.

Biochimica et Biophysica Acta 1377 1998 M1M11

Mini-Review
Regeneration and cancer
Jeremy P. Brockes
)
Ludwig Institute for Cancer Research, and Department of Biochemistry and Molecular Biology, Uniersity College London,
91 Riding House Street, London W1P 8BT, UK
Received 27 August 1997; accepted 28 August 1997
1. Introduction
The ability of an adult animal to regenerate large
sections of the body plan is quite common among the
invertebrate phyla, but it is restricted among verte-
. w x
brates to the urodele amphibians order Caudata 1 .

These include the newts aquatic members of the

.
Salamander family , and other salamanders such as
the axolotl. An adult newt can regenerate its tail and
limbs, as well as the upper and lower jaws, and
.
ocular tissues such as the lens Fig. 1A . Regenera-
tion of the jaws and appendages proceeds by local
formation of a mesenchymal growth zone or blastema
.
at the plane of amputation Fig. 1B , a mechanism
w x
referred to as epimorphic regeneration 2 . The mes-
enchymal cells in the blastema divide and undergo
differentiation and morphogenesis to replace the
structures lost on amputation. The mechanisms of
urodele regeneration are studied first because they
exemplify fundamental issues such as the basis of
positional identity, and the stability of the differenti-
ated state, and second because they may shed light on
why this ability has been lost in other vertebrates. A
critical aspect of regenerative ability is reflected in
w x
the origin of the precursor cells 3 . Urodeles can
effect local reversals in the differentiated state of
tissues in response to amputation or tissue removal.
For example, in both lens and limb regeneration,
differentiated post-mitotic cells of the iris epithelium
)
Fax: q44 171 878 4040; E-mail: jerbro@ludwig.ucl.ac.uk
or the limb mesenchyme are able to re-enter the cell
cycle and lose their differentiated character. Such
de-differentiation is reversible in that after several
rounds of division the ocular precursor cells or limb
blastema cells arrest and undergo differentiation into
lens or into limb mesenchyme.
The mechanisms underlying the plasticity of dif-
ferentiation in urodeles are not yet understood, and
they are a major concern of this article. It is unclear,
for example, to what extent urodele cells are intrinsi-
cally different from their counterparts in other verte-
brates, and to what extent they encounter distinctive
signals during regeneration. The ability to stage these
episodes of extensive proliferation in the tissues of an
adult animal has obvious parallels and distinctions
with neoplasia, although it has long been recognised
that regenerative ability in urodeles is associated with
striking resistance to tumor formation, particularly in
tissues that show plasticity of the differentiated state.
In the pre-oncogene era, regeneration offered an in-
teresting developmental and evolutionary perspective
on cancer, as illustrated by the suggestion that a
tumor was the result of blastema formation in an
w x
animal that could not regenerate 4 . At present it is
cancer research that offers new insights into the
mechanism of regeneration, although the perspective
of regeneration continues to fascinate. Although tu-
mors seem to arise by mutations in dividing progeni-
tor cells rather than by reversals of differentiation, the
balance between proliferation and differentiation re-
mains central to any discussion of cancer and regen-
0304-419Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.
. PII S0304- 419X 97 00029- 2
( ) J.P. BrockesrBiochimica et Biophysica Acta 1377 1998 M1M11 M2
( ) J.P. BrockesrBiochimica et Biophysica Acta 1377 1998 M1M11 M3
eration. In this article I describe for cancer biologists
the events of lens and limb regeneration, and discuss
the culture-based studies in the two systems that have
helped to clarify the issues of cell cycle re-entry,
de-differentiation and trans-differentiation. In addi-
tion, I discuss a second and possibly related property
of blastemal cells the absence of replicative senes-
cence in culture, as well as the issue of resistance to
carcinogenesis. For a recent review of urodele regen-
eration that encompasses the additional problem of
w x
pattern formation in the regenerate see 5 .
2. Lens regeneration
The two key systems for analysing the mechanism
of de-differentiation in urodeles are the classical
problems of lens and limb regeneration. Regeneration
of the lens proceeds without the complex aspects of
pattern formation seen in the limb, and has the advan-
tage of proceeding through the transitions of a single
.
cell type the iris pigmented epithelial cell PEC
w x
6,7 . After removal of a newt lens, regeneration
proceeds from the dorsal margin of the iris; the new
lens is never observed to arise from the ventral iris.
The dorsal pigmented myoepithelial cells enter the
cell cycle, lose their pigment granules and de-dif-
.
ferentiate Fig. 2 . The entry of PECs into S phase is
w x
maximal at about five days after lentectomy 8 , and
is probably critical for the subsequent events. Some
of the de-differentiated cells subsequently trans-dif-
ferentiate into lens, while others reconstruct the local
architecture of the iris epithelium. The conversion of
newt iris PECs into lens cells was established by
critical experiments with clonal cell culture of pig-
mented cells, when at least 15% of the clonal colonies
examined underwent definitive lens differentiation
w x
9 .
Lens regeneration in the newt does not proceed by
a recapitulation of events involved in lens develop-
ment. The lens regenerates from the iris, and not
from the cornea which is a descendant of the embry-
onic ectoderm from which the lens develops; it is
interesting that in larval Xenopus, lens regeneration
proceeds from the cornea up to the time of metamor-
w x
phosis 10 . Although newts are the only adult verte-
brates that are able to regenerate the lens, the ability
of cultured PECs of iris or retina to de-differentiate
and trans-differentiate into lens cells in culture is
w x
quite widespread under appropriate conditions 11,12 .
These conditions include the use of phenylthiourea to
inhibit melanogenesis, and basic FGF to promote
de-differentiation and trans-differentiation. Thus chick
and even human PECs will trans-differentiate to form
lens cells in culture. These observations might sug-
gest that there is no intrinsic difference in responsive-
ness between urodele ocular epithelial cells and those
of other vertebrates, and furthermore that it is the
local environment of the lentectomised newt iris
which is distinctive. On the other hand the particular
combination of phenylthiourea and FGF-2 is not nec-
essarily indicative of, or simply related to, the stimuli
which normally trigger trans-differentiation in the
dorsal iris. Thus it is possible that in relation to these
latter stimuli there is a significant difference in re-
sponsiveness between ocular epithelial cells of newts
and other vertebrates.
3. Limb regeneration
After amputation of a urodele limb the wound
surface is covered within 24 h by epithelial cells that
.
migrate from the edge of the dermis Fig. 1B . The
resulting wound epidermis, or apical cap, is a tran-
sient secretory epithelium which is important for
regeneration, although the precise identity and role of
its products is still unclear. Cells in the mesenchymal
tissue underlying the wound epidermis enter the cell
cycle and lose their differentiated identity. The result-
. Fig. 1. Regenerative structures at the rostral end of an emperor newt Tylototriton errucosus 1, dorsal crest; 2, limb; 3, retina; 4, lens; 5,
. . upper and lower jaws. B Regeneration of the forelimb in a red-spotted newt Notopthalmus iridescens after amputation at distal
. . w x mid-radius and ulna; shown at left or proximal mid-humerus; shown at right sites 1 . The original limb is shown at the top and the
regenerated limb at the bottom of the series. The photographs were taken at 7, 21, 25, 28, 32, 42 and 70 days after amputation.
( ) J.P. BrockesrBiochimica et Biophysica Acta 1377 1998 M1M11 M4
Fig. 2. Schematic diagram of the events of newt lens regeneration. The top row illustrates the removal of the lens and the growth of the
regenerate from the dorsal margin of the iris. The second row illustrates the events in the dorsal margin at the different stages in the first
row. The pigmented iris cells are initially associated with macrophages, they re-enter the cell cycle, lose their pigment granules, and
trans-differentiate into lens cells.
ing blastemal cells express various markers that dis-
tinguish them from cells in a normal limb and also
w x
from cells in a developing limb bud 1315 . Limb
mesenchyme contains a number of cell types, particu-
larly cartilage, muscle and interstitial fibroblasts, and
although the anatomical descriptions of early stages
of regeneration are consistent with de-differentiation
w x
1618 , it is necessary to introduce a cell marker or
lineage tracer to follow the fate of differentiated cells.
If labelled cartilage is implanted beneath the wound
epidermis, the label is found in monucleate cells of
the blastema and subsequently in connective tissue
and cartilage of the regenerate, but not in muscle
w x
19 . Muscle is a particularly interesting case because
the multinucleate myofiber is a prototypical example
of a post-mitotic differentiated cell, and because there
is considerable information about muscle differentia-
tion and its regulation. Muscle regeneration in higher
vertebrates proceeds by mobilising mononucleate re-
serve or satellite cells that lie beneath the basal
lamina, rather than by reversal of the differentiated
state of the multinucleate myofiber. These issues
have been addressed by study of cultured newt my-
otubes which can be manipulated in culture, and also
w x
implanted into a limb blastema 20 .
When newt blastemal cells are propagated in cul-
ture, they are able to divide for at least two hundred
generations without evidence of crisis or senescence
w x
21 , a feature to be considered later. We have fo-
cussed on a particular isolate called A1 cells which
fuse extensively to form myotubes after lowering the
w x
serum concentration in the medium 20 . The my-
otubes express characteristic markers of muscle dif-
w x
ferentiation such as myosin heavy chain 22 . It is
( ) J.P. BrockesrBiochimica et Biophysica Acta 1377 1998 M1M11 M5
possible to remove most of the mononucleate cells by
sieving the myotubes with nylon mesh, and they can
be plated out at low density prior to microinjection of
isolated multinucleate cells with the lineage tracer,
.
rhodaminelysinedextran Fig. 3A and B . This
reagent is neither taken up by cells from the medium
nor transferred between them, and if the labelled
myotubes are left in culture for three weeks the tracer
is only detected in multinucleate cells. After implan-
tation under the wound epidermis of a limb blastema,
many labelled mononucleate cells are detected in the
.
blastema after 710 days Fig. 3C . The number of
labelled cells is such that a minimum of 1520% of
the implanted nuclei must have undergone a reversal
of the mononucleate to multinucleate transition. If the
labelled myotubes are marked in their nuclei by
incorporation of tritiated thymidine, in addition to the
lineage tracer in the cytoplasm, the mononucleate
progeny in the blastema exhibit both markers. Fur-
thermore, the number of labelled cells increases over
. . Fig. 3. Cultured newt myotube and mononucleate cells seen under A phase contrast optics, and B under fluorescence after intracellular
. microinjection of the lineage tracer rhodaminelysinedextran rld . Such injected multinucleate cells are stable for at least four weeks in
. culture and no labelled mononucleate cells are visible, even in high density culture. C Labelled mononucleate cells in sections of
. regenerating limbs 910 days after implantation of labelled myotubes such as that shown in B . Sections were counterstained with a
. w x DNA stain blue to shown nuclei. Four labelled mononucleate cells are visible. For details see 20 .
( ) J.P. BrockesrBiochimica et Biophysica Acta 1377 1998 M1M11 M6
the next two weeks, hence they are able to divide and
w x
contribute to the blastema 20 . When cellular differ-
entiation is detectable in the regenerate at 34 weeks
post-implantation, labelled cells are detected in mus-
.
cle and, in a few cases, in cartilage Fig. 4AD . The
latter is an interesting result since although trans-dif-
ferentiation of ocular epithelial cells is readily ob-
served both in situ and in culture, it has been much
harder to demonstrate that it occurs in the mesenchy-
mal lineages of the limb arising from the blastema.
The term trans-differentiation is used in this context
to refer to a switch between cartilage and muscle,
rather than to a switch between the lineages of con-
nective and cartilaginous tissues.
These and other experiments suggest that the envi-
ronment of the early blastema is able to destabilise
the differentiated state and promote return to the cell
cycle. In view of the parallels between lens and limb
regeneration in this respect, it is interesting to con-
sider the fate of dorsal iris tissue after transplantation
to a limb blastema. When iris tissue fragments were
transplanted to control sites such as the fin, brain or
normal limb, the epithelial cells retained their pig-
mented identity, but after transplanting to a limb
blastema, a high percentage of the implants formed a
w x
lens 23 . These studies, in conjunction with the
culture experiments on PEC trans-differentiation that
were discussed above, underline the importance of
Fig. 4. Muscle fiber and cartilage cells in sections of regenerating limbs after implantation of labelled myotubes. A rld-labelled muscle
. . fiber is shown at 9 days after implantation under phase contrast optics A , or fluorescence B . Two rld-labelled cells in a differentiating
. . cartilage nodule at 26 days after implantation are shown under phase contrast C and fluorescence D .
( ) J.P. BrockesrBiochimica et Biophysica Acta 1377 1998 M1M11 M7
external signals in promoting re-entry and de-differ-
entiation. Recent work on newt myotubes in culture
has identified an intrinsic difference in responsive-
ness between urodele cells and their mammalian
counterparts.
4. Re-entry to the cell cycle
When A1 myotubes are shifted from low serum
differentiation medium into high serum, the myotube
nuclei re-enter the cell cycle and traverse S phase
. w x
Fig. 5A and B 22 . Entry into S phase is somewhat
asynchronous but )80% of the myotubes will un-
dergo this response. The duration of S phase is
comparable to that in mononucleate A1 cells, and
nuclei with 4N DNA content accumulate stably in the
myotubes without any evidence of cytopathology or
w x
cell death 22 . The cells arrest before mitosis, and
this may reflect the absence in culture of a signal
which is present in the blastema, and which allows
mitosis and the generation of mononucleate cells by
cytokinesis. These results are important first because
they reveal a significant difference between the
urodele cells and those of other vertebrates. Avian
and mammalian myotubes enter a stable state of
. Fig. 5. Newt myotubes re-enter the cell cycle after phosphorylation of the Rb protein. A Two myotubes stained with antibody to muscle
. . . . . myosin green and a DNA stain blue . B The same myotubes showing myosin and bromodeoxyuridine BrdU positive nuclei yellow
. in the myotube on the right. It has entered S phase after serum stimulation whereas the myotube on the left is negative for BrdU. C
. . Profile of Rb phosphorylation in purified myotubes maintained in low serum lane 1 , high serum lane 2 , and in mononucleate cells
. w x lane 3 . The lower band is the hypophosphorylated form and the upper band is the hyperphosphorylated form. For details see 22 .
( ) J.P. BrockesrBiochimica et Biophysica Acta 1377 1998 M1M11 M8
post-mitotic arrest after fusion such that they are
refractory to growth factors that act on their mononu-
w x
cleate precursor, the myoblast 2426 . They can be
induced to enter S phase after transfection with viral
oncogenes such as T antigen that are capable of
.
sequestering the retinoblastoma Rb gene product,
although the subsequent events lead to aberrant mi-
toses and widespread cell death in the myotubes
w x
2729 .
The second significant aspect of serum-induced
re-entry is that it is found in one particular circum-
stance in rodent myotubes. In mice that are homozy-
.
gous null for the Rb gene Rbyry , muscle devel-
opment is apparently normal up to the time when the
embryos die, and when Rbyry myogenic cells are
allowed to fuse in culture they form myotubes ex-
pressing muscle markers. After exposure to high
serum concentrations the mouse myotubes enter S
w x
phase in the same way as the newt A1 cells 30 . This
correspondence points up the potential importance of
the Rb family and its interactions for our understand-
ing of urodele regeneration. It is clear that urodele
myotubes are not genetically Rbyry in that newt
Rb mRNA and protein are expressed in A1 my-
otubes. It is likely, however, that Rb is the end point
of the serum-induced pathway. In mammalian my-

otubes, Rb is found in the hypophosphorylated ac-

.
tive form after analysis by immunoprecipitation and
gel electrophoresis and this is not changed by expo-
sure to serum or other mitogens an index of the
w x
stability of the post-mitotic arrest in these cells 31 .
In newt myotubes the hypophosphorylated form is
found in low serum, but high serum evokes the
.
formation of the hyperphosphorylated inactive form
. w x
Fig. 5C 20 . The activity of Rb in inhibiting the
G1S transition is relieved by its phosphorylation by
cyclin dependent kinases 4r6. These activities are in
turn specifically inhibited by members of the INK 4
INK4
w x
family such as p16 32 . It is therefore interesting
that expression of mammalian p16 blocks serum in-
duced re-entry in the A1 myotubes, suggesting that
w x
Rb might be a target of this pathway 20 . This is
further supported by the finding that expression of
mammalian Rb somewhat inhibits re-entry, and this
inhibition is stronger after expression of a mutated
Rb that is no longer a substrate for phosphorylation
w x
20 . Although Rb might be the endpoint, these exper-
iments leave open the question of what is different
between urodele and mammalian myotubes. If in-
w x
hibitors of cyclin dependent kinases such as p18 33
w x
or p21 34 are critical players in maintaining the
differentiated state, it is possible that down regulation
of their level or activity could undermine the post-
mitotic arrest.
In addition to the phenomenon of re-entry into S
phase, serum-stimulated newt myotubes and mouse
Rbyry myotubes share the property of G2 arrest,
suggesting that an Rb-independent mechanism may
restrict entry of differentiated muscle cells into mito-
w x
sis 35 . In other respects, however, the two cases are
quite different. Loss of Rb in mouse myoblasts or
myo D-transfected fibroblasts leads to incomplete
w x
myogenic differentiation 35 , and other events such
as apoptosis and polyploidy in the animal. In the
Rbyry myotubes it is suggested that the normal
role of Rb in maintaining the post-mitotic arrest is
mediated by the pocket protein p107, and that serum
stimulation leads to down-regulation of this compo-
w x
nent 30 . If so, this is distinct from the mechanism of
action of serum on the urodele cells which has phos-
w x
phorylation of Rb as its end point 22 .
It is interesting that the A1 myotubes are refractory
to the action of growth factors such as PDGF, FGF or
EGF which are all active at stimulating division of
w x
the A1 mononucleate cells 20 . This aspect of the
post-mitotic arrest is therefore intact it is perhaps
.
not surprising that the signal s provoking re-entry
shows some selectivity since it is important that
differentiated tissues manifest plasticity only in the
local context of amputation or injury. In this sense it
is not envisaged that the differentiated state is neces-
sarily different between urodeles and mammals, but
rather that one or more signal transduction pathways
in urodeles are open to permit selective re-entry.
It may be, for example, that the events associated
with wounding or clotting produce a signal that acts
on myotubes and other differentiated cells to trigger
an intracellular pathway leading to phosphorylation
of Rb. Such a signal would be consistent with the
regenerative response in other newt tissues such as
the heart. Removal of part of the atrial or ventricular
wall provokes formation of a clot, and re-entry of
cardiomyocytes into S phase occurs in a zone sur-
w x
rounding the clot 36 . Although this stimulus is
conjectural at present, it is clear that the response of
the myotubes offers new possibilities for analysing
( ) J.P. BrockesrBiochimica et Biophysica Acta 1377 1998 M1M11 M9
the transduction pathways, including the extracellular
signals that provoke de-differentiation.
5. Tumors, cell senescence and regeneration
The potential involvement of tumor suppressor
genes such as Rb in re-entry raises the issue of tumor
formation in urodeles. Most studies on urodeles with
chemical carcinogenesis have concluded that the inci-
dence of tumors is low, particularly after local appli-

cation to tissues during regeneration for review see

w x.
37 . A variety of toxic effects can be observed after
administration to the limb blastema but although
there are several reports of epithelial tumors, tumors
w x
of mesenchymal cells are not observed 3840 . It
has been suggested that the incidence may be higher
in adult anurans, which have lost regenerative ability,
w x
as compared to urodeles 41 . There are a number of
reports where tumors of non-regenerating tissue have
been evoked in urodeles, although the overall inci-
dence is again considered to be relatively low com-
pared to mammals. Although there is a rather exten-
sive literature of experiments of this sort, it is diffi-
cult to evaluate the basis of this resistance, for exam-
ple whether it reflects the metabolism of the chemi-
cals concerned, or some aspect of the response to
oncogene activation. It is interesting in relation to this
latter possibility that in the contexts of both limb and
lens regeneration there is evidence for a significant
incidence of supernumerary regenerates after carcino-
genic treatments. For example application of the alky-
lating carcinogen N-methyl-N
X
-nitro-n-nitroso-
guanidine to lentectomised eyes of the newt evoked
supernumerary lens formation in both the dorsal and
w x
ventral iris 42 . The latter result is particularly note-
worthy since it is never observed in normal lens
.
regeneration Fig. 2 . If the ventral lenses were re-
moved one year later they regenerated successfully,
indicating that the carcinogen had evoked a stable
change in cellular properties. The local exposure of
w x
larval salamander limbs to UV light 43 was able to
induce accessory limb formation, as was local appli-
cation of several chemical carcinogens to the newt
w x
limb 44 . It is possible that oncogene activation or
loss of tumor suppressor function in regenerative
tissue leads to cell cycle re-entry, de-differentiation
and participation in regeneration, rather than to for-
mation of a tumor. This might be related to the
mechanism of normal blastema formation after ampu-
tation. It will certainly be of interest to document the
effects of oncogene expression in urodele cells in
culture, and to examine their behaviour after implan-
tation into a blastema.
It is noteworthy in this context that cultured limb
blastemal cells from the newt do not undergo crisis or
replicative senescence even after more than 200 gen-
w x
erations 21 . Such cells retain expression of blastemal
cell markers and are incorporated into a regenerate
after implantation. The indefinite lifespan in culture
is not a feature of differentiated newt cell types such
as hepatocytes, splenocytes, pneumocytes or car-
diomyocytes, and may be consequent on events asso-
w x
ciated with the formation of blastemal cells 21,45 .
Regeneration, unlike development, is a process that
can occur repeatedly. It is possible to amputate a
newt limb at least 20 times, and to observe regenera-
tion with essentially the same time course on each
occasion. It can be estimated that a single cycle of
regeneration involves 510 cell generations. If the
nucleus of a differentiated mesenchymal cell is re-
cruited to contribute to the blastema on the first
cycle, and its progeny are recruited repeatedly in
subsequent cycles, it is obvious that the absence of
replicative senescence in blastemal cells may be criti-
cal to underpin this ability. Although the basis for the
indefinite lifespan is not known, it is interesting that
S phase entry by the newt myotubes is so strongly
inhibited by ectopic expression of mammalian p16
w x
22 . Loss of p16 in knockout mice leads to absence
w x
of senescence in cultured fibroblasts 46 , and hence
the earlier suggestion that serum stimulation leads to
down regulation of CDK inhibitors such as p16 takes
on added appeal in relation to the immortal pheno-
type.
This consideration is not unique to urodeles since
the ability to sustain multiple episodes of regenera-
tion is found in other contexts. Many invertebrates
have the ability to regenerate the primary body axis,
sometimes in a bidirectional fashion. Certain annelid
species, for example, respond to transection by growth
of a head on the tail fragment, and a tail on the head
w x
fragment 1 . It has often been remarked that this
ability may have arisen in association with asexual
reproduction in some species a single segment can
w x
give rise to secondary individuals 47 . In marine
( ) J.P. BrockesrBiochimica et Biophysica Acta 1377 1998 M1M11 M10
annelids that show a single episode of sexual repro-
duction termed a semelparous life cycle this
episode is associated with the withdrawal of en-
docrine influences which maintain regenerative abil-
w x
ity 48 . The more common iteroparous reproductive
strategy, where episodes of sexual reproduction occur
throughout the lifespan, is associated with continued

regenerative ability. In freshwater triclads Platyhel-

.
minthes such differences between long lived
iteroparous and short lived semelparous forms are
associated with a marked reduction in cell division
w x
and acceleration of senescence 49 . The recent
demonstration of direct entry into the senescent state
after acute expression of the activated ras oncogene
in rodent fibroblasts is most significant in this context
w x
50 , since it illustrates that there is not an obligatory
requirement to complete a certain number of divi-
sions prior to senescence. It is possible, therefore,
that the acute control of cell lifespan originally arose
in relation to the regulation of sexual reproduction
with respect to regeneration and asexual reproduction
in particular at the origin of the semelparous
option rather than as an anti-tumour mechanism.
This speculation illustrates that regeneration contin-
ues to provide a distinctive perspective on cell regula-
tory mechanisms in metazoa.
6. Conclusions
I have outlined two critical aspects of regeneration
as it occurs in urodeles and in other metazoans.
Re-entry to the cell cycle from the differentiated state
ensures a local supply of dividing progenitor cells,
while absence of replicative senescence in these cells
ensures that regeneration can occur repeatedly. It is
provocative that both aspects appear to countermand
mechanisms thought to restrict tumor formation. If
the post-mitotic arrest is undermined in rodent cells,
for example by mutation of Rb or cyclin dependent
kinase inhibitors, then the occurrence of cell cycle
re-entry in differentiated cells leads to apoptosis. An
example of this behavior is seen in lens development
w x
in mutant mice lacking Rb 51 or the CDK inhibitor
w x
p57kip2 52 , when inappropriate S phase entry in
lens fiber cells is associated with an increase in
apoptotic nuclei. Cellular senescence is thought to
prevent the accumulation of multiple mutations re-
quired for tumor formation by restricting the number
of generations that a cell may undergo. Since
blastemal cells have indefinite proliferative potential
yet are resistant to tumor formation, they may employ
alternative means of cell cycle regulation that some-
how by-pass this requirement. It is possible, as sug-
gested above, that if tumorigenic mutations arise, the
cells are somehow constrained within the regulatory
framework of epimorphic regeneration. The way for-
ward, as with many of the issues raised in this article,
is to understand the molecular basis of the distinct
aspects of urodele cell regulation.
Acknowledgements
I thank Professor G. Eguchi of Kumamoto Univer-
sity for Fig. 2, D. Stark and A. Kumar for help with
figures, A. Gann for stimulating discussions on the
subject matter of this article, two of the reviewers for
their helpful suggestions, and the Ludwig Institute for
Cancer Research for their support of my work on
urodele regeneration.
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