Survival of skin cancer stem cells requires the Ezh2 polycomb group protein.

Polycomb group proteins, including Ezh2, are important candidate stem cell maintenance proteins in epidermal squamous cell carcinoma. We previously showed that epidermal cancer stem cells (ECS cells) represent a minority of cells in tumors, are highly enriched in Ezh2 and drive aggressive tumor formation. We now show that Ezh2 is required for ECS cell survival, migration, invasion and tumor formation and that this is associated with increased histone H3 trimethylation on lysine 27, a mark of Ezh2 action. We also show that Ezh2 knockdown or treatment with Ezh2 inhibitors, GSK126 or EPZ-6438, reduces Ezh2 level and activity, leading to reduced ECS cell spheroid formation, migration, invasion and tumor growth. These studies indicate that epidermal squamous cell carcinoma cells contain a subpopulation of cancer stem (tumor-initiating) cells that are enriched in Ezh2, that Ezh2 is required for optimal ECS cell survival and tumor formation and that treatment with Ezh2 inhibitors may be a strategy for reducing ECS cell survival and suppressing tumor formation.

The Bmi-1 helix-turn and ring finger domains are required for Bmi-1 antagonism of (-) epigallocatechin-3-gallate suppression of skin cancer cell survival.

The Bmi-1 Polycomb group (PcG) protein is an important epigenetic regulator of chromatin status. Elevated Bmi-1 expression is observed in skin cancer and contributes to cancer cell survival. (-) Epigallocatechin-3-gallate (EGCG), an important green tea-derived cancer prevention agent, reduces Bmi-1 level resulting in reduced skin cancer cell survival. This is associated with increased p21(Cip1) and p27(Kip1) expression, reduced cyclin, and cyclin dependent kinase expression, and increased cleavage of apoptotic markers. These EGCG-dependent changes are attenuated by vector-mediated maintenance of Bmi-1 expression. In the present study, we identify Bmi-1 functional domains that are required for this response. Bmi-1 expression reverses the EGCG-dependent reduction in SCC-13 cell survival, but Bmi-1 mutants lacking the helix-turn-helix-turn-helix-turn (Bmi-1ΔHT) or ring finger (Bmi-1ΔRF) domains do not reverse the EGCG impact. The reduction in Ring1B ubiquitin ligase activity, observed in the presence of mutant Bmi-1, is associated with reduced ability of these mutants to interact with and activate Ring1B ubiquitin ligase, the major ligase responsible for the ubiquitination of histone H2A during chromatin condensation. This results in less chromatin condensation leading to increased tumor suppressor gene expression and reduced cell survival; thereby making the cells more susceptible to the anti-survival action of EGCG. We further show that these mutants act in a dominant-negative manner to inhibit the action of endogenous Bmi-1. Our results suggest that the HT and RF domains are required for Bmi-1 ability to maintain skin cancer cell survival in response to cancer preventive agents.

Identification of a population of epidermal squamous cell carcinoma cells with enhanced potential for tumor formation.

Epidermal squamous cell carcinoma is among the most common cancers in humans. These tumors are comprised of phenotypically diverse populations of cells that display varying potential for proliferation and differentiation. An important goal is identifying cells from this population that drive tumor formation. To enrich for tumor-forming cells, cancer cells were grown as spheroids in non-attached conditions. We show that spheroid-selected cells form faster growing and larger tumors in immune-compromised mice as compared to non-selected cells. Moreover, spheroid-selected cells gave rise to tumors following injection of as few as one hundred cells, suggesting these cells have enhanced tumor-forming potential. Cells isolated from spheroid-selected tumors retain an enhanced ability to grow as spheroids when grown in non-attached culture conditions. Thus, these tumor-forming cells retain their phenotype following in vivo passage as tumors. Detailed analysis reveals that spheroid-selected cultures are highly enriched for expression of epidermal stem cell and embryonic stem cell markers, including aldehyde dehydrogenase 1, keratin 15, CD200, keratin 19, Oct4, Bmi-1, Ezh2 and trimethylated histone H3. These studies indicate that a subpopulation of cells that possess stem cell-like properties and express stem cell markers can be derived from human epidermal cancer cells and that these cells display enhanced ability to drive tumor formation.

Biochemistry of epidermal stem cells.

BACKGROUND: The epidermis is an important protective barrier that is essential for maintenance of life. Maintaining this barrier requires continuous cell proliferation and differentiation. Moreover, these processes must be balanced to produce a normal epidermis. The stem cells of the epidermis reside in specific locations in the basal epidermis, hair follicle and sebaceous glands and these cells are responsible for replenishment of this tissue. SCOPE OF REVIEW: A great deal of effort has gone into identifying protein epitopes that mark stem cells, in identifying stem cell niche locations, and in understanding how stem cell populations are related. We discuss these studies as they apply to understanding normal epidermal homeostasis and skin cancer. MAJOR CONCLUSIONS: An assortment of stem cell markers have been identified that permit assignment of stem cells to specific regions of the epidermis, and progress has been made in understanding the role of these cells in normal epidermal homeostasis and in conditions of tissue stress. A key finding is the multiple stem cell populations exist in epidermis that give rise to different structures, and that multiple stem cell types may contribute to repair in damaged epidermis. GENERAL SIGNIFICANCE: Understanding epidermal stem cell biology is likely to lead to important therapies for treating skin diseases and cancer, and will also contribute to our understanding of stem cells in other systems. This article is part of a Special Issue entitled Biochemistry of Stem Cells.

A proteasome inhibitor-stimulated Nrf1 protein-dependent compensatory increase in proteasome subunit gene expression reduces polycomb group protein level.

The polycomb group (PcG) proteins, Bmi-1 and Ezh2, are important epigenetic regulators that enhance skin cancer cell survival. We recently showed that Bmi-1 and Ezh2 protein level is reduced by treatment with the dietary chemopreventive agents, sulforaphane and green tea polyphenol, and that this reduction involves ubiquitination of Bmi-1 and Ezh2, suggesting a key role of the proteasome. In the present study, we observe a surprising outcome that Bmi-1 and Ezh2 levels are reduced by treatment with the proteasome inhibitor, MG132. We show that this is associated with a compensatory increase in the level of mRNA encoding proteasome protein subunits in response to MG132 treatment and an increase in proteasome activity. The increase in proteasome subunit level is associated with increased Nrf1 and Nrf2 level. Moreover, knockdown of Nrf1 attenuates the MG132-dependent increase in proteasome subunit expression and restores Bmi-1 and Ezh2 expression. The MG132-dependent loss of Bmi-1 and Ezh2 is associated with reduced cell proliferation, accumulation of cells in G(2), and increased apoptosis. These effects are attenuated by forced expression of Bmi-1, suggesting that PcG proteins, consistent with a prosurvival action, may antagonize the action of MG132. These studies describe a compensatory Nrf1-dependent, and to a lesser extent Nrf2-dependent, increase in proteasome subunit level in proteasome inhibitor-treated cells and confirm that PcG protein levels are regulated by proteasome activity.

Sulforaphane suppresses polycomb group protein level via a proteasome-dependent mechanism in skin cancer cells.

The polycomb group (PcG) genes encode a family of proteins that methylate and ubiquitinate histones to close chromatin and suppress gene expression. PcG proteins are present at elevated levels in cancer cells, and this is associated with reduced tumor suppressor protein level and enhanced cell survival. Agents that reduce PcG protein level are regarded as potentially cancer-preventative agents. Sulforaphane (SFN) is a biologically important isothiocyanate found in cruciferous vegetables that is an important candidate chemopreventive agent. However, the impact of SFN on the level and function of PcG proteins in skin cancer cells has not been assessed. We show that SFN treatment causes a concentration-dependent reduction in PcG protein (Bmi-1, Ezh2) expression in SCC-13 skin cancer cells and also reduces trimethylation of lysine 27 of histone H3. This is associated with accumulation of cells in G(2)/M phase; reduced levels of cyclin B1, cyclin A, cyclin dependent kinases 1 and 2; and increased p21(Cip1) expression. Sulforaphane treatment also increases cleavage of procaspase 3, 8, and 9 and enhances PARP cleavage and apoptosis. Similar results are observed in other skin-derived cell immortalized and transformed cell lines. Forced expression of the Bmi-1 polycomb protein in SCC-13 cells reverses these effects. The SFN-dependent loss of Bmi-1 and Ezh2 is due to proteasome-associated degradation. These results suggest that dietary isothiocyanates may suppress cancer progression by reducing PcG protein level via a proteasome-dependent mechanism, thereby inhibiting PcG-dependent pro-survival epigenetic events.

(-)-Epigallocatechin-3-gallate and DZNep reduce polycomb protein level via a proteasome-dependent mechanism in skin cancer cells.

Polycomb group (PcG) protein-dependent histone methylation and ubiquitination drives chromatin compaction leading to reduced tumor suppressor expression and increased cancer cell survival. Green tea polyphenols and S-adenosylhomocysteine (AdoHcy) hydrolase inhibitors are important candidate chemopreventive agents. Previous studies indicate that (-)-epigallocatechin-3-gallate (EGCG), a potent green tea polyphenol, suppresses PcG protein level and skin cancer cell survival. Inhibition of AdoHcy hydrolase with 3-deazaneplanocin A (DZNep) inhibits methyltransferases by reducing methyl group availability. In the present study, we examine the impact of EGCG and DZNep cotreatment on skin cancer cell function. EGCG and DZNep, independently and in combination, reduce the level of PcG proteins including Ezh2, eed, Suz12, Mel18 and Bmi-1. This is associated with reduced H3K27me3 and H2AK119ub formation, histone modifications associated with closed chromatin. Histone deacetylase 1 level is also reduced and acetylated H3 formation is increased. These changes are associated with increased tumor suppressor expression and reduced cell survival and are partially reversed by vector-mediated maintenance of Bmi-1 level. The reduction in PcG protein level is associated with increased ubiquitination and is reversed by proteasome inhibitors, suggesting proteasome-associated degradation.

Polycomb group proteins are key regulators of keratinocyte function.

The Polycomb group (PcG) proteins are epigenetic suppressors of gene expression that function through modification of histones to change chromatin structure and modulate gene expression and cell behavior. Recent studies show that PcG proteins are expressed in epidermis, that their levels change during differentiation and in disease states, and that PcG expression is regulated by agents that influence cell proliferation and survival. The results indicate that PcG proteins regulate keratinocyte cell-cycle progression, apoptosis, senescence, and differentiation. These proteins are expressed in progenitor cells, in the basal layer, and in suprabasal keratinocytes, and the level, timing, and distribution of expression suggest that the PcG proteins have a central role in maintaining the balance between cell survival and death in multiple epidermal compartments. Additional studies indicate an important role in skin cancer progression.

The Bmi-1 polycomb protein antagonizes the (-)-epigallocatechin-3-gallate-dependent suppression of skin cancer cell survival.

The polycomb group (PcG) proteins are epigenetic regulators of gene expression that enhance cell survival. This regulation is achieved via action of two multiprotein PcG complexes--PRC2 (EED) and PRC1 [B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1)]. These complexes modulate gene expression by increasing histone methylation and reducing acetylation--leading to a closed chromatin conformation. Activity of these proteins is associated with increased cell proliferation and survival. We show increased expression of key PcG proteins in immortalized keratinocytes and skin cancer cell lines. We examine the role of two key PcG proteins, Bmi-1 and enhancer of zeste homolog 2 (Ezh2), and the impact of the active agent in green tea, (-)-epigallocatechin-3-gallate (EGCG), on the function of these regulators. EGCG treatment of SCC-13 cells reduces Bmi-1 and Ezh2 level and this is associated with reduced cell survival. The reduction in survival is associated with a global reduction in histone H3 lysine 27 trimethylation, a hallmark of PRC2 complex action. This change in PcG protein expression is associated with reduced expression of key proteins that enhance progression through the cell cycle [cyclin-dependent kinase (cdk)1, cdk2, cdk4, cyclin D1, cyclin E, cyclin A and cyclin B1] and increased expression of proteins that inhibit cell cycle progression (p21 and p27). Apoptosis is also enhanced, as evidenced by increased caspase 9, 8 and 3 cleavage and increased poly(adenosine diphosphate ribose) polymerase cleavage. EGCG treatment also increases Bax and suppresses Bcl-xL expression. Vector-mediated enhanced Bmi-1 expression reverses these EGCG-dependent changes. These findings suggest that green tea polyphenols reduce skin tumor cell survival by influencing PcG-mediated epigenetic regulatory mechanisms.

Multiple PKCdelta tyrosine residues are required for PKCdelta-dependent activation of involucrin expression--a key role of PKCdelta-Y311.

Protein kinase C-delta (PKCdelta) is a key regulator of human involucrin (hINV) gene expression and is regulated by tyrosine phosphorylation. However, a comprehensive analysis of the requirement for individual PKCdelta tyrosine residues is lacking. We show that multiple tyrosine residues influence the ability of PKCdelta to increase hINV gene expression. Mutation of individual PKCdelta tyrosine residues 52, 64, 155, 187, or 565 does not reduce the ability of PKCdelta to increase hINV promoter activity; however, simultaneous mutation of these five tyrosines markedly reduces activity. Moreover, restoration of any one of these residues results in nearly full activity restoration. It is significant that phosphorylation of PKCdelta-Y(311) is reduced in the five-tyrosine mutant and that mutation of Y(311) results in reduced PKCdelta activity comparable to that observed in the five-tyrosine mutant. Restoration of any one of the tyrosine residues in the five-tyrosine mutant restores Y(311) phosphorylation and biological activity. In addition, reduced phosphorylation of endogenous PKCdelta-Y(311) is associated with reduced biological activity. These findings indicate that PKCdelta activity requires Y(311) and a second tyrosine residue; however, any one of the several tyrosine residues can serve in the secondary role.

Expression of Bmi-1 in epidermis enhances cell survival by altering cell cycle regulatory protein expression and inhibiting apoptosis.

The polycomb group (PcG) genes are epigenetic suppressors of gene expression that play an important role in development. In this study, we examine the role of Bmi-1 (B-cell-specific Moloney murine leukemia virus integration site 1) as a regulator of human epidermal keratinocyte survival. We identify Bmi-1 mRNA and protein expression in epidermis and in cultured human keratinocytes. Bmi-1 is located in the nucleus in cultured keratinocytes, and in epidermis it is expressed in the basal and suprabasal layers. Adenovirus-delivered Bmi-1 promotes keratinocyte survival and protects keratinocytes from stress agent-mediated cell death. This is associated with increased levels of cyclin D1 and selected cyclin-dependent kinases, and reduced caspase activity and poly(ADP-ribose) polymerase (PARP) cleavage. Bmi-1 may be involved in the maintenance of disease state, as Bmi-1 levels are elevated in transformed keratinocytes, skin tumors, and psoriasis. The presence of Bmi-1 in suprabasal non-proliferative cells of the epidermis and within a high percentage of cells within skin tumors suggests a non-stem cell pro-survival role for Bmi-1 in this tissue. Based on the suprabasal distribution of Bmi-1 in epidermis, we propose that Bmi-1 may promote maintenance of suprabasal keratinocyte survival to prevent premature death during differentiation. Such a function would help assure proper formation of the stratified epidermis.

Keratinocyte proliferation, differentiation, and apoptosis--differential mechanisms of regulation by curcumin, EGCG and apigenin.

We have proposed that it is important to examine the impact of chemopreventive agents on the function of normal human epidermal keratinocytes since these cells comprise the barrier that protects the body from a range of environmental insults. In this context, it is widely appreciated that cancer may be retarded by consumption or topical application of naturally occurring food-derived chemopreventive agents. Our studies show that (-)-epigallocatechin-3-gallate (EGCG), a green tea-derived polyphenol, acts to enhance the differentiation of normal human keratinocytes as evidenced by its ability to increase involucrin (hINV), transglutaminase type 1 (TG1) and caspase-14 gene expression. EGCG also stimulates keratinocyte morphological differentiation. These actions of EGCG are mediated via activation of a nPKC, Ras, MEKK1, MEK3, p38delta-ERK1/2 signaling cascade which leads to increased activator protein 1 (AP1) and CAATT enhancer binding protein (C/EBP) transcription factor expression, increased binding of these factors to DNA, and increased gene transcription. In contrast, apigenin, a dietary flavonoid derived from plants and vegetables, and curcumin, an agent derived from turmeric, inhibit differentiation by suppressing MAPK signal transduction and reducing API transcription factor level. Curcumin also acts to enhance apoptosis, although EGCG and apigenin do not stimulate apoptosis. In addition, all of these agents inhibit keratinocyte proliferation. These findings indicate that each of these diet-derived chemopreventive agents has a profound impact on normal human keratinocyte function and that they operate via distinct and sometimes opposing mechanisms. However, all are expected to act as chemopreventive agents.

Curcumin suppresses AP1 transcription factor-dependent differentiation and activates apoptosis in human epidermal keratinocytes.

The diet-derived cancer preventive agent, curcumin, inhibits skin cancer cell proliferation and tumor formation. However, its effect on normal human keratinocyte differentiation, proliferation, and apoptosis has not been adequately studied. Involucrin (hINV) is a marker of keratinocyte differentiation and a useful model for the study of chemopreventive agent action. We show that curcumin suppresses the differentiation agent-dependent activation of hINV gene expression and that an AP1 transcription factor DNA binding site in the hINV gene is required for this regulation. A protein kinase C, Ras, MEKK1, MEK3 signaling cascade controls hINV expression by regulating AP1 factor level. Curcumin treatment inhibits the novel protein kinase C-, Ras-, and MEKK1-dependent activation of hINV promoter activity and reduces the differentiation agent-dependent increase in AP1 factor level and DNA binding. This reduction requires proteasome function. In addition, curcumin treatment reduces cell number, which is associated with a reduced cyclin and cdk1 levels. Curcumin treatment also suppresses the Bcl-xL level, leading to reduced mitochondrial membrane potential and increased cleavage of procaspases and poly(ADP-ribose) polymerase. These studies provide important insights regarding the mechanism whereby curcumin acts as a chemopreventive agent in normal human epidermis.

Apigenin inhibition of involucrin gene expression is associated with a specific reduction in phosphorylation of protein kinase Cdelta Tyr311.

Apigenin is a plant-derived flavanoid that has significant promise as a skin cancer chemopreventive agent. In the present study, we examine the mechanism whereby apigenin regulates normal human keratinocyte differentiation. Expression of involucrin (hINV), a marker of keratinocyte differentiation, is increased by differentiating agents via a protein kinase Cdelta (PKCdelta), Ras, MEKK1, MEK3 cascade that increases AP1 factor level and AP1 factor binding to DNA elements in the hINV promoter. We show that apigenin inhibits this response. Apigenin suppresses the 12-O-tetradeconylphorbol-13-acetate-dependent increase in AP1 factor expression and binding to the hINV promoter and the increase in hINV promoter activity. Apigenin also inhibits the increase in promoter activity observed following overexpression of PKCdelta, constitutively active Ras, or MEKK1. The suppression of PKCdelta activity is associated with reduced phosphorylation of PKCdelta-Y311. The physiological importance of this phosphorylation event was confirmed by showing that the PKCdelta phosphorylation-defective mutant, PKCdelta-Y311F, is less able to increase hINV promoter activity. Activation of hINV promoter activity by the green tea polyphenol, (-)-epigellocathecin-3-gallate, is also inhibited by apigenin, suggesting that the two chemopreventive agents can produce opposing actions in keratinocytes. Additional studies show that the apigenin-dependent suppression of differentiation is associated with reduced cell proliferation but that there is no evidence of apoptosis.

Opposing action of curcumin and green tea polyphenol in human keratinocytes.

Persistent environmental insult can convert a normal cell into a cancer cell. However, various natural chemopreventive agents called antioxidants can retard this progression. We have recently explored the effects of several chemopreventive agents, including green tea polyphenol and curcumin, on normal human keratinocyte function. Our findings suggest that a bioactive polyphenol from green tea, (-)-epigallocatechin-3-gallate (EGCG), acts to increase involucrin gene expression, suggesting that EGCG treatment enhances normal human keratinocyte differentiation. Mechanistic studies indicate that EGCG alters mitogen-activated protein kinase cascade function to activate involucrin gene transcription via a Ras, MEKK1, MEK3, ERK1/2-p38delta cascade that targets AP1 and CAATT enhancer binding protein transcription factors. These findings suggest that EGCG may inhibit disease progression by promoting keratinocyte differentiation. Parallel studies indicate that not all antioxidants produce a similar response. Curcumin, an antioxidant derived from the turmeric, antagonizes the EGCG-dependent response by interfering in this signaling pathway. These studies suggest that different antioxidant may produce antagonistic effects in tissues.

A novel retinoid-related molecule inhibits pancreatic cancer cell proliferation by a retinoid receptor independent mechanism via suppression of cell cycle regulatory protein function and induction of caspase-associated apoptosis.

Retinoid-related molecules are important potential agents for the treatment of cancer. In the present study, we test the effect of a novel retinoid-related ligand, AGN193198 (4-[3-(1-heptyl-4,4-dimethyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)-3-oxo-prophenyl] benzoic acid), on pancreatic cancer cell proliferation and survival. AGN193198 treatment reduces BxPC-3 cell proliferation more efficiently than high-affinity retinoid acid receptor (RAR)- or retinoid X receptor (RXR)-selective retinoids. Moreover, AGN193198 does not activate transcription from RAR or RXR response elements and its effects on cell survival are not reversed by treatment with RAR- or RXR receptor-selective antagonists. These results suggest that the AGN193198-dependent inhibition of BxPC-3 cell function is not mediated via activation of the classical retinoid receptors. Cell cycle analysis of AGN193198-treated BxPC-3 cells indicates that AGN193198 causes accumulation of cells in G2/M. This change is associated with a marked reduction in regulators of S (cyclin A, cyclin-dependent kinase (cdk)2), G2/M (cyclin B1, cdk1, cdc25c) and G1 (cyclin D1, cyclin E, cdk2, cdk4) phase, and an increase in p21 and p27 level. Kinases assays reveal that cdk1, cdk2 and cdk4 activity are suppressed in AGN193198-treated cells. In addition, reduced cell proliferation is associated with enhanced procaspase (3, 8 and 9) and PARP cleavage. Z-VAD-FMK, a pancaspase inhibitor, inhibits AGN193198-dependent caspase activation and attenuates cell death. Z-VAD-FMK inhibits PARP cleavage, but does not alter the AGN193198-dependent reduction in cell cycle regulatory protein expression and activity, suggesting that caspase activation and suppression of cell cycle regulatory protein levels are independent processes. AGN193198 produces similar responses in other pancreatic cancer cell lines including AsPC-1 and MIA PaCa-2. These studies suggest that AGN193198 may be useful for the treatment of pancreatic cancer.

Human epidermal keratinocytes undergo (-)-epigallocatechin-3-gallate-dependent differentiation but not apoptosis.

Epigallocatechin-3-gallate (EGCG) is an important chemopreventive agent derived from green tea. We recently reported that EGCG treatment enhances keratinocyte differentiation as evidenced by increased human involucrin promoter activity [Balasubramanian,S., Efimova,T. and Eckert,R.L. (2002) J. Biol. Chem., 277, 1828-1836]. In the present paper, we extend these findings and show that EGCG also increases the expression of other differentiation markers-procaspase 14 and type I transglutaminase (TG1). Both TG1 mRNA and protein level, and activity are increased by treatment with EGCG. Increased TG1 activity is evidenced by a direct transglutaminase assay, and by the ability of EGCG to stimulate the covalent incorporation of fluorescein cadaverine substrate into crosslinked intracellular structures. In contrast, type II transglutaminase levels are not altered by EGCG treatment. We also assessed whether EGCG promotes keratinocyte apoptosis. We show that EGCG treatment does not promote the cleavage of procaspase-3, -8, -9 or poly(ADP-ribose) polymerase. Moreover, treatment with the pan-caspase inhibitor, Z-VAD-FMK, does not reverse the EGCG-associated reduction in cell viability. In addition, there is no increase in cells having sub-G(1)/S DNA content, and no evidence for the release of cytochrome c from the mitochondria. These findings confirm, using several endpoints, that EGCG treatment enhances normal keratinocyte differentiation but does not promote apoptosis.

Antioxidants regulate normal human keratinocyte differentiation.

Cancer begins with a normal cell that, due to persistent environmental insult, is transformed, via a series of progressively more insidious steps, into a cancer cell. A major goal of chemopreventive therapy is to alter the normal cell response to the environmental agent with the goal of inhibiting disease progression. (-)-Epigallocatechin-3-gallate (EGCG) is an important bioactive green tea antioxidant that possesses remarkable cancer chemopreventive properties. We have recently explored the hypothesis that EGCG prevents cancer by promoting keratinocyte differentiation. Based on our findings, we argue that EGCG acts to enhance the differentiation of normal keratinocytes. This is a potentially important finding, as it represents a novel mechanism of disease inhibition by EGCG--cancer preventive "differentiation therapy". However, not all antioxidant chemopreventive agents work by this mechanism. Curcumin, for example, inhibits the differentiation-promoting activity of EGCG. This report discusses the mechanism of EGCG and curcumin action in regulating expression of involucrin, a marker of keratinocyte differentiation.

Regulation of involucrin gene expression.

The epidermis is a dynamic renewing structure that provides life-sustaining protection from the environment. The major cell type of the epidermis, the epidermal keratinocyte, undergoes a carefully choreographed program of differentiation. Alteration of these events results in a variety of debilitating and life-threatening diseases. Understanding how this process is regulated is an important current goal in biology. In this review, we summarize the literature regarding regulation of involucrin, an important marker gene that serves as a model for understanding the mechanisms that regulate the differentiation process. Current knowledge describing the role of transcription factors and signaling cascades in regulating involucrin gene expression are presented. These studies describe a signaling cascade that includes the novel protein kinase C isoforms, Ras, MEKK1, MEK3, and a p38delta-extracellular signal regulated kinase 1/2 complex. This cascade regulates activator protein one, Sp1, and CCATT/enhancer-binding protein transcription factor DNA binding to two discrete involucrin promoter regions, the distal- and proximal-regulatory regions, to regulate involucrin gene expression.