12-hydroxy-5-8-10-14-eicosatetraenoic-acid and Cell-Transformation--Neoplastic

We performed a detailed and comprehensive study of the involvement of tumor suppressor genes in human prostate cancer. We utilized primers flanking either the restriction fragment length polymorphism (RFLP) or variable number of tandem repeat [VNTR; microsatellite or simple repeat site (SRS)] polymorphic sites to polymerase chain reaction (PCR) amplify the genomic DNA and detect loss of heterozygosity of the target genes. Quantitative reverse transcription (RT)-PCR was performed to measure the mRNA expression levels and PCR/single strand conformational polymorphism (SSCP) and DNA sequencing carried out to detect mutation of the tumor suppressor genes. We found that multiple tumor suppressor genes (e.g., p53, DCC, APC, MCC, BRCA1, and WAF1/CIP1) were inactivated at different frequencies via various mechanisms [e.g., loss of heterozygosity (LOH), loss of expression (LOE), mutation, and inactivation by cellular binding protein]. Several important and novel findings are as following: LOH and LOE of the DCC gene, LOH, LOE, and possible mutation of the APC/MCC genes, LOH of the BRCA1 locus, and mutation of the WAF1/CIP1 gene. For p53 tumor suppressor gene alone, multiple inactivation mechanisms (i.e., LOH, LOE, mutation, and amplification of the cellular inactivating protein MDM2) were identified. A possible involvement of genomic instability or mutator phenotype in human prostate cancer was investigated by microsatellite typing using PCR. A high frequency of microsatellite instability was detected and the microsatellite instability found to correlate with advanced stage and poor differentiation of prostate cancer, suggesting that genes functioning in DNA mismatch repair or general stabilization of the genome may be involved in prostate cancer. The results obtained in this study suggested that multiple tumor suppressor genes (both known and unknown genes) may share the role in prostate cancer; a pattern which has been found in a number of human malignancies such as cancers of the esophagus, colon and breast. In fact, we performed deletion studies aimed at localizing potential tumor suppressor loci on various chromosomal regions. A number of chromosomal regions (i.e., 6p12-24 and 17q21) were found to potentially harbor unidentified tumor suppressor genes. Detailed deletion mapping has localized the potential tumor suppressor loci to a < 2 Mb region centromeric to the BRCA1 gene on chromosome 17q. In addition, we identified a number of novel mechanisms of tumor s

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate 12-Lipoxygenase; Biomarkers, Tumor; Carcinoma; Cell Transformation, Neoplastic; Chromosome Mapping; Colorectal Neoplasms; Disease Progression; DNA, Neoplasm; Gene Expression Regulation, Neoplastic; Genes, APC; Genes, DCC; Genes, Tumor Suppressor; Humans; Lipoxygenase Inhibitors; Loss of Heterozygosity; Male; Neoplasm Proteins; Prognosis; Prostatic Neoplasms; Tumor Cells, Cultured

Accumulating evidence suggests that platelet-type 12-lipoxygenase (p12-LOX) plays an important role in tumor development. However, how p12-LOX contributes to tumorigenesis is still not understood. The role of p12-LOX was therefore examined in tumor promotion using mouse epidermal JB6 P+ cells that are sensitive to 12-O-tetradecanoylphorbol-13-acetate-induced transformation. The expression of p12-LOX was significantly higher in JB6 P+ cells than in JB6 P- cells that were resistant to transformation, and its expression was further increased by tumor necrosis factor (TNF)-alpha. Importantly, the inhibition of p12-LOX in JB6 P+ cells by baicalein, a specific inhibitor or small interfering RNA significantly suppressed TPA-induced transformation. Moreover, treatment with 12(S)-hydroxyeicosatetraenoic acid (HETE), a metabolite of p12-LOX, enhanced TPA-induced neoplastic transformation either in the presence or absence of baicalein. These results indicate that p12-LOX is required for tumor promotion of epidermal cells and that 12(S)-HETE functions as a rate-limiting factor. Notably, treatment with baicalein significantly suppressed the proliferation of JB6 P+ cells when cells were seeded at a low density in a culture plate. Moreover, the cloning efficiency of JB6 P+ cells was dramatically decreased by inhibition of p12-LOX. In contrast, baicalein treatment did not affect the cloning efficiency of most malignant cancer cells. These results indicate that p12-LOX is induced by the inflammatory cytokine TNF-alpha in the early stage of tumorigenesis, and is required for tumor promotion through enhancing efficient proliferation of a small number of initiated cells. The present results suggest that the p12-LOX pathway may be an effective target of chemoprevention for skin carcinogenesis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Blood Platelets; Cell Proliferation; Cell Transformation, Neoplastic; Cloning, Molecular; Enzyme Inhibitors; Epidermis; Epithelial Cells; Flavanones; Mice; Mice, Inbred BALB C; Skin Neoplasms; Tumor Necrosis Factor-alpha