Ras gene mutations occur relatively early during colorectal tumor development and have been observed in 40-50% of malignant colorectal tumors. Advances in endoscopic techniques have made it possible to detect small, flat colorectal tumors that could not be detected by standard examinations. To determine whether ras gene mutations are also involved in the genesis of small, flat colorectal tumors, we examined ras point mutations in 34 cases of small polypoid or flat elevated colorectal tumors (32 adenomas, 2 carcinomas) and in 26 cases of small, flat colorectal tumors (13 adenomas, 13 carcinomas) by means of the polymerase chain reaction (PCR) and dot-blot hybridization. Ras gene point mutations were observed in 16 of the 34 tumors of the former type (47%), but in none of the 26 tumors of the latter type, even though the grade of dysplasia was severe in the flat tumors. Our results suggest that different genetic pathways for tumor progression may exist for polypoid and for flat colorectal carcinomas.

The mechanism of DNA degradation and its clinical applications were examined.When purified A phage and extracted liver DNA were fixed in phosphate buffered formaldehyde, the DNA did not degrade, but there was incomplete digestion with endonuclease. Rat liver tissues were fixed under various conditions and DNA extracted. Immediate fixation with buffered formaldehyde at low temperature, or the addition of EDTA to buffered formaldehyde blocked the DNA degradation. Analysis of pulsed field gel electrophoresis also showed that DNA was degraded before extraction. These results suggest that tissue nuclease has an important role in DNA degradation in tissue. Furthermore, formaldehyde fixation at low temperature, which may take time and which decreases slightly the staining capacity, is useful for the extraction of intact DNA. For clinical application, the detection of provirus was examined. Genomic DNA was extracted from a necropsy sample of adult T cell leukaemia fixed in formaldehyde; human T cell leukaemia virus type-I (HTLV-I) provirus was successfully detected by Southern blotting. The polymerase chain reaction (PCR) facilitated the detection of specific genes from paraffin wax embedded tissues which contained relatively low molecular DNA.5 We also extracted DNA from formaldehyde fixed tissues, but the extracted DNA was degraded. We therefore examined the mechanism of DNA degradation. Furthermore, genomic DNA was extracted from a necropsy specimen fixed in formaldehyde and used for the detection of HTLV-I provirus. MethodsTo ascertain the direct effect of formaldehyde on DNA 100 ,ug of i phage DNA (Takara, Kyoto, Japan), salmon sperm DNA, and extracted rat liver DNA were fixed in phosphate buffered formaldehyde (formaldehyde concentration 433%, methanol concentration 0-70, 33 mM NaH2PO4, 45 7 mM Na2 HPO4, pH 7) for 24 hours at room temperature and dialysed once in TNE (10 mM TRIS-HCI, pH 8, 1 mM EDTA, 100 mM NaCl) to remove the formaldehyde.Closed colony Long-Evans (LE) rats were used to study the effect under different fixation conditions. After anaesthesia with ether and laparotomy buffered formaldehyde was immediately injected into the portal vein.

Degradation and extraction of high molecular weight DNA from formaldehyde fixed tissues suitable for gene analysis are presented. We previously reported that DNase might play an important role in the degradation of DNA extracted from formaldehyde fixed tissues (Tokuda et al. 1990). In the present study, DNase activity of the supernatant from rat tissues fixed in buffered formaldehyde at room temperature was negligible within 3 hr. Analysis of DNA extracted from reconstituted chromatin revealed that the degradation increased in the absence of DNase depending on the duration of the formaldehyde fixation. Furthermore, high molecular weight DNA could be extracted from tissues devoid of DNase activity fixed in buffered formaldehyde containing EDTA. These results demonstrated that DNA degradation was due mainly to a mechanism other than DNAse which was inhibited by EDTA. For clinical application, v-H-ras gene was successfully detected by Southern blotting from rat spleen tissues fixed in buffered formaldehyde especially at 4 C. Fixation at low temperature is useful for gene analysis.

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