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  • Title: DNA in situ sensitivity to denaturation: a new parameter for flow cytometry of normal human colonic epithelium and colon carcinoma.
    Author: Kunicka JE, Darzynkiewicz Z, Melamed MR.
    Journal: Cancer Res; 1987 Aug 01; 47(15):3942-7. PubMed ID: 3607742.
    Abstract:
    Modal DNA (ploidy) and sensitivity of DNA in situ to denaturation by acid have been analyzed by flow cytometry of 10 colorectal adenomas and 35 adenocarcinomas; 39 normal mucosa samples served as controls. A new method was developed to denature DNA in chromatin of the freshly isolated, intact, and unfixed individual cell nuclei from surgically resected material. The sensitivity of DNA denaturation (T alpha) was assayed by metachromatic staining with acridine orange and calculated as a ratio of the alpha t index of the tumor sample to the alpha t index of normal mucosa; the alpha t index is that fraction of DNA, following treatment at pH 1.4, that stains metachromatically with acridine orange at pH 2.6. All adenomas were diploid and in nine of 10 the T alpha value was close to 1.00, indicating no difference from control specimens in DNA sensitivity to denaturation. Forty-nine% of adenocarcinomas were aneuploid. Forty-six% of adenocarcinomas differed from normal in sensitivity of DNA to denaturation; the T alpha value was lower than 0.90 indicating that chromatin of the tumor cells was more resistant to denaturation than control cells. There was no correlation between sensitivity to denaturation of DNA and incidence of aneuploidy. However, there was a correlation between T alpha and the pathologically determined stage of disease. There was increased resistance to denaturation in 58% of tumors classified as Dukes' C/D stage, in 36% of tumors classified as Dukes' B, and in 20% classified as Dukes' A stage of the disease. Statistical analysis of these results revealed significant differences between distributions of T alpha in noninvasive (Adenomas and Dukes' A) versus invasive (Dukes' B and C/D) tumors with level of significance at P = 0.02. The data suggest that acid denaturation of DNA in situ may be a valuable adjunct in assessing the biology of colon cancer. The molecular basis for this phenomenon is discussed.
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