Hexosaminidase-altered aberrant crypts, carrying decreased hexosaminidase α and β 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 molecular analysis, it is necessary to avoid contamination with adjacent normal crypts and stromal cells. Decreased hexosaminidase activity in ACF, which has been histochemically demonstrated, was used in the present study to classify isolated crypts in combination with morphological changes. The length, rim diameter, and width (average±SD, μm) 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±45.7, and 97.3±34.7, the width being 1.69 times greater (P<0.0001). Crypts wider than 115 μm (approximately 2 times the average size of Hex+ crypts) were all from ACF, judging from hexosaminidase staining. To analyze transcription levels of Hex α and β subunits (Hexa and Hexb, respectively), real-time relative quantitative reverse transcription-polymerase chain 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 those in morphologically normal crypts, with β-actin as the internal standard. This decrease could be a molecular marker for precancerous enzyme-altered ACF.