Intestinal tumor suppression in ApcMin/+ mice by prostaglandin D2 receptor PTGDR.

Our earlier work showed that knockout of hematopoietic prostaglandin D synthase (HPGDS, an enzyme that produces prostaglandin D2) caused more adenomas in Apc(Min/+) mice. Conversely, highly expressed transgenic HPGDS allowed fewer tumors. Prostaglandin D2 (PGD2) binds to the prostaglandin D2 receptor known as PTGDR (or DP1). PGD2 metabolites bind to peroxisome proliferator-activated receptor γ (PPARG). We hypothesized that Ptgdr or Pparg knockouts may raise numbers of tumors, if these receptors take part in tumor suppression by PGD2. To assess, we produced Apc(Min/+) mice with and without Ptgdr knockouts (147 mice). In separate experiments, we produced Apc(Min/+) mice expressing transgenic lipocalin-type prostaglandin D synthase (PTGDS), with and without heterozygous Pparg knockouts (104 mice). Homozygous Ptgdr knockouts raised total numbers of tumors by 30-40% at 6 and 14 weeks. Colon tumors were not affected. Heterozygous Pparg knockouts alone did not affect tumor numbers in Apc(Min/+) mice. As mentioned above, our Pparg knockout assessment also included mice with highly expressed PTGDS transgenes. Apc(Min/+) mice with transgenic PTGDS had fewer large adenomas (63% of control) and lower levels of v-myc avian myelocytomatosis viral oncogene homolog (MYC) mRNA in the colon. Heterozygous Pparg knockouts appeared to blunt the tumor-suppressing effect of transgenic PTGDS. However, tumor suppression by PGD2 was more clearly mediated by receptor PTGDR in our experiments. The suppression mechanism did not appear to involve changes in microvessel density or slower proliferation of tumor cells. The data support a role for PGD2 signals acting through PTGDR in suppression of intestinal tumors.

Insulin-like growth factor II peptide fusion enables uptake and lysosomal delivery of α-N-acetylglucosaminidase to mucopolysaccharidosis type IIIB fibroblasts.

Enzyme replacement therapy for MPS IIIB (mucopolysaccharidosis type IIIB; also known as Sanfilippo B syndrome) has been hindered by inadequate mannose 6 phosphorylation and cellular uptake of rhNAGLU (recombinant human α-N-acetylglucosaminidase). We expressed and characterized a modified rhNAGLU fused to the receptor-binding motif of IGF-II (insulin-like growth factor 2) (rhNAGLU-IGF-II) to enhance its ability to enter cells using the cation-independent mannose 6-phosphate receptor, which is also the receptor for IGF-II (at a different binding site). RhNAGLU-IGF-II was stably expressed in CHO (Chinese-hamster ovary) cells, secreted and purified to apparent homogeneity. The Km and pH optimum of the fusion enzyme was similar to those reported for rhNAGLU. Both intracellular uptake and confocal microscopy suggested that MPS IIIB fibroblasts readily take up the fusion enzyme via receptor-mediated endocytosis that was inhibited significantly (P<0.001) by the monomeric IGF-II peptide. Glycosaminoglycan storage was reduced by 60% (P<0.001) to near background levels in MPS IIIB cells after treatment with rhNAGLU-IGF-II, with half-maximal correction at concentrations of 3-12 pM. A similar cellular uptake mechanism via the IGF-II receptor was also demonstrated in two different brain tumour-derived cell lines. Fusion of rhNAGLU to IGF-II enhanced its cellular uptake while maintaining enzymatic activity, supporting its potential as a therapeutic candidate for treating MPS IIIB.

Mannose 6-phosphate conjugation is not sufficient to allow induction of immune tolerance to phenylalanine ammonia-lyase in dogs.

The immune response to exogenous protein has been shown to reduce therapeutic efficacy in animal models of enzyme replacement therapy. A previously published study demonstrated an immunosuppressive regimen which successfully induced immune tolerance to α-L-iduronidase in canines with mucopolysaccharidosis I. The two key requirements for success were high-affinity receptor-mediated enzyme uptake, conferred by mannose 6-phosphate conjugation, and immunosuppression with low-dose antigen exposure. In this study, we attempted to induce immune tolerance to phenylalanine ammonia-lyase by producing a recombinant mannose 6-phosphate conjugated form and administering it to normal dogs according to the previously published tolerance induction regimen. We found that the recombinant conjugated enzyme was stable, could bind to the mannose 6-phosphate receptor with high affinity, and its uptake into fibroblast cells was mediated by this receptor. However, at the end of a tolerance induction period, all dogs demonstrated an antigen-specific immune response when challenged with increasing doses of unconjugated phenylalanine ammonia-lyase. The average time to seroconvert was not significantly different among three separate groups of test animals (n = 3 per group) and was not significantly different from one group of control animals (n = 3). None of the nine test group animals developed immune tolerance to the enzyme using this method. This suggests that high-affinity cellular uptake mediated by the mannose 6-phosphate receptor combined with a previously studied tolerizing regimen is not sufficient to induce immune tolerance to an exogenous protein and that other factors affecting antigen distribution, uptake, and presentation are likely to be important.

Hematopoietic prostaglandin D synthase (HPGDS): a high stability, Val187Ile isoenzyme common among African Americans and its relationship to risk for colorectal cancer.

Intestinal tumors in Apc(Min/+) mice are suppressed by over-production of HPGDS, which is a glutathione transferase that forms prostaglandin D(2) (PGD(2)). We characterized naturally occurring HPGDS isoenzymes, to see if HPGDS variation is associated with human colorectal cancer risk. We used DNA heteroduplex analysis and sequencing to identify HPGDS variants among healthy individuals. HPGDS isoenzymes were produced in bacteria, and their catalytic activities were tested. To determine in vivo effects, we conducted pooled case-control analyses to assess whether there is an association of the isoenzyme with colorectal cancer. Roughly 8% of African Americans and 2% of Caucasians had a highly stable Val187lle isoenzyme (with isoleucine instead of valine at position 187). At 37°C, the wild-type enzyme lost 15% of its activity in 1h, whereas the Val187Ile form remained >95% active. At 50°C, the half life of native HPGDS was 9min, compared to 42 min for Val187Ile. The odds ratio for colorectal cancer among African Americans with Val187Ile was 1.10 (95% CI, 0.75-1.62; 533 cases, 795 controls). Thus, the Val187Ile HPGDS isoenzyme common among African Americans is not associated with colorectal cancer risk. Other approaches will be needed to establish a role for HPGDS in occurrence of human intestinal tumors, as indicated by a mouse model.

Biochemical characterization of fluorescent-labeled recombinant human alpha-L-iduronidase in vitro.

In vivo tracking of the delivery of therapeutic proteins is a useful tool for preclinical studies. However, many labels are too large to use without disrupting the normal uptake, function, or other properties of the protein. Low-molecular-weight fluorescent labels allow in vivo and ex vivo tracking of the distribution of therapeutic proteins, and should not alter the protein's characteristics. We tested the in vitro properties of fluorescent-labeled recombinant human alpha-l-iduronidase (rhIDU, the enzyme deficient in Hurler syndrome) and compared labeled to unlabeled proteins. Labeled rhIDU retained full enzymatic activity and showed similar kinetics to nonlabeled rhIDU. Uptake of labeled rhIDU into human Hurler fibroblasts, measured by activity assay, was equivalent to unlabeled rhIDU enzyme and showed an uptake constant of 0.72 nM. Labeled rhIDU was also able to enter cells via the mannose 6-phospate receptor pathway and reduce glycosaminoglycan storage in Hurler fibroblasts. Subcellular localization was verified within lysosomes by confocal microscopy. These findings suggest that fluorescent labeling does not significantly interfere with enzymatic activity, stability, or uptake, and validates this method as a way to track exogenously administered enzyme.

High dietary niacin may increase prostaglandin formation but does not increase tumor formation in ApcMin/+ mice.

High doses of niacin (nicotinic acid) used to treat dyslipidemias cause flushing, due to high levels of prostaglandin D(2) (PGD(2)). GPR109A, a G-protein coupled receptor, triggers the flushing in the skin. In addition to boosting PGD(2), niacin binding to GPR109A activates the entire prostanoid cascade. We found that GPR109A occurs throughout the gastrointestinal tract. Mice that alternated between a 1% niacin diet and a control diet had higher urinary prostaglandin E(2) (PGE(2)) metabolite levels when on niacin (2.8-fold increase; 95% confidence interval, 1.8-3.9). PGE(2) promotes tumors in the intestines, whereas PGD(2) may have an opposite effect, on the basis of our report showing that transgenic hematopoietic prostaglandin D synthase suppresses intestinal adenomas in Apc(Min/+) mice. To determine if either tumor growth or tumor suppression prevails, we fed Apc(Min/+) mice a 1% niacin diet and assessed tumor development. A 1% niacin diet did not affect the number of tumors scored histologically in Apc(Min/+) mice at 14 wk (33 mice on niacin, 33 controls). Although niacin stimulates production of various prostaglandins, our results support an interpretation that very high intakes of niacin are safe in relation to intestinal tumors in this model.

Early versus late treatment of spinal cord compression with long-term intrathecal enzyme replacement therapy in canine mucopolysaccharidosis type I.

Enzyme replacement therapy (ERT) with intravenous recombinant human alpha-l-iduronidase (IV rhIDU) is a treatment for patients with mucopolysaccharidosis I (MPS I). Spinal cord compression develops in MPS I patients due in part to dural and leptomeningeal thickening from accumulated glycosaminoglycans (GAG). We tested long-term and every 3-month intrathecal (IT) and weekly IV rhIDU in MPS I dogs age 12-15months (Adult) and MPS I pups age 2-23days (Early) to determine whether spinal cord compression could be reversed, stabilized, or prevented. Five treatment groups of MPS I dogs were evaluated (n=4 per group): IT+IV Adult, IV Adult, IT + IV Early, 0.58mg/kg IV Early and 1.57mg/kg IV Early. IT + IV rhIDU (Adult and Early) led to very high iduronidase levels in cervical, thoracic, and lumber spinal meninges (3600-29,000% of normal), while IV rhIDU alone (Adult and Early) led to levels that were 8.2-176% of normal. GAG storage was significantly reduced from untreated levels in spinal meninges of IT + IV Early (p<.001), IT+IV Adult (p=.001), 0.58mg/kg IV Early (p=.002) and 1.57mg/kg IV Early (p<.001) treatment groups. Treatment of dogs shortly after birth with IT+IV rhIDU (IT + IV Early) led to normal to near-normal GAG levels in the meninges and histologic absence of storage vacuoles. Lysosomal storage was reduced in spinal anterior horn cells in 1.57mg/kg IV Early and IT + IV Early animals. All dogs in IT + IV Adult and IV Adult groups had compression of their spinal cord at 12-15months of age determined by magnetic resonance imaging and was due to protrusion of spinal disks into the canal. Cord compression developed in 3 of 4 dogs in the 0.58mg/kg IV Early group; 2 of 3 dogs in the IT + IV Early group; and 0 of 4 dogs in the 1.57mg/kg IV Early group by 12-18months of age. IT + IV rhIDU was more effective than IV rhIDU alone for treatment of meningeal storage, and it prevented meningeal GAG accumulation when begun early. High-dose IV rhIDU from birth (1.57mg/kg weekly) appeared to prevent cord compression due to protrusion of spinal disks.

Hematopoietic prostaglandin D synthase suppresses intestinal adenomas in ApcMin/+ mice.

Aspirin and other nonsteroidal anti-inflammatory drugs prevent some cases of colon cancer by inhibiting prostaglandin (PG) synthesis. PGE(2) promotes colon neoplasia, as shown by knockout mouse studies on enzymes and receptors in the PG cascade. A few experiments 20 to 30 years ago suggested that PGD(2) may suppress tumors, but a role for biosynthetic enzymes for PGD(2) in tumor development has not been studied. We report here that disruption of the gene for hematopoietic PGD synthase in Apc(Min/+) mice led to approximately 50% more intestinal adenomas compared with controls. Tumor size was not affected. By immunohistochemistry, we detected hematopoietic PGD synthase mainly in macrophages and monocytes of the gut mucosa. The mean number of tumors did not increase with knockout of the gene for the lipocalin type of the enzyme, which is not produced in the intestine. On the other hand, Apc(Min/+) mice with transgenic human hematopoietic PGD synthase tended to have 80% fewer intestinal adenomas. The transgene produced high mRNA levels (375-fold over endogenous). There was a suggestion of higher urinary excretion of 11beta-PGF(2alpha) and a lower excretion of a PGE(2) metabolite in transgenic mice, but differences (30-40%) were not statistically significant. The results support an interpretation that hematopoietic PGD synthase controls an inhibitory effect on intestinal tumors. Further studies will be needed to prove possible mechanisms, such as routing of PG production away from protumorigenic PGE(2) or inhibition of the nuclear factor-kappaB cascade by PGD(2) metabolites.