Tumor Promoter 12-O-tetradecanoylphorbol-13-acetate Treatment Research Articles

Abstract 3082: Epidermal-specific overexpression of T-cell protein tyrosine phosphatase in mouse attenuates chemically-induced skin carcinogenesis

Abstract Tyrosine phosphorylation signaling, which is regulated by the counter-activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), is critical in maintaining cellular homeostasis. The aberrant increase of tyrosine phosphorylation by mutation and/or overexpression of PTKs can contribute to skin carcinogenesis. While PTKs have been extensively studied in skin carcinogenesis, PTPs have not been studied due to their inactivation by environmental toxicants. Our recent studies showed that T-cell protein tyrosine phosphatase (TC-PTP; encoded by Ptpn2) deficiency in mouse epidermis significantly increased skin tumor formation, demonstrating TC-PTP deficiency predisposes mice to skin carcinogenesis. To further examine the tumor suppressive role of TC-PTP in skin carcinogenesis, we generated epidermal-specific TCPTP-overexpressing (K5HA.Ptpn2) transgenic mice. TC-PTP overexpression led to sensitization to tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA)-induced apoptosis both in vivo epidermis and in vitro keratinocytes. TC-PTP overexpression in epidermis significantly reduced epidermal thickness and hyperproliferation compared to wild-type control following treatment with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, TC-PTP overexpression significantly induced epidermal differentiation following TPA treatment as evidenced by the increased expression of epidermal differentiation markers loricrin, involucrin, keratin 10, and transglutaminase 5 in comparison to control. Two-stage skin carcinogenesis analysis using a DMBA/TPA regimen clearly revealed that K5HA.Ptpn2 mice exhibited delayed tumor development and significantly reduced tumor numbers compared to control mice. Taken together, our results suggest that TC-PTP is a potential therapeutic target for the prevention of skin cancer given its ability to promote epidermal apoptosis and differentiation and inhibit epidermal proliferation. Citation Format: Mihwa Kim, Liza D. Morales, Iksoon Jang, Andrew Tsin, Dae Joon Kim. Epidermal-specific overexpression of T-cell protein tyrosine phosphatase in mouse attenuates chemically-induced skin carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3082.

Targeted disruption of TC-PTP in the proliferative compartment augments STAT3 and AKT signaling and skin tumor development

Tyrosine phosphorylation is a vital mechanism that contributes to skin carcinogenesis. It is regulated by the counter-activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Here, we report the critical role of T-cell protein tyrosine phosphatase (TC-PTP), encoded by Ptpn2, in chemically-induced skin carcinogenesis via the negative regulation of STAT3 and AKT signaling. Using epidermal specific TC-PTP knockout (K14Cre.Ptpn2fl/fl) mice, we demonstrate loss of TC-PTP led to a desensitization to tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA)-induced apoptosis both in vivo epidermis and in vitro keratinocytes. TC-PTP deficiency also resulted in a significant increase in epidermal thickness and hyperproliferation following exposure to the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Western blot analysis showed that both phosphorylated STAT3 and phosphorylated AKT expressions were significantly increased in epidermis of TC-PTP-deficient mice compared to control mice following TPA treatment. Inhibition of STAT3 or AKT reversed the effects of TC-PTP deficiency on apoptosis and proliferation. Finally, TC-PTP knockout mice showed a shortened latency of tumorigenesis and significantly increased numbers of tumors during two-stage skin carcinogenesis. Our findings reveal that TC-PTP has potential as a novel target for the prevention of skin cancer through its role in the regulation of STAT3 and AKT signaling.

Abstract 1290: Investigations to evaluate the role of Twist1 during tumor promotion and epithelial carcinogenesis.

Abstract Recent studies have identified the involvement of receptor tyrosine kinases (RTKs) and their downstream signaling pathways, including MAPK, PI3K/Akt/mTOR, and Stat3, as key regulatory factors of epidermal proliferative capacity during skin carcinogenesis. Through development and study of transgenic mice with overexpression of a constitutively active form of Stat3 (Stat3C) in the basal cell layer, previous studies in the laboratory discovered that constitutive Stat3 activation accelerates tumor progression in the two-stage skin carcinogenesis model, which utilizes the tumor initiator 7,12-dimethylbenz[α]anthracene (DMBA) and tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Twist1, a well-known inducer of EMT, has been shown to be regulated by Stat3 in a number of systems. Western blot analysis of epidermal protein lysates from Stat3C transgenic mice indicated a two-fold induction of Twist1 expression as compared to wild type mice. In addition, immunofluorescence staining analysis of tumors from wild type mice [papillomas and squamous cell carcinomas (SCCs)] and Stat3C mice (SCCs) showed nuclear co-localization of Twist1 and Stat3. In the present study, we investigated the activation and subsequent downstream signaling of Twist1 during TPA-induced epidermal hyperproliferation. Western blot analysis of epidermal lysates from mice treated topically with multiple applications of TPA showed an initial decrease (4-6 h) followed by a subsequent rebound of Twist1 protein at 18 hours post TPA treatment. This initial decrease and then rebound coincided with that of the cell cycle regulatory proteins cyclin D1, cyclin E1, E2F-1, and c-myc. Interestingly, basal levels of Twist1 were reduced in epidermis specific Stat3 deficient mice. Furthermore, Stat3 deficient mice treated with TPA failed to show a rebound in expression of Twist1 in the epidermis. Immunofluorescence experiments of TPA treated epidermis revealed that nuclear localization of Twist1 was restricted to the proliferative compartment. Collectively, these results, combined with other data to be presented, suggest that Stat3 regulates basal levels of Twist1. This protein is localized in the nucleus of cells found primarily in the proliferative compartment of hyperplastic epidermis and skin tumors. The current results further suggest that Twist1 may play a role in cell cycle progression in keratinocytes during tumor promotion. Research supported by CA76520. Citation Format: Jaya Srivastava, Everardo Macias, Dharanija Rao, Kaoru Kiguchi, John DiGiovanni. Investigations to evaluate the role of Twist1 during tumor promotion and epithelial carcinogenesis. [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 1290. doi:10.1158/1538-7445.AM2013-1290

PKM2 inhibitor shikonin suppresses TPA‐induced mitochondrial malfunction and proliferation of skin epidermal JB6 cells

Chemoprevention has been a pivotal and effective strategy during the skin cancer treatment. Using human skin normal and tumor samples, we demonstrated that both the expression and activity levels of pyruvate kinase M2 (PKM2) were higher in skin tumor tissues than normal tissues, suggesting that PKM2, one of important metabolic enzyme, might serve as a target for skin cancer prevention and/or therapy. Shikonin, a small-molecule active chemical, has been studied as an anti-cancer drug candidate in human cancer models. However, the mechanism of action and the chemopreventive potential of shikonin are unclear. Herein, we used the skin epidermal JB6 P+ cells and demonstrated that shikonin suppressed the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) induced neoplastic cell transformation and PKM2 activation in the early stage of carcinogenesis. Mitochondrial functions were inhibited by TPA treatment, as indicated by reduced mitochondrial membrane potential and mitochondrial respiration, which were restored by shikonin. We also examined the levels of lactate as a glycolysis marker, and shikonin suppressed its increase caused by tumor promoter treatment. Modulation of cell metabolism by shikonin was associated with G2-M phase accumulation, and Fra-1 (a major subunit of activator protein 1 in skin tumorigenesis) downregulation. In addition, we demonstrated that AMP-activated protein kinase (AMPK), an energy sensor, which is inactivated by TPA, shikonin could reverse AMPK activity. These results suggest that shikonin bears chemopreventive potential for human skin cancers in which PKM2 is upregulated, which might be mediated by inhibiting oncogenic activation, PKM2 activation, and mitochondrial dysfunction.

Withaferin A suppresses tumor promoter 12‐O‐tetradecanoylphorbol 13‐acetate‐induced decreases in isocitrate dehydrogenase 1 activity and mitochondrial function in skin epidermal JB6 cells

Withaferin A (WA) is a bioactive compound derived from Withania somnifera. The antitumor activity of WA has been well studied in human cancer models; however, its chemopreventive potential is unclear. In the present study, we used the skin epidermal JB6 P+ cells, a well-established model for tumor promotion, and demonstrated that WA suppressed the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced cell transformation and cell proliferation. Interestingly, TPA inactivated isocitrate dehydrogenase 1 (IDH1), which was reversed by WA. Similar results were also observed in mouse skin tissue. Therefore, we focused on metabolism as the potential mechanism of action. We found that mitochondrial functions were downregulated by TPA treatment, as indicated by reduced mitochondrial membrane potential, complex I activity and mitochondrial respiration. However, all of these downregulations were inhibited by WA. In addition, we examined the levels of α-ketoglutarate, a product of IDH1, and WA blocked its reduction upon TPA treatment. Finally, we detected the lactate level as a glycolysis marker, and WA suppressed its elevation caused by tumor promoter treatment. Altogether, these results suggest that WA might exert its chemopreventive activity via inhibiting not only oncogenic activation, but also IDH1 inactivation and mitochondrial dysfunction in early tumorigenesis.

Abstract B59: A combination of resveratrol and ursolic acid produces synergistic inhibitory effects on markers of skin tumor promotion

Abstract Resveratrol (RES), found in grapes and Ursolic Acid (UA), found in rosemary and apples, delay the onset and reduce the number of experimentally-induced skin tumors in mice and inhibit the oncogenic Akt/mammalian target of rapamycin (Akt/mTOR) and Nuclear Factor kappa B (NF\#954;B) pathways. Based on their similar downstream effects via different proximal targets, we hypothesized that a combination treatment of RES and UA would synergize to produce a stronger inhibitory effect on skin tumor promotion. Methods: The dorsal skin of FVB mice (n=8) were shaved and treated topically twice per week for 2 weeks with sub-optimal doses of 2 μmol UA, 2 μmol RES, or a combination of both in 200 μL acetone. Each treatment was followed 30 minutes later with topical application of 3.4 nmol of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in 200 μL acetone. Animals were sacrificed either 6 h (Western blotting) or 48 h (histology) after the final TPA treatment. The skins of five mice per group were removed and the epidermis was collected by scraping for Western blotting. For immunohistochemical (IHC) analysis of epidermal proliferation three mice per group were i.p. injected with 100 mg/kg Bromodeoxyuridine (BrdU) in PBS 30 min. prior to sacrifice. Mouse skin from treated areas was fixed in formalin, paraffin embedded, and analyzed via IHC. Results: RES and UA in combination had a stronger effect on hallmarks of skin tumor promotion than was expected based on the sum of their individual inhibitory effects, indicating synergism between RES and UA. RES and UA in combination synergistically inhibited TPA-induced epidermal thickening (80.2% inhibition vs 66.1 % expected) and epidermal proliferation (100.0% inhibition vs 90.0% expected). Western blotting revealed that components of the NF\#954;B and Akt/mTOR pathways were synergistically downregulated by the RES + UA combination in mouse epidermis. TPA-induced Akt (thr308) phosphorylation (86.0% inhibition vs 26.6% expected) and NFkB subunit p65 (ser536) phosphorylation (100.0% inhibition vs 73.3% expected) were suppressed to a much stronger degree with the combination treatment than that predicted. Similar results were seen for other pathway components, such as p70S6K and ribosomal S6. The synergistic activities of RES and UA were also manifest in the HaCaT human keratinocyte cell line. Cells were treated with sub-optimal doses of RES, UA, or RES + UA and stimulated with 10 ng/ml human tumor necrosis factor alpha (TNFα) for 30 min. and cell lysates were analyzed via Western blotting. TNFα-mediated phosphorylation of p65 was not inhibited by 25 μM RES or 0.1 μM −1 μM UA, but combinations of RES + UA resulted in strong synergistic inhibition of p65 phosphorylation (>66% inhibition vs 0% expected). Also, when HaCaT cells were treated with NFkB- and Akt/mTOR-inhibitory doses of RES (150 μM-200 μM) and UA (5 μM-10 μM), a strong increase (5 fold) in phosphorylation/activation of the energy sensing AMP-activated protein kinase (AMPK) was observed by Western blotting. AMPK also inhibits NF\#954;B and Akt/mTOR activities in other systems, suggesting that AMPK activation may function as a mediator of the synergistic anti-tumor promoting effects of RES and UA. Conclusions: These results indicate that UA and RES synergize to inhibit markers of tumor promotion in both in vivo and in vitro skin models. Future studies will further elucidate the synergistic mechanism of RES and UA and also determine if RES and UA have a synergistic preventive effect on skin tumor promotion during two-stage skin carcinogenesis in vivo. Supported by NIH grants CA079065, supplemental CA079065-10S1 (ARRA), CA129409 and CA037111. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B59.

Inhibitory effects of oligonol on phorbol ester-induced tumor promotion and COX-2 expression in mouse skin: NF-κB and C/EBP as potential targets

Plant polyphenols possess anti-oxidant and anti-inflammatory activities and are hence potential candidates for preventing cancer. The present study was aimed at evaluating the anti-inflammatory and anti-tumor promoting activity of oligonol, a formulation of catechin-type oligomers, in mouse skin stimulated with a proto-type tumor promoter 12- O-tetradecanoylphorbol-13-acetate (TPA). Pretreatment of mouse skin with oligonol significantly inhibited TPA-induced expression of cyclooxygenase-2 (COX-2). Oligonol diminished nuclear translocation and DNA binding of nuclear factor-kappaB (NF-κB) via blockade of phosphorylation and subsequent degradation of IκBα in TPA-treated mouse skin. Moreover, oligonol suppressed TPA-induced DNA binding of CCAAT/enhancer-binding protein (C/EBP) in mouse skin. Oligonol pretreatment also attenuated the phosphorylation and/or catalytic activities of extracellular signal-regulated protein kinase-1/2 (ERK1/2) and p38 mitogen-activated protein (MAP) kinase. Moreover, p38 MAP kinase inhibitor SB203580, but not the MEK inhibitor U0126, negated TPA-induced DNA binding of C/EBP. In addition, oligonol reduced the incidence and the multiplicity of papillomas and squamous cell carcinomas in 7,12-dimethylbenz[ a]anthracene (DMBA)-initiated and TPA-promoted mouse skin, and prolonged the survival of tumor-bearing mice. Pretreatment with oligonol diminished the levels of proliferating cell nuclear antigen and expression of COX-2 in papillomas and carcinomas, respectively, as compared to DMBA plus TPA treatment alone. Taken together, the above findings suggest that oligonol inhibits TPA-induced COX-2 expression by blocking the activation of NF-κB and C/EBP via modulation of MAP kinases and suppresses chemically induced mouse skin tumorigenesis.

Chronic exposure to 12-O-tetradecanoylphorbol-13-acetate represses sod2 induction in vivo: the negative role of p50

It is well documented that the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) can activate manganese superoxide dismutase (MnSOD) expression. However, it is unclear how repeated exposure to TPA following a single application of tumor initiator 7,12-dimethylbenz-(a)-anthracene causes tumor development. We generated transgenic mice expressing human MnSOD promoter- and enhancer-driven luciferase reporter gene and used a non-invasive imaging system to investigate the effects of TPA on MnSOD expression in vivo. Our data indicate that TPA initially activates MnSOD expression, but this positive effect declines after repeated applications. Changes in MnSOD expression in vivo were verified by measuring MnSOD mRNA and protein levels. Using chromatin immunoprecipitation coupled to Western analysis of the transcription factors known to be essential for the constitutive and TPA-induced transcription of MnSOD, we found that TPA treatment leads to both activation and inactivation of MnSOD gene transcription. During the activation phase, the levels of p50, p65, specificity protein 1 (Sp1) and nucleophosmin (NPM) increase after TPA treatments. Sustained treatments with TPA lead to further increase of p50 but not p65, Sp1 or NPM, suggesting that excess p50 may have inhibitory effects leading to the suppression of MnSOD. Alteration of p50 levels by expressing p50 cDNA or p50 small interfering RNA in mouse epithelial (JB6) cells confirms that p50 is inhibitory to MnSOD transcription. These findings identify p50 as having a negative effect on MnSOD induction upon repeated applications of TPA and provide an insight into a cause for the reduction of MnSOD expression during early stages of skin carcinogenesis.

Resveratrol inhibits phorbol ester-induced expression of COX-2 and activation of NF-κB in mouse skin by blocking IκB kinase activity

Aberrant expression of cyclooxygenase-2 (COX-2) has been implicated in tumor promotion. Resveratrol, a phytoalexin present in grapes, was reported to inhibit multistage mouse skin carcinogenesis. In the present study, we found that topically applied resveratrol significantly inhibited COX-2 expression induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Resveratrol-suppressed phosphorylation and subsequent degradation of IkappaBalpha, thereby inhibiting activation of nuclear factor-kappaB (NF-kappaB) in TPA-stimulated mouse skin. Pretreatment with resveratrol also suppressed TPA-induced phosphorylation of extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein (MAP) kinase. Resveratrol blunted TPA-induced phosphorylation of p65 and its interaction with CBP/p300, rendering NF-kappaB transcriptionally inactive. To get further insights into the molecular basis of NF-kappaB inactivation by resveratrol, we examined the role of IkappaB kinase (IKK) in mediating TPA-induced activation of NF-kappaB and COX-2 expression. TPA treatment led to rapid induction of IKK activity in mouse skin, which was abolished either by resveratrol or an IKK inhibitor Bay 11-7082. Topical application of Bay 11-7082 also abrogated TPA-induced NF-kappaB activation and COX-2 expression, supporting the involvement of IKK in TPA-induced COX-2 expression. Taken together, the above findings suggest that resveratrol targets IKK in blocking TPA-induced NF-kappaB activation and COX-2 expression in mouse skin in vivo.

Topical Application of A Novel Immunomodulatory Peptide, RDP58, Reduces Skin Inflammation in the Phorbol Ester-Induced Dermatitis Model

RDP58 is the first lead compound in a series of immunomodulating decapeptides discovered through activity-based screening and computer-aided, rational design. RDP58 disrupts cellular responses signaled through the Toll-like and tumor necrosis factor (TNF) receptor families and occludes important signal transduction pathways involved in inflammation, inhibiting the production of tumor necrosis factor alpha (TNFalpha), interferon-gamma, interleukin (IL)-2, IL-6, and IL-12. These pro-inflammatory cytokines are thought to be involved in the pathogenesis of several inflammatory and autoimmune diseases, including atopic dermatitis and psoriasis. The goal of this study was to determine the ability of RDP58 to inhibit skin inflammation following exposure to the well-characterized protein kinase C activator and tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Topical application of RDP58 to the epidermis following TPA treatment resulted in the amelioration of the phorbol ester-induced irritant contact dermatitis. Substantial reductions were observed in skin thickness and tissue weight, neutrophil-mediated myeloperoxidase activity, inflammatory cytokine production, and various histopathological indicators. We also found RDP58 to be effective in reducing the compounding inflammatory damage brought on by chronic TPA exposure, and that it is capable of targeting inflammatory mediators specifically in the keratinocyte. These results demonstrate that topically applied RDP58 is an effective anti-inflammatory treatment in the phorbol ester-induced dermatitis model, and suggest that it may have therapeutic potential in a variety of immune-related cutaneous diseases.

P53 Translocation to Mitochondria Precedes Its Nuclear Translocation and Targets Mitochondrial Oxidative Defense Protein-Manganese Superoxide Dismutase

The tumor suppressor gene p53 is activated by reactive oxygen species-generating agents. After activation, p53 migrates to mitochondria and nucleus, a response that eventually leads to apoptosis, but how the two events are related is unknown. Herein, we show that p53 translocation to mitochondria precedes its translocation to nucleus in JB6 skin epidermal cells treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Translocation of p53 to mitochondria occurs within 10 minutes after TPA application. In the mitochondria, p53 interacts with the primary antioxidant enzyme, manganese superoxide dismutase (MnSOD), consistent with the reduction of its superoxide scavenging activity, and a subsequent decrease of mitochondrial membrane potential. In contrast to the immediate action on mitochondria, p53 transcriptional activity in the nucleus increases at 1 hour following TPA application, accompanied by an increase in the levels of its target gene bax at 15 hours following TPA treatment. Activation of p53 transcriptional activity is preventable by application of a SOD mimetic (MnTE-2-PyP5+). Thus, p53 translocation to mitochondria and subsequent inactivation of MnSOD explains the observed mitochondrial dysfunction, which leads to transcription-dependent mechanisms of p53-induced apoptosis.

A Mechanism-Based Antioxidant Approach for the Reduction of Skin Carcinogenesis

Studies in our laboratories showed that overexpression of manganese superoxide dismutase (MnSOD) reduced tumor incidence in a multistage skin carcinogenesis mouse model. However, reduction of MnSOD by heterozygous knockout of the MnSOD gene (MnSOD KO) did not lead to an increase in tumor incidence, because a reduction of MnSOD enhanced both cell proliferation and apoptosis. The present study extends our previous studies in the MnSOD KO mice and shows that apoptosis in mouse epidermis occurred prior to cell proliferation (6 versus 24 hours) when treated with tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). To investigate the possibility that a timed administration of SOD following apoptosis but before proliferation may lead to suppression of tumor incidence, we applied a SOD mimetic (MnTE-2-PyP(5+)) 12 hours after each TPA treatment. Biochemical studies showed that MnTE-2-PyP(5+) suppressed the level of protein carbonyls and reduced the activity of activator protein-1 and the level of proliferating cellular nuclear antigen, without reducing the activity of p53 or DNA fragmentation following TPA treatment. Histologic examination confirmed that MnTE-2-PyP(5+) suppressed mitosis without interfering with apoptosis. Remarkably, the incidence and multiplicity of skin tumors were reduced in mice that received MnTE-2-PyP(5+) before cell proliferation. These results show a novel strategy for an antioxidant approach to cancer intervention.

Haploinsufficiency for Odc Modifies Mouse Skin Tumor Susceptibility

Numerous studies have linked overexpression of ornithine decarboxylase (Odc) gene with enhanced susceptibility to mouse skin tumorigenesis. However, there is little experimental evidence suggesting that modest reductions in Odc expression might reduce tumor susceptibility. To address this issue, here we report the use of the Odc(+/-) haploinsufficiency model, in which one copy of the murine Odc gene has been inactivated by a homologous recombination. Compared with Odc(+/+) mice, Odc(+/-) mice exhibit reduced epidermal ODC enzyme activity and polyamine accumulation following treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, following chronic TPA treatment, the characteristic hyperplastic response of the epidermis was diminished in Odc(+/-) mice. Finally, when subjected to a two-stage initiation-promotion protocol, substantially fewer skin papillomas developed in Odc(+/-) mice compared with wild-type littermates. These results support the concept that differences in tissue polyamine levels, resulting from either overexpression or reductions in ODC, are important modifiers of tumor susceptibility.

The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) provokes a prolonged morphologic response and ERK activation in Tsc2-null renal tumor cells.

Loss of tumor suppressor function dramatically alters the cellular response to chemicals. The phorbol ester tumor promoter, 12-O-tetradecanoylphorbol 13-acetate (TPA), stimulates cell proliferation through rapid activation of protein kinase C (PKC), followed by gradual degradation of the kinase. TPA also activates the GTPase Rap1 in some cell types. The tumor suppressor protein Tsc2 has a proposed GTPase activating protein (GAP) function for Rap1, providing a common mechanistic target for Tsc2 and TPA. We compared the cellular response of Tsc2-null (ERC-18) and Tsc2-competent (NRK-52E) renal epithelial cells to TPA treatment. Treatment of ERC-18 cells with 100 ng/ml TPA for 24 h resulted in loss of cell-cell contact, retraction of the cell periphery and rounding. These changes were reversed 1 h after treatment in NRK-52E cells and were apparent 24 h after treatment of ERC-18 cells. Expression of Tsc2 in ERC-18 cells abrogated the prolonged morphologic response. TPA treatment rapidly increased phosphorylation of ERK, a reported downstream effector of both PKC and Rap1, in ERC-18 cells, but induced weak Rap1 activation. TPA-induced ERK phosphorylation was prolonged in ERC-18 cells compared to NRK-52E cells and expression of Tsc2 in ERC-18 cells did not inhibit prolonged ERK activation. The selective PKC inhibitor, bisindolylmaleimide VIII, however, inhibited TPA-induced changes in morphology and ERK activation. These results imply that TPA-induced changes in morphology and ERK activation are mediated primarily through PKC and not Rap1 in renal epithelial cells. These data also imply that Tsc2 expression modulates TPA-induced changes in renal epithelial cell morphology via an ERK-independent mechanism.

The chemopreventive compound curcumin is an efficient inhibitor of Epstein-Barr virus BZLF1 transcription in Raji DR-LUC cells.

To characterize the effects of inhibitors of Epstein-Barr virus (EBV) reactivation, we established Raji DR-LUC cells as a new test system. These cells contain the firefly luciferase (LUC) gene under the control of an immediate-early gene promoter (duplicated right region [DR]) of EBV on a self-replicating episome. Luciferase induction thus serves as an intrinsic marker indicative for EBV reactivation from latency. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induced the viral key activator BamH fragment Z left frame 1 (BZLF1) protein ("ZEBRA") in this system, as demonstrated by induction of the BZLF1 protein-responsive DR promoter upstream of the luciferase gene. Conversely, both BZLF1 protein and luciferase induction were inhibited effectively by the chemopreventive agent curcumin. Semiquantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) further demonstrated that the EBV inducers TPA, sodium butyrate, and transforming growth factor-beta (TGF-beta) increased levels of the mRNA of BZLF1 mRNA at 12, 24, and 48 h after treatment in these cells. TPA treatment also induced luciferase mRNA with similar kinetics. Curcumin was found to be highly effective in decreasing TPA-, butyrate-, and TGF-beta-induced levels of BZLF1 mRNA, and of TPA-induced luciferase mRNA, indicating that three major pathways of EBV are inhibited by curcumin. Electrophoretic mobility shift assays (EMSA) showed that activator protein 1 (AP-1) binding to a cognate AP-1 sequence was detected at 6 h and could be blocked by curcumin. Protein binding to the complete BZLF1 promoter ZIII site (ZIIIA+ZIIIB) demonstrated several specific complexes that gave weak signals at 6 h and 12 h but strong signals at 24 h, all of which were reduced after application of curcumin. Autostimulation of BZLF1 mRNA induction through binding to the ZIII site at 24 h was confirmed by antibody-induced supershift analysis. The present results confirm our previous finding that curcumin is an effective agent for inhibition of EBV reactivation in Raji DR-CAT cells (carrying DR-dependent chloramphenicol acetyltransferase), and they show for the first time that curcumin inhibits EBV reactivation mainly through inhibition of BZLF1 gene transcription.

Transforming JB6 cells exhibit enhanced integrin-mediated adhesion to osteopontin

Transformation of preneoplastic epidermal JB6 cells with tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is an in vitro model of late-stage tumor promotion. Osteopontin (OPN) is a secreted, adhesive protein that is highly expressed in JB6 cells with TPA treatment, and its expression persists for at least 4 days, which is the time required for subsequent expression of transformed phenotype. These observations suggest that OPN may play a role in promoting JB6 cell transformation. To function in transformation of JB6 cells, OPN must bind to the surface of the JB6 cell and subsequently signal within the cell. Therefore, we investigated whether JB6 cells adhere to OPN and, if so, to which surface receptors. TPA-treated JB6 cells had significantly (P < 0.05) increased adherence to OPN compared with dimethylsulfoxide-treated control cells. Enhanced attachment of JB6 cells to OPN was also observed after treatment with another tumor promoter phorbol dibutyrate but not with nontumor promoters (phorbol and 1alpha,25-dihydroxyvitamin D(3)), suggesting that tumor promoters specifically modulate attachment to OPN. The argininylglycylaspartic acid (RGD) cell-binding region of OPN mediates attachment of TPA-treated JB6 cells because RGD, but not argininylglycylglutamic acid (RGE), peptides inhibited adherence of these cells to OPN in a dose-dependent manner. Flow cytometric analyses, blocking adhesion assay using anti-alpha(v) antibody, and co-immunoprecipitation assay all indicated that TPA-treated cells had similar levels of alpha(v) and beta(5) but decreased levels of beta(1) compared with untreated cells and that cell adhesion to OPN is most likely mediated through the alpha(v)beta(5). Furthermore, calphostin C, a specific protein kinase C (PKC) inhibitor, decreased TPA-treated JB6 cell adhesion to OPN by 50%, suggesting that TPA increased integrin affinity or avidity for OPN through a PKC-mediated pathway. Collectively, these results indicate that transforming JB6 cells adhere to OPN through its RGD sequence. The most likely OPN receptor is the alpha(v)beta(5) integrin, which increases the affinity or avidity for OPN through a PKC-dependent pathway rather than increasing the number of receptors.

Phorbol-ester-induced formation of actin filaments and microtubules: role of protein kinase C

Cell division and motility are generated by the cytoskeletal structures contained in networks of actin filaments and microtubules. Among the Ca2+-activated protein kinases, protein kinase C (PKC) can be activated with tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA) (Kikkawaet al., 1983), which induces alterations in the cytoskeleton of cultured cells. To study the regulation of the cytoskeleton by PKC, we investigated the formation of actin filaments, microtubules, changes in cell shape and Ca2+signalling after treatment with TPA in unfertilised eggs of the sea urchinHemicentrotus pulcherrimus.Formation of actin filaments and microtubles was induced in the eggs by the TPA treatment. When the eggs were treated with an inactive TPA, 4α-PMA, these cytoskeletal changes did not occur. Moreover, elongation of actin filaments was inhibited by the Ca2+chelating agent BAPTA, but microtubule organisation was unaffected by intracellular Ca2+chelation in the TPA-treated egg. TPA is known to induce alkalisation in sea urchin eggs (Swann &amp; Whitaker, 1985) and when the eggs are treated with TPA in Na+-free seawater, the rise in intracellular pH (pHi) is depressed. However, elongation of actin filaments and microtubules occurred under these conditions. An increase in pHiis considered to be essential for triggering the bundle of actin filaments (Begget al., 1982) and microtubule assembly (Schattenet al., 1992). These results show that actin and microtubule assemblies require activation of PKC, and that the formation of actin and microtubules may be related to the type of PKC isoform: Ca2+-dependent or -independent. Moreover, this mechanism is essentially independent of cytoplasmic alkalisation.

Resistance to skin tumorigenesis in DNAPK-deficient SCID mice is not due to immunodeficiency but results from hypersensitivity to TPA-induced apoptosis.

Scid/scid mice have a mutation in the gene encoding the catalytic subunit of DNA-dependent protein kinase (DNAPK(cs)) and are defective in end joining of DNA double-strand breaks. As a consequence, they are radiosensitive, lack mature T and B lymphocytes and are predisposed to lymphomagenesis. To determine if this DNA repair defect also increased predisposition to skin tumor formation, we treated the dorsal skin of scid/scid mice with the carcinogen 7,12-dimethylbenz[a]anthracene followed by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Contrary to expectations, we observed a 5-fold reduction in skin tumor multiplicity in scid/scid mice. We addressed whether this was related to their immunodeficiency by similarly treating Rag1(-/-) and Rag2(-/-) knockout mice which also lack mature T and B lymphocytes. We observed no difference in skin tumor multiplicity for either strain compared with control littermates. This indicates a lack of a significant role for T or B lymphocyte mediated immunity on either papilloma or carcinoma formation. We observed a significant increase in apoptotic and necrotic cell death in follicular and interfollicular epithelial cells of scid/scid mice following TPA treatment. This hypersensitivity of SCID (severe combined immunodeficient) cells to TPA indicates that the resistance to skin tumor formation in scid/scid mice is due to loss of initiated cells through TPA-induced cell killing.

TGF-β3, but Not TGF-β1, Protects Keratinocytes against 12-O-Tetradecanoylphorbol-13-acetate-induced Cell Deathin Vitro and in Vivo

We have examined the role that individual TGF-beta isoforms, and in particular TGF-beta3, play in control of epidermal homeostasis. Mice with a knockout mutation of the TGF-beta3 gene die a few hours after birth. A full-thickness skin grafting approach was used to investigate the postnatal development and homeostatic control of the skin of these mice. Grafted skin of mice with a disruption of the TGF-beta3 gene developed similarly to grafts of wild type and TGF-beta1 knockout animals. However, a strikingly different response was observed after acute treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). When exposed to TPA, the grafted skin of wild type and TGF-beta1 knockout mice underwent a hyperplastic response similar to that of normal mouse skin. In marked contrast, TPA treatment of TGF-beta3 knockout grafts induced widespread areas of keratinocyte cell death. Analysis of cultured keratinocytes treated with purified TGF-beta isoforms revealed that TGF-beta3 plays a direct and specific function in protecting keratinocytes against TPA-induced cell death. The protective function of TGF-beta3 on TPA-induced cell death was not because of general suppression of the signaling pathways triggered by this agent, as ERK1/2 activation occurred to a similar if not greater extent in TGF-beta3-treated versus control keratinocytes. Instead, TGF-beta3 treatment led to a significant reduction in TPA-induced c-Jun N-terminal kinase activity, which was associated and possibly explained by specific counteracting effects of TGF-beta3 on TPA-induced disruption of keratinocyte focal adhesions.

Inhibition of tumor promoter activity toward mouse fibroblasts and their in vitro transformation by tissue inhibitor of metalloproteinases-1 (TIMP-1).

Tissue inhibitor of metalloproteinases-1 (TIMP-1), a natural inhibitor of matrix metalloproteinases (MMPs), is known to inhibit invasion and metastasis of tumor cells. In the present study we examined anti-tumor promoter activity of TIMP-1 and its effect on in vitro cell transformation using BALB/3T3 cells in low serum culture medium. In the dye transfer assay the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) continuously blocked gap-junctional intercellular communication (GJIC) of BALB/3T3 cells in confluent phase. TIMP-1 did not prevent transient inhibition of GJIC induced by TPA, but it quickly restored the reduced GJIC level to the control level. The recovery of GJIC was dependent on the concentration of TIMP-1 from 1 to 1000 ng/ml. In an in vitro two-stage transformation assay in which BALB/3T3 cells were treated with 0.5 microg/ml N-metyl-N'-nitro-N-nitrosoguanidine as initiator and 100 ng/ml TPA as promoter, TIMP-1 at concentrations > 10 ng/ml inhibited the focus formation of transformed cells by approximately 60%. TIMP-2 and a synthetic MMP inhibitor showed a similar inhibitory activity on in vitro cell transformation. Furthermore, zymographyic analysis showed that TPA treatment of BALB/3T3 cells induced secretion of gelatinase B and stromelysin-1 into the culture medium. These results indicate that TIMP-1 and TIMP-2 have inhibitory activity on in vitro transformation of cells. It seems likely that TPA-inducible MMPs are involved in carcinogenesis and TIMPs have a protective role against carcinogenesis in vivo.