Denise S. O'Keefe

Expression of prostate-specific membrane antigen (PSMA), increases cell folate uptake and proliferation and suggests a novel role for PSMA in the uptake of the non-polyglutamated folate, folic acid.

Prostate specific membrane antigen (PSMA) is a unique folate hydrolase that is significantly upregulated in prostate cancer. In a mouse model, PSMA is able to facilitate prostate carcinogenesis, however, little is known about the mechanism by which this occurs. As PSMA is able to hydrolyze polyglutamated folates, and cancer cells proliferate directly in response to available folate, we examined if expression of human PSMA in PC‐3 cells confers a proliferative advantage in a microenvironment with physiologically relevant folate levels.

Increased cancer cell proliferation in prostate cancer patients with high levels of serum folate.

A recent clinical trial revealed that folic acid supplementation is associated with an increased incidence of prostate cancer (Figueiredo et al., J Natl Cancer Inst 2009; 101(6): 432–435). As tumor cells in culture proliferate directly in response to available folic acid, the goal of our study was to determine if there is a similar relationship between patient folate status, and the proliferative capacity of tumors in men with prostate cancer.

DNA Methylation of the ABO Promoter Underlies Loss of ABO Allelic Expression in a Significant Proportion of Leukemic Patients

Background Loss of A, B and H antigens from the red blood cells of patients with myeloid malignancies is a frequent occurrence. Previously, we have reported alterations in ABH antigens on the red blood cells of 55% of patients with myeloid malignancies. Methodology/Principal Findings To determine the underlying molecular mechanisms of this loss, we assessed ABO allelic expression in 21 patients with ABH antigen loss previously identified by flow cytometric analysis as well as an additional 7 patients detected with ABH antigen changes by serology. When assessing ABO mRNA allelic expression, 6/12 (50%) patients with ABH antigen loss detected by flow cytometry and 5/7 (71%) of the patients with ABH antigen loss detected by serology had a corresponding ABO mRNA allelic loss of expression. We examined the ABO locus for copy number and DNA methylation alterations in 21 patients, 11 with loss of expression of one or both ABO alleles, and 10 patients with no detectable allelic loss of ABO mRNA expression. No loss of heterozygosity (LOH) at the ABO locus was observed in these patients. However in 8/11 (73%) patients with loss of ABO allelic expression, the ABO promoter was methylated compared with 2/10 (20%) of patients with no ABO allelic expression loss (P = 0.03). Conclusions/Significance We have found that loss of ABH antigens in patients with hematological malignancies is associated with a corresponding loss of ABO allelic expression in a significant proportion of patients. Loss of ABO allelic expression was strongly associated with DNA methylation of the ABO promoter.

Localization of the human growth arrest-specific gene (GAS1) to chromosome bands 9q21. 3-q22, a region frequently deleted in myeloid malignancies

The growth arrest-specific gene, Gas-1, was cloned from quiescent NIH3T3 mouse fibroblasts. Gas-1 mRNA accumulates when cells enter quiescence (Go) and expression is down-regulated by stimulation with serum or growth factors. DNA synthesis is inhibited when expression of Gas-1 is forced in normal or transformed NIH3T3 cell. Gas-1 encodes an integral membrane protein with two putative transmembrane domains flanking an extracellular region and with no significant similarities to any known proteins. The presence of an extracellular arginine-glycine-aspartic acid sequence suggests that the Gas-1 protein can associate with integrin-type receptors and may be involved in contact inhibition or in anchorage of the cells to the extracellular matrix. Since expression of Gas-1 is specific to quiescence, the Gas-1 protein may be required to sustain growth arrest or be involved in the control of differentiation. Thus, Gas-1 could act as a tumor suppressor gene by preventing uncontrolled proliferation. The authors report here the localization of GAS1 to human chromosome arm 9q at bands q21.3-q22.

Immunization with live amoebae, amoebic lysate and culture supernatant in experimental Naegleria meningoencephalitis.

Immunization with two doses of live Naegleria fowleri produced a survival of 34% of mice compared to 0% in unimmunized controls, whereas multiple doses of live N. fowleri resulted in loss of protective immunity. In contrast, multiple doses of N. fowleri lysate produced a survival of 30%, and multiple doses of N. fowleri culture supernatant produced a survival of 67 to 78%. Fractionation of the culture supernatant by column chromatography showed that all six fractions contained protective antigens, but the best protection occurred from immunization with the high molecular weight fraction (greater than 200,000 daltons).

Hypoexpression and Epigenetic Regulation of Candidate Tumor Suppressor Gene CADM-2 in Human Prostate Cancer

Purpose: Cell adhesion molecules (CADM) comprise a newly identified protein family whose functions include cell polarity maintenance and tumor suppression. CADM-1, CADM-3, and CADM-4 have been shown to act as tumor suppressor genes in multiple cancers including prostate cancer. However, CADM-2 expression has not been determined in prostate cancer. Experimental Design: The CADM-2 gene was cloned and characterized and its expression in human prostatic cell lines and cancer specimens was analyzed by reverse transcription-PCR and an immunohistochemical tissue array, respectively. The effects of adenovirus-mediated CADM-2 expression on prostate cancer cells were also investigated. CADM-2 promoter methylation was evaluated by bisulfite sequencing and methylation-specific PCR. Results: We report the initial characterization of CADM-2 isoforms: CADM-2a and CADM-2b, each with separate promoters, in human chromosome 3p12.1. Prostate cancer cell lines, LNCaP and DU145, expressed negligible CADM-2a relative to primary prostate tissue and cell lines, RWPE-1 and PPC-1, whereas expression of CADM-2b was maintained. Using immunohistochemistry, tissue array results from clinical specimens showed statistically significant decreased expression in prostate carcinoma compared with normal donor prostate, benign prostatic hyperplasia, prostatic intraepithelial neoplasia, and normal tissue adjacent to tumor (P < 0.001). Adenovirus-mediated CADM-2a expression suppressed DU145 cell proliferation in vitro and colony formation in soft agar. The decrease in CADM-2a mRNA in cancer cell lines correlated with promoter region hypermethylation as determined by bisulfite sequencing and methylation-specific PCR. Accordingly, treatment of cells with the demethylating agent 5-aza-2′-deoxycytidine alone or in combination with the histone deacetylase inhibitor trichostatin A resulted in the reactivation of CADM-2a expression. Conclusions: CADM-2a protein expression is significantly reduced in prostate cancer. Its expression is regulated in part by promoter methylation and implicates CADM-2 as a previously unrecognized tumor suppressor gene in a proportion of human prostate cancers. Clin Cancer Res; 16(22); 5390–401. ©2010 AACR.

Opposing Roles of Folate in Prostate Cancer

The US diet has been fortified with folic acid to prevent neural tube defects since 1998. The Physician Data Queries from the National Cancer Institute describe folate as protective against prostate cancer, whereas its synthetic analog, folic acid, is considered to increase prostate cancer risk when taken at levels easily achievable by eating fortified food or taking over-the-counter supplements. We review the present literature to examine the effects of folate and folic acid on prostate cancer, help interpret previous epidemiologic data, and provide clarification regarding the apparently opposing roles of folate for patients with prostate cancer. A literature search was conducted in Medline to identify studies investigating the effect of nutrition and specifically folate and folic acid on prostate carcinogenesis and progression. In addition, the National Health and Nutrition Examination Survey database was analyzed for trends in serum folate levels before and after mandatory fortification. Folate likely plays a dual role in prostate carcinogenesis. There remains conflicting epidemiologic evidence regarding folate and prostate cancer risk; however, there is growing experimental evidence that higher circulating folate levels can contribute to prostate cancer progression. Further research is needed to clarify these complex relationships.

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.

Novel nuclear localization of fatty acid synthase correlates with prostate cancer aggressiveness.

Fatty acid synthase is up-regulated in a variety of cancers, including prostate cancer. Up-regulation of fatty acid synthase not only increases production of fatty acids in tumors but also contributes to the transformed phenotype by conferring growth and survival advantages. In addition, increased fatty acid synthase expression in prostate cancer correlates with poor prognosis, although the mechanism(s) by which this occurs are not completely understood. Because fatty acid synthase is expressed at low levels in normal cells, it is currently a major target for anticancer drug design. Fatty acid synthase is normally found in the cytosol; however, we have discovered that it also localizes to the nucleus in a subset of prostate cancer cells. Analysis of the fatty acid synthase protein sequence indicated the presence of a nuclear localization signal, and subcellular fractionation of LNCaP prostate cancer cells, as well as immunofluorescent confocal microscopy of patient prostate tumor tissue and LNCaPs confirmed nuclear localization of this protein. Finally, immunohistochemical analysis of prostate cancer tissue indicated that nuclear localization of fatty acid synthase correlates with Gleason grade, implicating a potentially novel role in prostate cancer progression. Possible clinical implications include improving the accuracy of prostate biopsies in the diagnosis of low- versus intermediate-risk prostate cancer and the uncovering of novel metabolic pathways for the therapeutic targeting of androgen-independent prostate cancer.

Clearing up the confusion over the glutamate carboxypeptidase II gene

In the December 2003 issue of this journal, two letters were published discussing the validity of studies on the His475Tyr (C1561T) polymorphismof theGlutamateCarboxypeptidase II (GCPII) gene [Scala et al., 2003; Vieira, 2003]. In essence, both letters concluded (incorrectly we feel) that either an almost identical homolog of theGCPII gene exists on chromosome2, or that only one such homolog exists on chromosome 11. The H475Y polymorphism is thought to reduce the folate hydrolase activity of the enzyme by approximately 50% [Devlin et al., 2000], andas such is currently being tested for associationwith neural tube defects and hyperhomocysteinemia, among other folate metabolism related disorders [Vargas-Martinez et al., 2002; Vieira et al., 2002]. In fact, the human genome does contain another gene that is almost identical to the GCPII gene, and that can be amplified using the primers used by Devlin et al. [2000] and Vieira et al. [2002], if non-restrictive PCR conditions are used. In addition, the multiple names and substrates of the GCPII enzyme have appeared to cause confusion regarding the number of transcripts that arise from this gene. We seek to use this forum to clarify this situation. The cDNA and genomic sequences of this gene were originally characterized by our laboratory [Israeli et al., 1993; O’Keefe et al., 1998], and the gene was called Prostate-Specific MembraneAntigen (PSMA) due to its cell localization and very high expression in the prostate and prostate cancer. The single full-length transcript encodes for an enzyme that can reduce polyglutamated folates to the monoglutamyl forms, hence the gene and/or enzyme has been called folate hydrolase I (FOLH1) and folylpoly-gamma-glutamate carboxypeptidase (FGCP). However, the enzyme is also able to utilize the neurotransmitter N-acetyl-aspartyl-glutamate (NAAG) as a substrate, and is often calledNAALADase I by groups involved in neuroprotection studies. It is important to realize, however, that the enzymatic activity for both substrates is the same, leading to the encompassing term Glutamate Carboxypeptidase II [reviewed in O’Keefe et al., 2001]. While we were working on PSMA/GCPII as a diagnostic and therapeutic target for prostate cancer, we, like Scala et al. [2003], scanned the high-throughput-genome-sequence database for possible homologs of PSMA/GCPII. We also found sequences on chromosomes 2 and 13 that were highly homologous to the gene, and, therefore, tested to see if they truly existed by using PCR analysis of somatic cell hybrids containing either human chromosome 2 or 13. Neither the locus on chromosome 2 or 13 exists in the somatic cell hybrids we analyzed (provided by Coriell Cell Repositories, Camden, NJ). However as we have previously reported, a homolog to PSMA/ GCPII does reside on the long arm of chromosome 11 [O’Keefe et al., 1998]. We and others have since shown that the PSMA/ GCPII (GenBank accession NM_004476) and the recently discovered PSMA-like gene (GenBank accession AF261715) arose froma gene duplication event on chromosome 11q13 that occurred approximately 14million years ago, and that the copy called PSMA/GCPII along with other duplicated genes from 11q13,nowresides on the short armof chromosome11 [O’Keefe et al., 1998, 2004; Zhang et al., 2001]. During our genomic analysis of the homologous PSMA-like gene, we noted that the primers described by Devlin et al. [2000] and used by Vieira et al. [2002] could amplify both the PSMA/GCPII and PSMA-like genomic sequences (accession numbers AF007544 and AC024234 respectively—the latter sequence is from the high-throughput genome sequence database), although there are two non-contiguousmismatches in the reverse primer compared to the genomic sequence of the PSMA-like gene. Similarly, the Vargas-Martinez et al. [2002] forward primer is 100% homologous to the PSMA-like gene sequence, while the reverse primer has two non-contiguous mismatches but also mismatches with the third TAT of the (TAT)3 repeat that was present in the sequence we looked at. Given that a BLAST search of the PSMA and PSMA-like exon 13 and 300 bp of intron sequence either side reveals that the two genes have over 97% identity with each other, our suggestion would be to design primers based on regions where the two sequences differ. Alternatively, we used the primers described by Devlin et al. [2000], together with an annealing temperature of 608C and High-Fidelity Taq Polymerase (Roche Applied Science, Indianapolis, IN, USA). Using DNA from a somatic cell hybrid containing human chromosome 11q as a negative control, we were able to exclude amplification of the PSMA-like gene while successfully amplifying the H475Y polymorphism of the PSMA gene.