Epidermal Cell Populations Research Articles

Human Hair Follicles: “Bulging” with Neural Crest–Like Stem Cells

Several studies have reported the existence of precursor cells residing within various adult tissues that appear to either retain or recapitulate features of neural crest stem cells (NCSCs). In rodents, unique populations of both epidermal and dermal cells, resident within hair follicles, exhibit such characteristics, although the existence of equivalent NCSC-like cells in human tissues has remained uncertain. In this issue, Yu et al. show that NCSC-like cells also reside within the bulge region of adult human hair follicles.

Assaying proliferation and differentiation capacity of stem cells using disaggregated adult mouse epidermis

In this protocol, we describe how to isolate keratinocytes from adult mouse epidermis, fractionate them into different sub-populations on the basis of cell surface markers and examine their function in an in vivo skin reconstitution assay with disaggregated neonatal dermal cells. We also describe how the isolated keratinocytes can be subjected to clonal analysis in vitro and in vivo and how to enrich for hair follicle-inducing dermal papilla cells in the dermal preparation. Using these approaches, it is possible to compare the capacity of different populations of adult epidermal stem cells to proliferate and to generate progeny that differentiate along the different epidermal lineages. Isolating, fractionating and grafting cells for the skin reconstitution assay is normally spread over 2 d. Clonal growth in culture is assessed after 14 d, while evaluation of the grafts is carried out after 4-5 weeks.

Differential sensitivity of epidermal cell subpopulations to β-catenin-induced ectopic hair follicle formation

Wnt signalling is required for hair follicle development and for the growth phase (anagen) of postnatal follicles. When the pathway is activated at high levels in adult mouse epidermis, ectopic follicles form from existing follicles, interfollicular epidermis (IFE) and sebaceous glands, revealing a remarkable ability of the tissue to be reprogrammed. To compare the competence of different epidermal cell populations to form ectopic follicles, we expressed a 4-hydroxy-tamoxifen (4OHT) inducible, stabilised β-catenin transgene (ΔNβ-cateninER) under the control of two different promoters. We targeted the reservoir of stem cells in the hair follicle bulge via the keratin 15 (K15) promoter and targeted the sebaceous glands and base of the follicle (bulb) with a truncated K5 promoter (ΔK5). No ectopic follicles formed in the IFE in either model, establishing the autonomy of the IFE stem cell compartment in undamaged epidermis. Activation of β-catenin in the bulge stimulated proliferation and bulge expansion. Existing hair follicles entered anagen, but no ectopic follicles formed. ΔK5ΔNβ-cateninER expressing hair follicles also entered anagen on 4OHT treatment. In addition, a subpopulation of cells at the base of the sebaceous gland readily formed ectopic follicles, resulting in complete and reversible conversion of sebaceous glands into hair follicles. Combined activation of β-catenin and the vitamin D receptor enhanced differentiation of sebaceous gland-derived hair follicles and stimulated ectopic follicle formation in the hair follicle bulb, but not in the bulge. Our results suggest that the bulge and sebaceous gland are, respectively, non-permissive and permissive niches for Wnt induced hair follicle differentiation.

Are epidermal stem cells unique with respect to aging?

Epidermal stem cells are a population of somatic stem cells responsible for maintaining and repairing the epidermis of the skin. A malfunctioning epidermal stem cell compartment results in loss of the epidermis and death of the whole organism. Since the epidermis continually renews itself by sloughing a layer of cells every day, it is in a constant state of cellular turnover and requires continual cell replacement for life. Thus, maintaining a pristine epidermal stem cell population is of prime importance, even during aging. Unlike stem cells from internal tissues, epidermal stem cells show little response to aging. They do not appear to decrease in number or functionality with age, and do not show changes in gene expression, developmental responsiveness, or age-associated increases of reactive oxygen species. Thus, epidermal stem cells may be a unique somatic stem cell.

Epidermal stem cell diversity and quiescence

Mammalian epidermis is maintained by self-renewal of stem cells and terminal differentiation of their progeny. New data reveal a diversity amongst stem cells that was previously unrecognized. Different stem cell populations have different locations and differ in whether they are quiescent or actively cycling. During normal epidermal homeostasis, each stem cell population feeds a restricted number of differentiated lineages. However, in response to injury or genetic manipulation the different pools of stem cells demonstrate multi-lineage differentiation ability. While it is well established that Wnt signalling promotes hair follicle (HF) differentiation, new observations suggest a role for EGF receptor signalling in promoting differentiation of interfollicular epidermis. NFATc1 maintains quiescence in the HF, while Lrig1 exerts the same function in the junctional zone. The stage is now set for exploring the relationship between the different epidermal stem cell populations and between quiescence and lineage selection.

Isolation and identification of stem cells from adult cashmere goat skin

Continuously renewing epithelia are maintained by stem cells that slowly proliferate and remain in the tissues for life. It has been known for decades that mouse epithelial stem cells can be selected by adherence to specific integrins. The adherence of cashmere goat epidermal cells to collagen type IV for 10 min was used to obtain enriched epidermal stem cells. The characteristics of the rapidly adherent epidermal cells were determined. The rapidly adherent epidermal cells exhibited the stem cell characteristics of immaturity, were quiescent, showed a high colony formation efficiency, and expressed candidate surface markers for epidermal stem cells (keratin 15, keratin 19, p63, CD34, and beta1-integrin). The rapidly adherent epidermal cells represented the epidermal stem cell population.

Characterization of Bipotential Epidermal Progenitors Derived from Human Sebaceous Gland: Contrasting Roles of c-Myc and β-Catenin

The current belief is that the epidermal sebaceous gland (SG) is maintained by unipotent stem cells that are replenished by multipotent stem cells in the hair follicle (HF) bulge. However, sebocytes can be induced by c-Myc (Myc) activation in interfollicular epidermis (IFE), suggesting the existence of bipotential stem cells. We found that every SZ95 immortalized human sebocyte that underwent clonal growth in culture generated progeny that differentiated into both sebocytes and cells expressing involucrin and cornifin, markers of IFE and HF inner root sheath differentiation. The ability to generate involucrin positive cells was also observed in a new human sebocyte line, Seb-E6E7. SZ95 xenografts differentiated into SG and IFE but not HF. SZ95 cells that expressed involucrin had reduced Myc levels; however, this did not correlate with increased expression of the Myc repressor Blimp1, and Blimp1 expression did not distinguish cells undergoing SG, IFE, or HF differentiation in vivo. Overexpression of Myc stimulated sebocyte differentiation, whereas overexpression of beta-catenin stimulated involucrin and cornifin expression. In transgenic mice simultaneous activation of Myc and beta-catenin revealed mutual antagonism: Myc blocked ectopic HF formation and beta-catenin reduced SG differentiation. Overexpression of the Myc target gene Indian hedgehog did not promote sebocyte differentiation in culture and cyclopamine treatment, while reducing proliferation, did not block Myc induced sebocyte differentiation in vivo. Our studies provide evidence for a bipotential epidermal stem cell population in an in vitro model of human epidermal lineage selection and highlight the importance of Myc as a regulator of sebocyte differentiation.

GLI1 repression of ERK activity correlates with colony formation and impaired migration in human epidermal keratinocytes

Basal cell carcinoma (BCC) of the skin is a highly compact, non-metastatic epithelial tumour type that may arise from the aberrant propagation of epidermal or progenitor stem cell (SC) populations. Increased expression of GLI1 is a common feature of BCC and is linked to the induction of epidermal SC markers in immortalized N/Tert-1 keratinocytes. Here, we demonstrate that GLI1 over-expression is linked to additional SC characteristics in N/Tert-1 cells including reduced epidermal growth factor receptor (EGFR) expression and compact colony formation that is associated with repressed extracellular signal-regulated kinase (ERK) activity. Colony formation and repressed ERK activity remain evident when EGFR is increased exogenously to the basal levels in GLI1 cells revealing that ERK is additionally inhibited downstream of the receptor. Exposure to epidermal growth factor (EGF) to increase ERK activity and promote migration negates GLI1 colony formation with cells displaying an elongated, fibroblast-like morphology. However, as determined by Snail messenger RNA and E-cadherin protein expression this is not associated with epithelial-mesenchymal transition (EMT), and GLI1 actually represses induction of the EMT marker vimentin in EGF-stimulated cells. Instead, live cell imaging revealed that the elongated morphology of EGF/GLI1 keratinocytes stems from their being 'stretched' due to migrating cells displaying inefficient cell-cell detachment and impaired tail retraction. Taken together, these data suggest that GLI1 opposes EGFR signalling to maintain the epithelial phenotype. Finally, ERK activity was predominantly negative in 13/14 BCCs (superficial/nodular), indicating that GLI1 does not routinely co-operate with ERK to induce the formation of this common skin tumour.

Efficient In Vivo Targeting of Epidermal Stem Cells by Early Gestational Intraamniotic Injection of Lentiviral Vector Driven by the Keratin 5 Promoter

At the present time, no efficient in vivo method for gene transfer to skin stem cells exists. In this study, we hypothesized that early in gestation, specific epidermal stem cell populations may be accessible for gene transfer. To test this hypothesis, we injected lentiviral vectors encoding the green fluorescence protein marker gene driven by either the cytomegalovirus promoter or the keratin 5 (K5) promoter into the murine amniotic space at early developmental stages between embryonic days 8 and 12. This resulted in sustained green fluorescent protein (GFP) expression in both basal epidermal stem cells and bulge cells in the hair follicles of the skin. Transduction of stem cell populations was dependent on the developmental stage, and confirmed by the prolonged duration of GFP expression in all skin elements into adulthood. In addition, transduced stem cell populations responded to regenerative signals after wounding and actively participated in wound healing. Finally, we quantified the fraction of epidermal stem cells transduced, and the distribution of transduction related to the promoters utilized, confirming improved efficiency with the K5 promoter. This simple approach has possible biological applications in our study of gene functions in skin, and perhaps future clinical applications for treatment of skin based disorders.

Neuregulin3 alters cell fate in the epidermis and mammary gland

BackgroundThe Neuregulin family of ligands and their receptors, the Erbb tyrosine kinases, have important roles in epidermal and mammary gland development as well as during carcinogenesis. Previously, we demonstrated that Neuregulin3 (Nrg3) is a specification signal for mammary placode formation in mice. Nrg3 is a growth factor, which binds and activates Erbb4, a receptor tyrosine kinase that regulates cell proliferation and differentiation. To understand the role of Neuregulin3 in epidermal morphogenesis, we have developed a transgenic mouse model that expresses Nrg3 throughout the basal layer (progenitor/stem cell compartment) of mouse epidermis and the outer root sheath of developing hair follicles.ResultsTransgenic females formed supernumerary nipples and mammary glands along and adjacent to the mammary line providing strong evidence that Nrg3 has a role in the initiation of mammary placodes along the body axis. In addition, alterations in morphogenesis and differentiation of other epidermal appendages were observed, including the hair follicles. The transgenic epidermis is hyperplastic with excessive sebaceous differentiation and shows striking similarities to mouse models in which c-Myc is activated in the basal layer including decreased expression levels of the adhesion receptors, α6-integrin and β1-integrin.ConclusionThese results indicate that the epidermis is sensitive to Nrg3 signaling, and that this growth factor can regulate cell fate of pluripotent epidermal cell populations including that of the mammary gland. Nrg3 appears to act, in part, by inducing c-Myc, altering the proliferation and adhesion properties of the basal epidermis, and may promote exit from the stem cell compartment. The results we describe provide significant insight into how growth factors, such as Nrg3, regulate epidermal homeostasis by influencing the balance between stem cell renewal, lineage selection and differentiation.

Skin keratinocytes pre-treated with embryonic stem cell-conditioned medium or BMP4 can be directed to an alternative cell lineage.

In this study, we have investigated whether secreted factors from embryonic stem cells (ESCs) could reprogramme keratinocytes and increase their potential to be directed into alternative cell lineages. Contact and non-contact co-cultures of skin keratinocytes and murine ESCs were used initially to confirm any reprogramming ability of ESC-conditioned medium (CM). Immunofluoresence was used to assess nuclear expression of octamer-4 (Oct-4), as well as to confirm neuronal protein expression in neuroectodermally directed keratinocytes. Transcript expression changes were evaluated using semiquantitative reverse transcription-polymerase chain reaction. Western blotting, accompanied by densitometry analysis, was used to evaluate protein expression following morphology changes. We found that keratinocytes treated with ESC-CM changed their morphology and were stimulated to express the pluripotency regulator, Oct-4, and its target transcripts, Sox-2, Nanog, Utf1 and Rex-1. We demonstrate that at least one of the reprogramming factors is bone morphogenetic factor-4 (BMP4). Pre-treated keratinocytes could be specifically directed to differentiate into cells of the neuronal lineage. The majority of responsive keratinocytes were the epidermal stem cell population, with a small percentage of transit-amplifying cells also being affected. Our results suggest that ESC-CM contains a number of factors, including BMP4, which are capable of reprogramming mouse skin keratinocytes to make them more developmentally potent, as evidenced by their ability to be re-differentiated into cells of the neuronal lineage. Our findings also imply a continuum of differentiation within the basal keratinocyte population. An increase in developmental potential combined with directed differentiation could increase the therapeutic relevancy of somatic cells.

Double Duty for Rac1 in Epidermal Wound Healing

During cutaneous wound healing, increased proliferation and migration of epidermal keratinocytes is essential for efficient re-epithelialization of the wound and restoration of barrier function to the skin. Although numerous cell culture studies have identified intracellular signaling proteins that control proliferation and migration in response to extracellular cues from the wound microenvironment, confirming their importance in wound healing requires appropriate in vivo models. The Rho-family guanosine triphosphatase (GTPase) Rac1 is an effector of cellular responses to growth factors, cytokines, and adhesion proteins present in wounds, and it has long been suspected to be an important regulator of wound healing. Two different genetic models now confirm an essential role for Rac1 in wound healing and, further, identify a dual role for Rac1 in promoting keratinocyte migration and proliferation during wound re-epithelialization. This sets the stage for determining which of the known Rac1 pathways are critical for wound repair in vivo and for linking these pathways to specific integrin or growth factor receptors that mediate cellular responses to cues from the wound environment. Together with studies that implicate Rac1 in maintaining epidermal stem cell populations, these findings lay the foundation for identifying distinct epidermal compartments from which Rac1 controls different aspects of wound re-epithelialization.

Thermolysin in human cultured keratinocyte isolation

When treating extensively burned patients using cultured epidermal sheets, the main problem is the time required for its production. Conventional keratinocyte isolation is usually done using Trypsin. We used a modification of the conventional isolation method in order to improve this process and increase the number of colonies from the isolated epidermal cell population. To compare the action of trypsin and thermolysin in the keratinocyte isolation using newborn foreskin. This method used thermolysin as it selectively digests the dermo-epidermal junction. After dermis separation, the epidermis was digested by trypsin in order to obtain a cell suspension. Compared to the conventional procedure, these experiments demonstrated that in the thermolysin group, the epidermis was easily detached from the dermis, there was no fibroblast contamination and there were a larger number of keratinocyte colonies which had a significant statistical difference. The number of colonies in the thermolysin group was significantly greater than in the trypsin group.

P63 expression pattern during rat epidermis morphogenesis and the role of p63 as a marker for epidermal stem cells

Recent evidence has suggested that p63 plays a particular role in maintaining the population of epidermal stem cells (ESCs), but the expression pattern of p63 during normal rat epidermis development is still unknown. We investigated the expression of p63 during development and differentiation of rat epidermis and identified whether p63 is a specific marker of rat ESCs cultured in vitro. Skin biopsies were obtained from embryonic day (E) 12 rat embryo, newborn, and 4-week-old rats. Immunohistochemistry was used to monitor the expression profile of p63. ESCs were isolated from newborn rat epidermis by rapid adhesion to IV collagen and examined by immunofluorescence staining to detect p63 and k14 expression. P63 expression was first observed at E12 when the epidermis was only a simple-layer surface ectoderm. P63 continued to be expressed as the epidermis matured but restricted to the basal layer. In adult trunk epidermis, p63 was expressed in both the hair follicle and the sebaceous glands. Immunofluorescence staining for ESCs was double positive for p63 and k14. These expression data suggest a role of p63 in the development of rat epidermis. P63 transcription factor may be a specific marker of keratinocyte progenitor cells.

Imiquimod as an antiaging agent

Topical imiquimod therapy has proven to be effective for a variety of infectious, neoplastic, and inflammatory dermatologic diseases. Several published reports have validated the benefit of imiquimod therapy for actinic keratoses and superficial melanoma and nonmelanoma skin cancers. There is, however, limited evidence demonstrating the use of topical imiquimod application as an antiaging treatment. We examined the effectiveness of imiquimod 5% cream in the treatment of photoaging by evaluating pretreatment and posttreatment biopsy specimens and documenting the histologic changes. This study represents an extension of an earlier project in our department in which patients with biopsy-proven lesions of lentigo maligna (LM) were recruited from a university dermatology service, a hospital, and referrals from private practitioners for an open-labeled efficacy trial with daily topical application of 5% imiquimod for 3 months. Biopsy of clinically affected skin was carried out on all patients before and after treatment. Using a semiquantitative method, biopsy specimens were analyzed for changes in the dermal collagen table (solar elastosis vs papillary dermal fibroplasia). Additional parameters analyzed included epidermal changes (atrophy vs acanthosis, melanin content, and hypergranulosis) and inflammatory effects (epidermal and dermal cell populations along with presence of pigment incontinence). Variables were compared using paired Wilcoxan rank sums. Of 26 evaluable patients who completed 3 months of daily application, 24 (>92.3%) showed a significant increase in papillary dermal fibroplasia (P < .0001) with associated reduction in solar elastosis (P = .0036). Other noteworthy findings were restoration of normal epidermal thickness (P = .0073) and melanization (P < .0001). This study only evaluates the effect of imiquimod in the lesional skin of LM. It is not known whether the results are applicable to nonlesional, photoaged skin. Topical imiquimod appears to induce reparative changes to the epidermis and the dermal collagen table in chronically sun-damaged skin associated with LM, indicating its potential use as an antiaging treatment. These findings need to be confirmed in photodamaged skin not associated with LM.

A double-blind, randomized quantitative comparison of calcitriol ointment and calcipotriol ointment on epidermal cell populations, proliferation and differentiation

Calcitriol and calcipotriol are widely used in the topical treatment of psoriasis. However, studies comparing both treatment modalities are scarce. Especially, there are almost no studies comparing the effects on epidermal cell populations in a quantitative manner. The aim of this study was to quantitatively compare the effects of topical calcitriol and topical calcipotriol on clinical scores and epidermal subpopulations. From five patients with stable plaque psoriasis, skin biopsies were taken from two symmetrical regions on the trunk or extremities before and after treatment with either calcitriol or calcipotriol. Frozen sections were labelled immunofluorescently using direct immunofluorescence for beta-1 integrin and the Zenon labelling technique for keratin (K) 6, K10 and K15. The digital photographs of the stained sections were quantitatively analysed and the results of both treatments were compared. The clinical SUM-score improved significantly for both the calcitriol- and the calcipotriol-treated lesions. In the calcipotriol-treated group the expression of K10 and K15 increased and the expression of K6 decreased significantly. No changes were seen for the marker beta-1 integrin. In the calcitriol-treated group none of the markers changed significantly. A tendency towards significance was seen for the changes in the expression of K6 and K15 in favour of calcipotriol. Both calcitriol and calcipotriol gave a significant improvement in clinical scores. However, treatment with calcipotriol resulted in a normalization of K6, K10 and K15, whereas treatment with calcitriol did not. Comparison of both treatments showed a tendency towards significance for the above-mentioned markers for calcipotriol only.

Aberrant accumulation of p53 associates with Ki67 and mitotic count in benign skin lesions.

Sixty-two skin samples from patients with a variety of benign disorders (20 cases of psoriasis, 14 cases of chronic dermatitis, 11 seborrhoeic keratoses, 11 cases of lichen planus), and seven normal skin samples, were stained immunohistochemically with a polyclonal antibody (CM-1) to p53, and a monoclonal antibody to Ki67, using the avidin-biotin complex method. p53-positive keratinocytes could be found in most of these lesions. The percentage of p53-positive cells was, however, far lower than usually seen in p53-positive malignant tumours. No p53 reactivity was observed in the normal skin samples. Variable Ki67 reactivity was observed in all skin samples. Overall, the number of Ki67-positive cells was higher in skin samples in which the proportion of p53-positive cells was high (> 0.5% of total epidermal cell population) (P = 0.004). This also applied separately to psoriatic and non-psoriatic lesions (P = 0.028 and P = 0.033, respectively). In cases with > 10% of Ki67-positive cells, there were significantly more mitoses (P < 0.001). This association applied to both psoriasis and the other lesions studied (P = 0.024 and P < 0.001, respectively). The results show that immunohistochemically detectable accumulation of p53 is a frequent finding in non-neoplastic skin lesions. As p53 positivity was associated with the proliferation marker Ki67, the accumulation of p53 is possibly a response to an increased proliferation rate of the keratinocytes in these skin diseases, or alternatively it may be associated with apoptosis.

P63 Is an Essential Proapoptotic Protein during Neural Development

The p53 family member p63 is required for nonneural development, but has no known role in the nervous system. Here, we define an essential proapoptotic role for p63 during naturally occurring neuronal death. Sympathetic neurons express full-length TAp63 during the developmental death period, and TAp63 levels increase following NGF withdrawal. Overexpression of TAp63 causes neuronal apoptosis in the presence of NGF, while cultured p63-/- neurons are resistant to apoptosis following NGF withdrawal. TAp63 is also essential in vivo, since embryonic p63-/- mice display a deficit in naturally occurring sympathetic neuron death. While both TAp63 and p53 induce similar apoptotic signaling proteins and require BAX expression and function for their effects, TAp63 induces neuronal death in the absence of p53, but p53 requires coincident p63 expression for its proapoptotic actions. Thus, p63 is essential for developmental neuronal death, likely functioning both on its own, and as an obligate proapoptotic partner for p53.

Effect of calcipotriol on epidermal cell populations in alefacept‐treated psoriatic lesions

The effect of the established antipsoriatic treatment with topical calcipotriol (with a maximum of 100 g per week) in addition to systemic treatment with alefacept, a new biological agent for psoriasis, on epidermal cell populations in the psoriatic lesion was investigated using a combination of the Zenon labelling technique and microscopic image analysis. Epidermal cell populations were measured quantitatively with this sensitive method. Frozen sections of non-treated psoriatic epidermis and psoriatic epidermis treated with either alefacept intramuscular or alefacept intramuscular in combination with topical calcipotriol for 12 weeks were compared immunohistochemically. Antibodies against keratin 6, 10 and 15 were labelled with the Zenon technique, whereas antibodies against the Ki-67 antigen and beta-1 integrin were covalently Fluorescein Isothiocyanate (FITC)-labelled. Using image analysis, these markers were measured in the epidermis in a standardized manner. Treatment of psoriasis with alefacept resulted in a good clinical response in several patients and in a normalization of epidermal expression of the immunohistochemical parameters for differentiation and proliferation. The addition of topical calcipotriol resulted in a faster clinical improvement with a similar overall clinical response and a similar response of epidermal cell populations as compared to treatment with alefacept monotherapy after 12 weeks of treatment. This study also suggests that the appearance of keratin 15 has a predictive value for the duration of remission. It can be concluded that the addition of a low-dose calcipotriol treatment does not contribute to the clinical efficacy of alefacept, both at the clinical level and with respect to markers for epidermal proliferation and differentiation.

Dietary Feeding of Silibinin Prevents Early Biomarkers of UVB Radiation–Induced Carcinogenesis in SKH-1 Hairless Mouse Epidermis

Solar radiation is the causal etiologic factor in the development of nonmelanoma skin cancer (NMSC). Depletion of the stratospheric ozone layer leads to an increase in ambient UV radiation loads, which are expected to further raise skin cancer incidence in many temperate parts of the world, including the United States, suggesting that skin cancer chemopreventive approaches via biomarker efficacy studies or vice versa are highly warranted. Based on our recent study reporting strong efficacy of silibinin against photocarcinogenesis, we assessed here the protective effects of its dietary feeding on UVB-induced biomarkers involved in NMSC providing a mechanistic rationale for an early-on silibinin efficacy in skin cancer prevention. Dietary feeding of silibinin at 1% dose (w/w) to SKH-1 hairless mice for 2 weeks before a single UVB irradiation at 180 mJ/cm(2) dose resulted in a strong and significant (P < 0.001) decrease in UVB-induced thymine dimer-positive cells and proliferating cell nuclear antigen, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and apoptotic sunburn cells together with an increase (P < 0.001) in p53 and p21/cip1-positive cell population in epidermis. These findings suggest that dietary feeding of silibinin affords strong protection against UVB-induced damages in skin epidermis by (a) either preventing DNA damage or enhancing repair, (b) reducing UVB-induced hyperproliferative response, and (c) inhibiting UVB-caused apoptosis and sunburn cell formation, possibly via silibinin-caused up-regulation of p53 and p21/cip1 as major UVB-damage control sensors.