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ã Oncogene (2000) 19, 3278 ± 3289 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc Normal and c-Myc-promoted human keratinocyte dierentiation 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 dierentiation in human keratinocytes is poorly understood. It is believed that keratinocytes suppress DNA replication and cell cycle arrest in G0 before they initiate terminal dierentiation. However, a temporal separation between both events has not been established. Moreover, c-Myc promotes keratinocyte dierentiation without causing cell cycle arrest. To address these paradoxes we have analysed cell cycle control during normal and c-Mycpromoted dierentiation. Continuous activation of c-Myc or initiation of terminal dierentiation results in a block of G2/M, cellular growth, endoreplication and polyploidy. Keratinocytes abandon G1, continue replicating DNA as they dierentiate terminally and become polyploid. In fact, simply blocking mitosis with nocodazole resulted in increased cell size, terminal dierentiation and endoreplication. This indicates that terminal dierentiation 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 dierentiation 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 dierentiation 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 dierentiation 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 dierentiation (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 dierentiation in culture. Similarly, a subpopulation of epidermal keratinocytes has been found to express terminal dierentiation 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 dierentiation (Missero et al., 1996; Hauser et al., 1997). However, although constitutive expression of these molecules in keratinocytes causes cell cycle arrest, terminal dierentiation 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 eect on keratinocyte terminal dierentiation (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 dierentiation (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 dierentiation (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 dierentiation. c-Myc ®rst drove keratinocytes to leave the stem cell compartment and become transit amplifying cells, which subsequently initiated terminal dierentiation after four to ®ve rounds of cell division. This increase of terminal dierentiation, 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 dierentiation 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 dierentiation 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; Homan, 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 dierentiation 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 dierentiation marker involucrin and DNA content. The cell cycle distribution of involucrin positive or negative cells was then quantitated by ¯ow-cytometry (Table 1). Terminally dierentiating 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 dierentiating and non dierentiating cells expressing the empty vector puro-resistance (KpBabe) and in cells expressing the negative myc mutant (K106; Table 1). Therefore, the presence of dierentiating keratinocytes in S phase was not speci®c of c-Myc activation. There was, however, a slight accumulation of c-Myc dierentiating cells in G2/M. Keratinocytes can be induced to terminally dierentiate when they are placed in suspension in the absence of cell adhesion. When normal, control keratinocytes expressing the empty vector were induced to terminally dierentiate 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%). 3279 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 dierentiation (Gandarillas and Watt, 1997). We have explored whether constitutive activity of c-Myc stimulated the cell cycle of terminally dierentiating 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 dierentiation. At this stage bi- or tri-nucleate cells were frequently observed (Figure 1a). Confocal analyses of c-Myc staining showed stratifying, dierentiating keratinocytes to often contain more than one nucleus or a large single nucleus (Figure 1b). Multinucleate dierentiating 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 dierentiating or non-dierentiating 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 dierent 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 Oncogene c-Myc and endoreplication in keratinocyte differentiation A Gandarillas et al 3280 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 dierentiating, 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 dierentiate 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 dierentiation marker involucrin. The DNA content of involucrin negative or positive cells was determined Oncogene (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 dierentiating 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 dierentiation. This suggests that dierentiating 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 dierent 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 dierentiation (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 dierentiating 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 dierentiating 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-dierentiating compartment (23.5%; Figure 2c) a similar proportion of diploid and polyploid terminally dierentiating 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 dierentiating 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 dierentiation. Confocal analyses of double staining for DNA and a terminal dierentiation 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, dierentiating 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 dierentiation, we quantitated polyploid cells after stimulating terminal dierentiation c-Myc and endoreplication in keratinocyte differentiation A Gandarillas et al 3281 Figure 2 Flow-cytometry analyses of cell cycle and dierentiation 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-dierentiating or dierentiating 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 dierent 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 dierentiation. BrdU incorporation analyses in suspended cells indicated that DNA synthesis continued after 6 h, when cells are irreversibly committed to terminal dierentiation (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 dierentiation 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 dierentiation (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 dierentiation 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 dierentiation (see Gandarillas and Watt, 1997). Oncogene c-Myc and endoreplication in keratinocyte differentiation A Gandarillas et al 3282 Figure 3 Nuclear division (endomitosis) in endoreplicating, dierentiating primary keratinocytes. (a) multiple simultaneous anaphases in a dierentiating 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 dierentiation (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 dierentiating 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 dierentiation (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 dierentiating keratinocytes in G2/ M and the presence of endoreplication in suprabasal cells indicate that a mitosis blockade associates with terminal dierentiation. To further test this association, we studied it in an inverse fashion: we blocked keratinocyte mitosis continuously with nocodazole and determined the eect on dierentiation and endoreplication. Primary Oncogene keratinocytes were hard to synchronise completely, as they are dicult 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 dierentiation (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 dierentiation 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 dierentiation 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 dierentiation. (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; Homan, 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 dierentiation and cell size in keratinocytes (Figure 7). Suspension-induced dierentiation, 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 eect of c-Myc on cell size (Figure 7d,e). This eect was detected even 24 h after activation of c-mycER, when all other dierentiation markers were still unaected (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 dierentiation. However, we have observed a similar cell cycle distribution in dierentiating and non-dierentiated keratinocytes, even shortly after inducing terminal dierentiation in suspension. In Oncogene c-Myc and endoreplication in keratinocyte differentiation A Gandarillas et al 3284 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 dierentiate in suspension for 24 h; note the large size of cell and nucleus and the presence of two nuclei in some very dierentiated 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 dierentiation. It must thus be concluded that keratinocyte terminal dierentiation 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 dierentiating 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 dierentiation 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 dierentiating cells eventually abandon G1, but the diminution of the G1 peak as terminal dierentiation 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 Dierentiating 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 dierentiation. The results presented here indicate that terminal dierentiation tends to associate with mitosis-defective cell cycle progression, rather than with arrest in G0. That was the case during spontaneous or suspension-induced dierentiation, 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 dierentiation are to be elucidated. When proliferating basal cells detach from the basement membrane they produce the dierentiation 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 dierentiating 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 Eects of blocking mitosis with nocodazole on keratinocyte cell cycle and dierentiation. 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 dierentiating cells, and triggered terminal dierentiation and endoreplication; (b) constitutive expression of the dierentiation 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 dierentiation keratins or corni®ed envelope precursors and factors that promote dierentiation, 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 dierentiation. Furthermore, in the light of endoreplication, c-Mycpromoted keratinocyte dierentiation 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 dierentiation. In keratinocytes, endoreplication masked the eect 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 dierentiation and a G2/M blockade while DNA replication continues. The correct checkpoints in G2/M would then be lost in the onset of dierentiation. 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 dierentiation, 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 dierentiation 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 dierentiation 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 dierentiation (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 sucient to commit these cells to subsequent dierentiation, 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 dierentiating layers), occasional nuclear division in dierentiating 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 dierentiation? 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 dierentiation 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 dierentiation and after activating c-Myc. Interestingly, keratinocyte size correlates with dierentiation in culture and in vivo, so that beyond a certain volume they may initiate the expression of dierentiation markers (Watt and Green, 1981). Moreover, cell shape and the area of cell adhesion to the substrate tightly control keratinocyte dierentiation (Watt et al., 1988; Adams and Watt, 1989). The eect on cell size elicited by activating c-Myc was evident after 24 h, prior to any stimulation of dierentiation. It is tempting to speculate that an increased cell volume may ultimately trigger terminal dierentiation 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 dierentiation may be important as a selfdefence mechanism against oncogenic mutations and to keep a tight balance between proliferation and terminal dierentiation. In the event of loss of the cell cycle control, the initiation of terminal dierentiation upon loss of adhesion would block cell division without the need to suppress DNA replication. Linking dierentiation with cell cycle progression may ensure that whenever the latter is stimulated, terminal dierentiation 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 dierentiation 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 dierentiating 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 (Homan, 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 dierentiation 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 dierentiation. 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 dierent individuals were used (strains ka, kq, kz and kmb). To block keratinocytes at dierent 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 dierent 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 dierent 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. 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