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Cancer Research 67, 4732-4741, May 15, 2007. doi: 10.1158/0008-5472.CAN-06-3437
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Expression of p16Ink4a Compensates for p18Ink4c Loss in Cyclin-Dependent Kinase 4/6–Dependent Tumors and Tissues

Matthew R. Ramsey1,4, Janakiraman Krishnamurthy1, Xin-Hai Pei2, Chad Torrice1, Weili Lin3, Daniel R. Carrasco5,6, Keith L. Ligon6, Yue Xiong2 and Norman E. Sharpless1

Departments of 1 Medicine and Genetics, 2 Biochemistry and Biophysics, and 3 Neurology and 4 Curriculum in Genetics and Molecular Biology, The Lineberger Comprehensive Cancer Center, The University of North Carolina School of Medicine, Chapel Hill, North Carolina; 5 Department of Adult Oncology, Dana-Farber Cancer Institute, Harvard Medical School; and 6 Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts

Requests for reprints: Norman E. Sharpless, Departments of Medicine and Genetics, The Lineberger Comprehensive Cancer Center, The University of North Carolina School of Medicine, CB# 7295, Chapel Hill, NC 27599-7295. Phone: 919-966-1185; Fax: 919-966-8212; E-mail: nes{at}med.unc.edu.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell cycle progression from G1 to S phase depends on phosphorylation of pRb by complexes containing a cyclin (D type or E type) and cyclin-dependent kinase (e.g., cdk2, cdk4, or cdk6). Ink4 proteins function to oppose the action of cdk4/6-cyclin D complexes by inhibiting cdk4/6. We employed genetic and pharmacologic approaches to study the interplay among Ink4 proteins and cdk4/6 activity in vivo. Mouse embryo fibroblasts (MEF) lacking p16Ink4a and p18Ink4c showed similar growth kinetics as wild-type MEFs despite increased cdk4 activity. In vivo, germline deficiency of p16Ink4a and p18Ink4c resulted in increased proliferation in the intermediate pituitary and pancreatic islets of adult mice, and survival of p16Ink4a–/–;p18Ink4c–/– mice was significantly reduced due to aggressive pituitary tumors. Compensation among the Ink4 proteins was observed both in vivo in p18Ink4c–/– mice and in MEFs from p16Ink4a–/–, p18Ink4c–/–, or p16Ink4a–/–;p18Ink4c–/– mice. Treatment with PD 0332991, a specific cdk4/6 kinase inhibitor, abrogated proliferation in those compartments where Ink4 deficiency was associated with enhanced proliferation (i.e., islets, pituitary, and B lymphocytes) but had no effect on proliferation in other tissues such as the small bowel. These data suggest that p16Ink4a and p18Ink4c coordinately regulate the in vivo catalytic activity of cdk4/6 in specific compartments of adult mice. [Cancer Res 2007;67(10):4732–41]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Control of the retinoblastoma family proteins (pRb, p107, and p130) through phosphorylation by cyclin-dependent kinases (cdk) is essential for cell cycle regulation. Cdks require cyclin cofactors for their activity, and cyclin-cdk pairs have been shown to be active at the G1-S boundary. Cdk2 can bind to cyclin E1, cyclin E2, or cyclin A, whereas cdk4 and cdk6 have been found to pair with the D-type cyclins (reviewed in ref. 1). The activity of these complexes is tightly controlled by both periodic synthesis and destruction of cyclins as well as by cdk inhibitor proteins.

The Ink4 family of cdk inhibitors consists of four members [p16Ink4a (2), p15Ink4b (3), p18Ink4c (4), and p19Ink4d (5)], and all are specific inhibitors of cdk4/6-cyclin D complexes. Both p16Ink4a and p15Ink4b consist of four ankyrin repeats, with ~80% sequence similarity to each other, whereas p18Ink4c and p19Ink4d have a fifth ankryin repeat (reviewed in ref. 6). All four inhibit cdk4 and cdk6 by binding opposite the cyclin binding site, causing an allosteric shift and blocking cyclin binding and ATP hydrolysis (7, 8). Although there have been some reported differences in the regulation of different Ink4s (9–11), they seem to bind cdk4 and cdk6 with comparable affinity (12). In general, distinct roles for these proteins in vivo have not been delineated, although p16Ink4a in particular has been associated with senescence and tumor suppression (reviewed in ref. 13).

Despite the biochemical similarities, mice lacking individual Ink4 genes exhibit different phenotypes. Mice lacking p19Ink4d mice are overtly normal but have testicular atrophy and deafness (14, 15). Mice lacking p15Ink4b show increased proliferation in the lymphoid lineages and a low incidence of spontaneous tumor formation (16). On the other hand, both p16Ink4a–/– (17–19) and p18Ink4c–/– (16, 20) mice exhibit increased susceptibility to spontaneous and carcinogen-induced tumors, but with minimal overlap of tumor spectrum. Mice lacking p16Ink4a develop spontaneous lymphomas and sarcomas at low penetrance, whereas the majority of p18Ink4c–/– mice develop pituitary tumors of the intermediate lobe, suggesting different in vivo roles for these proteins.

Elucidation of the control of the proliferative cdks by the partially redundant cdk inhibitors remains a major hurdle in the clear understanding of the cell cycle, a problem made more difficult by the compensation seen among family members. In this study, we have investigated the effects of combined loss of p16Ink4a and p18Ink4c in vitro and in vivo. We show that in murine embryo fibroblasts (MEF), the loss of p18Ink4c results in compensatory increases in p16Ink4a, whereas loss of p16Ink4a is associated with increased expression of p15Ink4b. In vivo, the loss of p18Ink4c results in compensatory increases in p16Ink4a in various organs, which restrains proliferation. Additionally, using genetic and pharmacologic approaches, we show that specific in vivo compartments in the adult mouse are exquisitely dependent on cdk4/6 kinase activity for proliferation. These data suggest that p16Ink4a and p18Ink4c function to regulate physiologic and aberrant proliferation in specific compartments of adult mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mouse colony and cell culture. Animals were generated and genotyped as previously described (18, 20) and were N1 in FVB. Mice were housed and treated in accordance with protocols approved by the institutional care and use committee for animal research at the University of North Carolina. Murine p16Ink4a and p18Ink4c are located 18 cM apart on chromosome 4, requiring a modified breeding scheme of crossing two double heterozygotes in cis or two double heterozygotes in trans to generate the colony. For survival analyses, animals were examined thrice per week. Rare deaths of unknown cause (n = 5) were censored from tumor-free analysis, but their inclusion does not change the conclusions. Islet size was quantified using Image-Pro Express software on 115 to 215 individual islets from at least two mice per genotype at 35 weeks of age. To assess proliferation after cdk4/6 inhibition, mice were treated daily for 2 weeks by oral gavage with 150 mg/kg of 87.11 mmol/L PD 0332991 (Pfizer) dissolved in 50 mmol/L sodium lactate buffer (pH 4.0), or buffer alone as described (21).

MEFs were cultured from whole 13.5-day-old embryos as described (18) and grown at 21%O2, 5% CO2. Cumulative population doublings were calculated as Log2 (number of cells at harvest/number of cells plated). Transformations with H-RasV12G and SV40 TAg and high-density seeding assays were done as previously described (22) with duplicate samples from four to six lines of each genotype.

Western blotting and immunoprecipitation kinase assays. Western blot assays were done as described (19). Antibodies against p21Cip1 (F-8, Santa Cruz), p16Ink4a (M-156, Santa Cruz), actin (C-1, Santa Cruz), cyclin D1 (DCS-6, Cell Signaling), cyclin E (M-20, Santa Cruz), cdk2 (M2, Santa Cruz), and cdk4 (C-22, Santa Cruz) are commercially available. Antibodies to p15Ink4b, p18Ink4c, and p27Kip1 have been previously described (20). Antibody against p19Ink4d was raised against a COOH-terminal peptide, and affinity was purified.

Kinase assays were done on MEFs as described (23), with some modifications. Briefly, fresh cells were lysed for 45 min at 4°C in NP40 buffer [50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 0.5% NP40, phosphatase inhibitor cocktail I and II (Calbiochem)]; 100 µg (cdk2) or 900 µg (cdk4) total protein was immunoprecipitated with antibodies specified above. Lysates were precleared twice with 50 µL protein A agarose beads and immunoprecipitated for 12 h at 4°C. Beads were rinsed thrice with cold NP40 buffer, then twice with cold kinase assay buffer [50 mmol/L HEPES (pH 7.0), 10 mmol/L MgCl2, 5 mmol/L MnCl2, 1 mmol/L DTT, 5 µmol/L ATP]. Reactions were done in 30 µL kinase buffer with 5 µCi [{gamma}-32P]ATP (3,000 Ci/mmol) and 1 µL GST-Rb substrate (Santa Cruz, Rb769). Reactions were carried out for 30 min at 30°C. SDS sample buffer was then added; lysates were boiled for 3 min; and protein was separated on 12.5% bis-acrylamide gels.

Immunohistochemistry. Assistance in sample processing was provided by the University of North Carolina Center for Gastrointestinal Biology and Disease. Paraffin samples from indicated genetic backgrounds were stained in a uniform fashion using well-established methods within the clinical laboratory at the Brigham and Women's Hospital Pathology Immunohistochemical laboratory. Briefly, five micron sections were cut, and immunohistochemical staining for either Ki-67 (rabbit polyclonal, NCL-Ki67p, Novocastra), adrenocorticorticotropic hormone (ACTH; DAKO, N1531), prolactin (PRL; DAKO, N1549), growth hormone (GHR; DAKO, L1814), leutinizing hormone (LH; DAKO, L1827), follicle-stimulating hormone (FSH; DAKO, L1810), B220 (BD Biosciences/PharMingen, 557390), or CD3 (Serotec, MCA1477) was done using highly sensitive DAKO EnVision polymerized horseradish peroxidase detection methods. The proliferative index of the pituitary was calculated by counting only the cells in the intermediate pituitary: (total Ki67 + cells) / (total intermediate pituitary cells). The islet proliferative index was calculated on a per-islet basis as (total Ki67 + islet cells) / (total number of islet cells) per islet for each sample.

Taqman real-time PCR. Expression of mRNA was analyzed by quantitative Taqman real-time PCR as previously described, with some modifications (24). Reactions were carried out using cDNA equivalent to 80 ng RNA and done in triplicate for each sample. 18S rRNA was used as a loading control for all reactions. Primer sets for 18S (Hs99999901_s1), p15Ink4b (Mm00483241_m1), p18Ink4c (Mm00483243_m1), p19Ink4d (Mm00486943_m1), p21Cip1 (Mm00432448_m1), and p27Kip1 (Mm00438167_g1) were purchased from Applied Biosystems; p16Ink4a and p19Arf primers were designed as previously described (25).

Statistics. Analysis of Kaplan-Meier survival curves was done using the log-rank test for each genotype pair. Proliferative index and islet size were not distributed normally and, therefore, were compared using a nonparametric (Mann-Whitney) test. High-density seeding was evaluated using the Student's unpaired t test. All error bars represent SE.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vitro growth and transformation of MEFs lacking p16Ink4a and p18Ink4c. Wild-type MEFs show significant expression of p16Ink4a that markedly increases with serial passage (26, 27); yet, p16Ink4a-deficient MEFs enter senescence with identical kinetics as wild-type cells (17, 18). MEFs lacking p18Ink4c (16), p19Ink4d (14), or both (28) also show no differences in life span despite significant expression of these proteins in cultured MEFs (27). In contrast, p15Ink4b–/– MEFs show a modest extension of life span (16), and cells carrying the Ink4-insensitive cdkR24C mutation are immortal in culture (29, 30). Serial passage of p16Ink4a–/–;p18Ink4c–/– MEFs revealed identical proliferation and life span as wild-type MEFs (Fig. 1A ), and these cells entered senescence with identical kinetics as wild-type cells. Additionally, p16Ink4a–/–;p18Ink4c–/– MEFs showed similar cdk and cyclin D1 expression (Fig. 1B) with serial passage.


Figure 1
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  		Figure 1. p16Ink4a–/–;p18Ink4c–/– MEFs show enhanced cdk4 kinase activity. A, life span of wild-type (WT) and p16Ink4a–/–;p18Ink4c–/– (DKO) MEFs grown according to standard 3T9 protocols at 21% O2, 5% CO2. Four to six lines were assessed per genotype. Points, mean; bars, SE. B, levels of cyclin E, cdk2, cyclin D1, cdk4, and p16Ink4a in asynchronously growing wild-type and p16Ink4a–/–;p18Ink4c–/– MEFs of indicated passage. Actin serves as a loading control. C, in vitro cdk4 (left) and cdk2 (right) kinase activity of wild-type MEFs with passage (top). Cdk4 or cdk2 complexes were immunoprecipitated (IP) from MEFs using either 900 µg (cdk4) or 100 µg (cdk2) total cell lysate. Kinase activity was assessed against GST-Rb-769 substrate. NP, no primary antibody. NS, no Rb substrate. One sixth of the total cdk4 or cdk2 immunoprecipitated is shown as loading control (bottom). D, in vitro cdk4 kinase activity of wild-type and p16Ink4a–/–;p18Ink4c–/– MEFs (top). Cdk4 complexes were immunoprecipitated from MEFs of indicated passage using 900 µg total cell lysate. Kinase activity was assessed against GST-Rb-769 substrate. One sixth of total cdk4 immunoprecipitated is shown as loading control (bottom).

 
In an effort to understand the underlying mechanisms driving MEF proliferation, we assessed the kinase activity of cdk4 and cdk2 with serial passage. As expected, cdk2 kinase activity decreased with passage, correlating highly with the onset of senescence (Fig. 1C). Surprisingly, cdk4 activity actually increased with passage, with its highest level of activity when cells were entering senescence (Fig. 1C). This increased cdk4 activity is likely due to the increased cyclin D protein and mRNA levels seen at late passage (Fig. 1B; data not shown). Although p16Ink4a–/–;p18Ink4c–/– MEFs showed slightly increased cdk4 kinase activity compared with wild-type cells at all passages (Fig. 1D), there was little effect on proliferative life span (Fig. 1A). These data suggest that p16Ink4a and p18Ink4c regulate cdk4 kinase activity in MEFs, but these increases in cdk4 activity do not increase their proliferation, which seems to be predominantly determined by cdk2 kinase activity.

We additionally examined the effects of Ink4 deficiency on other aspects of MEF behavior in vitro. Growth arrest to high density in MEFs is partially mediated by p16Ink4a (22), but a role for p18Ink4c has not been explored. We assessed the ability of p18Ink4c to cooperate with p16Ink4a loss in the regulation of high-density growth. Plates were re-fed but not passaged for 21 days and then assessed for cell number. Compared with wild-type cells, p16Ink4a–/–;p18Ink4c–/– MEFs showed a 45% increase in cell number (P = 0.011; data not shown), comparable to the effect seen with loss of p16Ink4a alone (22). As has been previously reported for p16Ink4a–/– (18, 22) and p18Ink4c–/– (16) MEFs, transformation by H-rasV12G alone or H-RasV12G + SV40 T-Ag was not significantly different between wild-type, p16Ink4a–/–, p18Ink4c–/–, or p16Ink4a–/–;p18Ink4c–/– MEFs (data not shown). These data confirm an effect of p16Ink4a on density arrest in MEFs but suggest only at most a modest role for p16Ink4a and p18Ink4c in resisting Ras-mediated transformation in MEFs.

p16Ink4a–/–;p18Ink4c–/– mice develop aggressive pituitary tumors. To analyze the effects of combined p16Ink4a and p18Ink4c deficiency in vivo, we generated mice lacking p16Ink4a and/or p18Ink4c. Both p16Ink4a and p18Ink4c lie on the murine chromosome 4, requiring a modified breeding scheme similar to that reported for mice lacking p15Ink4b and p18Ink4c (16). p16Ink4a–/–;p18Ink4c–/– mice were produced in the expected ratio based on calculated frequency of recombination between p16Ink4a and p18Ink4c, were fertile, and exhibited normal behavior. No significant difference in body weight was found between p16Ink4a–/–;p18Ink4c–/– and wild-type or p16Ink4a+/–;p18Ink4c+/– mice in this mixed genetic background. With aging, p16Ink4a–/–;p18Ink4c–/– mice showed increased morbidity with a median tumor-free survival of 42.7 weeks, compared with 68.8 weeks for p18Ink4c–/– mice (P < 0.001) and >70 weeks for p16Ink4a–/– mice (P = 0.005; Fig. 2A ), showing strong cooperation between loss of p16Ink4a and p18Ink4c in promoting tumorigenesis.


Figure 2
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  		Figure 2. Mice lacking p16Ink4a and p18Ink4c develop pituitary tumors. A, tumor-free survival of mice lacking p16Ink4a and/or p18Ink4c. The control groups consisted of p16Ink4a+/+;p18Ink4c–/– (n = 14) or p16Ink4a–/–;p18Ink4c+/+ (n = 10) and were compared with the p16Ink4a–/–;p18Ink4c–/– (n = 27) group. Mean survival: p16Ink4a–/–;p18Ink4c–/– mice, 42.7 wks; p18Ink4c–/– mice, 68.8 wks; p16Ink4a–/– mice, >70 wks. B, magnetic resonance images of wild-type (left) and p16Ink4a–/–;p18Ink4c–/– (right) mice at 44 wks of age. T1-weighted images (top) are at the level of the pituitary (white arrow). T2-weighted images (bottom) are taken at the level of the ventricles. Note marked hydrocephalus (black arrow) in the mouse on right. Compare with normal image of wild-type mouse (left). C, representative gross samples and H&E staining of pituitaries of indicated genotypes at 35 wks of age. Pituitary in gross samples is outlined (dashed line). D, immunohistochemistry of hyperplastic intermediate lobe from p16Ink4a–/–;p18Ink4c–/– mouse at 35 wks of age. Note the tumor stains for ACTH but not PRL, GH, LH, or FSH, indicating that tumor cells are adrenocorticotroph in origin. ACTH and {alpha}-MSH are cleavage products derived from the same precursor molecule proopiomelanocortin.

 
Morbidity was associated with large pituitary tumors of the intermediate lobe, in some cases producing non-communicating hydrocephalus, hind limb paralysis, and/or pontine compression (Fig. 2B). Although pituitary tumors were found in all p16Ink4a–/–;p18Ink4c–/– mice examined over 30 weeks of age, p18Ink4c–/– mice developed pituitary tumors as previously reported (16, 20), with ~50% penetrance by 1 year. No pituitary tumors were found in p16Ink4a–/– mice at any age examined. p16Ink4a–/–;p18Ink4c–/– mice developed non-pituitary tumors (e.g., lymphomas and sarcomas) at a comparable frequency to that of p16Ink4a–/– mice (data not shown), suggesting that cooperation between p16Ink4a and p18Ink4c in tumor suppression was most marked in the pituitary. Although this observation is in accord with previous findings showing an important anticancer role of cdk4/6 regulation in tumorigenesis of the intermediate pituitary (20, 30), loss of p18Ink4c combined with loss of p15Ink4b (16) or p19Ink4d (28) did not appreciably accelerate pituitary formation nor increase incidence, suggesting that p16Ink4a plays a predominant role in the prevention of pituitary tumorigenesis in p18Ink4c–/– mice.

In an effort to understand the basis of this cooperation, we did gross, histologic, and molecular analysis of age-matched wild-type, p16Ink4a–/–, p18Ink4c–/–, and p16Ink4a–/–;p18Ink4c–/– mice. Inspection of pituitaries of 35-week-old mice revealed normal macroscopic and microscopic structure in wild-type and p16Ink4a–/– mice (Fig. 2C). Consistent with previous data (20), p18Ink4c–/– mice at this age showed hyperplasia of the intermediate lobe with modest associated disruption of the gland architecture. However, p16Ink4a–/–;p18Ink4c–/– mice showed a marked increase in the size of the pituitary and anatomic disarray of the intermediate lobe with near complete effacement of the posterior lobe (Fig. 2C). Similar to cdk4R24C knock-in mice (30), cystic degeneration, hemorrhage, and necrosis were commonly seen in the pituitaries of p16Ink4a–/–;p18Ink4c–/– mice, even in animals less than 20 weeks of age, but were rarely seen in p18Ink4c–/– mice. Pituitary tumors in p16Ink4a–/–;p18Ink4c–/– mice stained strongly for ACTH, but not for other peptide hormones (PRL, GH, LH, or FSH) made in the pituitary (Fig. 2D), suggesting tumors were of the adrenocorticotroph lineage. Additionally, blood serum levels of {alpha}-MSH increased with age in p16Ink4a–/–;p18Ink4c–/– mice (Supplementary Fig. S1), as was reported for Rb+/– mice (31). These data suggest that p16Ink4a serves to limit tumor progression in the intermediate pituitary of p18Ink4c–/– mice.

p16Ink4a functionally compensates for p18Ink4c loss in the pituitary. To determine the functional consequences of combined p16Ink4a and p18Ink4c loss, we determined the Ki67 proliferative index in the intermediate lobe of the pituitary in adult mice. The intermediate lobes of the pituitary in 35-week-old wild-type, p16Ink4a–/–, p18Ink4c–/–, and p16Ink4a–/–;p18Ink4c–/– mice were compared. The rate of proliferation was comparable in wild-type (0.4%) and p16Ink4a–/– (0.5%) mice but significantly increased in p18Ink4c–/– mice (1.9%, P < 0.05 for both pairwise comparisons). Mice lacking both p16Ink4a and p18Ink4c showed an even greater increase in proliferation (2.7%) above wild-type (P = 0.003), p16Ink4a–/– (P < 0.001), and p18Ink4c–/– mice (P = 0.09; Fig. 3A ). This result suggests that the increase in p16Ink4a expression partially compensates for p18Ink4c loss in the intermediate pituitary, even in animals of intermediate (35-week-old) age.


Figure 3
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  		Figure 3. p18Ink4c–/– mice have increased p16Ink4a mRNA levels in specific tissues. A, quantification of proliferative index in the pituitary of 35-wk-old wild-type, p16Ink4a–/–, p18Ink4c–/–, and p16Ink4a–/–;p18Ink4c–/– mice. At least two mice per genotype were used, and all cells in the intermediate lobe were counted for each pituitary. Columns, mean; bars, SE. B, relative mRNA expression by real-time PCR of p16Ink4a, Arf, p15Ink4b, p19Ink4d, and p21Cip1 in indicated tissue of 10.6-wk-old wild-type and p18Ink4c–/– mice. Ratio of expression of indicated gene in p18Ink4c–/– mice compared with wild-type littermates. At least two to six mice per genotype were assayed; samples were measured in triplicate. 18S RNA served as a loading control and normalization. Columns, mean; bars, SE. C, relative increase in mRNA expression of p16Ink4a, Arf, p18Ink4c, p15Ink4b, p19Ink4d, and p21Cip1 in the pituitary with aging. Ratio of fold increase in old (54–59 wks) versus young (10.6 wks) is shown by p18Ink4c genotype. At least two mice per genotype were assayed; samples were measured in duplicate. Columns, mean; bars, SE.

 
To understand the mechanistic basis for this cooperation in tumors, we did an analysis of cdk inhibitor expression in the pituitary of wild-type and p18Ink4c–/– mice. Although there was a modest increase in Arf (3.4-fold), p21Cip1 (2.3-fold), p15Ink4b (2.4-fold), and p19Ink4d (1.5-fold) mRNA in p18Ink4c–/– mice when compared with wild-type mice, expression of p16Ink4a mRNA was by far the most increased (18.2-fold) in the pituitary of young (10.6-week-old) p18Ink4c–/– mice (Fig. 3B). Consistent with previous findings (20), p18Ink4c–/– mice of this age exhibited only mild hyperplasia with no evidence of tumors and had overtly normal tissue architecture (data not shown). Although this analysis does not exclude an effect of p18Ink4c deficiency on the protein expression of other cell cycle regulators, such as p27Kip1 (20), it suggests a potent in vivo mechanism of compensation for germline p18Ink4c deficiency by increased transcription of p16Ink4a.

Previous studies have shown that p16Ink4a expression increases with age in most mammalian tissues (24, 25, 27), and we noted that total pituitary similarly exhibited an increase in p16Ink4a expression with aging (Fig. 3C). A comparable age-induced induction of p16Ink4a expression was noted in p18Ink4c–/– mice such that the ratio of p16Ink4a expression in old (~57-week-old) versus young (10.6-week-old) pituitary was the same for wild-type or p18Ink4c–/– mice (Fig. 3C). Therefore, old p18Ink4c–/– mice showed a >40-fold increase in the mRNA expression of p16Ink4a compared with young wild-type mice, indicating the additive induction of p16Ink4a in this compartment in response to aging and p18Ink4c deficiency.

p16Ink4a and p18Ink4c coordinately control proliferation in the islets. To determine if the effect of combined Ink4 loss was seen in other tissues, we considered the pancreatic islet from 35-week-old mice (29, 32–34). As previously reported for p18Ink4c–/– mice (34), we found that p16Ink4a–/–;p18Ink4c–/– mice showed a modest increase in median islet size compared with wild-type and p16Ink4a–/– mice (Supplementary Fig. S2). There was no discernable difference in islet size between p18Ink4c–/– and p16Ink4a–/–;p18Ink4c–/– mice in animals of this age. Double staining with Ki67 and insulin indicated that the majority (>80%) of proliferating islet cells were ß-cells (data not shown), whose replication is regulated by cdk4, p18Ink4c, and cyclin D2 in the adult (32–35). In mice of this age, no significant difference in proliferation was seen between wild-type (0.58%), p16Ink4a–/– (0.67%), or p18Ink4c–/– (0.71%) islets for any pairwise comparison (Supplementary Fig. S2). However, p16Ink4a–/–;p18Ink4c–/– islets had a significantly higher rate of proliferation (0.98%) than wild-type (P = 0.02), p16Ink4a–/– (P = 0.08), and p18Ink4c–/– (P = 0.02) islets. It is important to note that these studies were carried out in young mice, and we expect that the effects of p16Ink4a loss would be more pronounced in older mice, as p16Ink4a expression potently inhibits islet proliferation in an age-dependent manner (24, 25). In aggregate, these data suggest that p16Ink4a and p18Ink4c coordinately regulate cdk4/6-dependent proliferation in the pancreatic ß-cells of adult mice.

Ink4 family members can transcriptionally compensate for loss of other members. To determine if the increase in p16Ink4a mRNA in the pituitary was tissue specific, we examined other tissues from p18Ink4c–/– mice. p16Ink4a levels were elevated in the adrenal glands (32.3-fold), spleen (4.3-fold), and kidney (3.9-fold) of p18Ink4c–/– mice, but there was no discernable change in p16Ink4a in the lung (Fig. 3B). The pituitary, adrenal glands, and spleen also showed moderately elevated levels of Arf and p15Ink4b, but little change was seen in the kidney or the lung. These results suggest that loss of p18Ink4c induces expression of other cdk inhibitors, specifically p16Ink4a in a tissue specific fashion in vivo.

It is possible that changes in cdk inhibitor levels could be a non–cell-autonomous effect due increased in vivo proliferation from p18Ink4c loss. We thus turned our attention to a more purified cell system (MEFs), which express all four Ink4 proteins (27). An examination of wild-type MEFs showed a massive increase in expression in all three transcripts from the Ink4/Arf locus (p15Ink4b, p16Ink4a, and Arf) in the first few days of culture, which was not seen for p18Ink4c and p19Ink4d (Fig. 4A ). However, only p16Ink4a continued to increase throughout passage, whereas mRNA levels of Arf, p15Ink4b, p18Ink4c, p19Ink4d, p21Cip1, and p27Kip1 remained relatively constant.


Figure 4
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  		Figure 4. MEFs show transcriptional compensation between Ink4 proteins. A, relative mRNA expression of p16Ink4a, Arf, p15Ink4b, p18Ink4c, p19Ink4d, p21Cip1, and p27Kip1 in cultured wild-type MEFs. Fold change from E13.5 embryo, which has not been put in culture. At least two lines per genotype were assayed; samples were measured in triplicate. 18S RNA served as a loading control and normalization. Points, mean; bars, SE. B, real-time PCR of p16Ink4a, Arf, p15Ink4b, p18Ink4c, p19Ink4d, and p21Cip1 in MEFs. Mid–life span (~10 population doublings) MEFs lacking p16Ink4a or p18Ink4c or both (DKO) were assessed for mRNA levels of indicated cdk inhibitors. Fold change from wild-type levels. 18S RNA served as a loading control and normalization. Columns, mean; bars, SE. C, Western blot analysis of p16Ink4a, Arf, p15Ink4b, p18Ink4c, p19Ink4d, p21Cip1, and p27Kip1. Mid–life span (~10 population doublings) wild-type, p16Ink4a–/–, p18Ink4c–/–, and p16Ink4a–/–;p18Ink4c–/– MEFs were analyzed for protein expression. Actin serves as a loading control. Arrow, specific band in Arf blot. D, in vitro cdk4 kinase activity of wild-type, p16Ink4a–/–, p18Ink4c–/–, and p16Ink4a–/–;p18Ink4c–/– MEFs (top). Cdk4 complexes were immunoprecipitated from mid–life span (~10 population doublings) MEFs using 900 µg total cell lysate. Kinase activity was assessed against GST-Rb-769 substrate. One sixth of the total cdk4 immunoprecipitated is shown as loading control (bottom). Relative activity represents fold difference from wild type.

 
We then examined mid–life span MEFs (~10 population doublings) for cdk inhibitor expression. p16Ink4a–/– MEFs had a significant increase in p15Ink4b (5.5-fold), moderate increases in Arf (2.5-fold) and p21Cip1 (1.6-fold) and a slight increase in p19Ink4d (1.3-fold) at both the mRNA (Fig. 4B) and protein (Fig. 4C) levels when compared with wild-type MEFs. As was seen in the various tissues in vivo (Fig. 3B), p18Ink4c–/– MEFs showed the greatest increase in p16Ink4a levels (2.9-fold) and more modest change in Arf (1.9-fold), p21Cip1 (1.4-fold), and p15Ink4b (2.1-fold), with little change in p19Ink4d (0.9-fold). MEFs lacking both p16Ink4a and p18Ink4c showed a large difference in the expression of p15Ink4b (4.8-fold) and smaller increases for Arf (2.8-fold), p21Cip1 (1.8-fold), and p19Ink4d (2.0-fold). There were no detectable differences in p27Kip1 mRNA (data not shown) or protein (Fig. 4C) between genotypes. Interestingly, all changes in mRNA levels were closely mirrored by changes in protein expression, suggesting that protein and message correlate highly under these conditions (compare Fig. 4B and C). Importantly, the compensation among the ink4 proteins is not complete, as p16Ink4a–/–, p18Ink4c–/–, and p16Ink4a–/–;p18Ink4c–/– MEFs show elevated cdk4 kinase activity (Fig. 4D) compared with wild-type cells. In aggregate, these results suggest that increased cdk4 activity due to loss of Ink4 proteins can be partially compensated for through activation of either p15Ink4b or p16Ink4a both in vitro and in vivo.

Cdk4/6 kinase activity is required for proliferation in specific compartments in adult mice. Genetic data have suggested a role for cdk4 in the regulation of proliferation in the pituitary and the pancreatic islets (20, 29, 33–35). We have found that these tissues show increased proliferation in mice lacking the cdk4/6 inhibitors p16Ink4a and p18Ink4c (Fig. 3A; Supplementary Fig. S2), suggesting a role for cdk4/6 catalytic activity in the regulation of these tissues in adult mice. However, cdk4/6 kinase activity is required for development of certain embryonic tissues, leading to late embryonic lethality in cyclin D (36) or cdk4/6 (37) null animals, thus limiting the ability to examine adult tissues. To address this issue, we determined the effect of acute cdk4/6 inhibition in 26-week-old p16Ink4a–/–;p18Ink4c–/– mice using PD 0332991, a specific inhibitor of cdk4/6, but not cdk2 or other tested kinases (38). PD 0332991 is a pyrido(2,3-d)pyrimidin-7-one compound modified with a 2-aminopyridine side chain that has been shown to block the growth of pRB-competent human cell lines and xenografted tumors with potent activity at nanomolar concentrations. PD 0332991 shows good oral bioavailability and can be given to mice for prolonged treatment with minimal toxicity (21, 38). Comparable to its potency in human cells, PD 0332991 was shown to inhibit murine cdk4 activity at doses as low as 50 nmol/L, whereas cdk2 activity was not affected even at micromolar concentrations (Fig. 5A ).


Figure 5
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  		Figure 5. Inhibition of cdk4/6 blocks proliferation in some but not all tissues. A, PD 0332991 effectively inhibits murine cdk4 but not cdk2 kinase activity in p16Ink4a–/–;p18Ink4c–/– MEFs. In vitro cdk2 and cdk4 kinase assay of p16Ink4a–/–;p18Ink4c–/– MEFs after 24 h of treatment with varying concentrations of PD 0332991. Cdk2 or cdk4 complexes were immunoprecipitated, and kinase activity was assessed against GST-Rb-769 substrate. Note that more protein (900 versus 100 µg) was used for cdk4 immunoprecipitation than for cdk2 immunoprecipitation. B, inhibition of cdk4/6 blocks proliferation in some tissues. Ki67 staining of pancreatic islets, pituitary, lymph nodes, and small bowel of 26-wk-old p16Ink4a–/–;p18Ink4c–/– mice. Mice were treated with vehicle (top) or 150 mg/kg PD 0332991 (bottom) for 2 wks. PL, posterior lobe; IL, intermediate lobe; GC, germinal center. C, quantification of proliferation in the pancreatic islets, intermediate pituitary, and small bowel of 26-wk-old p16Ink4a–/–;p18Ink4c–/– mice with and without 2 wks treatment with 150 mg/kg PD 0332991. Cells were counted from at least two mice per genotype, with at least 400 cells counted for the small bowel. Proliferation in pancreatic ß-cells was determined as average proliferation per islet from at least 38 islets of at least two mice per genotype. Pituitary proliferation was assessed as proliferative index of the entire intermediate lobe of at least two mice. Columns, mean; bars, SE. Magnification, x20 (all photos). D, lymph nodes from 26-wk-old p16Ink4a–/–;p18Ink4c–/– mice treated for 2 wks with 150 mg/kg PD 0332991 were stained for Ki67 and CD3 or B220. Note that Ki67 positive cells (nuclear) do not stain for either CD3 or B220 (cell surface markers). Therefore, Ki67-positive cells in PD 0332991–treated mice are neither T cells nor B cells.

 
To investigate the effects of cdk4/6 inhibition in adults, animals were treated with 150 mg/kg PD 0332991 by oral gavage daily for 2 weeks. As previously reported (21), this dose and schedule were well tolerated by the mice without evident toxicity. In accord with the phenotype observed in the p16Ink4a–/–;p18Ink4c–/– mice, both the pituitary and pancreatic islets showed a marked reduction in proliferation after treatment with PD 0332991 (Fig. 5B and C). Consistent with the reported hyperproliferation in germinal center B cells in p18Ink4c-deficient animals (39), we found that the majority of proliferating cells in p16Ink4a–/–;p18Ink4c–/– lymph nodes express the B-cell marker B220 and not CD3, a T-cell marker (data not shown). As was seen in the islet and pituitary, treatment with PD 0332991 inhibited proliferation of these hyperproliferative B cells in the germinal centers of lymph node (Fig. 5B). These data suggest that the pancreatic islet, intermediate pituitary, and germinal center B lymphocytes require the catalytic activity of cdk4/6 for proliferation in adult mice.

Unlike the islet and pituitary, however, proliferation in other tissues was not affected by prolonged cdk4/6 inhibition. The high rate of proliferation seen in the small bowel was not changed after 2 weeks of PD 0332991 treatment (Fig. 5B and C). In addition, a subset of B220-negative and CD3-negative cells dispersed throughout the lymph nodes continued to proliferate despite cdk4/6 inhibition (Fig. 5D). The morphology, location, and frequency of these cells seem most consistent with inter-digitating dendritic cells of the lymph node. Although it is formally possible that PD 0332991 is excluded from certain proliferating compartments, a more likely explanation is that proliferation of some cell types in adult mice do not require cdk4/6 activity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
p16Ink4a and p18Ink4c cooperate to suppress tumorigenesis in mice. The elucidation of the redundant contributions of individual genes in a highly related family is a problem in the study of mammalian biology, particularly in the case of the Ink4 family of cdk inhibitors. All four proteins are capable of binding cdk4 and cdk6 (2–5), and there seems to be little biochemical distinction among the Ink4s with regard to this activity (12). Therefore, it is not surprising that there is significant compensation by remaining Ink4s in MEFs singly deficient for p16Ink4a and p18Ink4c. However, Ink4-null mice show marked phenotypic differences, indicating distinct roles for these proteins in vivo. An examination of p15Ink4b–/–;p18Ink4c–/– (16) and p19Ink4d–/–;p18Ink4c–/– (28) mice has led to elucidation of some of the unique functions of Ink4 proteins, and we have expanded on this data through generation of mice lacking both p16Ink4a and p18Ink4c.

Our data suggest that p16Ink4a and p18Ink4c are important determinants of physiologic and neoplastic proliferation in certain tissues in vivo. For example, p18Ink4c seemed to regulate homeostatic islet and pituitary proliferation in young mice, and this effect was constrained by p16Ink4a expression (Fig. 3, Fig. 4A, and Fig. 5B; Supplementary Fig. S2). Loss of both proteins potently cooperated in pituitary tumorigenesis in our study; whereas the tumor spectrum, frequency, and latency in p18Ink4c–/– mice was not augmented by additional loss of p15Ink4b (16) or p19Ink4d (28). Although high penetrance of lethal pituitary tumors limited the finding of new tumor types, the latency of lethal pituitary tumors in p16Ink4a–/–;p18Ink4c–/– mice was much shorter than either single knockout, with survival comparable to mice with cdk4R24C mutation, which does not bind Ink4 proteins (29, 30). Analysis of Ink4 levels in several tissues suggested an explanation for the cooperation seen between p16Ink4a and p18Ink4c loss: only p16Ink4a expression was significantly increased in the pituitary of p18Ink4c–/– mice, and not p15Ink4b or p19Ink4d. This compensation is not limited to the pituitary, where there was an obvious phenotype, but was also seen in other tissues to varying degrees and in cultured MEFs (Fig. 3B and Fig. 4B and C). These results suggest a potent feedback mechanism for regulating cdk4/6 in the response to increased kinase activity.

Control of p16Ink4a expression by Rb-family protein function has been noted in several systems where inactivation of Rb leads to a rapid increase in p16Ink4a expression (40, 41). Likewise, p16Ink4a can be activated by overexpression of E2F (42), arguing for the presence of a feedback loop between Rb and p16Ink4a. Clearly, however, physiologic proliferation with attendant Rb family phosphorylation is not generally sufficient to activate p16Ink4a expression. Instead, Rb loss only seems to activate p16Ink4a expression under specific circumstances (e.g., neoplastic growth). Recent work has suggested a possible explanation for the conditional regulation of p16Ink4a by Rb. Kotake et al. (43) have shown that the Polycomb group (PcG) protein Bmi-1, which durably represses of p16Ink4a expression in association with covalent histone modifications (44, 45), is targeted to the Ink4a/Arf locus by Rb family proteins. Additionally, Bmi-1 seems to require Rb family function to stably repress p16Ink4a expression in human and murine cells. Therefore, it is tempting to speculate that p16Ink4a expression (and possibly that of p15Ink4b and Arf as well) is increased in certain p18Ink4c-deficient tissues because of a relative hyperphosphorylation of pRb with attendant decrease in the PcG-mediated repression of the Ink4a/Arf locus.

Certain adult tissues require cdk4/6 activity. Recent genetic experiments have suggested that MEFs can proliferate without cdk4 (32, 33), cdk6 (37), or both (36, 37). Conversely, cdk2 kinase activity is also dispensable in MEFs, as cdk2–/– (46, 47) and cyclin E1–/–;cyclin E2–/– (48) cells can proliferate in culture, albeit more slowly. Although cdk4/6 activity was not a major determinant of MEF proliferation, this was not true in certain tissues in vivo such as the intermediate pituitary, germinal center B cells, and pancreatic ß-cell. In these tissues, the specific requirement of cdk4/6 catalytic activity was supported by our findings that genetic loss of p16Ink4a and p18Ink4c resulted in their increased proliferation, whereas acute pharmacologic inhibition abrogated their proliferation.

The dependence of pancreatic ß-cells cells on cdk4 for proliferation seems restricted to postnatal mice, as cdk4–/– mice are born with islets, but cannot sustain proliferation as the animal grows (32, 33). Conversely, loss of cdk4 regulation by p18Ink4c loss (34), p16Ink4a loss (25), or knock-in of cdk4R24C (29, 30, 33) results in aberrant proliferation in the pancreatic ß-cells. In aggregate, these results indicate an exclusive role for cdk4 in the regulation of pancreatic ß-cell proliferation in the adult mouse. Additionally, some tissues are regulated both prenatally and postnatally by cdk4/6. We have shown that B cells require cdk4/6 for proliferation in the adult animal, but they are also required in the developing embryo, as both D-deficient and cdk4/6-deficient mice die with greatly reduced common lymphoid progenitors (36, 37). Most importantly, cdk4/6–/– and cyclin-D1/D2/D3–/– mice show that the absolute requirement for cdk4/6 in development seems to hold in only a few select tissue compartments, as substantial development occurs in their absence (36, 37). Our data using a specific cdk4/6 inhibitor further these developmental observations to show that adult mice similarly only require cdk4/6 in a limited number of compartments, including B cells, pituitary adrenocortotrophs, and pancreatic ß-cells; compartments where p16Ink4a–/–;p18Ink4c–/– mice show the most marked phenotype.

In summary, we have shown that p16Ink4a compensates for p18Ink4c loss to regulate neoplastic and physiologic proliferation. Additionally, by both pharmacologic and genetic approaches, we have shown that p16Ink4a and p18Ink4c function in vivo to constrain cdk4/6 catalytic activity in highly specific compartments. The search for effective Cdk inhibitors has been hampered by lack of clarity over the best Cdk to target and the challenge of achieving appropriate levels of kinase selectivity. Our data show that cdk4/6 kinase activity is directly responsible for regulating proliferation of specific tissue compartments in adult mice and thus help to predict the toxicity of specific cdk4/6 inhibitors. Additionally, these data offer hope that certain established cancers (e.g., those characterized by cdk4 mutation, cyclin D amplification, or p16Ink4a loss) will also strictly require cdk4/6 activity for proliferation and therefore respond to specific pharmacologic inhibition of these kinases.


   Acknowledgments
 
Grant support: Sidney Kimmel Foundation for Cancer Research, NIH grants AG 024379 and DK34987, and NIH Cell and Molecular Biology Training program grant GM008581 (M.R. Ramsey).

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.

We thank Peter Toogood, Diego Castrillon, Lishan Su, Grigory Kovalev, Paula Miliani de Marval, Feng Bai, Stuart Shumway, Chad McCall, and Kathy Wilber for advice, reagents, and technical assistance and Robert Duronio and William Kim for critical reading of the manuscript.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 9/18/06. Revised 2/ 1/07. Accepted 3/12/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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