Skip to main content
NCBI home page
Japanese Journal of Cancer Research : Gann logoLink to Japanese Journal of Cancer Research : Gann
. 1996 Jun;87(6):575–582. doi: 10.1111/j.1349-7006.1996.tb00262.x

Early Alterations of Apoptosis and Cell Proliferation in Azoxymethane‐initiated Rat Colonic Epithelium

Yoshinobu Hirose 1,✉, Naoki Yoshimi 1, Hiroki Makita 1, Akira Kara 1, Takuji Tanaka 1, Hideki Mori 1
PMCID: PMC5921139  PMID: 8766520

Abstract

Alterations of apoptosis and cell proliferation in the colonic epithelium of rats after exposure to azoxymethane (AOM) were estimated by means of the terminal deoxynucleotidyl transferasemediated dUTP‐biotin nick end labeling (TUNEL) method, measurement of 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation, immunohistochemical staining for proliferating cell nuclear antigen (PCNA), and counting of raitotic cells. F344 male rats were given a single s.c. injection of AOM (15 mg/kg body weight) at 6 week of age, and killed 4 h, 8 h, 3 days, and 7 days after the AOM treatment. At 4 h after the treatment, many damaged cells were already observed in the colonic epithelium, and they were positive by TUNEL staining. At 8 h, the number of TUNEL‐positive cells was largest. The reduction of DNA synthesis in the colonic epithelium, confirmed by BrdU incorporation, was not distinct in comparison with the mitotic inhibition. There was no remarkable change in PCNA labeling index, except that strong expression of PCNA was detected in many damaged cells. On the 3rd day, the appearance of cell death became infrequent and an increase of cell proliferation occurred. On the 7th day, the expression of TUNEL and the cell proliferation biomarkers were at almost normal levels. These findings suggest that AOM induces apoptosis, which is associated with synchronous inhibition of mitosis. The data also indicate that PCNA immunostaining does not reflect the true proliferation state in the early phase after AOM exposure, probably due to the occurrence of cell cycle arrest or DNA repair.

Keywords: Apoptosis, Cell proliferation, Azoxymethane, Colon, Rat

Full Text

The Full Text of this article is available as a PDF (570.9 KB).

REFERENCES

  • 1. ) Cotter , T. G. , Glynn , J. M. , Echeverri , F. and Green , D. R.The induction of apoptosis by chemotherapeutic agents occurs in all phases of the cell cycle . Anticancer Res. , 12 , 773 – 779 ( 1992. ). [PubMed] [Google Scholar]
  • 2. ) Kerr , J. F. R. , Winterford , C. M. and Harmon , B. V.Apoptosis: its significance in cancer and cancer therapy . Cancer , 73 , 2013 – 2026 ( 1994. ). [DOI] [PubMed] [Google Scholar]
  • 3. ) Bedi , A. , Pasricha , P. J. , Akhtar , A. J. , Barber , J. P. , Bedi , G. C. , Giardiello , F. M. , Zehnbauer , B. A. , Hamilton , S. R. and Jones , R. J.Inhibition of apoptosis during development of colorectal cancer . Cancer Res. , 55 , 1811 – 1816 ( 1995. ). [PubMed] [Google Scholar]
  • 4. ) Zedeck , M. S. , Grab , D. J. and Sternberg , S. S.Differences in the acute response of the various segments of rat intestine to treatment with the intestinal carcinogen, methylazoxymethanol acetate . Cancer Res. , 37 , 32 – 36 ( 1977. ). [PubMed] [Google Scholar]
  • 5. ) Sunter , J. P. , Appleton , D. R. and Watson , A. J.Acute changes occurring in the intestinal mucosae of rats given a single injection of 1,2‐dimethylhydrazine . Virchows Arch. (Cell Pathol.) , 36 , 47 – 57 ( 1981. ). [DOI] [PubMed] [Google Scholar]
  • 6. ) Wargovich , M. J. , Medline , A. and Bruce , W. R.Early histopathologic events to evolution of colon cancer in C57 BL/6 and CF1 mice treated with 1,2‐dimethylhydrazine . J. Natl. Cancer Inst. , 71 , 125 – 131 ( 1983. ). [PubMed] [Google Scholar]
  • 7. ) Ijiri , K.Apoptosis (cell death) induced in mouse bowel by 1,2‐dimethylhydrazine, methylazoxymethanol acetate, and 7‐rays . Cancer Res. , 49 , 6342 – 6346 ( 1989. ). [PubMed] [Google Scholar]
  • 8. ) Potten , C. S. , Li , Y. Q. , O'Connor , P. J. and Winton , D. J.A possible explanation for the differential cancer incidence in the intestine, based on distribution of the cytotoxic effects of carcinogens in the murine large bowel . Carcinogenesis , 13 , 2305 – 2312 ( 1992. ). [DOI] [PubMed] [Google Scholar]
  • 9. ) Thurnherr , N. , Deschner , E. E. , Stonehill , E. H. and Lipkin , M.Induction of adenocarcinomas of the colon in mice by weekly injections of 1,2‐dimethylhydrazine . Cancer Res. , 33 , 940 – 945 ( 1973. ). [PubMed] [Google Scholar]
  • 10. ) Ward , J. M. , Yamamoto , R. S. and Brown , C. A.Pathology of intestinal neoplasms and other lesions in rats exposed to azoxymethane . J. Natl. Cancer Inst. , 51 , 1029 – 1039 ( 1973. ). [DOI] [PubMed] [Google Scholar]
  • 11. ) Zedeck , M. S. and Sternberg , S. S.A model system for studies of colon carcinogenesis: tumor induction by a single injection of methylazoxymethanol acetate . J. Natl. Cancer Inst. , 53 , 1419 – 1421 ( 1974. ). [DOI] [PubMed] [Google Scholar]
  • 12. ) Zedeck , M. S.Hydrazine derivatives, azo and azoxy compounds, and methylazoxymethanol and cycasin . In “ Chemical Carcinogens ,” ed. Searle C. E. , pp. 915 – 944 ( 1984. ). American Chemical Society; , Washington , D. C. . [Google Scholar]
  • 13. ) Saffhill , R. , Margison , G. P. and O'Connor , P. J.Mechanisms of carcinogenesis induced by alkylating agents . Biochim. Biophys. Acta , 823 , 111 – 145 ( 1985. ). [DOI] [PubMed] [Google Scholar]
  • 14. ) Rogers , K. J. and Pegg , A. E.Formation of O6‐methylguanine by alkylation of rat liver, colon, and kidney DNA following administration of 1,2‐dimethylhydrazine . Cancer Res. , 37 , 4082 – 4087 ( 1977. ). [PubMed] [Google Scholar]
  • 15. ) Swenberg , J. A. , Cooper , H. K. , Bucheler , J. and Kleihues , P.1,2‐Dimethylhydrazine‐induced methylation of DNA bases in various rat organs and the effect of pretreatment with disulfiram . Cancer Res. , 39 , 465 – 467 ( 1979. ). [PubMed] [Google Scholar]
  • 16. ) Lane , D. P.p53, Guardian of the genome . Nature , 358 , 15 – 16 ( 1992. ). [DOI] [PubMed] [Google Scholar]
  • 17. ) Sancar , A.Mechanisms of DNA excision repair . Science , 266 , 1954 – 1956 ( 1994. ). [DOI] [PubMed] [Google Scholar]
  • 18. ) Barnes , D. M. and Gillett , C. E.Determination of cell proliferation . J. Clin. Pathol. , 48 , M2 – M5 ( 1995. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19. ) Satoh , K. , Hatayama , I. , Tateoka , N. , Tamai , K. , Shimizu , T. , Tatematsu , M. , Ito , N. and Sato , K.Transient induction of single GST‐P positive hepatocytes by DEN . Carcinogenesis , 10 , 2107 – 2111 ( 1989. ). [DOI] [PubMed] [Google Scholar]
  • 20. ) Moolgavkar , S. H. , Luebeck , E. G. , de Gunst , M. , Port , R. E. and Schwarz , M.Quantitative analysis of enzymealtered foci in rat hepatocarcinogenesis experiments‐I. Single agent regimen . Carcinogenesis , 11 , 1271 – 1278 ( 1990. ). [DOI] [PubMed] [Google Scholar]
  • 21. ) Kastan , M. B. , Onyekwere , O. , Sidransky , D. , Vogelstein , B. and Craig , R. W.Participation of p53 protein in the cellular response to DNA damage . Cancer Res. , 51 , 6304 – 6311 ( 1991. ). [PubMed] [Google Scholar]
  • 22. ) Nichols , A. F. and Sancar , A.Purification of PCNA as a nucleotide excision repair protein . Nucleic Acids Res. , 20 , 2441 – 2446 ( 1992. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. ) Dietrich , D. R.Toxicological and pathological applications of proliferating cell nuclear antigen (PCNA), a novel endogenous marker for cell proliferation . Critical Rev. Toxicol. , 23 , 77 – 109 ( 1993. ). [DOI] [PubMed] [Google Scholar]
  • 24. ) Hall , P. A. , McKee , P. H. , Menage , H. D. , Dover , R. and Lane , D. P.High levels of p53 protein in UV‐irradiated normal human skin . Oncogens , 8 , 203 – 207 ( 1993. ). [PubMed] [Google Scholar]
  • 25. ) Gavrieli , Y. , Sherman , Y. and Ben‐Sasson , S. A.Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation . J. Cell Biol. , 119 , 493 – 501 ( 1992. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. ) Merritt , A. J. , Potten , C. S. , Kemp , C. J. , Hickman , J. A. , Balmain , A. , Lane , D. P. and Hall , P. A.The role of p53 in spontaneous and radiation‐induced apoptosis in the gastrointestinal tract of normal and p53‐deficient mice . Cancer Res. , 54 , 614 – 617 ( 1994. ). [PubMed] [Google Scholar]
  • 27. ) Tanaka , T. , Kojima , T. , Suzui , M. and Mori , H.Chemoprevention of colon carcinogenesis by the natural product of a simple phenolic compound protocatechuic acid: suppressing effects on tumor development and biomarkers expression of colon tumorigenesis . Cancer Res. , 53 , 3908 – 3913 ( 1993. ). [PubMed] [Google Scholar]
  • 28. ) Hirose , Y. , Tanaka , T. , Kawamori , T. , Ohnishi , M. , Makita , H. , Mori , H. , Satoh , K. and Kara , A.Chemoprevention of urinary bladder carcinogenesis by the natural phenolic compound protocatechuic acid in rats . Carcinogenesis , 16 , 2337 – 2342 ( 1995. ). [DOI] [PubMed] [Google Scholar]
  • 29. ) Kerr , J. F. R. and Harmon , B. V.Definition and incidence of apoptosis: an historical perspective . In “ Apoptosis: the Molecular Basis of Cell Death ,” ed. Tomei L. D. and Cope F. O. , pp. 5 – 29 ( 1991. ). Cold Spring Harbor Laboratory Press; , New York . [Google Scholar]
  • 30. ) Majno , G. and Joris , I.Apoptosis, oncosis, and necrosis: an overview of cell death . Am. J. Pathol. , 146 , 3 – 15 ( 1995. ). [PMC free article] [PubMed] [Google Scholar]
  • 31. ) Ryan , J. J. , Danish , R. , Gottlieb , C. A. and Clarke , M. F.Cell cycle analysis of p53‐induced cell death in murine erythroleukemia cells . Mol. Cell. Biol. , 13 , 711 – 719 ( 1993. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. ) Allday , M. J. , Inman , G. J. , Crawford , D. H. and Farrell , P. J.DNA damage in human B cells can induce apoptosis, proceeding from G1/S when p53 is transactivation competent and G2/M when it is transactivation defective . EMBO J. , 14 , 4994 – 5005 ( 1995. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33. ) Sunter , J. P. , Watson , A. J. , Wright , N. A. and Appleton , D. R.Cell proliferation at different sites along the length of the rat colon . Virchows Arch. B Cell Pathol. , 32 , 75 – 87 ( 1979. ). [DOI] [PubMed] [Google Scholar]
  • 34. ) Ward , J. M.Dose response to a single injection of azoxymethane in rats . Vet. Pathol. , 12 , 165 – 177 ( 1975. ). [DOI] [PubMed] [Google Scholar]
  • 35. ) Morris , G. F. and Mathews , M. B.Regulation of proliferating cell nuclear antigen during the cell cycle . J. Biol. Chem. , 264 , 13856 – 13864 ( 1989. ). [PubMed] [Google Scholar]
  • 36. ) Burford‐Mason , A. P. , MacKay , A. J. , Cummins , M. and Dardick , I.Detection of proliferating cell nuclear antigen in paraffin‐embedded specimens is dependent on preembedding tissue handling and fixation . Arch. Pathol. Lab. Med. , 118 , 1007 – 1013 ( 1994. ). [PubMed] [Google Scholar]
  • 37. ) Shivji , M. K. K. , Kenny , M. K. and Wood , R. D.Proliferating cell nuclear antigen is required for DNA excision repair . Cell , 69 , 367 – 374 ( 1992. ). [DOI] [PubMed] [Google Scholar]
  • 38. ) Smith , M. L. , Chen , I.‐T. , Zhan , Q. , Bae , I. , Chen , C.‐Y. , Gilmer , T. M. , Kastan , M. B. , O'Connor , P. M. and Fornace , Jr. , A. J.Interaction of the p53‐regulated protein Gadd45 with proliferating cell nuclear antigen . Science , 266 , 1376 – 1380 ( 1994. ). [DOI] [PubMed] [Google Scholar]
  • 39. ) Korsmeyer , S. J.Bc1‐2 initiates a new category of oncogenes: regulators of cell death . Blood , 80 , 879 – 886 ( 1992. ). [PubMed] [Google Scholar]

Articles from Japanese Journal of Cancer Research : Gann are provided here courtesy of Wiley

RESOURCES