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.     

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.     

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.     

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.     

Confirmation of the mapping of a 12-O-tetradecanoylphorbol-13-acetate promotion susceptibility locus, Psl1, to distal mouse chromosome 9.

Susceptibility to two-stage skin carcinogenesis in the mouse is affected by several genes. In addition, studies suggest that genes that modify the response of mice to skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) also may influence histologic changes in the skin as the result of TPA treatment. One TPA susceptibility locus, Psl1, previously was mapped to distal chromosome 9. The mapping of this locus was confirmed by marker-based genotypic selection. Furthermore, Psl1 or a gene closely linked to Psl1 influenced epidermal hyperplasia and epidermal labeling index of mice treated with TPA.     

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.     

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)     

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.     

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.     

Correlation of phorbol ester promotion in the resistant C57BL/6J mouse with sustained hyperplasia but not ornithine decarboxylase or protein kinase C

The activation of protein kinase C, induction of ornithine decarboxylase (ODC), and hyperplasia have been suggested to be linked, sequential processes resulting from phorbol ester application to mouse skin. However, evidence is presented indicating that these events are not necessarily linked or dependent on one another and that significant differences exist in these responses between phorbol ester promotion sensitive (SSIN) and resistant (C57BL/6J) mice. The epidermis from SSIN mice treated with a single application of 12-O-tetradecanoylphorbol-13-acetate (TPA) displayed a large induction of ODC and a subsequent extensive hyperplasia. A second TPA treatment at 24 or 48 h after the first did not result in ODC induction (refractory state), and protein kinase C was shown to be down-regulated at these times. By 72 h, however, a responsive state had returned even through protein kinase C remained down-regulated. The epidermis of C57BL/6J responds to a single application of TPA with a level of ODC induction similar to that of the SSIN mice. Protein kinase C was down-regulated by approximately 75% after 24 h and was virtually completely down-regulated at 48 and 72 h (95-97%). In contrast to the above findings for the sensitive mice, however, little, if any, hyperplasia was produced. In addition, while a second TPA treatment at 24 h did not result in ODC induction (refractory state), hyperplasia did occur within 24 to 48 h. When the second TPA application was given 48 h after the first, at a time when protein kinase C was down-regulated, an overinduction of ODC occurred, as well as subsequent hyperplasia. Furthermore, a significant number of papillomas resulted when these increased treatment frequencies, i.e., once a day or every other day, were used to promote dimethylbenz(a)anthracene-initiated C57BL/6J mice. It is concluded that, while hyperplasia remains an apparent requirement for tumor promotion, the ODC induction following an initial TPA treatment is insufficient for or not causally related to this hyperplasia. In addition, subsequent ODC induction, at least in the C57BL/6J mouse, is probably not mediated by protein kinase C.     

Sensitivity to tumor promotion of SENCAR and C57BL/6J mice correlates with oxidative events and DNA damage

Significant differences in sensitivity to multistage carcinogenesishave been noted between mice that are sensitive (SENCAR) andresistant (C57BL/6J) to 12-O-tetradecanoyl-phorbol-13-acetate(TPA). However, the mechanism of this sensitivity has not yetbeen established. Recent studies from this laboratory have shownthat TPA significantly enhances formation of hydrogen peroxide(H₂O₂) and oxidized DNA bases in SENCAR mouse skin, as it increasesthe infiltration of polymorphonuclear leukocytes (PMNs), asquantitated by myeloperoxidase (MPO). In the studies reportedhere, we compared SENCAR and C57BL/6J mice with respect to TPA-mediatededema, hyperplasia, PMN infiltration, oxidant formation andoxidative DNA damage in mouse skin. Topical application of twoTPA doses (2x2–40 µg, 20 h apart) dose-dependentlyincreased PMN infiltration and oxidant formation in both mousestrains, which was consistent with TPA-induced morphologicalalterations (edema and hyperplasia). However, at low TPA doses(2–4 µg), the increases over controls in the SENCARmice were significantly greater (P < 0.01) than those inC57BL/6J mice. Comparison of the net values indicated that 4µg TPA enhanced PMN infiltration (MPO units/cm²) and oxidantformation (nmol H₂O₂/cm²) in SENCAR mice by 7.7- and 11-foldrespectively over those present in TPA-treated C57BL/6J mouseskin. At the same dose, TPA also significantly increased formationof thymidine glycol (dTG; 5.5-fold), 5-hydroxymethyl-2'-deoxy-uridine(HMdU; 4.9-fold) and 8-hydroxyl-2-deoxyguanosine (8-OHdG; 11.4-fold)in SENCAR mouse epidermis. Then, the levels of all three declined.In C57BL/6J mice, there were virtually no increases at 4 µgTPA, but their levels gradually increased with higher TPA dosesand reached maxima at 10 µg TPA for dTG (1.9-fold increase),at 20 µg TPA for 8-OHdG (6.0-fold), and at 30 µgTPA for HMdU (1.8-fold). We conclude that the TPA-mediated oxidativeevents and oxidative DNA modification by different doses ofTPA correlate with the promoting potencies of those doses inboth mouse strains. Therefore, they could be, at least in part,responsible for the strain-dependent sensitivity to tumor promotion.     

Overexpression of a dominant-negative ornithine decarboxylase in mouse skin: effect on enzyme activity and papilloma formation

A transgenic mouse line expressing a truncated form of the ornithine decarboxylase (ODC) dominant-negative mutant K69A/C360A under the control of the keratin 6 promoter has been established (K6/ODCdn mice). These mice were backcrossed onto both the DBA/2J and C57BL/6J backgrounds for subsequent tumorigenesis experiments utilizing an initiation/promotion protocol. In short-term experiments, expression of the ODCdn protein product was induced in the epidermis within 24 h after application of the tumor promoter tetradecanoyl phorbol acetate (TPA) to the skin, and ODC activity in the epidermis of K6/ODCdn mice was reduced by at least 75% compared with littermate controls. However, in tumorigenesis experiments utilizing a variety of initiator (7,12-dimethylbenz[a]anthracene; DMBA) and promoter (TPA) concentrations, K6/ODCdn mice formed at least as many tumors as their littermate controls regardless of background strain. In experiments utilizing chrysarobin, a tumor promoter with a different mechanism of action than TPA, again there was no significant difference in tumor formation between K6/ODCdn mice and littermate controls. Similarly, when K6/ODCdn mice were crossed with K5/ODC mice, a transgenic line described previously which forms tumors without application of a promoting agent, double transgenic mice formed as many tumors as mice expressing the K5/ODC transgene alone. Analysis of epidermis following multiple TPA applications revealed a dramatic spike in ODC activity in both K6/ODCdn mice and non-transgenic mice after six applications, and western blot analysis suggested a stabilization of endogenous wild-type ODC in K6/ODCdn transgenic mice. ODC activity, endogenous protein and polyamines were also elevated in tumors from K6/ODCdn mice. The accumulation of endogenous ODC protein is most probably the result of competition from the transgene-derived ODCdn protein for binding of antizyme, which is known to regulate ODC activity by stimulating degradation of the ODC protein.     

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.     

Possible dissociation of the phorbol ester-induced oxidant response and tumor promotion in the F1 offspring of SSIN x C57BL/6J mice

The F₁ progeny of a cross between 12-O-tetradecanoyl-phor-bol-13-acetate(TPA) tumor promotion-sensitive SSIN mice and TPA promotion-resistantC57BL/6J mice were found to be sensitive to TPA as a tumor promoter.The tumor response was substantial, with an average of 15 papillomasper mouse and a 100% incidence following initiation with 400nmol dimethylbenz[a]anthracene and promotion with 6.5 nmol (4µg) TPA. To determine whether tumor promotability of theF₁ mice correlates with other parameters believed to be associatedwith TPA responsiveness, oxidant generation, epidermal hyperplasiaand edema were compared in the parents and F₁ hybrids. The SSINproduced a strong hyperplastic response to TPA, the C57BL/6Ja negligible response and the F₁ hybrids a moderate response.In the SSIN, 6.5 nmol (4 µg) TPA caused an 18% increasein the water content of the skin (edema) while no change wasseen for either the C57BL/6J or F₁ hybrids. The oxidant responseof the F₁ hybrids to either TPA or phospholipase C was markedlyless than that observed for the SSIN and was similar to theresponse previously observed for the C57BL/6J mice. These findingssuggest that the oxidant response may not be an essential aspectof TPA tumor promotion. It may be related to the edema response,suggesting that at least this aspect of inflammation is notnecessary for promotion.     

Phorbol ester induction of 8-lipoxygenase in inbred SENCAR (SSIN) but not C57BL/6J mice correlated with hyperplasia, edema, and oxidant generation but not ornithine decarboxylase induction

Several responses suggested to be critical components of phorbol ester tumor promotion were compared in 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion-sensitive SSIN and TPA promotion-resistant C57BL/6J mice. SSIN mice treated topically with 2 micrograms of TPA showed extensive hyperplasia accompanied by edema, measured as a 26% increase in water content of the skin. Only a very slight hyperplasia and 7% increased water content occurred after TPA treatment of C57BL/6J mice. The induction of ornithine decarboxylase was determined to be the same both in vivo and in vitro for SSIN and C57BL/6J mice, which does not correlate with the histological observations. Because hyperplasia and inflammation can be mediated by arachidonic acid metabolites, it was hypothesized that differences in this metabolic pathway would correlate with the histological responses. No significant qualitative or quantitative differences, however, were observed in the profiles of the major cyclooxygenase products between the strains of mice. Prostaglandin E2, the principal prostaglandin, was synthesized at a 3-fold greater level than prostaglandins D2 or F2 alpha in response to TPA. The most abundant lipoxygenase product was 12-hydroxyeicosatetraenoic acid followed by 8-, 15-, and 5-hydroxyeicosatetraenoic acid. 8-Lipoxygenase activity is elevated 24 h after TPA treatment in the SSIN mice by approximately 4-fold; no elevation is seen in C57BL/6J mice. A comparison of the oxidant response to TPA as well as to phospholipase C showed that SSIN epidermal cells generated a higher level, measured by chemiluminescence, than C57BL/6J cells. This suggests that oxidant generation or possibly 8-lipoxygenase activity may be the basis for the sensitivity or resistance to TPA as a hyperplasiogen and as a tumor promoter.     

Cancer modifier alleles inhibiting lung tumorigenesis are common in inbred mouse strains

Lung tumor susceptibility in inbred mouse strains is caused by the susceptibility allele at the pulmonary adenoma susceptibility 1 (Pas1(s)) locus. However, after urethane treatment, most strains carrying the Pas1(s) allele show an intermediate (1-4 tumors/mouse) instead of a highly susceptible (15-30 tumors/mouse) lung tumor phenotype. To test the hypothesis that strains displaying the intermediate lung tumor phenotype carry dominant or codominant resistance alleles at pulmonary adenoma resistance (Par) loci, we crossed mice of intermediate susceptibility or resistance to lung tumorigenesis with the highly susceptible A/J strain. Eleven F(1) hybrids were treated with urethane to induce lung tumorigenesis. The A/J strain developed 35.3 tumors/mouse, while its F(1) hybrid with C57BL/6J mice (null allele at Par loci) developed 22.8 tumors/mouse due to the Pas1 allele dosage effect. F(1) hybrids of strains 129/SvJ, CBA/J, ST/J and LP/J (Pas1(s)) and of SPW, DBA/2J and C57L/J (Pas1(r)) mice showed significant reduction in lung tumor multiplicity (i.e., 0.3-12.8 tumors/mouse) compared to A/J and (A/J x C57BL/6J)F(1) mice. These results indicate that resistance alleles at Par loci are common in inbred mouse strains and account for the lung tumorigenesis intermediate phenotype of strains carrying the Pas1(s) allele.     

Proto-oncogene activation in liver tumors of hepatocarcinogenesis-resistant strains of mice.

Activation of the ras family of oncogenes occurs frequently in liver tumors of the B6C3F1 mouse, a strain which is highly sensitive to hepatocarcinogenesis. Many other mouse strains are much more resistant to hepatocarcinogenesis; the aim of this study was to determine the frequency and pattern of oncogene activation in spontaneous and chemically induced liver tumors of three such strains, the C57BL/6J, the C57BL/6 x DBA/2 F1 hybrid (B6D2F1) and the C57BL/6 x Balb/c F1 hybrid (B6BCF1). The C57BL/6, DBA/2 and Balb/c strains are all relatively resistant to spontaneous hepatocarcinogenesis (1.5-3.6% of animals develop liver tumors in 2 years); with regard to chemically induced hepatocarcinogenesis the Balb/c is highly resistant, the C57BL/6 has low susceptibility and the DBA/2 has low to moderate susceptibility. The nude mouse tumorigenicity assay was used to search for activated oncogenes in 15 C57BL/6J liver tumors induced by a single neonatal dose of vinyl carbamate (VC, 0.15 mumol/g body weight). Three tumors contained H-ras genes activated by point mutations at codon 61 and one contained a non-ras oncogene. The polymerase chain reaction and allele-specific oligonucleotide hybridization were used to study H-ras mutations in spontaneous and VC-induced tumors from all three strains of mice. The frequency of H-ras codon 61 mutations in tumors induced by 0.15 mumol/g body weight VC in the C57BL/6J mouse (5/37) was similar to that in spontaneous tumors (2/9); surprisingly, tumors induced by a lower dose of VC (0.03 mumol/g body weight) had a higher frequency of H-ras mutations (12/28). The frequencies of H-ras activation detected in VC (0.03 mumol/g body weight)-induced tumors from the two F1 hybrids studied differed markedly. Only one VC-induced B6BCF1 tumor contained a mutated H-ras gene (1/10), whereas the majority of B6D2F1 tumors contained such mutations (23/33). Several spontaneous B6D2F1 liver tumors contained H-ras codon 61 mutations (6/15). Thus, H-ras activation frequency does not determine susceptibility to hepatocarcinogenesis in inbred mice and their F1 hybrids, since a relatively high frequency of H-ras mutations was observed in two resistant strains and a low frequency was found in the other strain.     

Dietary fat- and phenobarbital-induced alterations in hepatic antioxidant functions of mice

Inbred strains of mice with differential response to known tumorpromoters were compared with respect to their susceptibilityto modulation of hepatic antioxidant enzymes by long-term treatmentwith high fat diet (HF) and pheno-barbital (PB). Mice of theC57BL/6J (C57), C3H/HeOuJ (C3H) and DBA/2J (DBA) strains werefed diets containing low (5%) or high (15%) amounts of fat (sunfloweroil) for 26 weeks from the age of 6 weeks onwards. Groups ofmice on the 5% fat diet received 0.05% PB in their drinkingwater from 12 to 22 weeks of age. Mice of the C57 strain areknown to be refractory to promotion of hepatocarcinogenesis,the C3H strain has a high incidence of spontaneous tumors andis sensitive to promotion by HF and PB, and the DBA strain isespecially sensitive to promotion by PB. Within all strainsof mice, and in both dietary groups, the degree of oxidativestress in the liver was found to increase with age, as was indicatedby the increased amounts of TBA reactive material (lipid peroxidation)and decreased glutathione (GSH) and phospholipid contents ofthe tissue. HF elevated the amount of TBA reactive materialin the liver of C57 and C3H mice, induced GSH-peroxidase andMn-superoxide dismutase activities in the C3H strain, and depressedthe hexose monophosphate shunt activity within all mouse strains.PB drastically decreased the amount of TBA reactive materialin the liver in all mouse strains, increased catalase activityin all strains and the activity of GSH-peroxidase in the C3Hand DBA strains. The above strain differences in responses ofhepatic antioxidant functions to HF and PB parallel the differentialresponsiveness of these mouse strains to promotion of hepatocarcinogenesisby these agents, and the increased antioxidant capacity wasproportional to susceptibility to tumor promotion.     

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.