Prostatespecific Promoters

Recently, much effort has been made to develop tissue-specific delivery systems that eliminate the threat of harm to the patient. Several studies have demonstrated the importance of tissue-specific vectors, revealing systemic toxicity with the administration of high doses of nonspecific vectors (49,50). Viral vectors with broad tropisms can transduce any cell in the body, provided that the cell expresses the correct receptor specific to the virus. Through the use of prostate-specific promoters and enhancers, the expression of the therapeutic gene can be limited to cells that contain the appropriate activators and transcription factors. Several prostate-specific genes have been identified and well-characterized. Recently, chimeric promoters in which repressor elements have been deleted, activating mutations incorporated or enhancer sequences combined, have achieved greater prostate specificity and stronger transcriptional activity. No promoter is prostate cancer-specific; therefore, all prostate cells are susceptible to the delivered therapeutic gene. This however, is not significant, because the prostate is a nonvital organ in the postreproductive population.

3.1. PSA Promoter

PSA is released into the bloodstream when prostatic basement membrane is compromised, such as occurs in prostate cancer and, therefore, is used as a sensitive serum marker for the diagnosis and progression of prostate cancer (51). PSA, a serine protease, is encoded by the human Kallikrein 3 (hK3) gene (52). PSA expression is androgen receptor (AR)-dependent, and its transcript levels are significantly reduced in the absence of androgen (53). AR regulates PSA expression by binding to a 440-base pair (bp) androgen-responsive enhancer core (AREc) in the upstream 5' flanking region of the PSA gene (54,55). In vitro experiments have confirmed the tissue specificity and androgen dependency of this promoter (56). In addition, this promoter has been used in multiple gene therapy studies (57,58). Although this promoter confers high tissue specificity, its usefulness in men undergoing androgen ablation therapy is limited. To circumvent this problem, Gotoh et al. characterized the long (5837-bp) PSA promoter as less dependent on androgen and, therefore, more active than the short (631-bp) PSA promoter in the absence of androgen (57). Two cis-acting elements within the long PSA promoter, a 440-bp AREc and a 150-bp pN/H androgen-independent positive regulator, are responsible for this androgen-independent activity. A chimeric promoter with threefold higher activity than the native PSA promoter has been produced by juxtaposing both elements (59). Further attempts to enhance the activity while retaining specificity of the PSA promoter in Ad vectors include duplication of the AREc, which led to a 20-fold increase (60), or tandem duplication of the PSA promoter, which led to a 50-fold increase above basal promoter activity (61).

3.2. PSMA Promoter

PSMA was discovered by Horoszewicz et al. by a monoclonal antibody produced in mice immunized with cell membranes from LNCaP cells (62). PSMA is a type II integral membrane glycoprotein with folate hydrolase (63), N-acetylated a-linked acidic dipeptidase (64), and glutamate carboxypeptidase (65) activities. PSMA is expressed predominantly in prostate tissue and tumor neovasculature, with low levels detected in the gastrointestinal tract, salivary glands, kidney, and brain (66). Its expression is elevated higher in prostate cancer than in benign hyperplasia or normal prostate (67). In addition, serum PSMA levels are highest in patients with metastatic disease, suggesting enhanced PSMA expression as prostate cancer progresses (68). Unlike PSA, PSMA expression is upregulated under androgen-depleted conditions (69). A 1.2-kb PSMA promoter has been identified with high promoter activity (70); however, significant leaky activity in PSMA-negative cells limits its clinical usefulness as a gene therapy promoter. Recently, the PSMA enhancer (PSME) was discovered within the third intron of the PSMA gene, FOLH1 (71). Lee et al. have demonstrated PSMA activity mediated by NFATcl cooperatively binding at the AP-3 site within PSME (72). PSME has been used to transcriptionally target suicide (73) and oncolytic (74) gene therapies for prostate cancer under low androgen levels.

3.3. Prostate-Specific Enhancing Sequence

To achieve the highest transcriptional activity with strong prostate specificity, Lee et al. developed a novel chimeric promoter, PSES, under the hypothesis that AREc and PSME could function synergistically in any androgen environment. Through deletion and linker scan mutagenesis, the main prostate-specific enhancer activity of the PSA AREc and PSME were located in a 189-bp region called AREc3 and a 331-bp region called PSME(del2), respectively. PSES was developed by combining both AREc3 and PSME(del2) and placing AREc3 upstream from PSME(del2). AREc3 contains six GATA transcription factor-binding sites and three AR-binding sites leading to high enhancer activity once surrounding silencer regions were deleted. PSME(del2) contains eight AP-1 and three AP-3 binding sites acting as positive regulators in the absence of androgen, and a downstream deletion of an Alu transcription-silencing repeat. PSES showed significantly stronger transcriptional activity than either AREc3 or PSME(del2) alone in the presence or absence of andro-

gen. Furthermore, PSES demonstrated fivefold higher activity than universal promoter RSV and activity equal to CMV promoter. In vitro studies revealed that PSES is active in several PSA- and PSMA-positive prostate cancer cell lines, but not in PSA- and PSMA-negative prostate cells or nonprostate cell lines (75). A replication-competent Ad was created using the bidirectional PSES promoter to control both E1a and E4 adenoviral early genes (76). Because of its small size, high level of tissue specificity, and strong promoter activity regardless of androgen status, PSES is an ideal promoter for use in prostate cancer gene therapy.

3.4. OC Promoter

OC is a highly conserved bone y-carboxyglutamic acid protein that has been shown to be transcriptionally regulated by 1,25-dihydroxyvitamin D3 (77). This noncollagenous bone protein constitutes 1 to 2% of the total protein in bone, and its expression is limited to differentiated osteoblasts and osteotropic tumors, especially primary and metastatic prostate cancer (78). The osteoblastic nature of osseous prostate cancer metastases is well-characterized (79), and the mechanism is thought to be via its osteomimetic properties, specifically, its ability to express bone-related proteins such as OC (80). The human OC promoter contains numerous regulatory elements, including a vitamin D-responsive element, making it inducible by vitamin D3 administration (81,82), a glucocorticoid response element, an AP-1 binding site (83), and an AML-1 binding site, which has been shown to be responsible for 75% of OC expression (84). The OC promoter retained its tissue-specificity in a recombinant OC promoter-driven herpes simplex virus (HSV)-thymidine kinase (TK)-expressing adenoviral vector. Ko et al. developed a gene therapy for osteosarcoma in which coadministration of Ad-OC-TK and acyclovir resulted in osteoblast-specific cell toxicity (85). A similar strategy was developed for the intralesional injection of Ad-OC-TK to osseous prostate cancer metastases followed by administration of valacyclovir (VAL). In phase I clinical trials, this therapy induced apoptosis in every lesion treated, without serious adverse effects to the patients (86,87).

3.5. Human Telomerase Promoter

Telomeres are tandem repeat structures found at the termini of chromosomes that maintain chromosomal integrity by preventing DNA rearrangements, degradation, and end-to-end fusions. In most normal somatic cells, the telomeric cap is shortened with each cycle of DNA replication and cell division. When telomeres shorten to a critical length, cells progress toward irreversible arrest of growth and cellular senescence (88). In contrast, tumor cells have evolved a means to prevent telom-ere shortening through the activation of the catalytic component of human telomerase reverse tran-scriptase (hTERT) (89). The hTERT promoter region has been cloned and characterized, and contains a high GC content. Unlike most promoters, it does not contain TATA or CAAT boxes (90). Importantly, the hTERT promoter is active in most cancer cells, including prostate cancer cells (91) and inactive in most normal cells, thereby providing a unique approach to target cancer cells. Promising results have been reported using the hTERT promoter to deliver tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) (92) and Bax (93), inducers of apoptosis, to prostate cancer cells. Recently, the hTERT promoter was used to control adenoviral genes E1a and E1b to control the replication of an oncolytic Ad in a tumor-specific manner. This virus replicated efficiently in and killed a broad spectrum of cancer cells without harming normal human cells lacking telomerase activity (94). Clinical use of this promoter may be limited, however, to local intralesional gene therapy, because systemic delivery of an hTERT virus could have toxic effects on normal proliferating cells and stem cells in which telomerase is active.

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