Inhibition of mouse skin tumor promotion and of promoter-stimulated epidermal polyamine biosynthesis by alpha-difluoromethylornithine

Application of the tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) to mouse skin leads to a manifold induction of ornithine decarboxylase (ODC) activity within 5 hr and an increased accumulation of putrescine. The relevance of these TPA-induced changes to the mechanism of tumor promotion was investigated using alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. DFMO applied to mouse skin (0.3 mg in 0.2 ml of solvent) or administered in the drinking water (1%) in conjunction with skin tumor promotion by TPA inhibited the formation of mouse skin papillomas by 50 and 90%, respectively. TPA-induced ODC activity and the accumulation of putrescine were almost completely inhibited. DFMO given in the drinking water decreased spermidine levels, but DFMO treatment by any route did not alter the spermine levels of mouse epidermis. DFMO decreased TPA-induced hyperplasia by 25 to 40%, and the TPA-caused increases in DNA synthesis and mitotic index were inhibited by 60 and 50%, respectively. Therefore, in mouse epidermis, enhanced cell proliferation can be dissociated from ODC induction and the accumulation of putrescine. At the tested dose levels and routes of administration, DFMO did not inhibit the inflammatory response to TPA in several tissues. These results provide evidence for an essential role of ODC induction and the accumulation of putrescine in tumor promotion by TPA and add strength to the proposal that DFMO may be a promising drug for the prevention and treatment of cancer in human beings.     

Protein kinase Cdelta-mediated signal to ornithine decarboxylase induction is independent of skin tumor suppression

Protein Kinase Cdelta (PKCdelta), a Ca(2+)-independent, phospholipid-dependent serine/threonine kinase, is among the PKC isoforms expressed in mouse epidermis. We reported that FVB/N transgenic mice that overexpress ( approximately eightfold) PKCdelta protein in basal epidermal cells are resistant to skin tumor formation by the 7,12-dimethylbenz(a)anthracene (DMBA)-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion protocol. However, despite being resistant to skin tumor promotion by TPA, PKCdelta transgenic mice elicited a 3-4-fold increase in TPA-induced epidermal ODC activity and putrescine levels than their wild-type littermates. PKCdelta was observed to be the key component of the TPA signal transduction pathways to the induction of mouse epidermal ODC activity. To determine if TPA-induced ODC activity and associated putrescine levels in PKCdelta transgenic mice contributed to PKCdelta-mediated suppression of skin tumor promotion by TPA, the irreversible inhibitor of ODC, alpha-difluoromethylornithine (DFMO), was used. PKCdelta transgenic mice and their wild-type littermates were initiated with 100 nmol DMBA and then promoted twice weekly with 5 nmol TPA. The experimental group was given 0.5% DFMO in their drinking water, while the control group was given tap water. After 25 weeks, the number of papillomas (>2 mm) per mouse was counted. The DFMO treatment did not affect the skin tumor multiplicity of PKCdelta transgenic mice. These results indicate that PKCdelta-induced ODC activity is not involved in PKCdelta-mediated tumor suppression. Thus, the signaling pathways via PKCdelta to epidermal ODC induction and skin tumor suppression appear to be independent.     

Inhibition of mouse skin tumor promotion and of promoter-induced epidermal polyamine biosynthesis by methylglyoxal bis(butylamidinohydrazone)

Effects of methyiglyoxal bis(butylamidinohydrazone) (MGBB),a reversible inhibitor of ornithine decarboxylase (ODC) andS-adenosylmethionine decarboxylase (AdoMetDC), on 12-0-tetradecanoylphorbol-13-acetate(TPA)-induced increases of ODC and AdoMetDC activities, ODCmRNA level and polyamine contents in mouse skin were investigatedin connection with tumor formation. Formation of papillomasby applications of TPA to 7,12-dimethylbenz[a]anthracene (DMBA)-initiatedmouse skin was effectively inhibited by simultaneous topicalapplications of MGBB. MGBB also dose-dependently inhibited theability of TPA to induce increases of ODC activity, ODC mRNAlevel and the accumulation of putrescine and spermidine in mouseskin. Induction of AdoMetDC activity was not affected by thedrug. These inhibitory effects of MGBB on ODC induction andtumor promotion were more evident in multiple application experimentsthan with a single application of the drug.     

Inhibition of the development of metastatic squamous cell carcinoma in protein kinase C epsilon transgenic mice by alpha-difluoromethylornithine accompanied by marked hair follicle degeneration and hair loss

The role of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated polyamine biosynthesis in the development of metastatic squamous cell carcinoma (mSCC) in protein kinase C epsilon (PKC epsilon) transgenic mice was determined. TPA treatment induced epidermal ornithine decarboxylase (ODC) activity and putrescine levels approximately 3-4-fold more in PKC epsilon transgenic mice than their wild-type littermates. Development of mSCC by the 7,12-dimethylbenz(a)anthracene (100 nmol)-TPA (5 nmol) protocol in PKC epsilon transgenic mice was completely prevented by administration of the suicide inhibitor of ODC alpha-difluoromethylornithine (DFMO, 0.5% w/v) in the drinking water during TPA promotion. However, DFMO treatment led to marked hair loss in PKC epsilon transgenic mice. DFMO treatment-associated hair loss in PKC epsilon transgenic mice was accompanied by a decrease in the number of intact hair follicles. These results indicate that TPA-induced ODC activity and the resultant accumulation of putrescine in PKC epsilon transgenic mice are linked to growth and maintenance of hair follicles, and the development of mSCC. Severe hair loss observed in PKC epsilon transgenic mice on DFMO during skin tumor promotion has not been reported before in the prevention of cancer in other animal models or in human cancer prevention trials.     

Inhibition of both stage I and stage II skin tumor promotion in SENCAR mice by a polyphenolic fraction isolated from green tea: inhibition depends on the duration of polyphenol treatment

The polyphenolic fraction isolated from green tea (GTP) is apotent inhibitor of phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate(TPA)-induced tumor promotion in mouse skin. In this study,we assessed the effect of GTP on both stage I and stage II skintumor promotion, and also analyzed the effect of duration ofGTP treatment on skin tumor promotion in SENCAR mice. Topicalapplication of GTP (6mg/animal) concurrently with each applicationof either TPA (3.2 nmol) or mezerein (3.2 nmol) in stage I orstage II of the murine skin tumor promotion protocol, respectively,resulted in significant protection against skin papilloma formationin terms of both tumor multiplicity (42—50%) and tumorgrowth (43—54%). More profound and sustained protectiveeffects of GTP were evident when it was applied continuouslyduring both stage I and stage II of the skin tumor promotionprotocol concurrently with TPA or mezerein treatments, respectively.Under this treatment regimen, compared to non-GTP-treated positivecontrols, GTP application showed 71%, 37% and 74% protectionin terms of tumor multiplicity, tumor incidence and tumor growth,respectively. These data indicate that GTP inhibits both stageI and stage II of skin tumor promotion and that the inhibitionof tumor promotion depends on the duration of GTP treatment.     

Anti-mutagenesis and anti-promotion by apigenin, robinetin and indole-3-carbinol

We assessed the anti-mutagenic and anti-promotion propertiesof two flavones, apigenin and robinetin, and of indole-3-carbinol,because these compounds have been reported in vegetables, theconsumption of which has been associated with reduced ratesof cancer. However, the active components of these foods andtheir effects on carcinogenesis have not been established. Anti-mutagenicitywas determined in the Salmonella typhimurium assay by measuringthe effects of the test compounds on bacterial mutagenesis inducedby methyl-nitrosourea (MNU), methyl-n-nitro-N-nitrosoguanidine(MNNG), benzo[a]pyrene (BaP) or 2-aminoanthracene (2-AA). Inclusionof apigenin resulted in a 62% and a 43% inhibition of mutagenicitywith 13 nmol of 2-AA and 30 nmol BaP respectively. Robinetincaused an 87% inhibition of mutagenicity by 2-AA, but indole-3-carbinolhad little or no effect on the mutagenicity of any of the compounds.None of the three compounds inhibited mutagenesis by MNU orMNNG and none were mutagenic or toxic when tested in the absenceof mutagenic compounds at doses up to 20 µg/plate. Anti-promotionproperties were assessed by measuring the effects of apigenin,robinetin and indole-3-carbinol on induction of ornithine decarboxylaseactivity (ODC) in mouse epidermis by 17 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA). Pretreatment of the skin half an hourbefore TPA with apigenin, robinetin, butylated hydroxyanisole,13-cis-retinoic acid (all at 50 µol) or di-fluoromethylornithine(1.6 µmol) inhibited ODC induction at 6 h after TPA by67–80%. Pretreatment with 50 µmol indole-3-carbinolcaused a 78% elevation in the TPA induction at this time. Doseresponse measurements were conducted with apigenin, indole-3-carbinoland robinetin. Inhibition by 30–90% of TPA-induced ODCwas observed at 6 h after TPA in mice pre-treated with 12.5–100µmol apigenin. Pretreatment with 37.5 or 50 µmolindole-3-carbinol or 0.5, 12.5 or 25 µmol robinetin resultedin elevated induction of epidermal ODC by TPA at 6 h after TPA.However, treatment with 50 or 100 µmol robinetin diminishedODC induction at 6 h after TPA. Treatment with 100 µmolapigenin or 50 or 100 µmol indole-3-carbinol in non-TPA-treatedmouse skin caused elevations in epidermal ODC. In comparingthe time course of ODC induction, indole-3-carbinol (50 µmol)pretreatment shifted the induction of epidermal ODC to earliertimes, in addition to elevating ODC induction by TPA. However,apigenin (50 µmol) pretreatment inhibited TPA-inducedODC activity at 4, 6 and 8 h, indicating no shift in ODC induction.In conclusion, indole-3-carbinol showed no potential for inhibitionof mutagenesis in the present study and presented potentialfor enhancement of promotion. In contrast, the potential ofapigenin and robinetin as inhibitors of the initiation and promotionphases of carcinogenesis merits further study.     

Ionizing radiation enhances malignant progression of mouse skin tumors

Chemical carcinogenesis in mouse skin has been divided intothe process of initiation, promotion and progression. Recentlywe have shown that ionizing radiation acts as an initiator inthis model system. In this paper we describe a three-stage experimentusing ionizing radiation in the third stage of mouse skin carcinogenesis.CD-1 mice were initiated with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) followed by biweekly promotion with 12-O-tetradecanoylphorbol-13-acetate(TPA). After 20 weeks of promotion, the animals were treatedwith either acetone, TPA (twice a week for 2 weeks) or eightfractions of 1 MeV electrons (1 Gy/fraction over a period of10 days). The conversion of papillomas to squamous cell carcinomaswas 80% for animals treated with ionizing radiation comparedwith 25% for tumor-bearing animals treated with TPA. Ionizingradiation increased the number of cumulative carcinomas pergroup. The lack of an increase in the number of cumulative papillomasper group due to ionizing radiation suggests that the dose andfractionation protocol used in this study enhanced the progressionof pre-existing papillomas.     

Sequential studies of skin tumorigenesis in PGK mosaic mice: the effect of repeated exposure to a carcinogen on regressed mouse skin papillomas

Most mouse skin papillomas developing after 7,12-diinethylbenz[a]anthracene(DMBA) initiation followed by repeated 12-O-tetradecanoylphorbol-13-acetate(TPA) promotion are promoter-dependent; termination of promotionresults in their regression. Previous evidence, from mappingthe locations of papillomas and using the X-chromosome-linkedphosphoglycerate kinase cell markers, shows that regressionof promoter-dependent papillomas is permanent. Exposure to anothercourse of TPA promotion was found not to induce regenerationin the regressed papillomas. To determine whether repeated exposureto a carcinogen causes regeneration of regressed papilomas,the effects of weekly applications of low doses of DMBA weretested. The results reported here indicate that regressed promoter-dependentpapillomas do not regenerate when exposed repeatedly to DMBA.However, the treatment with DMBA induced many new tumors mostof which were promoter-independent papillomas or malignant carcinomas.These findings provide further support for the conclusion thattermination of promotion leads to permanent regression of mostpromoter-dependent mouse skin papillomas.     

Modulation of the co-promoting activity of gamma interferon in SENCAR and C57BL/6 mouse skin by difluoromethylornithine and the scheduling and duration of interferon treatment

The murine skin multistage carcinogenesis model was used to characterize the co-promoting and tumor progressing activities of i.p. administered recombinant DNA-derived murine gamma interferon (rMuIFN-gamma). The dorsal skins of female SENCAR mice were topically initiated with 7,12-dimethylbenz[a]anthracene (DMBA) and promoted twice a week for 20 weeks with 1 microgram of 12-O-tetradecanoylphorbol-13-acetate (TPA). Doses of rMuIFN-gamma that had no effect on papilloma multiplicities when administered 1 day prior to TPA treatment increased the numbers of papillomas per mouse by 33-38% when administered immediately prior (zero time) to TPA application. A minimum of 6 weeks of co-treatment with TPA and rMuIFN-gamma (zero time) were necessary for demonstration of rMuIFN-gamma-dependent co-promotion. The ad libitum administration of either 0.25 or 1% (w/v) solutions of alpha-difluoromethylornithine (DFMO) in the drinking water inhibited by 90% the TPA-dependent elevation of epidermal ornithine decarboxylase activity but had minimal effect on papilloma multiplicities in TPA-promoted mice. However, both doses of DFMO completely suppressed rMuIFN-gamma-dependent co-promotion. Carcinoma incidence and multiplicities by weeks 46-48 of the promotion-progression period were statistically indistinguishable for initiated mice treated with TPA, TPA + DFMO, TPA + IFN-gamma or TPA + DFMO + IFN-gamma. Similarly, i.p. administration of rMuIFN-gamma to papilloma-bearing mice in a tumor progression study, with and without simultaneous topical TPA treatment, did not affect carcinoma latency or carcinoma multiplicities. C57BL/6 mice initiated with DMBA developed few papillomas (0.2 paps/mouse) after 19 weeks of TPA promotion. The i.p. administration of rMuIFN-gamma to C57BL/6 mice at the time of TPA treatment, at doses that were co-promoting in SENCAR mice, did not increase papilloma multiplicities. Collectively, our studies suggest that the co-promoting activity of rMuIFN-gamma is exceptionally sensitive to inhibition by DFMO and dependent upon the scheduling and duration of rMuIFN-gamma treatment, and the mouse strain/stock employed for the studies.     

Differential effects of dietary {beta}-carotene on papilloma and carcinoma formation induced by an initiation-promotion protocol in SENCAR mouse skin

SENCAR mice were used to determine the effects of the provitaminA compound ß-carotene on papilloma formation and theconversion of papillomas to carcinomas in a two-stage protocolwith one application of the initiator 7,12-dimethyl-benz[a]anthracene(DMBA, 20 µg) and 20 weekly applications of the promoter12-O-tetradecanoylphorbol-13-acetate (TPA, 2 µg). A purifiedvitamin A-free diet was supplemented with ß-caroteneat four levels (0.6, 6, 60 and 600 µg/g of diet) for femalemice and two levels (60 and 600 µg/g) for male mice. Dietarysupplementations of ß-carotene did not result in significantchanges in body weight and survival of female and male mice.However, papillomas developed more rapidly and papilloma incidence(% mice with papillomas) reached its maximum (100%) sooner inmale mice fed 600 µg of ß-carotene/g of dietthan those fed 60 µg/g. There were smaller differencesin papilloma incidence among the dietary groups in female mice,but the papilloma incidence again reached 100% sooner in micefed 600 µg of ß-carotene/g of diet. Female andmale mice fed 600 µg of ß-carotene/g of diethad significantly higher papilloma yields (average number ofpapillomas/mouse) than other dietary groups and a very low percentageof these papillomas converted to carcinomas in these mice. Thus,B-carotene at 600 µg/g inhibited the conversion of papillomasto carcinomas in both sexes. In addition, papilloma yields werehigher in female mice and these papillomas regressed more quicklythan those in the corresponding groups of male mice. In conclusion,dietary ß-carotene caused differential effects onpapilloma and carcinoma yields and sex-dependent differencesin papilloma formation in female and male SENCAR mice treatedwith DMBA and TPA in a two-stage carcinogenesis protocol.     

Modulation of chrysarobin skin tumor promotion

The present study examined the effect of several prototypic inhibitors of phorbol ester skin tumor promotion on skin tumor promotion by chrysarobin, an anthrone tumor promoter. Retinoic acid (RA) inhibited skin tumor promotion by chrysarobin; however, the degree of inhibition was dependent on the treatment protocol. When RA (10 micrograms/mouse) was given 1 h after each twice-weekly application of chrysarobin (220 nmol/mouse), a marked inhibition of papilloma formation was observed (78%). In additional experiments, using a once-weekly application of chrysarobin, RA also inhibited skin tumor promotion but the magnitude of inhibition was less. Interestingly, RA (10 micrograms/mouse), given 1 or 6 h after the promoter, did not inhibit the induction of epidermal ornithine decarboxylase (ODC) activity induced by a single topical application of chrysarobin (220 nmol). Fluocinolone acetonide (1 microgram/mouse), given 5 min before each twice-weekly application of chrysarobin (220 nmol/mouse) effectively inhibited skin tumor promotion (88%). A 0.5 or 0.25% supplement of alpha-difluoromethylornithine (alpha-DFMO) in the drinking water inhibited the induction of epidermal ODC following chrysarobin (220 nmol/mouse) treatment by 85 or 70%, respectively. Supplements of both 0.25 and 0.5% of alpha-DFMO also led to a 50 and 61% inhibition, respectively, in the number of papillomas per mouse after 25 weeks of promotion with chrysarobin. Interestingly, 0.25% alpha-DFMO in the drinking water did not reduce the number of papillomas per mouse after 20 weeks of promotion with 1.7 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA). However, the number of papillomas per mouse that were greater than or equal to 4 mm in diameter was significantly reduced in both chrysarobin- and TPA-treated mice. The data indicate that RA, FA and alpha-DFMO may be general inhibitors of tumor promoter regardless of the chemical class of tumor promoter. The ability of these inhibitors of phorbol ester promotion to inhibit anthrone promotion indicates that some common biochemical pathways may exist for both classes of skin tumor promoters.     

FVB/N mice: an inbred strain sensitive to the chemical induction of squamous cell carcinomas in the skin

The widespread use of FVB/N mice for the establishment of transgeniclines containing active oncogenes suggested the importance oftesting the parent FVB/N mice for sensitivity to experimentalcarcinogenesis. After initiation of mouse skin by a single treatmentwith 7, 12-dimethylbenz[a]anthracene (DMBA) and promotion by20 weekly applications of 12-O-tetradecanoylphorbol-13-acetate(TPA), the skin tumor incidence was compared in FVB/N mice,TPA-sensitive (SENCAR and CD-I) and TPA-resistant mice (BALB/cand C57BL/6). Initiation by 25 µg DMBA followed by promotionwith a low dose of TPA (2 µg/week) induced one or morepapillomas in only 25% of FVB/N mice, compared with 100% inSENCAR, 53% in CD-I, 17% in BALB/c and 0% in C57BL/6 mice. Ata more effective dose of TPA (5 /ig/week), FVB/N mice initiatedby 5, 25 or 100µg DMBA developed 3.4, 6.9 and 11.8 papillomasper mouse. In contrast, the incidence of squamous cell carcinomas(SCCs) (17–18/30 mice) did not increase with DMBA dose.TPA promotion of non-initiated mice induced only six papillomas,but three progressed to SCCs, a high rate of malignant conversion.Skin tumor induction by 20 weekly treatments with 10 µgDMBA produced few papillomas, but 50.0% of the papillomas progressedto carcinomas in FVB/N mice, compared with 9.15% in SENCAR,37.5% in CD-I, 23.1% in BALB/c and 15.0% in C57CL/6 mice. Thefirst carcinomas appeared after 14 weeks in FVB/N, 24 weeksin SENCAR, 26 weeks in CD-I and C57BL/6 and 34 weeks in BALB/cmice. Thus, FVB/N mice develop an unusually high incidence ofSCCs after treatment with repeated DMBA, DMBA initiation-TPApromotion and even TPA alone.     

Lack of inhibition by retinoids of bis(2-oxopropyl)nitrosamine-induced carcinogenesis in Syrian hamsters

Syrian hamsters were treated with either a low (10 mg/kg bodyweight) or high (40 mg/kg body weight) single dose of bis(2-oxopropyl)nitrosamine(BOP) and beginning 1 week later fed either low (0.2 mmol/kgdiet) or high (0.4–1.0 mmol/kg diet) levels of one offour retinoids [13 cis retinoic acid (13-cis-RA), N-ethylretinamide(ERA), N-(2-hydroxy- ethyl)retinamide (OHERA) or N-(phenyl)retinamide(PRA)] for periods of 40 or 50 weeks. The high retinoid levels(0.4–1.0 mmol/kg diet) fed following the highest BOP treatmentenhanced pancreatic carcinoma yields (average number/effectiveanimal) in males fed all four retinoids, and in females fedERA and 13-cis-RA. Enhanced adenoma yields were also seen inall groups when high retinoid levels were fed following 40 mgBOP/kg body weight. However, these retinoid levels caused anincreased adenoma yield in male hamsters only and did not modifycarcinoma yields when fed following 10 mg HOP/kg body weight.Similarly, tumor yields at extra-pancreatic sites were elevatedin refinoid-fed hamsters of both sexes after 40 mg BOP/kg bodyweight and in males fed ERA and 13-cis-RA after 10 mg BOP/kgbody weight when retinoids were given at the high levels (0.4–1.0mmol/kg diet). Increased incidences of bile duct and liver tumorsin particular were found in hamsters given 40 mg BOP/kg bodyweight. Consumption of retinoid levels of 0.4 mmol/kg diet andabove was also associated with a high incidence of liver cellnecrosis, ovarian cysts and ovarian hemorrhage. Retinoids (ERA,OHERA, and PRA) fed at the low level (0.2 mmol/kg diet) followingthe low BOP dose did not enhance carcinogenesis in the pan creasor at other sites and did not cause alterations in morphologicobservations.     

Presence of a functionally altered ornithine decarboxylase activity in chrysarobin-promoted mouse epidermal papillomas

Recent work from this laboratory has demonstrated the presenceof a structurally and functionally different ornithine decarboxylase(ODC) in mouse epidermal tumors induced by a two-stage protocolinvolving initiation with 7,12-dimethylbenzanthracene (DMBA)and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA).In this report, the enzymatic properties of ODC present in DMBA-initiatedchrysarobin-promoted papillomas are compared to the enzyme inducedby chrysarobin in normal epidermis. Analyses of 13 individualtumor extracts indicated each had an elevated level of ODC activitycompared to uninduced normal epidermis. Addition of GTP to theenzyme assay caused a marked stimulation of ODC activity innine of 13 tumor extracts but had no effect on chrysarobin-inducedepidermal ODC. Enzyme kinetic analyses indicated that GTP loweredthe atypically high apparent K_m values for L-orni-thine of thepapilloma enzyme to values typical of epidermal ODC. The K forGTP activation of papilloma ODC was 7 x 10^–9 M. Whena series of nucleotides was tested, only GTP, the non-hydrolysableanaly GTPS, dGTP and GDP were capable of significant activationat 1 µM, while other derivatives including GMP, ATP andCTP were less effective. The ability of the tumor enzyme tobind GTP was confirmed by the results of GTP-agarose chromatography,in which the papilloma enzyme (but not chrysarobin-induced epidermalODC) bound to this affinity column and could be eluted by GTP.While some differences were observed in the properties of ODCfrom chrysarobin-promoted versus TPA-promoted papillomas, themajor conclusion of this study is that both agents cause theappearance of a functionally altered ODC in the majority ofpapillomas produced by a two-step protocol.     

Quantitation of early clonal expansion of two mutant 61st codon c-Ha-ras alleles in DMBA/TPA treated mouse skin by nested PCR/RFLP

It has been hypothesized that tumor promotion in mouse skininvolves clonal expansion of initiated cells with activatedc-Harvey (Ha)-ras oncogene to give rise to benign tumors. Wehave used the two stage mouse skin carcino-genesis model using7, 12-dimethylbenz[a]anthracene (DMBA) as the initiator and12-0-tetradecanoyl-phorbol-13-acetate (TPA) as the tumor promoterto quantitate the number of mutated c-Ha-ras alleles in mouseepidermal DNA. Epidermal samples were harvested over a 12-weekperiod before the appearance of papillomas. Three 61st codon(i.e. CAA) c-Ha-ras mutations, CTA (T2), CGA (G2) and CAT (T3)were quantitated by newly developed nested PCR/RFLP assays.During TPA promotion the number of T2 mutant copies showed aprogressive increase starting at 4 weeks after initiation andthe number of T3 mutant alleles showed an increase startingat 6 weeks. By 12 weeks after initiation, TPA-promoted mouseepidermis averaged     

Inhibition of the induction of ornithine decarboxylase activity by 12-O-tetradecanoylphorbol-13-acetate in mouse skin by sphingosine sulfate

We investigated the effect of sphingosine sulfate on the inductionof ODC (ornithine decarboxylase) activity by TPA (12-O-tetradecanoylphorbol-13-acetate)in mouse skin. When applied topically to the shaved skin ofSENCAR mice at dosages of 10–40 µunol per animal,30 min before the superficial application of 8.5 nmol of TPA,sphingosine sulfate dramatically inhibited the induction ofODC activity by the tumor promoter. Significant inhibition ofTPA-induced ODC activity was observed at 4, 6 and 8 h afterTPA treatment in separate studies. The results indicate thatsphingosine sulfate is an effective inhibitor of ODC inductionby TPA in mouse skin.     

Inhibitory effect of silymarin, an anti-hepatotoxic flavonoid, on 12-O-tetradecanoylphorbol-13-acetate-induced epidermal ornithine decarboxylase activity and mRNA in SENCAR mice

In recent years, considerable emphasis has been placed on identifyingnew cancer chemopreventive agents which could be useful forhuman populations. Silymarin, an anti-oxidant flavonoid isolatedfrom artichoke, has been shown to possess siginificant activityagainst hepatotoxicity and other pharmacological and physiologicaldisorders. Since many antioxidants inhibit tumor promotion,and because ornithine decarboxylase (ODC) is a well known biochemicalmarker of tumor promotion, we assessed the effect of skin applicationof silymarin on 12-O-tetradecanoylphorbol-13-acetate (TPA) inducedepidermal ODC activity and ODC mRNA levels in SENCAR mice. Applicationof silymarin at doses of 0.5–18 mg (1–37 µmol)/mouseprior to that of TPA (2.5 µg) treatment resulted in significantinhibition of TPA-induced epidermal ODC activity in a dose-and time-dependent manner. Northern blot analysis revealed thattopical application of silymarin at the dose of 2 mg/mouse resultedin almost complete inhibition of TPA-induced epidermal ODC mRNA.In other studies, silymarin also showed significant inhibitionof epidermal ODC activity induced by several other tumor promoters,including free radical-generating compounds. Our data suggestthat silymarin could be a useful anti-tumor promoting agentcapable of ameliorating the tumor promoting effects of a widerange of tumor promoters.     

Enhanced pancreatic and skin tumorigenesis in cabbage-fed hamsters and mice

Studies were conduded to evaluate the ability of dietary driedcabbage supplements to inhibit pancreatic carcinogenesis inhamsters and skin tumorigenesis in mice. Pancreatic cancer wasinduced by treatment with 40 mg/kg body wt N-nitrosobis-(2oxopropyl)amine(BOP). Cabbage was fed from before carcinogen treatment in lowfat diet and, beginning 1 week after BOP treatment, cabbagewas given in low fat and high fat diets in comparison with therespective non-cabbage containing diets. Dried cabbage was incorporatedat 9 and 11% levels into the low and high fat diets. Feedingcabbage in the high fat diet elevated the yield of BOP-inducedpancreatic ductular cardnoma (1.6 carcinomas/effedive animal)in comparison with that observed in hamsters fed cabbage ina low fat diet or in those given a high fat diet without cabbage, 0.6–0.8 carcinomaa/effedive animal (P 0.05). Furthermore,the incidence of BOP-induced gall bladder adenocadnomm was elevatedin cabbage-fed hamsters irrespedve of dietary fat intake. Effetsof dietary fat and cabbage on food consumption, body weight,and serum T₃ and T₄ values are described. Skin tumorigenesiswas induced in SENCAR mice by 10 nmd 7,12 dlmethylbenz[a]anthracene(DMBA) and promoted beginning 1 week later with twice weeklyapplications of 2 µg 12-O-tetradecanoyl-13-phorbol acetate(TPA). Dried cabbage was incorporated into AIN semi-purifieddiets from before DMBA treatment and throughout TPA treatment.Skin papilloma yield was elevated in DMBA-initiated TPA-promotedmice that were fed diets containing 10% cabbage. Mice fed cabbagedeveloped an average of 8.45 papillomas per mouse following22 weeks of promotion while mice given control diet developed7.25 papillomas per mouse (P < 0.001). Cabbage feeding didnot influence survival, food consumption or body weight of themice. These results suggest the need for further research onthe use of cabbage as a chemopreventive measure.     

SENCAR mouse skin tumors produced by promotion alone have A to G mutations in codon 61 of the c-rasHa gene

SENCAR mice, developed by selective breeding for high susceptibilityto skin carcinogenesis by initiation with 7, 12-dimethylbenz[a]anthraceneand promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA),form squamous papillomas in     

Effect of 13 cis retinoic acid on early precancerous antigenic goblet-cell modifications and induction of cancer during 1,2-dimethylhydrazine carcinogenesis in rats

The possible effect of oral 13 cis retinoic acid (13-cis-RA)on the carcinogenic process induced by 28 weekly s.c. injectionsof 1,2-dimethylhydrazine (DMH) in 34 Wistar rats was investigated.Using immunohistology, precancerous and cancerous stages werecompared with the same stages induced by DMH without additional13-cis-RA in 33 rats. M1 antigens, which characterize modificationsin goblet-cell differentiation occurring early in rat coloniccarcinogenesis, were used to investigate the possible effectof retinoids on differentiation during precancerous stages.From 3–20 weeks after the start of the experiment, nosignificant differences were observed in the timing of Ml antigensin the 2 groups of rats. It was also observed that 13-cis-RAhad no effect on histological lesions associated with precancerousmucosa, nor on the occurrence of intestinal adenocarcinomas.Thus, under these conditions, oral administration of 13-cis-RAdid not significantly inhibit precancerous or cancerous stagesof intestinal carcinoma development.