MOLECULAR AND CELLULAR BIOLOGY, July 2007, p. 5161–5171
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 27, No. 14
Cooperation between p27 and p107 during Endochondral Ossification
Suggests a Genetic Pathway Controlled by p27 and p130?
Nancy Yeh,1† Jeffrey P. Miller,1,2† Tripti Gaur,3‡ Terence D. Capellini,2‡ Janko Nikolich-Zugich,4
Carmen de la Hoz,1§ Licia Selleri,2Timothy G. Bromage,5Andre J. van Wijnen,3
Gary S. Stein,3Jane B. Lian,3Anxo Vidal,1* and Andrew Koff1,2*
Laboratory of Cell Cycle Regulation, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue,
New York, New York 100211; Program in Molecular Biology, Cornell University Weill Medical School, New York,
New York 100212; Department of Cell Biology and Cancer Center, University of Massachusetts Medical School,
Worcester, Massachusetts 016553; Vaccine and Gene Therapy Institute, Oregon Health and Science University,
Beaverton, Oregon 970064; and Hard Tissue Research Unit, Departments of Biomaterials and
Basic Sciences, New York University College of Dentistry, New York, New York 100105
Received 29 December 2006/Returned for modification 21 February 2007/Accepted 2 May 2007
Pocket proteins and cyclin-dependent kinase (CDK) inhibitors negatively regulate cell proliferation and can
promote differentiation. However, which members of these gene families, which cell type they interact in, and
what they do to promote differentiation in that cell type during mouse development are largely unknown. To
identify the cell types in which p107 and p27 interact, we generated compound mutant mice. These mice were
null for p107 and had a deletion in p27 that prevented its binding to cyclin-CDK complexes. Although a fraction
of these animals survived into adulthood and looked similar to single p27 mutant mice, a larger number of
animals died at birth or within a few weeks thereafter. These animals displayed defects in chondrocyte
maturation and endochondral bone formation. Proliferation of chondrocytes was increased, and ectopic
ossification was observed. Uncommitted mouse embryo fibroblasts could be induced into the chondrocytic
lineage ex vivo, but these cells failed to mature normally. These results demonstrate that p27 carries out
overlapping functions with p107 in controlling cell cycle exit during chondrocyte maturation. The phenotypic
similarities between p107?/?p27D51/D51and p107?/?p130?/?mice and the cells derived from them suggest
that p27 and p130 act in an analogous pathway during chondrocyte maturation.
Differentiation of mammalian cells depends on the estab-
lishment of a specific transcriptional program that leads to a
specialized function. Establishment of this program is gener-
ally coordinated with, or dependent upon, the cell exiting the
cell cycle. Cell cycle exit is governed by the interactions of
three families of proteins: the cyclin-dependent kinases
(CDKs), the CDK inhibitors (CKI), and the pocket proteins
(15, 65). CDKs promote proliferation in part by phosphorylat-
ing the pocket proteins (Rb, p107, and p130Rb2), leading to
changes in their ability to interact with E2F transcription fac-
tors or decreasing their stability (6, 12). Opposing the activity
of CDKs are the CKI. The Ink4 family proteins (p15, p16, p18,
and p19) bind to cyclin D-CDK complexes or compete with
cyclin D for binding to CDK subunits. Members of the Kip
family (p21, p27, and p57) bind to and inhibit cyclin-CDK2
The precise combinations of cyclins, CKI, and pocket pro-
teins used to initiate or maintain cell cycle exit is cell type and
signal specific. In some cases, a single protein is important,
despite the fact that other family members are expressed. For
example, p27-deficient oligodendrocyte progenitor cells and
osteoblasts fail to exit the cell cycle in a timely manner, leading
to a delay in differentiation (7, 8, 17). p21 and p57 are coex-
pressed in these cell types, respectively (8, 20, 62). In other
cases, similar family members act in a redundant manner, and
proliferative effects are more obvious when both are deleted.
For example, p107 and p130 collaborate to control chondro-
cyte proliferation and maturation (13, 33). In yet other cases,
deletion of members from different families is required to
unmask proliferative phenotypes. For example, neuronal cells
either fail to exit the cell cycle or reenter the cycle in p19 and
p27 double knockout mice (72). Additionally, codeletion of
p130 and p27 prevents cell cycle exit in angiogenic stem and
progenitor cells, hindering their export from the bone marrow
(67). Consequently, mouse knockouts are useful for identifying
the specific cell type in which particular combinations of CKI
and pocket proteins are required for proper development.
Cyclins, CKI, and pocket proteins can also affect the differ-
entiation program independently of any role in cell cycle exit.
p21 is required for the timely differentiation of oligodendro-
cyte progenitor cells, but not for growth arrest (70). In Xenopus
laevis Muller glia, a mutant of p21 that fails to interact with
RRL917C, Box 207, Memorial Sloan-Kettering Cancer Center, 1275
York Avenue, New York, NY 10021. Phone: (212) 639-2354. Fax:
(646) 422-2062. E-mail: email@example.com. Present address for
Anxo Vidal: Department of Physiology, School of Medicine,
University of Santiago de Compostela, S. Fancisco 1, 15782 Santiago
de Compostela, Spain. Phone: 34-981-582658, ext. 12290. Fax: 34-981-
574145. E-mail: firstname.lastname@example.org.
† N.Y. and J.P.M. contributed equally to the manuscript.
‡ T.G. and T.D.C. contributed equally to the manuscript.
§ Present address: Department of Cell Biology and Histology,
School of Medicine and Dentistry, University of the Basque Country,
E-48949 Leioa, Vizcaya, Spain.
?Published ahead of print on 14 May 2007.
at CNIO on July 3, 2007
cyclin D-CDK complexes can replace this differentiation-pro-
moting function in p27Xic1knockdown cells (44). Likewise,
p27Xic1plays a role in muscle and neuronal differentiation (63,
64). The carboxyl-half of p27, which does not bind cyclin-CDK
complexes, plays a role in neuronal differentiation and cell
migration (43). Finally, the interaction of Rb with Runx2/
Cbfa1 regulates osteoblast differentiation (60). Additional pos-
sible targets have also emerged from protein interaction stud-
ies. For example, cyclin D1 can interact with a corepressor
complex important in hormone receptor-dependent gene reg-
ulation (3, 46, 47, 73). Pocket proteins can interact with the Id
transcription factors, chromatin remodeling enzymes, and
other transcription factors in differentiating cells (22, 38). p21
can interact with Stat3, CBP/p300, and CEBP?, among a num-
ber of others (16, 45, 56). p57Kip2interacts with B-myb (27).
In this work, we identified cell types in which a genetic
interaction between p27 and p107 could be observed. We re-
port that p107?/?p27D51/D51mice display increased neonatal
lethality and defects in chondrocyte maturation both in vivo
and in vitro and that this is associated with a failure of cells to
appropriately exit the cell cycle and initiate a differentiation
program. The similarities between the phenotypes of p107?/?
p130?/?and p107?/?p27D51/D51mice suggest that p27 and
p130 carry out qualitatively similar functions during chondro-
MATERIALS AND METHODS
Mice. Generation of p130?/?(13), p107?/?(34), and p27D51/D51(29) mice has
been reported. Intercrossing p107?/?p27D51/?animals generated p107?/?
p27D51/D51mice. p130?/?p27D51/D51mice were generated by intercrossing
p130?/?p27D51/?animals. p107?/?p130?/?mice were generated by intercross-
ing p107?/?p130?/?mice. All these mice were in a mixed C57BL6/SvJ back-
Organ weights and thymocyte staining. The weights of the brain, spleen, and
kidney were determined as previously described (29). To determine the numbers
of thymocytes at each developmental stage, the thymus was surgically removed,
washed in phosphate-buffered saline (PBS), and mechanically disrupted. Pieces
of tissue were removed by centrifugation and further filtered through a nylon
mesh. Cell counts were performed in a hemocytometer. Single-cell suspensions
were subsequently incubated with fluorescent antibodies against CD4 and CD8
(BD Pharmingen), and the number of cells that were CD4?CD8?, CD4?CD8?,
CD4?CD8?, and CD4?CD8?was determined by fluorescence-activated cell
Morphometric analysis of long bone and sternal development. Newborn ani-
mal cartilage and bone were stained with alcian blue and alizarin red as described
elsewhere (25). Briefly, newborn animals were fixed overnight in 95% ethanol
and then stained in alcian blue (1 mg per ml of 40% ethanol–60% glacial acetic
acid) for 24 h at room temperature. These animals were then washed overnight
in 100% ethanol and rehydrated through a series of ethanol dilutions (75%, 50%,
and 25%; 2 h each), transferred to water, and then destained overnight in 0.5%
KOH. The alcian blue-stained embryos were counterstained for 24 h in alizarin
red (2 ml of a 0.1% solution of alizarin red in 8 ml 0.5% KOH) and then
destained again in 0.5% KOH. Prior to imaging, the embryos were cleared in
staged glycerol solutions (25% plus one drop of H2O2to aid clearing, and then
50% and 75%, 24 h each) and stored in 80% glycerol.
Forelimb measurements were acquired with a high-resolution three-dimen-
sional (3D) digitizer. This 3D measurement system consists of a three-axis
Mitutoyo measuring microscope (MTI Corporation, Los Angeles, CA) fitted
with high-precision linear scales in each x, y, and z axis, a noncontact measure-
ment video-based zoom optical head (RAM Optical Instrumentation, Inc., Hun-
tington Beach, CA) that included a through axis electronic crosshair, and a
Hitachi color charge-coupled device camera integrated to a PC. Digital readout
was transferred to 3D analytical measurement software (Measuregraph 1-2-3;
RAM Optical Instrumentation, Inc.). All measurements were made with an
accuracy of ?4 ?m (0.004 mm) and recorded to the nearest 0.01 mm. The 3D
capability of this instrument is essential for newborn mouse skeletal measure-
ments at this accuracy, as it allows refocusing in the z axis to ensure correct
positioning of the crosshairs, particularly on rounded surfaces. Each element was
dissected free of its articulations and measured. Measurements in millimeters
were taken as follows. The length of the humerus was taken as the maximum
proximal-distal distance parallel with the anterior border (from the head to the
trochlea), and the width was measured at midshaft (the 50% point) perpendic-
ular to the length axis (from anterior to posterior, thus avoiding the deltoid
tuberosity in the measurement). The length of the radius was taken as the
maximum proximal-distal distance parallel with the interosseous border (from
the head to the styloid process), and the width was measured at midshaft (the
50% point) perpendicular to the length axis. The length of the ulna was taken as
the maximum proximal-distal distance parallel with the interosseous border
(from the olecranon to the styloid process), and the width was measured at
midshaft (the 50% point) perpendicular to the length axis.
In vivo BrdU staining. Cell proliferation was assessed by incorporation of
bromodeoxyuridine (BrdU; Sigma). Pregnant females were injected intraperito-
neally with a 5-mg/ml BrdU solution in PBS (approximately 50 ?g/g body
weight). After 2 h, embryos were harvested and fixed in 4% paraformaldehyde,
embedded in paraffin, and sectioned at 8 ?m. Sections were stained with hema-
toxylin and eosin or immunostained with anti-BrdU antibody (Roche) as previ-
ously published (52). The number of BrdU-positive and the total number of
proliferating chondrocytes in the fourth and fifth sternabrae were counted.
Serum starvation and BrdU incorporation in primary MEFs. Mouse embryo
fibroblasts (MEFs) were isolated and cultured as described elsewhere (66). All
experiments were performed with MEFs from either passage 3 or 4 and plated
at approximately 104cells/cm2. For serum starvation, cells were cultured 24 h
after plating and then washed twice with PBS and incubated for 60 h in medium
containing 0.1% fetal bovine serum. For most experiments, a fraction of each
harvested culture was pulsed with [3H]thymidine (40 ?Ci/ml) to verify growth
To measure the percentage of proliferating cells, BrdU incorporation was
determined. In parallel to the cultures developed for protein and RNA extrac-
tion, cells were plated in two-well Permanox chamber slides (Nunc) and pulsed
with BrdU (100 ?M) for 4 h, washed once with PBS, and fixed with 70% ethanol
for 30 min in the dark. After washing with PBS, the DNA was denatured by
treatment with NaOH for 2 min. The cells were washed with 0.1 M Na2B4O7, pH
8.5, and then with PBS. The slides were then incubated for 30 min in the dark
with 20 ?l of fluorescein isothiocyanate-conjugated BrdU antibody (BD Phar-
mingen) diluted with 50 ?l PBS containing 0.5% bovine serum albumin and 0.5%
Tween 20. After several washes with PBS, the DNA was stained with 4?,6?-
diamidino-2-phenylindole (5 ?g/ml) for 5 to 10 min and washed with PBS again.
Slides were mounted, and BrdU-positive cells were visualized with a fluorescence
microscope. A total of at least 100 cells in five different fields were counted per
Immunoblotting and immunokinase assay. For the measurement of histone
H1 kinase activity, cells were lysed at a concentration of 8 ? 106per 100 ?l of
NP-40–RIPA buffer as we previously described (30). The equivalent of 2 ? 106
cells were immunoprecipitated with either cyclin E antiserum (30), cyclin A
antibodies (H432; Santa Cruz), or rabbit anti-mouse antibodies (Zymed), and
kinase assays were carried out as previously described (30).
To determine the amounts of CDK1 (C19), CDK2 (M2), CDK4 (C22), CDK6
(C21), cyclin D1 (72-13G), cyclin D3 (C16), cyclin E (M20), cyclin A2 (H432),
p21 (F5), and p27 (C19), we extracted proteins in 20 mM HEPES-KOH pH 7.6,
5 mM KCl, 0.5 mM MgCl2, 100 mM NaCl containing 2 mM phenylmethylsul-
fonyl fluoride and 0.5 mM dithiothreitol as we previously described (57). To
determine the amount of p130 (C20), p107 (C18), E2F4 (C20), and E2F5 (C20),
proteins were extracted in a buffer containing 100 mM HEPES-KOH pH 7.5, 500
mM NaCl, 10 mM EDTA, 0.2% Triton X-100, 1 mM dithiothreitol, and 1 mM
phenylmethylsulfonyl fluoride as described elsewhere (40). Ten to 80 ?g of
extract was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophore-
sis, and proteins were transferred to polyvinylidene difluoride membranes and
blotted as we previously described (57). All antibodies were from Santa Cruz
Biotechnology except for Rb (G3-245), which was purchased from Pharmingen.
Northern blotting. Total RNA was isolated using the RNeasy kit (QIAGEN).
Poly(A)?RNA was selected with the Poly(A) Pure kit (Ambion) following the
manufacturer’s instructions. Eight ?g of total RNA (for CDK1, cyclin A, and
ARPP0) or 0.8 ?g poly(A)?RNA (for thymidylate synthase [TS], dihydrofolate
reductase [DHFR], B-myb, E2F4, and p130) was denatured and resolved on
agarose gels using the Northern Max-Gly kit (Ambion). RNA was transferred to
a BrightStar-Plus nylon membrane (Ambion) overnight. Following UV cross-
linking, the membrane was prehybridized with ULTRAhyb (Ambion) in a roller
bottle for 30 min at 42°C. Full-length cDNA clones (ATCC) were excised from
the vector, gel purified, and random-prime labeled with [?-32P]dCTP (Amer-
5162YEH ET AL.MOL. CELL. BIOL.
at CNIO on July 3, 2007
sham) using the Prime-It II kit (Stratagene). Labeled probes were purified using
Centrisep spin columns (Princeton Separations), heated at 90°C for 10 min,
added to ULTRAhyb (106cpm/ml), and incubated with membranes overnight
Chondrogenic differentiation and expression analysis. High-density micro-
mass cultures from mouse embryonic fibroblasts were prepared, and chondro-
genic differentiation was induced by addition of BMP2 as previously described
(21, 37). Briefly, 1 ? 105passage 2 primary mouse embryo fibroblasts were plated
in 10-?l drops and cultured in the presence of recombinant human BMP2 (a kind
gift from John Wozney, Wyeth Research, Cambridge, MA). Cells were either
fixed with 2% formaldehyde for 10 min for alcian blue or alkaline phosphatase
staining as described previously (21, 37) or harvested in TRIzol reagent (Invitro-
gen) for RNA preparation following the manufacturer’s instructions. Protein
extracts were prepared by sonicating cells in DIP buffer (48) as we previously
RNA was purified by RNase-free DNase I treatment, and cDNA was prepared
from 1 ?g of RNA using a First Strand synthesis kit (Invitrogen). Relative
transcript levels were measured by quantitative reverse transcription-PCR (qRT-
PCR) using SYBR Green 2? master mix (Applied Biosystems) in a 25-?l
reaction volume. Primer sequences were as follows: collagen type 2a1 (5?CTG
GAATGTCCTCTGGCGA and 5?TGAGGCAGTCTGGGTCTTCAC); sox 9
(5?GAGGCCACGGAAGAGACTCA and 5?CAGCGCCTTGAAGATAGCA
TT); Indian hedgehog (5?TCACCCCCAACTACAATCCC and 5?CCGTGTTC
TCCTCGTCCTTG); collagen type 10a1 (5?CCTGCAGCAAAGGAAAACTC
and 5?TGTGGTAGTGGTGGAGGACA); Runx2 (5?CGGCCCTCCCTGAA
CTCT and 5?TGCCTGCCTGGGATCTGTA); alkaline phosphatase (5?TTGT
GCCAGAGAAAGAGAGAGA and 5?GTTTCAGGGCATTTTTCAAGGT);
osteocalcin (5?CTGACAAAGCCTTCATGTCCAA and 5?GCGGGCGAGTCT
GTTCACTA); histone H4 (5?CCAGCTGGTGTTTCAGATTACA3? and 5?AC
CCTTGCCTAGACCCTTTC3?); cyclin A2 (5?ACAGAGCTGGCCTGAGT
GA3? and 5?TTGACTGTTGGGCATGTTGT3?); CDK1 (5?GGCAGTTCATG
GATTCTTCACTC3? and 5?GCCAGTTTGATTGTTCCTTTGTC3?). Gene
expression for each gene was normalized to glyceraldehyde 3-phosphate dehy-
drogenase levels as detected by primers from Applied Biosystems.
The phenotypes of adult p27D51/D51and p107?/?p27D51/D51
mice are similar. To determine whether p107 and p27 act in a
redundant manner to control development of any particular
cell lineage in mice, we intercrossed p107?/?p27?/D51male
mice with p107?/?p27?/D51female mice. p107?/?mice had no
significant phenotype in a mixed 129SvJ/C57BL6 background
(13). On the other hand, p27D51/D51mice show enhanced
growth, multiorgan hyperplasia, female infertility, and the
spontaneous development of pituitary intermediate lobe ade-
noma in this background (29). The D51 allele encodes a trun-
cated protein that does not bind cyclin-CDK complexes, and
mice expressing this allele have phenotypes similar to those
described for p27-null animals (19, 41).
The phenotype of adult p107?/?p27D51/D51mice was gener-
ally similar to that observed in p27D51/D51animals. The weight
differences were more obvious in female mice than male mice
(Fig. 1). p107?/?p27D51/D51mice generally weighed between
the normal weight of wild-type (and p107?/?) mice and the
increased weight of p27D51/D51mice. However, a number of
p107?/?p27D51/D51mice weighed the same or less than their
siblings until approximately the third week of age, by which
time they begin to overtake them. Additionally, females were
infertile, and no litters were obtained from them despite their
being able to mate.
The average wet weights of the kidney and spleen were
significantly greater (P ? 0.05) in the p27 knockout and the
double mutant mice compared to wild-type or p107 single
FIG. 1. Weights of mice. (A) The weights of two representative male and female mice of each genotype are shown over the first 8 to 10 weeks
of age. (B) The average weight and standard deviation are indicated in the graphs for male and female mice of each genotype (n ? 10 for each
VOL. 27, 2007 SKELETAL DEFECTS IN p107?/?p27D51/D51MICE5163
at CNIO on July 3, 2007
mutant mice. The brain weights were comparable (Fig. 2A).
However, the weight of each individual organ relative to the
total weight of the animal from which it was obtained was
similar, indicating that combining p107 deficiency with the p27
mutation did not affect the proportional growth previously
reported (19, 29, 41).
The cellularity of the thymus, which was much greater and
disproportionate to the body weight of the animal, was in-
creased in female p107?/?p27D51/D51mice, almost to the same
extent as that seen in p27D51/D51mice (Fig. 2B). The number of
thymocytes was nearly equivalent in wild-type and p107?/?
mice (wild type, n ? 5; p107?/?, n ? 5). In p27D51/D51mice
(n ? 4), consistent with what we reported before, we saw a
roughly threefold increase in thymocyte number, and this was
comparable to the increase in p107?/?p27D51/D51mice (n ? 3).
Furthermore, the genotype did not affect the distribution of
thymocytes among the double-negative, double-positive, and
single-positive CD4 or CD8 subsets (data not shown). Similar
results were observed with 13- to 15-week-old female mice and
in 8- to 10-week-old and 13- to 15-week-old male mice. These
results suggest that there was limited genetic interplay between
p27 and p107 in control of body mass and T-cell lympho-
Additionally, both male and female p107?/?p27D51/D51mice
developed intermediate lobe pituitary adenomas reactive with
antibodies against adrenocorticotropin but not growth hor-
mone or prolactin. These tumors were histologically similar to
the tumors arising in p27D51/D51mice. In contrast to the
p130?/?p27D51/D51mice (67), there was no obvious change in
the vascular architecture of the pituitary.
Nevertheless, although p107 deficiency did not exacerbate
the phenotype of surviving p27D51/D51mice, there was some
interaction between these gene products, as the number of
p107?/?p27D51/D51animals identified at 3 weeks of age was
significantly lower than expected (Table 1).
Neonatal lethality is associated with the loss of p107 and
p27. Because of the reduced frequency at which p107?/?
p27D51/D51animals were identified at weaning, we collected the
dead and moribund newborns from 11 litters of the p107?/?
p27?/D51intercrosses. Of these pups, 95% were homozygous
for the mutant p27 allele, and the remaining were heterozy-
gous. Fostering newborn pups to wild-type mothers did not
increase survival, suggesting that lethality was an intrinsic de-
fect of the pups.
To determine when decreased viability first manifested, we
determined the genotypes of embryos at various days postcon-
ception using timed pregnancies. Double knockout embryos
were present at the expected Mendelian ratios from day 13.5 to
day 16.5, with a slight, albeit statistically insignificant, decline
at day 17.5 (Table 1). This phenotype was reminiscent of the
one reported for p107?/?p130?/?mice (13), raising the pos-
sibility that the combined loss of p107 and p27 was qualitatively
similar, but perhaps quantitatively weaker, than the loss of
p107 and p130.
Reducing p130 exacerbates neonatal lethality in p107?/?
p27D51/D51mice. The relationship between pocket proteins and
CKI is complex, with each family of proteins affecting the
expression levels of the other and compensating for each other
to control CDK activity (10, 11, 35, 65). For example, p130 can
be recruited into cyclin E-CDK2 complexes in Kip-deficient
MEFs (11). Additionally, CDK-dependent ubiquitin-depen-
dent pathways increase p130 turnover (4, 59).
Even though p107?/?p130?/?animals have diminished
long-term viability and a modest reduction in growth, p107 and
p130 act redundantly, and a single allele is sufficient to ensure
neonatal viability (13). As we were interested in whether p27
and p130 compensated for each other, we asked if reducing the
TABLE 1. Effect of p27 dose on survival of embryonic and
neonatal p107-deficient animals
No. of p107-deficient animals of
Neonatal day 21
Neonatal day 1
Embryonic day 17.5
Embryonic day 16.5
Embryonic day 15.5
Embryonic day 14.5
Embryonic day 13.5
aThe ?2test was based on an expected Mendelian inheritance pattern for the
intercrossing of p107?/?p27D51/?mice.
FIG. 2. (A) Organ weights. The organs indicated were dissected
from 23- to 41-week-old female mice, and the average weight and
standard deviation were determined. Black, wild type, n ? 5; light gray,
p27D51/D51, n ? 6; dark gray, p107?/?, n ? 5; white, p107?/?p27D51/D51,
n ? 3. (B) Thymocytes from 8- to 10-week-old mice were collected,
and the total cell number was determined. The mean number of
thymocytes and the standard deviation are shown. Asterisks indicate
those values significantly different (P ? 0.01) from wild-type mice
based on a two-tailed Student’s t test.
5164YEH ET AL.MOL. CELL. BIOL.
at CNIO on July 3, 2007
dose of p130 in p107?/?p27D51/D51mice would exacerbate the
viability phenotype. We did not recover any neonatal p107?/?
p130?/?p27D51/D51pups from nine crosses (n ? 35) (Table 2),
and the number of p107?/?p130?/?p27?/D51mice recovered
was also significantly lower than expected. In contrast, when we
reduced p107 in p130?/?p27D51/D51animals, we recovered
p107?/?p130?/?p27D51/D51pups at the expected ratio. This
suggests that p130 can compensate for the reduced amount of
p27 in the p107?/?p27D51/D51mice.
Loss of p107 and p27 affects morphology of the long bones.
The neonatal lethality in p107?/?p130?/?mice is related to
defects in endochondral ossification (13). Endochondral ossi-
fication occurs in the long bones of the developing limbs and
the sternum. During this process, chondrocytes in the cartilage
of developing long bones proliferate, become quiescent, un-
dergo a shift to hypertrophic growth, and are eventually re-
placed by bone. To determine if ossification was affected by the
loss of p107 and p27, we generated wild-type, p107?/?p27D51/D51,
p107?/?p130?/?, p27D51/D51, and p130?/?p27D51/D51day 1
neonatal animals and stained whole-mount embryo prepara-
tions with alcian blue and alizarin red. Alcian blue stains non-
mineralized cartilage, whereas alizarin red stains mineralized
bone. Representative images of the forelimbs of p107?/?,
p107?/?p130?/?, and p107?/?p27D51/D51mice are shown in
Fig. 3A. Measurements of the humerus, radius, and ulna from
all genotypes are compiled in Table 3.
Looking at the forelimbs, we noted that there were signifi-
cant changes in the length and width of the humerus, radius,
and ulna in p107?/?p27D51/D51mice, consistent with a defect in
endochondral ossification. This was reminiscent of the pheno-
type reported in p107?/?p130?/?mice and confirmed in this
work (13). Nevertheless, whereas the length of the bones was
more affected in p107?/?p130?/?animals, the width was more
affected in the p107?/?p27D51/D51mice. While loss of p27 or
p130 may be qualitatively similar, the latter finding may reflect
potential quantitative differences in the mechanisms by which
p130 and p27 control bone formation or remodeling.
We also noted phenotypic similarities between p107?/?
p130?/?and p107?/?p27D51/D51mice in the sternum (Fig. 3B).
Wild-type, p130?/?p27D51/D51, and p27D51/D51animals showed
a typical pattern of bony elements (the sternabrae) separated
by bands of cartilage (the sternal joints, points of fusion with
the costal cartilage). In contrast, ossification of costal junctions
was observed in one-third of the p107?/?animals (9/26; P ?
0.02) and in over three-fourths of the p107?/?p27D51/D51(15/
18; P ? 0.001) and p107?/?p130?/?(7/8; P ? 0.001) animals.
These findings are consistent with the idea that either p130 or
p27 could compensate for the normal function of p107 during
normal development of costal cartilage tissue.
Loss of p107 and p27 increases the S-phase fraction of
proliferating chondrocytes. p107 and p130 are required for
growth arrest and maturation of chondrocytes in vivo (13).
Thus, we asked whether p107 and p27 act redundantly to affect
cell cycle exit in the developing sternum. To avoid potential
problems associated with the developmental staging of differ-
ent embryos, we only looked at littermates from a p107?/?
p27?/D51cross. The fraction of proliferating chondrocytes in
the fourth and fifth sternal joints of day 16.5 embryos was
increased in p107?/?p27D51/D51mice (n ? 2) compared to
p107?/?mice (n ? 3) (Fig. 4). Distinctions between the chon-
drocytes of the rib, the proliferating chondrocytes, the prehy-
pertrophic chondrocytes, and the hypertrophic chondrocytes
were observed (data not shown). Thus, p107 and p27 can
cooperate to control proliferation in chondrocytes, suggesting
that p27 and p130 might be on a common pathway controlling
proliferation in these cells.
The combined loss of p107 and p27 does not affect cell
proliferation, the cell cycle machinery, or p107/p130-regulated
gene expression in mouse embryo fibroblasts. To further ex-
plore the possibility that p27 deficiency and p130 deficiency
were functionally equivalent, we began to look into cellular
models in which the redundancy of p107 and p130 was already
established. Specifically, we were interested in determining if
there was a similar effect on cell behavior when p107 and p27
were both lost as when p107 and p130 were both lost. MEFs
have a relatively neutral cellular phenotype that is not com-
mitted to a specific mesenchymal lineage. Combined loss of
FIG. 3. Endochondral ossification in newborn mice. Neonatal mice
were collected, and skeletal preparations were stained with alcian blue
and alizarin red to differentiate the chondrocytic and bony regions.
(A) Representative images of the forelimbs from p107?/?, p107?/?
p27D51/D51, and p107?/?p130?/?mice are shown. Red, bone; blue,
cartilage. The number of animals analyzed for this phenotype is indi-
cated in Table 3. (B) Representative images of the sternal regions of
mice. Note the presence of bone in the costal junctions in p107?/?
p130?/?mice and the p107?/?p27D51/D51deficient mice. The number
of mice afflicted with this phenotype is reported in the text.
TABLE 2. Effect of p130 dose on survival of neonatal p107?/?
% of mice p130?/?and:
Observed, 3 wk
aThe percentage of mice expected with Mendelian inheritance when inter-
crossing p107?/?p130?/?p27D51/D51and p107?/?p130?/?p27D51/?mice.
bThe X2test was based on a comparison between the expected and observed
percentages of each genotype.
VOL. 27, 2007SKELETAL DEFECTS IN p107?/?p27D51/D51MICE5165
at CNIO on July 3, 2007
either p107 and p130, or of p107 and p27, did not affect the
ability of MEFs to exit the cell cycle when deprived of serum
(Fig. 5A). Furthermore, quiescent p107?/?p27D51/D51MEFs
entered S phase at a rate similar to wild-type cells and mod-
estly slower than p107?/?p130?/?cells when serum was added
back to the medium (Fig. 5B).
CDK activity must surpass a threshold level to induce cell
proliferation, and pocket protein binding to these complexes
might inhibit activity to a subthreshold level in Kip-deficient
cells without reaching the nadir established by Kip binding
(65). Thus, we looked at whether CDK activity was higher in
the quiescent p107?/?p27D51/D51cells compared to wild-type
cells (Fig. 5C). Cyclin A2-associated kinase activity was mod-
estly increased in serum-starved p107?/?p27D51/D51MEFs.
Cyclin E-associated kinase activity was unaffected. This is con-
sistent with the CDK-inhibitory binding of p107 to cyclin A2 in
Kip-deficient cells (9).
To try and understand why cyclin A activity was slightly
higher, we looked at the expression of cell cycle regulators in
quiescent cells. Cyclin A and CDK1 proteins were modestly
increased, consistent with the increased kinase activity, and
there was no significant change in p21, the D-type cyclins,
cyclin E, CDK2, CDK4, CDK6, or E2F4 and E2F5 (Fig. 5D).
p130 accumulation was only occasionally affected (Fig. 5D). In
three of the eight clones examined, p130 levels decreased by
less than 50%. These data indicate that deficiency of both p107
and p27 caused only marginal differences in cyclin A2 and
CDK1 accumulation and in cyclin A-associated kinase activity,
the magnitudes of which were unable to affect quiescence
Although there was no significant effect on cell proliferation,
we wanted to determine if the modest amount of cyclin A2
kinase activity could affect the p107/p130-controlled quies-
cence-associated gene expression program in MEFs. This pro-
gram is associated with the repression of cyclin A2, CDK1,
B-myb, TS, and DHFR mRNA (26). We found that cyclin A2
and CDK1 mRNA were expressed at comparable levels to
those observed in wild-type cells and not at the increased levels
seen in p107?/?p130?/?cells (Fig. 5E). ARPP0 served as a
loading control. TS, DHFR, B-myb, and p130 mRNAs were
not increased in quiescent p107?/?p27D51/D51MEFs, either
(Fig. 5E). Thus, there is no evidence that the loss of p27 and
p107 function is sufficient to reduce the activity of p130 in
MEFs, but it can modestly enhance cyclin A accumulation and
activity in an apparently posttranscriptional manner.
Taken together, combined loss of p27 and p107 function
does not have major effects on the levels, associations, and/or
activities of any of the principal components of the cell cycle
regulatory machinery in MEFs as measured in our studies,
consistent with these cells retaining their normal dependence
on growth factors for the stimulation of cell proliferation.
Defects in chondrogenic differentiation in p107?/?p27D51/D51
cells. Chondrogenic differentiation in micromass cultures was
previously reported to be disturbed in p107?/?p130?/?cells
(33, 50). Thus, we wanted to examine chondrogenic differen-
tiation of p107?/?p27D51/D51cells ex vivo in micromass culture
as well (37). To accomplish this, we induced the commitment
of MEFs to the chondrogenic lineage by treating micromass-
cultured early passage MEFs obtained from wild-type, p107?/?
p27D51/D51, and p107?/?p130?/?embryos with BMP2. The
extent and rate of differentiation were analyzed histochemi-
cally by alcian blue staining and alkaline phosphatase staining
(a marker of hypertrophic chondrocytes). This revealed re-
duced deposition of extracellular matrix as well as delayed
induction of hypertrophy in p107?/?p27D51/D51and p107?/?
p130?/?cells compared to wild type (Fig. 6A). qRT-PCR of
chondrocyte-specific marker genes showed a normal induction
pattern for the chondrocyte marker collagen type 2a1. Sox9, a
marker of committed chondrogenesis, was also induced with
similar kinetics in the three genotypes, but its continued pres-
TABLE 3. Forelimb measurements
Length (mm)Width (mm)
4.68 ? 0.12
4.54 ? 0.33
3.85 ? 0.31a
2.79 ? 0.34a
4.41 ? 0.22
3.78 ? 0.15
3.57 ? 0.32
2.94 ? 0.22a
2.28 ? 0.19a
3.53 ? 0.21
4.93 ? 0.14
4.64 ? 0.42
4.16 ? 0.18a
3.26 ? 0.26a
4.54 ? 0.24
0.55 ? 0.06
0.53 ? 0.04
0.70 ? 0.04a
0.67 ? 0.05a
0.53 ? 0.02
0.40 ? 0.01
0.37 ? 0.03
0.52 ? 0.03a
0.44 ? 0.04
0.35 ? 0.02
0.44 ? 0.03
0.43 ? 0.03
0.59 ? 0.04a
0.48 ? 0.02
0.39 ? 0.06
aSignificantly different (P ? 0.005) from the wild type (two-tailed Student’s t test).
FIG. 4. Cell proliferation in prehypertrophic chondrocytes in costal
joints. Pregnant mice were injected with bromodeoxyuridine and sac-
rificed, and embryos were collected at day 16.5. BrdU incorporation
was determined immunohistochemically, and the percentage of posi-
tive cells was plotted. Only littermates were used for comparisons; in
one litter a single pair of p107?/?and p107?/?p27D51/D51was ob-
tained, and in another litter two p107?/?animals were paired with a
single p107?/?p27D51/D51sibling. Each tick mark represents the per-
centage of cells in one section that were BrdU positive. p107?/?,
3,5797 cells counted; p107?/?p27D51/D51, 2,7743 cells counted.
5166YEH ET AL.MOL. CELL. BIOL.
at CNIO on July 3, 2007
ence was reduced in the knockout cells. The differentiation
markers (Indian Hedgehog, collagen type 10a1, alkaline phos-
phatase, and osteocalcin) were not induced in p107?/?p27D51/
D51 and p107?/?p130?/?cells (Fig. 6B). Runx2 mRNA was
downregulated as expected in the wild-type cells (37). A similar
pattern, but at a reduced level, was observed for p107?/?
p130?/?mutant cells. In contrast, Runx2 levels in the p107?/?
p27D51/D51mutant cells showed a delayed profile as a result of
higher proliferation, as suggested by higher expression of his-
tone H4 (Fig. 6B). This suggests that p107?/?p27D51/D51and
p107?/?p130?/?MEFs committed to the chondrogenic lin-
eage appropriately following treatment with BMP2, but their
FIG. 5. p130 function is not disrupted in p107?/?p27D51/D51mouse embryo fibroblasts. (A) Cells deprived of serum exit the cell cycle. Passage
4 cells were serum starved for 60 h and pulsed with bromodeoxyuridine for 4 h. This experiment was repeated at least five times with different clones
each time. (B) Serum-induced S-phase progression. Serum was added to serum-deprived quiescent cells, and their progress into S-phase was
determined by incorporation of radiolabeled thymidine as described elsewhere (31). Two independent clones of each genotype were analyzed, as
indicated in the figure. (C) Histone H1 kinase assay. Extracts were prepared from quiescent serum-starved cells or from quiescent cells that had
been induced to reenter the cell cycle by serum addition 20 h previously, and the amount of immunoprecipitable cyclin E- and cyclin A-associated
histone H1 kinase activity was measured. The genotypes are indicated above the lanes, with individual clone numbers noted. As a control for kinase
activity, cyclin E-associated kinase activity was ascertained on wild-type clone 371 cells 20 h after serum addition to a quiescent culture. Cyclin
A-associated kinase activity was ascertained on double mutant clone 342. Similar results were obtained when other clones with the indicated
genotypes were used (wild type, n ? 7; p107?/?p27D51/D51, n ? 5). (D) Immunoblot assay. The protein noted on the left of the panel was
determined by direct immunoblotting of whole-cell extracts. As a control for protein expression, extracts were also prepared from serum-stimulated
wild-type clone 264 and double mutant clone 342. A nonspecific cross-reactive band is noted by the asterisk to the right of the p130Rb2image. The
positive sign to the right of the p27Kip1image denotes the protein encoded by the D51 allele. (E) Northern blotting. In the top panel, the
accumulation of CDK1 and cyclin A mRNA was determined in serum-starved and asynchronous cells (indicated by the ? and ? symbols below
each lane). Genotypes and individual clone numbers are indicated above the lanes. Note that the accumulation of these mRNAs was much more
pronounced in the serum-starved p107?/?p130?/?cells compared to the p107?/?p27D51/D51or wild-type cells. ARPP0, a message that is not
affected by serum starvation, was used as a loading control. In the bottom panel, we compared the expression of TS, DHFR, B-myb, and p130 in
two additional clones, one wild type (372) and one p107?/?p27D51/D51(345), and we found that they were similar.
VOL. 27, 2007 SKELETAL DEFECTS IN p107?/?p27D51/D51MICE 5167
at CNIO on July 3, 2007
differentiation was inhibited. Commitment and differentiation
of p107?/?cells was similar to that of wild-type cells (data not
shown). These ex vivo findings are consistent with the pheno-
type observed in the developing sternabrae of p107?/?p27D51/D51
To address if the loss of p27 collaborated with p107 defi-
ciency to affect p130 accumulation in these cells, we looked at
the accumulation of p130 and other cell cycle regulators in
pooled micromass cultures. We did not see any genotype-
specific differences in either the accumulation of p130 or p27
or the reduction of hyperphosphorylated Rb, p21, or cyclin B1
that accompany chondrogenic commitment (Fig. 7A). There
was a marginal increase in cyclin A2 and a more robust in-
crease in CDK1 in the double knockout cells (Fig. 7A). Thus,
a modest change in the amount of cyclin A2 and CDK1 was
observed in both the p107?/?p130?/?and p107?/?p27D51/D51
cells; however, there was no significant change in p130 expres-
sion when we compared wild-type, p107?/?, and p107?/?
p27D51/D51cells, suggesting that the cellular phenotype in p107/
p27 doubly deficient mice is not due to a direct loss of p130
We could not recover enough protein from pooled micro-
mass cultures to explore the interaction of p130 with other
proteins in p107?/?p27D51/D51cells; however, we reasoned
that we could monitor cyclin A2 and CDK1 mRNA as surro-
gates of p107/p130-regulated gene expression, as we did pre-
viously in MEFs. We noted that CDK1 mRNA was higher in
both double knockout cell types compared to either the wild-
type or p107?/?control cells (Fig. 7B and C). Cyclin A2
mRNA was marginally higher in the double knockouts as well.
ARPP0 served as a loading control (Fig. 7B). These results
were confirmed by qRT-PCR (Fig. 7C). Thus, p27, like p130,
can genetically interact with p107 to control differentiation and
a gene expression program of chondrocytes. This suggests that
p27 and p130 are on overlapping pathways that work with p107
to promote cell cycle exit and differentiation in the chondro-
genic lineage. We suggest that p27 and p130 are in a single
functional group, but they are unable to fully compensate for
each other when one is absent.
In this report we provide evidence for the involvement of
p27 in controlling chondrocyte differentiation and endochon-
dral bone development in mice. We have generated and ana-
lyzed the phenotype of p107?/?p27D51/D51mice. The combi-
nation of these deficiencies resulted in phenotypes similar to
those previously reported for p107?/?p130?/?mice and reca-
pitulated here, including neonatal lethality (Tables 1 and 2),
aberrant or ectopic endochondral ossification with increased
chondrocyte proliferation in cartilaginous regions of the limbs
and sternum (Table 3; Fig. 3 and 4) (13), and chondrocyte
maturation ex vivo (Fig. 6 and 7).
Endochondral ossification begins when mesenchymal cells
are induced to enter a chondrocytic lineage. Many of the sig-
naling molecules and the transcription factors involved in
chondrocyte maturation have been identified (28, 32). A fetal
cartilage mold is established and chondrocytes begin to pro-
duce PTHrP, which stimulates proliferation and further differ-
entiation into postmitotic hypertrophic chondrocytes. Trans-
forming growth factor ? and Ihh both positively regulate
chondrocytic PTHrP expression. On the other hand, fibroblast
growth factor (FGF) signaling induces differentiation and the
withdrawal of chondrocytes from the cell cycle. Ultimately, this
process is controlled by a variety of transcription factors. Sox5
and -6 are essential for the induction of the chondrocytic lin-
eage from mesenchymal cells (55). Sox9 is involved in the
expansion of chondrocytic cells (5). Runx2/Cbfa1 is a determi-
nant of the chondro-osteoprogenitor lineage in mesenchymal
cells but must be downregulated for perichondrial progenitors
to enter the chondrocyte lineage (24, 36). Runx2 is, however,
required for hypertrophic chondrocyte maturation. Runx2 ac-
tivation of vascular endothelial growth factor in hypertrophic
chondrocytes is essential for progression of endochondral bone
formation (69). Runx2 mediates PTH/PTHrP signaling in the
hypertrophic zone (23) and FGF signaling in the perichon-
FIG. 6. Ex vivo chondrogenic differentiation of mouse embryonic
fibroblasts from wild-type, p107?/?p27D51/D51, and p107?/?p130?/?
mice. (A) Chondrogenic differentiation was assessed by alcian blue and
alkaline phosphatase staining of micromass cultures at day 9.
(B) Quantitative real-time PCR analysis of chondrogenic marker genes
during differentiation. Relative transcript levels were determined and
plotted individually for the gene products indicated within each graph.
5168YEH ET AL.MOL. CELL. BIOL.
at CNIO on July 3, 2007
drium (24, 49). Thus, it is not surprising that skeletal mal-
formations arise when the chondrocytic differentiation pro-
gram is deregulated by either chronic activation or insufficient
activation of Runx 2 (61, 68).
Skeletal manifestations can arise as a consequence of
changes in chondrocyte proliferation. Skeletal defects in
p107?/?p130?/?(13), p57?/?(71), and E2F1 transgenic mice
(51) are associated with increased chondrocyte proliferation,
without disruption of the normal morphological patterning of
the growing cartilage column. We documented similar defects
in bone development in p107?/?p27D51/D51mice. The exag-
geration of the p107 skeletal phenotype by either p130 or p27
insufficiency suggests that these proteins might share a func-
tion controlling proliferation and enforcing a p107-dependent
cell cycle block.
How the cell cycle program is coordinated with chondrocyte
maturation is becoming increasingly clear. Transforming
growth factor ? and PTHrP stimulate chondrocyte prolifera-
tion by increasing cyclin D1 (2) and decreasing p57Kip2(39).
Consistent with this, cyclin D1-deficient mice have reduced
growth plate proliferation (54), and the proliferation of chon-
drocytes continues in p57?/?mice, leading to defects in endo-
chondral ossification (71). However, the most compelling piece
of evidence supporting a link between PTHrP signaling and
p57 accumulation is the observation that skeletal abnormalities
associated with PTHrP deficiency are not seen in a p57-defi-
cient background (39). Interestingly, these skeletal abnormal-
ities are also reversed in a p107?/?p130?/?background (36),
suggesting that p57 and p107/p130 may act in a common path-
way during PTHrP-regulated endochondral ossification.
Conversely, FGF induces chondrocyte growth arrest and
maturation by inducing p107 and p130 (33). p107- and p130-
deficient mice have defects in endochondral ossification asso-
ciated with increased chondrocyte proliferation (13, 50). It has
been suggested that p57 might act by inhibiting CDK activity,
allowing p107 and p130 to accumulate and support chondro-
cyte differentiation (36). However, dephosphorylation of p107
is rapid, occurring within 1 hour of FGF treatment, and is not
FIG. 7. Expression of cell cycle regulatory molecules during ex vivo chondrogenic differentiation of wild-type, p107?/?, p107?/?p27D51/D51, and
p107?/?p130?/?MEFs. (A) Immunoblot assay. The protein noted on the left of the panel was determined by direct immunoblotting of whole-cell
extracts. A 200-?g aliquot of extract was used to detect p130 and Rb, 20 ?g was used for CDK1, and 80 ?g was used for all others. The genotypes
of the cells from which extracts were prepared are indicated above each lane and were confirmed by immunoblotting with p107 and p27 antibodies.
BMP2 treatment was carried out for 24 h prior to harvest. (B) Northern blot assay. These results are arranged as for panel A, but the accumulation
of CDK1 and cyclin A2 mRNA was determined. (C) Quantitative real-time PCR analysis of cyclin A2 and CDK1 in BMP2-treated micromass
cultures was performed, and results are plotted as a percentage of the level in untreated micromass cultures of the same genotype. The mean and
standard deviation from three independent experiments are plotted.
VOL. 27, 2007SKELETAL DEFECTS IN p107?/?p27D51/D51MICE5169
at CNIO on July 3, 2007
dependent on gene transcription or protein translation (30).
Consequently, it is more likely that changes in the pocket
proteins precede cell cycle exit induced by p57.
The regulation of p27 levels in relation to p57 might account
for the differences in the penetrance of the skeletal phenotype
that arise from compound mutations in the p27 and p107 loci
(17, 20, 62). In osteoblasts, the level of p27 is correlated with
Runx2 and inversely related to proliferation. p57 levels are
increased as chondrocytes exit the cell cycle with decreased
Runx2, whereas p27 levels only increase in conjunction with
the reactivation of Runx2 in hypertrophic chondrocytes. There
are a number of reports that show that p27 accumulates in
hypertrophic chondrocytes (1, 33, 42, 58) However, as there is
little change in the spatial or temporal regulation of chondro-
cyte proliferation in p27-deficient mice, it was suggested that
its contribution might be minimal (18). Our crossing the p27
deficiency into the sensitized background of p107-deficient
mice clearly unmasks a significant contributory function for
p27. In the chondrogenic lineage, differences in the temporal
regulation of these two CDK inhibitors may cause either pro-
tein to become genetically indispensable for normal cartilage.
Thus, there are four important cell cycle regulators impli-
cated in chondrocyte maturation: p57, p27, p107, and p130. To
understand the relationships between these proteins, we find it
helpful to think of the impact of the cell cycle on chondrocytic
maturation in two stages (14). First, because the loss of either
p57 or p107 alone had the greatest impact on chondrocyte
maturation in both mice and primary cultures, we think these
are at the top of the heirarchy. Additionally, we suspect that
p107 is the major input through which signaling affects matu-
ration, because p107 accumulates before p130 and before cell
cycle exit occurs in FGF-treated RCS cells (14). In the absence
of p107, compensatory upregulation of p130 might substitute
for the initial decision to exit the cycle, as suggested previously
(13), but it is also plausible that a parallel pathway controlled
by increasing p57 as cells enter the G1phase would operate a
bit more inefficiently to induce the initial growth arrest. In-
deed, it is clear that p107 single mutant mice do have a subtle
phenotype, albeit subclinical in regards to viability. So what do
p27 and p130 do? Because any role for p27 and p130 is only
unmasked in p107-deficient cells, p27 accumulates during the
hypertrophic transition in vivo and in vitro, and p130 accumu-
lates in RCS cells once they exit the cell cycle, we suspect that
these proteins normally accumulate only when the cells have
exited the cell cycle. Thus, they might serve to buffer the cell
against continual mitogenic signaling which could otherwise
induce promiscuous proliferation. Thus, p130 and p27 may
simply reinforce the cell cycle exit decision made by p107 and
p57. Ultimately, collaboration between the molecules that in-
duce cell cycle exit and those that maintain quiescence might
be required to permit the elaboration of appropriate transcrip-
tional program leading to the hypertrophic fate.
We thank David Cobrinik (Cornell University) and Antonio Giordano
(Temple University) for stimulating discussions as the work progressed
(MSKCC), Sukhee Lee (University of Massachusetts Medical Center),
Scott Weatherbee (MSKCC), and Laura Meltzer (MSKCC) for experi-
mental advice and assistance during the course of the work.
This work was funded by grants from the NCI (CA89563) and the
Golfers Against Cancer Foundation to Andrew Koff and by Caja
Madrid and Cultek Foundations to Andrew Koff, Anxo Vidal, and
Timothy G. Bromage.
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