Abstract
The number of silver‐stained nucleolar proteins (AgNOR) was counted in preneoplastic and neoplastic rat liver lesions induced by N‐2‐fluorenylacetamide (FAA) and was compared with that of bromodeoxyuridine (BrdU)‐incorporating cells detected immunohistochemically using monoclonal antibody against BrdU. Male ACI/N rats were given diet containing 200 ppm FAA for 12, 16 or 20 weeks to induce hepatocellular foci and tumors. The mean numbers of AgNOR stained by a one‐step silver colloid method and BrdU‐labeling indices in various liver cell lesions were as follows: nontreated liver (n = 20), 1.20 and 0.08; nonlesional areas (n=20), 1.33 and 0.13; altered liver cell foci (n = 80), 2.04 and 4.05 [eosinophilic cell type (n = 20), 1.78 and 1.82; clear cell type (n=20), 1.45 and 1.77; basophilic cell type (n=20), 1.99 and 4.58; hyperbasophilic cell type (n=20), 2.94 and 8.02]; neoplastic nodules (n = 10), 3.11 and 2.99; hepatocellular carcinomas (n = 10), 7.22 and 8.29. Thus, the mean number of AgNOR and the value of BrdU‐labeling index were well correlated and both values showed a stepwise increase from normal liver cells to liver cell carcinoma, although some scatter was present. These data suggest that mean number of AgNOR may reflect the cellular kinetics in rat hepatocarcinogenesis, and the one‐step silver colloid method for demonstration of AgNOR may therefore be a simple and useful staining to examine the proliferative nature of cells.
Keywords: Nucleolar organizer region, One‐step silver colloid staining, Bromodeoxyuridine, Immunohistochemistry, Rat hepatocarcinogenesis
Full Text
The Full Text of this article is available as a PDF (322.0 KB).
REFERENCES
- 1. ) Fakan , S. and Hernandez‐Verdun , D.The nucleolus and the nucleolar organizer regions (collective review) . Biol. Cell. , 56 , 189 – 206 ( 1986. ). [DOI] [PubMed] [Google Scholar]
- 2. ) Goessens , G.Nucleolar structure . Int. Rev. Cytol. , 87 , 107 – 158 ( 1984. ). [DOI] [PubMed] [Google Scholar]
- 3. ) Hernandez‐Verdun , D. , Derenzini , M. and Bouteille , M.The morphological relationship in electron microscopy between NOR‐silver proteins and intra‐nucleolar chroma‐tin . Chromosoma , 85 , 461 – 473 ( 1982. ). [DOI] [PubMed] [Google Scholar]
- 4. ) Hernandez‐Verdun , D.The nucleolar organizer regions (collective review) . Biol. Cell , 49 , 191 – 202 ( 1983. ). [PubMed] [Google Scholar]
- 5. ) Busch , H.Nucleolar proteins: purification, isolation and functional analysis . In“Chromosomal Non‐histone Proteins ,” ed. Hnilica L. S. , pp. 233 – 286 ( 1984. ). CRC Press; , Boca Raton , Florida ). [Google Scholar]
- 6. ) Dimova , R. N. , Markov , D. V. , Gajdardjieva , K. C. , Dabeva , M. D. and Hadjiolov , A. A.Electron microscopic localization of silver staining NOR‐proteins in rat liver nucleoli upon D‐galactosamine block of transcription . Eur. J. Cell Biol. , 28 , 272 – 277 ( 1982. ). [PubMed] [Google Scholar]
- 7. ) Walker , R. A.The histopathological evaluation of nucleolar organizer region proteins . Histopathology , 12 , 221 – 223 ( 1988. ). [DOI] [PubMed] [Google Scholar]
- 8. ) Howell , W. M. and Black , D. A.Controlled silver staining of nucleolus organizer regions with a protective colloidal developer: a one‐step method . Experientia , 36 , 1014 ( 1980. ). [DOI] [PubMed] [Google Scholar]
- 9. ) Ploton , D. , Menager , M. , Jeannesson , P. , Himber , G. , Pigeon , F. and Adnet , J. J.Improvement in the staining and in the visualization of the argyrophilic proteins of the nucleolar organizer region at the optical level . Histochem. J. , 18 , 5 – 14 ( 1986. ). [DOI] [PubMed] [Google Scholar]
- 10. ) Sugihara , H. , Hattori , T. and Fukuda , M.Immunohisto‐chemical detection of bromodeoxyuridine in formalin‐fixed tissue . Histochemistry , 85 , 193 – 195 ( 1986. ). [DOI] [PubMed] [Google Scholar]
- 11. ) Stewart , H. L. , Williams , G. M. , Keysser , C. H. , Lombard , L. S. and Montali , R. J.Histologic typing of liver tumors of the rats . J. Natl. Cancer Inst. , 64 , 177 – 207 ( 1980. ). 6986002 [Google Scholar]
- 12. ) Fujimoto , Y. , Oyamada , M. , Hattori , A. , Takahashi , H. , Sawaki , M. , Dempo , K. , Mori , M. and Nagao , M.Accumulation of abnormally high ploid nuclei in the liver of LEG rats developing spontaneous hepatitis . Jpn. J. Cancer Res. , 80 , 45 – 50 ( 1989. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. ) Tatematsu , M. , Fukushima , S. , Aoki , T. , Mera , Y. , Inoue , T. and Ito , N.Patterns of epithelial proliferation revealed by continuous administration of bromodeoxyuridine during urinary bladder carcinogenesis in rats . Jpn. J. Cancer Res. , 78 , 879 – 882 ( 1987. ). [PubMed] [Google Scholar]
- 14. ) Hall , P. A. , Crocker , J. , Watts , A. and Stansfeld , A. G.A comparison of nucleolar organizer region staining and Ki‐67 immunostaining in non‐Hodgkin's lymphoma . Histopathology , 12 , 373 – 381 ( 1988. ). [DOI] [PubMed] [Google Scholar]
- 15. ) Crocker , J. , Macartney , J. C. and Smith , P. J.Correlation between DNA flow cytometry and nucleolar organizer region data in non‐Hodgkin's lymphomas . J. Pathol. , 154 , 151 – 156 ( 1988. ). [DOI] [PubMed] [Google Scholar]
- 16. ) Editorial. NORs—a new method for the pathologist . Lancet , i , 1413 – 1414 ( 1987. ). [PubMed] [Google Scholar]
- 17. ) Underwood , J. C. E. and Giri , D. D.Editorial: Nucleolar organizer regions as diagnostic discriminants for malignancy . J. Pathol. , 155 , 95 – 96 ( 1988. ). [DOI] [PubMed] [Google Scholar]
- 18. ) Crocker , J. and McGovern , J.Nucleolar organiser regions in normal, cirrhotic, and carcinomatous livers . J. Clin. Pathol. , 41 , 1044 – 1048 ( 1988. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19. ) Busch , H. , Daskal , Y. , Gyorkey , F. and Smetana , K.Silver staining of nucleolar granules in tumor cells . Cancer Res. , 39 , 857 – 863 ( 1979. ). [PubMed] [Google Scholar]
- 20. ) Murano , S. , Horita , Y. , Maeda , S. , Takahashi , R. , Kano , Y. and Sugiyama , T.Amplification and abnormal chromosomal distribution of ribosomal genes (rDNA) in rat erythroleukemia cells . Cancer Genet. Cytogenet. , 6 , 303 – 312 ( 1982. ). [DOI] [PubMed] [Google Scholar]
- 21. ) Miller , O. J. , Tantravahi , R. , Miller , D. A. , Yu , L‐C. , Szabo , P. and Prensky , W.Marked increase in ribosomal RNA gene multiplicity in a rat hepatoma cell line . Chromosoma , 71 , 183 – 195 ( 1979. ). [DOI] [PubMed] [Google Scholar]
- 22. ) Mori , H. , Tanaka , T. , Sugie , S. , Takahashi , M. and Williams , G. M.DNA content of liver cell nuclei of N‐2‐fluorenylacetamide‐induced altered foci and neoplasms in rats and human hyperplastic foci . J. Natl Cancer Inst. , 69 , 1277 – 1282 ( 1982. ). [PubMed] [Google Scholar]
- 23. ) Bannasch , P.Cytology and cytogenesis of neoplastic (hyperplastic) hepatic nodules . Cancer Res. , 36 , 2555 – 2562 ( 1976. ). [PubMed] [Google Scholar]
- 24. ) Williams , G. M.Functional markers and growth behavior of preneoplastic hepatocytes . Cencer Res. , 36 , 2540 – 2543 ( 1976. ). [PubMed] [Google Scholar]
- 25. ) Bannasch , P. , Moore , M. A. , Klimek , F , and Zerban , H.Biological markers of preneoplastic foci and neoplastic nodules in rodent liver . Toxicol. Pathol. , 10 , 19 – 34 ( 1982. ). [DOI] [PubMed] [Google Scholar]
- 26. ) Buys , C. H. C. M. and Osinga , J.Selective staining of the same set of nucleolar phosphoproteins by silver and Giemsa. A combined biochemical cytochemical study on staining of NORs . Chromosoma , 89 , 387 – 396 ( 1984. ). [DOI] [PubMed] [Google Scholar]
- 27. ) Lischwe , M. A. , Smetana , K. , Olson , M. O. J. and Busch , H.Proteins C23 and B23 are the major nucleolar silver staining proteins . Life Sci. , 25 , 701 – 708 ( 1979. ). [DOI] [PubMed] [Google Scholar]