Surface-epitope masking and expression cloning identifies the human prostate carcinoma tumor antigen gene PCTA-1 a member of the galectin gene family.

The selective production of monoclonal antibodies (mAbs) reacting with defined cell surface-expressed molecules is now readily accomplished with an immunological subtraction approach, surface-epitope masking (SEM). Using SEM, prostate carcinoma (Pro 1.5) mAbs have been developed that react with tumor-associated antigens expressed on human prostate cancer cell lines and patient-derived carcinomas. Screening a human LNCaP prostate cancer cDNA expression library with the Pro 1.5 mAb identifies a gene, prostate carcinoma tumor antigen-1 (PCTA-1). PCTA-1 encodes a secreted protein of approximately 35 kDa that shares approximately 40% sequence homology with the N-amino terminal region of members of the S-type galactose-binding lectin (galectin) gene family. Specific galectins are found on the surface of human and marine neoplastic cells and have been implicated in tumorigenesis and metastasis. Primer pairs within the 3' untranslated region of PCTA-1 and reverse transcription-PCR demonstrate selective expression of PCTA-1 by prostate carcinomas versus normal prostate and benign prostatic hypertrophy. These findings document the use of the SEM procedure for generating mAbs reacting with tumor-associated antigens expressed on human prostate cancers. The SEM-derived mAbs have been used for expression cloning the gene encoding this human tumor antigen. The approaches described in this paper, SEM combined with expression cloning, should prove of wide utility for developing immunological reagents specific for and identifying genes relevant to human cancer.

Prevention of cardiovascular and renal pathology of aging by the advanced glycation inhibitor aminoguanidine.

Human aging is impacted severely by cardiovascular disease and significantly but less overtly by renal dysfunction. Advanced glycation endproducts (AGEs) have been linked to tissue damage in diabetes and aging, and the AGE inhibitor aminoguanidine (AG) has been shown to inhibit renal and vascular pathology in diabetic animals. In the present study, the effects of AG on aging-related renal and vascular changes and AGE accumulation were studied in nondiabetic female Sprague-Dawley (S-D) and Fischer 344 (F344) rats treated with AG (0.1% in drinking water) for 18 mo. Significant increases in the AGE content in aged cardiac (P < 0.05), aortic (P < 0.005), and renal (P < 0.05) tissues were prevented by AG treatment (P < 0.05 for each tissue). A marked age-linked vasodilatory impairment in response to acetylcholine and nitroglycerine was prevented by AG treatment (P < 0.005), as was an age-related cardiac hypertrophy evident in both strains (P < 0.05). While creatinine clearance was unaffected by aging in these studies, the AGE/ creatinine clearance ratio declined 3-fold in old rats vs. young rats (S-D, P < 0.05; F344, P < 0.01), while it declined significantly less in AG-treated old rats (P < 0.05). In S-D but not in F344 rats, a significant (P < 0.05) age-linked 24% nephron loss was completely prevented by AG treatment, and glomerular sclerosis was markedly suppressed (P < 0.01). Age-related albuminuria and proteinuria were markedly inhibited by AG in both strains (S-D, P < 0.01; F344, P < 0.01). These data suggest that early interference with AGE accumulation by AG treatment may impart significant protection against the progressive cardiovascular and renal decline afflicting the last decades of life.

Rescue of adult mouse motoneurons from injury-induced cell death by glial cell line-derived neurotrophic factor.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to rescue developing motoneurons in vivo and in vitro from both naturally occurring and axotomy-induced cell death. To test whether GDNF has trophic effects on adult motoneurons, we used a mouse model of injury-induced adult motoneuron degeneration. Injuring adult motoneuron axons at the exit point of the nerve from the spinal cord (avulsion) resulted in a 70% loss of motoneurons by 3 weeks following surgery and a complete loss by 6 weeks. Half of the loss was prevented by GDNF treatment. GDNF also induced an increase (hypertrophy) in the size of surviving motoneurons. These data provide strong evidence that the survival of injured adult mammalian motoneurons can be promoted by a known neurotrophic factor, suggesting the potential use of GDNF in therapeutic approaches to adult-onset motoneuron diseases such as amyotrophic lateral sclerosis.

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.

Genetic engineering of vein grafts resistant to atherosclerosis.

Previously, researchers have speculated that genetic engineering can improve the long-term function of vascular grafts which are prone to atherosclerosis and occlusion. In this study, we demonstrated that an intraoperative gene therapy approach using antisense oligodeoxynucleotide blockage of medial smooth muscle cell proliferation can prevent the accelerated atherosclerosis that is responsible for autologous vein graft failure. Selective blockade of the expression of genes for two cell cycle regulatory proteins, proliferating cell nuclear antigen and cell division cycle 2 kinase, was achieved in the smooth muscle cells of rabbit jugular veins grafted into the carotid arteries. This alteration of gene expression successfully redirected vein graft biology away from neointimal hyperplasia and toward medial hypertrophy, yielding conduits that more closely resembled normal arteries. More importantly, these genetically engineered grafts proved resistant to diet-induced atherosclerosis. These findings establish the feasibility of developing genetically engineered bioprostheses that are resistant to failure and better suited to the long-term treatment of occlusive vascular disease.