Text]
  69. Zhang S. Y., Camano J., Cooper F., Guo X., Klein-Szanto A. J. Immunohistochemistry of cyclin D1 in human breast cancer. Am. J. Clin. Pathol., 102: 695-698, 1994.[Medline]
  70. Sweeney K., Sarcevic B., Southerland R. L., Musgrove E. A. Cyclin D2 activates cdk2 in preference to cdk4 in human breast epithelial cells. Oncogene, 14: 1329-1340, 1997.[Medline]
  71. Medina D., Kittrell F. S. Enhancement of tumorigenicity with morphological progression in BALB/c preneoplastic outgrowth line. J. Natl. Cancer Inst., 79: 569-576, 1987.
  72. Medina D. Preneoplasia in mammary tumorigenesis Dickson R. Lippman M. eds. . Mammary Tumor Cell cycle, Differentiation and Metastasis, : 37-69, Kluwer Academic Publishers New York 1996.
  73. Said T. K., Bonnette S., Medina D. Immortal, non-tumorigenic mouse mammary outgrowths express high levels of cyclin B1 and activation of cyclin B1/cdc2 kinase. Cell Prolif., 29: 623-639, 1996.[Medline]
  74. Medina D., Kittrell F. Establishment of mouse mammary cell lines. Methods in Mammary Gland Biology and Breast Cancer Research, 137-145, Ip and Kluwer Academic/Plenum Publishing Corp. New York 2000.
  75. Said T. K., Luo L., Medina D. Mouse mammary hyperplasias and neoplasias exhibit different patterns of cyclins D1 and D2 binding to cdk4. Carcinogenesis (Lond.), 16: 2507-2513, 1995.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
Mol Cancer ResHome page
F. Ladam, I. Damour, P. Dumont, Z. Kherrouche, Y. de Launoit, D. Tulasne, and A. Chotteau-Lelievre
Loss of a Negative Feedback Loop Involving Pea3 and Cyclin D2 Is Required for Pea3-Induced Migration in Transformed Mammary Epithelial Cells
Mol. Cancer Res., November 1, 2013; 11(11): 1412 - 1424.
[Abstract] [Full Text] [PDF]


Home page
IOVSHome page
J.-g. Yang, Y. Deng, L.-x. Zhou, X.-y. Li, P.-r. Sun, and N.-x. Sun
Overexpression of CDKN1B Inhibits Fibroblast Proliferation in a Rabbit Model of Experimental Glaucoma Filtration Surgery
, January 14, 2013; 54(1): 343 - 352.
[Abstract] [Full Text] [PDF]


Home page
Cancer Prev ResHome page
M. C. Cabrera, E. S. Diaz-Cruz, B. V. S. Kallakury, M. J. Pishvaian, C. J. Grubbs, D. D. Muccio, and P. A. Furth
The CDK4/6 Inhibitor PD0332991 Reverses Epithelial Dysplasia Associated with Abnormal Activation of the Cyclin-CDK-Rb Pathway
Cancer Prevention Research, June 1, 2012; 5(6): 810 - 821.
[Abstract] [Full Text] [PDF]


Home page
J Biol ChemHome page
J. S. Schaefer, Y. Sabherwal, H. Y. Shi, V. Sriraman, J. Richards, A. Minella, D. P. Turner, D. K. Watson, and M. Zhang
Transcriptional Regulation of p21/CIP1 Cell Cycle Inhibitor by PDEF Controls Cell Proliferation and Mammary Tumor Progression
J. Biol. Chem., April 9, 2010; 285(15): 11258 - 11269.
[Abstract] [Full Text] [PDF]


Home page
DevelopmentHome page
R. C. Moraes, H. Chang, N. Harrington, J. D. Landua, J. T. Prigge, T. F. Lane, B. J. Wainwright, P. A. Hamel, and M. T. Lewis
Ptch1 is required locally for mammary gland morphogenesis and systemically for ductal elongation
Development, May 1, 2009; 136(9): 1423 - 1432.
[Abstract] [Full Text] [PDF]


Home page
Mol. Cell. Biol.Home page
F. Gizard, R. Robillard, B. Gross, O. Barbier, F. Revillion, J.-P. Peyrat, G. Torpier, D. W. Hum, and B. Staels
TReP-132 Is a Novel Progesterone Receptor Coactivator Required for the Inhibition of Breast Cancer Cell Growth and Enhancement of Differentiation by Progesterone
Mol. Cell. Biol., October 15, 2006; 26(20): 7632 - 7644.
[Abstract] [Full Text] [PDF]


Home page
Mol. Cell. Biol.Home page
F. Gizard, R. Robillard, O. Barbier, B. Quatannens, A. Faucompre, F. Revillion, J.-P. Peyrat, B. Staels, and D. W. Hum
TReP-132 Controls Cell Proliferation by Regulating the Expression of the Cyclin-Dependent Kinase Inhibitors p21WAF1/Cip1 and p27Kip1
Mol. Cell. Biol., June 1, 2005; 25(11): 4335 - 4348.
[Abstract] [Full Text] [PDF]


Home page
Mol. Cell. Biol.Home page
L. K. Pierson-Mullany and C. A. Lange
Phosphorylation of Progesterone Receptor Serine 400 Mediates Ligand-Independent Transcriptional Activity in Response to Activation of Cyclin-Dependent Protein Kinase 2
Mol. Cell. Biol., December 15, 2004; 24(24): 10542 - 10557.
[Abstract] [Full Text] [PDF]


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
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
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Said, T. K.
Right arrow Articles by Medina, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Said, T. K.
Right arrow Articles by Medina, D.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cell Growth & Differentiation