SYMPOSIUM PAPER |
Correspondence to: Theresa P. Pretlow, Inst. of Pathology, Case Western Reserve Univ., 2085 Adelbert Rd., Cleveland, OH 44106.
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Summary |
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Microscopic evaluation of whole-mount colons stained with methylene blue and/or hexosaminidase has identified putative preneoplastic lesions in the colons of rodents treated with carcinogen and in the grossly normal colons of humans. Enzyme histochemistry with glycol methacrylate sections has permitted the identification of putative premalignant lesions in rodent livers, human and rodent colons, and human prostates. Immunohistochemistry with paraffin-embedded tissues has been used to identify and characterize putative premalignant lesions in human colons and prostates. (J Histochem Cytochem 46:577583, 1998)
Key Words: , enzyme histochemistry, immunohistochemistry, glycol methacrylate embedding, paraffin embedding, premalignant lesions, cancer, colon, prostate, whole-mount tissue
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Introduction |
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THE MALIGNANT TRANSFORMATION of normal cells to malignant ones is a multistep process that occurs over many months to years in most species. During the long period of time (called latency) between the initial administration of carcinogen and the final development of a macroscopically visible tumor, a large number of changes occur in these cells. Histochemistry provides an invaluable tool with which to identify specific cells involved in this process and many of the phenotypic alterations that are observed both in premalignant cells and in cancer cells. Rodents are widely used (
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Materials and Methods |
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Specimens and Animal Protocols
Grossly normal human colonic mucosa and sections of human prostate were obtained for us by the Western Division of the Cooperative Human Tissue Network of the National Cancer Institute located at Case Western Reserve University. For experimental animal protocols, the reader is referred to the original articles cited for the specific studies.
Identification of Aberrant Crypt Foci (ACF) in Whole-mount Colons
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Fixed, unembedded tissue can be stained for the demonstration of enzyme activity, such as hexosaminidase, by using the same substrates as used for enzyme histochemistry with histological sections (
Enzyme Histochemistry with Glycol Methacrylate-embedded Tissue
Tissues embedded in glycol methacrylate (JB-4 embedding kit, Polysciences, Warrington, PA; molds and block holders, Energy Beam Sciences, Agawam, MA) can be stained with water-soluble reagents and can be cut at 2 µm (
For demonstration of hexosaminidase activity and immunohistochemical demonstration of the incorporation of 5-bromo-2'-deoxyuridine (BUdR) (Sigma; St Louis, MO) in serial sections, the initial fixation of colon tissue was reduced to 15 min in 2% paraformaldehyde at 4C, followed by a 1-hr wash in 3% sucrose in 0.1 M phosphate buffer (pH 7.4), and fixation in 100% acetone for 2 hr under vacuum (
Immunohistochemistry with Paraffin-embedded Tissue
Many antigens can now be demonstrated in formalin-fixed, paraffin-embedded tissues with antigen retrieval methods (
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Results and Discussion |
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Enzyme-altered Foci in Liver Carcinogenesis
From the earliest studies of the induction of tumors in experimental animals, it was known that there is a long latency period between the first application of carcinogen and the final appearance of tumor(s) (reviewed in -glutamyl transpeptidase activity and a decrease in bile canalicular ATPase activity (
Enzyme-altered Foci in Colon Carcinogenesis
Knowing that enzyme-altered foci in liver had provided a tool that led to an increased understanding of liver carcinogenesis, we asked whether similar enzyme changes preceded morphological changes in the colon. For these studies we used serial sections of glycol methacrylate-embedded, grossly normal colons from F344 rats that had been treated with the colon carcinogen dimethylhydrazine (-naphthyl butyrate esterase activity (Figure 1E) and a reduction in goblet cells (Figure 1F). A decrease in hexosaminidase activity was also seen in colon tumors (
Aberrant Crypt Foci in Animals
While we were working on enzyme-altered foci in colons, -naphthyl butyrate esterase activity or increased periodic acidSchiff-reactive material (
Having established that ACF are enzyme-altered, we wondered if all enzyme-altered foci in colons are ACF or if some enzyme-altered foci are morphologically normal. Our earlier studies with enzyme-altered foci in glycol methacrylate sections had suggested that some enzyme-altered foci were not larger than their adjacent normal crypts, but this was difficult to confirm because of the artifacts inherent in embedded tissue. By incubating fixed, intact, whole-mount colons from carcinogen-treated rats in the same substrate as was used for the histochemical demonstration of hexosaminidase activity in slides, we (
Multiple studies of ACF in animals (reviewed in
Aberrant Crypt Foci in Humans
Our laboratory was the first to report ACF in whole-mount preparations of grossly normal human colon mucosa (
Enzyme-altered or Antigen-altered Foci in Human Prostate
The studies of putative premalignant lesions in human prostate in our laboratory are much more preliminary. , and nm23 (Figure 2A and Figure 2B) also has been observed in a small number of benign prostate epithelial glands (
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In conclusion, histochemistry, including enzyme histochemistry of glycol methacrylate-embedded tissues, enzyme histochemistry of segments of intact tissue, and immunohistochemistry, has provided invaluable tools to identify and study putative premalignant lesions from their earliest identification through their progression to cancer.
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Acknowledgments |
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Supported in part by NIH grants CA66725, CA48032, CA54031, CA57179, DK45770, DK51347, and CA43703.
We thank the many students, research associates, and research assistants who contributed to this work, including Betty Barrow, Christopher Cheyer, Leon Hudson, Lawrence Monger, M. Nagabhushan, Mary Ann O'Riordan, and I-Mei Siu.
Received for publication August 7, 1997; accepted August 7, 1997.
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