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

To explore further the genetics of susceptibility to skin tumor promotion in inbred mice, several aspects of responsiveness to 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined in C3H/He mice and segregating crosses between this mouse strain and C57BL/6 mice as well as BXD and BXH recombinant inbred (RI) strains. Dose-response relationships were established for skin tumor promotion by TPA following initiation with 7,12-dimethylbenz[a]anthracene in C3H/He and B6C3F1, as well as several other mouse stocks and strains included for comparison. The relative responsiveness to TPA skin tumor promotion was: SENCAR much greater than DBA/2 greater than C3H/He approximately B6D2F1 greater than B6C3F1 much greater than C57BL/6. Analyses of the susceptibility of B6C3F2 and B6C3F1 x C57BL/6 backcross mice suggested that a minimum of two dominant genetic loci control responsiveness to phorbol ester promotion in these mice. Further analysis of BXH and BXD RI strains suggested the presence of four distinct promotion-responsive phenotypes controlled by a minimum of two genetic loci. The existence of a 'hyper-responsive' phenotype in the sets of RI strains, however, suggests that a third, recessive locus also may play a role in controlling responsiveness to TPA promotion. At 48 h after the last of four applications of TPA, marked hyperplasia and an increase in dark basal keratinocytes were observed in C3H/He mice, whereas in B6C3F1 mice the response in these parameters was intermediate between C3H/He and C57BL/6 mice. A marked dermal inflammation, as determined by infiltration of polymorphonuclear cells, was observed in C3H/He and B6C3F1 mice, whereas little was noted in C57BL/6 mice. Furthermore, histological evaluations of selected BXD RI strains revealed a significant correlation between the magnitude of the hyperplasia response and the percentage of mice bearing tumors. The present data, in conjunction with our previous studies, confirm that the major gene(s) controlling susceptibility to tumor promoter induced by TPA in two sensitive strains (i.e. DBA/2 C3H/He) are similar or closely linked to those for induction of sustained hyperplasia. In addition, the present data provide new evidence for a model where allelic differences at a minimum of three loci contribute to gene differences in susceptibility to phorbol ester promotion DBA/2 and C3H/He versus C57BL/6 mice.

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.

sn-1,2-didecanoylglycerol effectively induces epidermal ornithine decarboxylase but only weak hyperplasia in mouse skin.

The abilities of sn-1,2-didecanoylglycerol (sn-1,2-DDG) to induce epidermal ornithine decarboxylase (ODC) activity and epidermal hyperplasia were tested using SENCAR, DBA/2 and C57BL/6 mice. Following a single application of 5000 nmol of sn-1,2-DDG, ODC activity reached a maximum at 4 h after treatment with a peak activity of 6.03 nmol CO2/mg protein/60 min in C57BL/6, 1.50 in SENCAR and 0.73 in DBA/2, respectively. The time course and magnitude for induction of ODC activity after multiple treatments was very similar to that after a single application in these three mouse lines. Interestingly, the induced ODC activity in C57BL/6 was always higher than that in SENCAR and DBA/2 mouse epidermis regardless of the treatment protocol. Induction of hyperplasia and dark basal keratinocytes (DCs) and changes in the labeling index (LI) of basal keratinocytes in DBA/2 and C57BL/6 mice following treatment with sn-1,2-DDG were investigated. Multiple treatments (twice weekly for 2 weeks) of 5000 nmol sn-1,2-DDG did not induce substantial increases in epidermal thickness or DCs 24 or 48 h after the last treatment. In contrast, TPA induced a marked increase in epidermal thickness in DBA/2 rather than C57BL/6 and a considerably higher induction of DCs in DBA/2 (37.3 +/- 2.2%) than in C57BL/6 (9.6 +/- 2.5%) 48 h after the last treatment. The LIs after topical application of sn-1,2-DDG were elevated at 24 h, but returned to basal levels by 48 h in both strains, whereas TPA treatment significantly elevated the LI in both strains at 48 h after the last application. In addition, the effects of various doses and frequencies of application of sn-1,2-DDG were investigated using SENCAR mice. High doses (20,000 nmol) or more frequent applications (5000 nmol once daily for 7 days) of sn-1,2-DDG still produced only weak hyperplasia. These results suggest that the induction of epidermal ODC activity can be dissociated from the induction of epidermal hyperplasia and may provide an explanation for the lack of complete promoting activity presently observed with membrane permeable diacylglycerol derivatives.

Modulation of chrysarobin skin tumor promotion.

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

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

Since current evidence suggests that the tumor promotion stage is a primary determinant in susceptibility to multistage carcinogenesis, we have characterized the genetics of susceptibility to phorbol ester skin tumor promotion in inbred mice. Susceptibility of hybrids (B6D2F1), between 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 mouse but clearly less when doses of 1.7-6.8 nmol per mouse were used. In addition, no significant differences were observed between male and female B6D2F1 mice in terms of tumor incidence although some differences were observed in tumor multiplicities between male and female F1 mice at the highest TPA dose. Reciprocal F1 mice initiated with DMBA (i.e. D2B6F1) were also responsive to TPA. Female D2B6F1 mice were of lower sensitivity at lower doses of TPA, compared to female DBA/2, a finding similar to that observed with B6D2F1 mice initiated with MNNG. Further analyses of the susceptibility of B6D2F2 and B6D2F1 X C57BL/6 backcross mice to TPA promotion indicated that more than one dominant genetic locus must account for the differences in promotion sensitivity between DBA/2 and C57BL/6 mice. To understand further the genes responsible for promotion sensitivity, histological evaluations were performed on DBA/2, C57BL/6 and B6D2F1 mice. Histological examination revealed that the epidermis of DBA/2 mice showed a marked hyperplasia and the presence of a much greater number of dark basal keratinocytes (DCs) compared with C57BL/6 mice 48 h after the last of four applications of TPA (doses greater than or equal to 3.4 nmol). A marked dermal infiltration of polymorphonuclear leukocytes (PMNs) was observed in DBA/2 mice, whereas little infiltration was observed in the skin of C57BL/6 mice. The hyperplasia in the skin of B6D2F1 mice was intermediate between DBA/2 and C57BL/6 mice at all TPA doses examined except the lowest dose (1.7 nmol), whereas the DC response, although significantly lower at doses of 6.8 nmol or below, was similar to DBA/2 mice at higher TPA doses (13.6 and 17.0 nmol). The infiltration of PMNs in the dermis of B6D2F1 mice was similar to or greater than DBA/2 mice at all doses of TPA tested.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification and chromosomal locations of aconitase gene loci in Triticeae species.

Two systems of monomeric aconitase (ACO) isozymes, designated ACO-1 and ACO-2, were identified in Triticum aestivum and in five diploid Triticeae species. The gene loci Aco-A1, Aco-B1, and Aco-D1 were located in T. aestivum cv. 'Chinese Spring' chromosome arms 6Aq, 6Bq, and 6Dq, respectively, and the gene loci Aco-A2, Aco-B2, and Aco-D2 in 5 Aq, 5 Bq, and 5Dq, respectively. Aco-1 gene loci were also identified in 6Eß of Elytrigia elongata, 6HL of Hordeum vulgare cv. 'Betzes', 6RL of Secale cereale 'PI 252003', 6S(1) of T. longissimum, and CSU-31 of T. umbellulatum. Other Aco-2 gene loci were identified in 5RL of S. cereale cv. 'King II' and 4EL of E. elongata. Conservation of synteny relationships is indicated among the species studied for the genes identified, with the exception of Aco-E2; the presence of this gene in 4EL suggests that E. elongata differs from 'Chinese Spring' and 'King II' by a translocation involving 4E and 5E.

Effect of application frequency on epidermal ornithine decarboxylase induction by chrysarobin in SENCAR mice.

Ornithine decarboxylase (ODC EC 4.1.1.17) induction in mouse epidermis after single or multiple topical applications of chrysarobin differed from that following topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA). Following a single application of 220 nmol chrysarobin, ODC activity began to rise at 24 h reaching a peak at 56 h and returned to normal after 96 h. When 5 separate applications of 220 nmol chrysarobin were applied in multiple application protocols, an alteration in the ODC induction response was observed. With a once per week or twice per week application protocol, ODC was elevated in a multiphasic manner giving multiple peaks of activity after the last application. Interestingly, the magnitude of ODC induction was greater using the once per week, compared to the twice per week, application protocol. Preliminary results indicate that a once per week application protocol is more effective than a twice per week protocol for promoting the development of skin papillomas in SENCAR mice. Thus, the magnitude of the induced ODC response with chrysarobin, although low compared to TPA, correlated with tumor promoting activity.