Carcinogenesis vol.21 no.4 pp.641–646, 2000
Development and initial characterization of several new inbred strains of SENCAR mice for studies of multistage skin carcinogenesis
Lezlee G.Coghlan, I.Gimenez-Conti1, Heather E.Kleiner1, Susan M.Fischer1, Joyce E.Rundhaug1, Claudio J.Conti1, Thomas J.Slaga and John DiGiovanni1,2 The University of Texas M.D. Anderson Cancer Center, Science Park– Department of Veterinary Sciences, Bastrop, TX 78602 and 1The University of Texas M.D. Anderson Cancer Center, Science Park–Research Division, Smithville, TX 78957, USA 2To
whom correspondence shopuld be addressed Email:
[email protected]
The development and initial characterization of five new inbred strains of SENCAR mice are described in this paper. Ten randomly selected pairs of outbred SENCAR mice were mated and offspring from each separately maintained parental line were sib mated at each successive generation to result in inbred strains. Due to poor reproductive performance only five of the original 10 lines were bred to homogeneity. Initial characterization of the five remaining lines (referred to as SL2/sprd, SL5/sprd, SL7/sprd, SL8/ sprd and SLl0/sprd) at F12 for their responsiveness to a two-stage carcinogenesis protocol (10 nmol 7,12-dimethylbenz[a]anthracene and 0.25 µg 12-O-tetradecanoylphorbol13 acetate) revealed three groups of responders in terms of the number of papillomas per mouse: SL2/sprd and SL8/sprd > SL7/sprd and SL10/sprd >> SL5/sprd. The papilloma responses in SL2/sprd and SL8/sprd were very similar to SENCAR B/Pt compared at the same doses. Papillomas induced on SL2/sprd had the highest propensity to progress to squamous cell carcinomas, similar to that observed in outbred SENCAR and SENCAR B/Pt mice. More detailed comparison of the responsiveness of SL2/ sprd and SL5/sprd at Fl5 showed that these two inbred strains differed in their sensitivity to TPA-induced epidermal hyperplasia and that the dose of TPA required to produce a tumor response in SL5/sprd in comparison with that in SL2/sprd was 4–20 times higher. Overall, the availability of the different inbred SENCAR strains will greatly aid mechanistic studies of multistage skin carcinogenesis as well as studies to understand the underlying genetic basis of resistance to tumor promotion and progression in this model system. Introduction The two-stage model of mouse skin carcinogenesis, in use for over 50 years (1), has provided an important framework for conceptualizing the multistage nature of tumor development in other animal models and in man (2–5). Evidence that this model is relevant to human cancers comes from recent studies of human colon (6–11) and other cancers (12–16). Therefore, while certain aspects of skin carcinogenesis differ between Abbreviations: BrdU, bromodeoxyuridine; DMBA, 7,12-dimethylbenz[a] anthracene; H&E, hematoxylin and eosin; TPA, 12-O-tetradecanoylphorbol13-acetate. © Oxford University Press
mouse and man, examination of the multistage process in this model is likely to be directly relevant to understanding the development of many human epithelial cancers. In this regard, further development and characterization of the model should continue to provide valuable tools and concepts for studying mechanisms of multistage carcinogenesis. In the mouse two-stage protocol, the irreversible initiation event is induced by the topical application of a single subcarcinogenic dose of a carcinogen, generally 7,12-dimethylbenz[a]anthracene (DMBA) (17,18). In outbred SENCAR and SSIN mice, it has been shown that DMBA induces a mutation in codon 61 of the c-Ha-ras gene (19,20). The initiation stage is phenotypically silent, i.e. tumors do not develop unless initiation is followed by promoting events, such as wounding or chronic treatment with various chemical promoting agents (4,5). The second stage, promotion, involves chronic topical treatment with non-carcinogenic agents, such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Genetically susceptible mice develop cutaneous papillomas that vary in number, latency and ability to undergo malignant conversion to squamous cell carcinomas, termed by some the stage of progression (4,5). The general order of susceptibility within mouse stocks and strains to epidermal two-stage carcinogenesis, using DMBA for initiation and TPA for promotion, has been summarized as follows: SENCAR ⬎ DBA/2 ⬎ CD1 ⬎ C3H ⬎ Balb/c ⬎ C57BL/6 (4). Within this paradigm, development of the SENCAR mouse led to the more widespread use and characterization of the mouse skin model within the field of carcinogenesis (1,4,21–25). The SENCAR outbred stock is responsive to both DMBA and TPA, has a short tumor latency and variable rate of malignant conversion (23–25). Despite the sensitivity of outbred SENCAR mice to the twostage protocol, however, its usefulness for certain studies, such as those related to tissue or tumor grafting or studies of genetic sensitivity to epidermal carcinogenesis, is hindered by its outbred nature. Clearly, the development of inbred mice with different sensitivities to two-stage carcinogenesis is an important goal. The first such strain, named STS and developed by Boutwell through selective breeding, exhibited poor reproductive performance but became a parental contributor to the present day SENCAR stock (1,26). A modern SENCAR-derived inbred strain, called SSIN, has been developed through selective breeding of animals sensitive to a two-stage carcinogenesis protocol in which DMBA was the initiator and TPA was the promoter (27). SSIN mice were more sensitive than SENCAR to TPA, but subsequently it was found that the high papilloma response of SSIN did not correlate with an increase in malignant conversion and, in fact, certain markers of premalignancy were markedly suppressed in SSIN papillomas (28). Three inbred SENCAR strains (designated SENCAR A/Pt, SENCAR B/Pt and SENCAR C/Pt), which are closely related, have been developed by Hennings et al. (29). All three strains have a similar high sensitivity to two-stage carcinogenesis and to 641
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malignant progression and are more sensitive to TPA than the outbred SENCAR (29–32). However, in our hands these inbred strains of SENCAR mice exhibited poor reproductivity, leading to insufficient numbers of animals for study and undue experimental expense. In contrast, although the SSIN strain has excellent reproductive indices, its ability to resist progression leaves investigators with at least two needs. First, a new inbred strain with good fecundity and a high propensity for progression is desirable. Furthermore, the development of a SENCARderived inbred strain that is relatively resistant to two-stage carcinogenesis, as originally described by Boutwell (1), remains an important goal as well. In order to address these problems, we elected to produce several new strains of inbred SENCAR mice. The present study describes the initial production process, selected characteristics and tumor responses of five new inbred strains of SENCAR that were derived simultaneously from the outbred SENCAR stock. Some comparisons with other stocks and strains of SENCAR are made as well. Materials and methods Animals SENCAR breeding stock, 6–10 weeks of age, was acquired from the National Cancer Institute Animal Production Program (Frederick, MD). In order to obtain a full representation of the existing outbred SENCAR gene pool and eliminate the possibility of sib matings in the P1 generations, males and females were ordered within a window of dates that precluded both sexes coming from identical mothers. Following receipt and quarantine, 10 randomly selected pairs were identified numerically (1–10) and set up as P1. Offspring from each separately maintained parental line were sib mated at each successive generation to result in the formation of inbred lines of mice. Pedigrees were maintained throughout the process. When mice reached F17–19, representatives from each line were killed and liver and kidney samples were collected for isozyme electrophoresis (Charles River Biotechnical, Wilmington, MA). Mice were housed in suspended polycarbonate caging (or in disposable polystyrene caging) (Lab Products, Maywood, NJ) for 2 weeks following DMBA treatment on autoclaved hardwood bedding (P.J. Murphy Inc., Montville, NJ) in an AAALAC accredited facility. Room conditions included a temperature range of 68–74°F, relative humidity of 60–70% and a diurnal light cycle of 14 h light, 10 h dark. Autoclaved water (reverse osmosis, pH 2.5) and standard rodent chow (Teklad) were provided ad libitum. SSIN and SENCAR B/Pt (obtained from Dr Michael Potter, NCI) mice were bred in our veterinary resource facility in Bastrop, TX. Chemicals All DMBA (Eastman Kodak Co., Rochester, NY) and TPA (Chemicals for Cancer Research, Eden Prairie, MN) solutions were prepared in reagent grade acetone and were topically applied in a volume of 0.2 ml/mouse as described (26,33,34). Tumor experiments When several partially inbred lines reached F12, we produced sufficient numbers of mice to allow for small tumor induction experiments. Due to the staggered variability of the lines each reaching F12, not all experiments were done concurrently. Additional tumor experiments were conducted in selected lines at F15 and F17. Between 7–10 weeks of age, young mice of both sexes were shaved of their dorsal fur and 2 days later were initiated with 10 nmol (2.56 µg) of DMBA; only mice in the resting phase of the hair growth cycle were used at the time of initiation. Two weeks after DMBA treatment, the mice began receiving twice weekly TPA treatments. In the first tumor experiment, a dose of 0.25 µg TPA was used in all five lines of mice. Also included for comparison in the first experiment were groups of SENCAR B/Pt and SSIN mice tested at the same dose of TPA and a group of SENCAR mice tested at a dose of 1.0 µg TPA. In the second tumor experiment, SL2/sprd groups were promoted with 0.4, 0.2 and 0.1 µg TPA and SL5/sprd groups were promoted with 1.0, 0.4 and 0.2 µg, as shown in the tables and figures. After the appearance of the first papilloma in each experiment, tumors were tabulated weekly, and TPA treatments were continued to 50⫹ weeks in most cases. Papillomas were scored as exophytic lesions arising from a stalk. Carcinomas were recorded grossly as centrally cratered, downward invading lesions. Animals were killed when multiple carcinomas were present that exceeded a
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combined diameter of ~1.5 cm, when a single carcinoma exceeded a diameter of ~1.5 cm or when otherwise indicated according to deteriorating health. All carcinomas were confirmed histologically. Dose–response comparison of TPA-induced epidermal hyperplasia Based on the striking differences between SL2/sprd and SL5/sprd seen in the initial tumor experiments, and to confirm that these lines differed in their response to TPA, F17 mice of both lines were selected for a subacute dose– response study of the effects of TPA. Non-initiated mice were treated with the various doses of TPA twice a week over a 4 week period. Forty-eight hours after the last TPA treatment, mice received an i.p. injection of 100 µg/ g body wt bromodeoxyuridine (BrdU) and were killed by CO2 exposure 30 min later. The dorsal skin was removed, fixed in 10% formalin, sectioned and stained with hematoxylin and eosin (H&E). Measurements of epidermal thickness and labeling index were performed as has been described (35). Statistical analysis Significance of differences between papilloma per mouse data for each mouse line was evaluated with the Mann–Whitney U-test. Significance was set at P 艋 0.05.
Results Development of inbred strains Several of the original 10 lines demonstrated reproductive difficulties as early as generation F7. These problems were typified by small litters and unsuccessful pair matings. In half of the 10 lines (SL1/sprd, SL3/sprd, SL4/sprd, SL6/sprd and SL9/sprd) some inbreeding depression occurred and these problems increased in severity until reproduction ceased entirely, despite remedial measures such as pairing at the time of weaning and parent–offspring matings. Presently, SL2/sprd, SL5/sprd, SL7/sprd and SL8/sprd have been carried to F20⫹ and SL10/sprd has reached F18. Isozyme profiles (14 loci scattered over 10 chromosomes) indicated that most of the five lines were distinguishable from one another and from SSIN/UTSP and SENCAR B/Pt, with a few exceptions (data not shown). The standard commercial isozyme profile (Charles Ryer Laboratories, Wilmington, DE) was used for this comparison. Mice of the SL2/sprd and SL8/sprd lines and mice from the SL5/sprd and SL7/sprd lines were indistinguishable at these loci; additional studies to determine methods of differentiation for these strains are in progress. We also report that SENCAR B/Pt, maintained by us since late 1992 and in the gnotobiotic state since 1993, has undergone subline divergence and, as such, is now genetically distinguishable from SENCAR B/Pt. However, the SENCAR B/Pt mice used in the studies reported here had not yet exhibited this drift; they are correctly identified as SENCAR B/Pt. The five new lines described in this study exhibited variable fecundity rates in the following order: SL7/sprd ⬎ SL8/sprd ⫽ SL5/sprd ⬎ SL10/sprd ⬎ SL2/ sprd 艌 SENCAR B/Pt. All lines showed lower fecundity rates than both outbred SENCAR and SSIN mice. Tumor induction experiments The first tumor experiment was carried out in the five remaining lines of mice at generation F12. Mice were initiated with 10 nmol DMBA and promoted with 0.25 µg of TPA twice a week. Even at this low promoting dose, the SL2/sprd line had a vigorous tumorigenic response, with 6.2 ⫾ 1.5 papillomas/ mouse and 100% incidence. Furthermore, 85% of the SL2/ sprd mice developed carcinomas. Line SL8/sprd had a very similar number of papillomas per mouse, 6.7 ⫾ 1.06, with an incidence of 91%, and 33% developed carcinomas. Lines SL10/sprd and SL7/sprd had lower papilloma responses, as can also be seen in Table I. Remarkably, in the SL5/sprd strain, only 6% of the mice developed papillomas and none of these mice developed carcinomas with this treatment regi-
Inbred SENCAR mice
Table I. Initial characterization of the response of partially inbred (F12) SENCAR lines to an initiation–promotion regimena Line
No. mice
Papillomas per mouse (mean ⫾ SEM)b
Mice with papillomas (%)
Mice with carcinomas (%)
SL2/sprd SL5/sprd SL7/sprd SL8/sprd SL10/sprd Line B/Pt SSIN SENCAR
20 20 25 27 21 60 300 321
6.2 ⫾ 1.5 0.06 ⫾ 0.06 2.6 ⫾ 0.73 6.7 ⫾ 1.06 3.8 ⫾ 0.88 6.8 ⫾ 0.80 11.8 ⫾ 0.38 9.4 ⫾ 0.39
100 6 65 91 88 92 100 100
85 0 32 33 21c 86 6 86
(11F,9M) (9F,11M) (15F,10M) (7F,20M) (11F,10M) (F) (F) (F)
(F, female; M, male) were initiated with 10 nmol DMBA and promoted with 0.25 µg TPA twice weekly. Papilloma response was assessed at 29 weeks of TPA treatment. Carcinoma response was measured at 53 weeks of TPA treatment. bAverage papillomas per mouse for SL2/sprd, Line B/Pt and SL8/sprd were not significantly different (P ⬎ 0.05); SL10/sprd and SL7/sprd were not significantly different from each other (P ⬎ 0.05) but both were significantly lower than all other lines (P ⬎ 0.05); SL5/sprd was significantly lower than all other lines (P 艋 0.0002). cSL10/sprd carcinoma response is given at 36 weeks and therefore does not represent maximal response. aMice
men. In comparison, the fully inbred SENCAR B/Pt mice developed an average of 6.8 ⫾ 0.8 papillomas/mouse and had a tumor incidence of 100% at 29 weeks, at the same doses of DMBA and TPA; ~86% developed carcinomas. Statistical analysis of the papilloma data supported the three categories of responders as follows: SL2/sprd ⫽ SL8/sprd ⫽ SENCAR B/Pt ⬎ SL10/sprd ⫽ SL7/sprd ⬎⬎ SL5/sprd. All differences in papilloma responses were significant at P ⬍ 0.05. Also included for comparison were concurrent groups of SSIN (0.25 µg TPA) and outbred SENCAR (1 µg TPA) mice. The SSIN mice, treated with the same dose of TPA, had a higher number of papillomas per mouse than all other lines tested, including SL2/sprd, SL8/sprd and SENCAR B/Pt (P ⬍ 0.05). Because the outbred SENCAR mice were tested at a different dose of TPA a direct comparison is problematical. However, based on historical data using SENCAR mice at lower doses of TPA and a published comparison of outbred SENCAR with SENCAR B/ Pt (29), SL2/sprd, SL8/sprd and SENCAR B/Pt are considerably more sensitive than outbred SENCAR. A subsequent tumor experiment was conducted in SL2/sprd and SL5/sprd mice at generation F15. In order to more firmly establish the relative sensitivity and/or resistance to promotion, three doses of TPA were selected for each line. Since no sex differences were observed in responsiveness to two-stage carcinogenesis, only female mice were used in subsequent studies. For SL2/sprd, which had responded well to the 0.25 µg TPA dose in the first experiment, doses of 0.4, 0.2 and 0.1 µg TPA were used. For SL5/sprd, which had not demonstrated a tumor response to the 0.25 µg dose, doses of 1.0 (a dose commonly used in the outbred SENCAR stock), 0.4 and 0.2 µg were selected. Results of this experiment at 25 weeks are shown in Table II. At the 0.4 µg TPA dose, the SL2/sprd mice began developing tumors at 6 weeks (data not shown) and exhibited a peak papilloma response of 11 papillomas/mouse after 25 weeks of promotion (Table II). Furthermore, 100% of the mice had tumors by 11 weeks (data not shown). In contrast, the SL5/sprd mice receiving 0.4 µg had a maximum papilloma response of only 0.9 papillomas/mouse at 25 weeks of promotion (Table II). When SL5/sprd mice were treated with 1.0 µg TPA, they began developing tumors as early as 4 weeks, reaching a peak count of 7.5 papillomas/mouse at 25 weeks. At this dose, 100% of the mice carried tumors by 14 weeks. Note that SL5/sprd F15 mice were completely non-responsive
Table II. Comparison of the responses of SL2/sprd and SL5/sprd to different TPA dosesa Line
TPA dose (µg)
Papillomas per mouse
Mice with papillomas (%)
SL2/sprd
0.4b 0.4 0.2 0.1 0.4b 1.0 0.4 0.2
0 11.0 3.9 2.2 0 7.5 0.9 0
0 100 77 44 0 100 29 0
SL5/sprd
a25–30
female mice (F15) were used for each experimental group. Mice were initiated with 10 nmol DMBA followed 2 weeks later by twice weekly applications of TPA. Data are given at 25 weeks of promotion. bMice in this group received acetone (0.2 ml) instead of DMBA at the time of initiation.
to the 0.2 µg dose (as in the first experiment where 0.25 µg TPA was used in F12 mice). Dose–response studies with TPA As can be seen by the results shown in Figure 1 and Table III, the extent of epidermal hyperplasia induced by TPA was remarkably different between the SL2/sprd and SL5/sprd lines. After only 4 weeks of TPA treatment, epidermal thickness and labeling index were dramatically increased over strain-matched controls (acetone treatment only) in SL2/sprd even at the lowest TPA dose (0.1 µg), whereas 10 times as much TPA was required to approximate these measurements in the SL5/ sprd strain. The values for epidermal thickness in SL2/sprd mice increased to maximum levels at the 0.2 µg dose while at this dose the SL5/sprd mice had only a very modest increase, compared with the corresponding SL5/sprd control mice. A significant increase in both parameters was observed in SL5/ sprd mice at the 1.0 µg TPA dose, which induced responses in SL5/sprd quantitatively similar to those induced with 0.2– 0.4 µg/mouse in SL2/sprd. These data correlate well with the tumor data shown in Tables I and II and indicate that SL5/ sprd is much less sensitive than SL2/sprd to TPA, requiring between 4 and 20 times as much TPA (depending on the endpoint used for comparison) to produce a similar response. 643
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Fig. 1. Comparison of TPA-induced hyperplasia and proliferation in SL2/sprd and SL5/sprd mice. The dorsal skins of non-initiated, generation F17 mice were treated twice a week with 0.4 µg TPA for 4 weeks and then the mice received i.p. injections of BrdU 30 min prior to death. H&E stained section of dorsal skin from (A) an SL2/sprd mouse and (B) an SL5/sprd mouse. BrdU stained section of dorsal skin from (C) an SL2/sprd mouse and (D) an SL5/sprd mouse.
Table III. Comparison of TPA-induced hyperplasia in SL2/sprd and SL5/ sprd micea Line
TPA dose (µg)
Epidermal thickness (µm)
SL2/sprd
1.0 0.4 0.2 0.1 Acetone 1.0 0.4 0.2 Acetone
42.6 44.9 44.2 31.0 15.5 42.5 23.8 16.7 15.1
SL5/sprd
aFour
⫾ ⫾ ⫾ ⫾
8.5 7.8 9.0 9.8
⫾ 4.0 ⫾ 2.7 ⫾ 2.2
Labeling index (%) 15.5 26.9 21.9 19.1 1.8 20.0 5.4 7.0 2.5
⫾ ⫾ ⫾ ⫾
1.4 3.0 2.3 5.9
⫾ 8.4 ⫾ 2.0 ⫾ 2.9
or five female mice were used for each group.
Discussion In order to better address the questions pertinent to the cellular and molecular events and genotypic features associated with the development of both benign and malignant lesions in the two-stage model of skin carcinogenesis, a spectrum of genotypically similar inbred mice expressing distinct patterns of disease susceptibility and resistance is highly desirable. Several investigators have developed inbred mice for these protocols using SENCAR stocks either with or without further selection (27,29). As noted in the Introduction, each of these inbred lines, while useful, have certain limitations. Bangrazi and colleagues reported the development of lines both sensitive and resistant to two-stage carcinogenesis through a complex process of crossing and bi-directional selection using eight widely diverged inbred strains (36). However, it is unclear 644
whether these mice were carried to inbred status and whether carcinomas developed in either line. Cope et al. (37) reported the establishment of inbred lines both sensitive and resistant to two-stage carcinogenesis. These lines were reportedly derived through selective breeding of CD-1 mice. However, these lines appear to have been lost. Thus, the availability of additional inbred SENCAR lines will be valuable tools for studies of skin carcinogenesis. In the present study, we have generated five new lines of SENCAR mice and performed an initial characterization of their responsiveness to two-stage carcinogenesis using DMBA as the initiator and TPA as the promoter. In the first experiment, lines SL2/sprd, SL5/sprd, SL7/sprd, SL8/sprd and SL10/sprd were compared at a single dose of DMBA and TPA. The order of sensitivity of these five lines to this treatment protocol was SL2/sprd ⫽ SL8/sprd ⬎ SL10/sprd ⫽ SL7/sprd ⬎⬎ SL5/ sprd, when using the maximum papillomas per mouse as the end-point. Further comparison showed that SL2/sprd and SL8/ sprd were similar in responsiveness to SENCAR B/Pt. In addition, these lines were only slightly less sensitive than SSIN in terms of papilloma response while all four of the highly responsive inbred lines (SSIN, SENCAR B/Pt, SL2/ sprd and SL8/sprd) were considerably more sensitive than outbred SENCAR mice. A particularly exciting observation was the finding that SL5/sprd was relatively resistant to twostage carcinogenesis in our initial experiment. A more detailed comparison of the responsiveness of SL5/sprd and SL2/sprd to two-stage carcinogenesis using different TPA doses indicated that 4–20 times more TPA was required to produce a similar tumor response (Table II). The differences in susceptibility to two-stage carcinogenesis between SL5/sprd and SL2/sprd correlated very well with differences in sensitivity to TPA-
Inbred SENCAR mice
induced epidermal hyperplasia and proliferation (Table III). These data indicate that SL5/sprd mice differ from SL2/sprd mice at one or more loci that control responsiveness to TPA. Further study of genetic crosses between these two inbred lines could ultimately yield the identification of TPA promotion susceptibility genes. The current data also indicate that SL2/sprd is highly sensitive to TPA promotion and, like outbred SENCAR and SENCAR B/Pt, this new inbred SENCAR line showed a strong propensity to develop carcinomas. Papillomas also progressed to squamous cell carcinomas in the other three lines (SL7/ sprd, SL8/sprd and SL10/sprd), although at a somewhat lower rate. While these data should be considered tentative, they raise the interesting possibility that at least lines SL7/sprd and SL8/sprd might represent lines intermediate between SSIN and SL2/sprd (and SENCAR B/Pt) in terms of papilloma progression. Further more detailed analysis of malignant conversion at different doses of DMBA and TPA will be required to substantiate this preliminary observation. In conclusion, five new inbred SENCAR lines have been developed that will be further characterized in future studies. The initial characterization suggests a spectrum of papilloma and carcinoma responses obtained from these five new inbred SENCAR strains that will provide investigators with new tools for studying the genetic basis of papilloma and squamous cell carcinoma development. In particular, the availability of a fertile inbred SENCAR mouse with high TPA sensitivity and whose papillomas progress to squamous cell carcinomas with a high propensity will be particularly valuable. In addition, the need for a SENCAR-derived inbred strain that is resistant to TPA promotion has been a long recognized void. SL5/sprd appears to represent a relatively resistant (compared with the other four inbred SENCAR strains developed in this study) inbred strain that may fill this void. Future studies will focus on a more detailed analysis of papilloma progression rates in the five inbred strains and a more detailed analysis of the cellular, biochemical and molecular differences between SL5/ sprd and the other inbred strains. Acknowledgements We gratefully acknowledge Dr Michael Potter for providing breeding pairs of his inbred lines of mice and Dale Weiss, Pam Kille, Donna Schutz and Carla Webb for technical assistance with maintenance of breeding colonies and mouse experiments. We would also like to acknowledge Jimi Lynn Rosborough for her assistance with general histology and epidermal hyperplasia assays and Cynthia Aranda and Melissa Bracher for manuscript preparation. This work was supported by National Institutes of Health Grant CA57596, ES08355, M.D. Anderson Cancer Center Core Grant P30-CA16672 and the Center for Research for Environmental Disease Grant P30-ES07784.
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