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BACKGROUND To evaluate their relative activity and specificity for prostate cells promoter and regulatory regions from three prostate-expressed genes–prostate-specific antigen (PSA), probasin, and relaxin H2–have been compared in prostate cell lines and in lines of breast, bladder, liver, kidney, lung, and ovarian origin. METHODS After transfection into different cell types, the activity of promoters was assayed using linked reporter genes and normalized against that of the Rous sarcoma virus. Activity was measured both in the presence and in the absence of co-transfected androgen receptor (AR). RESULTS PSA and probasin regulatory regions showed strong responsiveness to co-transfection of the AR in most cell types. The core PSA promoter region showed low activity and specificity, but the specificity and level of expression were substantially increased by inclusion of upstream sequences, particularly the enhancer region. Probasin promoter fragments showed specificity of expression for prostate cell lines but required AR for significant levels of expression. Relaxin promoter fragments directed significant AR-inducible expression in prostate cells but showed little specificity and variable AR responsiveness in other cell types. CONCLUSIONS Of regulatory regions tested, a 430-base pair probasin promoter and PSA enhancer/core promoter showed the best combination of AR-stimulated prostate cell expression with limited expression in other cell types. Prostate 35:18–26, 1998. © 1998 Wiley-Liss, Inc.

Recently our group completed a genome-wide linkage study investigating Australian and Spanish families with inherited risk of colorectal cancer (CRC). A minor linkage peak from that study located on chromosome 1 correlates with the location of a known CRC risk-modifying gene, prostaglandin synthase (PTGS2). PTGS2 encodes the inducible prostaglandin synthase enzyme cyclooxygenase-2 (COX-2). Prostaglandins are implicated in the initiation of carcinogenesis and progression of tumours. Sequencing of PTGS2 in a small subset of affected individuals identified a high frequency of the minor C allele of single nucleotide polymorphism rs5275. We then genotyped the rs5275 polymorphism in 183 affected and 223 unaffected individuals from our CRC predisposed families. Tests for association in the presence of linkage were made using family-based association tests. The C allele was found to be significantly associated (P

Up to 25% of colorectal cancer (CRC) may be caused by inherited genetic variants that have yet to be identified. Previous genome-wide linkage studies (GWLSs) have identified a new loci postulated to contain novel CRC risk genes amongst affected families carrying no identifiable mutations in any of the known susceptibility genes for familial CRC syndromes. To undertake a new GWLS, we recruited members from 54 non-syndromic families from Australia and Spain where at least two first-degree relatives were affected by CRC. We used single-nucleotide polymorphism arrays to genotype 98 concordant affected relative pairs that were informative for linkage analyses. We tested for genome-wide significance (GWS) for linkage to CRC using a quantile statistic method, and we found that GWS was achieved at the 5% level. Independently, using the PSEUDO gene-dropping algorithm, we also found that GWS for linkage to CRC was achieved (P=0.02). Merlin non-parametric linkage analysis revealed significant linkage to CRC for chromosomal region 10p15.3–p15.1 and suggestive linkage to CRC for regions on 14q and 9q. The 10p15.3–p15.1 has not been reported to be linked to hereditary CRC in previous linkage studies, but this region does harbour the Kruppel-like factor 6 (KLF6) gene that is known to be altered in common CRC. Further studies aimed at localising the responsible genes, and characterising their function will give insight into the factors responsible for susceptibility in such families, and perhaps shed further light on the mechanisms of CRC development.

The Escherichia coli enzyme (purine nucleoside phosphorylase, PNP) gene is delivered directly into PC3 tumors by one injection of replication-deficient human type-5 adenovirus (Ad5). Expressed PNP converts the systemically administered prodrug, 6MPDR, to a toxic purine, 6MP, causing cell death. We sought to increase the specificity of recombinant Ad vectors by controlling PNP expression with the promoter region from the androgen-dependent, prostate-specific rat probasin (Pb) gene. To increase its activity, the promoter was combined with the SV40 enhancer (SVPb). Cell lines were transfected with plasmids containing both a reporter gene, under SVPb control, and a reference gene cassette to allow normalization of expression levels. Plasmids expressed approximately 20-fold more reporter in prostate cancer than in other cells, but surprisingly, the SVPb element was both androgen-independent and retained substantial prostate specificity. Killing by Ad5-SVPb-PNP vector of cell lines cultured with 6MPDR for 6 days was 5- to 10-fold greater in prostate cancer than in liver or lung cells. In vivo, a single intratumoral injection of Ad5-SVPb-PNP (4 x 10(8) pfu), followed by 6MPDR administration twice daily for 6 days, significantly suppressed the growth of human prostate tumors in nude mice and increased their survival compared to control animals. Thus, the androgen-independent, prostate-targeting Ad5 vector reduces human prostate cancer growth significantly in vitro and in vivo. This first example of an androgen-independent vector points the way toward treatment of emerging androgen-independent prostate cancer in conjunction with hormone ablation therapy at a time when the tumor burden is low.

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.

Anna Abuli, Ceres Fernandez-Rozadilla, Virginia Alonso-Espinaco, Maria DoloresGiraldez, Jenifer Munoz, Xavier Bessa, Xavier Llor, Rodrigo Jover, Luis Carvajal-Carmona,Ian Tomlinson, Victor Moreno, Angel Carracedo, Antoni Castells, Montserrat Andreu,Clara Ruiz-Ponte, Sergi Castellvi-BelColorectal cancer (CRC) is the second most common cancer in Spain. Although the majorityof CRC is sporadic, inherited susceptibility is relevant in about 30-35% of cases, being dueto hereditary mutations in less than 5%. Much of the remaining genetic risk may beattributabletoalarge numberofcommon,low-penetrancegeneticvariants. ThereispreviousevidenceingeneticsusceptibilitytoCRCoftheinvolvementofgenesthatbelongtocolorectalcarcinogenesis pathways, genes involved in CRC susceptibility by studies in mice, and geneslocated on chromosomal regions (9q22 and 3q22) identified by genetic linkage analysis inCRC families. Objectives. To select genes from CRC pathways, mouse susceptibility studiesor chromosomal regions 9q22 and 3q22 with potential implications in CRC and its poly-morphisms with a putative functional effect, and to assess their implication in geneticsusceptibility toCRC by acase-control associationstudy. Patients andmethods. Case-controlgenetic association study in 2 stages in the EPICOLON cohort (stage 1, 515 cases vs.515 controls; replication stage 2, 900 cases vs. 900 controls). Results. We assessed 400polymorphisms in stage 1. Ten per cent showed significant association with CRC. In orderto validated these statistically significant associations, we replicated 42 polymorphisms inan independent cohort (stage 2), and 5 of them showed significant findings in both stages:[rs1444601, OR=0,85 (0,75-0,95, p=0,006); rs2071387, OR=0,56 (0,36-0,88, p=0,009);rs13088006, OR=0,67 (0,48-0,93, p=0,017); rs2297155, OR=0,35 (0,14-0,9, p=0,019);rs939453,OR=0,89(0,81-0,99,p=0,038)].Conclusions.Fivepolymorphismsshowedstatist-ically significant differences in both independent CRC cohorts, and they may correspondto new low-penetrance genetic components for CRC.