Activation of innate anti-viral immune response genes in symptomatic benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is the most common urologic disease in men over age 50. Symptoms include acute urinary retention, urgency to urinate and nocturia. For patients with severe symptoms, surgical treatment is used to remove the affected tissue. Interestingly, the presence of histologic BPH does not always correlate with symptoms. The molecular basis of symptomatic BPH and how it differs from asymptomatic BPH is unknown. Investigation into the molecular players involved in symptomatic BPH will likely give insight into novel therapeutic, and potentially preventative, targets. We determined the expression of genes involved in the innate anti-viral immune response in tissues from patients undergoing surgery to alleviate the symptoms of BPH, and compared the results with prostate tissue with histologic BPH, but from patients with few urinary issues (asymptomatic BPH). We found that expression of complement factor I, apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like protein 3G, oligoadenylate synthetase 2 and interferon-induced tetratricopeptide 1, four genes whose protein products are involved in the innate anti-viral immune response, was significantly transcriptionally upregulated in symptomatic BPH. Additionally, we observe hypomethylation and concomitant expression of ancient retroviral-like sequences, the long interspersed nuclear element 1 retrotransposons, in symptomatic BPH when compared with normal prostate tissue. These findings merit further investigation into the anti-viral immune response in symptomatic BPH.

DNA methylation paradigm shift: 15-lipoxygenase-1 upregulation in prostatic intraepithelial neoplasia and prostate cancer by atypical promoter hypermethylation.

Fifteen (15)-lipoxygenase type 1 (15-LO-1, ALOX15), a highly regulated, tissue- and cell-type-specific lipid-peroxidating enzyme has several functions ranging from physiological membrane remodeling, pathogenesis of atherosclerosis, inflammation and carcinogenesis. Several of our findings support a possible role for 15-LO-1 in prostate cancer (PCa) tumorigenesis. In the present study, we identified a CpG island in the 15-LO-1 promoter and demonstrate that the methylation status of a specific CpG within this island region is associated with transcriptional activation or repression of the 15-LO-1 gene. High levels of 15-LO-1 expression was exclusively correlated with one of the CpG dinucleotides within the 15-LO-1 promoter in all examined PCa cell-lines expressing 15-LO-1 mRNA. We examined the methylation status of this specific CpG in microdissected high grade prostatic intraepithelial neoplasia (HGPIN), PCa, metastatic human prostate tissues, normal prostate cell lines and human donor (normal) prostates. Methylation of this CpG correlated with HGPIN, PCa and metastatic human prostate tissues, while this CpG was unmethylated in all of the normal prostate cell lines and human donor (normal) prostates that either did not display or had minimal basal 15-LO-1 expression. Immunohistochemistry for 15-LO-1 was performed in prostates from PCa patients with Gleason scores 6, 7 [(4+3) and (3+4)], >7 with metastasis, (8-10) and 5 normal (donor) individual males. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect 15-LO-1 in PrEC, RWPE-1, BPH-1, DU-145, LAPC-4, LNCaP, MDAPCa2b and PC-3 cell lines. The specific methylated CpG dinucleotide within the CpG island of the 15-LO-1 promoter was identified by bisulfite sequencing from these cell lines. The methylation status was determined by COBRA analyses of one specific CpG dinucleotide within the 15-LO-1 promoter in these cell lines and in prostates from patients and normal individuals. Fifteen-LO-1, GSTPi and beta-actin mRNA expression in BPH-1, LNCaP and MDAPCa2b cell lines with or without 5-aza-2'-deoxycytidine (5-aza-dC) and trichostatin-A (TSA) treatment were investigated by qRT-PCR. Complete or partial methylation of 15-LO-1 promoter was observed in all PCa patients but the normal donor prostates showed significantly less or no methylation. Exposure of LNCAP and MDAPCa2b cell lines to 5-aza-dC and TSA resulted in the downregulation of 15-LO-1 gene expression. Our results demonstrate that 15-LO-1 promoter methylation is frequently present in PCa patients and identify a new role for epigenetic phenomenon in PCa wherein hypermethylation of the 15-LO-1 promoter leads to the upregulation of 15-LO-1 expression and enzyme activity contributes to PCa initiation and progression.

In vivo suicide gene therapy model using a newly discovered prostate-specific membrane antigen promoter/enhancer: a potential alternative approach to androgen deprivation therapy.

Prostate-specific membrane antigen (PSMA) is a type-2 membrane protein expressed in the prostate, and it is highly expressed in metastatic or poorly differentiated adenocarcinomas. Moreover, PSMA expression is upregulated by androgen deprivation. These advantages make PSMA a useful target for prostate cancer therapy, especially in combination with conventional hormonal treatment. We recently reported that a prostate-specific enhancer is present in the third intron of the PSMA gene. In this study, we have further analyzed the activity of PSMA promoter/enhancer in prostate cancer cells and cells of other tissue origins (breast cancer MCF-7, lung cancer H157, and colorectal cancer HCT8 cells), and we have examined whether this construct could be used for efficient expression of the suicide gene, cytosine deaminase (CD), in vivo. The PSMA promoter/enhancer expressed the luciferase reporter gene in the prostate cancer lines LNCaP and C4-2, with 8- to 20-fold higher expression than the simian virus 40 promoter/enhancer, although it was inactive in the other cell lines. This construct efficiently drove the suicide gene CD, sensitizing C4-2 cells to 5-fluorocytosine (5-FC) with the inhibitory concentration (IC(50)) <300 micromol/L in vitro. Athymic male nude mice bearing the transfected C4-2 cells were treated with intraperitoneal injections of either 5-FC (600 mg/kg) twice a day or saline solution for 3 weeks. C4-2 cell tumors were eliminated by 5-FC when they were expressing our therapeutic construct carrying CD under the regulatory control of the PSMA promoter/enhancer. Our results show the in vivo utility of the PSMA promoter/enhancer in a gene therapy situation targeting prostate cancer.

A tissue-specific enhancer of the prostate-specific membrane antigen gene, FOLH1.

Prostate-specific membrane antigen (PSMA) is an integral membrane protein that is highly expressed on the surface of prostate epithelial cells. It is also expressed on the vascular endothelium of a number of tumor types. We have used an enhancer trap approach with randomly cleaved overlapping DNA fragments from an approximately 55-kb P1 cosmid insert encompassing the 5' half and upstream sequences of the PSMA gene (FOLH1) to isolate an enhancer that strongly activates the FOLH1 core promoter region. The enhancer (PSME) is located in the third intron about 12 kb downstream from the start site of transcription and is characterized by a 72-bp direct repeat within a 331-bp core region. The PSME activates transcription from its own and heterologous promoters in prostate cell lines; enhancement is greatest in the PSMA-expressing cell line LNCaP (>250-fold). The PSME shows essentially no activity in five nonprostate cell lines. PSME-enhanced expression is repressed in the presence of androgen, mimicking the repression of the endogenous FOLH1 gene. The data demonstrate that both cell-type specificity and androgen regulation are intrinsic properties of the enhancer. These properties make the PSME an excellent candidate for regulation of gene expression in gene therapy approaches to prostate cancer.

Overview of evolving strategies incorporating prostate-specific membrane antigen as target for therapy.

Prostate-specific membrane antigen (PSMA) is a potential target in prostate cancer patients because it is very highly expressed and because it has been reported to be upregulated by androgen deprivation. This overview addresses the expression of the PSMA gene in terms of the promoter and enhancer and how that may play a role in gene therapy. We also review PSMA as a target for antibodies for imaging and treatment and the development of a novel hybrid T-cell receptor that combines the specificity of anti-PSMA antibodies with that of T-cell receptor activation when introduced into primary lymphocytes by retroviral-mediated gene transfer. We also discuss our recent findings on the expression of a PSMA-like gene and how that understanding allows specific targeting of PSMA.

Prostate-specific suicide gene therapy using the prostate-specific membrane antigen promoter and enhancer.

BACKGROUND: Prostate-specific membrane antigen (PSMA) is abundantly expressed in virtually 100% of prostate cancers and metastases. In addition, unlike prostate-specific antigen (PSA), PSMA is upregulated under conditions of androgen deprivation. Therefore, PSMA is an attractive therapeutic target for advanced prostate cancer. Recently, both the promoter and the enhancer driving prostate-specific expression of the PSMA gene were cloned. We describe here our analysis of the PSMA enhancer for the most active region(s) and present a way of using the enhancer in combination with the E. coli cytosine deaminase gene for suicide-driven gene therapy that converts the nontoxic prodrug 5-fluorocytosine (5-FC) into the cytotoxic drug 5-fluorouracil (5-FU) in prostate cancer cells. METHODS: Deletion constructs of the full-length PSMA enhancer were subcloned into a luciferase reporter vector containing either the PSMA or SV-40 promoter. The most active portion of the enhancer was then determined via luciferase activity in the C4-2 cell line. We then replaced the luciferase gene with the E. coli cytosine deaminase gene in the subclone that showed the most luciferase activity. The specificity of this technique was examined in vitro, using the prostate cancer cell line LNCaP, its androgen-independent derivative C4-2, and a number of nonprostatic cell lines. The toxicity of 5-FC and 5-FU on transiently transfected cell lines was then compared. RESULTS: The enhancer region originally isolated from the PSMA gene was approximately 2 kb. Deletion constructs revealed that at least two distinct regions seem to contribute to expression of the gene in prostate cancer cells, and therefore the best construct for prostate-specific expression was determined to be 1, 648 bp long. The IC(50) of 5-FC was similar in all cell lines tested (>10 mM). However, transfection with the 1648 nt PSMA enhancer and the PSMA promoter to drive the cytosine deaminase gene enhanced toxicity in a dose-dependent manner more than 50-fold, while cells that did not express the PSMA gene were not significantly sensitized by transfection. CONCLUSIONS: Suicide gene therapy using the PSMA enhancer may be of benefit to patients who have undergone androgen ablation therapy and are suffering a relapse of disease.

Prostate-specific membrane antigen is produced in tumor-associated neovasculature.

Prostate-specific membrane antigen (PSMA), a type II transmembrane protein, was originally thought to be strictly expressed in prostatic tissue, but recent studies have demonstrated PSMA protein expression in nonprostatic tumor neovasculature as well. Using immunohistochemistry, reverse transcription-PCR assays, and in situ hybridization, we have demonstrated PSMA mRNA transcripts and protein expression in the endothelium of tumor-associated neovasculature of multiple nonprostatic solid malignancies. In addition, we found no PSMA mRNA or protein expression in the vascular endothelial cells of corresponding benign tissue examples. Our findings expand the possible therapeutic role of PSMA and establish it as a unique biomarker specifically produced and expressed by tumor-associated neovasculature but not produced or expressed by normal vessels.

Characterization of the translated products of the alternatively spliced luteinizing hormone receptor in the ovine ovary throughout the oestrous cycle.

The luteinizing hormone receptor (LHR) is alternatively spliced. It is not known if the alternatively spliced mRNAs are translated in vivo, or indeed if they have any vital role to play. The B splice form has been detected in every species examined, and it encodes a putative protein with a high affinity LH/CG binding domain but no trans-membrane or intra-cellular domains. We raised antisera that recognize the putative protein of the B form, and the closely related G form, and showed that the B form mRNA is translated in the ovine ovary, but not kidney or liver. It localized to the luteal cytosolic and microsomal fractions and the levels declined during regression induced by treatment with prostaglandin F2alpha. We examined alternative splicing by RNase protection analyses and RT-PCR analyses of healthy pre-ovulatory follicles, atretic or steroidogenically-inactive follicles, and of newly formed, mid-luteal and regressing corpora lutea. There was approximately 5-fold more B form mRNA than A form. Thus we have evidence that the LHR B form is translated in vivo, but no evidence that alternative splicing of the LHR mRNA is differentially regulated, throughout the oestrous cycle.

Mapping, genomic organization and promoter analysis of the human prostate-specific membrane antigen gene.

Prostate-specific membrane antigen (PSMA) is a 100 kDa type II transmembrane protein with folate hydrolase and NAALAdase activity. PSMA is highly expressed in prostate cancer and the vasculature of most solid tumors, and is currently the target of a number of diagnostic and therapeutic strategies. PSMA is also expressed in the brain, and is involved in conversion of the major neurotransmitter NAAG (N-acetyl-aspartyl glutamate) to NAA and free glutamate, the levels of which are disrupted in several neurological disorders including multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease and schizophrenia. To facilitate analysis of the role of PSMA in carcinoma we have determined the structural organization of the gene. The gene consists of 19 exons spanning approximately 60 kb of genomic DNA. A 1244 nt portion of the 5' region of the PSMA gene was able to drive the firefly luciferase reporter gene in prostate but not breast-derived cell lines. We have mapped the gene encoding PSMA to 11p11-p12, however a gene homologous, but not identical, to PSMA exists on chromosome 11q14. Analysis of sequence differences between non-coding regions of the two genes suggests duplication and divergence occurred 22 million years ago.

A rapid and reliable PCR method for genotyping the ABO blood group. II: A2 and O2 alleles.

PCR permits direct genotyping of individuals at the ABO locus. Several methods have been reported for genotyping ABO that rely on differentiating the A, B, and O alleles at specific base substitutions. However, the O allele as defined by serology comprises at least two alleles (O1 and O2) at the molecular level, and most current ABO genotyping methods only take into account the O1 allele. Determining the presence of the O2 allele is critical, as this not-infrequent allele would be mistyped as an A or a B allele by standard PCR typing methods. Furthermore, none of the methods to date distinguish between the A1 and A2 alleles, even though 10% of all white persons are blood group A2. We have developed a method for genotyping the ABO locus that takes the O2 and A2 alleles into account. Typing for A2 and O2 by diagnostic restriction enzyme digestion is a sensitive, nonradioactive assay that provides a convenient method useful for forensic and paternity testing and for clarifying anomalous serological results.

A rapid and reliable PCR method for genotyping the ABO blood group.

The ABO blood group has been used extensively as a marker in population studies, epidemiology, and forensic work. However, until the cloning of the gene, it was not possible to determine the genotype of group A and B individuals without recourse to family studies. We have developed a method to determine the ABO genotype directly from human DNA using multiplex PCR and restriction enzyme analysis. Two PCR fragments spanning positions 258 and 700 of the cDNA sequence are amplified. The site at position 258 allows us to differentiate the O allele from the A and B alleles. The site at position 700 allows us to distinguish the B allele from the A and O alleles. Analysis at the two sites thus allows us to distinguish the three alleles. The multiplex PCR product is digested separately with four enzymes, two for each of the sites. The pair of enzymes for each site cut in a reciprocal fashion. Whereas one enzyme for each site is theoretically sufficient for genotyping, the use of complementary pairs of enzymes prevents the assignment of a false genotype as a result of false negative or partial digestion. This method is fast and reliable, does not rely on probing of blots, and should be widely applicable.