Identification of H-ras mutations in DNA from urine sediments facilitates the detection of low-grade bladder tumors and, in combination with cytology, increases the overall tumor detection from 33% to 60%. Preliminary results in patient follow-up suggest that detection of H-ras mutations may have some clinical utility in detecting the presence of abnormal cells in the absence of an overt lesion following cystoscopy or positive cytology.
A series of 111 human bladder tumors were screened using oligonucleotide mutant specific probes, restriction enzyme analysis and single-stranded confirmation polymorphism (SSCP) for the presence of H-ras activation events. Thirty-three tumors were found to harbor H-ras mutations where a glycine to valine (G-->T) change in codon 12 was the most common point mutation recorded (26 tumors). Additional mutations involved glycine to cysteine at codon 13 (2 tumors) and glutamine to arginine/lysine/leucine at codon 61 (3/1/1 tumors, respectively). Ambiguous signals recorded with oligonucleotide probes were further analyzed using SSCP analysis revealing the presence of H-ras mutations in restricted regions of some tumors. The apparent sensitivity of SSCP enabled us to extend this study to DNA isolated from urine sediments where 4 of the 9 patients studied showed representation of mutant H-ras. Our study demonstrates a sensitive, non-invasive assay for the screening of urine-borne cells, with no requirement for prior knowledge of the mutational change at the H-ras locus.
pSVCT3 is a cytoplasmic-localization mutant of simian virus 40 (SV40) isolated from the SV40 adenovirus 7 hybrid virus (PARA) and cloned into plasmid PBR. The large T antigen of pSVCT3 accumulates in the cytoplasm of infected monkey cells instead of being transported to the nucleus. The sole change in CT3 large T antigen is amino acid residue 128 (Lys->Asn). Transformation of precrisis rodent cells by pSVCT3 is negligible, whereas the frequency of transformation of established rodent cell lines by pSVCT3 is comparable to that of wild-type SV40. According to the model, in which transformation of precrisis cells involves the combined oncogenic action of both nuclear and cytoplasmic gene products, we predicted that pSVCT3 would localize in the cytoplas..-of human cells and would therefore at most only partially and rarely transform precrisis human cells. We have found that pSVCT3 is able to transform precrisis human cells at high frequency. Furthermore, pSVCT3-transformed human precrisis cells relocalized T antigen to their nuclei. The relocalization of large T antigen was not dependent on cell growth. Wild-type and pSVCT3-transformed human cell lines both have about five copies of integrated SV40 DNA. SV40 virus-specific proteins, including the 100,000-molecular-weight super large T antigen, were expressed in pSVCT3-transformed human cells. Our results suggest that molecules in precrisis human cells, but not cells of other species, are able to complement the cytoplasmic-localization defect of the CT3 mutant large T antigen.
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