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.