Effects of beta-carotene on chemically-induced skin tumors in HRA/Skh hairless mice

In order to evaluate the role of beta-carotene as an inhibitor of skin carcinogenesis, hairless female HRA/Skh mice were treated with the initiator 7,12-dimethylbenz[a]anthracene (DMBA) and with the promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), and were fed a balanced diet free from vitamin A either with or without gavage-administered beta-carotene. There was no evidence of avitaminosis A or differences in body weight in mice deprived of beta-carotene and vitamin A, compared with those given 290 or 1430 IU beta-carotene/kg per day. Mice fed with normal animal feed pellets displayed a significantly higher body weight (28.5 +/- 1.95 g) compared with mice on the special diet (25.7 +/- 1.9 g), and also displayed a higher papilloma yield. However animals on the special diet, fed with beta-carotene from weaning, displayed significantly lower numbers of papillomas per mouse. This lower papilloma yield was evident particularly between 12-24 weeks after commencement of the study, which coincided with the period of maximum tumor yield in DMBA/TPA-treated mice. The characteristic regression of papillomas after that time points to the reversibility of many earlier papillomas, and their dependence on continued TPA administration. Evaluation of carcinomas in mice on the various dietary regimes showed there was no significant difference between any group, including those fed with beta-carotene continuously from weaning. The present results demonstrate that a sustained dietary intake of beta-carotene from 3 weeks of age partially suppressed the growth of papillomas, but did not affect the course of malignant progression in DMBA/TPA-treated HRA/Skh mice. It is evident that beta-carotene predominantly affects TPA-dependent papillomas, which possess reversible properties and have a low probability of progression to form carcinomas.     

Further characterization of skin tumor promotion and progression by mezerein in SENCAR mice

This study evaluated the skin tumor-promoting activity of mezerein in SENCAR mice. The effect of initiation dose of 7,12-dimethylbenz(a)anthracene (DMBA) on tumor promotion by mezerein was examined. Excellent dose-response relationships were observed for initiation with DMBA at 0.2-20 micrograms per mouse with mezerein as a complete promoter. None of the mezerein-only promotion groups had papilloma responses similar to those of the corresponding groups receiving two-stage promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA) followed by mezerein, even when a 40-micrograms initiating dose of DMBA was used. The effect delaying promotion with mezerein for 10 weeks was also examined in mice initiated with either 0.2, 2, 20, or 40 micrograms of DMBA per mouse. The 10-week delay led to a slight increase in the number of papillomas per mouse in some but not all treatment groups. Again, none of the delayed-mezerein-treatment groups had papilloma responses similar to those of the corresponding two-stage promotion (TPA-mezerein) groups at any corresponding initiating dose of DMBA. Finally, the progression of papillomas to carcinomas during promotion with mezerein was examined in groups of mice initiated with either 2 or 20 micrograms of DMBA. Higher ratios of carcinomas to papillomas were observed in mice promoted with mezerein than in mice receiving TPA promotion or two-stage promotion (TPA-mezerein). However, the presence of two to four times more papillomas in some mezerein-treated groups did not lead to greater numbers of carcinomas than in the groups with fewer papillomas. The data do not support the idea that spontaneous stage I promotion can be induced by delaying mezerein treatment for 10 weeks. Furthermore, the data suggest that the higher ratio of carcinomas to papillomas observed with mezerein promotion may be a function of the lower tumor burdens obtained after promotion with this compound rather than a specific property of the chemical.     

Enhancement of mezerein-promoted papilloma formation by treatment with 12-O-tetradecanoylphorbol-13-acetate or mezerein prior to initiation

The effects of promoter treatments prior to initiation on subsequent promotion by mezerein were examined in SENCAR mice. Groups of mice received two applications of various complete as well as first and second stage promoters given at various time intervals prior to initiation ranging from 3 days to 10 weeks. The mice were then initiated with 2 micrograms of 7,12-dimethylbenz[a]anthracene (DMBA) followed 2 weeks later by twice-weekly treatments with 2 micrograms of mezerein. The papilloma response in mice, receiving pretreatments with 2 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA) either 3 days, 1, 2, 3 or 5 weeks before initiation, was similar to that seen when TPA was given after initiation during stage I of promotion followed by stage II of promotion with mezerein (4-5 papillomas per mouse in all groups). Surprisingly, pretreatment with the stage II promoter, mezerein (2 micrograms), either 2 or 5 weeks prior to initiation, also gave papilloma responses similar to that induced with the standard two-stage promotion protocol (4.7 and 6.4 papillomas per mouse, respectively). The papilloma response was less than that in the standard two-stage promotion protocol when pretreatments with the stage I promoter A23187 (80 micrograms/mouse) were given either 2 or 5 weeks before initiation (2.6 and 2.3 papillomas per mouse, respectively). However, a repeat experiment (currently in progress) with a higher dose of A23187 (160 micrograms/mouse) given 2 weeks prior to initiation indicates that it is more effective than the 80 micrograms dose. When the time interval between pretreatment and initiation was increased to 10 weeks, the papilloma response with TPA and A23187 pretreatment was reduced to below two papillomas per mouse and with mezerein pretreatment to below three papillomas per mouse, indicating the effect was reversible. Histological changes in epidermis of mice which received two applications of these compounds correlated with the tumor response. In this regard, treatment with two applications of TPA and mezerein resulted in an epidermal hyperplasia of similar magnitude (epidermal thickness of 53.5 +/- 1.5 and 50.0 +/- 1.1 microns, respectively). The hyperplasia produced by treatment with two applications of 80 micrograms A23187 (39.4 +/- 1.8 microns) was significantly less. The ability of pretreatments with benzoyl peroxide (20 mg) and chrysarobin (50 micrograms) to affect the subsequent promoting activity of mezerein was also examined.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Promoter independence as a feature of most skin papillomas in SENCAR mice

In the present study, the fate of individual papillomas induced by initiation-promotion on the backs of SENCAR mice was monitored after discontinuation of limited promoter treatment. Groups of 40 SENCAR mice each were initiated by a single topical application of 7,12-dimethylbenz[a]anthracene (DMBA) at 2, 1, 0.5, or 0.25 micrograms/mouse. Animals were promoted with 2 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA) twice weekly during 10 weeks. At that time point, 10 papilloma bearing mice from each group were randomly selected to follow the growth of their existing tumors. Animals and their individual tumors were identified, charted, and photographed weekly. After an initial increase, the average number of papillomas/mouse remained constant after discontinuation of TPA in all the groups except the group receiving the highest DMBA dose (Group 1) and with highest tumor load. Twenty-one weeks after TPA was discontinued, only 10-20% of the papillomas had regressed and no statistically significant differences were found among the different DMBA dose groups. On the other hand, Group 1 showed the highest percentage of coalescing tumors which was apparently a function of tumor load. In addition, no differences were observed in the proportion of positive tumors with activating point mutations at codon 61 of the Ha-ras gene when comparing samples of papillomas from the highest DMBA initiation dose group (2 micrograms) versus the lowest DMBA initiation dose group (0.25 micrograms). Our present data suggest that papillomas induced with low doses of DMBA in SENCAR mice are no more TPA dependent than those induced by higher initiating doses. Furthermore, in SENCAR mice at the doses used in the present study (0.25-2 micrograms/mouse), the number of so-called "promoter dependent" papillomas represents only a small percentage of the total papillomas produced using the initiation-promotion protocol.     

Dietary fat effects on the initiation and promotion of two-stage skin tumorigenesis in the SENCAR mouse

We investigated the influence of dietary corn oil on initiation of skin tumors in SENCAR mice with 7,12-dimethylbenz(a)anthracene (DMBA) (10 nmol at 8 to 9 wk of age) and the promotion of these tumors with 12-O-tetradecanoylphorbol-13-acetate (TPA) (3.2 nmol twice weekly for 20 wk). Diet high in corn oil (24.6%) was fed, in comparison with control diet (5%), to mice during two time schedules: (a) high-fat diet was fed preceding and for 1 wk following DMBA to assess the effects of high corn oil diet on initiation; and (b) high-fat diet was fed starting at the time of the first TPA treatment (1 wk following DMBA initiation) until the end of the experiment to assess effects of high corn oil diet on promotion. Mice were trained to consume equivalent caloric allotments of the low- and high-fat diets to ensure that the observed effects on tumor development were for dietary fat at constant calorie intake. Feeding high corn oil diet during DMBA treatment did not influence the incidence of skin papilloma or carcinoma, but the number of papillomas per effective mouse was reduced in mice fed the high-fat diet during initiation. Consumption of the high corn oil diet during and following TPA treatment resulted in an increase in the incidence of papillomas up until Wk 14 of the experiment, an increase in the number of papillomas per effective mouse throughout the experiment, and an increase in the number of carcinomas per effective mouse during Wk 25 to 34. However, cumulative carcinoma yield (Wk 25-44) did not differ between the diet groups. Dietary treatment did not influence food consumption, body weight, or survival in the mice treated with DMBA and TPA. Northern blot hybridization studies were carried out on RNA purified from tumors of high- and low-fat mice to determine if diet influenced the pattern of Ha-ras oncogene expression. The results of this experiment indicated that elevated levels of Ha-ras-specific RNA, in comparison with normal epidermal RNA, were present in papillomas and carcinomas from DMBA-initiated, TPA-promoted mice irrespective of the diet the mice were fed.     

Inhibitory effect of apigenin, a plant flavonoid, on epidermal ornithine decarboxylase and skin tumor promotion in mice

This investigation studied the effect of topical application of apigenin on skin tumorigenesis initiated by 7,12-dimethylbenz(a)anthracene (DMBA) and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA) in SENCAR mice. Apigenin was a potent inhibitor of epidermal ornithine decarboxylase induction by TPA in a dose-dependent manner from 1 to 20 mumol. Two tumorigenesis studies were conducted. In the first study, 20 mumol of apigenin was applied topically and no effect on body weight was observed. By week 33 after DMBA initiation, 48% of DMBA/TPA-treated mice developed carcinomas, while none occurred in DMBA/apigenin/TPA-treated groups. In the second study, doses of 5 and 20 mumol of apigenin were used. The papilloma incidence for 0, 5, and 20 mumol apigenin at 26 weeks after DMBA was 93.3, 58, and 39.3%, and papilloma numbers per mouse were 7.5, 2.5, and 1.8, respectively. Apigenin prolonged by 3 weeks the latency period of tumor appearance. In addition, apigenin significantly inhibited the incidence of carcinoma and the numbers of carcinomas. The incidence of carcinomas per tumor-bearing animal and the ratio of carcinomas/papillomas in two apigenin-treated groups decreased although there were no significant differences between the three groups. These data indicate that apigenin inhibited skin papillomas and showed the tendency to decrease conversion of papillomas to carcinomas.     

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.     

Skin tumorigenesis by initiators and promoters of different chemical structures in lines of mice selectively bred for resistance (Car-r) or susceptibility (Car-s) to two-stage skin carcinogenesis.

Carcinogenesis-resistant (Car-R) and carcinogenesis-susceptible (Car-S) mice were obtained applying a bi-directional selective breeding approach to a two-stage skin carcinogenesis protocol, using 9,10-dimethyl-1,2-benzanthracene (DMBA) as initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as promoter. Sixteen generations of selection produced a remarkable interline difference in responsiveness to two-stage skin carcinogenesis between Car-R and Car-S: identical DMBA (25 microgram) and TPA (5 microgram) doses induced papillomas in 100% of Car-S compared with 3.3% of Car-R mice and maximal responses of 14.3 or 0.03 papillomas/mouse, respectively, despite the shorter promotion applied to Car-S (49 vs. 208 days). To define the factors determining this great difference, Car-R and Car-S mice were challenged by initiators/promoters chemically unrelated to those used for selection. Both lines were subjected to either initiation by N-methyl-N-nitrosourea (MNU) followed by TPA promotion, or promotion by benzoyl peroxide, or 1,8-dihydroxy-3-methyl-9-anthrone (chrysarobin) following DMBA initiation. Initiation with MNU induced a 10-fold tumour incidence in Car-S compared with Car-R mice, and a 32-fold difference in tumour induction rate. The 2 lines also differed markedly in susceptibility to benzoyl peroxide promotion: Car-S mice initiated with 25 microgram DMBA and promoted with 7.5 mg benzoyl peroxide showed a 12-fold tumour incidence and a 103-fold tumour induction rate compared with the corresponding Car-R group. Both lines, however, were refractory to chrysarobin promotion. The progression of papillomas to carcinomas was examined in all Car-S groups. The incidence of mice that developed carcinomas was 57% in MNU-initiated mice. Benzoyl peroxide was also able to promote carcinoma development in Car-S mice, though with a lower incidence (30.4%) than TPA.     

Further studies on the influence of initiation dose on papilloma growth and progression during two-stage carcinogenesis in SENCAR mice

The present study was designed to further evaluate the growthand progression of papillomas to squamous cell carcinomas (SCCs)in groups of animals receiving initiating doses of 7,12-dimethylbenz[a]anthracene(DMBA) producing relatively low papilloma yields following longterm promotion (60 weeks) with 12-O-tetradecanoylphorbol-13-acetate(TPA). For comparison, groups of animals were initiated withvarious doses of DMBA and then promoted with mezerein (MEZ),benzoyl peroxide (BzPo) and chrysarobin (CHRY). Following initiation,groups of female SENCAR mice received the following promoterdoses: TPA (1.0 or 2.0 µg per mouse); MEZ (2.0 µgper mouse); BzPo (20.0 mg per mouse); and CHRY (52.8 µgper mouse). The maximum papilloma to SCC conversion ratio obtainedwith TPA in the current study was 0.32. This value was in therange of maximum conversion ratios obtained with the other compounds:MEZ, 0.40; CHRY, 0.32 and BzPo, 0.19. In general, the highestpapilloma to SCC conversion ratios observed with TPA as thepromoter were obtained in groups that received the lowest dosesof DMBA and had relatively low papilloma burdens. A comparisonof papilloma to SCC conversion in groups of mice promoted withTPA, MEZ or CHRY and having similar papilloma yields, revealedvery similar conversion ratios. Comparison of the BzPo groupwith a similar papilloma yield indicated that the conversionratio was slightly lower with this promoter. The present resultsindicate that in mice promoted with TPA and having relativelylow papilloma numbers, a larger proportion of these papillomasprogress to SCCs during continued promoter treatment. Furthermore,the results suggest that papillomas behave similarly in theirability to progress to SCCs regardless of the promoter usedwhen comparing groups of mice with similar tumor numbers. Thedata are discussed in terms of possible mechanisms for the observedresults.     

Effects of dose and duration of treatment with the tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate on mouse skin carcinogenesis

The effects of dose and duration of treatment with the potenttumor-promoting agent 12-O-tetra-decanoylphorbol-13-acetate(TPA) on the formation of skin tumors in Charles River CD-1mice was studied. Mice were initiated with a single applicationof 0.2 µmol of 7, 12-dimethylbenz[a]anthracene (DMBA)in 0.2 ml acetone. Beginning two weeks after initiation, micewere treated twice weekly with various doses (0.01 – 20nmol) of TPA in 0.2ml acetone. Application of either 0.01 or0.1 nmol of TPA did not elcity tumors during the 50 weeks durationof treatment. A dose-dependent increase in the number of papillomaswas observed through the range of 1 to 10 nmol of TPA. Twiceweekly applications of 20nmol of TPA did not further enhancethe papilloma incidence. A good correlation was observed betweenthe induction of ornithine decarboxylase (ODC) activity andthe formation of skin tumors by various doses of TPA. To determine the effect of promotion duration on the incidenceof papillomas and carcinomas, mice were treated with 10 nmolof TPA for various durations (6,12,18,24,30, or 36 weeks) beginning2 weeks after initiation with 0.2 µmol of DMBA. Mice promotedfor only 6 weeks developed papilllomas and carcinomas afterpromotion had been discontinued. There was an intermediate incidenceof tumors in the group treated for 12 weeks. Promotion for 18,24, 30, or 36 weeks elicited virtually identical yields of papillomas.The incidence of carcinomas was proportional to promotion durationtimes of 6, 12, and 18 weeks, but carcinoma incidence was lessthan maximal in mice promoted for 24 weeks or longer. The results indicate that a) the incidence of papillomas servesas a rapid (18 weeks) index for subsequent appearance of carcinomas,b) twice weekly applications of 10 nmol of TPA for 18 weeksfollowing initiation of female CD-1 mice with 0.2 µmnolof DMBA is an appropriate protocol for maximum tumor yield ininitiation-promotion experiments, and c) ODC induction may bean important component of the mechanism of skin tumor promotionby TPA.     

Elevated expression and point mutation of the Ha-ras proto-oncogene in mouse skin tumors promoted by benzoyl peroxide and other promoting agents

The two-stage skin carcinogenesis model of initiation and promotion in SENCAR mice has been used to examine the effects of various tumor-promoting agents on the expression of the Ha-ras oncogene in early stages of tumorigenesis in vivo. Papillomas were induced in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated SENCAR mouse epidermis by (i) complete promotion with benzoyl peroxide; (ii) complete promotion with 12-O-tetradecanoyl phorbol-13-acetate (TPA); and (iii) two-stage promotion with TPA for 2 weeks followed by mezerein for 9 weeks. Results of Northern blot hybridization analyses show that early papillomas contain significantly elevated levels of Ha-ras polyadenylated [poly(A)+] RNA, irrespective of the type of tumor promotion regimen used. This pattern holds for promoters of the phorbol ester class as well as for the free radical generating agent benzoyl peroxide. Furthermore, digestion of tumor DNA with diagnostic restriction endonucleases demonstrated that 9-week-old papillomas induced by DMBA contained a point mutation in the 61st codon of one allele of the Ha-ras gene. The results represent the earliest stage in the development of a papilloma at which a Ha-ras point mutation has been reported.     

Increased sensitivity to two-stage skin carcinogenesis of mice heterozygous for the repeated epilation mutation (Er)

Mice heterozygous for repeated epilation mutation (Er) have cutaneous abnormalities that result in repeated loss of hair. Skin papillomas and carcinomas occur spontaneously in such Er/+ mice. BALB/c mice are generally resistant to induced skin cancers. We investigated whether Er/+ heterozygous mice of BALB/c genetic background exhibit increased susceptibility to spontaneous and induced skin tumors. Although none of the Er/+ CXB(N5) mice spontaneously developed skin tumors, they exhibited increased sensitivity to the development of skin papillomas induced by an initiation-promotion regimen. Er/+ mice developed papillomas after 20 micrograms DMBA initiation in the absence of TPA promotion, but the same dose of DMBA was subtumorigenic in +/+ (sibling) mice. Although 15 weeks of TPA promotion resulted in similar tumor susceptibilities, tumor latencies and tumor frequencies in the 2 groups of initiated mice, the papillomas were qualitatively different. Er/+ mice developed more papillomas of the delayed promoter-independent type, which occur after termination of promotion. In contrast, +/+ mice developed more promoter-dependent papillomas, which regress after termination of promotion. Therefore Er/+ mice had a significantly higher number of papillomas than +/+ mice at the termination of the experiment. These results suggest that Er-mutation-induced skin defects not only lead to the repeated loss of hair, but also influence the mode of development of skin papillomas from carcinogen-initiated cells.     

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

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

Inhibition of mouse skin tumor promotion by adriamycin and daunomycin in combination with verapamil or palmitoylcarnitine

The anti-cancer drugs Adriamycin (ADR) and Daunomycin (DAU) alone were unable to inhibit the promotion of skin papillomas by repeated applications of 8.5 nmol of 12-O-tetradecanoylphorbol-13-acetate (TPA) in 7,12-dimethylbenz(a)anthracene (DMBA)-initiated mice. Pretreatments with 50 micrograms of ADR also failed to alter the tumor-promoting activities of smaller doses of TPA. Therefore, the effects of the anthracycline antibiotics on skin tumor promotion were evaluated in combination with the Ca2+ antagonist verapamil (VRP) and the protein kinase C (PKC) inhibitor palmitoylcarnitine (PC), compounds known to circumvent drug resistance. When applied simultaneously with each promotion treatment with 8.5 nmol of TPA, 2.5 mg of VRP inhibited the number of papillomas/mouse by 26%. But the combination of VRP + 50 micrograms of ADR or DAU inhibited the yields of papillomas by 50 or 47%, respectively, suggesting that VRP was required to reveal the antitumor-promoting activities of otherwise ineffective drugs. Similarly, the promotion of skin tumors by TPA was inhibited synergistically by the combinations of 2 mumol of PC + 50 micrograms of ADR or DAU. For instance, ADR and DAU had no effects alone but inhibited the incidence of skin papillomas by 78 and 86%, respectively, in the presence of PC, a compound which alone inhibited the tumor incidence by only 44%. The results indicate that ADR and DAU are effective against the promoting component of skin carcinogenesis only if they are applied in combination with Ca2+ antagonists or PKC inhibitors at a time when they can inhibit the early biochemical effects induced by TPA.     

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.     

Analysis of c-Ha-ras gene mutations in skin tumors induced in carcinogenesis-susceptible and carcinogenesis-resistant mice by different two-stage protocols or tumor promoter alone

In the present study we describe the molecular analysis of c-Ha-ras gene mutations in 47 papillomas and 17 carcinomas developed in two lines of mice, carcinogenesis-susceptible (Car-S) and carcinogenesis-resistant (Car-R), selectively bred for extreme susceptibility or resistance to chemical skin carcinogenesis initiated and promoted with different doses of 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). This study also presents the analysis of c-Ha-ras gene mutations in 22 papillomas and 22 carcinomas in Car-S mice initiated with DMBA and promoted with benzoyl peroxide (BzPo) and in seven papillomas and one carcinoma from a group of uniniated Car-S mice that received only BzPo treatment. The data showed that a A(182)-->T transversion in the c-Ha-ras gene was present in 100% and 81% of the skin tumors developed in Car-S and Car-R mice, respectively, after DMBA initiation and TPA promotion, suggesting that differences in genetic susceptibility can influence the frequency of c-Ha-ras mutations in the skin tumors produced. The same A(182)-->T mutation with an incidence of 68% was found in papillomas from DMBA-initiated and BzPo-promoted Car-S mice. The difference in the mutation frequency between DMBA/BzPo and DMBA/TPA papillomas suggested that the promotion step contributes to the final mutation pattern. The tumor induction experiment with BzPo alone showed that this compound can induce tumor development in 26% of Car-S mice, and the molecular analysis of the tumors showed a broad mutation spectrum, including mutations in codons 12, 13, and 61 of the c-Ha-ras gene. Mol. Carcinog. 30:111-118, 2001.     

Characterization of skin tumor promotion and progression by chrysarobin in SENCAR mice

The characteristics of the skin tumor promotion response with anthrone derivatives has been further examined in SENCAR mice. Chrysarobin (1,8-dihydroxy-3-methyl-9-anthrone) was an effective skin tumor promoter when applied twice weekly with dose-dependent increases in both papillomas and squamous cell carcinomas between 25 and 100 nmol/mouse. A similar dose-response relationship for papilloma and carcinoma formation was observed when chrysarobin was applied once weekly. Interestingly, chrysarobin was approximately twice as active as a skin tumor promoter when applied once weekly versus twice weekly. Doses of 25,100, and 220 nmol/mouse gave maximal papilloma responses of 2.90, 8.15, and 9.38 versus 0.73, 4.70, and 5.42 papillomas/mouse, respectively, in mice initiated with 25 nmol 7,12-dimethylbenz(a)anthracene. Thus, unlike 12-O-tetradecanoylphorbol-13-acetate (TPA), where a twice weekly application frequency is optimal, application of anthrone promoters such as chrysarobin once weekly is a more optimal frequency for papilloma development. Chrysarobin was also a much more effective skin tumor promoter when the start of promotion was delayed by an additional 10 weeks. Thus, groups of mice initiated with 10 nmol 7,12-dimethylbenz(a)anthracene and having promotion started in either the 3rd or the 13th week after initiation had maximal responses of 5.6 or 11.0 papillomas/mouse, respectively. In addition, the rate of papilloma development was faster in the delayed promotion group. The progression of papillomas to carcinomas was examined in all chrysarobin-treated groups and compared with three groups of mice treated with 3.4 nmol TPA. After 60 weeks of promotion, the anthrone promoter-treated groups had carcinoma:papilloma ratios 2.5 to 5.0 times higher than the TPA-treated groups. This was due primarily to the fact that similar carcinoma responses were observed in both anthrone- and TPA-treated mice at optimal promoting doses whereas the papilloma responses were significantly lower in the former groups. The data suggest that anthrone derivatives are very efficient tumor promoters. The results are further discussed in terms of mechanisms of skin tumor promotion.     

Induction of terminal differentiation-resistant epidermal cells in mouse skin and in papillomas by different initiators during two-stage carcinogenesis

Carcinogen treatment of normal mouse epidermal cells causes some cells, if cultured under the appropriate conditions, to continue to proliferate instead of terminally differentiate, forming foci at 37 degrees C in medium with a calcium level above 0.1 mM. We have examined these Calcium (Ca)-resistant cells formed in the skin of SENCAR mice after treatment with the carcinogen initiator 7,12-dimethylbenz[a]anthracene (DMBA) followed by tumor promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Although in our previous studies TPA promotion initially increased the size but reduced the number of foci caused by the carcinogen initiator N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), TPA promotion of DMBA-treated mice increased the size but had no effect on the number of foci. Papillomas resulting from DMBA plus TPA treatment contained many rapidly growing Ca-resistant cells, corroborating our earlier results with MNNG. Permanent cell lines prepared from papilloma-derived foci formed squamous cell carcinomas in nude mice after relatively short periods in culture. These data provide further evidence that Ca-resistant cells may be papilloma (and perhaps carcinoma) precursors in vivo. In addition, since TPA tends to reduce the number of early Ca-resistant cells caused by MNNG but not by DMBA, this may at least partially explain why treatment with DMBA plus TPA is much more effective in producing papillomas in SENCAR mice than is treatment with MNNG plus TPA.     

Comparison of the tumor-initiating activity of 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene in female SENCAR and CD-1 mice

The derivation of mice resistant and susceptible to skin tumorigenesisusing the initiation-promotion regimen is described. Dose-responserelationships for tumor-initiating activities of 7,12-dimethylbenz[a]-anthracene(DMBA) and benzo[a]pyrene (BP) in the susceptible line (SENCAR)are presented. A single topical dose of either 0.1, 1.0, 10or 100 nmol DMBA, followed one week later by twice weekly applicationsof 8.5 nmol 12–0-tetradecanoylphorbol-13-acetate (TPA)for 19 weeks, produced 0, 3.3, 4.9 and 23.1 papillomas per mouse,respectively. Single topical initiating doses of either 50,100 or 200 nmol BP produced 1.7, 3.8 or 7.8 papillomas per mouse,respectively, after 28 weeks of promotion with 8.5 nmol TPA.SENCAR mice were compared with CD-1 mice for the initiatingactivity of DMBA and BP. Initiating doses of 0.1, 1.0, 10 and100 nmol DMBA produced 0.6, 3.8, 7.0 and 24 papillomas per mouse,respectively, in SENCAR mice and in CD-1 mice produced 0, 0.2,3.0 and 5.6 papillomas per mouse, respectively, after 25 weeksof promotion with TPA. With BP as the initiator, 10, 50, 100and 200 nmol doses produced 0.9, 1.6, 3.8 and 8.3 papillomasper mouse, respectively, in SENCAR mice and in CD-1 mice produced0.1, 0.7,1.8 and 3.8 papillomas per mouse, respectively, after25 weeks of promotion with TPA. SENCAR mice were compared with CD-1 mice for possible differencesin the oxidative metabolism of DMBA using epidermal homogenatesas the enzyme source. Basal levels of monooxygenase activitytoward DMBA were similar in both mouse stocks. Epidermal monooxygenaseactivities following pre-treatment with inducers including DMBA,3-methylcholanthrene, dibenz[a,c]anthracene, Aroclor 1254 and2,3,7,8-tetrachlorodibenzo-p-dioxin, also were quite similarin both mouse stocks. High-pressure liquid chromatographic profilesof ethyl acetate/ acetone (2:1) extractable metabolites revealeda close similarity in the patterns as well as the rates of formationof specific metabolites. Metabolites of DMBA tentatively identifiedbased on cochromato-graphy with purified reference standardsincluded phenols, 12-hydroxymethyl-7-methylbenz[a]anthracene,7-hydroxymethyl-12-methylbenz[a]antnracene, 7,12-dihydroxymethylbenz[a]anthracene,(±)-trans-8,9-dihydro-8,9-dihydroxy-7,12-dimethylbenz[a]anthraceneand (±)-trans-5,6-dihydro-5,6-dihydroxy-7,12-dimethylbenz[a]anthracene.The results suggested that differences in oxidative metabolismof DMBA were not responsible for the differences in sensitivityto tumor-initiation between SENCAR and CD-1 mice.