Figure 1.
Polyploid cells and nuclei in epidermal cell suspensions.
(a–c): representative flow-cytometry analysis of freshly isolated keratinocytes (a–b) or nuclei (c) from human epidermis. Side (SSC) and Forward (FSC) Scatter reflect cell size and complexity. FL2 reflects propidium iodide (PI, DNA content: 2C, 4C, >4C or polyploidy). Coloured dots are cells in the different phases of the cell cycle (G1: green; S/G2/M: blue; polyploidy: red). Numbers show percent of cells within each region for each histogram. a: Keratinocytes from foreskin were double labelled for postmitotic keratin K1 and DNA. Cells were sorted on basis of low K1 (R1), high (R2) and very high K1 (R3) and the DNA content of the different subpopulations analysed. The dot Plot represents K1 versus cell size expression. Histograms represent DNA content of all cells (TOTAL) or of K1 negative cells (R1), according to negative antibody b: Keratinocytes from breast skin were fixed and double stained for involucrin and DNA. Dot plots represent SSC and FSC (left), PI versus cell size (middle) or involucrin expression versus cell size (right), as indicated. The photograph shows keratinocytes stained with involucrin (green) and PI (red); note the smaller nuclei negative for involucrin (arrows). c: Freshly isolated nuclei from human breast epidermis dot plots represent light Scatter (left), FL2-Width versus FL2-Area (first right: typical analyses to exclude aggregates) or nuclear size (light scatter) versus DNA content (second-right). Red histograms represent DNA content of all nuclei (TOTAL) or nuclei with higher size and complexity (R2), on basis of the light scattering. Photographs show FISH on diploid (left) or polyploid (right) nuclei stained with probes for centromere 20 (red; 2 dots on the left, 4 dots on the right) or Her2Neu locus (green; two dots on the left, four dots on the right; also note the difference in nuclear size). The data are representative of at least three independent experiments.
Figure 2.
Chromosome amplifications and binucleate cells in suprabasal human epidermis.
a,b,c: Fluorescence In Situ Hybridisation (FISH) for centromeres of chromosome 8 (a, red), 18 (b, green) or 20 (c, green) on human skin sections from healthy breast (a,b) or foreskin (c). Nuclei stained with the Dapi compound in blue. Similar results were obtained for chromosomes 8,9,18 and 20. d: isolated nuclei from breast epidermis hybridised for centromere of chromosome 9 (red), Her2Neu locus (green) and DNA (blue). Arrows indicate nuclei with cromatide amplifications. e, f: hematoxylin/eosin (e) or giemsa (f) staining of human skin sections from scalp (e; slightly cross-cut) or breast (f). Arrows indicate binucleated cells. The white or black line represents the basement membrane that links the epidermis with the underlying dermis. SS: skin surface.
Figure 3.
In situ quantitation of nuclear DNA of human epidermis in situ.
a,b,f,g: 3D reconstruction of epidermis of foreskin (a,b) or scalp (f,g) stained for DNA with the Dapi compound, showing the nuclei in grey scale (a,f) or in rainbow colours corresponding to the estimated DNA content (b,g). Grey nuclei in b,g were incomplete and therefore disregarded. c,d,h,i: dot plots showing volume versus sum of fluorescence intensity values of nuclei in the dermis as control (c, h) or in the epidermis (d, i). e,j: approximate distribution of nuclei according to their estimated DNA content (left axis). Values in red are the means of the sum of intensities of the corresponding group of nuclei (right axis). Bars are standard deviations. White lines: basement membrane. SS: skin surface. Two different fields per individual were analysed.
Figure 4.
Expression of cell cycle regulators of S phase in normal human epidermis.
Expression of cyclin E (Cyc E;a,c), cyclin A (Cyc A;b–e), or phospho-Rb ser780 (P-Rb780;f–h,j) or ser807-811 (P-Rb807-11;i) and differentiation markers keratin 1 (K1;a,b,d), keratin 10 (K10;f–h,j) or keratin 5 (K5;e) as indicated. Colours as indicated. Nuclei stained with Dapi, blue. Circle in b highlight patch of positive cyclin A cells. More details are shown in Fig. S1. d,e,h,i: amplified areas of the basal layers. g,j: amplified area of suprabasal layers. Arrows point at ‘mushroom cells’, arrowheads at mitotic figures. g shows in gray scale the DNA staining of metaphasic figures. Dotted lines indicate the basement membrane; broken lines, metaphasic cells. SS: skin surface. Results are representative of at least four different individuals.
Figure 5.
In situ DNA replication in human epidermis.
Human skin sections were subject to DNA synthesis and stained for digoxigenin (Dig is incorporated in neo-synthesised DNA (see Mat. and Meth.) and keratin 10 (K10) or cyclin A, as indicated. Colours as indicated. Nuclear DNA is stained in blue (Dapi) for conventional microscopy (a,b,h), or in red (propidium iodide) for confocal microscopy (c–g). c: negative control where Dig was not added to the reaction (Ctr; see also Fig. S5). a,b,h: sections of neonatal foreskin; c–g: adult foreskin. Arrows in d–f point at DNA synthesising basal cells (see also Fig. S3). g: amplified area of d. h: double labelling for DNA synthesis and cyclin A, a marker of S phase. Arrowhead in g: nucleolus. Broken line indicates the basement membrane. SS: skin surface. Note that DNA synthesis spread in more suprabasal layers, generally (not always) showing a sparser pattern. Results are representative of foreskin from more than 5 individuals.
Figure 6.
In situ DNA replication in human epidermis of various body sites.
Digoxigenin incorporation (Dig., green) and keratin 10 (blue). DNA stained in red (propidium iodide) for confocal microscopy. a: negative control with Dig, without other nucleotides(Ctr); a–b: scalp; c: breast; d: thigh; e,f: eyelid. Inset in b shows an amplified suprabasal nucleus. Arrows: DNA synthesising basal cells; arrowhead: nucleolus (b, inset). Dotted line basement membrane. SS: skin surface. Results are representative of at least four different individuals.
Figure 7.
Expression of G2/M markers in human skin.
Normal human skin sections were immunostained for Cyclin B (Cyc B;a–e, i), phospho-histone H3 (P-H3;f-i), keratin 1 (a–c,f–h) or keratin 5 (d) or γ-tubuline (γ-tub; j,k; to detect centrosomes; arrows), coloured as indicated. Nuclei were stained with Dapi (a–j, blue) or propidium iodide (confocal microscopy, k, red). Dotted line indicates the basement membrane. SS: skin surface. Arrows: in a–i point at mitotic cells; arrow in f,g at a typical metaphasic “mushroom” cell; in j,k centrosome duplications (j: conventional microscopy; k: confocal microscopy). Images are representative of skin from foreskin or breast of at least three different individuals.
Table 1.
Distribution of cell cycle markers and DNA replication in human epidermis.
Figure 8.
Expression of late Mitosis regulators in human skin.
Normal human skin sections were immunostained for cdc14A (a,b), Cyclin A (b), HEC1 (c) or Aurora Kinase B (AURB; d,e), and keratins 1 and 10, colours as indicated. Nuclei were stained with Dapi (blue, a–e). Dotted line indicates the basement membrane. SS: skin surface. Arrows point at suprabasal, differentiating positive cells. Images are representative of foreskin from two different individuals.
Figure 9.
Blocking mitosis by genotoxic insult triggers epidermal differentiation and endoreplication.
Keratinocytes freshly isolated from skin treated for 48 h with a: DMSO as control; b: Bleomycin; c: ICRF-193; d: statistics of a representative experiment. Morphology, involucrin expression, DNA synthesis (BrdU incorporation) and DNA content were analysed by flow-cytometry as in Fig. 1. SSC: side scatter; FSC: forward scatter; red circle gates for basal cells; black square gates for differentiating cells. The involucrin positive region (p) was determined by a negative isotype antibody control (α-human CD8). Small bars in histograms show s.e.m. of duplicates. Results are representative of experiments with cells from three different individuals.
Figure 10.
Model for the relationship between cell cycle and differentiation in human epidermis.
Stem cells within the basal layer (SC; yellow) by division give rise to actively cycling cells that proliferate rapidly (ACC; pink). ACC lose adherence to the basement membrane, block mitosis, initiate terminal differentiation, migrate into suprabasal layers and continue DNA replication (red). Some of them undergo endomitosis in peribasal layers and become binucleate. Suprabasal keratinocytes lose mitotic cyclins A and B and reinitiate DNA replication in the absence of nuclear division (endoreduplication; punctuated red), the nuclei becoming polyploid. Endomitosis and endoreduplication are different forms of endoreplication.