Differential gene expression in epidermis of mice sensitive and resistant to phorbol ester skin tumor promotion

Previous data from two-stage carcinogenesis studies in mouse skin demonstrated that genetic control of susceptibility to skin tumor promotion by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), in crosses between susceptible DBA/2J and resistant C57BL/6J mice is a multigenic trait. Utilizing a cDNA microarray approach, we compared global gene expression profiles in the epidermis of these two mouse strains treated with TPA or vehicle (acetone). Gene expression in the epidermis was analyzed after the treatment to identify global effects of TPA, as well as potential candidate genes that modify susceptibility to skin tumor promotion. DBA/2J and C57BL/6J mice were treated topically four times with 3.4 nmol TPA or acetone over a 2-wk period, and RNA was extracted from epidermis 6 h after the final treatment. Labeled cDNA generated from each group was hybridized to commercial cDNA microarrays (Agilent) containing more than 8000 targets. More than 450 genes were significantly influenced, directly or indirectly, by TPA treatment in the epidermis of either strain. Notably, 44 genes exhibited differential expression between the tumor promotion sensitive and resistant mouse strains. Several genes that were differentially expressed in DBA/2J versus C57BL/6J epidermis after TPA treatment were located in chromosomal regions linked to TPA promotion susceptibility. Three genes, Gsta4, Nmes1 (MGC58382), and Serpinb2, located within promotion susceptibility loci Psl1 (chr 9), Psl2 (chr 2), and Psl3 (chr 1), respectively, were identified in this analysis as potential candidates for modifiers of susceptibility to skin tumor promotion by TPA.     

Identification of novel genetic loci contributing to 12-O-tetradecanoylphorbol-13-acetate skin tumor promotion susceptibility in DBA/2 and C57BL/6 mice

Genetic differences in susceptibility to two-stage skin carcinogenesis have been known for many years. Studies of genetic crosses of sensitive DBA/2 with resistant C57BL/6 mice suggested that multiple autosomal genes determine the sensitivity of these mice to 12-O-tetradecanoylphorbol-13-acetate (TPA) skin tumor promotion. Previous studies mapped one promotion susceptibility locus, Psl1, to distal chromosome 9. Analysis of TPA promotion susceptibility in (C57BL/6 x DBA/2)F(1) x C57BL/6 mice and B x D recombinant inbred mouse strains suggested tentative associations of promotion susceptibility with several other chromosomal regions. To confirm these associations (C57BL/6 x BxD27)F(2) mice analyzed for TPA promotion susceptibility were genotyped for polymorphic genetic markers mapping to chromosomal regions for which tentative associations had been previously detected. BxD27 mice are sensitive to TPA skin tumor promotion but carry the C57BL/6 allele of Psl1. Because Psl1 does not segregate in this cross, its effect on TPA promotion susceptibility is the same for all mice in the cross. The results of this analysis support the mapping of three novel promotion susceptibility loci to chromosomes 1, 2, and 19. Psl2 maps near D2Mit229 on distal chromosome 2, and inheritance of the dominant DBA/2 allele results in increased sensitivity to TPA. Psl3 maps near D1Mit511 on distal chromosome 1. Interestingly, inheritance of an allele from the resistant C57BL/6 parent results in increased sensitivity to TPA. Psl3 appears to have an additive affect, with heterozygous mice having a stronger response to TPA than mice homozygous for the DBA/2 allele and a weaker response to TPA than mice homozygous for the C57BL/6 allele. Psl4 maps near D19Mit38 on distal chromosome 19 and inheritance of the dominant C57BL/6 allele results in decreased TPA sensitivity. Analysis of the combined effects of these loci on TPA promotion susceptibility indicates that they contribute independently to the overall sensitivity to TPA.     

DBA/2 mice are as sensitive as SENCAR mice to skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate

Mice of the inbred strain DBA/2 responded to a two-stage, initiation-promotiontumorigenesis protocol when high initiating doses (400 nmol/mouse)of 7,12-dimethylbenz[a]- anthracene were utilized. They alsoresponded when N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) wasused as the initiating agent. The tumor response in both caseswas characterized by a rapid rate of tumor development withthe maximal tumor responses reached on or before the 15th weekof promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA).When DBA/2 mice were compared with SENCAR mice for promotionsensitivity following initiation with MNNG, the two mouse stocksresponded with a nearly identical tumor response. C57BL/6 micewere essentially resistant to TPA promotion regardless of theinitiator or the dose of initiator used. A preliminary studywas conducted to determine how susceptibility to tumor promotionby TPA was inherited in F₁ mice derived from DBA/2 (sensitive)and C57BL/6 (resistant) parents. The B6D2F₁ mice were as sensitiveas the DBA/2 parent, suggesting that susceptibility in thesetwo inbred mouse strains is inherited as an autosoma1 dominanttrait. The results show that these two inbred mouse strainsmay provide a model system for studying genetic factors controllingsusceptibility to phorbol ester skin tumor promotion.     

The covalent binding of polycyclic hydrocarbons to DNA in the skin of mice of different strains.

The binding of three tritium-labelled carcinogenic polycyclic hydrocarbons, 7,12-dimethylbenz (a) anthracene (DMBA), benzo (a) pyrene (BP) and 3-methylcholanthrene (MCA) to DNA in mouse skin has been studied in C57BL, DBA/2 and Swiss mice following topical application of the hydrocarbons. DNA isolated from the treated areas was hydrolysed to deoxyribonucleosides and chromatographed on Sephadex LH20 columns. The levels of binding of hydrocarbon to DNA were determined from the amount of radioactivity eluted from Sephadex LH20 columns in those fractions containing hydrocarbon-DNA adducts and the radioactivity shown, by rechromatography on AG5OWX4 columns, to be due to tritium incorporation into normal deoxyribonucleosides was not included. C57BL mice were treated with doses of DMBA ranging from 0.025 μmol to 1 μmol/mouse and the levels of hydrocarbon bound to DNA 19 h after treatment were determined; there was no evidence for a threshold dose below which no binding to DNA occurs, and the same hydrocarbon-DNA product peaks were obtained at all doses. The levels of binding of DMBA (1 μmol/mouse) to DNA in skin were compared in C57BL, DBA/2 and Swiss mice at times varying from 6 h to 8 days after treatment. DMBA became bound to similar extents in Swiss and C57BL mice and to a slightly greater extent in DBA/2 mice; the rate of disappearance of bound DMBA from DNA was similar in all three strains. DMBA (0.1 μmol/mouse) was bound to DNA in C57BL and DBA/2 mice to similar extents 19 h after treatment and to a slightly lesser extent in Swiss mice. The ratios of the sizes of the hydrocarbon-DNA product peaks varied with the time after treatment, but were similar at any given time for the three strains. Both BP (1 μmol/mouse) and MCA (1 μmol/mouse) were bound to DNA to similar extents 19 h and 48 h after treatment in all three strains. BP (0.1 μmol/mouse) was bound to DNA in the order DBA/2>C57BL> Swiss 19 h after treatment. The levels of binding for all three hydrocarbons in the different strains do not show a correlation with the reported susceptibilities of the three strains to polycylic hydrocarbon carcinogenesis.     

Effect of polycyclic aromatic compounds and phorbol esters on ornithine decarboxylase and aryl hydrocarbon hydroxylase activities in mouse liver

Single i.p. injections of 3-methylcholanthrene (MC; 50 mg/kg) administered to inbred C57BL/6 mice or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 100 micrograms/kg) to DBA/2 mice gave an increase in the hepatic activities of ornithine decarboxylase (ODC) and aryl hydrocarbon hydroxylase (AHH) with peaks occurring by 12 and 48 hr, respectively. A single i.p. dose of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA; 100 micrograms/kg) enhanced the activity of ODC about 70-fold within 12 hr in C57BL/6 mice and 18-fold within 24 hr in DBA/2 mice without affecting AHH activity markedly. 4-O-Methyl-12-O-tetradecanoylphorbol-13-acetate (100 micrograms/kg) raised ODC activity to about 25% of the TPA-treated value in C57BL/6 mice; in DBA/2 mice, TPA and 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate induced ODC activity to roughly the same level. Benzo(e)pyrene (50 mg/kg) failed to affect ODC and AHH activities significantly in either strain. The inducing effect of TPA on ODC activity was potentiated by a simultaneous administration of MC to C57BL/6 mice; combined TPA and TCDD to DBA/2 mice exerted an additive effect on hepatic ODC activity. Difluoromethylornithine administered i.p. effectively inhibited the induction of ODC activity elicited by TPA, MC, or TCDD either alone or in various combinations but did not interfere with AHH induction. These data indicate that different regulatory factors are involved in the ODC induction process elicited by TPA and polycyclic aromatic compounds and that MC and TCDD may induce ODC activity by different mechanisms. The results also confirm our earlier findings in rat skin and cells in culture which suggest that the ODC and AHH induction processes can occur independently of each other. Additionally, there is a strain-related difference in sensitivity with regard to ODC-inducing activity of TPA in the livers of C57BL/6 and DBA/2 mice.     

Susceptibility to phorbol ester skin tumor promotion in (C57BL/6xDBA/2) F1 mice is inherited as an incomplete dominant trait: evidence for multi-locus involvement

Since current evidence suggests that the tumor promotion stageis a primary determinant in susceptibility to multistage carcinogenesis,we have characterized the genetics of susceptibility to phorbolester skin tumor promotion in inbred mice. Susceptibility ofhybrids (B6D2F₁), beween DBA/2 (sensitive) and C57BL/6 (resistant)parents, initiated with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) and promoted with 12-O-tetradecanoylphorbol-13-acetate(TPA) was similar to DBA/2 mice at doses of 13.6 nmol per mousebut clearly less when doses of 1.7–6.8 nmol per mousewere used. In addition, no significant differences were observedbetween male andfemale B6D2F₁ mice in terms of tumor incidencealthough some differences were observed in tumor multiplicitiesbetween male and female F₁ mice at the highest TPA dose. ReciprocalF₁ mice initiated with DMBA (i.e. D2B6F₁) were also responsiveto TPA. Female D2B6F₁ mice were of lower sensitivity at lowerdoses of TPA, compared to female DBA–2, a finding similarto that observed with B6D2F₁ mice initiated with MNNG. Furtheranalyses of the susceptibility of B6D2F₂ and B6D2F₁xC57BL–6backcross mice to TPA promotion indicated that more than onedominant genetic locus must account for the differences in promotionsensitivity between DBA/2 and C57BL/6 mice. To understand furtherthe genes responsible for promotion sensitivity, histologkalevaluations were performed on DBA/2, C57BL/6 and B6D2F₁ mice.Histological examination revealed that the epidermis of DBA/2mice showed a marked hyperplasia and the presence of a muchgreater number of dark basal keratinocytes (DCs) compared withC57BL/6 mice 48 h after the last of four applications of TPA(doses 3.4 nmol). A marked dermal infiltration of polymorphonuclearleukocytes (PMNs) was observed in DBA/2 mice, whereas littleinfiltration was observed in the skin of C57BL/6 mice. The hyperplasiain the skin of B6D2F₁ mice was intermediate between DBA/2 andC57BL/6 mice at all TPA doses examined except the lowest dose(1.7 nmol), whereas the DC response, although significantlylower at doses of 6.8 nmol or below, was similar to DBA/2 miceat higher TPA doses (13.6 and 17.0 nmol). The infiltration ofPMNs in the dermis of B6D2F₁ mice was similar to or greaterthan DBA/2 mice at all doses of TPA tested. Our results suggestthat (i) susceptibility to TPA promotion is inherited as anincomplete dominant trait; (ii) neither cytoplasmic geneticdeterminants nor the X-chromosome appear to play a significantrole in susceptibility to TPA; and (iii) the degree of sustainedepidermal hyperplasia and especially the induction of DCs aftermultiple applications of TPA show an excellent correlation withinherited susceptibility to promotion. Our data are consistentwith a model where allelic differences at more than one geneticlocus contribute to the sensitivity of DBA/2 mice to phorbolester promotion.     

Enhanced induction of epidermal ornithine decarboxylase activity in C57BL/6 compared to DBA/2 mice by protein kinase C-activating skin tumor promoters: relevance to genetically mediated differences in promotion susceptibility.

Previous work from our laboratory demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) or a synthetic diacylglycerol induced significantly higher epidermal ornithine decarboxylase (ODC) activity in C57BL/6 than in DBA/2 mice. To understand further the genetic basis for this strain difference, two tumor promoters were evaluated for their effects on epidermal ODC activity: teleocidin, which activates protein kinase C (PKC); and 1,8-dihydroxyl-3-methyl-9-anthrone (chrysarobin), which does not. In addition, the ODC induction response in B6D2F1 offspring and BXD recombinant inbred (RI) strains was examined following multiple treatments with TPA. A single topical application of teleocidin to mouse dorsal skin led to the hyperinduction of epidermal ODC activity in C57BL/6 mice. In contrast, while chrysarobin induced epidermal ODC activity, no significant differences in the magnitude of this response were observed in SENCAR, DBA/2 or C57BL/6 mice. Consistent with our previous findings, the magnitude of ODC induction by teleocidin in these three mouse lines (C57BL/6 greater than SENCAR greater than DBA/2) did not correlate with their susceptibility to tumor promotion by TPA (SENCAR greater than DBA/2 greater than C57BL/6). ODC activity induced by multiple application of TPA in B6DF1 mice, whose susceptibility to phorbol ester tumor promotion is inherited as an incomplete dominant trait, was comparable to that induced in C57BL/6 mice at all the doses examined. Cluster analysis of TPA-induced ODC activity in BXD RI strains allowed us tentatively to group them into four or five phenotypes and to estimate a minimum of two genetic loci controlling TPA-induced ODC activity. Furthermore, in BXD RI strains, there was no apparent relationship between the magnitude of ODC induction and responsiveness to tumor promotion or sustained hyperplasia. Collectively, these results suggest that hyperinducibility of ODC in response to PKC-activating tumor promoters is inherited as an autosomal dominant trait, and that genetic determinants for ODC induction, at least in C57BL/6 and DBA/2 mice, appear completely independent of those controlling tumor promotion susceptibility.     

Further genetic analyses of skin tumor promoter susceptibility using inbred and recombinant inbred mice

To explore further the genetics of susceptibility of skin tumorpromotion in inbred mice, several aspects of responsivenessto 12-O-etradecanoylphorbol-13-acetate (TPA) were examined inC3H/He mice and segregating crosses between this mouse strainand C57BL/6 mice as well as BXD and BXH recombinant inbred (RI)strains. Dose-response relationships were established for skintumor promotion by TPA following initiation with 7, 12-dimethylbenz(a)anthracenein C3H/He and B6C3F1, as well as several other mouse stocksand strains included for comparison. The relative responsivenessto TPA skin tumor promotion was: SENCAR > > DBA/2 >C3H/He = B6D2F1 > B6C3F1 > > C57BL/6. Analyses of thesusceptibility of B6C3F2 and B6C3F1 x C57BL/6 backcross micesuggested that a minimum of two dominant genetic loci controlresponsiveness to phorbol ester promotion in these mice. Furtheranalysis of BXH and BXD RI strains suggested the presence offour distinct promotion-responsive phenotypes controlled bya minimum of two genetic loci. The existence of a ‘hyper-responsive’phenotype in the sets of RI strains, however, suggests thata third, recessive locus also may play a role in controllingresponsiveness to TPA, promotion. At 48 h after the last offour applications of TPA, marked hyperplasia and an increasein dark basal keratino-cytes were observed in C3H/He mice, whereasin B6C3F1 mice the response in these parameters was intermediatebetween C3H/He and C57BL/6 mice. A marked dermal inflamation,as determined by infiltration of polymorpho-nuclear cells, wasobserved in C3H/He and B6C3F1 mice, whereas little was notedin C57BL/6 mice. Furthermore, histological evaluations of selectedBXD RI strains revealed a significant correlation between themagnitude of the hyperplasia response and the percentage ofmice bearing tumors. The present data, in conjunction with ourprevious studies, confirm that the major gene(s) controllingsusceptibility to tumor promoter induced by TPA in two sensitivestrains (i. e. DBA/2 C3H/He) are similar of closely linked tothose for induction of sustained hyperplasia. In addition, thepresent data provide new evidence for a model where allelicdifferences at a minimum of three loci contribute to geneticdifferences in susceptibility to phorbol ester promotion inDBA/2 and C3H/He versus C57BL/6 mice.     

Murine susceptibility to two-stage skin carcinogenesis is influenced by the agent used for promotion

Several approaches were employed to investigate whether murinestock and strain differences in susceptibility to two-stageskin carcinogenesis are due to differences in the metabolismof the initiating aromatic hydrocarbons, or the consequencesof the agents used for promotion. A cellmediated mutagenesisassay was used to quantitatively compare the abilities of culturednewborn SENCAR, DBA/2, C57BL/6 and BALB/c keratinocytes to metabolizedimethyl-benz[a]anthracene (DMBA) to mutagenic and cytotoxicmetabolites. At equivalent concentrations of DMBA, throughouta 25-fold range in promutagen concentration, C57BL/6, BALB/cand SENCAR keratinocyte-dependent mutant frequencies were verysimilar and approximately twice DBA/2 keratinocyte-dependentmutant frequencies. In in vivo tumor studies, C57BL/6 mice weremore sensitive than SENCAR mice to complete skin carcinogenesisprotocols employing repetitive weekly treatments with DMBA andbenzo[a]pyrene (BP). At equivalent concentrations of eitherDMBA or BP, C57BL/6 mice developed carcinomas sooner, and hada greater number of carcinomas per animal. SENCAR mice werevery sensitive to two-stage skin carcinogenesis protocols employingBP and DMBA as initiators and benzoyl peroxide and 12-O-tetradecanoylphorbol-13-acetate(TPA) as promoters. C57BL/6 mice were relatively refractoryto TPA promotion but sensitive to promotion with benzoyl peroxide.These findings suggest that murine stock and strain-dependentdifferences in sensitivity to two-stage skin carcinogenesismay not be due to major differences in the metabolism of theinitiating hydrocarbons, but are partially the consequencesof the agents used for promotion.     

Comparison of the histological changes in the skin of DBA/2 and C57BL/6 mice following exposure to various promoting agents

The effects of multiple applications of 12-O-tetradecanoyl-phorbol-13-acetate(TPA, 6.8 nmol), teleocidin (6.8 nmol), 1,8-dihydroxy-3-methyl-9-anthrone(chrysarobin, 220 nmol), mezerein (6.8 nmol), 4-O-Methyl-TPA(4-O-Me-TPA, 150µg) and benzoyl peroxide (BzP, 20 mg)on the skin of DBA/2 and C57BL/6 mice were studied histologically.After four applications of TPA given over a 2-week period, theepidermis of DBA/2 mice showed a marked epidermal hyperplasiaand the presence of a much greater number of dark basal kerntinocytes(DCs) 48 h after the last treatment compared with C57BL/6 micetreated with a similar dose and protocol. A marked dermal infiltrationof polymorphonuclear leukocytes (PMNs) was observed in DBA/2mice 48 h after the last application of TPA, whereas littlePMN infiltration was observed in skin of C57BL/6 mice. At 96h after the last application of TPA, DBA/2 mice still showeda much greater degree of epidermal hyperplasia than C57BL/6mice. PMNs were virtually absent in the dermis of both DBA/2and C57BL/6 mice by 96 h after the last TPA treatment. Interestingly,treatment of both strains of mice with multiple applicationsof teleocidin induced a marked epidermal hyperplasia, a highpercentage of DCs and a high labeling index (LI), similar tothat observed in DBA/2 mice 48 h after the last treatment. Chrysarobin(given once-weekly for 4 weeks) induced a moderate sustainedhyperplasia and DC response 48 h after the last treatment inboth DBA/2 and C57BL/6 mice; however, C57BL/6 mice showed agreater epidermal hyperplasia than DBA/2 mice. Chrysarobin induceda significant infiltration of PMNs into the dermis of DBA/2mice whereas in C57BL/6 mice there only a slight dermal infiltrationof PMNs. Mezerein (given twice-weekly for 2 weeks) induced amoderate epidermal hyperplasia, DC response and LI of similarmagnitude in both DBA/2 and C57BL/6 mice, but did not inducePMN infiltration in either strain. BzP and 4-O-Me-TPA (giventwiceweekly for 2 weeks) induced only a weak sustained epidermalhyperplasia, DC response and LI of similar magnitude in bothstrains of mice, and there was little, if any, dermal infiltrationof PMNs either 48 or 96 h after the last treatment. Examinationof the relationship between the extent of induced hyperplasiaand the DC response showed an excellent linear correlation whereasthe extent of PMN infiltration into the dermis was not wellcorrelated with either parameter. The results suggest that theinduction of both sustained hyperplasia and DCs correlate wellwith the skin papillomapromoting ability of the various promotingagents examined, and that C57BL/6 mice are somewhat peculiarin their resistance to the induction of these parameters byphorbol esters.     

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.     

Association of a murine chromosome 9 locus (Psl1) with susceptibility to mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate

It has been known for many years that there are dramatic differences in the susceptibility of mouse stocks and strains to two-stage skin carcinogenesis and that these differences are due to the animals' responsiveness to tumor-promoting agents. In earlier studies using several inbred mouse strains, we found that susceptibility to skin tumor promotion by phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) is a multigenic trait. To extend this work, we conducted a genome scan of (C57BL/6 × DBA/2)F₁ × C57BL/6 mice previously scored for sensitivity to skin tumor promotion by TPA. As a result of this scan, we now report an association of increased TPA promotion susceptibility with inheritance of the DBA/2 alleles of markers on the distal portion of mouse chromosome 9. Additional linkage analyses using (C57BL/6 × DBA/2)F₂ and B×D recombinant inbred mice confirmed this association and suggested that a TPA promotion susceptibility locus maps near D9Mit51 (LOD_w = 4.1). We designated this locus promotion susceptibility locus 1 (Psl1). Mol. Carcinog. 20:162–167, 1997. © 1997 Wiley-Liss, Inc.     

Initiating potential of 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and 3,3',4',5,7-pentahydroxyflavone (quercetin) in two-stage mouse skin carcinogenesis

Tumor initiating activity of 3,3',4',5,7-pentahydroxyflavone (quercetin), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and 2-(2-furyl)-3-(5-nitro-2-furyl) (AF-2) acrylamide) were tested in a two-stage mouse skin carcinogenesis model using 12-O-tetradecanoyl phorbol-13-acetate (TPA) as a promoter. These compounds dissolved in dimethyl sulfoxide were topically applied twice weekly for 5 weeks on the dorsal skin, and then followed by TPA for 47 weeks. The total initiating dose was 100 mg for each compound. 7,12-Dimethylbenz(a)anthracene (DMBA) at a total dose of 100 micrograms was used as a positive control compound. AF-2 induced skin tumors in 35% of the mice (average of 0.4 tumors/mouse), HBA in 15% in (0.2/mouse), BHT in 13% (0.13/mouse) and quercetin in 5% (0.1/mouse). No tumors appeared in the groups treated with either test chemicals alone or TPA alone. Statistical analysis according to either Fisher's exact test or Peto's trend test revealed significant differences for tumor appearance in the AF-2/TPA and BHA/TPA followed by TPA groups as compared to in the DMSO/TPA group. The results indicate that AF-2 and BHA have weak tumor initiating activity on mouse skin, but such effects are not apparent for BHT or quercetin.     

Induction of cutaneous mast cell tumors by N-methyl-N'-nitro-N-nitrosoguanidine followed by TPA in female mice of 4 out of 5 strains tested

Appreciable yields of cutaneous mast cell tumors were induced in a two-stage skin carcinogenesis protocol comprising N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) initiation followed by 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion in 4 of 5 strains of mice. Only female mice of each of the 5 strains were studied. The incidences of benign and/or malignant lesions differed considerably between strains; 27% in DBA/2, 22% in BDF1, 11% in BALB/c, 10% in CDF1 and 0% in C57BL/6 mice and no mast cell tumors were detected in any of the strains when treated with the initiator alone. First found in a DBA/2 mouse at week 50, most tumors were observed after 100 weeks of promotion, and were usually small in size (less than 2 mm in diameter) and predominantly located within the corium, although they occasionally extended into the subcutaneous tissue. Histologically, the benign mast cell tumors were composed of non-encapsulated, well circumscribed densely packed sheets of discrete cuboidal or rhomboid cells. Metachromatic granules were clearly visible in the cytoplasm by Toluidine Blue staining. Two of the tumors induced in DBA/2 mice were diagnosed as malignant mast cell tumors on the twin bases of cellular atypia and deep infiltration into the muscular layer. The cutaneous mast cell tumors were constantly accompanied by subepidermal mast cell aggregations which were also commonly observed in tumor-free skin of mice receiving the initiation-promotion procedure.     

Comparison of epidermal protein kinase C activity, ornithine decarboxylase induction and DNA synthesis stimulated by TPA or dioctanoylglycerol in mouse strains with differing susceptibility to TPA-induced tumor promotion

The purpose of this study was to examine the activity and associated kinetic parameters of epidermal protein kinase C (PKC) following stimulation by sn-1,2-dioctanoylglycerol (DIC8) or 12-O-tetradecanoylphorbol-13-acetate (TPA) and to examine the relationship between levels of epidermal PKC activity and the induction of ornithine decarboxylase by these agents, utilizing various stocks and strains of mice. Importantly, the mouse strains and stock used in this study have known differing susceptibilities to undergo TPA-induced tumor promotion: the CD-1 stock and the DBA/2 strain (both sensitive to TPA-induced tumor promotion) and the C57BL/6 strain (resistant to TPA-induced tumor promotion). TPA-stimulated protein kinase C activity was measured in the 10(5)g supernatant fraction of epidermal homogenates using lysine-rich histone as a phosphate acceptor substrate. The maximal velocities for TPA-stimulated epidermal PKC activity in CD-1, DBA/2 and C57BL/6 were 0.28, 0.29 and 0.27 nmol PO4-histone/mg 10(5)g protein/min, respectively. TPA-stimulated epidermal PKC from CD-1, DBA/2 and C57BL/6 had similar theoretical Vmax values and the apparent concentrations of TPA yielding half-maximal stimulation of PKC were also similar. DiC8-stimulated PKC activity to a greater Vmax; however, the concentration required to yield half-maximal stimulation of PKC was one thousand times greater than that of TPA. There were no strain differences in these parameters when the enzyme was stimulated with DiC8. Thus, the levels of epidermal PKC activity in CD-1, DBA/2 and C57BL/6 mice exhibit no strain differences when stimulated by TPA or DiC8 using lysine-rich histone as a phosphate acceptor substrate. Since sn-1,2-diacylglycerols are known effective inducers of epidermal ornithine decarboxylase (ODC) activity, the induction of epidermal ODC was examined in each mouse strain 5 h after topical application of 2 nmol TPA, 5 nmol TPA or 2.5 mumol DiC8. After topical treatment with TPA, C57BL/6 demonstrated an unexpected 2- and 4-fold increase in ODC activity over CD-1 and DBA/2 mice. After treatment with DiC8, C57BL/6 demonstrated a 6- and 10-fold increase in ODC activity over CD-1 and DBA/2, respectively. Thus, the resistant strain (C57BL/6) demonstrated a 'hyperinducibility' of epidermal ODC activity by TPA or DiC8. The time course for the induction of epidermal ODC was examined in each strain, and at every time point measured (3-15 h), the C57BL/6 strain exhibited this 'hyperinducibility' of ODC relative to the other strains. Epidermal DNA synthesis was stimulated to a similar extent in C57BL/6 and CD-1 mice.(ABSTRACT TRUNCATED AT 400 WORDS)     

N-nitrosocimetidine as a modifier of chemically-initiated tumors in mice

N-Nitrosocimetidine (NCM) is a nitrosation product of cimetidine, a commonly-prescribed pharmaceutical agent. In spite of its known genotoxicity, NCM has failed to cause tumors in assays with rats and mice, but has given indications of enhancing or suppressive effects on tumor development. This possibility was tested by administering NCM topically to the skin or in the drinking water to mice in which tumors had been initiated by treatment with chemical carcinogens. Sencar mouse skin papillomas initiated by 7,12-dimethylbenzanthracene (DMBA) and promoted by 12-O-decanoylphorbol-13-acetate (TPA), progressed more rapidly to carcinoma on mice given treatment during stage 3 (after TPA) with NCM (1 mg/week) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG, 120 micrograms/week) [corrected] than on stage 3 acetone controls. Oral NCM (1 g/l drinking water) did not have this effect but rather suppressed development of keratoacanthomas, as did stage 3 MNNG or TPA. Primary lung tumors initiated in BALB/c mice by i.p. injection of urethane; and tumors of forestomach, lung, mammary, lymphoid and skin tissues caused in (C57BL/6 X DBA/2)F1 mice by oral DMBA were not markedly affected by NCM given in drinking water (1000-1800 ppm) until 14-16 months of age. These results confirm NCM's general lack of activity as an in vivo toxicant, but show that under certain circumstances it may enhance or suppress tumor development.     

Levels and membrane localization of the c-K-ras p21 protein in lungs of mice of different genetic strains and effects of 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) and Aroclor 1254

Mutational activation of the K-ras oncogene often occurs in human and mouse lung adenocarcinomas. Since K-ras p21 functions in trans-membrane signaling, we have investigated whether the amount of this protein in lung cell membranes is a variable that could influence lung tumorigenesis, either due to genetic differences or in response to tumor promoters. The six mouse strains assessed showed little difference in the total lung K-ras p21 after immunoprecipitation and immunoblotting. However, amount of ras p21 in the membrane fraction showed significant differences, with C57BL/6 and BALB/c having 3-5-fold more than NIH Swiss, AKR and DBA mice. Interestingly, a congenic AKR strain having the Ahr(b-1) Ah receptor allele from C57BL/6 mice (designated AKR.B6Ah) had high lung membrane K-ras p21 similar to that of C57BL/6. To test for possible changes related to lung tumor promotion, mice were treated with a promotional dose of TCDD (5 nmol/kg). After 48 h C57BL/6 lungs showed an increase in p21 in both total and membrane fractions. BALB/c, DBA and Swiss mice showed an increase only in membranes. There was no change in the AKR and AKR.B6Ah. Aroclor 1254 (250 mg/kg) caused an increase in membrane/cytosol ratio in Swiss mice. Thus the membrane:cytosol K-ras p21 ratio may be influenced by the Ahr phenotype, and TCDD and PCBs can induce p21 or increase its membrane level in certain strains, but these properties are not fully dependent on Ahr receptor type. In confirmation of the relevance of these findings for the tumor target cell type, the immortalized alveolar type 2 E10 cell line presented K- ras p21 in membrane, and this was increased 4-fold by treatment with 10 nM TCDD.      

Evidence for a common genetic pathway controlling susceptibility to mouse skin tumor promotion by diverse classes of promoting agents

The present study has compared different mouse stocks and strains with known sensitivity to phorbol ester skin tumor promotion for their sensitivities to skin tumor promotion by a prototypic organic peroxide (benzoyl peroxide, BzPo) and anthrone (chrysarobin, Chr) tumor promoter. Following initiation with either 7,12-dimethylbenz(a)anthracene and/or N-methyl-N'-nitro-N-nitrosoguanidine, groups of mice were promoted with several different doses of each promoting agent. Among mice selectively bred for sensitivity to phorbol ester promotion, the order of sensitivity to BzPo was inbred SENCAR (SSIn) greater than SENCAR greater than CD-1. With Chr as the promoter, the order of sensitivity was SENCAR greater than SSIn greater than CD-1. Concurrent tumor promotion experiments examined the responsiveness of two common inbred mouse strains, DBA/2 and C57BL/6. The phorbol ester-responsive mouse strain, DBA/2, was more sensitive to skin tumor promotion by Chr than was C57BL/6 at all doses tested but was clearly less sensitive than both SENCAR and SSIn mice. Finally, DBA/2 and C57BL/6 mice were similar in their responsiveness to BzPo promotion, but again both of these inbred strains were significantly less sensitive than were SSIn and SENCAR mice to this organic peroxide type of skin tumor promoter. Histological evaluations comparing SENCAR and C57BL/6 mice revealed that a major difference between these strains in response to multiple Chr and BzPo treatments was in the inflammatory response (measured by edema formation). Unlike 12-O-tetradecanoylphorbol-13-acetate, Chr and BzPo did not induce dramatic differences in the epidermal hyperplasia (as measured by epidermal thickness) in these two mouse lines. The results presented in this paper suggest that there is a common pathway controlling susceptibility to skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate, BzPo, and chrysarobin. These results are discussed in terms of a possible genetic model(s) for skin tumor promotion in mice.     

Sensitivity of HRA/Skh hairless mice to initiation/promotion of skin tumors by chemical treatment

HRA/Skh hairless mice were investigated for their sensitivity to initiation and promotion by chemicals because of (a) the known sensitivity of these mice to photocarcinogenesis, (b) their low background papilloma incidence (2/3000 mice under 1 year of age) and (c) ease of treatment and identification of tumors, in the absence of hair. Employing a variety of treatments with 7,12-dimethylbenz[a]anthracene (DMBA) as initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as promoter, it was found that the strain was susceptible to both initiation and promotion. Papilloma incidence was at least equivalent to that observed with other sensitive mouse strains. Following initiation with 2.56 micrograms DMBA, papilloma development was promoter-concentration-dependent, resulting in 22.5 papillomas/mouse at 20 weeks in animals administered 5 micrograms TPA. In the absence of DMBA initiation, TPA treatment was weakly carcinogenic in HRA/Skh mice. This treatment induced a dose-dependent increase in papillomas, one of which progressed to a keratoacanthoma-like tumor after 65 weeks. These results show that HRA/Skh mice are highly sensitive, not only to UV carcinogenesis, but also to chemical initiation and promotion of skin papillomas.     

Persistence of carcinogenic effect in intact progeny of mice treated transplacentally with 7,12-dimethylbenz[a]anthracene

Pregnant SHR mice were treated once with 7,12-dimethylbenz[a]anthracene (DMBA) on days 17-19 of gestation. F1 and F2 descendants of these mice received multiple skin applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) twice a week for 24 weeks beginning at 12 weeks of age, or applications of solvent alone. The increase in the frequency of skin tumours in F1 and F2 descendants was reported elsewhere. In addition, we report here an increase in overall numbers of tumor-bearing animals, independently of TPA treatment both in F1 and F2 groups compared to respective control groups. Separate statistical analyses were performed for lung tumours, mammary gland tumours, leukaemias and lymphomas. In both generations of descendants of DMBA-treated mothers lung tumour incidence was considerably increased and differed significantly (maximal P-value = 0.003) from control values. Local applications of TPA resulting in strong skin tumour promoting effect described in our previous paper (Napalkov et al., Carcinogenesis, 8(3) (1987) 381) did not produce any significant change in the rates of other types of tumours. The results of the present study provide additional evidence in support of the hypothesis on possibility of hereditary transmission of carcinogenic action of certain chemical compounds.