The amino-terminal domain of desmoplakin binds to plakoglobin and clusters desmosomal cadherin-plakoglobin complexes.

The desmosome is a highly organized plasma membrane domain that couples intermediate filaments to the plasma membrane at regions of cell-cell adhesion. Desmosomes contain two classes of cadherins, desmogleins, and desmocollins, that bind to the cytoplasmic protein plakoglobin. Desmoplakin is a desmosomal component that plays a critical role in linking intermediate filament networks to the desmosomal plaque, and the amino-terminal domain of desmoplakin targets desmoplakin to the desmosome. However, the desmosomal protein(s) that bind the amino-terminal domain of desmoplakin have not been identified. To determine if the desmosomal cadherins and plakoglobin interact with the amino-terminal domain of desmoplakin, these proteins were co-expressed in L-cell fibroblasts, cells that do not normally express desmosomal components. When expressed in L-cells, the desmosomal cadherins and plakoglobin exhibited a diffuse distribution. However, in the presence of an amino-terminal desmoplakin polypeptide (DP-NTP), the desmosomal cadherins and plakoglobin were observed in punctate clusters that also contained DP-NTP. In addition, plakoglobin and DP-NTP were recruited to cell-cell interfaces in L-cells co-expressing a chimeric cadherin with the E-cadherin extracellular domain and the desmoglein-1 cytoplasmic domain, and these cells formed structures that were ultrastructurally similar to the outer plaque of the desmosome. In transient expression experiments in COS cells, the recruitment of DP-NTP to cell borders by the chimera required co-expression of plakoglobin. Plakoglobin and DP-NTP co-immunoprecipitated when extracted from L-cells, and yeast two hybrid analysis indicated that DP-NTP binds directly to plakoglobin but not Dsg1. These results identify a role for desmoplakin in organizing the desmosomal cadherin-plakoglobin complex and provide new insights into the hierarchy of protein interactions that occur in the desmosomal plaque.

The proapoptotic tumor suppressor protein kinase C-delta is lost in human squamous cell carcinomas.

Protein kinase C (PKC)-delta is proapoptotic in human keratinocytes, and is downregulated or inactivated in keratinocytes expressing the activated Ha-ras oncogene, making it a candidate tumor suppressor gene for squamous cell carcinoma (SCC). We evaluated the significance of PKC-delta loss in transformed human keratinocytes using tumorigenic HaCaT Ras II-4 cells that have significantly reduced PKC-delta levels. Re-expression of PKC-delta by retrovirus transduction caused an increase in apoptosis and growth inhibition in culture. The growth inhibition induced by PKC-delta could be partially reversed by Bcl-x(L) expression, indicating that apoptosis was in part responsible for PKC-delta-induced growth inhibition. PKC-delta re-expression suppressed the tumorigenicity of HaCaT Ras II-4 cells in nude mice (P<0.05), and the small tumors that did form contained elevated levels of activated caspase-3, indicating increased apoptosis. In addition, we found that 29% (12/42) of human Bowen's disease (squamous carcinoma in situ) or SCC cases had absent or reduced PKC-delta when compared to the surrounding normal epidermis. These results indicate that PKC-delta inhibits transformed keratinocyte growth by inducing apoptosis, and that PKC-delta may function as a tumor suppressor in human SCCs where its loss in cells harboring activated ras could provide a growth advantage by conferring resistance to apoptosis.

Protein kinase C family: on the crossroads of cell signaling in skin and tumor epithelium.

The protein kinase C (PKC) family represents a large group of phospholipid dependent enzymes catalyzing the covalent transfer of phosphate from ATP to serine and threonine residues of proteins. Phosphorylation of the substrate proteins induces a conformational change resulting in modification of their functional properties. The PKC family consists of at least ten members, divided into three subgroups: classical PKCs (alpha, betaI, betaII, gamma), novel PKCs (delta, epsilon, eta, theta), and atypical PKCs (zeta, iota/lambda). The specific cofactor requirements, tissue distribution, and cellular compartmentalization suggest differential functions and fine tuning of specific signaling cascades for each isoform. Thus, specific stimuli can lead to differential responses via isoform specific PKC signaling regulated by their expression, localization, and phosphorylation status in particular biological settings. PKC isoforms are activated by a variety of extracellular signals and, in turn, modify the activities of cellular proteins including receptors, enzymes, cytoskeletal proteins, and transcription factors. Accordingly, the PKC family plays a central role in cellular signal processing. Accumulating data suggest that various PKC isoforms participate in the regulation of cell proliferation, differentiation, survival and death. These findings have enabled identification of abnormalities in PKC isoform function, as they occur in several cancers. Specifically, the initiation of squamous cell carcinoma formation and progression to the malignant phenotype was found to be associated with distinct changes in PKC expression, activation, distribution, and phosphorylation. These studies were recently further extended to transgenic and knockout animals, which allowed a more direct analysis of individual PKC functions. Accordingly, this review is focused on the involvement of PKC in physiology and pathology of the skin.

A caspase-resistant mutant of PKC-delta protects keratinocytes from UV-induced apoptosis.

Keratinocyte apoptosis induced by UV radiation is a major protective mechanism from skin photocarcinogenesis. The induction of apoptosis by UV radiation, as well as a variety of genotoxic stimuli, involves the activation of PKC-delta by caspase-3-mediated cleavage in its hinge region, thus generating a constitutively active catalytic fragment. To determine the role of PKC-delta cleavage in UV apoptosis signaling, we introduced a caspase-resistant PKC-delta mutant (D330A) into human keratinocytes by retrovirus transduction. Overexpression of PKC-delta(D330A) protected keratinocytes from UV-induced apoptosis and enhanced long-term survival. PKC-delta(D330A) partially prevented the release of cytochrome c from the mitochondria and the loss of Mcl-1, a key antiapoptotic protein downregulated during UV apoptosis. Thus, the cleavage and activation of PKC-delta are critical components of UV-induced apoptosis in human keratinocytes, and the inactivation of PKC-delta can promote the survival of keratinocytes exposed to UV radiation.

Expression of human chromosome 2 ornithine decarboxylase gene in ornithine decarboxylase-deficient Chinese hamster ovary cells.

Ornithine decarboxylase (ODC) belongs to a multigene family and some of these may very well be nonfunctional (pseudogenes). We isolated an ODC gene from a human chromosome 2-specific library and transfected the gene into ODC-deficient Chinese hamster ovary cells to directly demonstrate that this ODC gene is functional and ODC is essential for cell proliferation. After screening 2.5 X 10(5) plaques using a human ODC complementary DNA probe, a typical clone with a 5.4-kilobase insert was isolated and then cloned into the HindIII site of the pGem-1 vector. One (phODC 2B1) of these clones containing a 5.4-kilobase ODC gene insert was identified. Restriction enzyme analysis and partial sequencing data revealed that phODC 2B1 contained the full length protein-coding sequences but lacked first exon and 3'-polyadenylation sequences. Primer extension analysis indicated that human ODC mRNA has homologous sequences with the ODC gene from human chromosome 2. To determine that the chromosome 2 ODC gene is functional, ODC-deficient Chinese hamster ovary cells were transfected with the ODC expression vector (phSV2B1-neo) and several G418-resistant transfectants were isolated which expressed 70- to 400-fold more ODC activity than parental or wild-type Chinese hamster ovary cells. Furthermore, these stable transfectants exhibited a higher growth rate than wild-type cells. These results indicate that the ODC gene from human chromosome 2 encodes functional ODC protein, and ODC (and its product putrescine) is required for cell growth.

Alterations in murine keratinocyte differentiation induced by activated rasHa genes are mediated by protein kinase C-alpha.

Primary mouse keratinocytes expressing the v-rasHa oncogene (v-rasHa keratinocytes) produce squamous papillomas when grafted onto nude mice and respond abnormally to signals for terminal differentiation both in vivo and in vitro. Since protein kinase C (PKC) activators and v-rasHa induce similar phenotypic changes in cultured keratinocytes, and cellular diacylglycerol levels are constitutively elevated in ras-transformed keratinocytes, we tested whether PKC is a downstream target for oncogenic ras in this cell type. Ca(2+)-dependent PKC activity was increased in lysates from cultured v-rasHa keratinocytes when compared to control cells; in contrast, Ca(2+)-independent activity decreased. Similar to PKC activators, v-rasHa blocked Ca(2+)-mediated expression of the early epidermal differentiation markers keratins K1 and K10 while inducing aberrant expression of K8. Pretreatment of v-rasHa keratinocytes with bryostatin to block PKC function restored Ca(2+)-mediated expression of K1 and K10 and blocked abnormal expression of K8, suggesting that these responses are mediated by the PKC pathway. Furthermore, expression of K1 is restored at bryostatin doses which specifically down-modulate PKC-alpha, the only Ca(2+)-dependent PKC isozyme detected in cultured keratinocytes. In contrast to the inhibition of K1 and K10, Ca(2+)-induced expression of the late epidermal differentiation markers loricrin, filaggrin, and keratinocyte transglutaminase was accelerated by v-rasHa, as previously reported in normal keratinocytes treated with PKC activators. Pretreatment of v-rasHa keratinocytes with bryostatin blocked expression of late markers in these cells, and this response was correlated with down-regulation of PKC-alpha. The results of this study suggest that oncogenic ras alters keratinocyte differentiation by altering the function of the PKC signaling pathway, and that PKC-alpha is the specific isozyme involved in down-modulating expression of keratins K1 and K10 and up-regulating expression of loricrin, filaggrin, and keratinocyte transglutaminase.

Targeted disruption of the epidermal growth factor receptor impairs growth of squamous papillomas expressing the v-ras(Ha) oncogene but does not block in vitro keratinocyte responses to oncogenic ras.

We have assessed the role of epidermal growth factor receptor (EGFR) signaling in biological responses to the v-ras(Ha) oncogene using primary keratinocytes from Egfr -/- mice and wild-type littermates. On the basis of several criteria, Egfr -/- keratinocytes were unresponsive to either acute or chronic exposure to several EGFR ligands but were stimulated to proliferate in response to several other mitogens. Although conditioned medium from primary keratinocytes transduced with v-ras(Ha) retrovirus (v-ras(Ha) keratinocytes) was a potent mitogen for wild-type but not Egfr -/- keratinocytes, v-ras(Ha) transduction of primary keratinocytes of either genotype resulted in a strong mitogenic response, arguing against an obligatory role for EGFR activation in v-ras(Ha)-mediated stimulation of keratinocyte proliferation. Infection with high-titer v-ras(Ha) retrovirus altered the keratin expression pattern in keratinocytes of both genotypes, suppressing differentiation-specific keratins K1 and K10 while activating aberrant expression of K8 and K18. In wild-type but not Egfr -/- cultures, K1 and K10 were also suppressed following infection at lower retroviral titers, presumably as a result of paracrine EGFR activation on uninfected cells present in these cultures. Squamous papillomas produced by grafting Egfr -/- v-ras(Ha) keratinocytes onto nude mice were only 21% of the size of wild-type v-ras(Ha) tumors, and a striking redistribution of S-phase cells was detected by immunostaining for bromodeoxyuridine. In Egfr -/- v-ras(Ha) papillomas, the fraction of total labeled nuclei detected in suprabasal layers was increased from 19 to 39%. In contrast, the basal layer labeling index of Egfr -/- papillomas was reduced to 34%, compared to 43% in wild-type tumors. Our results indicate that, although autocrine EGFR signaling is not required for keratinocyte responses to oncogenic ras in culture or benign tumor formation in nude mouse grafts, disruption of this pathway impairs growth of v-ras(Ha) papillomas by a mechanism that may involve alterations in keratinocyte cell cycle progression and/or migration in vivo.

Protein kinase Cdelta-mediated phosphorylation of alpha6beta4 is associated with reduced integrin localization to the hemidesmosome and decreased keratinocyte attachment.

In mammalian epidermis, expression of the alpha6beta4 integrin is restricted to the hemidesmosome complexes, which connect the proliferative basal cell layer with the underlying basement membrane. Keratinocyte differentiation is associated with down-regulation of alpha6beta4 expression and detachment of keratinocytes from the basement membrane. Neoplastic keratinocytes delay maturation, proliferate suprabasally, and retain the expression of the alpha6beta4 integrin in suprabasal cells disassociated from the hemidesmosomes. We now show that the alpha6beta4 integrin is a substrate for serine phosphorylation by protein kinase C in keratinocytes. Furthermore, protein kinase C-mediated phosphorylation of alpha6beta4 is associated with redistribution of this integrin from the hemidesmosome to the cytosol. Specifically, in vitro kinase assays identified the protein kinase Cdelta as the primary isoform phosphorylating alpha6 and beta4 integrin subunits. Using recombinant protein kinase C adenoviruses, overexpression of protein kinase Cdelta but not protein kinase Calpha in primary keratinocytes increased beta4 serine phosphorylation, decreased alpha6beta4 localization to the hemidesmosome complexes, and reduced keratinocyte attachment. Taken together, these results establish a link between protein kinase Cdelta-mediated serine phosphorylation of alpha6beta4 integrin and its effects on alpha6beta4 subcellular localization and keratinocyte attachment to the laminin underlying matrix.

Caspase activation and disruption of mitochondrial membrane potential during UV radiation-induced apoptosis of human keratinocytes requires activation of protein kinase C.

The induction of apoptosis in human keratinocytes by UV radiation involves caspase-mediated cleavage and activation of protein kinase C delta (PKCdelta). Here we examined the role of PKC activation in caspase activation and disruption of mitochondria function by UV radiation. Inhibition of PKC partially blocked UV radiation-induced cleavage of PKCdelta, pro-caspase-3, and pro-caspase-8, and the activation of these caspases. PKC inhibition also blocked the UV-induced loss of mitochondria membrane potential, but did not block the release of cytochrome c from mitochondria. Expression of the active catalytic domain of PKCdelta was sufficient to induce apoptosis and disrupt mitochondrial membrane potential, however a kinase inactive PKCdelta catalytic domain did not. Furthermore, the PKCdelta catalytic fragment generated following UV radiation localized to the mitochondria fraction, as did ectopically expressed PKCdelta catalytic domain. These results identify a functional role for PKC activation in potentiating caspase activation and disrupting mitochondrial function during UV-induced apoptosis.

Jagged-1 mediated activation of notch signaling induces complete maturation of human keratinocytes through NF-kappaB and PPARgamma.

Establishing an effective epidermal barrier requires a series of coordinated molecular events involving keratinocytes (KCs) within a stratified epithelium. Epidermal maturation depends on convergence of pathways involving components of NF-kappaB and peroxisome proliferator activated receptor (PPAR) signaling systems that promote terminal differentiation and production of a stratum corneum. The Notch-1 receptor and its ligand Delta-1 have been proposed by others to participate in early events in KC differentiation. Here, we establish differential expression patterns for several Notch receptors and ligands in normal human skin. These immunolocalization findings, together with functional studies demonstrating increased levels of Notch ligand/receptors occurring during the onset of differentiation, prompted use of a soluble Notch ligand, a peptide derived from the most conspicuously expressed ligand in skin, Jagged-1. Exposing submerged KC monolayers to this peptide (JAG-1) in co-presence of elevated calcium ion concentration, produced stratification with loricrin expression. Using a living human epidermal equivalent (EE) model system, when submerged cultures were raised to an air/liquid interface to generate a fully mature epidermis, activation of Notch signaling was detected. Addition of JAG-1 peptide to submerged EEs was sufficient to induce epidermal maturation. Moreover, a soluble decoy Notch inhibitor prevented such differentiation and corneogenesis in human EEs exposed to either an air/liquid interface or to the JAG-1 peptide. In KC monolayers, addition of JAG-1 peptide induced IKKalpha mediated NF-kappaB activation, as well as increased PPARgamma expression. Immunoprecipitation/Western blot analysis revealed a physical association between the p65 subunit of NF-kappaB and PPARgamma. These results indicate that activation of Notch signaling is necessary for maturation of human epidermis, and activation by a soluble Notch ligand is sufficient to trigger complete KC differentiation including cornification.

Inhibition of tyrosine kinase activity induces caspase-dependent apoptosis in anaplastic large cell lymphoma with NPM-ALK (p80) fusion protein.

OBJECTIVE: The t(2;5)(p23;q35) translocation creates a fusion gene NPM-ALK (p80) that encodes a product with tyrosine kinase activity believed to play an important role in development of anaplastic large cell lymphoma (ALCL). Our study was aimed to analyze tyrosine kinase activity and phosphotyrosine in ALCLs. We were also interested in determining the effect of tyrosine kinase inhibitors on survival of ALCL. METHODS: Eleven cases of ALCL and three ALCL cell lines with t(2;5)(Karpas-299, SUPM2, SU-DHL-1) and 10 Hodgkin's disease (HD) samples were stained with anti-phosphotyrosine antibody. The tyrosine kinase activity, p80 phosphorylation, and the apoptotic effects of two tyrosine kinase inhibitors, herbimycin A and STI-571, were determined on ALCL cell lines. RESULTS: Herbimycin A had showed both a time- and dose-dependent apoptotic effect on all three cell lines, while STI-571 demonstrated a minimal effect. Following herbimycin A treatment, a decrease in tyrosine kinase activity in the ALCL cell lines and inhibition in NPM-ALK (p80) autophosphorylation was demonstrated by immunoprecipitation and Western blotting. Herbimycin A-induced apoptosis was accompanied by caspase-3 activation. Furthermore, apoptosis induced by herbimycin A was blocked by both z-VAD-FMK and z-DEVD-FMK, suggesting a critical role of caspases. CONCLUSIONS: These findings indicate that tyrosine kinase activity is a common characteristic of ALCLs and necessary for ALCL cell survival. These findings further suggest that therapies targeting tyrosine kinases, including p80, may have clinical utility.

TACC3 deregulates the DNA damage response and confers sensitivity to radiation and PARP inhibition.

Deregulation of the transforming acidic coiled-coil protein 3 (TACC3), an important factor in the centrosome-microtubule system, has been linked to a variety of human cancer types. We have recently reported on the oncogenic potential of TACC3; however, the molecular mechanisms by which TACC3 mediates oncogenic function remain to be elucidated. In this study, we show that high levels of TACC3 lead to the accumulation of DNA double-strand breaks (DSBs) and disrupt the normal cellular response to DNA damage, at least in part, by negatively regulating the expression of ataxia telangiectasia mutated (ATM) and the subsequent DNA damage response (DDR) signaling cascade. Cells expressing high levels of TACC3 display defective checkpoints and DSB-mediated homologous recombination (HR) and non-homologous end joining (NHEJ) repair systems, leading to genomic instability. Importantly, high levels of TACC3 confer cellular sensitization to radiation and poly(ADP-ribose) polymerase (PARP) inhibition. Overall, our findings provide critical information regarding the mechanisms by which TACC3 contributes to genomic instability, potentially leading to cancer development, and suggest a novel prognostic, diagnostic and therapeutic strategy for the treatment of cancer types expressing high levels of TACC3.

Staurosporine induces a sequential program of mouse keratinocyte terminal differentiation through activation of PKC isozymes.

Staurosporine (stsp) induces assembly of cornified envelopes in mouse keratinocyte cultures. To clarify whether this effect is the consequence of a coordinated differentiation program similar to that observed in epidermis, we assessed the expression of multiple differentiation-specific markers in stsp-treated keratinocytes. In medium containing 0.05 mM Ca2+, in which the basal cell phenotype is normally maintained, stsp induced dose-dependent increases in keratin 1, epidermal and keratinocyte transglutaminases, SPR-1, loricrin, and profilaggrin mRNA. Based on nuclear run-on analysis, stsp-mediated marker expression was found to be due at least in part to increased transcription. Since protein kinase C (PKC) activation is required for keratinocyte differentiation, we tested whether stsp influenced this signaling pathway. Stsp induced the translocation of multiple PKC isoforms from the cytosol to membrane and/or cytoskeletal fractions, inducing isozyme downregulation within 24 h. Moreover, AP-1 DNA binding activity was elevated in stsp-treated keratinocytes, consistent with the notion that this agent influences keratinocyte-specific gene expression via the PKC pathway. Stsp-mediated marker expression was inhibited by the PKC inhibitor GF 109203X. In cells pre-treated with bryostatin 1 to selectively down-modulate specific PKC isoforms, stsp-induced loricrin, filaggrin, and SPR-1 expression was suppressed when PKC alpha, epsilon, and/or delta were downregulated, suggesting that these isozymes may be necessary for marker expression in response to this agent. Thus, in addition to its effects on cornified envelope assembly, stsp induces a coordinate program of differentiation-specific keratinocyte gene expression that is mediated at least in part by the PKC signaling pathway.

Role of oncogenes and tumor suppressor genes in multistage carcinogenesis.

The introduction of the techniques of molecular biology as tools to study skin carcinogenesis has provided more precise localization of biochemical pathways that regulate the tumor phenotype. This approach has identified genetic changes that are characteristic of each of the specific stages of squamous cancer pathogenesis: initiation, exogenous promotion, premalignant progression, and malignant conversion. Initiation can result from mutations in a single gene, and the Harvey allele of the ras gene family has been identified as a frequent site for initiating mutations. Heterozygous activating mutations in c-rasHa are dominant, and affected keratinocytes hyperproliferate and are resistant to signals for terminal differentiation. An important pathway impacted by c-rasHa activation is the protein kinase C (PKC) pathway, a major regulator of keratinocyte differentiation. Increased activity of PKC alpha and suppression of PKC delta by tyrosine phosphorylation contribute to the phenotypic consequences of rasHa gene activation in keratinocytes. Tumor promoters disturb epidermal homeostasis and cause selective clonal expansion of initiated cells to produce multiple benign squamous papillomas. Resistance to differentiation and enhanced growth rate of initiated cells impart a growth advantage when the epidermis is exposed to promoters. The frequency of premalignant progression varies among papillomas, and subpopulations at high risk for progression have been identified. These high-risk papillomas overexpress the alpha 6 beta 4 integrin and are deficient in transforming growth factor beta 1 and beta 2 peptides, two changes associated with a very high proliferation rate in this subset of tumors. The introduction of an oncogenic rasHa gene into epidermal cells derived from transgenic mice with a null mutation in the TGF beta 1 gene have an accelerated rate of malignant progression when examined in vivo. Thus members of the TGF beta gene family contribute a tumor-suppressor function in carcinogenesis. Accelerated malignant progression is also found with v-rasHa transduced keratinocytes from skin of mice with a null mutation in the p53 gene. The similarities in risk for malignant conversion by initiated keratinocytes from TG beta 1 and p53 null geneotypes suggest that a common, growth-related pathway may underly the tumor-suppressive functions of these proteins in the skin carcinogenesis model.

Phosphorylation of murine homeodomain protein Dlx3 by protein kinase C.

The Dlx3 homeodomain gene is expressed in terminally differentiated murine epidermal cells. As demonstrated for differentiation-specific granular markers, Dlx3 is activated in primary mouse keratinocytes cultured in vitro by increasing the level of the extracellular Ca(2+). This activation is mediated through a protein kinase C-dependent (PKC) pathway. In this study, we investigated whether PKC can modulate the activity of murine Dlx3 protein. Using in vitro kinase assays, we show that PKC enzymes phosphorylate the Dlx3 protein. Using keratinocyte nuclear extracts for the kinase reaction, we determined that Dlx3 protein is phosphorylated, and the phosphorylation is inhibited by the PKC-specific inhibitor GF109203X, suggesting that Dlx3 is phosphorylated by PKC in vivo. Of the PKC isoforms present in the epidermis, we tested alpha, delta, epsilon and zeta. Dlx3 is primarily phosphorylated by PKC alpha. By deletion and mutational analysis, we show that the serine residue S(138), located in the homeodomain of Dlx3 protein, was specifically phosphorylated by PKC. The phosphorylation of purified Dlx3 proteins by PKC partially inhibited formation of complexes between Dlx3 protein and DNA. These results suggest that Dlx3 protein can be directly phosphorylated by PKC and this affects the DNA binding activity of Dlx3.

Abnormal NF-kappaB signaling pathway with enhanced susceptibility to apoptosis in immortalized keratinocytes.

The transcriptional activation and proper regulation of NF-kappaB is known to be important to the apoptotic resistant phenotype of epidermal-derived keratinocytes. By comparing and contrasting the responses of normal foreskin-derived keratinocytes versus an immortalized skin-derived keratinocyte cell line (i.e. HaCaT cells), several molecular defects involving NF-kappaB signaling pathway were delineated in the immortalized keratinocytes. While exposure to IFN-gamma plus TPA produces growth arrest in both normal and immortalized keratinocytes, with rapid phosphorylation of MEKKI and recruitment of distinctive protein kinase C isoforms into the signalosome complex, subsequent molecular events necessary for NF-kappaB activation were abnormal in HaCaT cells. This disrupted NF-kappaB activation in HaCaT cells was accompanied by enhanced susceptibility to UV-light induced apoptosis, which was associated with elevated levels of E2F-1 and decreased TRAF1/TRAF2 levels. Additional defects in HaCaT cells included markedly diminished levels of IKKbeta (and lack of induction of kinase activity) in response to inflammatory stimuli, a failure of p21(WAF1/CIP1) to associate with CDK2, and a decreased association between p65 and p300. These studies suggest caution in using HaCaT cells as a substitute for normal keratinocytes to study apoptosis in the skin. Thus, it appears that while the immortalized cells can escape cell cycle checkpoints by elevated levels of E2F-1, an adverse biological consequence of such dysregulated cell cycle control is the inability to activate the anti-apoptotic NF-kappaB signaling pathway. Therefore, exploiting this apoptosis vulnerability in pre-malignant, or immortalized cells, prior to acquiring a death-defying phenotype characteristic of more advanced malignant cell types, provides the basis for an early interventional therapeutic strategy for cutaneous oncologists.

Crosstalk between PKCα and Notch-4 in endocrine-resistant breast cancer cells.

The Notch pathway is functionally important in breast cancer. Notch-1 has been reported to maintain an estrogen-independent phenotype in estrogen receptor α (ERα)+ breast cancer cells. Notch-4 expression correlates with Ki67. Notch-4 also plays a key role in breast cancer stem-like cells. Estrogen-independent breast cancer cell lines have higher Notch activity than estrogen-dependent lines. Protein kinase Cα (PKCα) overexpression is common in endocrine-resistant breast cancers and promotes tamoxifen (TAM)-resistant growth in breast cancer cell lines. We tested whether PKCα overexpression affects Notch activity and whether Notch signaling contributes to endocrine resistance in PKCα-overexpressing breast cancer cells.Analysis of published microarray data from ERα+ breast carcinomas shows that PKCα expression correlates strongly with Notch-4. Real-time reverse transcription PCR and immunohistochemistry on archival specimens confirmed this finding. In a PKCα-overexpressing, TAM-resistant T47D model, PKCα selectively increases Notch-4, but not Notch-1, expression in vitro and in vivo. This effect is mediated by activator protein-1 (AP-1) occupancy of the Notch-4 promoter. Notch-4 knockdown inhibits estrogen-independent growth of PKCα-overexpressing T47D cells, whereas Notch-4IC expression stimulates it. Gene expression profiling shows that multiple genes and pathways associated with endocrine resistance are induced in Notch-4IC- and PKCα-expressing T47D cells. In PKCα-overexpressing T47D xenografts, an orally active γ-secretase inhibitor at clinically relevant doses significantly decreased estrogen-independent tumor growth, alone and in combination with TAM. In conclusion, PKCα overexpression induces Notch-4 through AP-1. Notch-4 promotes estrogen-independent, TAM-resistant growth and activates multiple pathways connected with endocrine resistance and chemoresistance. Notch inhibitors should be clinically evaluated in PKCα- and Notch-4-overexpressing, endocrine-resistant breast cancers.

Involvement of retinoic acid nuclear receptors in retinoic acid-induced tissue transglutaminase gene expression in rat tracheal 2C5 cells.

The involvement of retinoic acid nuclear receptors (RARs) in the induction of tissue transglutaminase (TG) by retinoic acid in rat tracheal 2C5 cells was determined. The levels of RAR alpha and RAR beta were altered in 2C5 cells by transfection with RAR expression vectors. Increased expression of RAR alpha increased the induction of tissue TG by retinoic acid. In contrast, decreased RAR alpha expression, using an antisense RAR alpha expression vector, diminished the normal level of tissue TG induction caused by retinoic acid. Transfectants overexpressing RAR beta were also more responsive to retinoic acid for the induction of tissue TG, although the magnitude of TG induction was not as great as resulted from RAR alpha overexpression. These results indicate that the levels of the RAR alpha and RAR beta dictate the magnitude of tissue TG induction by retinoic acid.

The mechanism of the inhibition of squamous differentiation of rat tracheal 2C5 cells by retinoic acid.

Retinoic acid (RA) plays an essential role in maintaining normal differentiation of tracheal epithelial cells. During vitamin A deficiency, tracheocytes undergo squamous metaplasia, an abnormal differentiation that can be reversed by RA. We used rat tracheal 2C5 cells to study the mechanism of inhibition of squamous differentiation by RA. 2C5 cells grown to confluence in the presence of serum underwent squamous differentiation as marked by an increase in their level of crosslinked envelope formation. The serum-induced crosslinked envelope formation was blocked by RA in 2C5 cells with an ED50 < 0.1 nM. However, the activity of the crosslinking enzyme, keratinocyte transglutaminase, did not correlate with the formation of crosslinked envelopes in 2C5 cells. Changes in biochemical markers of squamous differentiation such as an altered expression of specific cytokeratins also accompanied serum-induced squamous differentiation of 2C5 cells. The expression of the keratin squamous differentiation markers (i.e. K13) were inhibited by RA, although the ED50 for K13 expression was > 1 nM. The different dose responses for RA inhibiting differentiation markers suggests multiple mechanisms of regulation by RA. These results indicate that 2C5 cells remain responsive to differentiation factors such as RA and serum despite being an immortalized cell line.