To 12-O-tetradecanoylphorbol-13-acetate Treatment Research Articles

Lgr4 is crucial for skin carcinogenesis by regulating MEK/ERK and Wnt/β-catenin signaling pathways

Lgr4 is a member of the leucine-rich, G protein–coupled receptor family of proteins, and has recently been shown to augment Wnt/β-catenin signaling via binding to Wnt agonists R-spondins. It plays an important role in skin development, but its involvement in skin tumorigenesis is unclear. Here, we report that mice deficient for Lgr4 are resistant to 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced keratinocyte proliferation and papilloma formation. We show that TPA treatment activates MEK1, ERK1/2 and downstream effector AP-1 in wild-type (WT) epidermal cells and mice, but not in cells or mice where Lgr4 is depleted. Wnt/β-catenin signaling is also dramatically activated by TPA treatment, and this activation is abolished when Lgr4 is deleted. We provide evidences that blocking both MEK1/ERK1/2 and Wnt/β-catenin pathways prevents TPA-induced increase in the expression of Ccnd1 (cyclin D1), a known Wnt/β-catenin target gene, and that the activation of MEK1/ERK1/2 pathway lies upstream of Wnt/β-catenin signal pathway. Collectively, our findings identify Lgr4 as a critical positive factor for skin tumorigenesis by mediating the activation of MEK1/ERK1/2 and Wnt/β-catenin pathways.

Abstract 5425: Epidermal respiratory uncoupling blocks skin carcinogenesis by inhibiting Akt.

Abstract Metabolic reprogramming, characterized by increased glycolysis and decreased oxygen consumption, is now considered a hallmark of malignancy. Mitochondrial uncoupling proteins oppose this phenotype by driving futile mitochondrial respiration that is uncoupled from ATP synthesis, and mice expressing a keratin 5 - uncoupling protein 3 (K5-UCP3) transgene in the epidermis are completely protected from the formation of chemically induced skin carcinomas. We crossed these mice with Tg.AC mice that express a cutaneous oncogenic v-Ha-Ras transgene, bypassing tumor initiation. Bi-transgenic Tg.AC/K5-UCP3 mice were still remarkably resistant to 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumorigenesis, indicating that UCP3 likely inhibits tumor promotion. Accordingly, K5-UCP3 mice exhibited striking resistance to epidermal proliferation induced by TPA as measured using a bromodeoxyuridine (BrdU) based labeling assay. In agreement with these data, K5-UCP3 epidermis failed to show activation of Akt (Ser473 & Thr308 phosphorylation) and phosphorylation of downstream signaling molecules in response to TPA treatment. This effect corresponded to decreased expression of cyclins D1 and A, and increased expression of the cell cycle inhibitory proteins p21 and p27. Decreased Akt activation was also observed in vitro in response to epidermal growth factor (EGF) treatment of serum starved, isolated K5-UCP3 primary keratinocytes. To test the notion that the lack of Akt signaling was responsible for the lack of TPA induced proliferation and tumorigenesis, we bred bi-transgenic K5-UCP3/K5-Akt mice, and found that over-expression of wild type Akt recovered proliferation following TPA treatment. Future experiments will elucidate the mechanism underlying UCP3-induced inhibition of Akt activation and signaling. Citation Format: Sara M. Nowinski, Joyce E. Rundhaug, Okkyung Rho, Cory U. Lago, Susan M. Fischer, John DiGiovanni, Edward M. Mills. Epidermal respiratory uncoupling blocks skin carcinogenesis by inhibiting Akt. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5425. doi:10.1158/1538-7445.AM2013-5425

Resistance to phorbol ester-induced differentiation in human myeloid leukemia cells: A hypothetic role for the mRNA stabilization process

UM384 cells, derived from the human myeloid leukemia U937 cell line, fail to differentiate in response to 12- O-tetradecanoylphorbol-13-acetate (TPA). Using cDNA microarray and real-time quantitative PCR (RT-QPCR) approaches, we observed a difference in the response to TPA treatment: all the genes from U937 cells were continuously modulated from 2 to 24 h. In UM384 cells, 60% of the genes were transiently modulated at 2 h, then returned to control levels at 24 h. Moreover, HuR, an AU-rich element-binding protein (ARE-BP), was differentially located in the two cell lines. Therefore, a defect of mRNA stabilization could be responsible for the resistance of UM384 cells to TPA-induced differentiation, suggesting a possible role for the post-transcriptional regulation in the leukemogenesis.

Notch Signals Inhibit the Development of Erythroid/Megakaryocytic Cells by Suppressing GATA-1 Activity through the Induction of HES1

The effects of Notch signals on the erythroid/megakaryocytic differentiation of hematopoietic cells were examined. Activation of Notch signals by the intracellular Notch1 or an estradiol-inducible form of Notch1/ER suppressed the expression of the erythroid marker glycophorin A in an erythroid/megakaryocytic cell line K562. Although Mock-transfected K562 cells underwent megakaryocytic differentiation in response to 12-O-tetradecanoylphorbol-13-acetate (TPA), estradiol-activated Notch1/ER induced apoptosis during TPA treatment in the transfectant, which was accompanied by the reduced expression of an antiapoptotic molecule Bcl-XL. Even when apoptosis was prevented by the overexpression of Bcl-XL, activated Notch signals still inhibited TPA-induced megakaryocytic differentiation. As for this mechanism, Notch1/recombination signal binding protein J-kappa-induced HES1 but not HES5 was found to inhibit the function of an erythroid/megakaryocytic lineage-specific transcription factor GATA-1. Although HES1 did not affect the DNA binding activity of GATA-1 in gel shift and chromatin immunoprecipitation assays, it directly bound to GATA-1 and dissociated a critical transcriptional cofactor, p300, from GATA-1. Furthermore, overexpressed HES1 inhibited the development of erythroid and megakaryocytic cells in colony assays. Also, the Notch ligand Jagged1 expressed on NIH3T3 cells suppressed the development of erythroid and megakaryocytic cells from cocultured Lin-Sca-1+ hematopoietic stem/progenitor cells. These results suggest that Notch1 inhibits the development of erythroid/megakaryocytic cells by suppressing GATA-1 activity through HES1.

Involvement of desmoplakin phosphorylation in the regulation of desmosomes by protein kinase C, in HeLa cells.

In the present study, we have examined how modulation of protein kinase C (PKC) activity affected desmosome organization in HeLa cells. Immunofluorescence and electron microscopy showed that PKC activation upon short exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in a reduction of intercellular contacts, splitting of desmosomes and dislocation of desmosomal components from the cell periphery towards the cytoplasm. As determined by immunoblot analysis of Triton X-100-soluble and -insoluble pools of proteins, these morphological changes were not correlated with modifications in the extractability of both desmoglein and plakoglobin, but involved almost complete solubilization of the desmosomal plaque protein, desmoplakin. Immunoprecipitation experiments and immunoblotting with anti-phosphoserine, antiphosphothreonine and anti-phosphotyrosine antibodies revealed that desmoplakin was mainly phosphorylated on serine and tyrosine residues in both treated and untreated cells. While phosphotyrosine content was not affected by PKC activation, phosphorylation on serine residues was increased by about two-fold. This enhanced serine phosphorylation coincided with the increase in the protein solubility, suggesting that phosphorylation of desmoplakin may be a mechanism by which PKC mediates desmosome disassembly. Consistent with the loss of PKC activity, we also showed that down-modulation of the kinase (in response to prolonged TPA treatment) or its specific inhibition (by GF 109203X) had opposite effects and increased desmosome formation. Taken together, these results clearly demonstrate an important role for PKC in the regulation ofdesmosomal junctions in HeLa cells, and identify serine phosphorylation of desmoplakin as a crucial event in this pathway.

Desmosomes are regulated by protein kinase C in primary rat epithelial cells.

In the present study, we addressed the possible relevance of protein kinase C (PKC) in the regulation of intracytoplasmic desmosome assembly. Treatment of cultured rat lingual and epidermal keratinocytes with a potent and highly selective PKC inhibitor (GF109203X) induced an increase in granular labelling for major desmosomal proteins, desmoplakins, desmoglein and plakoglobin, both intracellularly and at the cell surface. This was associated with the formation of ultrastructurally recognizable desmosomes deep in the cytoplasm and increase in intercellular desmosome number. In contrast, PKC activation upon short exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in altered cell morphology, loss of intercellular contact and accumulation of desmosomal proteins in the juxtanuclear zone. On the other hand, PKC depletion by long term TPA treatment re-established cell-cell contact, where desmosomal markers were exclusively redistributed. Taken together, these results suggest that inhibition of PKC is required for intracytoplasmic as well as intercellular desmosome assembly, whereas its activation may regulate disassembly process.

Cytoskeletal architecture in mouse lung epithelial cells is regulated by protein-kinase C-alpha and calpain II.

Brief exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) caused a uniformly flattened population of mouse lung epithelial cells to become more heterogeneous; some cells rounded up, and others detached to overlap with flatter cells. Actin stress fiber organization was disrupted, and F-actin accumulated in lemellipodia. Vinculin dissociated from the focal adhesion plaques to diffuse throughout the cytoplasm. Inhibition of protein kinase C (PKC) activity blocked these effects of TPA. After 8 h of TPA exposure, actin filaments reassembled and vinculin again localized to the cell periphery. Calpain inhibition attenuated the decrease of PKC-alpha protein and PKC activity from the membrane fraction, and prevented the redistribution of cytoskeletal elements. Talin immunostaining was widespread throughout control cells but was localized to the periphery 8 h after treatment with TPA or with inhibitors of PKC and calpain. Both vinculin and talin concentrations increased with prolonged TPA treatment. PKC-zeta and calpain II were not appreciably affected by TPA exposure. Translocation of PKC-alpha to the membrane, followed by its calpain-induced downmodulation, is apparently required for the reversible pattern of cytoskeletal changes caused by TPA.

Evidence for Protein Tyrosine Kinase Involvement in CD6-Induced T Cell Proliferation

Several studies have demonstrated that addition of soluble anti-CD6 mAbs to 12-O-tetradecanoylphorbol 13-acetate (TPA)-treated naive T cells can induce cell proliferation. We showed in the present study that cell proliferation in TPA-treated T cell cultures can be enhanced several fold when the anti-CD6 mAbs are either immobilized or crosslinked with rabbit anti-mouse immunoglobulins (RAM Ig). Using a src family protein tyrosine kinase (PTK) inhibitor, herbimycin A, the cell proliferation induced by the anti-CD6 mAb, IOR-T1, in TPA-treated T cells were effectively abolished. Analysis of the cellular proteins in these cells after crosslinking the CD6 receptor with IOR-T1 (followed by RAM Ig) in the presence of TPA resulted in an increased level of tyrosine phosphorylation. Pretreatment of naive T cells with herbimycin A (0.5 and 1 μg/ml) for 18 hr completely inhibited the tyrosine phosphorylation on cellular substrates in T cell cultures stimulated with IOR-T1/RAM Ig and TPA. Similar concentrations of herbimycin A also inhibited the increase in IL-2 mRNA expression and cell proliferation in T cell cultures after IOR-T1/RAM Ig and TPA treatment. Furthermore, the increase in cytosolic free Ca2+concentration in naive T cells after crosslinking of the CD6 receptor with IOR-T1/RAM Ig was also inhibited by herbimycin A. Taken together, our results suggest that CD6-mediated T cell proliferation is IL-2 dependent, and involves tyrosine kinase activity which is strictly dependent on protein kinase C activation.

Genes regulating glutathione concentrations in X-ray-transformed rat embryo fibroblasts: changes in gamma-glutamylcysteine synthetase and gamma-glutamyltranspeptidase expression.

The concentration of glutathione (GSH) and the expressions of gamma-glutamylcysteine synthetase and gamma-glutamyltranspeptidase (GGT) were assessed in rat embryo fibroblasts (REF) displaying various stages of X-ray-induced transformation. A secondary culture of REF cells was irradiated, and a normal-immortalized cell line (X-REF-23) was isolated. Chronic exposure of X-REF-23 cells to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) yielded cells (X-REF-23-TP) capable of benign tumor formation in nude mice. These cells exhibited GSH concentrations and gamma-glutamylcysteine synthetase heavy subunit mRNA levels that were approximately 50% less than those measured in X-REF-23 cells. Neither X-REF-23 nor X-REF-23-TP cells exhibited detectable GGT mRNA or activity. Administration of 3 Gy of X-rays followed by chronic TPA treatment yielded cells (X-REF-23-TPX) capable of malignant tumor formation in nude mice. These cells expressed GGT mRNA and Concanavalin-A minus GGT activity. One TPX clone (X-REF-23-TPX.1) was chosen for further characterization. Northern blotting of X-REF-23-TPX.1 cells indicated that gamma-glutamylcysteine synthetase heavy subunit mRNA levels were similar to those of X-REF-TP cells. X-REF-23-TPX.1 cells contained nearly the same amount of GSH as X-REF-23 cells. However, the ability of diethylmaleate (DEM) to deplete GSH was diminished in X-REF-23-TPX.1 cells compared with X-REF-23 cells. Furthermore, exposure of X-REF-23-TPX.1 cells to DEM stimulated GSH resynthesis such that the GSH concentration exceeded control values during exposure. The resynthesis of GSH during a DEM exposure was found to be dependent upon the expression of GGT, as demonstrated by inhibition with AT-125. These experiments indicate that ionizing radiation can lead to elevated constitutive expression of GGT in transformed REF cells and that expression of GGT activity was responsible for the increased rate of GSH repletion observed in X-REF-23-TPX.1 cells.

Neurite outgrowth and protein phosphorylation in chick embryonic sensory ganglia induced by a brief exposure to 12-O-tetradecanoylphorbol 13-acetate.

An exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) at 20 nM for as short as 30 min was sufficient to elicit neurite outgrowth from explanted chick embryonic sensory ganglia. Attachment of the ganglia to the collagen-coated substratum during exposure to TPA was essential for subsequent neurite outgrowth. Pulse-labeling with [35S]-methionine indicated no significant difference in protein synthesis between control and TPA-treated ganglia. In vitro phosphorylation assay revealed a prominent protein kinase C substrate with an apparent molecular mass of 66,000 dalton (66 kDa) in chick embryo ganglia extracts. Treatment of intact ganglia with TPA for 30 min also specifically stimulated the phosphorylation of the same protein. When staurosporine, a potent inhibitor of protein kinase C, was present during TPA treatment, both neurite outgrowth and the phosphorylation of the 66-kDa protein were blocked. Biochemical analysis of the phosphorylated 66-kDa protein indicated that (1) phosphorylation was only in serine residue, (2) the pI value was 4.5, (3) after V8 protease digestion, two phosphorylated peptide fragments, 6.0 and 7.5 kDa in size, were produced, and (4) it cross-reacted with an antiserum raised against a 66-kDa neurofilament subunit from rat spinal cord. These results suggest that early activation of protein kinase C and the phosphorylation of the 66-kDa protein may be involved in neuritogenesis.

Chemically induced skin carcinogenesis in a transgenic mouse line (TG.AC) carrying a v-Ha-ras gene.

A transgenic mouse line (TG.AC) created in the FVB/N strain, carries a v-Ha-ras gene fused to a zeta-globin promoter gene. These trangenic mice have the properties of genetically initiated skin and have been shown to be sensitive to 12-O-tetradecanoylphorbol-13-acetate (TPA), a well-described promoter of skin papillomas in the two-stage mouse skin tumorigenesis model. It was of interest to determine whether the TG.AC mouse strain was also responsive to other known promoters. Groups of heterozygous or homozygous TG.AC mice were treated topically, 2x/week, for up to 20 weeks with benzoyl peroxide (BPO), 2-butanol peroxide (2-BUP), phenol (PH), acetic acid (AA), TPA and acetone (ACN), the vehicle control. Skin papillomas were induced in all groups treated with TPA, BPO and 2-BUP. Papillomas were observed in some treatment groups as early as 3 weeks. The relative activity of the promoters was TPA > 2-BUP > BPO > PH = AA = ACN. No papillomas were observed in any of the uninitiated FVB/N mice treated in a similar manner and which served as treatment control groups. Studies to determine the sensitivity of TG.AC mice to TPA, indicated that a total dose of 25-30 micrograms of TPA administered in 3 or 10 applications, was sufficient to induce an average incidence of 11-15 papillomas per mouse. The papilloma incidence continued to increase and was maintained up to 15 weeks after TPA treatment was terminated. The short latency period and high incidence of papilloma induction indicate that TG.AC mice have a high sensitivity to known skin promoters. The TG.AC line should prove to be a sensitive model for identifying putative tumor promoters or complete carcinogens.

Differential responsiveness to phorbol esters correlates with differential expression of protein kinase C in KG-1 and KG-1a human myeloid leukemia cells

The KG-1 myeloid leukemia cell line differentiates into macrophages in response to 12- O-tetradecanoylphorbol 13-acetate (TPA), while its spontaneous variant, the KG-1a cell line, is resistant to the differentiative effects of TPA. To determine the mechanism underlying these differential responses to TPA, experiments were performed to determine the relative numbers of TPA binding sites, protein kinase C (PKC) enzyme activity levels, PKC translocation responses and PKC gene expression in these cell lines. KG-1a cells exhibited 40% fewer high affinity binding sites for TPA than KG-1 cells. Although PKC translocation from cytosol to the membrane fraction was observed in both cell types, total PKC activity, measured in vitro using type III-S histone as substrate, was reduced by 70% in KG-1a cells. These biochemical differences between the parental line and the phorbol ester non-responsive variant were correlated with the depressed level of PKC-β RNA abundance in KG-1a cells. Both lines expressed PKC-α RNA at comparable levels. Chronic exposure to TPA resulted in down-regulation of PKC enzyme activity in both cell lines, and a selective decrease in PKC-β RNA transcripts in both cell types. In contrast, chronic TPA treatment had no effect on the levels of PKC-α RNA in KG-1 and KG-1a cells. Our results indicate a correlation between the level of PKC-β expression and the responsiveness of myeloid lineage precursor cells to the differentiative effects of TPA.

Down-regulation of protein kinase C in rat glioma C6 cells: Effects on the β-adrenergic receptor-coupled adenylate cyclase

Continuous exposure of rat glioma C6 cells to 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a time and dose dependent loss of [3H]phorbol dibutyrate binding sites and protein kinase C activity. Thus, by 24 h, the cells were essentially depleted of protein kinase C activity. In agreement with previous studies, TPA treatment caused a reduction in isoproterenol-stimulated adenylate cyclase activity and a sequestration of beta-adrenergic receptors. Cells were treated with TPA for 24-48 h to completely down-regulate protein kinase C and then exposed to isoproterenol. Agonist-mediated desensitization of adenylate cyclase and sequestration of beta-adrenergic receptors occurred at similar rates in control and TPA-treated cells. In addition, agonist-mediated down-regulation of beta-adrenergic receptors was not impaired by the absence of protein kinase C activity. Although both agonists and phorbol esters cause desensitization of the beta-adrenergic receptor-coupled adenylate cyclase, agonist-mediated events can occur independently of protein kinase C.

Effects of short-term exposure to the tumor promoter, 12- O-tetradecanoylphorbol-13-acetate on skin carcinogenesis in SENCAR mice

Skin tumor promotion after a short-term exposure to 12- O-tetradecanoylphorbol-13-acetate (TPA) was studied in female SENCAR mice. Mice were dosed once by the topical application of 20 μg of dimethylbenz[ a]anthracene (DMBA) in 0.2 ml acetone. A week later, they received topical applications of TPA (2 or 4 μg per 0.2 ml acetone) once or twice a week for periods of 1–10 weeks and were killed at 30 weeks. Skin tumors were counted and measured for size weekly. When TPA was applied once a week for 10 weeks or only twice a week for 2 weeks, there was significant promotion of papilloma formation in a large proportion of mice initiated with DMBA. Mice that received one or two applications had a few skin tumors. The total number of papillomas decreased considerably and the majority appeared to regress after 20 weeks in mice that received TPA treatment for 10 weeks. In mice that received only 4 TPA treatments, however, the majority of the papillomas grew progressively in size and did not regress during the entire experimental period. A greater proportion of these tumors progressed to carcinoma than did those in mice receiving TPA for 10 weeks. Thus, a short-term exposure was effective in causing certain changes in skin of SENCAR mice that led to tumor development and progression.

Effects of tumor promoters on the rate and commitment to terminal differentiation of subpopulations of murine keratinocytes

The effects of skin-tumor-promoting and -nonpromoting agents on the kinetics of terminal differentiation of subpopulations of keratinocytes differing in buoyant density isolated from mice (SENCAR) that are very sensitive to 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion were investigated. Topical pretreatment of dorsal skin with complete (TPA), first-stage (calcium ionophore A23187) and second-stage (mezerein) tumor promoters, but not the hyperplastic agent ethylphenylpropiolate, accelerated the rate of terminal differentiation of keratinocytes with densities less than 1.074 g/cm 3, but had little effect on cells with a greater density. Within 8.5 hr of TPA treatment, a period preceding mitosis, a large percentage of the most dense basal-cell keratinocytes (⩾1.074 g/cm 3) were converted to cells with a lower density, with a reduced plating efficiency and with an increased rate of differentiation, suggesting that TPA induces a subpopulation of basal cells to commit to terminal differentiation, and accelerates the rate of differentiation of committed cells.