Novel human prostate-specific cDNA: molecular cloning, expression, and immunobiology of the recombinant protein.
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.
Naz
Rajesh K
RK
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. Rnaz@mco.edu
Santhanam
Ramasamy
R
Tyagi
Neetu
N
D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm / Q000096:biosynthesis* / Q000235:genetics*; D000954:Antigens, Surface / Q000096:biosynthesis* / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D020869:Gene Expression Profiling; D015723:Gene Library; D006801:Humans; D006965:Hyperplasia / Q000473:pathology; D008297:Male; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D018076:DNA, Complementary; C468775:NPSA protein, human; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D011994:Recombinant Proteins