Recently, we published a study demonstrating that a
deletion of the gene p21CIP/WAF converts a non-regener-
ating strain of mouse to one capable of epimorphic
regeneration and has provided a unique opportunity to
uncover some of the unknowns of this process in
mammals. Since p21 is involved intricately in so many
cellular processes, it is not clear at this time how deletion
of this gene results in such a healing phenotype. Th is
review will discuss our results, how our fi ndings relate to
other studies, and speculation as to the role of p21 in
A mammalian model of regeneration, the MRL
In 1998, the MRL (Murphy Roths Large) mouse,
generated from cross-breeding AKR, C3H, C57BL/6(B6),
and LG strains of mice , was shown to be able to close
ear punches without showing residual signs of injury or
scarring . Multiple tissues were perfectly replaced,
cartilage re-grew, and hair follicles reappeared. Further-
more, this type of perfect multi-tissue healing, known as
epimorphic regeneration, occurred with the formation of
a blastema-like structure that had been shown to be key
to amphibian limb regeneration [3-5]. Th is phenomenon
had earlier been seen in rabbit ear holes [6-8], and
furthermore, a blastema-derived structure had also been
described during antler re-growth . Th e amphibian
and mammalian ear hole regeneration processes have
many features in common, including rapid re-
epithelialization of the wound , elimination of the
basement membrane between the epidermal and dermal
tissue layers [10,11], blastema formation, re-growth of
cartilage and hair follicles, and scarless healing [2,12,13].
However, the existence of an inbred mouse model
allowed this process to be genetically approachable. It
was also determined that one of the strains used to
generate the MRL mouse, the LG/J mouse, contributed
the regeneration phenotype .
Ear hole closure has lent itself exceedingly well to
genetic studies as this is a wound that is easy to access
and measure and has proven to be a highly quantitative
trait [15-17]. Recently, making use of an advanced
intercross line (LG, SM F34 AIL) employing 1,200 mice
and 3,600 single nucleotide polymorphisms , 18
quantitative trait loci were identifi ed for ear hole closure
with small intervals from 0.661 to 7.141 Mb in length,
which essentially reduced the healing intervals 10- to
50-fold from studies using F2 mice  (JM Cheverud et
al., manuscript in preparation). Th is has allowed a more
focused analysis of candidate genes. Further narrowing of
The MRL (Murphy Roths Large) mouse has provided
a unique model of adult mammalian regeneration
as multiple tissues show this important phenotype.
Furthermore, the healing employs a blastema-like
structure similar to that seen in amphibian
regenerating tissue. Cells from the MRL mouse
display DNA damage, cell cycle G2/M arrest, and
a reduced level of p21CIP1/WAF. A functional role for
p21 was confi rmed when tissue injury in an adult
p21-/- mouse showed a healing phenotype that
matched the MRL mouse, with the replacement
of tissues, including cartilage, and with hair follicle
formation and a lack of scarring. Since the major
canonical function of p21 is part of the p53/p21 axis,
we explored the consequences of p53 deletion. A
regenerative response was not seen in a p53-/- mouse
and the elimination of p53 from the MRL background
had no negative eff ect on the regeneration of the
MRL.p53-/- mouse. An exploration of other knockout
mice to identify p21-dependent, p53-independent
regulatory pathways involved in the regenerative
response revealed another signifi cant fi nding showing
that elimination of transforming growth factor-β1
displayed a healing response as well. These results are
discussed in terms of their eff ect on senescence and
© 2010 BioMed Central Ltd
The role of p21 in regulating mammalian
Larry Matthew Arthur and Ellen Heber-Katz*
The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA
Arthur and Heber-Katz Stem Cell Research & Therapy 2011, 2:30
© 2011 BioMed Central Ltd
these loci and testing of candidates using gene knockouts
should lead to the fi nal identifi cation of these genes.
Besides ear hole closure, multiple organ and injury
systems have extended the MRL mouse’s unusual healing
properties. Th ey include regenerative studies in the heart
[19-21], central nervous system stem cells and tissue
[22-24], cartilage , cornea , digit [27,28] and myo-
metrial healing . Dorsal skin wound healing, which
involves skin contracture, has been reported to be no
diff erent or even worse in the MRL compared to controls
[30,31]. However, a recent study shows that if the wound
has a syngeneic or allogenic skin transplant, the MRL
shows far better healing than the control . One
possible explanation for the healing diff erences in
diff erent systems is that wound contracture, involving
myofi broblasts or cells expressing Sma-1 (smooth muscle
actin), known to be responsible for scarring, is diff erent
in the MRL. Preliminary studies suggest this  (D
Gourevitch, K Bedelbaeva, unpublished data). Th us, the
wound site and type of wound need to be considered in
the MRL’s healing properties.
G2/M cell cycle accumulation of regenerating cells
Th e cells derived from the ear of regenerating and non-
regenerating mice also show signifi cant diff erences from
each other and represent what is seen in vivo. MRL
fi broblast-like cells from uninjured ears display an
uncommon metabolic profi le characteristic of an
embryonic-type aerobic glycolysis, a feature of the adult
MRL mouse itself, versus the more common metabolic
state - oxidative phosphorylation - as seen in the B6
mouse . Th ese cells express stem cell markers similar
to adult MRL tissue that expresses these markers . In
a separate study, cells derived from the injured MRL ear
blastema expressed stem cell markers as found in vivo
 and displayed highly proliferative and migratory
responses in vitro similar to human multipotential
progenitor cells in this study .
Th e rapid growth rate of fi broblast-like cells from the
uninjured MRL ear was noted early on and examination
of cell cycle regulation comparing healer MRL to non-
healer B6 cells showed that the healer cells had an
unusual accumulation of cells in G2/M . A likely
explanation of such G2/M accumulation or potential
arrest was a DNA damage response and this was
supported by an increased p53 response in the MRL 
and confi rmed with data showing that foci of γH2AX and
TopBP1, a phosphorylated histone and a protein re-
cruited to sites of DNA damage, respectively, were highly
increased in MRL cells and tissue . DNA damage
itself was tested using the comet assay and found in
nearly 90% of healer cells compared to 5% of non-healer
cells, showing both single-strand and double-strand
breaks. Furthermore, the DNA repair protein RAD51
was increased in healer cells, suggesting that error-free
homologous recombination was being used . Th e
cause of the DNA damage is still unclear, but the lack of
the cell cycle protein p21Cip1/Waf1 discussed below suggests
a replicative stress mechanism.
Th ese results agree with many reports in the literature
that G2/M accumulation is associated with regeneration
in examples ranging from hydra  to amphibian  to
mammalian liver [39,40]. Th e literature also shows that
cells undergoing blastema formation synthesize DNA but
have a low mitotic index, indicating an accumulation
between S and M and implicating G2 [41-47]. Multiple in
vitro studies have carefully explored cell cycle arrest and
the factors involved in the re-entry of cells into S phase of
the cell cycle and accumulation in G2, as seen in
multinucleated muscle myotubes and myofi bers from
regenerating amphibian limbs , in multinucleated
mammalian myotubes generated from rat C2C12 cell line
myoblasts, and in primary mouse myoblasts [49-51].
In MRL ear-derived cells, the fact that DNA damage
was so widespread made one question why an accumu-
lation of cells was seen in G2/M and not in G1/S. Th is led
to an examination of G1 cell cycle regulatory proteins.
Th e fi rst to be examined, the CDKN1A or p21Cip1/Waf1
protein , was found to be repressed in these cultured
cells. Examination of similar ear-derived cells from a
CDKN1A-defi cient mouse  showed the same
phenotype as MRL cells with increased DNA damage,
γH2AX expression, and G2/M accumulation. But most
striking was the fact that this mouse could fully close ear-
hole injuries at least as well as the MRL mouse .
Th ere have been other mice that possess the ability to
partially heal ear holes, including nude mice , mice
expressing the transgene AGF (angiopoietin-related
growth factor) in keratinocytes , and mice selected for
infl ammatory potential . However, what was surprising
to us was that deletion of this single gene, as predicted
from our in vitro ear dermal cell model, could actually
result in the full MRL epimorphic regeneration phenotype.
The role of p21CIP1/Waf1, regeneration, and the
Earlier studies have examined the role of p21 in regenera-
tion of the mammalian liver. Gene expression of p21
plays a role in hepatic regeneration by both p53-
dependent and p53-independent control mechanisms
. Transgenic mice that over-express p21 produced
large polyploid nuclei in a portion of the hepatocytes and
the regenerative capacity of the livers was halted .
Over-expression of STAT-3 with resulting p21 upregu-
lation impairs regeneration in fatty livers . Consistent
with this picture, repression of the p53/p21 pathway was
shown to enhance liver regeneration . Such studies
parallel our recent fi ndings .
Arthur and Heber-Katz Stem Cell Research & Therapy 2011, 2:30
Page 2 of 7
Th e overall understanding of the functions of p21 can be
quite overwhelming considering the complexity of
functions in which this protein has been implicated. p21 is
involved in the response to cellular stresses, such as DNA
damage, oxidative stress, cytokines, mitogens, tumor
viruses, and anti-cancer agents, and can have tumor
suppressive activities and oncogenic capabilities depend-
ing on the cell type and context [60,61]. For example, p21
is transcriptionally regulated by p53 for tumor suppressor
activity and as an inhibitor of cell cycle progression
through the inhibition of cyclin-dependent kinase (CDK)-
cyclin complexes and proliferating cell nuclear antigen,
which can lead to diff erentiation, apop tosis, or senescence.
Increasing this complexity is the fact that p21 can regulate
gene expression and other cellular events, such as
autophagy and a DNA damage repair response, through
protein-protein interactions that depend on the cell type,
subcellular localization, expres sion levels, protein stability,
and post-translational modifi cations [62-66].
So which of these functions are involved in the re-
genera tion phenotype seen in the p21-/- mice? Some
indication may come from in vitro studies in other re-
generating systems. For example, adult urodele amphi-
bians can regenerate limbs through a process that
involves loss of diff erentiation markers, cell cycle re-
entry, proliferation, formation of a blastema, and diff er-
en tiation into adult tissue . In an amphibian in vitro
model of skeletal muscle regeneration, retinoblastoma
(Rb) protein plays a predominant role in cell cycle re-
entry through phosphorylation by CDK4/6 . Th is
process requires serum to stimulate entry of the
quiescent nuclei of multinuclear myotubes into S-phase
with a serum-derived thrombin-activated factor being
necessary for Rb hyperphosphorylation, resulting in its
‘inactivation’ [48,68]. Th ese cells enter S phase but arrest
and do not separate into single cells, which would allow
further progression of the cell cycle through mitosis.
However, there are confl icting reports about mammalian
cells. Myotubes from an Rb-/- mouse are capable of cell
cycle re-entry and show DNA synthesis upon serum
stimulation but no mitosis in one study  but no cell
cycle re-entry in another . In a separate study using
mammalian myotubes generated from the rat C2C12
myoblast line, newt regeneration blastema extract led to
myotube cellularization to smaller myotubes and pro-
liferating mononucleate cells, suggesting de-diff eren-
tiation with reduced expression of mature muscle cell
markers . In addition, a recent report using primary
myoblasts  suggests that another factor in addition to
Rb, p19arf, must be inactivated for cell cycle re-entry and
de-diff erentiation in postmitotic mammalian muscle. Th e
tumor suppressor protein p19arf acts as a regeneration
suppressor and is not found in regenerative vertebrates,
suggesting that it has interesting potential as a key to
mammalian regeneration. Th us, Rb inactivation has been
shown to be important in both amphibian and mam-
malian regeneration in vitro.
Th e p21 protein, its major role being a CDK inhibitor
found on chromosome 17 in the mouse, is known to
block proliferation by preventing the phosphorylation of
Rb and the transcription of cell cycle-regulated pro-
proliferative proteins. Th e p21 protein binds to cyclin-
CDK (2/4) complexes, not allowing them to function as
kinases. Th ey in turn cannot phosphorylate Rb, which
remains bound to E2F, a transcription factor responsible
for proliferation, eff ectively blocking E2F function. Th us,
p21 activity directly leads to suppression of cell cycle
transit and the loss of p21 should promote E2F activity,
lead to enhanced DNA synthesis and potentially to de-
diff erentiation. Rb function, then, in the studies above
should be directly aff ected by p21 activity.
Not surprisingly, p53 and p21 have been shown to
prevent the transition from fi broblasts to induced
pluripotent stem cells [70-72]. Th e level of de-diff er-
entiation in the p21-/- mouse is being further explored,
although we have previously reported that stem cell
markers are over-expressed in MRL tissue .
The role of p53, senescence, and transforming
growth factor-β in regeneration
As mentioned above, we found that p53 was up-regulated
in MRL mouse ears, though p21 was absent. Is there a
role for p53 in regeneration? Unlike the p21-/- mouse,
which is a complete regenerator, p53-/- mice show no
regenerative capacity . Th is fi nding established a p53-
independent function of p21 that is important for
regeneration. However, MRL.p53-/- crosses showed not
only healing rates similar to or better than the MRL itself
but also showed enhanced diff erentiation in the form of
increased chondrogenesis and adipogenesis . Th e
major role played by p53 as the ‘guardian’ of the genome
is due to its ability to respond to DNA damage and
cellular stress by inhibiting cell cycle progression and
then regulating DNA repair, cell cycle control, apoptosis,
diff erentiation, autophagy induction, and senescence. It is
not clear which of these functions or lack thereof could
be responsible for the enhanced diff erentiation observed
in MRL.p53-/- mice [64,71,74-79]. One study suggests that
removal of p53 allows for an accumulation of cells with
elevated levels of DNA damage (on a repair-defi cient
background mouse), which delays hair follicle renewal
and regeneration [80,81]. However, we observed hair
follicle formation in our MRL/p53-/- mice . Further
regeneration studies on diff erent tissue types need to be
performed in order to determine the role of p53 in
One potential area of interest are the roles of p21 and
p53 in both diff erentiation and cellular senescence at
Arthur and Heber-Katz Stem Cell Research & Therapy 2011, 2:30
Page 3 of 7
wound sites. It has been shown that elimination of p21 in
mouse stem cells with dysfunctional telomeres, a marker
for senescence induction, increases stem cell function
and the life span of these mice without an increase in
cancer formation, providing a direct role for p21 in both
stem cell diff erentiation and senescence . One direct
link for p21 in diff erentiation and senescence is sup pres-
sion by the Twist proteins, major regulators of embryo-
genesis . Th e Twist proteins inhibit p21 in a p53-
independent manner and promote epithelial-mesen-
chymal transition and suppress cellular senescence .
Th e two major pathways for inducing senescence in
cells of multiple tissues are p53/p21 [85-91] and p16ink4a
[75,92-95]. In an earlier paper, we suggested that senes-
cence was not a factor in MRL regeneration because of
the lack of p53 requirement . However, there is, in
fact, evidence that p21 can induce senescence in the
absence of p53 [87,96-98] as well as p53-mediated p21-
independent activation of senescence [99-101]. It has
been suggested that reactive oxygen species are necessary
to maintain the senescence phenotype and that both p16
and p21 are involved [99,102,103]. Actually, we previously
reported that reactive oxygen species levels are decreased
in the MRL mouse , consistent with an aerobic
glycolytic metabolism, which argues against senescence
playing a functional role. In addition, the protein RhoD,
which is required for transformation by the oncogenic
protein Ras, is responsible for suppressing p21 induction
and subsequent senescence [104,105]. Th e gene ID1 has
been shown to repress HRAS-mediated senescence in
the presence of increased amounts of p21 , arguing
the other way. Recently, a publication showed that the
matricellular protein CCN1, which is expressed at the
sites of wounds, induces senescence through p53 and
actually helps to prevent fi brosis during tissue repair
. In this case, however, the healing is tissue repair
with scarring and not blastema-induced scarless re-
genera tion. Th us, the connection between senescence
and regeneration, and its diff erence compared to onco-
genesis, is yet to be determined.
Another major regulator of p21 is transforming growth
factor (TGF)-β1, which is involved in anti-proliferation
and diff erentiation . TGF-β1 controls proliferation,
diff erentiation, migration, and apoptosis in embryonic
and adult tissue through the Smad3 pathway [109-113].
Multiple studies in mutant mice lacking the TGF-β1/
Smad3 pathway have implicated a regeneration pheno-
type in mice: mice lacking TGF-β1 show an increase in
wound closure and epithelialization ; transgenic
mice null for Smad3 show increased re-epithelialization
and tissue renewal ; and Smad7 over-expression
leads to Smad3 down-regulation and to enhanced liver
regeneration through the TGF-β/Smad3/p21 pathway
. Smad3 has been implicated as a candidate gene in
our genetic mapping studies of healer MRL and parental
LG mice . Contrary to these results, other transgenic
studies on TGF-β1-null mice showed malfunctions in the
repair of excisional back skin wounds due to altered
infl ammatory responses [117-119]. Our studies have
shown that a TGF-β1/Rag1 double knockout mouse is a
partial healer . An interesting fact is that TGF-β1
enhances Sma-1 production
associated with scarring  and reduces regenerative
healing, whereas the TGF-β isoform TGF-β3 enhances
scar-free healing .
and myofi broblasts
Th e MRL mouse is the fi rst genetically dissectible and
molecularly tractable mammalian model of regeneration
of multiple tissues in a single organism. It establishes the
fact that regenerative capacity has not been lost to
mammals through evolution but remains as a cryptic
trait, which can be activated by the deletion of a single
gene, p21. Th us, the p21-null mouse now should become
a ‘single gene’ standard model for mammalian regenera-
Th e lack of p21 may act to enhance the regenerative
response in various ways. It could alter DNA damage and
checkpoint responses, leading to enhanced proliferation.
It could reduce TGF-β signaling, leading to reduced scar
formation, and alter diff erentiation patterns. It could lead
to lack of senescence and reduced cytokine responses. It
could support progenitor cell stability as seen in induced
pluripotent stem cell formation.
Besides determining exactly which function of p21 and
its absence is responsible for enhanced ear hole closure, it
will also be important to defi ne the critical pathways in
the MRL mouse that actually lead to p21 down-regulation
CDK, cyclin-dependent kinase; MRL, Murphy Roths Large; Rb, retinoblastoma;
Sma-1, smooth muscle actin; TGF, transforming growth factor.
The authors declare that they have no competing interests.
These studies were supported by the FM Kirby Foundation, G Harold and Leila
Y Mathers Foundation, and an NIH ARRA grant from NIGMS. The work was also
supported by an NCI Cancer Center Grant (P30 CA10815). LMA was supported
by the Training Program Grant in Basic Cancer Biology 5T32CA09171. We
would like to thank Larry O Arthur, Andrew Snyder, and John Leferovich for
their useful comments and insight.
Published: 29 June 2011
This article is part of a review series on Epigenetics and regulation.
Other articles in the series can be found online at http://stemcellres.
Arthur and Heber-Katz Stem Cell Research & Therapy 2011, 2:30
Page 4 of 7
1. Murphy ED: Lymphoproliferation (lpr) and other single-locus models for
murine lupus. In Immunologic Defects in Laboratory Animals. Volume 2. Edited
by Gershwin ME, Merchant B. New York: Plenum Publishing Corp.;
2. Clark LD, Clark RK, Heber-Katz E: A new murine model for mammalian
wound repair and regeneration. Clin Immunol Immunopathol 1998,
3. Stocum DL: The urodele limb regeneration blastema. Determination and
organization of the morphogenetic fi eld. Diff erentiation 1984, 27:13-28.
4. Brockes JP, Kumar A: Appendage regeneration in adult vertebrates and
implications for regenerative medicine. Science 2005, 310:1919-1923.
5. Gardiner DM, Bryant SV: Molecular mechanisms in the control of limb
regeneration: the role of homeobox genes. Int J Dev Biol 1996, 40:797-805.
6. Goss RJ, Grimes LN: Tissue interactions in the regeneration of rabbit ear
holes. Am Zool 1975, 12:151-157.
7. Price J, Faucheux C, Allen S: Deer antlers as a model of mammalian
regeneration. Curr Top Dev Biol 2005, 67:1-48.
8. Kierdorf U, Kierdorf H: Deer antlers- a model of mammalian appendage
regeneration: an extensive review. Gerontology 2011, 57:53-65.
9. Goss RJ: Tissue diff erentiation in regenerating antlers. Biol Deer Production
10. Gourevitch D, Clark L, Chen P, Seitz A, samulewicz SJ, Heber-Katz E: Matrix
metalloproteinase activity correlates with blastema formation in the
regenerating MRL mouse ear hole model. Dev Dyn 2003, 226:377-387.
11. Stocum DL, Crawford K: Use of retinoids to analyze the cellular basis of
positional memory in regenerating amphibian limbs. Biochem Cell Biol
12. Nye HL, Cameron JA, Chernoff EA, Stocum DL: Regeneration of the urodele
limb: a review. Dev Dyn 2003, 226:280-294.
13. Brockes JP: Amphibian limb regeneration: rebuilding a complex structure.
Science 1997, 276:81-87.
14. Kench JA, Russell DM, Fadok VA, Young SK, Worthen GS, Jones-Carson J,
Henson JE, Nemazee D: Aberrant wound healing and TGF-beta production
in the autoimmune-prone MRL/+ mouse. Clin Immunol 1999, 92:300-310.
15. Blankenhorn E, Bryan G, Kossenkov A, Clark L, Zhang XM, Chang C, Horng W,
Pletscher L, Cheverud J, Showe L, Heber-Katz E: Genetic loci that regulate
healing and regeneration in LG/J and SM/J mice. Mamm Genome 2009,
16. Heber-Katz E, Chen P, Clarck L, Zhang X-M, Troutman S, Blankenhorn EP:
Regeneration in MRL mice: further genetic loci controlling the ear hole
closure trait using MRL and M.m. Castaneus mice. Wound Repair Regen
17. Yu H, Mohan S, Masinde G, Baylink D: Mapping the dominant wound
healing and soft tissue regeneration QTL in MRL x CAST. Mamm Genome
18. Norgard E, Lawson H, Pletscher L, Wang B, Brooks V, Wolf J, Cheverud J:
Genetic factors and diet aff ect long-bone length in the F34 LG,SM
advanced intercross. Mamm Genome 2011, 22:178-196.
19. Leferovich JM, Bedelbaeva K, Samulewicz S, Zhang XM, Zwas D, Lankford EB,
Heber-Katz E: Heart regeneration in adult MRL mice. Proc Natl Acad Sci U S A
20. Haris Naseem R, Meeson AP, Michael DiMaio J, White MD, Kallhoff J,
Humphries C, Goetsch SC, De Windt LJ, Williams MA, Garry MG, Garry DJ:
Reparative myocardial mechanisms in adult C57BL/6 and MRL mice
following injury. Physiol Genomics 2007, 30:44-52.
21. Alfaro MP, Pagni M, Vincent A, Atkinson J, Hill MF, Cates J, Davidson JM,
Rottman J, Lee E, Young PP: The Wnt modulator sFRP2 enhances
mesenchymal stem cell engraftment, granulation tissue formation and
myocardial repair. Proc Natl Acad Sci U S A 2008, 105:18366-18371.
22. Baker KL, Daniels SB, Lennington JB, Lardaro T, Czap A, Notti RQ, Cooper O,
Isacson O, Frasca S, Conover JC: Neuroblast protuberances in the
subventricular zone of the regenerative MRL/MpJ mouse. J Comp Neurol
23. Thuret S, Toni N, Aigner S, Yeo GW, Gage FH: Hippocampus-dependent
learning is associated with adult neurogenesis in MRL/MpJ mice.
Hippocampus 2009, 19:658-669.
24. Balu DT, Hodes GE, Anderson BT, Lucki I: Enhanced sensitivity of the MRL/
MpJ mouse to the neuroplastic and behavioral eff ects of chronic
antidepressant treatments. Neuropsychopharmacology 2009, 34:1764-1773.
25. Fitzgerald J, Rich C, Burkhardt D, Allen J, Herzka AS, Little CB: Evidence for
articular cartilage regeneration in MRL/MpJ mice. Osteoarthritis Cartilage
26. Ueno M, Lyons BL, Burzenski LM, Gott B, Shaff er DJ, Roopenian DC, Shultz LD:
Accelerated wound healing of alkali-burned corneas in MRL mice is
associated with a reduced infl ammatory signature. Invest Ophthalmol Vis Sci
27. Chadwick RB, Bu L, Yu H, Hu Y, Wergedal JE, Mohan S, Baylink DJ: Digit tip
regrowth and diff erential gene expression in MRL/Mpj, DBA/2, and
C57BL/6 mice. Wound Repair Regen 2007, 15:275-284.
28. Gourevitch DL, Clark L, Bedelbaeva K, Leferovich J, Heber-Katz E: Dynamic
changes after murine digit amputation: The MRL mouse digit shows
waves of tissue remodeling, growth, and apoptosis. Wound Repair Regen
29. Buhimschi CS, Zhao G, Sora N, Madri JA, Buhimschi IA: Myometrial wound
healing post-cesarean delivery in the MRL/MpJ mouse model of uterine
scarring. Am J Pathol 2010, 177:197-207.
30. Beare AHM, Metcalfe AD, Ferguson MWJ: Location of injury infl uences the
mechanisms of both regeneration and repair within the MRL/MpJ mouse.
J Anat 2006, 209:547-559.
31. Davis TA, Amare M, Naik S, Kovalchuk AL, Tadaki D: Diff erential cutaneous
wound healing in thermally injured MRL/MPJ mice. Wound Repair Regen
32. Tolba RH, Schildberg FA, Decker D, Abdullah Z, Büttner R, Minor T, Von
Ruecker A: Mechanisms of improved wound healing in Murphy Roths
Large (MRL) mice after skin transplantation. Wound Repair Regen 2010,
33. Bedelbaeva K, Snyder A, Gourevitch D, Clark L, Zhang XM, Leferovich J,
Cheverud JM, Lieberman P, Heber-Katz E: Lack of p21 expression links cell
cycle control and appendage regeneration in mice. Proc Natl Acad Sci U S A
34. Naviaux RK, Le TP, Bedelbaeva K, Leferovich J, Gourevitch D, Sachadyn P,
Zhang XM, Clark L, Heber-Katz E: Retained features of embryonic
metabolism in the adult MRL mouse. Mol Genet Metab 2009, 96:133-144.
35. Samulewicz SJ, Seitz A, Clark L, Heber-Katz E: Expression of preadipocyte
factor-1(Pref-1), a delta-like protein, in healing mouse ears. Wound Repair
Regen 2002, 10:215-221.
36. Reing JE, Zhang L, Myers-Irvin J, Cordero KE, Freytes DO, Heber-Katz E,
Bedelbaeva K, McIntosh D, Dewilde A, Braunhut SJ, Badylak SF: Degradation
products of extracellular matrix aff ect cell migration and proliferation.
Tissue Eng Part A 2009, 15:605-614.
37. Schmidt T, David CN: Gland cells in Hydra: cell cycle kinetics and
development. J Cell Sci 1986, 85:197-215.
38. Stocum DL, Cameron JA: Looking proximally and distally: 100 years of limb
regeneration and beyond. Dev Dyn 2011, 240:943-968.
39. Michalopoulos GK, DeFrances MC: Liver regeneration. Science 1997,
40. Celton-Morizur S, Desdouets C: Polyploidization of liver cells. Adv Exp Med
Biol 2010, 2010:123-135.
41. Tassava RA, Bennett LL, Zitnik GD: DNA synthesis without mitosis in
amputated denervated forelimbs of larval axolotls. J Exp Zool 1974,
42. Mescher AL, Tassava RA: Denervation eff ects on DNA replication and
mitosis during the initiation of limb regeneration in adult newts. Dev Biol
43. Tassava RA, Mescher AL: Mitotic activity and nucleic acid precursor
incorporation in denervated and innervated limb stumps of axolotl larvae.
J Exp Zool 1976, 195:253-262.
44. McCullough WD, Tassava RA: Determination of the blastema cell cycle in
regenerating limbs of the larval axolotl, Ambystoma mexicanum. Ohio J Sci
45. Tassava RA, McCullough WD: Neural control of cell cycle events in
regenerating salamander limbs. Am Zool 1978, 18:843-854.
46. Maden M: Neurotrophic control of the cell cycle during amphibian limb
regeneration. J Embryol Exp Morphol 1978, 48:169-175.
47. Tassava RA, Garling DJ: Regenerative responses in larval axolotl limbs with
skin grafts over the amputation surface. J Exp Zool 1979, 208:97-110.
48. Tanaka EM, Drechsel DN, Brockes JP: Thrombin regulates S-phase re-entry
by cultured newt myotubes. Curr Biol 1999, 9:792-799.
49. McGann CJ, Odelberg SJ, Keating MT: Mammalian myotube
dediff erentiation induced by newt regeneration extract. Proc Natl Acad Sci
U S A 2001, 98:13699-13704.
Arthur and Heber-Katz Stem Cell Research & Therapy 2011, 2:30
Page 5 of 7
50. Schneider JW, Gu W, Zhu L, Mahdavi V, Nadal-Ginard B: Reversal of terminal
diff erentiation mediated by p107 in Rb-/- muscle cells. Science 1994,
51. Huh MS, Parker MH, Scimè A, Parks R, Rudnicki MA: Rb is required for
progression through myogenic diff erentiation but not maintenance of
terminal diff erentiation. J Cell Biol 2004, 166:865-876.
52. El Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D,
Mercer WE, Kinzler KW, Vogelstein B: WAF1, a potential mediator of p53
tumor suppression. Cell 1993, 75:817-825.
53. Gawronska-Kozak B: Regeneration in the ears of immunodefi cient mice:
identifi cation and lineage analysis of mesenchymal stem cells. Tissue Eng
54. Oike Y, Yasunaga K, Ito Y, Matsumoto Si, Maekawa H, Morisada T, Arai F,
Nakagata N, Takeya M, Masuho Y, Suda T: Angiopoietin-related growth
factor (AGF) promotes epidermal proliferation, remodeling, and
regeneration. Proc Natl Acad Sci U S A 2003, 100:9494-9499.
55. De Franco M, Carneiro P, Peters L, Vorraro F, Borrego A, Ribeiro O, Starobinas N,
Cabrera W, Ibanez O: Slc11a1 (Nramp1) alleles interact with acute
infl ammation loci to modulate wound-healing traits in mice. Mamm
Genome 2007, 18:263-269.
56. Albrecht JH, Meyer AH, Hu MY: Regulation of cyclin-dependent kinase
inhibitor p21WAF1/Cip1/Sdi1 gene expression in hepatic regeneration.
Hepatology 1997, 25:557-563.
57. Wu H, Wade M, Krall L, Grisham J, Xiong Y, Van Dyke T: Targeted in vivo
expression of the cyclin-dependent kinase inhibitor p21 halts hepatocyte
cell-cycle progression, postnatal liver development and regeneration.
Genes Dev 1996, 10:245-260.
58. Torbenson M, Yang SQ, Liu HZ, Huang J, Gage W, Diehl AM: STAT-3
overexpression and p21 up-regulation accompany impaired regeneration
of fatty livers. Am J Pathol 2002, 161:155-161.
59. Stepniak E, Ricci R, Eferl R, Sumara G, Sumara I, Rath M, Hui L, Wagner EF:
c-Jun/AP-1 controls liver regeneration by repressing p53/p21 and p38
MAPK activity. Genes Dev 2006, 20:2306-2314.
60. Weiss RH: p21Waf1/Cip1 as a therapeutic target in breast and other
cancers. Cancer Cell 2003, 4:425-429.
61. Cazzalini O, Scovassi AI, Savio M, Stivala LA, Prosperi E: Multiple roles of the
cell cycle inhibitor p21CDKN1A in the DNA damage response. Mutat Res
62. Jung YS, Qian Y, Chen X: Examination of the expanding pathways for the
regulation of p21 expression and activity. Cell Signal 2010, 22:1003-1012.
63. Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D: p21 is a
universal inhibitor of cyclin kinases. Nature 1993, 366:701-704.
64. El Deiry WS: p21/p53, cellular growth control and genomic integrity. Curr
Top Microbiol Immunol 1998, 227:121-137.
65. Abbas T, Dutta A: p21 in cancer: intricate networks and multiple activities.
Nat Rev Cancer 2009, 9:400-414.
66. Gartel AL: p21WAF1/CIP1 and cancer: A shifting paradigm? BioFactors 2009,
67. Tanaka EM, Gann AAF, Gates PB, Brockes JP: Newt myotubes reenter the cell
cycle by phosphorylation of the retinoblastoma protein. J Cell Biol 1997,
68. Straube WL, Tanaka EM: Reversibility of the diff erentiated state:
regeneration in amphibians. Artif Organs 2006, 30:743-755.
69. Pajcini KV, Corbel SY, Sage J, Pomerantz JH, Blau HM: Transient inactivation
of Rb and ARF yields regenerative cells from postmitotic mammalian
muscle. Cell Stem Cell 2010, 7:198-213.
70. Hanna J, Saha K, Pando B, van Zon J, Lengner CJ, Creyghton MP, van
Oudenaarden A, Jaenisch R: Direct cell reprogramming is a stochastic
process amenable to acceleration. Nature 2009, 462:595-601.
71. Hong H, Takahashi K, Ichisaka T, Aoi T, Kanagawa O, Nakagawa M, Okita K,
Yamanaka S: Suppression of induced pluripotent stem cell generation by
the p53/p21 pathway. Nature 2009, 460:1132-1135.
72. Yamanaka S: A fresh look at iPS cells. Cell 2009, 137:13-17.
73. Arthur LM, Demarest RM, Clark L, Gourevitch D, Bedelbaeva K, Anderson R,
Snyder A, Capobianco AJ, Lieberman P, Feigenbaum L, Heber-Katz E:
Epimorphic regeneration in mice is p53-independent. Cell Cycle 2010,
74. El Deiry WS: Regulation of p53 downstream genes. Semin Cancer Biol 1998,
75. Beausejour CM, Krtolica A, Galimi F, Narita M, Lowe SW, Yaswen P, Campisi J:
Reversal of human cellular senescence: roles of the p53 and p16
pathways. EMBO J 2003, 22:4212-4222.
76. Sheahan S, Bellamy CO, Treanor L, Harrison DJ, Prost S: Additive eff ect of p53,
p21 and Rb deletion in triple knockout primary hepatocytes. Oncogene
77. Kuribayashi K, El Deiry WS: Regulation of programmed cell death by the
p53 pathway. Adv Exp Med Biol 2007, 615:201-221.
78. Vousden KH, Prives C: Blinded by the light: the growing complexity of p53.
Cell 2009, 137:413-431.
79. Maiuri MC, Galluzzi L, Morselli E, Kepp O, Malik SA, Kroemer G: Autophagy
regulation by p53. Curr Opin Cell Biol 2010, 22:181-185.
80. Ruzankina Y, Schoppy DW, Asare A, Clark CE, Vonderheide RH, Brown EJ:
Tissue regenerative delays and synthetic lethality in adult mice after
combined deletion of Atr and Trp53. Nat Genet 2009, 41:1144-1149.
81. Schoppy DW, Ruzankina Y, Brown EJ: Removing all obstacles: a critical role
for p53 in promoting tissue renewal. Cell Cycle 2010, 9:1313-1319.
82. Choudhury AR, Ju Z, Djojosubroto MW, Schienke A, Lechel A, Schaetzlein S,
Jiang H, Stepczynska A, Wang C, Buer J, Lee HW, von Zglinicki T, Ganser A,
Schirmacher P, Nakauchi H, Rudolph KL: Cdkn1a deletion improves stem cell
function and lifespan of mice with dysfunctional telomeres without
accelerating cancer formation. Nat Genet 2007, 39:99-105.
83. Ansieau S, Bastid J, Doreau A, Morel AP, Bouchet BP, Thomas C, Fauvet F,
Puisieux I, Doglioni C, Piccinin S, Maestro R, Voeltzel T, Selmi A, Valsesia-
Wittmann S, Caron de Fromentel C, Puisieux A: Induction of EMT by twist
proteins as a collateral eff ect of tumor-promoting inactivation of
premature senescence. Cancer Cell 2008, 14:79-89.
84. Smit MA, Peeper DS: Deregulating EMT and senescence: double impact by
a single twist. Cancer Cell 2008, 14:5-7.
85. Zhang X, Li J, Sejas DP, Pang Q: The ATM/p53/p21 pathway infl uences cell
fate decision between apoptosis and senescence in reoxygenated
hematopoietic progenitor cells. J Biol Chem 2005, 280:19635-19640.
86. Lee AC, Fenster BE, Ito H, Takeda K, Bae NS, Hirai T, Yu ZX, Ferrans VJ, Howard
BH, Finkel T: Ras proteins induce senescence by altering the intracellular
levels of reactive oxygen species. J Biol Chem 1999, 274:7936-7940.
87. Fang L, Igarashi M, Leung J, Sugrue MM, Lee SW, Aaronson SA: p21Waf1/
Cip1/Sdi1 induces permanent growth arrest with markers of replicative
senescence in human tumor cells lacking functional p53. Oncogene 1999,
88. Wang Y, Blandino G, Givol D: Induced p21waf expression in H1299 cell line
promotes cell senescence and protects against cytotoxic eff ect of
radiation and doxorubicin. Oncogene 1999, 18:2643-2649.
89. Dirac AM, Bernards R: Reversal of senescence in mouse fi broblasts through
lentiviral suppression of p53. J Biol Chem 2003, 278:11731-11734.
90. Xue W, Zender L, Miething C, Dickins RA, Hernando E, Krizhanovsky V,
Cordon-Cardo C, Lowe SW: Senescence and tumour clearance is triggered
by p53 restoration in murine liver carcinomas. Nature 2007, 445:656-660.
91. Coppe JP, Patil CK, Rodier F, Sun Y, Munoz DP, Goldstein J, Nelson PS, Desprez
PY, Campisi J: Senescence-associated secretory phenotypes reveal cell-
nonautonomous functions of oncogenic RAS and the p53 tumor
suppressor. PLoS Biol 2008, 6:e301
92. Vijayachandra K, Higgins W, Lee J, Glick A: Induction of p16ink4a and
p19ARF by TGFbeta1 contributes to growth arrest and senescence
response in mouse keratinocytes. Mol Carcinog 2009, 48:181-186.
93. Dai CY, Enders GH: p16 INK4a can initiate an autonomous senescence
program. Oncogene 2000, 19:1613-1622.
94. Takahashi A, Ohtani N, Yamakoshi K, Iida S, Tahara H, Nakayama K, Nakayama
KI, Ide T, Saya H, Hara E: Mitogenic signalling and the p16INK4a-Rb pathway
cooperate to enforce irreversible cellular senescence. Nat Cell Biol 2006,
95. Takahashi A, Ohtani N, Hara E: Irreversibility of cellular senescence: dual
roles of p16INK4a/Rb-pathway in cell cycle control. Cell Division 2007, 2:10.
96. Lin HK, Chen Z, Wang G, Nardella C, Lee SW, Chan CH, Yang WL, Wang J, Egia
A, Nakayama KI, Cordon-Cardo C, Teruya-Feldstein J, Pandolfi PP: Skp2
targeting suppresses tumorigenesis by Arf-p53-independent cellular
senescence. Nature 2010, 464:374-379.
97. Aliouat-Denis CcM, Dendouga N, Van den Wyngaert I, Goehlmann H, Steller
U, van de Weyer I, Van Slycken N, Andries L, Kass S, Luyten W, Janicot M,
Vialard JE: p53-independent regulation of p21Waf1/Cip1 expression and
senescence by Chk2. Mol Cancer Res 2005, 3:627-634.
98. Brown JP, Wei W, Sedivy JM: Bypass of senescence after disruption of
p21CIP1/WAF1 gene in normal diploid human fi broblasts. Science 1997,
Arthur and Heber-Katz Stem Cell Research & Therapy 2011, 2:30
Page 6 of 7
99. Macip S, Igarashi M, Fang L, Chen A, Pan ZQ, Lee SW, Aaronson SA: Inhibition
of p21-mediated ROS accumulation can rescue p21-induced senescence.
EMBO J 2002, 21:2180-2188.
100. Pantoja C, Serrano M: Murine fi broblasts lacking p21 undergo senescence
and are resistant to transformation by oncogenic Ras. Oncogene 1999,
101. Castro ME, Guijarro MdV, Moneo V, Carnero A: Cellular senescence induced
by p53-ras cooperation is independent of p21waf1 in murine embryo
fi broblasts. J Cell Biochem 2004, 92:514-524.
102. Fiorentino FP, Symonds CE, Macaluso M, Giordano A: Senescence and p130/
Rbl2: a new beginning to the end. Cell Res 2009, 19:1044-1051.
103. Passos JF, Nelson G, Wang C, Richter T, Simillion C, Proctor CJ, Miwa S,
Olijslagers S, Hallinan J, Wipat A, Saretzki G, Rudolph KL, Kirkwood TBL, von
Zglinicki T: Feedback between p21 and reactive oxygen production is
necessary for cell senescence. Mol Syst Biol 2010, 6:347.
104. Khosravi-Far R, Solski PA, Clark GJ, Kinch MS, Der CJ: Activation of Rac1,
RhoA, and mitogen-activated protein kinases is required for Ras
transformation. Mol Cell Biol 1995, 15:6443-6453.
105. Qiu RG, Chen J, McCormick F, Symons M: A role for Rho in Ras
transformation. Proc Natl Acad Sci U S A 1995, 92:11781-11785.
106. Swarbrick A, Roy E, Allen T, Bishop JM: Id1 cooperates with oncogenic Ras to
induce metastatic mammary carcinoma by subversion of the cellular
senescence response. Proc Natl Acad Sci U S A 2008, 105:5402-5407.
107. Jun JI, Lau LF: The matricellular protein CCN1 induces fi broblast
senescence and restricts fi brosis in cutaneous wound healing. Nat Cell Biol
108. Moustakas A, Pardali K, Gaal A, Heldin CH: Mechanisms of TGF-[beta]
signaling in regulation of cell growth and diff erentiation. Immunol Lett
109. Bierie B, Moses HL: Tumour microenvironment: TGF[beta]: the molecular
Jekyll and Hyde of cancer. Nat Rev Cancer 2006, 6:506-520.
110. Pardali K, Moustakas A: Actions of TGF-[beta] as tumor suppressor and
pro-metastatic factor in human cancer. Biochim Biophys Acta 2007,
111. Massague J: TGF[beta] in cancer. Cell 2008, 134:215-230.
112. Moustakas A, Kardassis D: Regulation of the human p21/WAF1/Cip1
promoter in hepatic cells by functional interactions between Sp1 and
Smad family members. Proc Natl Acad Sci U S A 1998, 95:6733-6738.
113. Pardali K, Kurisaki A, Moren A, ten Dijke P, Kardassis D, Moustakas A: Role of
Smad proteins and transcription factor Sp1 in p21Waf1/Cip1 Regulation
by transforming growth factor-beta. J Biol Chem 2000, 275:29244-29256.
114. Koch RM, Roche NS, Parks WT, Ashcroft GS, Letterio JJ, Roberts AB: Incisional
wound healing in transforming growth factor-beta1 null mice. Wound
Repair Regen 2000, 8:179-191.
115. Ashcroft GS, Yang X, Glick AB, Weinstein M, Letterio JJ, Mizel DE, Anzano M,
Greenwell-Wild T, Wahl SM, Deng C, Roberts AB: Mice lacking Smad3 show
accelerated wound healing and an impaired local infl ammatory response.
Nat Cell Biol 1999, 1:260-266.
116. Zhong Z, Tsukada S, Rehman H, Parsons CJ, Theruvath TP, Rippe RA, Brenner
DA, Lemasters JJ: Inhibition of transforming growth factor-beta/Smad
signaling improves regeneration of small-for-size rat liver grafts. Liver
Transpl 2010, 16:181-190.
117. Grose R, Werner S: Wound-healing studies in transgenic and knockout
mice. Mol Biotechnol 2004, 28:147-166.
118. O’Kane S, Ferguson MWJ: Transforming growth factor [beta]s and wound
healing. Int J Biochem Cell Biol 1997, 29:63-78.
119. Crowe MJ, Doetschman T, Greenhalgh DG: Delayed wound healing in
immunodefi cient TGF-[beta]1 knockout mice. J Invest Dermatol 2000,
120. Xu G, Bochaton-Piallat ML, Andreutti D, Low RB, Gabbiani G, Neuville P:
Regulation of alpha-smooth muscle actin and CRBP-1 expression by
retinoic acid and TGFbeta-1 in cultured fi broblasts. J Cell Physiol 2001,
121. J.Shaw T, Kishi K, Mori R: Wound-associated skin fi brosis: mechanisms and
treatments based on modulating the infl ammatory response. Endocr
Metab Immune Disord Drug Targets 2010, 10:320-330.
Cite this article as: Arthur LM, Heber-Katz E: The role of p21 in regulating
mammalian regeneration. Stem Cell Research & Therapy 2011, 2:30.
Arthur and Heber-Katz Stem Cell Research & Therapy 2011, 2:30
Page 7 of 7