P704P, P712P, and P775P: A genomic cluster of prostate-specific genes.

BACKGROUND: Discovery of prostate cancer- and tissue-specific genes will lead to an increased understanding of the molecular events associated with the malignant transformation and tumorigenesis of prostate cells. Such understanding will likely result in the development of promising new markers for screening, diagnosis, and prognosis, as well as potential therapeutic approaches for combating this disease. METHODS: A PCR-based subtraction method was combined with a high-throughput microarray screening approach to identify prostate tissue- and/or cancer-specific genes. Northern blot and quantitative real-time PCR were used to confirm prostate specificity. Bioinformatics analysis was performed to determine gene localization and to identify the open reading frame of novel genes. RESULTS: Three novel cDNA clones, P704P, P712P, and P775P, were identified and characterized to be specific for normal and malignant prostate tissues. Furthermore, P712P mRNA expression was found to be androgen responsive in LNCaP cells. Sequences for all three cDNAs were localized to an 80 kb genomic region on chromosome 22. Attempts to identify full-length transcripts did not reveal any apparent open reading frames, indicating that P704P, P712P, and P775P may belong to a novel class of transcripts with specific patterns of gene expression that do not code for translated proteins. CONCLUSIONS: A genomic cluster of prostate-specific genes with no apparent open reading frame has been discovered using a high-throughput approach combining subtraction with microarray. This may represent an important genomic region having possible connections to prostate biology with potential applications in prostate diagnostics and therapy.

Characterization of KLK4 expression and detection of KLK4-specific antibody in prostate cancer patient sera.

The ability to identify prostate tumor or prostate tissue specific genes that are expressed at high levels and use their protein products as targets could greatly aid in the diagnosis and treatment of prostate cancer. Using a polymerase chain reaction (PCR)-based subtraction technique, we have recovered the recently described KLK4 (prostase) gene from human prostate cDNA. In this study, KLK4 gene expression in human prostate tumors was further characterized using cDNA quantitative PCR and immunohistochemistry, demonstrating that the gene is specifically expressed at both the mRNA and protein levels in normal human prostate tissue, and in both primary and metastatic prostate tumor samples. Quantitative mRNA analysis also demonstrated low level expression including adrenal gland, salivary gland and thyroid. Finally, it was demonstrated that prostate cancer patient sera contain antibodies that bind specifically to recombinant KLK4 protein. This antibody has been used to detect KLK4-specific peptides in epitope mapping experiments. The relatively specific expression profile and elevated level of KLK4 mRNA and protein in both tumor and normal prostate tissues, in addition to detectable KLK4-specific antibody in cancer patient sera, supports additional efforts to determine if KLK4 can play a role in the diagnosis of prostate cancer, the monitoring of residual disease, or act as a target for immunotherapy.

Use of multiepitope polyproteins in serodiagnosis of active tuberculosis.

Screening of genomic expression libraries from Mycobacterium tuberculosis with sera from tuberculosis (TB) patients or rabbit antiserum to M. tuberculosis led to the identification of novel antigens capable of detecting specific antibodies to M. tuberculosis. Three antigens, Mtb11 (also known as CFP-10), Mtb8, and Mtb48, were tested together with the previously reported 38-kDa protein, in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies in TB patients. These four proteins were also produced as a genetically fused polyprotein, which was tested with two additional antigens, DPEP (also known as MPT32) and Mtb81. Sera from individuals with pulmonary and extrapulmonary TB, human immunodeficiency virus (HIV)-TB coinfections, and purified protein derivative (PPD)-positive and PPD-negative status with no evidence of disease were tested. In samples from HIV-negative individuals, the ELISA detected antibodies in >80% of smear-positive individuals and >60% smear-negative individuals, with a specificity of approximately 98%. For this group, smears detected 81.6% but a combination of smear and ELISA had a sensitivity of approximately 93%. The antigen combination detected a significant number of HIV-TB coinfections as well as antibodies in patients with extrapulmonary infections. Improved reactivity in the HIV-TB group was observed by including the antigen Mtb81 that was identified by proteomics. The data indicate that the use of multiple antigens, some of which are in a single polyprotein, can be used to facilitate the development of a highly sensitive test for M. tuberculosis antibody detection.