Dipeptidyl peptidase 9 enzymatic activity influences the expression of neonatal metabolic genes.

The success of dipeptidyl peptidase 4 (DPP4) inhibition as a type 2 diabetes therapy has encouraged deeper examination of the post-proline DPP enzymes. DPP9 has been implicated in immunoregulation, disease pathogenesis and metabolism. The DPP9 enzyme-inactive (Dpp9 gene knock-in; Dpp9 gki) mouse displays neonatal lethality, suggesting that DPP9 enzyme activity is essential in neonatal development. Here we present gene expression patterns in these Dpp9 gki neonatal mice. Taqman PCR arrays and sequential qPCR assays on neonatal liver and gut revealed differential expression of genes involved in cell growth, innate immunity and metabolic pathways including long-chain-fatty-acid uptake and esterification, long-chain fatty acyl-CoA binding, trafficking and transport into mitochondria, lipoprotein metabolism, adipokine transport and gluconeogenesis in the Dpp9 gki mice compared to wild type. In a liver cell line, Dpp9 knockdown increased AMP-activated protein kinase phosphorylation, which suggests a potential mechanism. DPP9 protein levels in liver cells were altered by treatment with EGF, HGF, insulin or palmitate, suggesting potential natural DPP9 regulators. These gene expression analyses of a mouse strain deficient in DPP9 enzyme activity show, for the first time, that DPP9 enzyme activity regulates metabolic pathways in neonatal liver and gut.

A rare variant in human fibroblast activation protein associated with ER stress, loss of enzymatic function and loss of cell surface localisation.

Fibroblast activation protein (FAP) is a focus of interest as a potential cancer therapy target. This membrane bound protease possesses the unique catalytic activity of hydrolysis of the post-proline bond two or more residues from the N-terminus of substrates. FAP is highly expressed in activated fibroblastic cells in tumours, arthritis and fibrosis. A rare, novel, human polymorphism, C1088T, encoding Ser363 to Leu, occurring in the sixth blade of the ß propeller domain, was identified in a family. Both in primary human fibroblasts and in Ser363LeuFAP transfected cells, we showed that this single substitution ablates FAP dimerisation and causes loss of enzyme activity. Ser363LeuFAP was detectable only in endoplasmic reticulum (ER), in contrast to the distribution of wild-type FAP on the cell surface. The variant FAP showed decreased conformational antibody binding, consistent with an altered tertiary structure. Ser363LeuFAP expression was associated with upregulation of the ER chaperone BiP/GRP78, ER stress sensor ATF6, and the ER stress response target phospho-eIF2α, all indicators of ER stress. Proteasomal inhibition resulted in accumulation of Ser363LeuFAP, indicating the involvement of ER associated degradation (ERAD). Neither CHOP expression nor apoptosis was elevated, so ERAD is probably important for protecting Ser363LeuFAP expressing cells. These data on the first loss of function human FAP gene variant indicates that although the protein is vulnerable to an amino acid substitution in the ß-propeller domain, inactive, unfolded FAP can be tolerated by cells.

GSH facilitates the binding and inhibitory activity of novel multidrug resistance protein 1 (MRP1) modulators.

MRP1 (ABCC1) is a membrane transporter that confers multidrug resistance in cancer cells by exporting chemotherapeutic agents, often in a reduced glutathione (GSH)-dependent manner. This transport activity can be altered by compounds (modulators) that block drug transport while simultaneously stimulating GSH efflux by MRP1. In MRP1-expressing cells, modulator-stimulated GSH efflux can be sufficient to deplete GSH and increase sensitivity to chemotherapy, enhancing cancer cell death. Further development of clinically useful MRP1 modulators requires a better mechanistic understanding of modulator binding and its relationship to GSH binding and transport. Here, we explore the mechanism of two MRP1 small molecule modulators, 5681014 and 7914321, in relation to a bipartite substrate-binding cavity of MRP1. Binding of these modulators to MRP1 was dependent on the presence of GSH but not its reducing capacity. Accordingly, the modulators poorly inhibited organic anion transport by K332L-mutant MRP1, where GSH binding and transport is limited. However, the inhibitory activity of the modulators was also diminished by mutations that limit E2 17ßG but spare GSH-conjugate binding and transport (W553A, M1093A, W1246A), suggesting overlap between the E2 17ßG and modulator binding sites. Immunoblots of limited trypsin digests of MRP1 suggest that binding of GSH, but not the modulators, induces a conformation change in MRP1. Together, these findings support the model, in which GSH binding induces a conformation change that facilitates binding of MRP1 modulators, possibly in a proposed hydrophobic binding pocket of MRP1. This study may facilitate the structure-guided design of more potent and selective MRP1 modulators.

Dual targeting of polyamine synthesis and uptake in diffuse intrinsic pontine gliomas.

Diffuse intrinsic pontine glioma (DIPG) is an incurable malignant childhood brain tumor, with no active systemic therapies and a 5-year survival of less than 1%. Polyamines are small organic polycations that are essential for DNA replication, translation and cell proliferation. Ornithine decarboxylase 1 (ODC1), the rate-limiting enzyme in polyamine synthesis, is irreversibly inhibited by difluoromethylornithine (DFMO). Herein we show that polyamine synthesis is upregulated in DIPG, leading to sensitivity to DFMO. DIPG cells compensate for ODC1 inhibition by upregulation of the polyamine transporter SLC3A2. Treatment with the polyamine transporter inhibitor AMXT 1501 reduces uptake of polyamines in DIPG cells, and co-administration of AMXT 1501 and DFMO leads to potent in vitro activity, and significant extension of survival in three aggressive DIPG orthotopic animal models. Collectively, these results demonstrate the potential of dual targeting of polyamine synthesis and uptake as a therapeutic strategy for incurable DIPG.

Targeting metabolic activity in high-risk neuroblastoma through Monocarboxylate Transporter 1 (MCT1) inhibition.

Amplification of the MYCN oncogene occurs in ~25% of primary neuroblastomas and is the single most powerful biological marker of poor prognosis in this disease. MYCN transcriptionally regulates a range of biological processes important for cancer, including cell metabolism. The MYCN-regulated metabolic gene SLC16A1, encoding the lactate transporter monocarboxylate transporter 1 (MCT1), is a potential therapeutic target. Treatment of neuroblastoma cells with the MCT1 inhibitor SR13800 increased intracellular lactate levels, disrupted the nicotinamide adenine dinucleotide (NADH/NAD+) ratio, and decreased intracellular glutathione levels. Metabolite tracing with 13C-glucose and 13C-glutamine following MCT1 inhibitor treatment revealed increased quantities of tricarboxylic acid (TCA) cycle intermediates and increased oxygen consumption rate. MCT1 inhibition was highly synergistic with vincristine and LDHA inhibition under cell culture conditions, but this combination was ineffective against neuroblastoma xenografts. Posttreatment xenograft tumors had increased synthesis of the MCT1 homolog MCT4/SLC16A, a known resistance factor to MCT1 inhibition. We found that MCT4 was negatively regulated by MYCN in luciferase reporter assays and its synthesis in neuroblastoma cells was increased under hypoxic conditions and following hypoxia-inducible factor (HIF1) induction, suggesting that MCT4 may contribute to resistance to MCT1 inhibitor treatment in hypoxic neuroblastoma tumors. Co-treatment of neuroblastoma cells with inhibitors of MCT1 and LDHA, the enzyme responsible for lactate production, resulted in a large increase in intracellular pyruvate and was highly synergistic in decreasing neuroblastoma cell viability. These results highlight the potential of targeting MCT1 in neuroblastoma in conjunction with strategies that involve disruption of pyruvate homeostasis and indicate possible resistance mechanisms.

CCI52 sensitizes tumors to 6-mercaptopurine and inhibits MYCN-amplified tumor growth.

The antimetabolite 6-mercaptopurine (6-MP) is an important component in the treatment of specific cancer subtypes, however, the development of drug resistance and dose-limiting toxicities can limit its effectiveness. The therapeutic activity of 6-MP requires cellular uptake, enzymatic conversion to thio-GMP and incorporation of thio-GTP into RNA and DNA, as well as inhibition of de novo purine synthesis by methyl-thio-IMP. Mechanisms that prevent 6-MP entry into the cell, prevent 6-MP metabolism or deplete thiopurine intermediates, can all lead to 6-MP resistance. We previously conducted a high-throughput screen for inhibitors of the multidrug transporter MRP4 using 6-MP sensitivity as the readout. In addition to MRP4-specific inhibitors, we identified a compound, CCI52, that sensitized cell lines to 6-MP independent of this transporter. CCI52 and its more stable analogue CCI52-14 also function as effective chemosensitizers in vivo, substantially extending survival in a transgenic mouse cancer model treated with 6-MP. Chemosensitization was associated with an increase in thio-IMP, suggesting that CCI52 functions directly on 6-MP uptake or metabolism. In addition to its chemosensitizing effects, CCI52 and CCI52-14 inhibited the growth of MYCN-amplified high-risk neuroblastoma cell lines and delayed tumor progression in a MYCN-driven, transgenic mouse model of neuroblastoma. These multifunctional inhibitors may be useful for the further development of anticancer agents and as tools to better understand 6-MP metabolism.

Inhibitor selectivity in the clinical application of dipeptidyl peptidase-4 inhibition.

DPP-4 (dipeptidyl peptidase-4) degrades the incretin hormones GLP-1 (glucagon-like peptide-1) and GIP (gastric inhibitory polypeptide), decreasing their stimulatory effects on beta-cell insulin secretion. In patients with Type 2 diabetes, meal-related GLP-1 secretion is reduced. DPP-4 inhibitors (alogliptin, saxagliptin, sitagliptin and vildagliptin) correct the GLP-1 deficiency by blocking this degradation, prolonging the incretin effect and enhancing glucose homoeostasis. DPP-4 is a member of a family of ubiquitous atypical serine proteases with many physiological functions beyond incretin degradation, including effects on the endocrine and immune systems. The role of DPP-4 on the immune system relates to its extra-enzymatic activities. The intracytosolic enzymes DPP-8 and DPP-9 are recently discovered DPP-4 family members. Although specific functions of DPP-8 and DPP-9 are unclear, a potential for adverse effects associated with DPP-8 and DPP-9 inhibition by non-selective DPP inhibitors has been posed based on a single adverse preclinical study. However, the preponderance of data suggests that such DPP-8 and DPP-9 enzyme inhibition is probably without clinical consequence. This review examines the structure and function of the DPP-4 family, associated DPP-4 inhibitor selectivity and the implications of DPP-4 inhibition in the treatment of Type 2 diabetes.

MRP1 modulators synergize with buthionine sulfoximine to exploit collateral sensitivity and selectively kill MRP1-expressing cancer cells.

Members of the ABC transporter family, particularly P-glycoprotein (P-gp, ABCB1), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance protein 1 (MRP1, ABCC1) are well characterized mediators of multidrug resistance, however their pharmacological inhibition has so far failed as a clinical strategy. Harnessing collateral sensitivity, a form of synthetic lethality where cells with acquired multidrug resistance exhibit hypersensitivity to unrelated agents, may be an alternative approach to targeting multidrug resistant tumour cells. We characterized a novel small molecule modulator that selectively enhanced MRP1-dependent efflux of reduced glutathione (GSH), an endogenous MRP1 substrate. Using cell lines expressing high levels of endogenous MRP1 from three difficult to treat cancer types-lung cancer, ovarian cancer and high-risk neuroblastoma-we showed that the MRP1 modulator substantially lowered intracellular GSH levels as a single agent. The effect was on-target, as MRP1 knockdown abolished GSH depletion. The MRP1 modulator was synergistic with the GSH synthesis inhibitor buthionine sulfoximine (BSO), with the combination exhausting intracellular GSH, increasing intracellular reactive oxygen species (ROS) and abolishing clonogenic capacity. Clonogenicity was rescued by the ROS scavenger N-acetylcysteine, implicating GSH depletion in the effect. The MRP1 modulator in combination with BSO also strongly sensitized cancer cells to MRP1-substrate chemotherapeutic agents, particularly arsenic trioxide, and was more effective than either the MRP1 modulator or BSO alone. GSH-depleting MRP1 modulators may therefore provide an enhanced therapeutic window to treat chemo-resistant MRP1-overexpressing pediatric and adult cancers.

Inhibition of polyamine synthesis and uptake reduces tumor progression and prolongs survival in mouse models of neuroblastoma.

Amplification of the MYCN oncogene is associated with an aggressive phenotype and poor outcome in childhood neuroblastoma. Polyamines are highly regulated essential cations that are frequently elevated in cancer cells, and the rate-limiting enzyme in polyamine synthesis, ornithine decarboxylase 1 (ODC1), is a direct transcriptional target of MYCN. Treatment of neuroblastoma cells with the ODC1 inhibitor difluoromethylornithine (DFMO), although a promising therapeutic strategy, is only partially effective at impeding neuroblastoma cell growth due to activation of compensatory mechanisms resulting in increased polyamine uptake from the surrounding microenvironment. In this study, we identified solute carrier family 3 member 2 (SLC3A2) as the key transporter involved in polyamine uptake in neuroblastoma. Knockdown of SLC3A2 in neuroblastoma cells reduced the uptake of the radiolabeled polyamine spermidine, and DFMO treatment increased SLC3A2 protein. In addition, MYCN directly increased polyamine synthesis and promoted neuroblastoma cell proliferation by regulating SLC3A2 and other regulatory components of the polyamine pathway. Inhibiting polyamine uptake with the small-molecule drug AMXT 1501, in combination with DFMO, prevented or delayed tumor development in neuroblastoma-prone mice and extended survival in rodent models of established tumors. Our findings suggest that combining AMXT 1501 and DFMO with standard chemotherapy might be an effective strategy for treating neuroblastoma.

Suppression of the ATP-binding cassette transporter ABCC4 impairs neuroblastoma tumour growth and sensitises to irinotecan in vivo.

The ATP-binding cassette transporter ABCC4 (multidrug resistance protein 4, MRP4) mRNA level is a strong predictor of poor clinical outcome in neuroblastoma which may relate to its export of endogenous signalling molecules and chemotherapeutic agents. We sought to determine whether ABCC4 contributes to development, growth and drug response in neuroblastoma in vivo. In neuroblastoma patients, high ABCC4 protein levels were associated with reduced overall survival. Inducible knockdown of ABCC4 strongly inhibited the growth of human neuroblastoma cells in vitro and impaired the growth of neuroblastoma xenografts. Loss of Abcc4 in the Th-MYCN transgenic neuroblastoma mouse model did not impact tumour formation; however, Abcc4-null neuroblastomas were strongly sensitised to the ABCC4 substrate drug irinotecan. Our findings demonstrate a role for ABCC4 in neuroblastoma cell proliferation and chemoresistance and provide rationale for a strategy where inhibition of ABCC4 should both attenuate the growth of neuroblastoma and sensitise tumours to ABCC4 chemotherapeutic substrates.

Extraenzymatic functions of the dipeptidyl peptidase IV-related proteins DP8 and DP9 in cell adhesion, migration and apoptosis.

The dipeptidyl peptidase IV gene family contains the four peptidases dipeptidyl peptidase IV, fibroblast activation protein, dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Dipeptidyl peptidase IV and fibroblast activation protein are involved in cell-extracellular matrix interactions and tissue remodeling. Fibroblast activation protein is upregulated and dipeptidyl peptidase IV is dysregulated in chronic liver disease. The effects of dipeptidyl peptidase 8 and dipeptidyl peptidase 9 on cell adhesion, cell migration, wound healing and apoptosis were measured by using green fluorescent protein fusion proteins to identify transfected cells. Dipeptidyl peptidase 9-overexpressing cells exhibited impaired cell adhesion, migration in transwells and monolayer wound healing on collagen I, fibronectin and Matrigel. Dipeptidyl peptidase 8-overexpressing cells exhibited impaired cell migration on collagen I and impaired wound healing on collagen I and fibronectin in comparison to the green fluorescent protein-transfected controls. Dipeptidyl peptidase 8 and dipeptidyl peptidase 9 enhanced induced apoptosis, and dipeptidyl peptidase 9 overexpression increased spontaneous apoptosis. Mechanistic investigations showed that neither the catalytic serine of dipeptidyl peptidase 8 or dipeptidyl peptidase 9 nor the Arg-Gly-Asp integrin-binding motif in dipeptidyl peptidase 9 were required for the impairment of cell survival, cell adhesion or wound healing. We have previously shown that the in vitro roles of dipeptidyl peptidase IV and fibroblast activation protein in cell-extracellular matrix interactions and apoptosis are similarly independent of catalytic activity. Dipeptidyl peptidase 9 overexpression reduced beta-catenin, tissue inhibitor of matrix metalloproteinases 2 and discoidin domain receptor 1 expression. This is the first demonstration that dipeptidyl peptidase 8 and dipeptidyl peptidase 9 influence cell-extracellular matrix interactions, and thus may regulate tissue remodeling.

MYCN promotes neuroblastoma malignancy by establishing a regulatory circuit with transcription factor AP4.

Amplification of the MYCN oncogene, a member of the MYC family of transcriptional regulators, is one of the most powerful prognostic markers identified for poor outcome in neuroblastoma, the most common extracranial solid cancer in childhood. While MYCN has been established as a key driver of malignancy in neuroblastoma, the underlying molecular mechanisms are poorly understood. Transcription factor activating enhancer binding protein-4 (TFAP4) has been reported to be a direct transcriptional target of MYC. We show for the first time that high expression of TFAP4 in primary neuroblastoma patients is associated with poor clinical outcome. siRNA-mediated suppression of TFAP4 in MYCN-expressing neuroblastoma cells led to inhibition of cell proliferation and migration. Chromatin immunoprecipitation assay demonstrated that TFAP4 expression is positively regulated by MYCN. Microarray analysis identified genes regulated by both MYCN and TFAP4 in neuroblastoma cells, including Phosphoribosyl-pyrophosphate synthetase-2 (PRPS2) and Syndecan-1 (SDC1), which are involved in cancer cell proliferation and metastasis. Overall this study suggests a regulatory circuit in which MYCN by elevating TFAP4 expression, cooperates with it to control a specific set of genes involved in tumor progression. These findings highlight the existence of a MYCN-TFAP4 axis in MYCN-driven neuroblastoma as well as identifying potential therapeutic targets for aggressive forms of this disease.

ABC transporters and neuroblastoma.

Neuroblastoma is the most common cancer of infancy and accounts for 15% of all pediatric oncology deaths. Survival rates of high-risk neuroblastoma remain less than 50%, with amplification of the MYCN oncogene the most important aberration associated with poor outcome. Direct transcriptional targets of MYCN include a number of ATP-binding cassette (ABC) transporters, of which ABCC1 (MRP1), ABCC3 (MRP3), and ABCC4 (MRP4) are the best characterized. These three transporter genes have been shown to be strongly prognostic of neuroblastoma outcome in primary untreated neuroblastoma. In addition to their ability to efflux a number of chemotherapeutic drugs, evidence suggests that these transporters also contribute to neuroblastoma outcome independent of any role in cytotoxic drug efflux. Endogenous substrates of ABCC1 and ABCC4 that may be potential candidates affecting neuroblastoma biology include molecules such as prostaglandins and leukotrienes. These bioactive lipid mediators have the ability to influence biological processes contributing to cancer initiation and progression, such as angiogenesis, cell signaling, inflammation, proliferation, and migration and invasion. ABCC1 and ABCC4 are thus potential targets for therapeutic suppression in high-risk neuroblastoma, and recently developed small-molecule inhibitors may be an effective strategy in treating aggressive forms of this cancer, as well as other cancers that express high levels of these transporters.

Identification of new MRP4 inhibitors from a library of FDA approved drugs using a high-throughput bioluminescence screen.

Multidrug resistance protein 4 (MRP4) effluxes a wide variety of drugs and endogenous signaling molecules from cells and has been proposed as an attractive therapeutic target in several solid tumors, including neuroblastoma and colorectal cancer. MRP4 also regulates the pharmacokinetics of its drug substrates and its absence can increase their tissue penetration. We observed that MRP4 can efflux the bioluminescence substrate d-luciferin, and exploited this phenomenon to develop a robust, high throughput, live cell-based bioluminescent screen to identify new MRP4 inhibitors. We applied this screen to a combined library of 3600 compounds, all of which were either FDA-approved drugs or bioactive compounds with defined mechanisms of action. From the primary screen, 36 compounds effectively inhibited MRP4 (>4-fold increase in bioluminescence), with inhibitors of receptor tyrosine kinases and phosphodiesterases highly over-represented. Selected compounds were tested for their ability to sensitize MRP4-overexpressing cell lines to the MRP4 substrate drugs 6-mercaptopurine and SN-38, with sensitization up to 6.5-fold with the ryanodine receptor antagonist dantrolene. These newly identified MRP4 inhibitors are readily available and are either established drugs or well-characterized bioactive compounds. As such, they should be immediately useful as investigative tools, and suitable for testing both in vitro and in vivo.

Calcitonin-specific transcription and splicing targets gene-directed enzyme prodrug therapy to medullary thyroid carcinoma cells.

Recurrent and metastatic medullary thyroid carcinoma (MTC) remains difficult to treat due to its limited responsiveness to chemotherapy, radiotherapy, and imaging. To investigate an alternative therapeutic approach, we examined the feasibility of targeting gene-directed enzyme/prodrug therapy delivered by adenoviral vectors to MTC. We previously described a modified human calcitonin (CT)/CT gene-related peptide promoter that produced increased expression while maintaining specificity for MTC cells. In this study, we introduced an additional level of specificity by using cell-specific splicing and examined whether the selectivity of the gene-directed enzyme/prodrug therapy for MTC was enhanced when both the promoter and splicing features were combined in a single transcription unit. Two replication-defective adenoviruses were constructed that expressed the Escherichia coli purine nucleoside phosphorylase (PNP) gene under the transcriptional control of a modified T2 promoter (Ad.T2-PNP) or the T2 promoter in combination with a CT minigene cassette in which the PNP gene was imbedded within the CT gene exon 4 (Ad.T2-CT/PNP). The specificity of PNP expression by Ad.T2-PNP, Ad.T2-CT/PNP, and control viruses in the MTC cell line, TT, and in a panel of non-MTC cell lines was evaluated. The highest level of PNP gene expression and the most effective cell killing in the presence of prodrug occurred in TT cells infected with Ad.T2-PNP, followed by Ad.T2-CT/PNP. Infection of most non-MTC cell lines, even with high multiplicities of Ad.T2-PNP, produced only low-level PNP expression that resulted in minimal cell killing in the presence of prodrug. High-level expression of PNP and effective cell killing was observed with both adenoviral gene constructs. The highest level of cell specificity was achieved with the combined use of promoter and splicing regulation in the Ad.T2-CT/PNP virus.

High-throughput screening identifies Ceefourin 1 and Ceefourin 2 as highly selective inhibitors of multidrug resistance protein 4 (MRP4).

Multidrug resistance protein 4 (MRP4/ABCC4), a member of the ATP-binding cassette (ABC) transporter superfamily, is an organic anion transporter capable of effluxing a wide range of physiologically important signalling molecules and drugs. MRP4 has been proposed to contribute to numerous functions in both health and disease; however, in most cases these links remain to be unequivocally established. A major limitation to understanding the physiological and pharmacological roles of MRP4 has been the absence of specific small molecule inhibitors, with the majority of established inhibitors also targeting other ABC transporter family members, or inhibiting the production, function or degradation of important MRP4 substrates. We therefore set out to identify more selective and well tolerated inhibitors of MRP4 that might be used to study the many proposed functions of this transporter. Using high-throughput screening, we identified two chemically distinct small molecules, Ceefourin 1 and Ceefourin 2, that inhibit transport of a broad range of MRP4 substrates, yet are highly selective for MRP4 over other ABC transporters, including P-glycoprotein (P-gp), ABCG2 (Breast Cancer Resistance Protein; BCRP) and MRP1 (multidrug resistance protein 1; ABCC1). Both compounds are more potent MRP4 inhibitors in cellular assays than the most widely used inhibitor, MK-571, requiring lower concentrations to effect a comparable level of inhibition. Furthermore, Ceefourin 1 and Ceefourin 2 have low cellular toxicity, and high microsomal and acid stability. These newly identified inhibitors should be of great value for efforts to better understand the biological roles of MRP4, and may represent classes of compounds with therapeutic application.

Targeted inactivation of dipeptidyl peptidase 9 enzymatic activity causes mouse neonate lethality.

Dipeptidyl Peptidase (DPP) 4 and related dipeptidyl peptidases are emerging as current and potential therapeutic targets. DPP9 is an intracellular protease that is regulated by redox status and by SUMO1. DPP9 can influence antigen processing, epidermal growth factor (EGF)-mediated signaling and tumor biology. We made the first gene knock-in (gki) mouse with a serine to alanine point mutation at the DPP9 active site (S729A). Weaned heterozygote DPP9 (wt/S729A) pups from 110 intercrosses were indistinguishable from wild-type littermates. No homozygote DPP9 (S729A/S729A) weaned mice were detected. DPP9 (S729A/S729A) homozygote embryos, which were morphologically indistinguishable from their wild-type littermate embryos at embryonic day (ED) 12.5 to ED 17.5, were born live but these neonates died within 8 to 24 hours of birth. All neonates suckled and contained milk spots and were of similar body weight. No gender differences were seen. No histological or DPP9 immunostaining pattern differences were seen between genotypes in embryos and neonates. Mouse embryonic fibroblasts (MEFs) from DPP9 (S729A/S729A) ED13.5 embryos and neonate DPP9 (S729A/S729A) mouse livers collected within 6 hours after birth had levels of DPP9 protein and DPP9-related proteases that were similar to wild-type but had less DPP9/DPP8-derived activity. These data confirmed the absence of DPP9 enzymatic activity due to the presence of the serine to alanine mutation and no compensation from related proteases. These novel findings suggest that DPP9 enzymatic activity is essential for early neonatal survival in mice.

Quantitation of fibroblast activation protein (FAP)-specific protease activity in mouse, baboon and human fluids and organs.

The protease fibroblast activation protein (FAP) is a specific marker of activated mesenchymal cells in tumour stroma and fibrotic liver. A specific, reliable FAP enzyme assay has been lacking. FAP's unique and restricted cleavage of the post proline bond was exploited to generate a new specific substrate to quantify FAP enzyme activity. This sensitive assay detected no FAP activity in any tissue or fluid of FAP gene knockout mice, thus confirming assay specificity. Circulating FAP activity was ∼20- and 1.3-fold less in baboon than in mouse and human plasma, respectively. Serum and plasma contained comparable FAP activity. In mice, the highest levels of FAP activity were in uterus, pancreas, submaxillary gland and skin, whereas the lowest levels were in brain, prostate, leukocytes and testis. Baboon organs high in FAP activity included skin, epididymis, bladder, colon, adipose tissue, nerve and tongue. FAP activity was greatly elevated in tumours and associated lymph nodes and in fungal-infected skin of unhealthy baboons. FAP activity was 14- to 18-fold greater in cirrhotic than in non-diseased human liver, and circulating FAP activity was almost doubled in alcoholic cirrhosis. Parallel DPP4 measurements concorded with the literature, except for the novel finding of high DPP4 activity in bile. The new FAP enzyme assay is the first to be thoroughly characterised and shows that FAP activity is measurable in most organs and at high levels in some. This new assay is a robust tool for specific quantitation of FAP enzyme activity in both preclinical and clinical samples, particularly liver fibrosis.