Identification of the human prostatic carcinoma oncogene PTI-1 by rapid expression cloning and differential RNA display.
Elucidating the relevant genomic changes mediating development and evolution of prostate cancer is paramount for effective diagnosis and therapy. A putative dominant-acting nude mouse prostatic carcinoma tumor-inducing gene, PTI-1, has been cloned that is expressed in patient-derived human prostatic carcinomas but not in benign prostatic hypertrophy or normal prostate tissue. PTI-1 was detected by cotransfecting human prostate carcinoma DNA into CREF-Trans 6 cells, inducing tumors in nude mice, and isolating genes displaying increased expression in tumor-derived cells by using differential RNA display (DD). Screening a human prostatic carcinoma (LNCaP) cDNA library with a 214-bp DNA fragment found by DD permitted the cloning of a full-length 2.0-kb PTI-1 cDNA. Sequence analysis indicates that PTI-1 is a gene containing a 630-bp 5' sequence and a 3' sequence homologous to a truncated and mutated form of human elongation factor 1 alpha. In vitro translation demonstrates that the PTI-1 cDNA encodes a predominant approximately 46-kDa protein. Probing Northern blots with a DNA fragment corresponding to the 5' region of PTI-1 identifies multiple PTI-1 transcripts in RNAs from human carcinoma cell lines derived from the prostate, lung, breast, and colon. In contrast, PTI-1 RNA is not detected in human melanoma, neuroblastoma, osteosarcoma, normal cerebellum, or glioblastoma multiforme cell lines. By using a pair of primers recognizing a 280-bp region within the 630-bp 5' PTI-1 sequence, reverse transcription-PCR detects PTI-1 expression in patient-derived prostate carcinomas but not in normal prostate or benign hypertrophic prostate tissue. In contrast, reverse transcription-PCR detects prostate-specific antigen expression in all of the prostate tissues. These results indicate that PTI-1 may be a member of a class of oncogenes that could affect protein translation and contribute to carcinoma development in human prostate and other tissues. The approaches used, rapid expression cloning with the CREF-Trans 6 system and the DD strategy, should prove widely applicable for identifying and cloning additional human oncogenes.
Shen
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Department of Pathology, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA.
Su
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Olsson
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Fisher
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D000328:Adult; D000595:Amino Acid Sequence; D001483:Base Sequence; D002277:Carcinoma / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D015723:Gene Library; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D015513:Oncogene Proteins / Q000235:genetics*; D009857:Oncogenes / Q000235:genetics*; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000235:genetics; D016133:Polymerase Chain Reaction; D011470:Prostatic Hyperplasia / Q000235:genetics; D011471:Prostatic Neoplasms / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; C094814:EEF1A1 protein, human; D015513:Oncogene Proteins; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors