Hexosaminidase-altered aberrant crypts, carrying decreased hexosaminidase alpha and beta subunit mRNAs, in colon of 1,2-dimethylhydrazine-treated rats

Aberrant crypt foci (ACF), consisting of morphologically irregular crypts, are thought to be precancerous lesions for colon cancers. For their molecul ar analysis, it is necessary to avoid contamination with adjacent normal cr ypts and stromal cells, Decreased hexosaminidase activity in ACF, which has been histochemically demonstrated, was used in the present study to classi fy isolated crypts in combination with morphological changes. The length, r im diameter, and width (average+/-SD, mum) of hexosaminidase-positive (Hex) crypts were 238.6+/-40.4, 89.5+/-22.9, and 57.6+/-14.0, respectively. For hexosaminidase-negative (Hex+) crypts, the values were 314.4+/-77.8, 140.3 +/-45.71 and 97.3+/-34.7, the width being 1.69 times greater (P<0.0001). Cr ypts wider than 115 <mu>m (approximately 2 times the average size of Hex+ c rypts) were all from ACF, judging from hexosaminidase staining. To analyze transcription levels of Hex alpha and beta subunits (Hexa and Herb, respect ively), real-time relative quantitative reverse transcription-polymerase ch ain reaction (RT-PCR) analysis was performed using the LightCycler system. In aberrant crypts, both Hexa and Hexb were significantly down-regulated to 0.266 (P<0.002) and 0.131 (P<0.001) units, respectively, compared with tho se in morphologically normal crypts, with beta -actin as the internal stand ard. This decrease could be a molecular marker for precancerous enzyme-alte red ACF.