Cloning and characterization of UROC28, a novel gene overexpressed in prostate, breast, and bladder cancers.

A novel gene, designated UROC28, was identified by an agarose gel-based differential display technique, and it was found to be up-regulated in prostate, breast, and bladder cancer. Expression of UROC28 was also up-regulated in prostate cancer cells in the presence of androgens as demonstrated by relative quantitative reverse transcription-PCR. The elevated expression of this gene was observed to increase in surgically removed tissues concomitantly with rising Gleason grade and was most elevated in metastatic tissue. UROC28 protein was detected in serum by Western slot blot analyses, and a significant higher UROC28 protein level was found in sera of prostate cancer individuals compared with normal individuals and individuals with nonmalignant prostatic hyperplasia. Northern analyses in normal tissues showed that the UROC28 cDNA hybridizes to two mRNAs at about 2.1 and 2.5 kb. Nucleic acid sequence analyses indicated that these two alternatively spliced mRNA variants differ only at the 3' untranslated region. These two mRNAs encode the same protein with 135 amino acids. Bioinformation analyses suggest that there is a possible transmembrane domain from amino acid aa34 to aa50, three protein kinase-C phosphorylation sites at aa62 (SQK), aa89 (TMK), and aa94 (SMK), and one myristylation site at aa118 (GLECCL). Genomic Southern hybridization and chromosomal mapping demonstrated that UROC28 is encoded by a single copy of gene at chromosome 6q23-24. In situ hybridization and immunohistochemistry experiments further confirmed up-regulation of this gene in prostate and breast cancers with the expression localizing to the glandular epithelium. This gene did not demonstrate increased expression in lung and colon cancer tissues.

Human prostate-specific transglutaminase gene: promoter cloning, tissue-specific expression, and down-regulation in metastatic prostate cancer.

OBJECTIVES: To investigate the tissue-specific and differential expression of the human prostate-specific transglutaminase (pTGase) gene in metastatic prostate cancer (CaP) and to study how this gene is regulated in the prostate. METHODS: Northern blot hybridization and polymerase chain reaction (PCR) were performed using RNA from a variety of organs to confirm prostate-specific expression of the gene. Relative quantitative reverse transcriptase-PCR (RT-PCR) was performed to investigate the differential expression of the gene among normal prostates and prostates with CaP and metastatic CaP. The pTGase gene promoter was cloned using genomic library screening and sequencing. Transfection experiments and chloramphenicol acetyltransferase (CAT) assays were performed to study the regulation of the gene. RESULTS: Northern hybridization and RT-PCR confirmed that the gene is only expressed in the prostate. Relative quantitative RT-PCR demonstrated a loss of expression of the pTGase gene among men with CaP and higher Gleason grades. In metastatic CaP tissue from various sites, 86% of the samples lost expression of the gene. We cloned and sequenced a 1.4-kilobase promoter region of the pTGase gene. Transfection and CAT assay results supported the theory that certain elements in the -1 to -520 region are sufficient to direct prostate-specific expression of the gene. Additional elements in the -520 to -1400 region may also contribute to its prostate-specific expression. CONCLUSIONS: The results of our study demonstrate that the human pTGase gene is only expressed in prostate tissue and that its expression is inhibited in most metastatic CaP. Prostate-specific expression of the gene is controlled by elements in the promoter region. The observed preferential loss of pTGase gene expression in metastatic CaP may be important to the pathogenesis and progression of this disease.