While DTPA may not represent the ideal chelate structure for 99mTc(CO)3, the data provides proof-of-concept support for the development of a next-generation phosphoramidate-based PSMA inhibitor-conjugates for use as SPECT imaging agents.

BackgroundThis study is a comparative epigenetic evaluation of the methylation status of the DLC1 tumor suppressor gene in naturally-occurring canine lymphoma. Canine non-Hodgkin's lymphoma (NHL) has been proposed to be a relevant preclinical model that occurs spontaneously and may share causative factors with human NHL due to a shared home environment. The canine DLC1 mRNA sequence was derived from normal tissue. Using lymphoid samples from 21 dogs with NHL and 7 normal dogs, the methylation status of the promoter CpG island of the gene was defined for each sample using combined bisulfite restriction analysis (COBRA), methylation-specific PCR (MSP), and bisulfite sequencing methods. Relative gene expression was determined using real-time PCR.ResultsThe mRNA sequence of canine DLC1 is highly similar to the human orthologue and contains all protein functional groups, with 97% or greater similarity in functional regions. Hypermethylation of the 5' and 3' flanking regions of the promoter was statistically significantly associated with the NHL phenotype, but was not associated with silencing of expression or differences in survival.ConclusionThe canine DLC1 is constructed highly similarly to the human gene, which has been shown to be an important tumor suppressor in many forms of cancer. As in human NHL, the promoter CpG island of DLC1 in canine NHL samples is abnormally hypermethylated, relative to normal lymphoid tissue. This study confirms that hypermethylation occurs in canine cancers, further supporting the use of companion dogs as comparative models of disease for evaluation of carcinogenesis, biomarker diagnosis, and therapy.

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Abstract. Prostate-specific membrane antigen (PSMA), a well-known biomarker of prostate cancer, has also been found to be highly expressed in the neovasculature of multiple non-prostatic solid tumors. As a consequence, it has the potential to become a biomarker for tumor-associated vasculature. Herein, we describe an in vitro model for assessing PSMA expression associated with tube formation by primary human umbilical vein endothelial cells (HUVECs) cultured in Matrigel and induced by tumor-conditioned medium (TCM) derived from human breast cancer cells (MDA-MB-231). In contrast to vascular endothelial growth factor (VEGF)-containing endothelial cell medium, TCM induced higher expression of PSMA in HUVECs. The vessel-like tubes were detected by imaging with fluorescent PSMA inhibitors. Consequently, this in vitro model is expected to enable subsequent studies aimed at determining the role of PSMA in angiogenesis and factors that induce it.

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Introduction: A preliminary study was performed to identify aberrant gene promoter and CpG island DNA methylation with associated modification of gene expression in canine non-Hodgkin lymphoma (NHL) to develop the naturally-occurring model of human NHL. Methods: The canine OSW cell-line or native CLL cells were competitively hybridized against pooled DNA from two normal lymph nodes on a novel Nimblegen microarray and singly on the Affymetrix Canine Genome 2.0 expression array. The hypothesis was that promoter and CpG island DNA methylation and gene expression will be altered in canine NHL compared to normal, and that changes in canine NHL are similar to those in human NHL. DNA was sheared to 300bp and precipitated with an anti-methylcytosine antibody. Cy3- and Cy5-labeled samples were competitively hybridized on a three-plex, custom Nimblegen CpG-island and promoter array with a genomic backbone using a dye-swap. Quality assurance was performed per subarray by probe, intensity, probe length, CpG content, and GC content. Lowess normalization within GC-bin was performed per subarray. A-quantile normalization was performed across arrays. Statistical comparison was by Z score, and Q value was calculated, adjusting for multiple comparisons, determining final significance at Q<0.05. Quality of total RNA was confirmed using the BioRad Experion. Each node and cancer sample was individually hybridized using standard Affymetrix procedures. Results were normalized and > 2-fold changes compared. Results: Compared to normal, a 2-fold change in hypermethylation was present in 226 regions for OSW and 37 regions for CLL, and hypomethylation in 585 regions for OSW and 33 regions for CLL. Differential methylation was seen in 25 regions in common between the NHL samples. At least 2-fold change in gene expression was seen in 806 genes for OSW and 660 genes in CLL with 148 genes altered in common. Hypermethylated and silenced genes included TNF and KLHL14. Downregulated gene families included transcription factors, complement components, and receptors. Common upregulated genes included CDA, FADS1, Embigin, NR4A2, POLR1C, PSMA5, and SAG. ABCB1 was upregulated in OSW. Altered methylation was seen in gene families that included transcription factors, cytoskeletal and neural regulators, TGF-b signaling, and surface receptors. Hypomethylation in CpG islands in chromosomal termini was present in the NHL samples. Conclusions: Promoters and CpG islands are aberrantly hypermethylated and hypomethylated in canine NHL samples compared to normal lymphoid samples. Gene expression is also altered, but does not globally correlate directly to methylation changes, as seen in human NHL. Hypomethylation of terminal chromosomal, non-gene CpG islands appears to be present in canine NHL. Evaluation of these changes for biomarker development, therapeutic target identification, and carcinogenesis elucidation is warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4812. doi:10.1158/1538-7445.AM2011-4812