The cell-cycle inhibitor p27 is a potential tumor suppressor, but its gene has never been found inactivated in human tumors. Because cell-cycle regulation of p27 cellular abundance occurs at the post-transcriptional level, we analyzed p27 protein expression and degradation in human colorectal carcinomas. Proteasome-mediated degradation activity of p27 was compared with its protein levels in a subset of tumor samples. We found that carcinomas with low or absent p27 protein displayed enhanced proteolytic activity specific for p27, suggesting that low p27 expression can result from increased proteasome-mediated degradation rather than altered gene expression. Patients whose tumors expressed p27 had a median survival of 151 months, whereas patients who lacked p27 (10%) had a median survival of 69 months. By multivariate analysis, p27 was found to be an independent prognostic marker. Lack of p27 was associated with poor prognosis (2.9 risk ratio for death; P = 0.003). The absence of p27 protein expression is thus a powerful negative prognostic marker in colorectal carcinomas, particularly in stage II tumors, and thereby may help in the selection of patients who will benefit from adjuvant therapy. These data suggest that aggressive tumors may result from the selection of a clone or clones that lack p27 due to increased proteasome-mediated degradation.

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Tumor cells that express a fusion gene of Escherichia coli cytosine deaminase ( CD ) and herpes simplex virus type 1 thymidine kinase ( TK ) sequences activate and are subsequently killed by the nontoxic prodrugs 5 -fluorocytosine and ganciclovir. We have previously developed a recombinant adenovirus containing the CD -TK fusion gene controlled by the human inducible heat shock protein 70 promoter so that heat at 418C for 1 hour induces therapeutic gene expression. This adenovirus effectively transduces heat -inducible expression of the CD -TK gene into human prostate carcinoma cells. However, because a limited number of cells in a tumor can actually be infected, we created a replicating adenoviral vector to increase CD -TK gene expression. This vector is a replication -competent, E1B -attenuated adenoviral vector containing the hsp70 promoter ± driven CD -TK gene ( Ad.E1A( + )HS -CDTK ). When human prostate adenocarcinoma DU -145 cells ( mutant p53 ) were infected with the virus at a multiplicity of infection ( MOI ) of 1 or 10, the viral replication was detected within 2 days at both MOIs. Similar results were observed in human colorectal carcinoma CX -1 cells. When DU -145 cells were infected with the virus at an MOI of 10, incubated for 24 hours, heated at 418C for 4 hours, and then harvested 20 hours later, Western blot analysis demonstrated that this virus successfully produced viral E1A proteins and heat shock stimulated the CD -TK gene expression by 12.3 -fold. In addition, Ad.E1A( + )HS -CDTK effectively suppressed cell proliferation by viral cytopathic effect ). Unlike with a replication -incompetent virus ( Ad.HS -CDTK ), the cytopathic effect of the virus and cytotoxicity in the presence of the prodrugs were still observed even at low MOI ( MOI = 1.0 ). Cancer Gene Therapy ( 2001 ) 8, 397 ± 404

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