Two-stage Carcinogenesis Experiment Research Articles

Protective role of p53 in skin cancer: Carcinogenesis studies in mice lacking epidermal p53.

p53 is a protein that causes cell cycle arrest, apoptosis or senescence, being crucial in the process of tumor suppression in several cell types. Different in vitro and animal models have been designed for the study of p53 role in skin cancer. These models have revealed opposing results, as in some experimental settings it appears that p53 protects against skin cancer, but in others, the opposite conclusion emerges. We have generated cohorts of mice with efficient p53 deletion restricted to stratified epithelia and control littermates expressing wild type p53 and studied their sensitivity to both chemically-induced and spontaneous tumoral transformation, as well as the tumor types originated in each experimental group. Our results indicate that the absence of p53 in stratified epithelia leads to the appearance, in two-stage skin carcinogenesis experiments, of a higher number of tumors that grow faster and become malignant more frequently than tumors arisen in mice with wild type p53 genotype. In addition, the histological diversity of the tumor type is greater in mice with epidermal p53 loss, indicating the tumor suppressive role of p53 in different epidermal cell types. Aging mice with p53 inactivation in stratified epithelia developed spontaneous carcinomas in skin and other epithelia. Overall, these results highlight the truly protective nature of p53 functions in the development of cancer in skin and in other stratified epithelia.

Abstract 2106: Twist1 regulates keratinocyte stem cell proliferation and migration and is required for skin tumor formation

Abstract Twist1 is a basic helix-loop-helix transcription factor and is known to play a role in tumor progression through regulation of genes involved in epithelial-mesenchymal transition. Twist1 is a known transcriptional target of signal transducer and activator of transcription-3 (Stat3). In previous studies, Stat3 was shown to play a role in skin tumor progression in the two-stage skin carcinogenesis model, at least in part, by regulating the levels of Twist1. Recent studies using keratinocyte-specific knockout (KO) of Twist1 indicate that Twist1 regulates keratinocyte proliferation during skin tumor promotion. Western blot analysis of lysates from both Twist1 deficient mouse primary keratinocytes and from epidermis isolated from BK5.Cre x Twist1flox/flox mice indicated that Twist1 KO leads to reduced levels of the cell cycle proteins Cyclin E1, E2F1, and Cdk2 and increased expression of p21 following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). ChIP analysis of epidermal lysates from BK5.Cre x Twist1flox/flox mice revealed that Twist1 bound to the promoter regions of Cyclin E1, E2F1, and c-Myc, indicating direct transcriptional regulation of these cell cycle regulators. Moreover, Twist1 KO in keratinocytes impeded cell cycle progression by reducing the number of cells that advanced to S-phase. Further analyses have demonstrated that deletion of Twist1 either in primary keratinocytes or in vivo leads to an upregulation of p53, which is responsible for the increased expression of p21 observed in these cells. Keratinocyte specific deletion of Twist1 in vivo suppressed epidermal proliferation induced by TPA treatment compared to that observed in wild-type (WT) mice. To further characterize the potential role of Twist1 during epithelial carcinogenesis, we analyzed the impact of Twist1 deletion in vivo on bulge region keratinocyte stem cells (KSCs). Keratinocyte specific deletion of Twist1 in vivo led to a significant reduction in the number of label-retaining cells as well as the number of α6-integrin+/CD34+ cells in the hair follicles of untreated mice. An ongoing two-stage skin carcinogenesis experiment shows that BK5.Cre x Twist1flox/flox mice have significantly reduced tumor development as observed by decreased tumor multiplicity and delayed latency compared WT mice. Furthermore, tumors that developed in BK5.Cre x Twist1flox/flox mice have significantly reduced size compared to tumors on WT mice. Collectively, these findings suggest that Twist1 has a novel role in epithelial carcinogenesis by regulating proliferation and migration of keratinocytes and KSCs. Research supported by RP140108, CA76520, and T32 ES007247. Citation Format: Jaya Srivastava, Okkyung Rho, John DiGiovanni. Twist1 regulates keratinocyte stem cell proliferation and migration and is required for skin tumor formation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2106. doi:10.1158/1538-7445.AM2015-2106

Abstract 1899: Anti-skin tumor promoting effects of pentacyclic triterpenes found in Perilla frutescens

Abstract In this study, we conducted a comprehensive examination of a series of pentacyclic tritperpenes found in P. frutescens, including ursolic acid (UA) oleanolic acid (OA), augustic acid (AA), corosolic acid (CA), 3-epi-corosolic acid (3-epiCA), maslinic acid (MA), and 3-epi-maslinic acid (3-epiMA) for their effects on epidermal cell signaling, proliferation, and skin inflammation in relation to their ability to inhibit skin tumor promotion by TPA. For these experiments, female ICR mice (7-9 weeks of age) were treated with either acetone or 2 μmol of UA, OA, AA, CA, 3-epiCA, MA, or 3-epiMA 30 min prior to TPA (6.8 nmol) treatment The treatment protocol involved twice weekly treatments over a two week period. Mouse epidermis (6 hr time point) and whole skin (48 hr time point) were collected for Western blot analysis and histological analysis, respectively. Pretreatment with these compounds inhibited the activation of JNK1/2, Src, and Stat3 as well as the induction of Cox-2 protein. Pretreatment with the compounds also reversed the effect of TPA on PDCD4 and p27, and increased the activation of AMPK and LKB1. We also examined the effect of the compounds on TPA-induced epidermal hyperproliferation as assessed by epidermal thickness and epidermal labeling index (LI). All of the compounds examined significantly inhibited TPA-induced epidermal hyperproliferation (significant reductions in both epidermal thickness and LI). In addition, the effect of the triterpenes on TPA-induced infiltration of mast cells in dermis was evaluated. UA significantly inhibited the infiltration of dermal mast cells induced by TPA. Notably, OA, AA, CA, 3-epiCA, MA, and 3-epiMA produced a greater inhibitory effect than that seen with UA on the number of infiltrated mast cells. Thus, UA and a series of related triterpenes differentially inhibited multiple signaling pathways, epidermal hyperproliferation, and a marker of skin inflammation (infiltrated mast cells) induced by TPA. Several of the compounds including CA, 3-epiCA, MA, and 3-epiMA were particularly effective in these experiments. We are currently testing the ability of this group of compounds to inhibit skin tumor promotion by TPA in a two-stage skin carcinogenesis experiment. Collectively, the current data suggest that several pentacyclic triterpenes, in addition to UA, may have potent chemopreventive activity. Research supported by CA164159. Citation Format: Jiyoon Cho, Okkyung Rho, Jacob Junco, Thomas J. Slaga, Andrew M. Camelio, Dionicio Siegel, John DiGiovanni. Anti-skin tumor promoting effects of pentacyclic triterpenes found in Perilla frutescens. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1899. doi:10.1158/1538-7445.AM2015-1899

Tumor Promoters - Microcystin-LR, Nodularin and TNF-α and Human Cancer Development

Microcystin-LR and nodularin, along with okadaic acid, are potent inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A). The mechanisms of action of microcystin-LR and nodularin in the liver and that of okadaic acid, a potent tumor promoter on mouse skin, have attracted the attention of the scientists. This paper reviews several topics: new inhibitors of PP1 and PP2A with new chemical structures, structure-function relationships for both receptor binding and inhibition of protein phosphatases, the crystal structure of PP1 or PP2A-toxin complex, induction of gene expression and apoptosis. These subjects were studied by using in vitro and in vivo experimental systems. Two-stage carcinogenesis experiments with microcystin-LR and nodularin for the first time demonstrated that microcystin-LR is a new tumor promoter in rat liver initiated with diethylnitrosamine (DEN), and that nodularin is a potent tumor promoter associated with weak initiating activity in rat liver initiated with DEN. A working group of WHO (IARC) concluded that microcystin-LR is "possibly carcinogenic to humans" and that nodularin is "not classifiable as to carcinogenicity". Our studies revealed that chemical tumor promoters are inducers of TNF-α in the cells of target tissues and that TNF-α is an endogenous tumor promoter. This advance in carcinogenesis made it possible to look for the link between chemical tumor promoters and endogenous tumor promoters, such as TNF-α and IL-1. The carcinogenic features of TNF-α are described in this review, and the TNF-α inducing protein (Tipα) of Helicobacter pylori genome is presented as an example of a tumor promoter of human stomach cancer development.

Proprotein Convertase Inhibition Results in Decreased Ski Proliferation, Tumorigenesis, and Metastasis

PACE4 is a proprotein convertase (PC) responsible for cleaving and activating proteins that contribute to enhance tumor progression. PACE4 overexpression significantly increased the susceptibility to carcinogenesis, leading to enhanced tumor cell proliferation and premature degradation of the basement membrane. In the present study, we sought to evaluate a novel approach to retard skin tumor progression based on the inhibition of PACE4. We used decanoyl-RVKR-chloromethylketone (CMK), a small-molecule PC inhibitor, for in vitro and in vivo experiments. We found that CMK-dependent blockage of PACE4 activity in skin squamous cell carcinoma cell lines resulted in impaired insulin-like growth factor 1 receptor maturation, diminished its intrinsic tyrosine dnase activity, and decreased tumor cell proliferation. Two-stage skin chemical carcinogenesis experiments, together with topical applications of CMK, demonstrated that this PC inhibitor markedly reduced tumor incidence, tumor multiplicity, and metastasis, pointing to a significant delay in tumor progression in wild-type and PACE4 transgenic mice. These results identify PACE4, together with other PCs, as suitable targets to slow down or block tumor progression, suggesting that PC inhibition is a potential approach for therapy for solid tumors.

Inhibition of CREB Function in Mouse Epidermis Reduces Papilloma Formation

We used a double transgenic tetracycline system to conditionally express A-CREB, a dominant negative protein that prevents the DNA binding and function of cAMP-responsive element binding protein (CREB) family members, in mouse basal epidermis using the keratin 5 promoter. There was no phenotype in the adult. However, following a 7,12-dimethylbenz(a)anthracene (DMBA)/phorbol-12-myristate-13-acetate two-stage skin carcinogenesis experiment, A-CREB-expressing epidermis develop 5-fold fewer papillomas than wild-type controls. However, A-CREB expression one month after DMBA treatment does not prevent papilloma formation, suggesting that CREB functions at an early stage of papilloma formation. Oncogenic H-Ras genes with A-->T mutations in codon 61 were found in wild-type skin but not in A-CREB-expressing skin 2 days after DMBA treatment, suggesting that A-CREB either prevents DMBA mutagenesis or kills oncogenic H-Ras cells. In primary keratinocyte cultures, A-CREB expression induced apoptosis of v-Ras(Ha)-infected cells and suppressed the expression of cell cycle proteins cyclin B1 and cyclin D1. These results suggest that inhibiting CREB function is a valuable cancer prevention strategy.

Susceptibility of pRb‐deficient epidermis to chemical skin carcinogenesis is dependent on the p107 allele dosage

Functional inactivation of the pRb-dependent pathway is a general feature of human cancer. However, only a reduced spectrum of tumors displays inactivation of the Rb gene. This can be attributed, at least partially, to the possible overlapping functions carried out by the related retinoblastoma family members p107 and p130. We observed that loss of pRb in epidermis, using the Cre/LoxP technology, results in proliferation and differentiation defects. These alterations are partially compensated by the elevation in the levels of p107. Moreover, epidermis lacking pRb and p107, but not pRb alone, develops spontaneous tumors, and double deficient primary keratinocytes are highly susceptible to Ha-ras-induced transformation. Two-stage chemical carcinogenesis experiments in mice lacking pRb in epidermis revealed a reduced susceptibility in papilloma formation and an increase in the malignant conversion. We have now explored whether the loss of one p107 allele, inducing a decrease in the levels of p107 up to normal levels could restore the susceptibility of pRb-deficient skin to two-stage protocol. We observed partial restoration in the incidence, number, and size of tumors. However, there is no increased malignancy despite sustained p53 activation. We also observed a partial reduction in the levels of proapoptotic proteins in benign papillomas. These data confirm our previous suggestions on the role of p107 as a tumor suppressor in epidermis in the absence of pRb.

Differential expression of genes associated with cell proliferation and apoptosis induced by okadaic acid during the transformation process of BALB/c 3T3 cells

Okadaic acid (OA) is a tumor promoter in two-stage carcinogenesis experiments. Nevertheless, the effects of OA on cell transformation, cell proliferation and apoptosis vary widely, and the molecular events underlying these effects of OA are not well understood. In the present study, we examined the promoting activity and the associated effects on cell growth and apoptosis mediated by OA in BALB/c 3T3 cells, and evaluated alterations of gene transcriptional expression by microarray analysis. The promoting activity of OA was estimated by a two-stage transformation assay, in which cells were treated first with a low dose of the initiator N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) and then with OA for 14 days. It showed that OA, at concentrations of 7.8–31.3 ng/ml, enhanced the transformation of MNNG-treated cells. In the promotion phase, cells exposed to OA (7.8 ng/ml) grew slowly for the first 2 days and subsequently died. As determined by Hoechst 33342 fluorescent dye and Annexin-V/PI dual-colored flow cytometry, OA induced morphologically apoptotic cells and increased the percentage of early apoptotic cells. The gene expression profile induced by OA at five time points in the promotion phase was determined by use of a specific mouse toxicological microarray containing 1796 clones, and a total of 177 differentially expressed genes were identified. By gene ontology analysis, 31 of these were determined to be functionally involved with cell growth and/or maintenance. In this group, numerous genes associated with the cell proliferation and cell cycle progression were down-regulated at early and/or middle time points. Among these was a subset of genes associated with apoptosis, in which Bnip3, Cycs, Casp3 and Bag1 genes are involved in the mitochondrial pathway of apoptosis. Ier3, Mdm2 and Bnip3 genes may be p53 targets. Furthermore, real-time PCR confirmed the expression changes of five genes selected at random from the differentially expressed genes. We conclude that OA induces cell growth inhibition and apoptosis in the two-stage, MNNG-initiated transformation of BALB/c 3T3 cells. The results of gene expression profile analysis imply that multiple molecular pathways are involved in OA-induced proliferation inhibition and apoptosis. Mitochondrial and p53-associated apoptotic pathways also may contribute to OA-induced apoptosis.

Forced expression of a constitutively active form of Stat3 in mouse epidermis enhances malignant progression of skin tumors induced by two-stage carcinogenesis

Recently, our laboratory demonstrated that Stat3 is required for the de novo development of chemically-induced skin tumors. We have further investigated the role of Stat3 in epithelial carcinogenesis using mice in which the expression of a constitutively active/dimerized form of Stat3 (Stat3C) is targeted to the proliferative compartment of epidermis (referred to as K5.Stat3C transgenic mice). Keratinocytes from K5.Stat3C mice showed increased survival following exposure to 7,12-dimethylbenz[a]anthracene (DMBA) and enhanced proliferation following exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA). In two-stage chemical carcinogenesis experiments using DMBA as the tumor initiator and TPA as the promoter, K5.Stat3C mice developed skin tumors with a shorter latency and in much greater number compared to non-transgenic littermates. Remarkably, 100% of the skin tumors that developed in K5.Stat3C transgenic mice bypassed the premalignant stage and were initially diagnosed as carcinoma in situ which rapidly progressed to squamous cell carcinoma (SCC). These tumors were highly vascularized, poorly differentiated and invasive and loss of expression of K10, filaggrin and E-cadherin was observed by 20 weeks. Finally, overexpression of Stat3C in a papilloma cell line led to enhanced cell migration and enhanced invasion through Matrigel in both the absence and presence of growth factors. In addition to its critical role in early stages of epithelial carcinogenesis, the current study reveals a novel role for Stat3 in driving malignant progression of skin tumors in vivo.

Ahnak/Desmoyokin Is Dispensable for Proliferation, Differentiation, and Maintenance of Integrity in Mouse Epidermis

Desmoyokin was first isolated from bovine muzzle epidermis and thought to be an epidermal desmosome-related protein. We previously demonstrated that the Desmoyokin gene is identical to the Ahnak gene, which is expressed ubiquitously and downregulated in neuroblastomas. It was assumed Ahnak/Desmoyokin was associated with epidermal cell adhesion, tumorigenesis, cell proliferation and differentiation, and embryonic development. To determine the precise biological function of Ahnak/Desmoyokin, we generated a null mutation in ES cells and mice. The resultant Ahnak/Desmoyokin-deficient ES cells normally differentiated into embryoid bodies and neural cells. The mutant mice were viable and fertile and showed no gross developmental defects. Electron microscopic examination of skin sections demonstrated that the ultrastructure of epidermal intercellular junctions, including desmosomes, of the mutant mice was indistinguishable from that of wild-type mice. Two-stage chemical skin carcinogenesis experiments showed no difference in frequency or onset of cutaneous tumor formation between wild-type and mutant mice. Moreover, no tumorigenesis was observed in other tissues and organs of mutant mice up to 2 y of age. These results lead us to conclude that Ahnak/Desmoyokin deficiency has only a minimal effect on epidermal cell adhesion, tumorigenesis, cell proliferation and differentiation, and overall mouse development.

New TNF-α releasing inhibitors as cancer preventive agents from traditional herbal medicine and combination cancer prevention study with EGCG and sulindac or tamoxifen

Herbal medicines are now attracting attention as potential sources of cancer preventive agents. Using inhibition of tumor necrosis factor-α (TNF-α) release assay, we studied Acer nikoense, Megusurino-ki in Japanese. Inhibitory potential was found in the leaf extract, and the main active principles were identified as geraniin and corilagin. The IC 50 values for TNF-α release inhibition were 43 μM for geraniin and 76 μM for corilagin, whereas that for (−)-epigallocatechin gallate (EGCG), the green tea polyphenol, as control was 26 μM. Furthermore, treatment with geraniin inhibited okadaic acid tumor promotion in a two-stage carcinogenesis experiment on mouse skin. Geraniin and corilagin are present in another well-known Japanese traditional herb, Geranium thunbergii, Genno-shoko in Japanese. Considering seasonal variations of the agents and sites of cultivation of herbs, this paper reviews the significance of geraniin as a new cancer preventive agent. In addition, based on accumulated results of green tea as a cancer preventive, we review two important results with EGCG: the synergistic effects of EGCG with sulindac or tamoxifen on cancer preventive activity in PC-9 cells, and cancer prevention of intestinal tumor development in multiple intestinal neoplasia (Min) mice by cotreatment using EGCG with sulindac. We report here new findings on additional gene expression resulting from cotreatment with EGCG and sulindac in PC-9 cells compared with gene expression by EGCG alone or sulindac alone. Overall, our results indicate that, with the continuing spread of cancer chemoprevention as a fundamental medical strategy, both clinicians and researchers should take a closer look at herbal medicine.

Promoting Effect of Sodium L-Ascorbate on N-Butyl-N-(4-hydroxybutyl)nitrosamine-induced Renal Pelvic Carcinogenesis in SD/cShi Rats of Both Sexes

Susceptibility to the promoting effects of sodium L-ascorbate (Na-AsA) on the development of pelvis and urinary bladder tumors in male and female SD/cShi rats, featuring spontaneous hydronephrosis, was investigated. Rats received 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in their drinking water for 4 weeks and subsequently given basal diet with or without a 5% Na-AsA supplement for 32 weeks. Histopathological examination revealed the promoting effect of Na-AsA on not only the development of urinary bladder tumors but also renal pelvic tumors in the animals of both sexes in this two-stage carcinogenesis experiment, the effect being more prominent in males. Administration of either BBN or Na-AsA alone also induced papillomas and papillary or nodular hyperplasia of renal pelvis or urinary bladder, respectively, in male but not female rats. However, the 5-bromo-2'-deoxyuridine-labeling index of urothelium in the pelvis and bladder increased slightly in male rats and significantly in female rats given Na-AsA alone for 8 weeks. N-butyl-N-(3-carboxypropyl)nitrosamine, which is a metabolite of BBN and proximate carcinogen, was found more in the urine of urinary bladder than that of renal pelvis. These results indicate that the urothelium of the renal pelvis and urinary bladder in SD/cShi rats is susceptible to promoting effects of Na-AsA in the present two stage model urinary tract carcinogenesis, with the urinary bladder of male rats as the most sensitive organ.

Discrete roles of cytokines, TNF-α, IL-1, IL-6 in tumor promotion and cell transformation

Based on our previous results, which pointed to tumor necrosis factor-alpha (TNF-alpha) as the essential cytokine in tumor promotion in mouse skin, we present here three principal findings related to the specific roles of TNF-alpha, interleukin-1 (IL-1) and IL-6 in tumor promotion (using TNF-alpha- and IL-6-deficient mice) and in BALB/3T3 cell transformation: i) The previously reported residual tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) in TNF-/- mice was confirmed by experiments with TNF+/+ and TNF-/- 129/Svj mice of the same strain, using two-stage carcinogenesis experiments. TPA produced tumors in 100% of TNF+/+ and 78% of TNF-/- mice at 20 weeks, and the average number of tumors per mouse was 11.1 in the former group and 2.1 in the latter. Judging from the expression of various inflammatory cytokine genes in TNF+/+ and TNF-/- mice, the residual tumor promoting activity of TPA in TNF-/- mice may be dependent on expression of IL-1alpha and IL-1beta genes. ii) Tumor promotion by TPA and okadaic acid in IL-6+/+ and IL-6-/- C57/BL6 mice was studied, with TPA producing tumors in 57.1% of IL-6+/+ and 40.0% of IL-6-/- mice at 20 weeks, and okadaic acid in 40.0% of IL-6+/+ and 53.3% of IL-6-/- mice. Thus, there was no significant difference between TPA or okadaic acid tumor promotion in either group. In addition, expression of IL-6 gene in skin of both types of mice suggested that IL-6 is not the essential cytokine in tumor promotion, since it can be replaced by other cytokines. iii) In transformed clones of BALB/3T3 cells induced by TNF-alpha alone, IL-1alpha gene expression was induced after transformation by TNF-alpha had occurred, which did not occur in parental cells. Expression patterns of TNF-alpha, IL-1beta, IL-6 and IL-10, along with TGF-beta, were similar in both parental and transformed cells. Considering all these results, we conclude that various cytokines have discrete roles in tumor promotion and cell transformation.

Overexpression of bone morphogenetic protein-6 (BMP-6) in murine epidermis suppresses skin tumor formation by induction of apoptosis and downregulation of fos/jun family members

Bone morphogenetic protein-6 (BMP-6) is a member of the transforming growth factor-beta superfamily. In murine skin, BMP-6 is highly expressed in postmitotic keratinocytes from day 15.5 p.c. till day 6 p.p. Expression in adult skin remains at very low levels, but pathological conditions such as wounding induce the expression of BMP-6. We demonstrate that tumor promotion by TPA (12-O-tetradecanoylphorbol-13-acetate) also induces expression of BMP-6 in suprabasal keratinocytes. This induction is due to post-transcriptional regulation since the level of BMP-6 mRNA remained unchanged. We performed two-stage skin carcinogenesis experiments with transgenic mice epidermally overexpressing BMP-6. These mice display augmented mitotic indices in normal and TPA-treated epidermis when compared to controls. Despite this hyperproliferation, BMP-6 transgenics showed a delayed development and strong suppression of benign and malignant skin tumor formation. In order to resolve this paradox we determined apoptotic frequencies as well as the expression of constituents of AP-1 (activator protein-1) which is essential for tumor promotion. A higher rate of apoptotic keratinocytes was detectable in transgenic mice versus controls and a downregulation of mRNA for jun/fos family members in transgenic skin after TPA-treatment. Thus expression of BMP-6 augments apoptosis and downregulates the transcription of AP-1 family members thereby establishing tumor resistance.

Role of ‘platelet-type’ 12-lipoxygenase in skin carcinogenesis

Murine and human skin express an abundance of lipoxygenase isoforms whose functions are not understood. Substantial data have implicated a role for the ‘platelet-type’ 12-lipoxygenase (P-12LO) metabolite, 12(S)-hydroxy-eicosatetraenoic acid (12-HETE), in a variety of tumor functions. Using P-12LO deficient mice, we sought to examine the role of the P-12LO pathway in tumor initiation and progression. Two distinct genetic strains of P-12LO deficient and wild-type mice, B6/129 Sv and SENCAR, were evaluated in two-stage carcinogenesis experiments. Carcinoma incidence was significantly reduced in the P-12LO deficient mice of the B6/129 Sv background but not the SENCAR-backcrossed mice. In contrast, papilloma incidence was reduced on the SENCAR background but not in the B6/129 Sv strain mice. A separate experiment employing a complete carcinogenesis protocol failed to find any difference in papilloma or carcinoma incidence. Overall, these data suggest that the P-12LO pathway may contribute to tumor incidence and progression in two-stage, but not complete, carcinogenesis, depending on the genetic background.

Development of spontaneous hyperplastic skin lesions and chemically induced skin papillomas in transgenic mice expressing human papillomavirus type 16 E6/E7 genes

Human papillomavirus type 16 (HPV16) has been known to be the major factor for the development of uterine cervical carcinomas. We have developed a line of transgenic mice that express the HPV16 E6 and E7 genes in certain organs using a fusion gene which consists of the tyrosinase promoter and E6/E7 of HPV16, and have chosen the tyrosinase minigene as a co-injected visual marker for the identification of transgenic mice. Our transgenic mice (1) expressed E6/E7 transgene mainly in skin and heart, and (2) showed skin and eye pigmentation profiles, and (3) raised incidence of hyperplastic skin lesions. We had performed two-stage skin carcinogenesis experiment to detect the susceptibility of skin papilloma development in our transgenic mice, using dimethylbenz-anthracene (DMBA) as a initiating agent and 12- O-tetradecanoyl-phorbol-13-acetate (TPA). After 1 week of DMBA treatment (25 μg dissolved in 0.2 ml acetone) and 15 consecutive weeks of TPA treatment (2.5 μg dissolved in 0.2 ml acetone) on the back of transgenic and non-transgenic control mice ( Fv-1 b strain mice which are Friend virus B-type susceptible (FVB)/N), papilloma incidence was increased in our transgenic mice ∼2-fold higher than in control (in female mice, 69.2 vs. 30%, respectively). Thus our transgenic mice may be useful for the development of immunological or other therapies for HPV-associated cancers.

Saponins isolated from Allium chinense G. Don and antitumor-promoting activities of isoliquiritigenin and laxogenin from the same drug.

Investigation of the Chinese crude drug "Xiebai," the bulbs of Allium chinense G. Don (Liliaceae), led to the isolation of 2 saponins, xiebai-saponin I (laxogenin 3-O-beta-xylopyranosyl (1-->4)-[alpha-arabinopyranosyl (1-->6)-beta-glucopyranoside) (1) and laxogenin 3-O-alpha-arabinopyranosyl (1-->6)-beta-glucopyranoside (2), and the aglycone, laxogenin (3), together with 2 chalcones, isoliquiritigenin (4) and isoliquiritigenin-4-O-glucoside (5), and beta-sitosterol glucoside (6). Compounds 1-5 were tested in vitro for their inhibitory effect on the 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated 32Pi-incorporation into phospholipids of HeLa cells. In addition to this, laxogenin (3) was proven to have an antitumor-promoting activity in a two-stage lung carcinogenesis experiment.

Inhibitory effect of euphol, a triterpene alcohol from the roots of Euphorbia kansui, on tumour promotion by 12-O-tetradecanoylphorbol-13-acetate in two-stage carcinogenesis in mouse skin.

The anti-inflammatory activity of euphol, twelve other triterpene alcohols and sitosterol-beta-D-glucopyranoside, isolated from the dichloromethane extract of the roots of Euphorbia kansui, has been evaluated in mice with inflammation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). TPA (1.7 nmol; 1.0 microg/ear) was dissolved in acetone and 10 microL delivered to the inner and outer surfaces of the right ear of ICR mice. A triterpene alcohol, sterol glucoside or vehicle (20 microL; chloroform-methanol 1:1), was applied topically approximately 30 min before each TPA treatment. The ear thickness was measured before treatment and then oedema was measured 6 h after TPA treatment. For the two-stage carcinogenesis experiment, initiation was accomplished by administration of a single topical application of 7,12-dimethylbenz[a]anthracene (DMBA; 195 nmol; 50 microg/mouse) to the shaved backs of mice. Promotion was with 1.7 nmol (1.0 microg) TPA, applied twice weekly to the same shaved area, begun one week after the initiation. Euphol (2.0 micromol; 853 microg), or its vehicle (acetone-dimethylsulphoxide, 9:1; 100 microL), was applied topically 30 min before each TPA treatment. The number and diameter of skin tumours were measured every other week for 20 weeks. All the compounds were found to possess marked inhibitory activity and their 50% inhibitory dose for TPA-induced inflammation was 0.2-1.0 mg/ear. Topical application of euphol (2.0 micromol; 853 microg/mouse) markedly suppressed the tumour-promoting effect of TPA (1.7 nmol; 1.0 microg/mouse) in mouse skin initiated with DMBA.

Okadaic Acid Stimulates the Expression of Vascular Endothelial Growth Factor Gene

Vascular endothelial growth factor (VEGF) is a specific mitogen for vascular endothelial cells and has been implicated in tumor angiogenesis. Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, is a non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoter in two-stage carcinogenesis experiments in mouse skin. To elucidate the role of VEGF in the angiogenesis of these experimental tumors, the effect of okadaic acid on VEGF gene expression was examined. In NIH 3T3, Rat1, HeLa, and A431 cells, VEGF mRNA was upregulated by 5- to 10-fold after incubation with okadaic acid. Furthermore, the amount of VEGF protein in the culture medium was significantly increased after stimulation with okadaic acid. Interestingly, okadaic acid-induced upregulation of VEGF mRNA was not suppressed by protein kinase C (PKC) inhibitor or by tumor necrosis factor α blocking antibody, although TPA-induced VEGF upregulation was strongly suppressed by PKC inhibitor. Our results indicate that okadaic acid is a new and potent inducer of VEGF, suggesting the involvement of VEGF as an angiogenic factor during multistep carcinogenesis in vivo.

Transgenic mice demonstrate AP-1 (activator protein-1) transactivation is required for tumor promotion.

Activator protein-1 (AP-1) is a transcription factor that consists of either a Jun-Jun homodimer or a Jun-Fos heterodimer. Transactivation of AP-1 is required for tumor promoter-induced transformation in mouse epidermal JB6 cells and for progression in mouse and human keratinocytes. Until now, the question of whether AP-1 transactivation is required for carcinogenesis in vivo has remained unanswered, as has the issue of functionally significant target genes. To address these issues we have generated a transgenic mouse in which transactivation mutant c-jun (TAM67), under the control of the human keratin-14 promoter, is expressed specifically in the basal cells of the epidermis where tumor induction is initiated. The keratin-14-TAM67 transgene was expressed in the epidermis, tongue, and cervix, with no apparent abnormalities in any tissue or organ. TAM67 expression blocked 12-O-tetradecanoylphorbol 13-acetate (TPA, phorbol 12-tetradecanoate 13-acetate) induction of the AP-1-regulated luciferase in AP-1 luciferase/TAM67 mice, but did not inhibit induction of candidate AP-1 target genes, collagenase-1 or stromelysin-3. More interestingly, TAM67 expression did not inhibit TPA-induced hyperproliferation. In two-stage skin carcinogenesis experiments, the transgenic animals showed a dramatic inhibition of papilloma induction. We conclude that transactivation of a subset of AP-1-dependent genes is required for tumor promotion and may be targeted for cancer prevention.