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Cancer Research 67, 4130, May 1, 2007. doi: 10.1158/0008-5472.CAN-07-0499
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

p21 Loss Cooperates with INK4 Inactivation Facilitating Immortalization and Bcl-2–Mediated Anchorage-Independent Growth of Oncogene-Transduced Primary Mouse Fibroblasts

Christopher J. Carbone1, Xavier Graña1,2, E. Premkumar Reddy1,2 and Dale S. Haines1,2

1 Fels Institute for Cancer Research and Molecular Biology and 2 Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania

Requests for reprints: Dale S. Haines, Fels Institute for Cancer Research and Molecular Biology and Department of Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140. Phone: 215-707-5765; Fax: 215-707-2102; E-mail: dhaines{at}temple.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The INK4 and CIP cyclin-dependent kinase (Cdk) inhibitors (CKI) activate pocket protein function by suppressing Cdk4 and Cdk2, respectively. Although these inhibitors are lost in tumors, deletion of individual CKIs results in modest proliferation defects in murine models. We have evaluated cooperativity between loss of all INK4 family members (using cdk4r24c mutant alleles that confer resistant to INK4 inhibitors) and p21Waf1/Cip1 in senescence and transformation of mouse embryo fibroblasts (MEF). We show that mutant cdk4r24c and p21 loss cooperate in pRb inactivation and MEF immortalization. Our studies suggest that cdk4r24c mediates resistance to p15INK4B/p16INK4A that accumulates over passage, whereas loss of p21 suppresses hyperoxia-induced Cdk2 inhibition and pRb dephosphorylation on MEF explantation in culture. Although cdk4r24c and p21 loss cooperate in H-rasV12/c-myc–induced foci formation, they are insufficient for oncogene-induced anchorage-independent growth. Interestingly, p21–/–; cdk4r24c MEFs expressing H-rasV12 and c-myc display detachment-induced apoptosis and are transformed by c-myc, H-rasV12, and Bcl-2. We conclude that the INK4 family and p21 loss cooperate in promoting pRb inactivation, cell immortalization, and H-rasV12/c-myc–induced loss of contact inhibition. In addition, absence of pRb function renders H-rasV12 + c-myc–transduced fibroblasts prone to apoptosis when deprived of the extracellular matrix, and oncogene-induced anchorage-independent growth of pocket protein–deficient cells requires apoptotic suppression. [Cancer Res 2007;67(9):4130–7]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Quiescent cells require exposure to mitogenic growth factors to enter the cell cycle and pass through the restriction point. The key effectors promoting cell cycle entry and progression through the G1-S transition are the cyclin-dependent kinase (Cdk) 4/Cdk6-cyclin D and Cdk2-cyclin E kinase complexes (1). Cdk4/Cdk6 associates with mitogen-induced D-type cyclins in early G1, whereas Cdk2 pairs with cyclin Es that accumulate in late G1. These kinase complexes phosphorylate pocket proteins pRb, p107, and p130, leading to release of the E2F family of transcriptional regulators and transcription of genes required for cell cycle progression and DNA synthesis. Cdk activity is negatively regulated by the INK4 (p16INK4A, p15INK4B, p18INK4C, and p19INK4D) and CIP/Kip (p21Waf1/Cip1, p27Kip1, and p57Kip2) family of inhibitors (2). Induction of these proteins by stress, growth-inhibitory, or differentiation-inducing signals provides a means of halting cell cycle progression to allow for repair or promoting exit from the cell cycle.

It was initially thought that Cdk4/Cdk6 and Cdk2 work sequentially on pRb and play essential and nonredundant roles in promoting cell cycle progression (3). However, the generation of knockout mouse models has challenged this notion and recent results indicate that, at least for Cdk4 and Cdk2, they play overlapping roles in pRb phosphorylation and modulation of E2F-dependent gene expression in mouse fibroblasts (4). Cdk inhibitors (CKI) from the INK4 and CIP/Kip families also possess overlapping activities in regulating pocket protein function, although in contrast to Cdks, they promote pocket protein dephosphorylation and positively regulate their activities (5).

Considering the fundamental roles of Cdks and CKIs in proliferation control, it is not surprising to find that the expression and activity of these proteins are deregulated in cancer cells. For example, human tumors frequently overexpress cyclin D1, Cdk4, and/or cyclin E.

Conversely, expression of CKIs p15INK4B, p16INK4A, p18INK4C, and p27Kip1 are down-regulated in tumors (6). Consistent with the expression data, transgenic mice harboring elevated levels of cyclin D1 or cyclin E are tumor prone, whereas those lacking individual CKIs are predisposed to cancer (7–9). For CKIs, however, the cancer predisposition phenotypes of single gene knockout are generally very mild and more dramatic phenotypes are observed in mice lacking multiple CKIs (6, 10–12). These studies reinforce the idea of functional overlap between CKI proteins and suggest that inactivation or down-regulation of multiple CKIs will be required for cellular transformation and tumor development.

Because of the difficulties in studying the cellular transformation process in vivo, cell-based models using primary mouse embryo fibroblasts (MEF) have been extensively used to define genes regulating "normal" cell proliferation and how gene disruption affects various growth characteristics in culture. Although there are clear differences between the proliferative characteristics of human and murine fibroblasts, studies have defined genes that are required for the escape of culture-induced growth arrest (or senescence), oncogene-induced proliferation, and anchorage-independent growth (13). For example, p53–/– MEFs do not undergo culture or oncogenic H-rasV12–induced proliferation arrest and are susceptible to H-rasV12–mediated anchorage-independent growth (14–16). Interestingly, deletion of pRb and p107, or all three pocket proteins, allows MEFs to proliferate on introduction of mutant RAS alone yet confers inefficient (at least when compared with p53–/– MEFs) anchorage-independent proliferation after transduction with this and other oncogenes, including c-myc (17). In terms of CKIs, MEFs lacking individual INKs or CIPs/Kips display only modest, if any, predisposition to immortalization in culture, H-rasV12–induced proliferation, or H-rasV12 + c-myc–mediated anchorage-independent growth (6, 10). Thus, a common theme that emerges from these studies is that in murine fibroblasts, the CKIs are likely to play overlapping roles in suppressing culture and oncogene-induced proliferative arrest and cells lacking pRb function will require inactivation or activation of other pathways to allow for oncogene-induced anchorage-independent proliferation.

Mutation of residue 24 from an arginine to cysteine in Cdk4 occurs in familial cases of melanomas and confers resistance of the protein to inhibition by INK4 family members (18, 19). Mouse modeling experiments have shown that mice harboring a knock-in cdk4r24c mutation develop tumors at an accelerated rate (18–20). Moreover, cdk4r24c MEFs are resistant to culture-induced senescence and are prone to transformation by oncogenes. However, these phenotypes are again mild, especially when compared with MEFs lacking all pocket proteins (21, 22), raising the possibility of functional redundancy between INK4 proteins and the CIP family of inhibitors in these processes. The goal of studies described here was to investigate cooperativity between the cdk4r24c mutation and loss of p21 in MEF senescence and oncogene-induced proliferation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and MEF isolations. Mice harboring the cdk4r24c mutation (mixed 129SV, CD-1, and C57BL/6J) have been described previously (18). p21–/–(FVB) and p53–/+ mice (mixed 129SV and C57BL/6J) were obtained from P. Leder (Harvard Medical School, Boston, MA) and S. Jones (University of Massachusetts, Worcester, MA), respectively. Homozygous cdk4r24c were crossed with p21–/– and p53–/+ animals. Pups were genotyped by PCR and resulting heterozygous siblings were mated to generate mice homozygous for one or two mutant alleles. MEFs were prepared from 13.5 days postcoitus embryos as described previously (23). Cells were grown ~1 to 2 days until the culture was 90% confluent, harvested, viably frozen, and labeled as passage 0. For experiments using cells cultured in 20% or 3% oxygen, torsos were dissected longitudinally and cultured in either 3% oxygen/5% CO2 or 20% oxygen/5% CO2.

3T3 assays. MEFs (3 x 105) of the indicated genotypes derived from passage 0 were plated in 10-cm dishes, grown for 3 days, trypsinized, and counted, and 3 x 105 cells were reseeded. This was repeated for each passage.

Western blotting. Proteins were resolved by SDS-PAGE, transferred to membranes, and probed with commercially available antibodies (see Supplementary Materials and Methods). Proteins (except for p16INK4A) were detected using secondary antibodies [horseradish peroxidase (HRP)–linked sheep anti-mouse IgG (Amersham) for monoclonal antibodies and HRP-linked sheep anti-goat IgG (Chemicon) for the anti-actin polyclonal antibody] and reagents provided in a Western Lighting Chemiluminescence Reagent kit from Amersham. p16INK4A was detected using an antimouse secondary antibody and solutions were provided in the Lumi-LightPLUS Western blotting kit (Roche).

Cell cycle analysis and Cdk2 kinase assays. Cells were harvested, fixed with 70% ethanol, and incubated on ice for an hour. After centrifugation, pellets were treated with 20 mg/mL RNase A and stained with 5 mg/mL propidium iodide. DNA content was determined using a flow cytometer and samples were analyzed with ModFit LT 3.1 SP2 software. Cdk2 kinase activity was determined from immunopurified complexes as described previously (24).

Senescence-associated ß-galactosidase assay. ß-galactosidase activity was detected as described previously (25) with slight modifications. MEFs from passage 10 of the 3T3 protocol were washed once with PBS, fixed with 0.5% glutaraldehyde (PBS), and washed in PBS supplemented with 1 mmol/L MgCl2. MEFs were stained with X-gal solution [1 mg/mL X-gal, 0.12 mmol/L K3Fe(CN)6, 0.12 mmol/L Fe4(CN)6, and 1 mmol/L MgCl2 in PBS (pH 6.0)] at 37°C.

Oncogene-induced foci and soft agar assays. Phoenix cells were transfected with 10 µg of each retroviral plasmid using the CalPhos Mammalian Transfection kit (BD Biosciences) and 25 µmol/L chloroquine (Sigma). The medium was changed 24 h after the transfection. Forty-eight hours after transfection, the retrovirus-containing medium was collected, filtered, supplemented with 5 µg/mL polybrene (Sigma), and used to infect early-passage MEFs growing in 3% oxygen/5% CO2 (done in duplicate for each genotype). Infected cell populations were selected with 2 µg/mL puromycin. For foci formation assays, 3 x 105 puromycin-selected cells were seeded into 10-cm plates. Cells were maintained for 10 days in a standard tissue culture environment and the medium was changed every 2 days. For soft agar assays, MEFs were sequentially transduced with the indicated viruses, and puromycin-selected and 4 x 104 cells were placed into 0.38% Nobel agar in DMEM containing 10% fetal bovine serum. This was overlaid on solidified 0.7% Nobel agar containing culture medium in a 6-cm dish. The cells were fed weekly and colonies were evaluated after 2 weeks. Viable colonies were stained with 0.05% nitroblue tetrazolium.

Detachment-induced apoptosis studies. The detachment-induced apoptosis assays were done as described previously (26). Briefly, equal numbers of cells were seeded onto 10-cm dishes containing solidified 0.7% Nobel agar supplemented with culture medium. After 24 h, cells were either processed for Western blotting analysis using the anti-poly(ADP-ribose) polymerase (PARP) antibody or resuspended in 1x binding buffer (PharMingen Annexin V-Phycoerythrin Apoptosis Detection kit). Cells were analyzed by flow cytometry.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
p21 deletion cooperates with cdk4r24c in suppression of culture-induced senescence immortalization and pRb inactivation. The INK4 and CIP family of CKIs activate pocket protein function by suppressing the activities of Cdk4 and Cdk2, respectively. The goal of this study was to investigate cooperativity between loss of INK4 family function (by using a knock-in mouse model where Cdk4 is replaced by a mutant cdk4r24c whose encoded product is resistant to the action of all INK4 proteins) and p21 (using p21–/– mice) in MEF proliferation. It deserves to be noted that the other target of INK4 proteins, Cdk6, shows little, if any, expression in MEFs, and in contrast to Cdk2–/–;Cdk4–/– MEFs, Cdk2–/–;Cdk6–/– MEFs do not display cell cycle defects over Cdk2–/– MEFs (1). Mice harboring mutant cdk4r24c alleles were bred with p21–/–mice to generate p21–/–; cdk4r24c animals. p21–/–; cdk4r24c MEFs were prepared from subsequent matings and their growth characteristics in culture were compared with cdk4r24c, p21–/–, and WT fibroblasts (these experiments were done with MEFs isolated from two independent embryos per genotype) using a 3T3 protocol. We used a 3T3 protocol (i.e., 3 x 105 cells seeded every 3 days in 10-cm dishes) where cells are seeded at 30% to 40% confluency. This was done to accentuate any proliferation differences between the various genotypes that could be masked by MEFs reaching contact inhibition. As shown in Fig. 1A , WT MEFs undergo senescence very rapidly on low-density plating and we terminated culturing the cells when the number of cells harvested at day 3 was less than that initially plated. Interestingly, p21–/– MEFs possess an increased proliferative capacity over WT and even cdk4r24c cells at low passages when cells are seeded at low densities. However, their proliferation rate declined over increasing passage. cdk4r24c MEFs also possess an extended life span when compared with WT MEFs, and unlike p21–/– MEFs, their growth rate is consistent until passage 20, where immortalized variants begin to dominate the culture.3 Figure 1A also shows that p21–/–; cdc4r24c MEFs do not undergo a significant crises period, display a marked increased proliferative capacity at all passages, and proliferate indefinitely. In addition, ß-galactosidase staining (a senescence marker) of cells at passage 10 shows little, if any, ß-galactosidase–positive cells in p21–/–; cdk4r24c MEFs, whereas numerous ß-galactosidase–positive cells were detected in p21–/– and cdk4r24c cultures (Fig. 1B).


Figure 1
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  		Figure 1. p21 deletion extends MEF life span in culture and cooperates with mutant Cdk4 in suppression of culture-induced senescence. A, MEFs of the indicated genotypes (each derived from two independent embryos) were propagated in culture by seeding 3 x 105 cells every 3rd day into 10-cm dishes. N3/NO, the number of cells present 3 d after seeding/3 x 105. B, an in situ ß-galactosidase assay was done with passage 10 MEFs of the indicated genotypes.

 
To determine the affects of combined INK4 family and p21Waf1/Cip1 loss on pRb expression and activity over passage, we assessed pRb phosphorylation status and levels of E2F target gene products cyclin A, p107, and E2F1 in WT, p21–/–, cdk4r24c, and p21–/–; cdk4r24c MEFs. As shown in Fig. 2 , the ratio of hyperphosphorylated to hypophosphorylated pRb (as better evidenced by the short exposure) was dramatically elevated in p21–/–; cdk4r24c MEFs when compared with p21–/– and cdk4r24c fibroblasts, especially at later passages. We also noticed elevated levels of total pRb in late passage p21–/–; cdk4r24c MEFs. The reason for the increase is unclear and under investigation. Nevertheless, Fig. 2 shows that hyperphosphorylated pRb correlates with higher levels of E2F-regulated proteins cyclin A, p107, and E2F1 at passage 5 and later in p21–/–; cdk4r24c/r24c MEFs. We conclude from these studies that loss of INK4 family and p21Waf1/Cip1 cooperates in promoting pocket protein inactivation and escape from culture-induced senescence and immortalization. Moreover, unlike immortalized p21–/– (10) and cdk4r24c MEFs, immortal p21–/–; cdk4r24c MEFs retain WT p53 alleles and p19ARF (see Supplementary Fig. S1), indicating that p53 functions upstream of both Cdk2 and Cdk4.


Figure 2
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  		Figure 2. p21 deletion cooperates with mutant Cdk4 in suppression of culture-induced pocket protein inactivation. Levels of pRb (both long and short exposures of the film are presented) and E2F target genes p107, E2F1, and cyclin A in the indicated MEFs and passages were determined by Western blotting. Hypophosphorylated and hyperphosphorylated of pRb (pRb-P). The actin blot was used as a loading control.

 
Mutant Cdk4 renders cells resistant to p15INK4B and p16INK4A that accumulate on cell passaging, whereas loss of p21Waf1/Cip1 suppresses hyperoxia-induced Cdk2 down-regulation and pRb activation. We next investigated the mechanism of cooperativity between the cdk4r24c mutation and p21 loss in MEF proliferation. It is well established that the levels of p15INK4B and p16INK4A accumulate as WT MEF cells are propagated in culture (27, 28). This is also the case for p21–/–, cdk4r24c, and p21–/–; cdk4r24c MEFs (see Fig. 3A ). Moreover, accumulation of p15INK4B and p16INK4A is associated with a sharp decline (from passage 1 to 15) in pRb phosphorylation on a Cdk4-specific site (29, 30) in p21–/– MEFs. Only a slight decrease was observed in cdk4r24c MEFs and no decline was evident in p21–/–; cdk4r24c MEFs (Fig. 3B). These, as well as the biological data presented in Fig. 1, suggest that cdk4r24c renders MEFs resistant to the action of INK4 proteins that accumulate on successive passaging of cells in culture.


Figure 3
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  		Figure 3. Expression levels of p15INK4B, p16INK4A, p21Waf1/Cip1 and phosphorylated pRb at Thr826 in MEFs defective in INK4 and/or p21Waf1/Cip1 function. A, Western blotting for p21Waf1/Cip1, p16INK4A, and p15INK4B was done with extracts prepared from the indicated genotypes and passage. B, pRb phosphorylation at Thr826 was determined by Western blotting. The amount of extract analyzed was the same as in the previous figure where equivalent loading was verified.

 
In contrast to p15INK4B and p16INK4A, p21Waf1/Cip1 is modestly induced, if at all, as MEFs are passaged in culture (10) and this is also the case in cells harboring cdk4r24c (Fig. 3A). Thus, it is possible that p21Waf1/Cip1 activity, but not expression, is differentially regulated. However, a previous study showed that p53-responsive transcripts of the MDM2 promoter are robustly induced in WT MEFs immediately after placement in tissue culture, peak at or around passage one, and remain high over passaging (31). Considering this result and that p21 is also a p53 target gene (32), we tested if p21Waf1/Cip1 is induced on MEF explantation into tissue culture. As shown in Fig. 4A , p21Waf1/Cip1 levels markedly increase on introduction of cells into the standard tissue culture environment. In contrast, the expression of other CKIs remains constant. To verify that p21Waf1/Cip1 induction during this process is p53 dependent, we measured the amount of p21Waf1/Cip1 in p53–/– MEFs after explantation. As depicted in Fig. 4B, p21Waf1/Cip1 is not induced in p53–/– MEFs when placed into culture. We conclude from these experiments that p21Waf1/Cip1 is up-regulated via a p53-dependent manner on explantation of MEFs into cell culture.


Figure 4
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  		Figure 4. p21Waf1/Cip1 is induced via a p53-dependent mechanism and p21 loss confers resistance to hyperoxia-induced Cdk2 down-regulation and pRb activation on explantation of MEFs into culture. A, CKI levels were assessed by Western blotting before placing cells in culture (0) and 24 and 48 h later. B, p21Waf1/Cip1 and actin were measured by Western blotting before (0) and 4 and 8 h after being placed into culture. C, p21Waf1/Cip1 and actin were assessed by Western blotting in MEFs before placing cells in culture (0) and at the indicated time points after exposure to 3% or 20% oxygen. D, passage 1 MEFs (derived from two independent embryos) were plated at 2 x 105 cells per 60-mm diameter dish (done in triplicate) and grown in 20% or 3% oxygen. Cells were counted 1, 2, and 3 d after plating. E, fluorescence-activated cell sorting analysis of DNA content was done with the cells harvested 3 d after plating. F, Cdk2 kinase activity was measured in WT and p21–/– MEFs 3 d after platting. Bottom, input of histone H1 and equivalent amounts of IgG in each immunoprecipitation. G, pRb phosphorylation status and levels of E2F-responsive proteins cyclin A, p107, and E2F1 were assessed by Western blotting using the same extracts used in the kinase assays. All of the above described experiments were done with MEFs from multiple embryos (at least three from each genotype) and show reproducible results.

 
Primary cells encounter oxidative stress when placed into the standard tissue culture environment of 20% oxygen. It is well known that p53 activity is induced by oxidative damage and recent work has shown that culturing of MEFs in atmospheric oxygen promotes DNA damage and senescence (33). To determine if p21Waf1/Cip1 is differentially regulated when MEFs are cultured in 20% atmospheric versus normoxic 3% oxygen, Western blotting was done with extracts prepared from early-passage MEFs grown at these two different oxygen concentrations. As shown in Fig. 4C, p21Waf1/Cip1 increases when MEFs are cultured at 20%, but not 3% oxygen. In addition, we found that WT MEFs cultured in 3% oxygen displayed increased (a) proliferative capacity (Fig. 4D), (b) number of cells in the S/G2-M phases of the cell cycle (Fig. 4E), (c) Cdk2 kinase activity (Fig. 4F), and (d) pRb phosphorylation and the expression of E2F target gene products (Fig. 4G) when compared with cells grown in 20% oxygen. In contrast to WT MEFs, p21–/– MEFs displayed increases in these measured variables, regardless of whether they were cultured in 3% or 20% oxygen. These studies suggest that p21 loss confers resistance to hyperoxia-induced Cdk2 down-regulation and pRb activation that occurs on explanation of MEFs in culture.

p21 loss cooperates with cdk4r24c in oncogene-induced foci formation. Another characteristic of cells with altered proliferation control characteristics is that they undergo deregulated growth (as assessed by loss of contact inhibition or anchorage-independent growth) in response to oncogenes. For example, introduction of activated Ras (i.e., H-rasV12) into primary WT fibroblasts induces senescence, although it promotes loss of contact inhibition in cells lacking pocket protein function and both loss of contact inhibition and anchorage-independent growth in p53–/– MEFs (17, 34). In addition, p21–/– MEFs lose contact inhibition when transduced with H-rasV12 and c-myc oncogenes (35). Thus, we next determined how p21–/–; cdk4r24c MEFs respond upon H-rasV12 and c-myc transduction. As shown in Fig. 5 , the proliferation of p21–/–; cdk4r24c MEFs transduced with H-rasV12 was not restrained when cells reached confluency and cells continued to proliferate to form multiple layers or foci. Similarly, p21–/–; cdk4r24c MEFs transduced with c-myc alone resulted in foci composed of rounded, refractory cells (Fig. 5). p21–/–; cdk4r24c cells containing c-myc and H-rasV12 exhibited a profound increase in proliferation, as evidenced by the presence of multiple layers of cells in over 50% of the tissue culture dish (Fig. 5). These phenotypes were not observed in any of the WT, p21–/–, and cdk4r24c transductions with individual or both oncogenes. These results show that loss of INK4 and p21Waf1/Cip1 cooperate in oncogene-induced loss of contact inhibition.


Figure 5
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  		Figure 5. Loss of p21Waf1/Cip1 and INK4 function allows for unrestrained proliferation in MEFs on oncogene transduction. MEFs of the indicated genotypes were transduced with a recombinant H-rasV12 and/or c-myc retroviruses. Cells were seeded and grown in standard tissue culture conditions for 10 d. Black outline, cells that are present in layers.

 
Bcl-2 confers anchorage-independent growth of oncogene-transduced p21–/–; cdk4r24c MEFs. Besides doing foci formation assays, we also tested if stably transduced H-rasV12 and c-myc p21–/–; cdk4r24c MEFs are capable of anchorage-independent growth. Growth in soft agar was not apparent in H-rasV12 + c-myc–transduced p21–/–; cdk4r24c, p21–/–, or cdk4r24c MEFs (see Supplementary Fig. S2). In contrast, control p53–/– MEFs formed colonies in soft agar on introduction of H-rasV12 and H-rasV12 + c-myc (Supplementary Fig. S2). Interestingly, when we looked at these plates, 1 week after seeding, we noticed colonies of very small size (8–10 cells) and the comprised cells displayed visible apoptotic characteristics.3 Based on this observation, we wanted to test if oncogene-transduced p21–/–; cdk4r24c cells display increased apoptosis when deprived of attachment and if suppression of an apoptotic response via the introduction of the antiapoptotic oncogene Bcl-2 enhances transformation by H-rasV12 + c-myc. Figure 6A shows that when compared with WT MEFs, p21–/–; cdk4r24c MEFs transduced with H-rasV12 + c-myc undergo increase apoptosis as assessed by PARP cleavage (Fig. 6A, left) and Annexin staining (Fig. 6A, right) when cells are deprived of anchorage. Moreover, p21–/–; cdk4r24c fibroblasts are efficiently transformed on sequential transduction with c-myc, HrasV12, and Bcl-2 (Fig. 6B). These results suggest that suppression of apoptotic pathways is required for oncogene-induced anchorage-independent proliferation of cells lacking INK4 and p21Waf1/Cip1 functions.


Figure 6
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  		Figure 6. p21–/–; cdk4r24c MEFs undergo enhanced detachment-induced apoptosis and proliferate in soft agar on transduction with c-myc, H-rasV12, and Bcl-2. A, MEFs stably transduced with H-rasV12 and c-myc oncogenes were harvested and either pelleted (0) or placed in dishes containing solidified Nobel agar for 24 h. MEFs were collected and apoptosis was assessed by PARP cleavage or Annexin V positivity. B, p21–/–; cdk4r24c and WT MEFs were sequentially transduced with empty vector (P) or the indicated oncogenes. After selection, cells were suspended in Nobel agar and the colonies were visualized by staining 2 wks later.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we show for the first time the cooperativity between loss of INK4 family function (via the use of cells that harbor the cdk424c mutation) and p21 in culture-induced senescence, pRb inactivation, and immortalization of MEFs. In addition, we show that p21Waf1/Cip1 is robustly induced via a p53-dependent manner on explantation of MEFs into the standard tissue culture environment, but not when MEFs are grown in physiologic oxygen concentration. The atmospheric hyperoxia-induced p21Waf1/Cip1 expression inhibits Cdk2 activity, pRb phosphorylation, and E2F-dependent gene expression. In addition, although INK4 and p21 loss also cooperate in H-rasV12/c-myc–induced foci formation, these alterations are not sufficient to confer H-rasV12 + c-myc–induced anchorage-independent growth, as lack of attachment triggers apoptosis. In keeping with these observations, INK4 and p21–/– MEFs are transformed by expression of H-rasV12, c-myc, and the antiapoptotic oncogene Bcl-2.

These studies provide a working model for mechanisms of cooperativity between INK4 and CIP family members in regulation of pocket protein function and perhaps more generalized proliferation control. Because INK4s (via down-regulation of Cdk4/Cdk6) and CIPs (via suppression of Cdk2) work upstream of pocket proteins, cooperativity between individual CKIs on regulating their function will be defined by their relative expression/activity within individual cell types or during specific biological processes. For example, we propose based on our expression studies that absence of INK4 activity renders cdk4r24c cells resistant to p15INK4B and p16INK4A that are up-regulated as cells are propagated in culture. However, these cells are sensitive to the growth-inhibitory activity of p21Waf1/Cip1 that is induced by culturing of cells in atmospheric oxygen. In contrast, p21–/– MEFs are resistant to hyperoxia-induced inhibition and proliferate well at early passages. Yet, they likely become sensitive to the action of high levels of p15INK4B and p16INK4A at later passages and lose their proliferative capacity. Cells lacking both INK4 and p21Waf1/Cip1 functions are resistant to growth-inhibitory signals that arise early and late during the culturing process; thus, they display an overall increased proliferative and immortalization rates over cells that harbor single alterations. This model does not rule out the possibility that p18INK4C, p19INK4D, and even p27Kip1 also cooperate in the culture-induced senescence process because these proteins are expressed in MEFs. Notwithstanding, it will be of interest to define how deletion of individual INK4s and CIPs in varying combinations affects MEF proliferation. The large number of INK4s and CIPs and their disparity in regulation provide a means for precisely regulating proliferation rates in context of numerous signals.

The "cooperativity-based on expression model" may explain why synergy of loss between various INK4s and CIPs is tissue specific (6). It could also reconcile differences in the relative contribution of individual proteins to the senescence process in human versus murine cells and even the disparate results obtained when the cells of the same type and species are cultured differently. For example, although the cdk4r24c mutation promotes escape from senescence in murine cells, it does not have a major affect in human cells (36). In contrast, p21 loss has a dramatic effect on senescence in human fibroblasts (37). Considering that the stress signals that drive senescence in human versus mouse cells are quite different, it is likely that the relative contribution of CKIs are dictated by the type and strength of stress signals that induce their expression in human and mouse cells. Similarly, whereas a previous study has shown that p21–/– MEFs proliferate similarly to WT MEFs using a 3T3 protocol where cells are seeded at high densities, we show here using a different 3T3 protocol where cells are seeded at lower densities that p21 loss promotes proliferation. Thus, it is likely that p21Waf1/Cip1 plays a more dominant role (either alone or in cooperation with other factors that are differentially present) when cells are seeded at lower confluency.

Besides showing cooperativity in suppression of MEFs senescence, we also show that cdk4r24c and p21 loss cooperate in oncogene-induced proliferation and allow cells to overcome contact inhibition. Previous studies have shown that H-rasV12 up-regulates INK4s and p21Waf1/Cip1, raising the likelihood that loss of the function of the proteins in p21–/–; cdk4r24c MEFs promotes H-rasV12–induced proliferation (34). In addition, H-rasV12 has been shown to override c-myc–induced apoptosis in p21–/– MEFs, pointing to synergy between H-rasV12 + c-myc in proliferation of p21–/–; cdk4r24c MEFs (35). Interestingly and in contrast to p53–/– MEFs, H-rasV12 + c-myc–transduced p21–/–; cdk4r24c MEFs do not proliferate in soft agar. These results are similar to what has been reported with MEFs lacking multiple pocket proteins (17). Thus, although deregulation of pRb or pocket protein function (via loss of the proteins themselves or the function of upstream regulators, such as those reported here) confers loss of contact inhibition by these oncogenes, our results are consistent with the idea that pocket protein loss is not sufficient for oncogene-induced anchorage-independent proliferation. Data presented here indicate that p21–/–; cdk4r24c cells harboring H-rasV12 + c-myc are prone to detachment-induced apoptosis and are transformed on cotransduction with Bcl-2. Although the mechanisms of cooperativity between cdk4r24c, p21 loss, Bcl-2, and H-rasV12 + c-myc obviously need to be better defined and extended to other pRb-deficient models, it is tempting to speculate that pocket protein loss renders H-rasV12 + c-myc–transduced fibroblasts prone to apoptosis when deprived of signals provided by the extracellular matrix. If this speculation is correct, it would explain why pocket protein–deficient fibroblasts are not as sensitive as p53–/– MEFs (defective on both G1-S checkpoint and apoptotic pathways) to oncogene-induced anchorage-independent proliferation.

Interestingly, p21Waf1/Cip1 has been implicated as both a positive and a negative regulator of cell proliferation. In addition, mouse modeling experiments have convincingly showed that p21 loss can accelerate or delay tumorigenesis (38). Results presented here suggest that under conditions where the actions of the INK4 family of inhibitors are perturbed, p21Waf1/Cip1 functions as a potent growth inhibitor. Interestingly, p21–/–; cdk4r24c MEFs do display an increased apoptotic index compared with WT MEFs when deprived of attachment in the presence of oncogenes and studies of MEFs deficient of pRb–/–, p107–/–, p130 have shown that they undergo increased apoptosis on serum withdrawal (39). Thus, although it is conceivable in certain situations, p21Waf1/Cip1 does not directly promote proliferation, but absence of its function leads to a defective G1-S checkpoint and enhances cell death to stimuli, resulting in a lower tumor incidence. An obvious question that will arise from these studies is will the major conclusions put forth be restricted to cell culture models. It is well appreciated that genes (e.g., p53 and pRb) involved in mediating culture-induced proliferative arrest and suppression of oncogene-mediated transformation also possess critical tumor suppressor properties in vivo. As mentioned above, p21 disruption does predispose mice to tumors late in life and facilitates tumorigenesis provoked by oncogene activation and loss of other tumor suppressors, including pRb (38, 40, 41). In addition, mice harboring the cdk4r24c mutation are prone to a wide range of tumors and display cooperativity with p27 deletion in formation of pituitary tumors (18, 19). In addition, loss of both INK4 (via mutation or promoter methylation) and p21Waf1/Cip1 (via p53-dependent and p53-independent mechanisms) function is a relatively common event in human malignancies. Thus, we predict based on these data and the ex vivo studies presented here that cdk4r24c and p21 loss will cooperate in promoting tumorigenesis in vivo, and this will be accelerated in models that are deficient in apoptotic pathways.


   Acknowledgments
 
Grant support: AG22022 (E.P. Reddy) and CA095569 (E.P. Reddy, X. Graña, and D.S. Haines). C.J. Carbone is a recipient of Department of Defense Breast Cancer Research Program predoctoral fellowship grant DAMD17-03-1-0412. Tobacco Settlement Funds, Fels Foundation, and the W.W. Smith Charitable Trust (D.S. Haines).

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 P. Leder for p21–/– mice, Steve Jones for p53–/+ mice, Scott Lowe (Cold Spring Harbor Laboratories, Cold Spring Harbor, NY) for pBabe-puro-H-rasV12, D. Liebermann (Temple University, Philadelphia, PA) for MSCVpac, and G. Nolan (Stanford University, Stanford, CA) for Phoenix cells.


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

3 C.J. Carbone and D.S. Haines, unpublished data. Back

Received 2/ 5/07. Accepted 2/14/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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