Different instability of nuclear DNA at acid hydrolysis in cancerous and noncancerous cells as revealed by fluorescent staining with acridine orange

Abstract

Ehrlich cancer cells and inflammatory cells in mouse ascitic fluid were hydrolyzed and stained with acridine orange (AO). The AO hydrolysis curves for G1/G2 + M phase cancer cells and inflammatory cells were differentially determined using flow cytometry by monitoring the metachromatic red-shifted fluorescence of the fluorochrome bound to the single-stranded DNA produced by acid hydrolysis. By computer fitting of the Bateman function to the hydrolysis curves, the kinetic parameters k1 (rate constant for the production of single-stranded DNA), k2 (rate constant for the degradation of the produced single-stranded DNA), and y0 (theoretical value of the single-stranded DNA present initially) were determined. It was found that the k2 value, which reflects the degree of DNA instability, was much higher for cancer cells in both the G1 and G2 + M phases than for inflammatory cells. This finding led us to develop a method for the differential AO staining of cancer cells and non-cancerous cells utilizing the different degree of DNA instability at acid hydrolysis. AO staining after hydrolysis with 2N HCl at 30 degrees C for 8.5 min was found to be the optimal method. In the 60 cases of human malignant epithelial and nonepithelial tumors tested, all of the malignant tumor cells emitted metachromatic red fluorescence, while all of the nonmalignant tumor cells (5 cases of benign tumor) and normal cells emitted orthochromatic green fluorescence when observed with a violet excitation light under a fluorescence microscope. This new technique can be a useful tool for the screening of malignancy in exfoliative cytology and also for basic cancer research.

Demonstration of Biological Aggressiveness of Bone Giant Cell Tumor by the Comparative Study of Immunohistochemical Detection of DNA-instability and Cortical Bone Destruction by CT.

“…Fukuda et al [13,14] reported that DNA-instability is a specific and common marker of malignancy irrespective of epithelial or mesenchymal origin. Based on this finding, they developed the method of differential staining of malignant cells with acridine orange or anti-singlestranded DNA antiserum by utilizing the phenomenon of increased production of denatured single-stranded DNA in cancerous cells with respect to normal cells after acid hydrolysis.…”

“…Immunohistochemical staining of paraffin-embedded sections with monoclonal anti-single-stranded DNA antiserum after acid hydrolysis to demonstrate DNAinstability (DNA-instability test) [13,14] Materials Paraffin-embedded 4 pm sections were prepared from surgically resected specimens of 4 osteochondromas, 4 enchondromas, 23 BGCT (only the bone tumors that consisted of typical stromal cells and multinucleated giant cells were diagnosed as BGCT. Those with bone formation or other cellular components such as histocytes, foamy cells and fibroblasts were not included), 6 chondrosarcoma and 6 osteosarcoma cases after fixation with buffered 10% formalin for 24 hr at room temperature.…”

“…Fukuda et al [13,14] found that nuclear DNA of malignant neoplastic cells is always and unexceptionally much more unstable than that of comparable benign tumor cells and comparable normal cells, irrespective of epithelial or mesnechymal origin, and developed the method of differential fluorescent or immunohistochemical staining of malignant neoplastic cells after denaturation of DNA by acid hydrolysis.…”

Correlation of fixation, HCL hydrolysis and proteolytic digestion in bromodeoxyuridine immunohistochemistry of human stomach labeled by extracorporial perfusion system.

“…The differences in the demonstrability of nuclear BrdU among the various kinds of cells might be based on the instability of the nuclear DNA for HC1 hydrolysis as reported by Fukuda et al (5). Sugihara et al (16) compared the immunohistochemical result with the autoradiographic one in mouse pyloric mucosa double labeled with BrdU and 3H-thymidine, and showed that most of the BrdU-positive cells have the nuclei labeled with 3H-thymidine.…”

The DNA-instability test as a specific marker of malignancy and its application to detect cancer clones in borderline malignancy

“…By quantitative analysis of the Feulgen hydrolysis curve, it was found that the extent to which nuclear DNA exposed to acid is denatured reflects the degree of DNA instability, which depends on the chromatin structure (Darzynkiewicz et al, 1979;Fukuda et al, 1985Fukuda et al, , 1986. The Bateman function was found to be appropriate for the Feulgen hydrolysis curve analysis (Böhm and Seibert, 1966;Pöppe et al, 1979;Madokoro et al, 1985).…”