Human T‐cell leukemia virus type I (HTLV‐I) encodes a 40 kDa trans‐acting protein, Tax, that regulates transcription of both the proviral and cellular genes, and can transform rat fibroblasts. To determine the functional importance of its trans‐acting capacities in cell transformation, we have examined two representative pathways of transcriptional activation–HTLV‐I long terminal repeat (LTR) mediated and NF‐kappa B dependent–by mutational analysis of Tax. In contrast to a previous report, mutants lacking the ability to activate an NF‐kappaB‐dependent promoter failed to transform rat fibroblasts, whereas a mutation which abolishes the HTLV‐I LTR‐mediated trans‐activation demonstrated a wild‐type capacity for cell transformation. Stable expression of Tax competent for transformation caused enhanced DNA binding of NF‐kappa B in rat fibroblasts. We also demonstrate that stable co‐expression of the NFKB2 precursor, known as a member of the I kappa B proteins, with wild‐type Tax blocked transformation as well as eliminated aberrant NF‐kappaB activation by Tax without interference with the HTLV‐I LTR‐mediated trans‐activation. Our results indicate that constitutive activation of NF‐kappa B is essential for Tax‐mediated transformation of rat fibroblasts.

Microspectrophotometric evaluation of differentially stained sister chromatids made it possible to analyse precisely the factors involved in the Giemsa methods. The concentration of Hoechst 33258, pH of the mounting medium temperature during UV-exposure and the quality (wavelength)of UV-light influenced the differential staining. Exposure of blacklight of 10(-5) M Hoechst 33528-stained brdU-labeled chromosome specimens mounted in McIlvaine buffer (pH 8.0) at 50 degrees C reproducibly allowed differential staining of sister chromatids within 15 min. On the other hand, Korenberg-Freedlender's method using no Hoechst 33258 was also UV-light-dependent. Thus, photolysis of BrdU-substituted DNA was considered the basic mechanism of the Giemsa methods where the photosensitive Hoechst 33258 played a role as a sensitizer.

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To investigate the roles of allelic loss in the development of urothelial cancer, loss of heterozygosity was examined on 7 chromosomal arms in 49 cases of urothelial cancer of various grades and stages. Loss of heterozygosity was found in alleles in order of frequency as follows: 9q (21/38, 55%), 11p (20/44, 45%), 17p (18/42, 43%), 13q (10/39, 26%), 3p (8/41, 20%), 10q (2/29, 7%), and 1p (1/36, 3%). Invasive (high-grade or > or = pT2) tumors showed the loss of 17p (13/16, 81%) and the loss of 13q (7/16, 44%) with significantly higher frequencies than non-invasive (grade 1-2 < or = pT1) tumors. Although the loss of 3p and the loss of 11p were also more frequently associated with the invasive phenotypes, the loss of 11p was detected in a considerable number (9 of 26, 35%) of non-invasive tumors. Our results indicate that the loss of 11p might generally occur at an earlier stage before the loss of 3p, 13q or 17p in tumor progression. Since no correlation was found between the loss of 9q and the tumor grade or stage, this genetic alteration appears to be unrelated to invasiveness, and could be one of the initial events in tumorigenesis. Although accumulated allelic losses of 3p, 11p, 13q and 17p are considered to be involved in the development of the invasive type of urothelial cancers, these multiple genetic alterations may have already occurred in some pathologically non-invasive urothelial cancers. Furthermore, there appears to be some variation in the pattern of cumulative allelic loss.

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.