Resistance of Copenhagen rats to hepatocarcinogenesis does not involve T-cell immunity

Geoffrey A. Wood, James E. Korkola, and Michael C. Archer,,

1 Department of Medical Biophysics and
2 Department of Nutritional Sciences, University of Toronto, Toronto, Canada M5S 3E2


    Abstract
 Top
 Abstract
 Introduction
 References
 
Previously, we have shown that Copenhagen (Cop) rats are highly resistant, compared with susceptible F344 rats, to the growth of glutathione S-transferase 7-7 (GST 7-7) positive preneoplastic liver lesions following treatment with a modified resistant hepatocyte (RH) protocol. Donryu rats, a strain with a level of susceptibility similar to F344, have a reduced T-cell response compared with the closely related, but highly resistant, DRH rat. Cop and DRH rats share several characteristics in their resistance to preneoplastic liver lesion growth and this study, therefore, was designed to examine whether T-cells play a role in Cop resistance. Cop rats were crossed with an athymic (nude) rat to produce F1s that were then interbred to produce F2 animals, some of which were nude with a partial Cop background. A comparison of the susceptibility of nude F2 animals and their euthymic (non-nude) littermates allowed us to determine what role, if any, T-cells play in Cop resistance. We treated 11 Cop, 11 F344, 19 nude F2s, and 18 non-nude F2s with diethylnitrosamine (DEN), followed 3 weeks later by a modified RH protocol. As expected, F344 rats were highly susceptible, having 41.9 ± 3.3% (mean ± SEM) of their liver section areas occupied by GST 7-7-positive lesions and Cop rats were highly resistant, having only 4.7 ± 1.1% of their liver section areas occupied by lesions. Both nude and non-nude F2s were, like Cop rats, highly resistant (1.8 ± 0.29 and 2.7 ± 0.45%, respectively). These results show that T-cells are unnecessary for Cop rat resistance, or only play a minor role, and that the nude parental strain is also likely to be resistant to the growth of preneoplastic liver lesions.

Abbreviations: Cop, Copenhagen; DEN, diethylnitrosamine; RH, resistant hepatocyte; GST 7-7, glutathione S-transferase 7-7.


    Introduction
 Top
 Abstract
 Introduction
 References
 
Previously, we have shown that Copenhagen (Cop) rats are highly resistant to the formation of preneoplastic liver lesions initiated by diethylnitrosamine (DEN) and promoted using the modified resistant hepatocyte (RH) protocol (1,2). Compared with susceptible F344 rats, Cop rats had ~10-fold less of their liver volumes occupied by glutathione S-transferase 7-7 (GST 7-7)-positive lesions 3 weeks following treatment with the RH protocol. DRH rats are also highly resistant to the growth of preneoplastic liver lesions (35). This strain was developed from the susceptible Donryu strain and a recent study has shown that Donryu rats have a reduced T-cell response compared with DRH rats (6). The authors proposed that this immunological impairment might be one of the factors that account for the difference in susceptibility of the two strains. Cop and DRH rats share several characteristics in their resistance to preneoplastic liver lesion growth. Both strains are resistant to promotion but not to initiation (2,3), neither strain has a defect in the metabolism of the carcinogens used (1,7) and the rats are physiologically normal. We decided, therefore, to examine whether T-cells play a role in Cop resistance. We crossed Cop rats with an athymic nude rat to produce F1s that were then interbred to produce F2 animals, some of which were athymic with a partial Cop background. A comparison of the growth of preneoplastic liver lesions following the RH protocol between athymic (nude) F2 animals and their euthymic (non-nude) littermates allowed us to determine what role, if any, T-cells play in Cop resistance.

Female Cop rats (all rats from Harlan Sprague–Dawley, Madison, WI) were crossed to a nude male rat to yield F1s. Interbreeding of the F1s yielded F2s that were either athymic (nude) or euthymic (non-nude). To prove that the nude F2 rats were indeed athymic, we examined their peripheral blood by flow cytometry as described previously (8). White blood cells from F2 rats were isolated from blood collected from the retro-orbital sinus and stained using a FITC-labeled anti-CD5 pan T-cell specific antibody (Cedarlane, Ontario, Canada). Gated cells (104/sample) were then counted on a FACscan flow cytometer (Becton Dickinson, CA). We found that F2 nude rats were athymic having no T-cells, whereas non-nude F2s had normal levels of T-cells (data not shown, see ref. 8 for representative results).

We treated 7–8 week-old male rats (11 Cop, 11 F344, 19 nude F2s and 18 non-nude F2s) with an i.p. injection of 200 mg/kg DEN (Eastman Kodak, Rochester, NY), followed 3 weeks later by a modified RH protocol as previously described (1,9). Briefly, this consisted of 3 daily gavages of 20 mg/kg 2-acetylaminofluorene (Sigma, St Louis, MO), followed the next day by 2 ml/kg CCl4 by gavage. All rats were killed 3 weeks later and sections of their livers were immunostained for GST 7-7-positive lesions as previously described (1).

As expected, F344 rats were highly susceptible to promotion using the RH protocol having 41.9 ± 3.3% of their liver section areas (mean ± SEM) occupied by GST 7-7-positive lesions and Cop rats were highly resistant, having only 4.7 ± 1.1% of their liver section areas occupied by lesions. Both nude and non-nude F2s were highly resistant (Figure 1). Neither of the F2 groups were significantly different from each other, or from the Cop rats, but they were significantly different from F344 rats (P < 0.001, one-way ANOVA with a Tukey post-test). Also, no grossly visible lesions were present in any of the Cop or F2 rats, but all the F344 rats had grossly visible lesions virtually covering the surfaces of their livers. The numbers of lesions per mm2 of liver section area were not different between Cop, nude F2s and non-nude F2s (Cop, 0.60 ± 0.04; nude, 0.85 ± 0.08; non-nude, 0.75 ± 0.09; mean ± SEM), indicating that factors involved in initiation were similar in these groups. Since F344 rat lesions were coalescing, their number could not be measured.

From the results of this study it is clear that compared with F344 rats, Cop, F2 nude and F2 non-nude rats are all highly resistant to the growth of GST 7-7-positive liver lesions initiated by DEN and promoted using the RH protocol. Previously, we have shown that Cop rats carry a minimum of four incompletely dominant resistance genes (G.Wood and M.Archer, submitted for publication). Some of these genes would be inherited by the F2 rats. If any of the resistance genes require T-cells to produce a resistant phenotype, then nude F2 rats would be more susceptible as a group than non-nude F2s. If T-cells are not necessary or play only a minor role in resistance, then the susceptibility of nude F2s would not be different from non-nude F2s. Since the lack of T-cells clearly had no effect on resistance, we may conclude that T-cells are unnecessary for Cop rat resistance or only play a minor role.

The susceptibility of the nude parental strain to hepatocarcinogenesis is unknown. F344 rats have a susceptible phenotype and CopxF344 F1 rats are of intermediate susceptibility (G.Wood and M.Archer, submitted for publication). If nude rats are susceptible like F344s, then Copxnude F1 rats would likewise be expected to be of intermediate susceptibility. The F2 rats that result from interbreeding Copxnude-derived F1s would, therefore, be expected to have susceptibilities ranging from resistant to susceptible. Since the F2 rats do not exhibit a range of susceptibilities, but are instead all highly resistant, they must have inherited at least one resistance gene from the nude parental rat. These data suggest that the nude parental strain is, like the Cop rat, resistant to preneoplastic liver lesion growth. Cop rats have long been known to be highly resistant to mammary tumorigenesis (10). Previously, we have shown through similar breeding experiments that nude rats are also resistant to mammary tumorigenesis (8). Taken together, these results make the interpretation of experiments that use nude rats as models of tumor development or metastasis potentially difficult. For example, although nude rats develop tumors and metastases after the injection of cancer cells (11,12), if the mechanism of resistance of nude rats involves stromal factors, this could influence the growth of lesions and their response to experimental manipulation. Since susceptible Donryu rats were found to have a reduced T-cell response compared with resistant DRH rats (6), our results suggest that the resistance of DRH and Cop rats is caused by different mechanisms. This may not be the case, however, since Mise-Omata et al. (6) found that Donryu rats had a lower T-cell response than DRH rats in some, but not all, of their assays. Indeed, some individual Donryu rats had higher T-cell responses than DRH rats. Furthermore, this study showed that the T-cell response of DRH rats is similar to Sprague–Dawley and Wistar rats. These two latter strains, however, are not resistant to liver lesion growth, but are of intermediate susceptibility (13). This would indicate that T-cell function does not correlate with the level of resistance. Thus, it is possible that T-cells do not play a significant role in DRH rat resistance, and Cop and DRH rats may, therefore, share a common resistance mechanism(s).

Previously, we have shown that Cop rat resistance to mammary tumorigenesis does not involve T-cells (8). In the current study, we show that T-cells are also not involved in the resistance of Cop rats to hepatocarcinogenesis. Copxnude F2 rats are highly resistant to the growth of GST 7-7-positive liver lesions initiated with DEN and promoted with the RH protocol. Furthermore, there was no difference in the level of resistance of nude and non-nude F2s. These results show that T-cells are unnecessary for Cop rat resistance, or only play a minor role, and that the nude parental strain is also likely to be resistant to the growth of preneoplastic liver lesions.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1. Percent of liver section areas occupied by GST 7-7-positive lesions following initiation with DEN and promotion using the RH protocol. *F344 rats were significantly different from the other groups (P < 0.01).

 

    Notes
 
3 Present address: University of California San Francisco Cancer Center, 2340 Sutter Street, San Francisco, CA 94143-0808, USA Back

4 To whom correspondence should be addressed at: Department of Nutritional Sciences, Faculty of Medicine, FitzGerald Building, 150 College Street, Toronto, Canada M5S 3E2 Back


    Acknowledgments
 
This investigation was supported by a grant from the Canadian Breast Cancer Research Initiative. M.C.A. is the recipient of a National Sciences and Engineering Research Council of Canada Industrial Research Chair, and acknowledges support from the member companies of the Program in Food Safety (University of Toronto).


    References
 Top
 Abstract
 Introduction
 References
 

  1. Wood,G.A., Korkola,J.E., Lee,V.M., Sarma,D.S.R. and Archer,M.C. (1997) Resistance of Copenhagen rats to chemical induction of glutathione S-transferase 7-7-positive liver foci. Carcinogenesis, 18, 1745–1750.[Abstract]
  2. Wood,G.A., Sarma,D.S.R. and Archer,M.C. (1999) Resistance to the promotion of glutathione S-transferase 7-7-positive liver lesions in Copenhagen rats. Carcinogenesis, 20, 1169–1175.[Abstract/Free Full Text]
  3. Denda,A., Kitayama,W., Konishi,Y. et al. (1999) Genetic properties for the suppression of development of putative preneoplastic glutathione S-transferase placental form-positive foci in the liver of carcinogen-resistant DRH strain rats. Cancer Lett., 140, 59–67.[ISI][Medline]
  4. Yan,Y., Higashi,K., Tanimoto,A., Fukamachi,Y., Itoh,H., Abe,T. and Higashi,T. (1998) Remarkable difference in the incidence of liver tumors by N-nitrosodimethylamine in carcinogen-resistant DRH rat and its parental strain Donryu rat. Sangyo Ika Daigaku Zasshi, 20, 307–314.[Medline]
  5. Yang,M., Yamamura,K., Yan,Y., Fukamachi,Y., Hirano,T., Kawamoto,T. and Higashi,K. (1997) Studies on the microsomal cytochrome P450s in the livers of carcinogen-resistant and sensitive rats during long-term administration of 3'-methyl-4-dimethylaminoazobenzene [published erratum appears in Sangyo Ika Daigaku Zasshi, 20, 79]. Sangyo Ika Daigaku Zasshi, 19, 277–286.[Medline]
  6. Mise-Omata,S., Sugiura,T., Higashi,K. and Yamashita,U. (1999) Reduced T cell response in carcinogen-sensitive Donryu rats compared with carcinogen-resistant DRH rats. Jpn J. Cancer Res., 90, 1277–1284.[ISI][Medline]
  7. Gotoh, S., Todaka,N., Yan,Y., Fukamachi,Y., Abe,T. and Higashi,K. (1999) Studies on the multidrug resistance gene expression in the livers of rats associated with different susceptibility of hepatocarcinogenesis. Sangyo Ika Daigaku Zasshi, 21, 265–276.[Medline]
  8. Korkola,J.E., Wood,G.A. and Archer,M.C. (1997) Resistance to chemically-induced mammary tumors in Copenhagenxnude-derived F2 athymic rats: evidence that T-cell immunity is not involved in Copenhagen resistance. Carcinogenesis, 18, 53–57.[Abstract]
  9. Semple-Roberts,E., Hayes,M.A., Armstrong,D., Becker,R.A., Racz,W.J. and Farber,E. (1987) Alternative methods of selecting rat hepatocellular nodules resistant to 2-acetylaminofluorene. Int. J. Cancer, 40, 643–645.[ISI][Medline]
  10. Isaacs,J.T. (1986) Genetic control of resistance to chemically induced mammary adenocarcinogenesis in the rat. Cancer Res., 46, 3958–3963.[Abstract]
  11. Gershwin,M.E., Ruebner,B.H. and Ikeda,R.M. (1982) Transplantation and metastasis of NB rat prostate neoplasia in congenitally athymic (nude) mice, nude mice treated with antilymphocyte sera and congenitally athymic rats. Exp. Cell Biol., 50, 145–154.[ISI][Medline]
  12. Vogel,I., Shen,Y., Soeth,E., Juhl,H., Kremer,B., Kalthoff,H. and Henne-Bruns,D. (1998) A human carcinoma model in athymic rats reflecting solid and disseminated colorectal metastases. Langenbecks Arch. Surg., 383, 466–473.[ISI][Medline]
  13. Blanck,A., Hansson,T., Eriksson,L.C. and Gustafsson,J.A. (1984) On mechanisms of sex differences in chemical carcinogenesis: effects of implantation of ectopic pituitary grafts on the early stages of liver carcinogenesis in the rat. Carcinogenesis, 5, 1257–1262.[Abstract]
Received October 13, 2000; revised November 21, 2000; accepted November 27, 2000.