A common therapy for nonorgan-confined prostate cancer involves androgen deprivation. To develop a better understanding of the effect of androgen on prostatic cells, we have analyzed gene expression changes induced by dihydrotestosterone (DHT) in the androgen responsive prostate cancer line LNCaP, at both RNA and protein levels. Changes at the RNA level induced by DHT were determined by means of serial analysis of gene expression (SAGE), and protein profiling was done by means of quantitative two-dimensional polyacrylamide gel electrophoresis. Among 123,371 transcripts analyzed, a total of 28,844 distinct SAGE tags were identified representing 16,570 genes. Some 351 genes were significantly affected by DHT treatment at the RNA level (p < 0.05), of which 147 were induced and 204 repressed by androgen. In two independent experiments, the integrated intensity of 32 protein spots increased and 12 decreased at least two-fold in response to androgen, out of a total of 1031 protein spots analyzed. The change in intensity for most of the affected proteins identified could not be predicted based on the level of their corresponding RNA. Our study provides a global assessment of genes regulated by DHT and suggests a need for profiling at both RNA and protein levels for a comprehensive evaluation of patterns of gene expression.

Increased expression of proteases has been correlated with the malignant progression of a variety of tumors. We found a significant increase in cathepsin H expression in high-grade prostatic intraepithelial neoplasia and carcinoma of the prostate. Two forms of cathepsin H, the full-length form (CTSH) and a truncated form with a 12-amino acid deletion in its signal peptide region (CTSH⌬10 -21), were identified by cDNA sequence analysis. This deletion occurred not at the genomic level but likely at the RNA processing level. Both forms are expressed in prostate tissues as well as LNCaP, PC-3, and DU-145 prostate cancer cell lines. The deletion within the signal peptide region affected the trafficking of cathepsin H. Fluorescence microscopy, subcellular fractionation, and activity data indicated that the truncated form was perinuclear and secreted and had a reduced lysosomal association as compared with the full-length cathepsin H. Furthermore, the truncated cathepsin H was enzymatically active. Therefore, an increase in overall cathepsin H expression, particularly in the truncated form with a high secretion propensity, may affect cell biological behaviors such as those associated with tumor progression.

Tissue-type transglutaminase (TG-2) has been implicated in neurodegenerative diseases. In this study, induction of TG-2 was studied in rats following transient middle cerebral artery occlusion. Alterations in 2,3,5-triphenylterazolium chloride staining revealed maximum infarction 3 days after injury. Measurement of mRNA transcript levels by real-time PCR analysis showed both forms of TG-2 mRNA peaking on day 5 after injury in ipsilateral cortex, with greater induction of the full-length TG-2 (TG-L) transcript than the truncated form of the TG-2 (TG-S) transcript. However, in the ipsilateral hippocampus, peak induction of both forms of TG-2 mRNA peaked 1 day after injury and to a lesser extent than observed in the ipsilateral cortex. Western blot analysis demonstrated that TG-L protein expression progressively increased from 1 to 7 days after ischemia, with greater expression in cortex than hippocampus (525 ± 10% vs. 196 ± 8% of control, respectively). However, expression of TG-S was not detected. These results demonstrate that increased TG-2 mRNA and protein expression occurs in a delayed fashion following ischemic injury. The temporal profile of TG-2 induction after ischemia was similar to that observed after traumatic brain injury (previously described), suggesting a similar role of TG-2 in both pathological conditions.

SUMMARY We propose that, in prostate cancer, serine proteases occupy the apex of the proteolytic cascade and initiate the process of matrix degradation. Subsequently, collagenases are recruited after activation of procollagenases by another serine protease plasmin, which is formed by the activation of plasminogen by urokinase (u-PA). We show that u-PA alone can degrade fibronectin but not laminin. Serum free-conditioned medium (SF-CM) from DU-145 human prostatic carcinoma cells can degrade both fibronectin and laminin. Urokinase and gelatinase secretion was compared and correlated with the invasive ability of several human prostatic epithelial cell lines. DU-145 and LNCaP carcinoma cell lines and the following cell lines developed in our laboratories were used: PWR-1E (Ad12-SV40 immortalized, nontumorigenic), RWPE-1 (HPV-18 immortalized, nontumorigenic), and RWPE-2 (HPV-18 immortalized, Ki-ras-transformed, tumorigenic). Secreted urokinase levels were markedly increased in the malignant DU-145 and RWPE-2 cells and correlated with their invasive ability in vitro. Secreted gelatinase activity was undetectable in SF-CM from DU-145, trace in LNCaP, but detectable in the other three cell lines.The tumorigenic RWPE-2 cells secrete higher levels of gelatinases than their parent RWPE-1 cells. all-trans Retinoic acid (RA) caused a decrease in the extracellular u-PA and gelatinase activity and invasive ability of DU-145 and RWPE-2 cells. Treatment of cultures with RA reduced the ability of SF-CM to degrade fibronectin and laminin. These results may explain one mechanism by which retinoids inhibit invasion and metastasis. These studies have important translational value in the chemoprevention of prostatic intraepithelial neoplasia (PIN) and intervention in the progression of human prostate cancer.

A common therapy for nonorgan-confined prostate cancer involves androgen deprivation. To develop a better understanding of the effect of androgen on prostatic cells, we have analyzed gene expression changes induced by dihydrotestosterone (DHT) in the androgen responsive prostate cancer line LNCaP, at both RNA and protein levels. Changes at the RNA level induced by DHT were determined by means of serial analysis of gene expression (SAGE), and protein profiling was done by means of quantitative two-dimensional polyacrylamide gel electrophoresis. Among 123,371 transcripts analyzed, a total of 28,844 distinct SAGE tags were identified representing 16,570 genes. Some 351 genes were significantly affected by DHT treatment at the RNA level (p < 0.05), of which 147 were induced and 204 repressed by androgen. In two independent experiments, the integrated intensity of 32 protein spots increased and 12 decreased at least two-fold in response to androgen, out of a total of 1031 protein spots analyzed. The change in intensity for most of the affected proteins identified could not be predicted based on the level of their corresponding RNA. Our study provides a global assessment of genes regulated by DHT and suggests a need for profiling at both RNA and protein levels for a comprehensive evaluation of patterns of gene expression.