Nuclear DNA content of isolated crypts of background colonic mucosa from patients with familial adenomatous polyposis and sporadic colorectal cancer

Abstract

The DNA content of the upper one third of the crypt epithelium was compared with that of the lower two thirds in the background colorectal mucosa of eight cases of familial adenomatous polyposis (FAP) and eight control cases of sporadic colorectal cancer (SCRC). Intact crypts were isolated by incubating fresh lesion free colorectal mucosa in calcium and magnesium free Hanks' balanced salt solution (CMFH) containing 30 mM EDTA for 30 minutes at 37 degrees C and then agitating in CMFH. The crypts were then separated from the lamina propria, fixed in 70% ethanol and under a dissecting microscope divided manually into upper and lower portions. Each portion was digested with pepsin to obtain a suspension of single nuclei, and smears of the nuclei were stained with 4',6,-diamidino-2-phenylindole dihydrochloride (DAPI). Nuclear DNA was determined using a cytophotometric microscope. Results showed that the DNA content of the epithelium of the upper one third of crypts was diploid in both FAP and SCRC cases, and that proliferative fractions with diploid peaks were present in the lower two thirds of the crypts in both groups. These results support our previous finding that the proliferative compartment of background crypts is confined to the lower two thirds and does not extend to the upper parts of the crypts.