RESUMO
BACKGROUND: Transcription factor Stat5a/b is highly critical for the viability of human prostate cancer cells in vitro and for prostate tumor growth in vivo. Stat5 is constitutively active in clinical prostate cancers but not in the normal human prostate epithelium. Moreover, Stat5a/b activation in prostate cancer is associated with high histological grade of prostate cancer. However, the molecular mechanisms underlying constitutive activation of Stat5a/b in prostate cancer are unclear. The receptor-associated tyrosine kinase Jak2 is a known key activator of Stat5a/b in prostate cancer cells in response to ligand stimulation. Recently, a single gain-of-function point mutation of JAK2 was described in myeloproliferative diseases leading to constitutive Jak2 kinase activity, subsequent Stat5a/b activation and involvement of V617F Jak2 in the pathogenesis of myeloproliferative disorders. MATERIALS AND METHODS: We determined whether JAK2 undergoes the V617F activating mutation during clinical progression of human prostate cancer using a highly sensitive assay (amplification refractory mutation system) and a unique material of fresh specimens from organ-confined or castration-resistant prostate cancers. RESULTS: The JAK2 V617F mutation was not found in any of the normal or malignant prostate samples analyzed in this study. CONCLUSIONS: Future work should focus on determining the molecular mechanisms other than V617F mutation of Jak2 resulting in continuous Stat5 activation in clinical prostate cancers.
Assuntos
Janus Quinase 2/genética , Neoplasia Prostática Intraepitelial/genética , Neoplasias da Próstata/genética , Antagonistas de Androgênios/uso terapêutico , Antineoplásicos/uso terapêutico , Análise Mutacional de DNA , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Masculino , Mutação , Orquiectomia , Reação em Cadeia da Polimerase , Neoplasia Prostática Intraepitelial/enzimologia , Neoplasia Prostática Intraepitelial/patologia , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismoRESUMO
A newly developed real-time RT-polymerase chain reaction assay for quantitation of hepatitis C virus (HCV) RNA in human plasma and serum was applied. A pair of primers and a probe (molecular beacon) were designed that are specific for the recognition of a highly conservative 5'-non-coding region (5'-NCR) in HCV genome. HCV real-time RT-PCR assay had a sensitivity of 1000 RNA copies per reaction, with a dynamic range of detection between 10(3) and 10(7) RNA copies. The coefficient variation of threshold cycle (Ct) values in intra- and inter-runs were less than 1.37 and 4.66%, respectively. The real-time RT-PCR assay on the HCV sero-positive samples yielded reproducible data, with less than 2.09% of the inter-assay variation. In order to determine its potential for clinical diagnosis, real-time RT-PCR was used to examine the HCV RNA levels in plasma from sero-positive and negative subjects, showing that the assay is highly sensitive and has specificity of 100%. It was demonstrated that the real-time RT-PCR was able to amplify HCV RNA in reference sera with seven genotypes (1A, 1B, 2B, 3A, 4, 5A and 6A) that include six major HCV genotypes circulated in the world. Since HCV is a major pathogen of post-transfusion and community-transmitted non-A, non-B hepatitis, this assay has a broad application for basic and clinical investigations.