Wild-type P16ICDKNZ MTSI) cDNA, directed by the cytomegalovirus (CMV) immediate early promoter, was transfected into RT4 and RTI I 2 bladder-carcinoma cell lines bearing a mutated endogenous P16ICDKNZ gene and lacking endogenous P 16lCDKN2 respectively. In both cases, only trans- Bladder carcinoma is one of the most common malignancies, occurring as the fourth most frequent neoplasm in men and the ninth most frequent in women in the United States. Cytogenetic and molecular genetic analyses have documented the involvement of oncogenes, such as c-Ha-ras, c-myc and c-erb-2-neu, of epidermal growth factor and of tumor-suppressor genes in bladder carcinoma (Cordon-Cardo et at., 1994). A large proportion (60%) of bladder cancers shows deletion or mutational inactivation of the p53 gene on chromosome 1 7~1 3 .Likewise, alterations in the RB gene on chromosome 13q14 are observed in about 30% of bladder cancers. Alterations in both p53 and RB genes correlate with advanced stages in the progression of the disease. Although no other known tumorsuppressor genes have yet been involved in bladder carcinoma, several studies have reported cytogenetic deletions or nonrandom LOH for alleles located on several human chromosomes. Monosomy of chromosome 9 is a frequent change, reaching 94% of cases in some investigations (Cairns et al., 1993). LOH at various loci on chromosome 9 was observed not only in tumors of high grade or stage but also in more than 50% of superficial tumors of Ta and Tis initial stages, indicating that allelic loss on chromosome 9 is an early genetic event in the development of bladder cancer. Deletion of loci at 91321-22 have been described in malignant mesothelioma, head-andneck cancer, non-small-cell and small-cell lung cancer, melanoma, glioma and acute lymphoblastic leukemia, glioblastoma and bladder carcinoma. The P161CDxN2 (plQNK4"", MTSI) gene has recently been cloned and mapped to chromosome 9p21 (Serrano et al., 1993; Kamb et al., 1994). The gene encodes p16, a cell-cycle regulatory protein that binds to CDK4 and inhibits phosphorylation of the RB protein by the CDK41cyclin D complex. Hornozygous or hemizygous deletions of the P161CDKN2 gene were frequently detected in primary glioblastoma and pancreatic adenocarcinoma, as well as in tumor cell lines derived from several tumor histotypes, including bladder carcinoma (Kamb et al., 1994;Nobori et al., 1994;Okamoto et al., 1994), but intragenic mutations of this gene were rare both in primary tumors and in tumor cell lines (Okamoto et al., 1994;Cairns et al., 1994;Spruck et al., 1994). The only exceptions were pancreatic adenocarcinoma (Caldas et al., 1994) and esophageal squamous-cell carcinoma (Mori et al., 1994) where sequence changes were detected in 38% and 52% of primary cases respectively. P16/CDKN2-gene alterations, however, were generally found to be 2 to 3 times more common in tumor cell lines than in primary tumors. This observation suggests that, although PI6ICDKN2 may play a role in tumorigenesis, its inactivation is selected by in vitro ...