Showing papers by "Thomas D. Schmittgen published in 2003"

Expression of prostate specific membrane antigen and three alternatively spliced variants of PSMA in prostate cancer patients

Abstract: Prostate specific membrane antigen (PSMA) is a folate gamma glutamyl carboxypeptidase that is oriented on the plasma membrane of normal and prostate cancer cells. A cytosolic version of PSMA, PSM', results from alternative splicing of the PSMA gene. Two additional alternatively spliced variants of PSMA, PSM-C and PSM-D, have been described recently. The ratio of PSMA to PSM' mRNA was higher in a small number of prostate cancer specimens compared to normal prostate cancer and benign prostatic hypertrophy (Su et al. Cancer Res 1995;55:1441). The intent of our study was to measure the gene expression of PSMA and the 3 PSMA splice variants in a large number of patient's tissues. A real-time, quantitative PCR assay was developed to quantify PSMA, PSM', PSM-C and PSM-D. Discrimination among the variants was achieved by designing unique primers and TaqMan probes for each gene. Amplification and detection was specific for the desired splice variant and was sensitive to one gene copy per reaction. The assay was used to quantify the gene expression in specimens of normal, benign, primary and metastatic prostate cancer from 72 patients. The mean PSMA expression (relative to 18S rRNA) was 2- to 3-fold lower in normal prostate (n = 4) compared to primary (n = 55, p = 0.31) and metastatic (n = 20, p = 0.33) prostate cancer. There was no difference in the PSMA expression between benign and cancerous prostate tissue from the same patients (n = 35). The ratio of PSMA to PSM' was lowest in the normal prostate and increased with increasing Gleason score (p < 0.001). The increased ratio in these tissues was a reflection of both increasing PSMA levels and decreasing PSM' mRNA. The expression of PSM-C did not differ in any of the tissue categories studied. The expression of PSM-D was similar in normal and primary prostate cancer but was 2-fold higher in lymph node (p < 0.005) and bone metastases (p < 0.05) compared to the primary tumors. Our results of the first detailed quantitative analysis of PSMA mRNA expression in patient's tissues demonstrate that PSMA and the 3 PSMA splice variants are expressed in normal, benign, cancerous and metastatic prostate cancer. We note increased PSMA expression in some malignant tissues, however, these increases are modest in magnitude. We also report that the expression of a novel splice variant, PSM-D, is elevated in prostate cancer metastases.

Expression pattern of mouse homolog of prostate-specific membrane antigen (FOLH1) in the transgenic adenocarcinoma of the mouse prostate model.

Abstract: BACKGROUND Prostate specific membrane antigen (PSMA) is expressed on the plasma membrane of normal prostate and in primary and metastatic prostate cancer in humans. Recently, a mouse homolog of PSMA (FOLH1) was identified that shares an 85% sequence homology with human PSMA. The transgenic adenocarcinoma of the mouse prostate (TRAMP) model displays spontaneous tumor development with age and metastasizes to tissues similar to human prostate cancer. This study characterized the expression of Folh1 in the TRAMP model to determine if the TRAMP would be a useful model system to evaluate PSMA-directed targeting strategies. METHODS A sensitive, real-time quantitative PCR assay was developed to measure Folh1 cRNA copy number in various tissues of 30–32-week-old TRAMP+ and age-matched, nontransgenic controls (TRAMP−). RESULTS Of the tissues studied, the highest expression of Folh1 was observed in the kidney and brain of both TRAMP+ and TRAMP− mice. Low levels of Folh1 cRNA (1–2 copies/ng total RNA) were detected in the tumor and lymph nodes of TRAMP + mice and in the seminal vesicles and lung of the TRAMP + and TRAMP− mice. The expression of Folh1 mRNA was sixfold higher in the prostate of 32-week-old TRAMP− mice compared to the tumor of 32-week-old TRAMP + mice. The rank order of the Folh1 expression in the tissues studied was kidney > brain > prostate > tumor > lymph nodes > lung > seminal vesicles > liver. Folh1 mRNA was undetectable in the bone marrow of both TRAMP+ and TRAMP− mice. Folate hydrolase activity assayed in the kidney, brain, lung, and liver paralleled the expression of Folh1 mRNA in these tissues. CONCLUSIONS We demonstrate that Folh1 is expressed at very high levels in some normal mouse tissues including the prostate gland and that the expression is not upregulated in the tumor of 32-week-old TRAMP + mice. Prostate 55: 308–316, 2003. © 2003 Wiley-Liss, Inc.

Inhibition of pre-mRNA splicing by cisplatin and platinum analogs.

Abstract: Our previous study demonstrated that the anticancer agent cis-diamminedichloroplatinum (II) (cis-DDP) inhibited the self-splicing activity of the Tetrahymena rRNA. The present study investigated the effects of cis-DDP on pre-mRNA splicing using a HeLa cell nuclear extract. A 2-h exposure of cis-DDP inhibited the splicing of the human B-globin pre-mRNA in a concentration-dependent manner. The concentration required for 50% inhibition of splicing (IC50) was 51 microM. Complete inhibition of spliceosome assembly occurred when the extracts were incubated with 150 microM cis-DDP. The inhibition of splicing by cis-DDP occurred at early events during spliceosome formation and to a greater extent if the extract was pre-incubated with cis-DDP in the absence of pre-mRNA. Splicing was inhibited when both pre-mRNA and cis-DDP were added simultaneously to the reaction mixture but not when cis-DDP was added 30 min after splicing was initiated with pre-mRNA. Clinically useful platinum analogs (ormaplatin, carboplatin, cis-tetraplatin and iproplatin) as well as the clinically ineffective Pt(dien)C1+, compound were tested for their ability to inhibit pre-mRNA splicing. The Pt(dien)C1+ compound, which acts in a monofunctional manner only, failed to inhibit splicing. A varying degree of splicing inhibition was observed for the other platinum analogs studied; the inhibitory activity decreased in the following order: ormaplatin > cis-tetraplatin > cis-DDP > iproplatin > carboplatin. We describe a novel mechanism that may be involved in the activity and/or toxicity of platinum agents.