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Oncogene (2000) 19, 3278 ± 3289
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Normal and c-Myc-promoted human keratinocyte di€erentiation both
occur via a novel cell cycle involving cellular growth and endoreplication
Alberto Gandarillas*,1, Derek Davies2 and Jean-Marie Blanchard1
1
2
Institut de GeÂneÂtique MoleÂculaire, Centre National de la Recherche Scienti®que (CNRS), F34293 Montpellier Cedex 5, France;
FACS Laboratory, Imperial Cancer Research Fund, London, WC2A 3PX, UK
The relationship between cell cycle and di€erentiation in
human keratinocytes is poorly understood. It is believed
that keratinocytes suppress DNA replication and cell
cycle arrest in G0 before they initiate terminal
di€erentiation. However, a temporal separation between
both events has not been established. Moreover, c-Myc
promotes keratinocyte di€erentiation without causing
cell cycle arrest. To address these paradoxes we have
analysed cell cycle control during normal and c-Mycpromoted di€erentiation. Continuous activation of c-Myc
or initiation of terminal di€erentiation results in a block
of G2/M, cellular growth, endoreplication and polyploidy. Keratinocytes abandon G1, continue replicating
DNA as they di€erentiate terminally and become
polyploid. In fact, simply blocking mitosis with nocodazole resulted in increased cell size, terminal di€erentiation and endoreplication. This indicates that terminal
di€erentiation associates with defective cell cycle progression and provides a novel insight into c-Myc biology.
Oncogene (2000) 19, 3278 ± 3289.
Keywords: epidermis; mitosis; cell size; ¯ow cytometry;
cell fate; endoreduplication
Introduction
Human epidermis is a strati®ed epithelium in which
proliferation and terminal di€erentiation are tightly
compartmentalised. Within the basal layer continuous
proliferation throughout adult life accounts for the loss
of cells that shed as squames from the surface of skin
(Watt, 1989; Fuchs and Byrne, 1994). A constant
balance between both processes is crucial to maintain
the normal structure of epidermis and its alteration
results in skin diseases. Proliferation is sustained by
interaction with the basement membrane via integrins
(Watt and Jones, 1993) and keratinocytes undergo
terminal di€erentiation when this interaction is interrupted, unlike endothelial or simple epithelial cells that
undergo apoptosis in suspension (Adams and Watt,
1989; Frisch and Francis, 1994; Gandarillas et al.,
1999; reviewed in Gandarillas, 2000). As keratinocytes
cease proliferation and migrate into the suprabasal
layers they switch from a cytoskeleton formed by
keratins K5 and K14 to another one formed by
keratins K1 and K10. Concomitantly, they enlarge
and initiate the expression of precursors and other
components of the corni®ed envelope, such as
*Correspondence: A Gandarillas
Received 10 January 2000; revised 4 April 2000; accepted 12 April
2000
involucrin, loricrin and ®laggrin (Watt and Green,
1981; Fuchs and Byrne, 1994).
Within the basal layer, epidermal stem cells are
mainly quiescent but have an unlimited potential to
self-renewal (Lavker and Sun, 1983; Hall and Watt,
1989). After division their progeny stays as stem cells
or enter an intermediate state, the transit amplifying
cell, which proliferates continuously but undergoes
terminal di€erentiation after a small number of cell
divisions. Although little is known about the control of
the keratinocyte cell cycle, basal keratinocytes are
believed to withdraw from the cell cycle, switch o€
DNA synthesis and arrest in G0 before they initiate
terminal di€erentiation (Fuchs, 1993; Gandarillas and
Watt, 1995; Hurlin et al., 1995; Hauser et al., 1997;
Harvat et al., 1998). However, some ®ndings do not
satisfy this model. Dover and Watt (1987) found no
temporal separation between proliferation and terminal
di€erentiation in culture. Similarly, a subpopulation of
epidermal keratinocytes has been found to express
terminal di€erentiation markers and yet undergo
S phase (Regnier et al., 1986; Bata-Csorgo et al.,
1993; Dover and Watt, 1987). Suprabasal mitotic
®gures or thymidine incorporation have also been
reported (Pinkus and Hunter, 1966; Penneys et al.,
1970).
Analyses of cell cycle regulators in keratinocytes
have also provided confusing results. Cell cycle
inhibitors P16, P21, P27 have been proposed to have
a role in keratinocyte di€erentiation (Missero et al.,
1996; Hauser et al., 1997). However, although
constitutive expression of these molecules in keratinocytes causes cell cycle arrest, terminal di€erentiation is
not stimulated (Harvat et al., 1998; Di Cunto et al.,
1998). Similarly, inhibition of cell cycle by c-myc
antisense oligonucleotides or TGFb does not have a
positive e€ect on keratinocyte terminal di€erentiation
(Reiss and Sartorelli, 1987; Pietenpol et al., 1990;
Hashiro et al., 1991).
A further paradoxical line of evidence has arisen
from our recent study of the role of c-Myc in
epidermal di€erentiation (Gandarillas and Watt,
1997). c-Myc promotes proliferation and apoptosis in
a variety of cell types and is frequently ampli®ed in
human malignancies (Henriksson and LuÈscher, 1996).
However, the precise mechanisms by which c-Myc
performs its functions are unclear. c-Myc promotes
entry of cells in S phase, possibly through inducing
positive regulators of cell cycle (Amati et al., 1998).
Recently, it has been suggested that c-Myc may induce
the expression of molecules involved in protein
synthesis and cellular growth (see Grandori and
Eisenman, 1997; Dang, 1999). Endogenous c-Myc is
hardly detectable in epidermis, and it is down-regulated
c-Myc and endoreplication in keratinocyte differentiation
A Gandarillas et al
at late stages of in vitro-induced keratinocyte di€erentiation (see Gandarillas and Watt, 1997). We
constitutively expressed wild type or conditional cMyc in all layers of primary keratinocytes by targeting
stem cells by retroviral infection (Gandarillas and
Watt, 1997). In both cases, and unexpectedly, activation of c-Myc for more than 5 days did not stimulate
proliferation or apoptosis but di€erentiation. c-Myc
®rst drove keratinocytes to leave the stem cell
compartment and become transit amplifying cells,
which subsequently initiated terminal di€erentiation
after four to ®ve rounds of cell division. This increase
of terminal di€erentiation, however, did not associate
with a DNA replication arrest.
Aiming to solve these paradoxes and to understand
this novel function of c-Myc, we have investigated the
relationship between cell cycle and di€erentiation in
human primary, normal or c-Myc expressing keratinocytes. The application of ¯ow-cytometry techniques to
keratinocytes (Nakatani et al., 1992; Bata-Csorgo et
al., 1993; Jones and Watt, 1993; Gandarillas and Watt,
1997; Harvat et al., 1998) has provided a powerful tool
to study these processes in detail. The results
consistently show that the initiation of keratinocyte
terminal di€erentiation precedes cell cycle arrest,
resulting in cycles of DNA replication without
completion of mitosis. This phenomenon has been
referred to as endoreplication or endoreduplication in
other biological systems (Gra®, 1998; Traas et al.,
1998; Hawkins et al., 1996) including some human
tissues (Kirk and Clingan, 1980; Ho€man, 1989; Jack
et al., 1990; Solari et al., 1996). Continuous activation
of c-Myc stimulated keratinocyte endoreplication and
produced an increase of cell size, as a result of a
blockade in mitosis. The results reveal a novel
relationship between keratinocyte cell cycle and
di€erentiation and provide new insights into the
biological consequences of c-Myc activation.
Dmyc106-143 that lacks the transactivation domain of
c-Myc; Littlewood et al., 1995; Gandarillas and Watt,
1997). Primary keratinocytes were cultured in the
presence of a feeder layer of mouse ®broblasts and
1.5 mM calcium. In such conditions keratinocytes
proliferate and stratify, mimicking epidermis in vivo
(Rheinwald, 1989; Watt, 1989). c-mycER was activated
in primary keratinocytes upon 4-OH-tamoxifen (OHT)
for 9 days and cells double stained for the terminal
di€erentiation marker involucrin and DNA content.
The cell cycle distribution of involucrin positive or
negative cells was then quantitated by ¯ow-cytometry
(Table 1). Terminally di€erentiating keratinocytes
expressing activated c-mycER were found in every
phase of the cell cycle, and a signi®cant proportion of
involucrin positive cells were undergoing S phase. The
numbers however, were similar in di€erentiating and
non di€erentiating cells expressing the empty vector
puro-resistance (KpBabe) and in cells expressing the
negative myc mutant (K106; Table 1). Therefore, the
presence of di€erentiating keratinocytes in S phase was
not speci®c of c-Myc activation. There was, however, a
slight accumulation of c-Myc di€erentiating cells in
G2/M.
Keratinocytes can be induced to terminally di€erentiate when they are placed in suspension in the
absence of cell adhesion. When normal, control
keratinocytes expressing the empty vector were induced
to terminally di€erentiate in suspension for 24 h, they
were found to contain a proportion of cells in S phase
(9%, Table 1) and to accumulate in G1 (62.8%) or G2
(26.0%). Interestingly, during 24 h in suspension a
proportion of pBabe keratinocytes abandoned G1 and
completed S phase to accumulate in G2 (total cells in
G1 decreased from 75.1% to 62.8%; total cells in
S phase from 11.3% to 9%; total cells in G2+M
increased from 12.2% to 26.0%).
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Normal and Myc differentiating keratinocytes continue
DNA synthesis and become polyploid
Results
Differentiating normal and Myc keratinocytes are found
in any phase of the cell cycle
Both the ratio of DNA synthesis and the proportion of
cells in S phase were only slightly decreased during cMyc-promoted terminal di€erentiation (Gandarillas
and Watt, 1997). We have explored whether constitutive activity of c-Myc stimulated the cell cycle of
terminally di€erentiating cells. For this purpose we
have made use of two conditional mycER fusion
proteins and constitutively expressed them in keratinocytes by retroviral infection: c-mycER (containing wildtype c-Myc) or 106ER (containing deletion mutant
Continuous activation of c-mycER in keratinocytes
drives stem cells to become transit amplifying cells and
leave the proliferative compartment after 5 days
(Gandarillas and Watt, 1997), when they initiate
terminal di€erentiation. At this stage bi- or tri-nucleate
cells were frequently observed (Figure 1a). Confocal
analyses of c-Myc staining showed stratifying, di€erentiating keratinocytes to often contain more than one
nucleus or a large single nucleus (Figure 1b). Multinucleate di€erentiating keratinocytes were also found
in normal cultures (Figure 1a) but not as frequently.
Multinucleate cells must be a consequence of a
defective mitosis that results in polyploidy. To
investigate this possibility, we analysed normal,
Table 1 Cell cycle distribution of terminally di€erentiating or non-di€erentiating keratinocytes as quantitated by ¯ow-cytometry
Invol7
G1
S
G2+M
75.4 (0.1)
11.8 (0.1)
11.9 (0.3)
KpBabe
Invol+19.3 (0.4)
74.4 (1.3)
10.8 (0.5)
13.7 (0.6)
Invol7
72.7 (0.4)
16.1 (0.5)
10.1 (0.0)
K106ER
Invol+17.1 (0.6)
70.8 (1.3)
16.0 (1.0)
10.8 (0.3)
Invol7
77.6 (1.8)
12.4 (0.8)
9.2 (1.1)
KmycER
Invol+50.5 (1.5)
71.2 (1.8)
11.6 (0.4)
16.3 (1.4)
Ksusp
Total+55.4 (1.2)
62.8 (1.3)
9.0 (0.4)
26.0 (1.2)
Numbers next to Invol+ are the percentage of involucrin positive cells within the whole population. Numbers in table are percentages of cells in
the di€erent phases of the cell cycle with respect to the involucrin positive or negative subpopulations. Data are means of triplicate samples;
s.e.m. are shown in parentheses. Ksusp: Cells in suspension for 24 h. All other cells were OHT-treated for 9 days
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Figure 1 Multinucleation in c-Myc expressing or normal
primary human keratinocytes. (a) Top and middle panels: cultures
of mycER-expressing keratinocytes in the absence of OHT (top
panel) or 5 days after activation of c-mycER with OHT (middle
panel; some of the frequent binucleate cells are highlighted with
arrows). Bottom panel: normal culture of primary keratinocytes
containing some large, terminally di€erentiating, multinucleate
cells (arrows). Bar: 200 mm. (b) Con¯uent keratinocytes expressing
mycER that had been activated with OHT for 5 days were stained
for c-Myc and analysed by confocal microscopy. Bottom panel
focuses on the top of the basal layer (B) and ®rst suprabasal layer
(S1) upper panels show two suprabasal layers (S2, S3) containing
tri-nucleate cells (arrows) and a gigantic nucleus (arrowhead).
Sections: 0.3 mm. Bar: 40 mm
stratifying cultures of primary keratinocytes by ¯owcytometry, after staining DNA with propidium iodide
and without gating out events beyond G2/M (Figure
2). Cell aggregates were excluded on basis of the
Width/Area (Figure 2a; see e.g., Ormerod, 1990). We
have found that normal, stratifying cultures of primary
human keratinocytes contain around 8% of polyploid
cells, with a slight variation depending on the
keratinocyte strain (Figure 2a). A peak of cells with
8 N DNA content and a small proportion of cells with
12 N DNA content were also observed. Events beyond
4 N were sorted and visualized with a conventional
¯uorescence microscope to con®rm that they were
single polyploid cells (see below). Moreover, cell
suspensions that had been stained for DNA were
placed on slides and analysed with a Laser Scanning
Cytometer, which detected a signi®cant proportion of
single, polyploid nuclei (see below).
As keratinocytes terminally di€erentiate and stratify
they progressively increase their cell size (BanksSchlegel and Green, 1981) and it is possible to sort
them on basis of their Side Scatter pro®le (Jones and
Watt, 1993). Polyploid keratinocytes were restricted to
the suprabasal population (Figure 2b). Parallel samples
were double stained for DNA and the terminal
di€erentiation marker involucrin. The DNA content
of involucrin negative or positive cells was determined
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(Figure 2b, third panel). Polyploid cells were restricted
to the involucrin positive population. At a given time,
they constituted more than a fourth of terminally
di€erentiating cells (26.3% on basis of light scatter;
29.3(+1.6%) on basis of involucrin staining; Figure
2a,b). Therefore polyploidy was not a random
consequence of the conditions in culture, but it was
tightly associated with terminal di€erentiation. This
suggests that di€erentiating keratinocytes undergo
endoreplication (cycles of DNA replication in the
absence of mitosis; see Introduction). Interestingly,
keratinocyte size correlated with DNA content, as
observed in endoreplicating tissues (Kirk and Clingan,
1980; Traas et al., 1998). Keratinocytes in the di€erent
phases of the cell cycle could be identi®ed on basis of
their light scatter properties (three colours in Figure
2a). Finally, the majority of very large cells, that are at
a late stage of terminal di€erentiation (Banks-Schlegel
and Green, 1981; Watt and Green, 1981) were
polyploid (red dots in Figure 2a,b), and only a small
proportion were in G1 (green dots in Figure 2a,b).
To con®rm that di€erentiating keratinocytes are able
to synthesise DNA, stratifying cultures were incubated
with BrdU, for 2.5 h, and double stained for DNA
content and BrdU (Figure 2c). BrdU incorporation in
basal or terminally di€erentiating cells was then
determined by means of physical parameters as in
Figure 2a,b. Although slightly more DNA-synthesising
cells were found within the basal, non-di€erentiating
compartment (23.5%; Figure 2c) a similar proportion
of diploid and polyploid terminally di€erentiating cells
were positive for DNA synthesis (21.3%; Figure 2c).
23.1% of DNA synthesising cells were suprabasal (on
basis of light scatter; Figure 2c), compared to 22.2%
suprabasal cells in the whole population (Figure 2a).
These observations suggest that the capacity of
di€erentiating cells to replicate DNA is not signi®cantly lower than that of basal cells. It is also worth
noting that BrdU or thymidine incorporation techniques are misleading to estimate keratinocyte proliferation, unless only the basal population is considered.
Keratinocytes thus become polyploid and occasionally multinucleate as they undergo terminal di€erentiation. Confocal analyses of double staining for DNA
and a terminal di€erentiation marker that is expressed
at the plasma membrane (CD44-V3; Hudson et al.,
1996), showed multiple simultaneous anaphases in
single, strati®ed keratinocytes (Figure 3a). Simultaneous multiple anaphases occur in some multinucleate
cells undergoing endomitosis (see e.g. Kirk and Clingan, 1980). To monitor this phenomenon in live cells,
activated-c-mycER or normal keratinocytes were
recorded for 4 days using time-lapse video microscopy.
These ®lms showed stratifying, di€erentiating keratinocytes undergoing nuclear divisions in the absence of
cell division (Figure 3b). This phenomenon was also
observed in normal cultures (not shown) but was more
frequent in c-mycER cells after activating c-Myc for 5
days (Figure 3b).
Induction of terminal differentiation and c-Myc action
both trigger keratinocyte endoreplication
To explore whether endoreplication is a direct consequence of terminal di€erentiation, we quantitated
polyploid cells after stimulating terminal di€erentiation
c-Myc and endoreplication in keratinocyte differentiation
A Gandarillas et al
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Figure 2 Flow-cytometry analyses of cell cycle and di€erentiation in primary human keratinocytes. (a,b) Single cell suspensions
were ®xed, stained for DNA with propidium iodide (PI; FL2) or double stained for DNA and involucrin. Aggregates were excluded
on basis of PI Area/Width (a). Light scatter (SSC-H and FSC-H) re¯ects cell size and complexity. Non-di€erentiating or
di€erentiating keratinocytes were gated on basis of the light scatter dot plot and their DNA content determined (R2 or R3 in a,b).
Third panel in b plots Side Scatter (SSC) versus involucrin expression (FITC). Numbers show the quantitation of every region with
respect to the number of cells within each plot. Three colours in third panels of a,b: green dots are total cells in G1 (M1 in a), blue
dots total cells in S+G2/M (M4 in a) and red dots polyploid cells (M3 in a). M2: cells in G2/M. (c) Brdu incorporation of primary
keratinocytes over 2.5 h to analyse DNA synthesis. Cells in R3 are positive for BrdU as determined with non-BrdU control samples.
BrdU incorporation of basal or suprabasal cells was determined on basis of light scatter as in a,b. The far-right panel shows the
light scatter properties of cells that incorporated BrdU. Data are from multiple samples of three di€erent strains of foreskin
keratinocytes. Numbers in brackets are the s.e.m
in normal keratinocytes in suspension or upon constitutive activation of c-Myc. As shown in Figure 4a ± c,
the proportion of polyploid cells increased signi®cantly
in keratinocytes that were suspended for 14 h (8.2 ±
16.4%), concomitantly with the increase of involucrin
expression (insets in Figure 4a ± c). While G2 and
polyploidy increased, G1 and S phases decreased, as
observed previously (Table 1), despite the fact that after
14 h cells had no longer the capacity to proliferate when
replated (not shown). After 24 h polyploidy was
reduced (13.4%), possibly as a consequence of DNA
degradation that takes place at late stages of terminal
di€erentiation. BrdU incorporation analyses in suspended cells indicated that DNA synthesis continued
after 6 h, when cells are irreversibly committed to
terminal di€erentiation (Adams and Watt, 1989) and up
to 24 h (Figure 4e). After 24 h DNA synthesis is greatly
reduced (not shown) possibly due to the lack of
anchorage. Promotion of terminal di€erentiation after
7 days of activating c-Myc did not suppress DNA
synthesis either (Figure 4f), and provoked an increase in
polyploidy that tightly correlated with the degree of
terminal di€erentiation (Figure 4g). It must be noted
that after activation of c-Myc for 7 days a higher
proportion of DNA synthesizing cells were in G1 and
S phases, as compared to the suspension situation. This
is due to the fact that suspension-induced di€erentiation
is a terminal process, whereas a proportion of c-Myc
cells in stratifying cultures continue to proliferate and
shed into the culture medium as they undergo late
stages of terminal di€erentiation (see Gandarillas and
Watt, 1997).
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3282
Figure 3 Nuclear division (endomitosis) in endoreplicating, di€erentiating primary keratinocytes. (a) multiple simultaneous
anaphases in a di€erentiating keratinocyte as revealed by confocal analyses of double staining for DNA (propidium iodide; left
panels) and a variant of CD44 that is speci®cally expressed during terminal di€erentiation (CD44V3; right panels). Lower panels
focus on a suprabasal layer (S); upper panels focus on a rounded-up cell within the same layer; broken lines highlight the edge of
the cell and straight lines the position of the three endomitotic spindles. (B) fainter, out of focus basal nuclei are visible; (S)
suprabasal nuclei; sections of 0.2 mm; bar: 20 mm. (b) six consecutive sequences of time-lapse ®lming of primary cultured
keratinocytes. The di€erentiating cell pointed-out with arrows goes from two to four nuclei during the 26 min of the sequence and
its edge has been stressed with black broken lines in sequence 6. Bar: 160 mm
Events with more than 4 N DNA content from the
DNA stainings in Figure 2a,b were sorted on slides
and visualized to con®rm that they were single,
polyploid cells (Figure 5a ± c). Note the presence of
three nuclei in a cell that begun to lose the involucrin
epitope (Figure 5b; involucrin is undetectable when it is
incorporated into the corni®ed envelope). Polyploid
cells often had a single, very bright gigantic polyploid
nucleus (Figure 5b,c). Very large cells and nuclei were
frequently observed when keratinocytes were placed in
suspension to induce terminal di€erentiation (Figure
5d,e). Figure 5f shows individual polyploid cells as
identi®ed using a Laser Scanning Cytometer to analyse
normal keratinocytes stained for DNA and placed and
mounted on slides. Note that either big individual
nuclei, binucleate cells (arrow-heads) or multinucleate
cells (arrows) were found within the polyploid fraction.
Blocking keratinocyte mitosis triggers terminal
differentiation and endoreplication
The accumulation of di€erentiating keratinocytes in G2/
M and the presence of endoreplication in suprabasal cells
indicate that a mitosis blockade associates with terminal
di€erentiation. To further test this association, we
studied it in an inverse fashion: we blocked keratinocyte
mitosis continuously with nocodazole and determined
the e€ect on di€erentiation and endoreplication. Primary
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keratinocytes were hard to synchronise completely, as
they are dicult to growth arrest in the absence of serum
and growth factors (Watt et al., 1991; unpublished
observations). DNA synthesis continued in the presence
of nocodazole (compare BrdU incorporation in Figure
6e with untreated control cells in Figure 6a), consistent
with observations that a nocodazole block still permits
DNA synthesis in some cells (Kung et al., 1990).
Nevertheless, a 48 h treatment with nocodazole provoked an accumulation in G2/M, an increase in
polyploidy (compare M2 and M1, respectively, in
`DNA' Figure 6a,b) and interestingly, a dramatic
increase of cell-size (`scatter' in Figure 6a,b) and terminal
di€erentiation (involucrin expression; Figure 6a,b).
After nocozadol treatment peaks beyond 4 N were more
evident, likely due to a higher degree of synchronisation.
The morphology of cells treated with nocodazole was
also indicative of an increased strati®cation and shedding
(compare Figure 6d with c showing con¯uent areas), a
great increase of cell size and premature terminal
di€erentiation in the basal layer (compare non-con¯uent
areas in Figure 6e and f).
Induction of differentiation, activation of c-Myc or a
mitosis block, all provoke an increase of cellular size
Terminal di€erentiation associates with an increase of
cell size (Banks-Schlegel and Green, 1981; Watt and
c-Myc and endoreplication in keratinocyte differentiation
A Gandarillas et al
3283
Figure 4 DNA content and DNA synthesis in primary keratinocytes during suspension- or c-Myc-promoted terminal
di€erentiation. (a ± c) involucrin expression and DNA content of primary keratinocytes before (a) or after 14 h (b) or 24 h (c) in
suspension was analysed by ¯ow-cytometry. Small insets show involucrin staining and a negative control antibody staining (human
CD8; broken line; a). Percentages show the proportion of cells in G2/M phase (R3) or polyploidy (R4); numbers are means of three
independent experiments and standard deviations are shown in brackets. (d ± f) keratinocytes placed in suspension for 24 h were
incubated with no BrdU (d) or with BrdU for the last 18 h (e). Note that adherent keratinocytes were incubated with BrdU for 1.5 h
after activation of c-Myc for 7 days (f). (g) show quantitations of involucrin positive cells (left panel) or polyploid cells (right panel)
in primary keratinocytes expressing the constructs indicated, after addition of OHT for the times indicated. Data in g are
representative of three independent experiments; small bars are the s.e.m
Green, 1981; see also Figure 2a,b). In addition,
polyploidy has been found associated with larger nuclei
and cells in endoreplicating systems (Traas et al., 1998;
Edgar, 1995; Ho€man, 1989; Jack et al., 1990). We
have observed that keratinocytes become larger after
activation of c-Myc in conventional cultures and in
vitro reconstituted epidermis (Gandarillas and Watt,
1997). We determined the relationship between DNA
content, terminal di€erentiation and cell size in
keratinocytes (Figure 7). Suspension-induced di€erentiation, nocodazole treatment or activation of c-Myc,
all provoked an accumulation of keratinocytes in G2/
M and a signi®cant increase of cell size. This increase
also took place in G1 cells, but was more marked in
G2/M and polyploid cells (Figure 7a ± c). Consistently,
a 48 h treatment with hydroxy-urea (HU) that blocked
keratinocytes in G1/S and reduced polyploidy, caused
a limited increase of cell size compared to that
provoked by nocodazole. In contrast, a 48 h TGF-b
treatment, that inhibits the G1/S transition, proliferation and c-Myc expression in keratinocytes (see e.g.
Pietenpol et al., 1990), did not cause a signi®cant
increase of cell size (not shown). Due to the conditional
ER fusion protein we could monitor the kinetics of the
e€ect of c-Myc on cell size (Figure 7d,e). This e€ect
was detected even 24 h after activation of c-mycER,
when all other di€erentiation markers were still
una€ected (Figure 7e; Gandarillas and Watt, 1997).
Discussion
Differentiating keratinocytes initiate terminal
differentiation from any point in the cell cycle and
undergo endoreplication
Epidermal keratinocytes have been believed to withdraw from the cell cycle and arrest in G0 before
initiating terminal di€erentiation. However, we have
observed a similar cell cycle distribution in di€erentiating and non-di€erentiated keratinocytes, even shortly
after inducing terminal di€erentiation in suspension. In
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Figure 5 Polyploid cells and nuclei in keratinocyte primary cultures. (a ± c) photographs of polyploid keratinocytes sorted by ¯owcytometry from the double staining involucrin (green)/DNA (red) in Figure 2b. Note multinucleate cells in (a arrowheads), a cell
with three nuclei in (b) and a gigantic, very bright single nucleus in (c). (d,e) double staining involucrin (green)/DNA (red) of
primary keratinocytes that were induced to di€erentiate in suspension for 24 h; note the large size of cell and nucleus and the
presence of two nuclei in some very di€erentiated cells. (f) single polyploid events were identi®ed, after staining DNA as in Figure
2a, by a Laser Scanning Cytometer on cell suspensions that were placed on slides. These events were single cells with either a single
big nucleus, two nuclei (arrowheads) or several nuclei (arrows). Bar: (a) 100 mm; (b ± f) 20 mm
addition, the proportion of cells in G1 was signi®cantly
reduced and the proportion in G2/M increased, in the
onset of di€erentiation. It must thus be concluded that
keratinocyte terminal di€erentiation initiates from any
point within the cell cycle, con®rming previous
suggestions in a similar direction (Dover and Watt,
1987; Kartasova et al., 1992; Nakatani et al., 1992;
Harvat et al., 1998). Moreover, terminally di€erentiating cells continued DNA synthesis and we have found
a signi®cant proportion of keratinocytes that are
polyploid and restricted to the suprabasal population.
Keratinocyte cell cycle and terminal di€erentiation
are therefore uncoupled and this results in endoreplication: extra rounds of DNA replication in the absence
of cell division. Some polyploid cells undergo nuclear
division (endomitosis), and become multinucleate, as
observed in other endoreplicating systems (e.g. Kirk
and Clingan, 1980; Jack et al., 1990). More than a
fourth of the suprabasal population was polyploid at a
given time and about a third was in G2/M or G1. It is
technically complex to determine whether all di€erentiating cells eventually abandon G1, but the
diminution of the G1 peak as terminal di€erentiation
progresses suggests that they tend to do so. A
proportion of Keratinocytes, however, accumulated in
G1 after 24 h in complete absence of cell adhesion, but
DNA synthesis was also suppressed in that situation.
Oncogene
Di€erentiating keratinocytes may or may not leave G1
depending on time and/or molecular signals, but it is
clear that a G1 or G0 arrest is not required for
initiation of terminal di€erentiation. The results
presented here indicate that terminal di€erentiation
tends to associate with mitosis-defective cell cycle
progression, rather than with arrest in G0. That was
the case during spontaneous or suspension-induced
di€erentiation, upon activation of c-Myc or after
blocking mitosis with nocodazole.
Mechanisms of keratinocyte endoreplication and c-Myc
function
The molecular mechanisms that suppress cell division
and trigger endoreplication in the onset of terminal
di€erentiation are to be elucidated. When proliferating
basal cells detach from the basement membrane they
produce the di€erentiation cytoskeleton and subsequently, the corni®ed envelope (Fuchs and Byrne,
1994). Our model is that this new cytoskeleton might
be too rigid and create a physical constraint that would
prevent the cell from splitting up even when the DNA
replication machinery is still functional. Time-lapse
video recording showed di€erentiating cells initiating
cell division, but failing to perform cytokinesis. Several
lines of evidence support this model: (a) in our
c-Myc and endoreplication in keratinocyte differentiation
A Gandarillas et al
3285
Figure 6 E€ects of blocking mitosis with nocodazole on keratinocyte cell cycle and di€erentiation. Untreated, control
keratinocytes (a) or keratinocytes that had been treated with nocodazole for 48 h (b), were analysed for DNA synthesis (BrdU:
FL1), DNA content (Propidium Iodide), light Scatter (SSC/FSC) and involucrin expression. Numbers show the proportions of
BrdU positive cells (R2), cells in G2/M (M2) or polyploid (M1) and are means of three independent experiments. S.e.m. are shown
in brackets. (c ± f) morphology of untreated cells (c,e) or those treated with nocodazol (d,f). (c,d) show cells in con¯uent areas; (e,f)
show individual colonies. Note the massive strati®cation at con¯uence in d and the large size of cells in f compared to control
keratinocytes. Bar: 200 mm
experiments suppression of mitosis by nocodazole
mimicked the cell cycle distribution of spontaneously
di€erentiating cells, and triggered terminal di€erentiation and endoreplication; (b) constitutive expression of
the di€erentiation cytoskeleton keratins K1 or K10 in
keratinocyte cell lines does not sustain proliferation
and yet allows DNA synthesis (Kartasova et al., 1992;
Paramio et al., 1999) and (c) interestingly, the skin of
transgenic mice expressing K10 under the K5 promoter
(speci®cally expressed in the basal layer) consists of a
single layer of ¯attened keratinocytes (JL Jorcano,
personal communication).
Endoreplication thus explains why expression of
di€erentiation keratins or corni®ed envelope precursors
and factors that promote di€erentiation, either in
culture (Kartasova et al., 1992; Gandarillas and Watt,
1997) or in vivo (Penneys et al., 1970; Morasso et al.,
1996) coexist with DNA synthesis. It may also explain
why inhibition of the G1/S transition in keratinocytes
by anti-c-myc antisense oligonucleotides, TGFb (Reiss
and Sartorelli, 1987; Pietenpol et al., 1990; Hashiro et
al., 1991), overexpression of cell cycle inhibitors P16,
P21 or P27 (Harvat et al., 1998; Di Cunto et al., 1998)
or inhibition of S phase kinase cdk2 (Ben-Bassat et al.,
1997; Martinez et al., 1999) did not result in an
increased terminal di€erentiation.
Furthermore, in the light of endoreplication, c-Mycpromoted keratinocyte di€erentiation appears compatible with its known role in driving cell cycle
progression. Constitutive activation of c-Myc produced
an increase of endoreplication that associated with
increased cell size and di€erentiation. In keratinocytes,
endoreplication masked the e€ect of c-Myc on cell
cycle when the whole population was considered
(Gandarillas and Watt, 1997). However, we have
observed a transient increase of BrdU incorporation
24 h after activating c-Myc, when only basal cells were
analysed (unpublished observations). Continuous stimulation of cell cycle may ®rst drive stem cells to
become transit amplifying cells and eventually, trigger
terminal di€erentiation and a G2/M blockade while
DNA replication continues. The correct checkpoints in
G2/M would then be lost in the onset of di€erentiation. Interestingly, the phenotype caused by a 48 h
nocodazole treatment was very similar to that observed
after activation of c-Myc for 5 days (Figure 6d,f;
Oncogene
c-Myc and endoreplication in keratinocyte differentiation
A Gandarillas et al
3286
Figure 7 Analyses of cell size and DNA content in human keratinocytes after suspension-induced di€erentiation, continuous
activation of c-Myc or blocking cell cycle. (a ± c) plot Side Scatter (SSC) versus DNA (PI) of normal primary keratinocytes after
24 h in suspension (a), activation of c-mycER upon OHT for 6 days (c-myc+) or a treatment with either nocodazole or hydroxyurea for 48 h (c). Parallel controls of untreated, adherent, normal primary keratinocytes are also shown. In b the control is c-mycER
expressing cells cultured in the absence of OHT. Numbers are the per cents of cells with a high SSC value (R1); in brackets are
s.e.m. of three independent experiments. (d) comparison of Side Scatter levels of primary keratinocytes expressing empty vector
pBabe, inactive c-myc mutant 106ER or c-mycER cultured in the presence of tamoxifen (OHT) for 9 days; (e) similar analyses in cmycER keratinocytes before or after activating c-Myc with OHT for the number of days indicated
Gandarillas and Watt, 1997). Four very recent reports,
together with our results, suggest that continuous
activity of c-Myc may uncouple cell growth and cell
division: (a) overexpression of c-Myc promoted endoreplication in ®broblasts and epithelial cells that
were arrested in metaphase (Li and Dang, 1999); (b) dmyc, the drosophila homologue of c-Myc, stimulated
cellular growth rather than proliferation, due to mitosis
being independently controlled (Johnston et al., 1999)
and (c) continuous c-Myc activity provoked an increase
in protein synthesis and cell size in a B-cell line and
during lymphocyte development of transgenic mice
when stimulating cell growth but not cell division
(Iritani and Eisenman, 1999; Schuhmacher et al., 1999).
By driving cellular growth and cell cycle progression,
c-Myc may promote proliferation when mitosis is
Oncogene
correctly executed, but di€erentiation and endoreplication when mitosis is impaired (see also Iritani and
Eissenman, 1999; Schuhmacher et al., 1999). It is worth
noting that c-Myc activity caused both polyploidy and
apoptosis in Rat-1 cells in the absence of mitosis (Li
and Dang, 1999), and that terminal di€erentiation in
keratinocytes may be the counterpart of apoptosis in
other cell types (reviewed in Gandarillas, 2000).
Endogenous c-Myc is hardly detectable in epidermis
and is down-regulated during in vitro-induced terminal
di€erentiation (see Gandarillas and Watt, 1997). A
sustained increase of c-Myc function in individual stem
cells may drive them into the actively proliferative
compartment. This may be sucient to commit these
cells to subsequent di€erentiation, when c-Myc activity
would no longer be required (see Gandarillas and
c-Myc and endoreplication in keratinocyte differentiation
A Gandarillas et al
Watt, 1997). Interestingly, constitutive activity of cMyc in mouse transgenic epidermis provoked hyperproliferation and hyperkeratosis (thickening of di€erentiating layers), occasional nuclear division in
di€erentiating cells and angiogenesis, but not apoptosis
(Pelengaris et al., 1999; Waikel et al., 1999). Mitogenic
soluble factors produced in vivo during vascularisation,
in¯ammation or tumorigenesis may favour mycinduced proliferation by alleviating the mitosis blockade.
What mechanisms link a G2/M blockade with
initiation of terminal di€erentiation? Successive cycles
of DNA replication in the absence of mitosis are known
to allow cells to become bigger (see e.g. Traas et al., 1998;
HuÈlskamp et al., 1999) and we have found that
keratinocyte terminal di€erentiation correlates with
DNA content. In our experiments, the increase of cell
size caused when blocking G2/M with nocodazole was
greater than that caused when blocking G1/S with
hydroxy-urea. This was also observed during terminal
di€erentiation and after activating c-Myc. Interestingly,
keratinocyte size correlates with di€erentiation in culture
and in vivo, so that beyond a certain volume they may
initiate the expression of di€erentiation markers (Watt
and Green, 1981). Moreover, cell shape and the area of
cell adhesion to the substrate tightly control keratinocyte
di€erentiation (Watt et al., 1988; Adams and Watt,
1989). The e€ect on cell size elicited by activating c-Myc
was evident after 24 h, prior to any stimulation of
di€erentiation. It is tempting to speculate that an
increased cell volume may ultimately trigger terminal
di€erentiation by reducing positive signalling via cell
adhesion. Interestingly, we have previously shown that cMyc activity down-regulates integrin expression (Gandarillas and Watt, 1997; see also Judware and Culp,
1997). A link between cell volumen, integrin signalling,
cell cycle and cell fate has been proposed elsewhere (see
e.g., Meyerowitz, 1994; Chicurel et al., 1998; Neufeld and
Edgar, 1998; Conlon and Ra€, 1999).
Physiological significance
In a stady-state epidermis, uncoupling cell cycle and
terminal di€erentiation may be important as a selfdefence mechanism against oncogenic mutations and to
keep a tight balance between proliferation and terminal
di€erentiation. In the event of loss of the cell cycle
control, the initiation of terminal di€erentiation upon
loss of adhesion would block cell division without the
need to suppress DNA replication. Linking di€erentiation with cell cycle progression may ensure that whenever
the latter is stimulated, terminal di€erentiation also
increases. Interestingly, this is what occurs in skin
hyperproliferative lesions such as psoriasis or woundhealing. Endoreplication may also account for apparently paradoxycal reported coexpression of S phase and
di€erentiation markers and suprabasal `mitotic' ®gures
in epidermis (see Introduction; Regnier et al., 1986; BataCsorgo et al., 1993; Pinkus and Hunter, 1966; Penneys et
al., 1970). Consistently, we have found a signi®cant
proportion of di€erentiating polyploid keratinocytes
isolated from normal human epidermis (Gandarillas et
al., unpublished observations).
Endoreplication is known to occur during Drosophila
development (Edgar, 1995) and in human megakariocytes (Ho€man, 1989), osteoclasts (Solari et al., 1996),
endometrium (Kirk and Clingan, 1980) and liver (Jack et
al., 1990). In all these cases it associates with increased
cell size and a requirement for a specialized function.
Very interestingly, endoreplication is known to be
important in plant epidermal di€erentiation to the
production of large single cell structures (trichomes;
Traas et al., 1998; HuÈlskamp et al., 1999).
We are currently investigating the molecular mechanisms connecting cell cycle, cell size, endoreplication and terminal di€erentiation. These should be key
issues to better understand how tissue homeostasis is
regulated.
3287
Materials and methods
Cell culture
Primary keratinocytes were isolated from neonatal human
foreskin and cultured in the presence of a feeder layer of
mouse J2-3T3 ®broblasts in FAD medium as described
previously (Rheinwald, 1989; Gandarillas and Watt, 1997).
Early passages (from 1 to 4) of Keratinocytes from four
di€erent individuals were used (strains ka, kq, kz and kmb).
To block keratinocytes at di€erent phases of the cell cycle,
stratifying cultures were treated with 2 mM hydroxy-urea,
10 ng/ml TGFb or 20 mM nocodazole for the length of time
indicated.
Expression of myc conditional forms
Primary keratinocytes expressing empty retroviral vector
pBabe, or pBabe containing conditional c-mycER, D106143ER (Littlewood et al., 1995) were obtained by retroviral
infection as previously described (Gandarillas and Watt,
1997) and cultured as normal keratinocytes. Activation of cMyc conditional forms was achieved by adding 100 nM 4hydroxytamoxifen (variant Z; Research Biochemicals International) to the culture medium every 48 h. Primary
keratinocytes from two di€erent individuals (strains ka and
kq) expressing conditional myc forms were used in this study.
Confocal analyses of stratifying keratinocytes
Keratinocytes expressing mycER were immunostained with
anti-myc mAb 9E10 (Evan et al., 1985) as previously
described (Gandarillas and Watt, 1997). Normal cultures
were ®xed in 3.8% formaldehyde and permeabilised in
7208C-cold methanol and double stained for CD44 with
mAb CD44V3 (kind gift of F Watt; Hudson et al., 1996) as
for 9E10 and for DNA with 40 mg/ml propidium iodide for
5 min at room temperature. Stainings were then analysed
with a Microphot FX microscope (Nikon UK Ltd) equipped
with an MRC-600 laser scanning confocal microscope
attachment (Bio Rad Microscience, Hemel Hempstead,
UK). To focus on di€erent layers of keratinocyte cultures
optical sections were 0.2 or 0.3 mm thick.
Quantitation of DNA content, differentiation and DNA
synthesis by flow cytometry
Trypsinised keratinocytes were washed once with PBS and
®xed. For involucrin staining, cells were ®xed in 1%
paraformaldehyde for 5 min, washed in PBS and stained
for involucrin as previously described (Gandarillas and Watt,
1997). Monoclonal anti-involucrin antibody SY5 was a kind
gift of F Watt. For DNA content, un®xed keratinocytes or
keratinocytes that had been stained for involucrin were ®xed
and permeabilized, or just permeabilized, respectively, in 70%
cold ethanol for at least 30 min at 48C, with vortexing for the
®rst minute. Cells were then washed twice with PBS and
Oncogene
c-Myc and endoreplication in keratinocyte differentiation
A Gandarillas et al
3288
resuspended in PBS containing 20 mg/ml ribonuclease and
40 mg/ml propidium iodide. Flow cytometry analysed were
then performed as before.
For DNA synthesis analyses, keratinocytes that had been
cultured in the presence of 10 mm BrdU were trypsinized,
washed once in PBS and ®xed in ethanol as above. BrdU
staining was then performed as described (Gandarillas and
Watt, 1997) followed by RNAse treatment and propidium
iodide for DNA content as above.
Keratinocytes stained for involucrin, or those double
stained for involucrin or BrdU and DNA, were ®rmly
resuspended and ®ltered through a 70 mM mesh to minimize
the presence of aggregates and then analysed by ¯ow
cytometry on a Becton Dickinson FACScan. 10 000 ± 50 000
events were gated on the basis of PI area/width to exclude
cell agregates as in (Ormerod, 1990), and adquired in list
mode for every sample.
Time-lapse videomicroscopy
Normal or c-mycER expressing primary keratinocytes were
recorded for up to 4 days using an inverted microscope
containing a CO2 chamber (Olympus IMTI or IMT2).
Frames were taken every 2 min utilizing video equipment as
described previously (Gandarillas and Watt, 1997).
Cell sorting and laser scanning cytometer
Cultured or freshly isolated keratinocytes were ®xed and
stained for involucrin and DNA as above and cells having
more than 4 N DNA content were sorted using a BectonDickinson FACStar Plus and visualized on a Zeiss Axiophot
¯uorescence microscope.
To quantitate DNA of single nuclei, two methods were
used: (a) 15 ml of single cell suspensions of cultured or freshly
isolated keratinocytes after trypsin treatment were air-dried
on glass slides at 378C, ®xed in 3.8% formaldehyde and
washed in PBS. Cells were then stained for DNA for 5 min as
before; (b) single cell suspensions were stained for DNA as
before and then 20 ml were placed on slides. Cells from either
method were covered with coverslips, sealed with nail varnish
and analysed with a Laser Scanning Cytometer (CompuCyte,
Cambridge, MA, USA).
Acknowledgements
The initial observations of this work were made in the
laboratory of F Watt in ICRF of London, and A
Gandarillas is especially grateful to her generosity and
helpful suggestions. We thank C Brooks for some technical
assistance, D Fisher for constructive suggestions and M
Gomez and H Land for helpful comments at an early stage
of the work. Thanks to JP Moles the data obtained from
English skin could be displayed on French computers. A
Gandarillas was funded by Bristol-Myers Squibb, ARC
and EMBO. This research received ®nancial support from
CNRS and Ligue Nationale Contre le Cancer.
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