Naz Rajesh K, Santhanam Ramasamy, Tyagi Neetu
Division of Research, Department of Obstetrics and Gynecology, Health Education Building, Rm. 211, Medical College of Ohio, 3055 Arlington Avenue, Toledo, OH 43614-5806, USA.
Biochem Biophys Res Commun. 2002 Oct 11;297(5):1075-84. doi: 10.1016/s0006-291x(02)02349-5.
The differential display-polymerase chain reaction technique was employed to obtain a prostate-specific approximately 300-bp cDNA fragment. On screening the human prostate-lambdagt10 library with this fragment, a full-length approximately 1.5-kb cDNA encoding for a prostate antigen, designated as human novel prostate-specific antigen (hNPSA), was found. Extensive database searches revealed that the hNPSA cDNA is a novel sequence. It has an open reading frame (ORF) of 735-bp encoding for 245 amino acids (aa), with a calculated molecular mass of approximately 27kDa. Hydrophilicity analysis of the deduced aa sequence indicated that hNPSA is a membrane-anchored peptide. Analysis for tissue-specificity by Northern blot and RT-PCR-Southern blot procedures indicated that hNPSA is specifically expressed only in human prostate. The hNPSA (ORF) was subcloned into pET22b(+) vector and expressed using the histidine-tagged gene fusion system. The recombinant (r) protein of approximately 27kDa was purified and antibodies (Ab) were raised in rabbits. The rhNPSA Ab recognized a specific protein band of approximately 35kDa in solubilized human prostate tissue and not in any of the other 10 human tissues tested in the Western blot procedure. The hNPSA expression is upregulated 2.5- to 3-fold, both at the mRNA and protein levels in androgen-dependent LNCaP cells, as compared to normal whole prostate tissue. Antisense, but not the sense, phosphothiorate-conjugated oligonucleotides based on the hNPSA cDNA sequence significantly (p<0.001) inhibited proliferation of LNCaP cells in a concentration-dependent manner. Thus, the novel hNPSA, which has prostate-specific expression and seems to be involved in carcinogenesis, may have applications in the specific diagnosis and treatment of prostate cancer.
采用差异显示-聚合酶链反应技术获得了一个约300 bp的前列腺特异性cDNA片段。用该片段筛选人前列腺-lambdagt10文库时，发现了一个编码前列腺抗原的全长约1.5 kb的cDNA，命名为人类新型前列腺特异性抗原（hNPSA）。广泛的数据库搜索显示，hNPSA cDNA是一个新序列。它有一个735 bp的开放阅读框（ORF），编码245个氨基酸（aa），计算分子量约为27 kDa。对推导的氨基酸序列进行亲水性分析表明，hNPSA是一种膜锚定肽。通过Northern印迹和RT-PCR-Southern印迹法分析组织特异性表明，hNPSA仅在人前列腺中特异性表达。将hNPSA（ORF）亚克隆到pET22b(+)载体中，并使用组氨酸标签基因融合系统进行表达。纯化了约27 kDa的重组（r）蛋白，并在兔体内制备了抗体（Ab）。在蛋白质印迹实验中，rhNPSA Ab在溶解的人前列腺组织中识别出一条约35 kDa的特异性蛋白条带，而在其他10种人组织中均未识别出。与正常全前列腺组织相比，在雄激素依赖性LNCaP细胞中，hNPSA在mRNA和蛋白质水平上的表达均上调2.5至3倍。基于hNPSA cDNA序列的反义而非正义硫代磷酸酯缀合寡核苷酸以浓度依赖性方式显著（p<0.001）抑制LNCaP细胞的增殖。因此，具有前列腺特异性表达且似乎参与致癌作用的新型hNPSA可能在前列腺癌的特异性诊断和治疗中具有应用价值。
