The authors have declared that no competing interests exist.
Conceived and designed the experiments: IM AS HN. Performed the experiments: HW. Analyzed the data: HW AS HN. Wrote the paper: HW IM AS HN.
Epidermal human keratinocytes are exposed to a wide range of environmental genotoxic insults, including the UV component of solar radiation. Epidermal homeostasis in response to cellular or tissue damage is maintained by a population of keratinocyte stem cells (KSC) that reside in the basal layer of the epithelium. Using cell sorting based on cell-surface markers, we have identified a novel α6 integrinhigh+/CD44+ sub-population of basal keratinocytes. These α6 integrinhigh+/CD44+ keratinocytes have both high proliferative potential, form colonies in culture that have characteristics of holoclones and have a unique pattern of resistance to apoptosis induced by UVB radiation or by agents that induce single- or double strand DNA breaks. Resistance to UVB induced apoptosis in the α6 integrinhigh+/CD44+ cells involved increased expression of TAp63 and was overcome by PI-3 kinase inhibition. In marked contrast, the α6 integrinhigh+/CD44+ cells were sensitive to apoptosis induced by the cross-linking agent cisplatin, and imatinib inhibition of c-Abl blocked the ability of cisplatin to kill α6 integrinhigh+/CD44+ cells. Our findings reveal a population of basal keratinocytes with long-term proliferative properties that display specific patterns of apoptotic resistance that is dependent upon the genotoxic stimulus, and provide insights into how these cells can be targeted with chemotherapeutic agents.
The human epidermis is a stratified epithelium that maintains its integrity through a process of constant regeneration, driven by a population of keratinocyte stem cells (KSCs) in the basal layer
The epidermis is frequently exposed to sunlight, with the ultra-violet B (UVB) fraction of the radiation able to penetrate the surface of the epidermis, directly damaging the DNA of the keratinocytes via formation of cyclobutane pyrimidine dimers (CPDs) and 6–4 photoproducts
In cancer cell lines CD44-positive cells have been shown to display a resistance to apoptosis following treatment with chemotherapy agents
Primary human keratinocytes were isolated from redundant facelift skin and passage 2–3 cells were used in all experiments unless otherwise stated. Keratinocytes were grown on mitomycin-treated 3T3 feeder layers in growth factor-containing medium (DMEM-F12 modified medium, PAA, Yeovil, UK) supplemented with 10% foetal bovine serum (FBS) (Biowest, East Sussex, UK), 1% (vol/vol) glutamine, 5 µg/ml insulin, 0.4 µg/ml hydrocortisone, 5 µg/ml transferrin, 10−10 M cholera toxin, 0.013 µg/µl lyothyronine (Sigma, Poole, UK) and 10 ng/ml EGF (Serotec, Oxford, UK) at 37°C in 10% CO2.
Frozen skin sections and primary human keratinocytes were fixed with 4% paraformaldehyde for 10 min at room temperature, washed with PBS, permeablised by incubating with 0.2% (v/v) Triton X in PBS for 5 min at RT if required and blocked with 5% donkey serum (Sigma, Poole, UK) at room temperature for 1 hour. Primary antibodies against human α-6 integrin (CD49f, BD Bioscience, Oxford, UK), human CD44 (AbD Serotec, Oxford, UK) or human total p63 (Santa Cruz Biotechnology, Heidelberg, Germany) were incubated at 4°C overnight with a further 3 washes completed before incubation with the relevant donkey secondary antibodies (Molecular Probes, Paisley, UK) at room temperature for 1 hour. Cells were then mounted with coverslips using Vectashield-DAPI mounting medium (Vectorlabs, Petersfield, UK) and analysed using a Nikon Eclipse TC2000S microscope (Nikon, Kingston, UK).
Cells were grown at clonal density until they reached 60% confluence (5 days), washed with PBS and removed from the culture dish using trypsin-EDTA (PAA, Yeovil, UK). Following neutralisation and washing with PBS the cells were resuspended at 1×106/ml in PBS/1% FBS with 5×105 cells used per incubation and analysis. Incubation was at 4°C for 30 min using CD44-FITC and CD49f-PE-Cy5 conjugated antibodies (BD Bioscience, Oxford, UK). Simultaneously cells were incubated with the corresponding IgG isotype controls (BD Bioscience, Oxford, UK). Cells were then washed in PBS/1% FBS and either recorded as live cells on a BD LSRII FACS machine or fixed cells following treatment with 1% paraformaldehyde. FACSDiva software was used to analyse all samples, with isotype and single-colour negative controls used to establish compensation settings, and a minimum of 30,000 events recorded per sample. For sorting of the cells a BD FACSAria machine was used. Other live cell-surface flow cytometry analysis used the BD antibodies β1 integrin (CD29-APC), transferrin receptor (CD71) with biotynilated-PE secondary, and appropriate IgG isotype controls (BD Bioscience, Oxford, UK). The FITC, PE and PE-Cy5 antibodies were excited using an argon laser (488 nm) and collected using a 530/30 nm filter (FITC), 575/26 nm filter (PE), and 675/20 nm filter (PE-Cy5). The APC antibody was excited using the 633 nm red diode and collected using a 660/20 nm filter.
To determine the proliferative capacity
Cells plated in dishes were grown to 50% confluence (4 days) and washed in PBS. A thin layer of PBS was added prior to UVB irradiation at increasing doses from 1 to 40 mJ/cm2. UVB irradiation was from a UVP Multiple Ray Lamp (Ultra-violet Products, Cambridge, UK) (MRL-58 model) fitted with F8T5 bulbs producing a sharp emission at 312 nm corresponding to mid-range UVB, calibrated before each experiment with a UVX Radiometer (UVP, Cambridge, UK). The PBS was then replaced with medium and cells grown for a further 6 to 48 hours dependent upon apoptosis assay. Chemical induction involved addition of chemotherapeutic drugs to the cell culture medium at increasing final doses; Etoposide 10–100 µg/ml, Cisplatin 25–200 µg/ml, Camptothecin 0.5–4 µM and Bleomycin 25–200 µg/ml (Sigma, Poole, UK) for 24 to 72 hours. The PI-3 kinase inhibitors Wortmannin (1 µM) and LY294002 (50 µM) and the tyrosine kinase inhibitor imatinib (1 µM) were added 2 hr prior to apoptosis induction. Doses of all drugs were chosen following preliminary dose-response assays, IC50 values of the drugs and previous relevant studies.
Live cells were analysed for Annexin-V binding (Annexin V-FITC antibody, BD Bioscience, Oxford, UK) using the cell surface staining method described above followed by an incubation with the antibody in Annexin-V Binding Buffer (BD Bioscience, Oxford, UK) for 15 min at room temperature, a wash with PBS and the addition of 200 ng/ml DAPI directly prior to flow cytometry analysis to distinguish non-viable cells.
For Caspase-3 detection cells grown in 6-well plates were fixed with 4% paraformaldehyde, washed with PBS and permeablised with 0.2% Triton-X 100 in PBS for 5 min at room temperature. Cells were blocked using 5% goat serum/0.1% Tween 20 in PBS for 2 hours and washed ×1 in PBS. Rabbit anti-human Anti-Active Caspase-3 (Promega, Southampton, UK) was incubated at 1∶250 in 5% goat serum/1% Tween 20/PBS overnight at 4°C. Cells were washed in PBS (10 min×2), in 1% Tween 20/PBS (10 min×2) and in PBS (10 min×2). The 2° antibody Alexa Fluor 488 goat anti-rabbit IgG (H+L) (Invitrogen) was incubated 1∶500 in PBS for 2 hours at room temperature. Cells were washed in PBS (5 min×2), 1% Tween 20/PBS (5 min×1) and PBS (5 min×1). Cells were mounted using DAPI mounting medium and coverslips with fluorescence viewed using the Nikon Eclipse TE 2000-S microscope.
Cells analysed for TdT-mediated dUTP nick end-labelling (TUNEL) were removed from the dish, incubated with cell surface antibodies and fixed overnight as described earlier. Cells were then washed in PBS (x2), incubated in TUNEL (Promega) at 37°C for 1 hour, washed in PBS x1 and Triton-X 100 (5 min x1) before addition of 1 µg/ml DAPI in PBS 30 min before flow cytometry analysis.
Total RNA was extracted from keratinocytes using the RNeasy Micro Kit (Qiagen, West Sussex, UK) according to manufacturer’s instructions. cDNA was generated using the SuperScript III First-Strand Synthesis SuperMix Kit (Invitrogen, Paisley, UK). Positive controls were transcribed from total human RNA (Stratagene QPCR Human Reference Total RNA, Agilent Technologies UK Limited, Stockport, UK) and negative controls with the omission of sample RNA (no template control, NTC) or omission of RT Enzyme Mix (no reverse transcriptase control, -RT). qRT/PCR was performed with Syber Green PCR master mix (Applied Biosystems, Warrington, UK) and results analysed using 7500 System Software (Applied Biosystems). The reaction was run at 95°C for 10 min followed by 45 cycles at 95°C for 15 s, 60°C for 30 s and 72°C for 40 sec and then one cycle at 95°C for 1 min. Forward (3′ to 5′) and reverse (5′ to 3′) primers were designed for ΔNp63 (Forward
In line with previous studies, expression of CD44 was observed in both the basal and suprabasal layers of the human epidermis
(A) Immunofluorescence histochemistry of frozen normal human skin probed with an antibody against CD44 (red) or α6 integrin (green). Negative controls probed with blue nuclear stain (DAPI) only. Representative images of n = 3; scale bar = 100 µm. (B) Primary keratinocytes analysed for expression of α6 integrin using flow cytometry showed an average of ∼30% α6 integrin+ cells and ∼10% CD44+ cells (n = 5). (C) Primary keratinocytes analysed for co-expression of both α6 integrin and CD44 using FACS Diva software. The CD44+ population that also highly expressed α6 integrin could be manually gated at about ∼5% of the whole cell population (n = 25). ‘Excluded’ cells are 3T3 mouse fibroblasts remaining from the feeder layer used in the culture process. (D) The four populations identified in (C) were separated using FACS and grown
To establish if there were measurable differences in the long-term growth of the α6 integrinhigh+/CD44+ cells compared to the rest of the keratinocyte population, the total cell output was quantified (
Cultured keratinocytes exposed to low doses of UVB (≤10 mJ/cm2) revealed very little morphological change at 24 hr post-irradiation compared to control cells (
(A) Keratinocytes were grown in culture to form large colonies and photographed 24 hours following UVB irradiation. (B) Quantification of apoptotic cells using flow cytometry. Annexin-V+ cells (DAPI+ and DAPI−) were totalled in both α6 integrinhigh+/CD44+ and remainder populations and plotted as a change in percentage from the control level (0 mJ/cm2 which averaged ∼8% apoptosis) to negate the necrotic cells present before treatment. Results show apoptosis levels 6, 16 and 24 hours after UVB irradiation of colonies at 50% confluency. Keratinocytes were also UVB irradiated as single cells 2 hours after plating, and apoptosis levels are shown after 24 hours. Data represents the mean ±SEM where n = 3. Statistical significance was determined using the paired Student’s
The chemotherapeutic agents etoposide and camptothecin are classified as Topoisomerase inhibitors. Etoposide binds to Topoisomerase II causing both single and double strand DNA breaks
Keratinocytes at 50% confluency treated were with genotoxic agents at increasing doses and analysed for apoptotic cells using flow cytometry. Annexin-V+ cells (DAPI+ and DAPI−) were totalled in both the α6 integrinhigh+/CD44+ and remainder cell populations and plotted as a change in percentage from the control (vehicle only control that averaged ∼10% apoptosis). Results show apoptosis levels 24 hours after treatment with Etoposide (A), Camptothecin (B) Bleomycin (C) and Cisplatin (D). Data represents the mean ±SEM where n = 3. Statistical significance was determined using the paired Student’s
Treatment with the platinum DNA cross-linking agent cisplatin produced an altered apoptotic response in the keratinocytes. As the concentration of cisplatin (25 to 200 µg/ml) administered to the keratinocytes was increased, the level of Annexin-V binding rose in both the remainder and α6 integrinhigh+/CD44+ populations (
Immunofluorescence staining to visualise the later-stage apoptotic marker of active Caspase-3 revealed a greater number of active Caspase-3 positive cells were present at 48 hr in keratinocytes treated with either UVB or cisplatin compared to untreated control cells (
(A) Keratinocytes were grown for 5 days to form colonies, treated with UVB (20 mJ/cm2) or cisplatin (200 µg/ml) or vehicle-only (control). After 24 hr the cells were stained with an antibody against Active Caspase-3 and photographed. Nuclei are stained blue with DAPI. (B) Keratinocytes were grown for 5 days, treated with UVB (30 mJ/cm2), cisplatin (50 µg/ml) or vehicle-only (control) for 24 hours, then fixed and analysed for TUNEL+ cells using flow cytometry. Data represents the mean where n = 3.
Flow cytometry was used to analyse the number of TUNEL+ cells in the last stage of programmed cell death, 24 hr after UVB or cisplatin treatment. The change in apoptotic DNA fragmentation from the control was significantly higher in the remainder fraction than the α6 integrinhigh+/CD44+ fraction after UVB irradiation (p<0.05,
(A) Immunofluorescence detection of phosphorylated Akt (pAktSer473) in FACS-separated α6 integrinhigh+/CD44+ and remainder keratinocytes treated with 30 mJ/cm2 UVB irradiation. The positive control is the unfractionated total keratinocyte population probed with both the primary and secondary antibodies and the negative control prepared with the secondary antibody only. Images are representative of n = 3 and cell nuclei were detected with DAPI (blue). The scale bar is equal to 100 µm. (B) Sub-confluent primary keratinocytes probed for detection of phosphorylated Akt (pAktSer473) following treatment with 1 µM wortmannin or 50 µM LY294002 prior to UVB irradiation (30 mJ/cm2). Control cells were treated with the vehicle-only and negative controls prepared by omission of the primary antibody. Cell nuclei were detected using DAPI (blue) and images are representative of n = 3. The scale bar is equal to 100 µm. (C) Primary keratinocytes treated with 1 µM Wortmannin, 50 µM LY294002 for 2 hr, or no drug (vehicle-only control), were analysed for positive TUNEL expression 24 hr post-irradiation with UVB (30 mJ/cm2). The graph shows TUNEL+ cells in the α6 integrinhigh+/CD44+ and remainder populations plotted as a change in percentage from control level (0 mJ/cm2, ∼ 3.5% apoptosis). Data represents the mean ± SEM where n = 4. Statistical significance was determined using the paired Student’s t-test, *P<0.05.
To identify if the PI3-kinase cell survival pathway is protecting the α6 integrinhigh+/CD44+ fraction, the cells were probed for expression of the activated downstream effector pAKT. An increase in positive nuclear expression of pAktSer473 was observed following UVB irradiation, but no difference between the fractions could be identified using immunofluorescence (
(A) Primary keratinocytes analysed for p63 gene expression in the α6 integrinhigh+/CD44+ and remainder populations using QRT-PCR. Data represents the log mean fold change of the α6 integrinhigh+/CD44+ population from the remainder population ± SEM of three replicates, normalised against GAPDH housekeeping gene. Keratinocytes analysed for gene expression after cisplatin (B) or UVB (C) where data represents the log mean fold change from the control (vehicle-only) ± SEM of three replicates, normalised against 28S-r-RNA housekeeping gene. Statistical significance was determined using the paired Student’s t-test, *P<0.05.
The full length TAp63 isoform of the proposed keratinocyte stem cell marker p63
(A) Primary keratinocytes treated with 50 µg/ml cisplatin, or 1 µM imatinib, or 1 µM imatinib for 2 hr and then 50 µg/ml cisplatin were analysed for TUNEL expression 24 hour post-treatment. TUNEL-positive cells were totalled in the α6 integrinhigh+/CD44+ and remainder populations and plotted as a percentage change from control level. Data represents the mean ± SEM where n = 3. Statistical significance was determined using the paired Student’s t-test, *P<0.05.) (B) QRT-PCR quantification of p63 gene expression in α6 integrinhigh+/CD44+ and remainder keratinocyte populations 16 hr after treatment with 1 µM imatinib and 50 µg/ml cisplatin. Data represents the log mean fold change from the control (vehicle-only) for each population ± SEM of three replicates. All results were normalised against the 28s-r-RNA housekeeping gene. Statistical significance was determined using the paired Student’s t-test (P>0.05 in all cases). (C) FACS-separated α6 integrinhigh+/CD44+ cultured keratinocytes were probed for p63 expression (green) using immunofluorescence 16 hr following treatment with 50 µg/ml cisplatin and/or 1 µM imatinib. The control cells were treated with vehicle-only and the negative control was prepared with the secondary antibody only. Nuclei were detected with DAPI (blue) and images are representative of n = 2. The scale bar is equal to 100 µm.
Addition of the tyrosine kinase inhibitor imatinib prior to cisplatin treatment significantly reduced the amount of cell death seen in the α6 integrinhigh+/CD44+ keratinocytes compared to treatment with cisplatin alone (p<0.05,
Gene expression of the ΔNp63 and TAp63 isoforms did not alter from the control (vehicle-only) level in the remainder keratinocyte population when treated with imatinib prior to cisplatin (
Analysis of the location of the total p63 protein in the α6 integrinhigh+/CD44+ keratinocytes revealed positive nuclear expression evident in the control cells (
Here we report the identification of a small basal fraction of cultured human primary keratinocytes that express CD44 and display a high growth capacity coupled with an enhanced resistance to apoptosis induced by UVB and specific DNA damaging agents.
Separation of cultured cells expressing high levels of the adhesion molecule α6 integrin enabled selection of the less-differentiated basal keratinocyte population. Analysis of the integrinhigh+ cells for positive CD44 expression revealed a co-expressing population that could be manually gated at ∼5% of the total keratinocyte population (α6 integrinhigh+/CD44+ cells). The morphology of the colonies formed in culture by this fraction of cells were predominantly holoclone-like in appearance and when passaged until exhaustion, the total cell output was of an equivalent level to that produced by recognised KSC phenotypes β1 integrinhigh and α6 integrinbri/CD71dim. When directly analysed for β1 integrin expression or low levels of CD71, the α6 integrinhigh+/CD44+ fraction contained a large number of cells previously identified as KSCs as well as other un-differentiated transient-amplifying cells.
To determine if this population displayed a different apoptotic capability compared to the rest of the cell population, the response of the cells to UVB irradiation was explored. The α6 integrinhigh+/CD44+ fraction displayed a significantly decreased level of apoptosis compared to the rest of the keratinocytes. Interestingly, α6 integrin has also been shown to be required for the growth and survival of breast cancer cells
The α6 integrinhigh+/CD44+ keratinocytes were also significantly resistant to DNA damage induced by the chemotherapeutic agents etoposide and camptothecin. The striking sensitivity to cisplatin was in contrast to the resistance noted in epithelial cancer stem cell lines
In conclusion, we have identified a sub-population of cultured human keratinocytes expressing high levels of CD44 and α6 integrin that display a high growth capacity. These α6 integrinhigh+/CD44+ cells display a resistance to undergo programmed cell death in response to UVB irradiation and chemotherapeutic drugs, except in the case of cisplatin treatment where a sensitivity to undergo apoptosis in line with the rest of the cell population is observed. The resistance to UVB-induced apoptosis could be an important property that allows the regenerative capacity of the undifferentiated cells in the human skin to be maintained despite acute UVB-exposure. The mechanism of this resistance appears to be attributed to a high level of baseline TAp63 gene expression linked to the PI 3-Kinase cell survival pathway. The contrasting sensitivity to cisplatin treatment appears to be due to the ability of the drug to cause a loss of this TAp63-conferred protection. This loss is likely to be due to the cisplatin-induced activation of a tyrosine kinase causing a reduction in TAp63 gene expression and nuclear p63 protein levels. Addition of a tyrosine kinase inhibitor reduces the apoptotic sensitivity of the cells to cisplatin, so they are able to demonstrate a resistance to the platinum agent-induced apoptosis. Correspondingly, the high baseline TAp63 mRNA expression does not decrease and the nuclear expression of the p63 protein is not lost.
The notion that a network of TAp63- linked apoptotic protection is present in a population of undifferentiated human keratinocytes may provide important information regarding the behaviour of long-lived cells in the human epidermis. If the resistance to undergo cell death following UVB irradiation is repeated
We would like to acknowledge the BBSRC for their PhD studentship, Luke Gammon and Lisa Harper for technical assistance and Gary Warnes for flow cytometry expertise and operation of the cell sorter.