CYP3A4-Catalyzed Simvastatin Metabolism as a Non-Invasive Marker of Small Intestinal Health in Celiac Disease.
American journal of gastroenterology
2013; 108 (8): 1344-1351
Dietary Vitamin D-3 and 1,25-Dihydroxyvitamin D-3 (Calcitriol) Exhibit Equivalent Anticancer Activity in Mouse Xenograft Models of Breast and Prostate Cancer
2012; 153 (6): 2576-2587
Histological examination of duodenal biopsies is the gold standard for assessing intestinal damage in celiac disease (CD). A noninvasive marker of disease status is necessary, because obtaining duodenal biopsies is invasive and not suitable for routine monitoring of CD patients. As the small intestine is a major site of cytochrome P450 3A4 (CYP3A4) activity and also the location of the celiac lesion, we investigated whether patients with active CD display abnormal pharmacokinetics of an orally administered CYP3A4 substrate, simvastatin (SV), which could potentially be used for noninvasive assessment of their small intestinal health.Preclinical experiments were performed in CYP3A4-humanized mice to examine the feasibility of the test. Subsequently, a clinical trial was undertaken with 11 healthy volunteers, 18 newly diagnosed patients with CD, and 25 celiac patients who had followed a gluten-free diet (GFD) for more than 1 year. The maximum concentration (Cmax) of orally administered SV plus its major non-CYP3A4-derived metabolite SV acid (SV equivalent (SVeq)) was measured, and compared with clinical, histological, and serological parameters.In CYP3A4-humanized mice, a marked decrease in SV metabolism was observed in response to enteropathy. In the clinical setting, untreated celiac patients displayed a significantly higher SVeq Cmax (46±24?nM) compared with treated patients (21±16?nM, P<0.001) or healthy subjects (19±11?nM, P<0.005). SVeq Cmax correctly predicted the diagnosis in 16/18 untreated celiac patients, and also the recovery status of all follow-up patients that exhibited normal or near-normal biopsies (Marsh 0-2). All patients with abnormal SVeq Cmax showed a reduction in the value after 1 year of following a GFD.SVeq Cmax is a promising noninvasive marker for assessment of small intestinal health. Further studies are warranted to establish its clinical utility for assessing gut status of patients with CD.
View details for DOI 10.1038/ajg.2013.151
View details for PubMedID 23732466
Activation and Inhibition of Transglutaminase 2 in Mice
2012; 7 (2)
1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3) or calcitriol], the hormonally active vitamin D metabolite, exhibits anticancer actions in models of breast cancer and prostate cancer. Because CYP27B1 (1?-hydroxylase), the enzyme catalyzing 1,25(OH)(2)D(3) formation in the kidney, is also expressed in extrarenal tissues, we hypothesize that dietary vitamin D(3) will be converted to 25(OH)D(3) in the body and then to 1,25(OH)(2)D(3) locally in the cancer microenvironment in which it will exert autocrine/paracrine anticancer actions. Immunocompromised mice bearing MCF-7 breast cancer xenografts showed significant tumor shrinkage (>50%) after ingestion of a vitamin D(3)-supplemented diet (5000 IU/kg) compared with a control diet (1000 IU/kg). Dietary vitamin D(3) inhibition of tumor growth was equivalent to administered calcitriol (0.025, 0.05, or 0.1 ?g/mouse, three times a week). Both treatments equivalently inhibited PC-3 prostate cancer xenograft growth but to a lesser extent than the MCF-7 tumors. Calcitriol at 0.05 ?g and 0.1 ?g caused modest but statistically significant increases in serum calcium levels indicating that the dietary vitamin D(3) comparison was to a maximally safe calcitriol dose. Dietary vitamin D(3) did not increase serum calcium, demonstrating its safety at the concentration tested. The vitamin D(3) diet raised circulating 1,25 dihydroxyvitamin D levels and did not alter CYP27B1 mRNA in the kidney but increased it in the tumors, suggesting that extrarenal sources including the tumors contributed to the elevated circulating 1,25 dihydroxyvitamin D(3). Both calcitriol and dietary vitamin D(3) were equipotent in suppressing estrogen synthesis and signaling and other proinflammatory and growth signaling pathways. These preclinical data demonstrate the potential utility of dietary vitamin D(3) supplementation in cancer prevention and therapy.
View details for DOI 10.1210/en.2011-1600
View details for Web of Science ID 000304370700010
View details for PubMedID 22454149
Inhibitory effects of calcitriol on the growth of MCF-7 breast cancer xenografts in nude mice: selective modulation of aromatase expression in vivo.
Hormones & cancer
2011; 2 (3): 190-202
Transglutaminase 2 (TG2) is an allosterically regulated enzyme with transamidating, deamidating and cell signaling activities. It is thought to catalyze sequence-specific deamidation of dietary gluten peptides in the small intestines of celiac disease patients. Because this modification has profound consequences for disease pathogenesis, there is considerable interest in the design of small molecule TG2 inhibitors. Although many classes of TG2 inhibitors have been reported, thus far an animal model for screening them to identify promising celiac drug candidates has remained elusive. Using intraperitoneal administration of the toll-like receptor 3 (TLR3) ligand, polyinosinic-polycytidylic acid (poly(I?C)), we induced rapid TG2 activation in the mouse small intestine. Dose dependence was observed in the activation of TG2 as well as the associated villous atrophy, gross clinical response, and rise in serum concentration of the IL-15/IL-15R complex. TG2 activity was most pronounced in the upper small intestine. No evidence of TG2 activation was observed in the lung mucosa, nor were TLR7/8 ligands able to elicit an analogous response. Introduction of ERW1041E, a small molecule TG2 inhibitor, in this mouse model resulted in TG2 inhibition in the small intestine. TG2 inhibition had no effect on villous atrophy, suggesting that activation of this enzyme is a consequence, rather than a cause, of poly(I?C) induced enteropathy. Consistent with this finding, administration of poly(I?C) to TG2 knockout mice also induced villous atrophy. Our findings pave the way for pharmacological evaluation of small molecule TG2 inhibitors as drug candidates for celiac disease.
View details for DOI 10.1371/journal.pone.0030642
View details for Web of Science ID 000301979000009
View details for PubMedID 22319575
Comparison of rectal and tympanic core body temperature measurement in adult Guyanese squirrel monkeys (Saimiri sciureus sciureus)
JOURNAL OF MEDICAL PRIMATOLOGY
2011; 40 (2): 135-141
Calcitriol (1,25-dihydroxyvitamin D(3)), the hormonally active metabolite of vitamin D, exerts many anticancer effects in breast cancer (BCa) cells. We have previously shown using cell culture models that calcitriol acts as a selective aromatase modulator (SAM) and inhibits estrogen synthesis and signaling in BCa cells. We have now examined calcitriol effects in vivo on aromatase expression, estrogen signaling, and tumor growth when used alone and in combination with aromatase inhibitors (AIs). In immunocompromised mice bearing MCF-7 xenografts, increasing doses of calcitriol exhibited significant tumor inhibitory effects (~50% to 70% decrease in tumor volume). At the suboptimal doses tested, anastrozole and letrozole also caused significant tumor shrinkage when used individually. Although the combinations of calcitriol and the AIs caused a statistically significant increase in tumor inhibition in comparison to the single agents, the cooperative interaction between these agents appeared to be minimal at the doses tested. Calcitriol decreased aromatase expression in the xenograft tumors. Importantly, calcitriol also acted as a SAM in the mouse, decreasing aromatase expression in the mammary adipose tissue, while increasing it in bone marrow cells and not altering it in the ovaries and uteri. As a result, calcitriol significantly reduced estrogen levels in the xenograft tumors and surrounding breast adipose tissue. In addition, calcitriol inhibited estrogen signaling by decreasing tumor ER? levels. Changes in tumor gene expression revealed the suppressive effects of calcitriol on inflammatory and growth signaling pathways and demonstrated cooperative interactions between calcitriol and AIs to modulate gene expression. We hypothesize that cumulatively these calcitriol actions would contribute to a beneficial effect when calcitriol is combined with an AI in the treatment of BCa.
View details for DOI 10.1007/s12672-011-0073-7
View details for PubMedID 21686077
Uterine Leiomyoma in a Guyanese Squirrel Monkey (Saimiri sciureus sciureus)
JOURNAL OF THE AMERICAN ASSOCIATION FOR LABORATORY ANIMAL SCIENCE
2010; 49 (2): 226-230
Measuring core body temperature in a manner that is safe for animals and veterinary personnel is an important part of a physical examination. For nonhuman primates, this can involve increased restraint, additional stress, as well as the use of anesthetics and their deleterious effects on body temperature measurements. The purpose of this study was to compare two non-invasive methods of infrared tympanic thermometry to standard rectal thermometry in adult squirrel monkeys.Tympanic temperatures were collected from 37 squirrel monkeys and compared to rectal temperatures using a human and veterinary infrared tympanic thermometer.Compared with rectal temperature measurements, the human tympanic thermometer readings were not significantly different, while the veterinary tympanic thermometer measurements were significantly higher (P<0.05). There were no differences between sexes.The tympanic thermometer designed for use in humans can be used in adult squirrel monkeys as an alternative to rectal thermometry for assessing core body temperature.
View details for DOI 10.1111/j.1600-0684.2010.00449.x
View details for Web of Science ID 000287965500009
View details for PubMedID 20946145
Profiling Human Androgen Receptor Mutations Reveals Treatment Effects in a Mouse Model of Prostate Cancer
MOLECULAR CANCER RESEARCH
2008; 6 (11): 1691-1701
An adult female squirrel monkey (Saimiri sciureus) presented with a 3.0 x 2.5 cm firm mass palpable within the caudal abdomen. Differentiation of the organs or structures involved with the mass could not be achieved with radiography or ultrasonography. Exploratory laparotomy revealed a mass within the lumen of the uterus; the mass was removed by partial hysterectomy. On gross examination, the mass was a focally extensive, unencapsulated, firm, solitary tumor. Histologic examination revealed that the mass was composed of interlacing bundles of smooth muscle cells with little fibrous stroma. The cells were elongated with poorly delineated borders and cigar-shaped nuclei, each containing a single, small nucleolus. Fewer than 1 mitosis per 20 high-power (magnification, x 400) fields were present. These gross and histologic findings supported a diagnosis of uterine leiomyoma. Although leiomyomas are the most common tumor of the reproductive tract in nonhuman primates, to our knowledge the current lesion is the first uterine leiomyoma reported to occur in a squirrel monkey.
View details for Web of Science ID 000275882100017
View details for PubMedID 20353700
X-linked inhibitor of apoptosis deficiency in the TRAMP mouse prostate cancer model
CELL DEATH AND DIFFERENTIATION
2008; 15 (5): 831-840
Gain-of-function mutations in the androgen receptor (AR) are found in prostate cancer and are implicated in the failure of hormone therapy. Most studies have emphasized the ligand-binding domain (LBD) where mutations can create promiscuous receptors, but mutations in the NH(2)-terminal transactivation domain have also been found. To assess AR alteration as a mechanism of treatment resistance, a mouse model (h/mAR-TRAMP) was used in which the murine AR coding region is replaced by human sequence and prostate cancer initiated by a transgenic oncogene. Mice received either no treatment, androgen depletion by castration, or treatment with antiandrogens, and 20 AR transcripts were sequenced per end-stage tumor. All tumors expressed several mutant alleles, although most mutations were low frequency. Some mutations that occurred multiple times within the population were differentially located dependent on treatment. Mutations in castrated or antiandrogen-treated mice were widely dispersed but with a prominent cluster in the LBD (amino acids 736-771), whereas changes in intact mice centered near the NH(2)-terminal polymorphic glutamine tract. Functional characterization of selected LBD mutant alleles showed diverse effects on AR activity, with about half of the mutations reducing transactivation in vitro. One receptor, AR-R753Q, behaved in a cell- and promoter-dependent manner, although as a germ-line mutation it causes androgen insensitivity syndrome. This suggests that alleles that are loss of function during development may still activate a subset of AR targets to become gain of function in tumorigenesis. Mutant ARs may thus use multiple mechanisms to evade cancer treatment.
View details for DOI 10.1158/1541-7786.MCR-08-0273
View details for Web of Science ID 000261134500004
View details for PubMedID 19010817
Androgen receptor variants and prostate cancer in humanized AR mice
JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY
2008; 108 (3-5): 230-236
Deregulation of apoptotic pathways plays a central role in cancer pathogenesis. X-linked inhibitor of apoptosis protein (XIAP), is an antiapoptotic molecule, whose elevated expression has been observed in tumor specimens from patients with prostate carcinoma. Studies in human cancer cell culture models and xenograft tumor models have demonstrated that loss of XIAP sensitizes cancer cells to apoptotic stimuli and abrogates tumor growth. In view of these findings, XIAP represents an attractive antiapoptotic therapeutic target for prostate cancer. To examine the role of XIAP in an immunocompetent mouse cancer model, we have generated transgenic adenocarcinoma of the mouse prostate (TRAMP) mice that lack XIAP. We did not observe a protective effect of Xiap deficiency in TRAMP mice as measured by tumor onset and overall survival. In fact, there was an unexpected trend toward more aggressive disease in the Xiap-deficient mice. These findings suggest that alternative mechanisms of apoptosis resistance are playing a significant oncogenic role in the setting of Xiap deficiency. Our study has implications for XIAP-targeting therapies currently in development. Greater understanding of these mechanisms will aid in combating resistance to XIAP-targeting treatment, in addition to optimizing selection of patients who are most likely to respond to such treatment.
View details for DOI 10.1038/cdd.2008.15
View details for Web of Science ID 000254960500004
View details for PubMedID 18259199
Glutamine tract length of human androgen receptors affects hormone-dependent and -independent prostate cancer in mice
HUMAN MOLECULAR GENETICS
2008; 17 (1): 98-110
Androgen, acting via the androgen receptor (AR), is central to male development, differentiation and hormone-dependent diseases such as prostate cancer. AR is actively involved in the initiation of prostate cancer, the transition to androgen independence, and many mechanisms of resistance to therapy. To examine genetic variation of AR in cancer, we created mice by germ-line gene targeting in which human AR sequence replaces that of the mouse. Since shorter length of a polymorphic N-terminal glutamine (Q) tract has been linked to prostate cancer risk, we introduced alleles with 12, 21 or 48 Qs to test this association. The three "humanized" AR mouse strains (h/mAR) are normal physiologically, as well as by cellular and molecular criteria, although slight differences are detected in AR target gene expression, correlating inversely with Q tract length. However, distinct allele-dependent differences in tumorigenesis are evident when these mice are crossed to a transgenic prostate cancer model. Remarkably, Q tract variation also differentially impacts disease progression following androgen depletion. This finding emphasizes the importance of AR function in androgen-independent as well as androgen-dependent disease. These mice provide a novel genetic paradigm in which to dissect opposing functions of AR in tumor suppression versus oncogenesis.
View details for DOI 10.1016/j.jsbmb.2007.09.002
View details for Web of Science ID 000253964600008
View details for PubMedID 17936615
Androgen-dependent pathology demonstrates myopathic contribution to the Kennedy disease phenotype in a mouse knock-in model
JOURNAL OF CLINICAL INVESTIGATION
2006; 116 (10): 2663-2672
The androgen receptor (AR) is involved in the initiation and progression of prostate cancer and its transition to androgen independence. Genetic variation in AR may contribute to disease risk and has been studied for a polymorphic N-terminal glutamine (Q) tract that shows population heterogeneity. While the length of this tract is known to affect AR in vitro, association with disease is complicated by genetic and environmental factors that have led to discordant epidemiological findings. To clarify the effect of Q tract polymorphism on prostate cancer, we created mice bearing humanized AR genes (h/mAr) varying in Q tract length. ARs with short Q tracts (12Q), which are transcriptionally more active, induce earlier disease in the transgene-induced TRAMP prostate cancer model than alleles with median (21Q) or long (48Q) tracts. Disease length varies within each genotype, with greater differentiation and AR expression in slower growing tumors. Remarkably, following androgen ablation, Q tract length has effects that are also allele-dependent and in directions opposite to those in hormone intact mice. Differences in AR activity conferred by Q tract length thus appear to direct distinct pathways of androgen-independent as well as androgen-dependent progression, and highlight substantial risk that may be associated with alterations in the androgen axis. This AR allelic series in humanized mice provides an experimental paradigm to dissect the role of AR in prostate cancer initiation and progression, to model response to treatment and to test therapies targeted specifically to the human AR.
View details for DOI 10.1093/hmg/ddm287
View details for Web of Science ID 000251866600008
View details for PubMedID 17906287
Replacing the mouse androgen receptor with human alleles demonstrates glutamine tract length-dependent effects on physiology and tumorigenesis in mice
2006; 20 (6): 1248-1260
Kennedy disease, a degenerative disorder characterized by androgen-dependent neuromuscular weakness, is caused by a CAG/glutamine tract expansion in the androgen receptor (Ar) gene. We developed a mouse model of Kennedy disease, using gene targeting to convert mouse androgen receptor (AR) to human sequence while introducing 113 glutamines. AR113Q mice developed hormone and glutamine length-dependent neuromuscular weakness characterized by the early occurrence of myopathic and neurogenic skeletal muscle pathology and by the late development of neuronal intranuclear inclusions in spinal neurons. AR113Q males unexpectedly died at 2-4 months. We show that this androgen-dependent death reflects decreased expression of skeletal muscle chloride channel 1 (CLCN1) and the skeletal muscle sodium channel alpha-subunit, resulting in myotonic discharges in skeletal muscle of the lower urinary tract. AR113Q limb muscles show similar myopathic features and express decreased levels of mRNAs encoding neurotrophin-4 and glial cell line-derived neurotrophic factor. These data define an important myopathic contribution to the Kennedy disease phenotype and suggest a role for muscle in non-cell autonomous toxicity of lower motor neurons.
View details for DOI 10.1172/JCI28773
View details for Web of Science ID 000240965700016
View details for PubMedID 16981011
Abnormalities of germ cell maturation and sertoli cell cytoskeleton in androgen receptor 113 CAG knock-in mice reveal toxic effects of the mutant protein
AMERICAN JOURNAL OF PATHOLOGY
2006; 168 (1): 195-204
Polymorphism in the length of the N-terminal glutamine (Q) tract in the human androgen receptor (AR) has been implicated in affecting aspects of male health ranging from fertility to cancer. Extreme expansion of the tract underlies Kennedy disease, and in vitro the AR Q tract length correlates inversely with transactivation capacity. However, whether normal variation influences physiology or the etiology of disease has been controversial. To assess directly the functional significance of Q tract variation, we converted the mouse AR to the human sequence by germline gene targeting, introducing alleles with 12, 21, or 48 glutamines. These three "humanized" AR (h/mAR) mouse lines were grossly normal in growth, behavior, fertility, and reproductive tract morphology. Phenotypic analysis revealed traits that varied subtly with Q tract length, including body fat amount and, more notably, seminal vesicle weight. Upon molecular analysis, tissue-specific differences in AR levels and target gene expression were detected between mouse lines. In the prostate, probasin, Nkx3.1, and clusterin mRNAs trended in directions predicted for inverse correlation of Q tract length with AR activation. Remarkably, when crossed with transgenic adenocarcinoma of mouse prostate (TRAMP) mice, striking genotype-dependent differences in prostate cancer initiation and progression were revealed. This link between Q tract length and prostate cancer, likely due to differential activation of AR targets, corroborates human epidemiological studies. This h/mAR allelic series in a homogeneous mouse genetic background allows examination of numerous physiological traits for Q tract influences and provides an animal model to test novel drugs targeted specifically to human AR.
View details for DOI 10.1210/me.2006-0021
View details for Web of Science ID 000237806900009
View details for PubMedID 16601069
Shape-defining scaffolds for minimally invasive tissue engineering
2004; 77 (12): 1798-1803
An unresolved question in the study of the polyglutamine neurodegenerative disorders is the extent to which partial loss of normal function of the mutant protein contributes to the disease phenotype. To address this, we studied Kennedy disease, a degenerative disorder of lower motor neurons caused by a CAG/glutamine expansion in the androgen receptor (Ar) gene. Signs of partial androgen insensitivity, including testicular atrophy and decreased fertility, are common in affected males, although the underlying mechanisms are not well understood. Here, we describe a knock-in mouse model that reproduces the testicular atrophy, diminished fertility, and systemic signs of partial androgen insensitivity that occur in Kennedy disease patients. Using this model, we demonstrate that the testicular pathology in this disorder is distinct from that mediated by loss of AR function. Testes pathology in 113 CAG knock-in mice was characterized by morphological abnormalities of germ cell maturation, decreased solubility of the mutant AR protein, and alterations of the Sertoli cell cytoskeleton, changes that are distinct from those produced by AR loss-of-function mutation in testicular feminization mutant mice. Our data demonstrate that toxic effects of the mutant protein mediate aspects of the Kennedy disease phenotype previously attributed to a loss of AR function.
View details for DOI 10.2353/ujpath.2006.050619
View details for Web of Science ID 000234449400023
View details for PubMedID 16400023
Minimally invasive surgical procedures are increasingly important in medicine, but biomaterials consistent with this delivery approach that allow one to control the structure of the material after implantation are lacking. Biomaterials with shape-memorizing properties could permit minimally invasive delivery of cell transplantation constructs and enable the formation of new tissues or structures in vivo in desired shapes and sizes.Macroporous alginate hydrogel scaffolds were prepared in a number of predefined geometries, compressed into significantly smaller, different "temporary" forms, and introduced into immunocompromised mice by means of minimally invasive surgical delivery through a small catheter. Scaffolds were rehydrated in situ with a suspension of cells (primary bovine articular chondrocytes) or cell-free medium and delivered through the same catheter. Specimens were harvested at 1 hr to evaluate the efficacy of cell delivery and the recovery of scaffold geometry, and at 8 and 24 weeks to evaluate neotissue formation.A high percentage (88%) of scaffolds that were introduced with a catheter and rehydrated with cells had recovered their original shape and size within 1 hr. This delivery procedure resulted in cartilage structures with the geometry of the original scaffold by 2 months and histologically mature appearing tissue at 6 months.Shaped hydrogels, formed by covalently cross-linking, can be structurally collapsed into smaller, temporary shapes that permit their minimally invasive delivery in vivo. The rapid recovery of scaffold properties facilitates efficient cell seeding in vivo and permits neotissue formation in desired geometries.
View details for DOI 10.1097/01.tp.0000131152.71117.0e
View details for Web of Science ID 000222300000004
View details for PubMedID 15223894