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Journal of Histochemistry and Cytochemistry, Vol. 50, 1633-1640, December 2002, Copyright © 2002, The Histochemical Society, Inc.

ARTICLE

Evaluation of Thymidylate Synthase Protein Expression by Western Blotting and Immunohistochemistry on Human Colon Carcinoma Xenografts in Nude Mice

Massimo Derenzinia, Lorenzo Montanaroa, Alessandra Chillàa, Elena Tostia, Claudio Ceccarellib, Fabio Dall'Olio, Dietmar Öfnerc, and Davide Treréa
a Department of Experimental Pathology, University of Bologna, Bologna, Italy
b Department of Oncology and Hematology, S. Orsola Hospital, University of Bologna, Bologna, Italy
c Department of General Surgery, University of Innsbruck, Innsbruck, Austria

Correspondence to: Massimo Derenzini, Alma Mater Studiorum, Università di Bologna, Dipartimento di Patologia Sperimentale, Via San Giacomo 14, 40126 Bologna, Italy. E-mail: derenzin@med.unibo.it

In this study we investigated the relationship between thymidylate synthase (TS) protein expression, evaluated by Western blotting analysis and by immunohistochemistry (IHC), and growth rate in human colon xenograft tumors in nude mice. Human colon cancer cell lines were used to induce xenograft tumors and the tumor mass growth rate was calculated by measuring tumor size variations over time. TS 106 monoclonal antibody was used for both Western blotting and IHC TS detection. Tumor cell growth fraction was measured by Ki67/MIB1 immunolabeling and tumor cell growth rate by evaluating the mean nucleolar size in silver-stained sections. TS Western blotting values were related to tumor mass growth rate (p<0.001) and cell growth rate (p=0.002) but not to cell growth fraction (p=0.676). The degree of the IHC staining showed only a trend to be associated with TS protein expression measured on Western blotting, and was not related either to tumor mass growth or cell proliferation rate. Tumor xenografts were also characterized for TS promoter tandem repeat and p53 status. No relationship was observed between these variables and TS expression evaluated by both Western blotting and IHC analysis. Our results demonstrate that TS expression evaluated by Western blotting analysis is directly related to the tumor mass growth rate and question the use of the IHC approach to obtain precise quantitative information on TS expression in tumor samples.

(J Histochem Cytochem 50:1633–1640, 2002)

Key Words: colorectal cancer, thymidylate synthase, tumor growth rate, cell kinetics, silver-stained nucleolus, Western blotting, immunohistochemistry, nude mice


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