Elsevier

Gene

Volume 285, Issues 1–2, 20 February 2002, Pages 247-256
Gene

Upregulation of prostate specific membrane antigen/folate hydrolase transcription by an enhancer

https://doi.org/10.1016/S0378-1119(02)00397-9Get rights and content

Abstract

Prostate specific membrane antigen (PSMA), also known as folate hydrolase (FOLH1), is a 100 kDa glycoprotein with elevated expression in prostate epithelial tissue. Expression of PSMA is upregulated as prostate tumor grade increases and is found in the vasculature of many tumors, with no presence in benign tissues. Due to the potential of the regulatory elements of the PSMA promoter and enhancer to be used in gene therapy and as biomarkers for prostate cancer under conditions of androgen ablation during treatment, we sequenced and analyzed the ability of 5.5 kb of PSMA promoter/leader region to promote transcription. A recently discovered enhancer, found in the third intron of the PSMA gene, FOLH1, was also studied. The promoter/leader region sequence provided basal expression in transcription assays, while addition of the enhancer activated transcription 41-fold in transient transfections and 144-fold in stable transfections of the LNCaP prostate cell line. This enhancement of transcription was not found in nonprostate cell lines or prostate cell lines that do not express PSMA. An analysis of the ability of androgens to act via the PSMA promoter/leader region and enhancer to activate transcription in transiently transfected LNCaP cells revealed no significant androgen response using the FOLH1 promoter/leader region and a downregulation of 42% with addition of the enhancer. In stably transfected LNCaP cells, the FOLH1 promoter/leader region produced a 21% downregulation in response to androgens, while addition of the enhancer resulted in a 45% downregulation. These results demonstrate significant upregulation of transcription by the PSMA promoter/enhancer, with specificity for the LNCaP prostate cell line, and downregulation of transcription in response to physiological levels of androgen.

Introduction

Prostate specific membrane antigen (PSMA), also referred to as folate hydrolase 1 (FOLH1), is a 100 kDa glycoprotein which was initially isolated from the cell membranes of LNCaP cells (Horoszewicz et al., 1983, Horoszewicz et al., 1987). Although a function for PSMA in the prostate is not known, three enzymatic activities have been demonstrated: folate hydrolase activity by cleavage of γ-linked glutamates (Pinto et al., 1996), N-acetylated α-linked acidic dipeptidase (NAALADase) activity by cleavage of α-linked glutamates (Carter et al., 1996) from N-acetyl-l-aspartyl-l-glutamate (NAAG), and dipeptidyl peptidase IV activity by cleavage of the bond between proline and amido methylcoumarin in glycine-proline-7-amido-4-methylcoumarin (Pangalos et al., 1999). Due to PSMA's folate hydrolase activity, the gene that encodes for PSMA has been designated FOLH1. The FOLH1 gene, located at chromosome 11p11-p12, contains 19 exons and spans approximately 60 kb (O'Keefe et al., 1998). Recently, an enhancer discovered in the third intron has been shown to have specificity for LNCaP cells when linked to the PSMA promoter or to other promoters (Watt et al., 2001), which makes the PSMA promoter and enhancer increasingly attractive for consideration in the treatment of prostate cancer.

Prostate cancer is the most frequently diagnosed cancer and it is the second leading cause of death in men, after lung cancer. In 2001, an estimated 198,100 men were diagnosed with and 31,500 died from prostate cancer in the United States (American Cancer Society, Cancer Facts & Figures, 2001). Prostate specific antigen (PSA) tests are routinely performed on men to detect, by immunoassay of the PSA level in serum, the presence of prostate cancer. Currently, androgen deprivation is the most effective treatment for advanced prostate cancer (Wright et al., 1996), but androgen deprivation downregulates PSA serum levels (Young et al., 1991, Gleave et al., 1992, Montgomery et al., 1992, Wolf et al., 1992), decreasing the utility of PSA as a tumor marker. Like PSA, PSMA has elevated expression in prostate cancer tissue, as opposed to normal tissue (Wright et al., 1995, Kawakami and Nakayama, 1997, Bostwick et al., 1998). However, unlike PSA, PSMA expression has been reported to increase under conditions of androgen deprivation (Israeli et al., 1994, Wright et al., 1996, Silver et al., 1997), potentially making it a more useful tool in tracking a patient's response during the prostate cancer treatment regimen.

Ribonuclease protection and Western blot analysis have shown that PSMA is most strongly expressed in the prostate, with lower levels detected in the brain, salivary gland, and small intestine (Israeli et al., 1994, Troyer et al., 1995). Immunohistochemistry has consistently detected strong PSMA expression in prostate epithelia, with weaker expression in the colon, small intestine, and kidney tubules (Lopes et al., 1990, Wright et al., 1995, Liu et al., 1997, Silver et al., 1997, Chang et al., 1999, Dumas et al., 1999). Weak expression in breast (Chang et al., 1999), cardiac muscle, and sweat glands (Lopes et al., 1990) has also been observed, while strong reactivity with skeletal muscle has only been seen with the 7E11-C5.3 antibody which recognizes an epitope of PSMA's cytoplasmic domain (Lopes et al., 1990, Chang et al., 1999). Of considerable importance is the observation of expression in the neovasculature of a wide range of tumors, while immunohistochemistry has not detected PSMA in the vasculature of benign tissue samples (Liu et al., 1997, Silver et al., 1997, Chang et al., 1999, Dumas et al., 1999). PSMA expression in tumor tissue increases as prostate tumor grade increases (Wright et al., 1995, Kawakami and Nakayama, 1997, Bostwick et al., 1998). Repression of PSMA steady state expression by androgens has been demonstrated by immunohistochemistry (Wright et al., 1996, Silver et al., 1997) and ribonuclease protection assays (Israeli et al., 1994).

Previously, we had analyzed the ability of 2 kb of sequence upstream of the FOLH1 gene to promote transcription (Good et al., 1999). Transient transfections of a luciferase expression construct, pGL3-Basic, containing 2 kb of FOLH1 upstream sequence, and subsequent luciferase assays revealed approximately a 2-fold increase in transcription over transfections with the vector alone. In an effort to discover elements that enhance transcription or are androgen-responsive, we decided to search further upstream of the FOLH1 gene. In this paper, we investigate the ability of 5527 bp of FOLH1 promoter/leader region sequence and an enhancer located in the third intron of the FOLH1 gene to promote transcription and we investigate their role in androgen-responsiveness of PSMA expression.

Section snippets

Reagents and supplies

Dulbecco's MEM, fetal bovine serum (FBS), non-essential amino acids, sodium pyruvate, l-glutamine, penicillin-streptomycin, and Geneticin (G418 Sulfate) were from Gibco BRL (Gaithersburg, MD). Charcoal-stripped FBS was from Cocalico Biologicals (Reamstown, PA); dihydrotestosterone (DHT), Taq DNA polymerase and PCR buffer were from Sigma (St. Louis, MO). PCR primers were from Sigma Genosys (The Woodlands, TX) and PCR nucleotide mix was from USB Corporation (Cleveland, OH). Human placental DNA

The LNCaP prostate cell line expresses PSMA and PSA and is androgen-responsive

The LNCaP cell line has been the model cell line for prostate cancer research. LNCaP cells express androgen receptor and exhibit androgen-responsiveness. Prostatic acid phosphatase (PAP), PSMA, and PSA are all produced by LNCaP cells. The presence of PSMA mRNA was confirmed in LNCaP cells versus the androgen-insensitive, PSMA-nonexpressing PC3 and DU145 prostate cell lines by Northern blotting. Total RNA was prepared from LNCaP, PC3, and DU145 cells and analyzed on a denaturing gel. Ethidium

Discussion

Northern blot analysis of PSMA mRNA levels indicates that PSMA transcription is specific for the LNCaP prostate cell line, which expresses PSMA, versus other prostate cell lines, DU145 and PC3, which do not express PSMA (Fig. 1A). In a previous study of the regulatory elements responsible for this specificity for LNCaP cells, up to 2 kb of sequence upstream of the PSMA gene was analyzed for its ability to regulate transcription (O'Keefe et al., 1998, Good et al., 1999). Normalization using a

Acknowledgements

The authors thank the Department of Pathology and Laboratory Medicine of the Overton Brooks VA Medical Center (Shreveport, LA) and Susan Dauenhauer for performance of the PSA immunoassays. This research was supported by grant HD29381 from the National Institutes of Health and a merit review grant from the Department of Veterans Affairs to S.R.G.

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