GNAS mutation detection is related to disease severity in girls with McCune-Albright syndrome and precocious puberty.

BACKGROUND: McCune-Albright syndrome (MAS) is characterized by a triad of gonadotropin-independent precocious puberty, café au lait skin pigmentation and fibrous dysplasia of bone. MAS is due to activating mutations of GNAS, the gene encoding Gsalpha. Interest exists in the use of GNAS mutation analysis to make a definitive diagnosis when the phenotype is not diagnostic, i.e. in partial forms of MAS. The utility of using peripheral blood for mutation analysis in this setting has not been thoroughly evaluated. OBJECTIVE: We performed a systematic analysis of genomic DNA for the detection of GNAS activating mutations in girls with MAS who presented with precocious puberty to evaluate whether identification of an activating mutation in peripheral blood is related to the presence of other features of MAS. STUDY DESIGN: Genomic DNA was isolated from blood from 13 girls with gonadotropin-independent precocious puberty. A polymerase chain reaction (PCR)-based technique was performed for GNAS mutation identification. RESULTS: GNAS activating mutations were identified in 4 patients, all of whom had classic MAS based on clinical evidence. CONCLUSIONS: Detection of activating mutations in leukocyte genomic DNA extracted from peripheral blood samples from girls with gonadotropin-independent precocious puberty was associated with the presence of other phenotypic manifestations of MAS. Until improvements in the diagnostic utility of GNAS activating mutation analysis from leukocyte genomic DNA occur, such testing in patients with atypical forms of MAS should continue to be reserved for research settings.

RNA interference (RNAi) for extracellular signal-regulated kinase 1 (ERK1) alone is sufficient to suppress cell viability in ovarian cancer cells.

While ovarian cancer is a leading cause of death in females today, the molecular, genetic, and environmental factors that initiate and support the progression of this disease are still only partially understood. The extracellular signal-regulated kinase (ERK) signaling pathway is a major contributor to cellular growth, differentiation and survival. Recently, we reported that this pathway is constitutively activated in ovarian cancer cells, and that by using RNA interference (RNAi) for ERK1 and ERK2, we were able to significantly suppress the number of viable tumor cells. In the present study, we have further investigated the mechanisms by which RNAi for the ERK kinases decreased viability in these cancer cells. It was determined that treatment of the cancer cells with small inhibitory RNAs (siRNAs) directed against ERK1 and ERK2 leads to the induction of apoptosis and necrosis by four hours following treatment. Additionally, we found that primary, nonmalignant ovarian cells do not respond similarly to ERK siRNA treatment and that these cells fail to die following treatment. Data presented show that ERK2 expression is more difficult to silence, depending upon cell type being examined and that silencing ERK1 expression alone is sufficient to significantly decrease tumor cell viability.

Functional imaging in small animals using X-ray computed tomography--study of physiologic measurement reproducibility.

X-ray computed tomography (CT) has been traditionally used for morphologic analysis and in the recent past has been used for physiology imaging. This paper seeks to demonstrate functional CT as an effective tool for monitoring changes in tissue physiology associated with disease processes and cellular and molecular level therapeutic processes. We investigated the effect of noise and sampling time on the uncertainty of tissue physiologic parameters. A whole body compartmental model of mouse was formulated to simulate tissue time density curves and study the deviation of tissue physiologic parameters from their true values. These results were then used to determine the appropriate scanning protocols for the experimental studies. Dynamic contrast enhanced CT (DCE-CT) was performed in mice following the injection of hydrophilic iodinated contrast agent (CA) at three different injection rates, namely 0.5 ml/min, 1 ml/min, and 2.0 ml/min. These experiments probed the Nyquist sampling limit for reproducibility of tissue physiologic parameters. Separate experiments were performed with three mice at four different X-ray tube currents corresponding to different image noise values. A two-compartment model (2CM) model was formulated to describe the contrast kinematics in the kidney cortex. Three different 2CMs were implemented namely the 4-parameter (4P), 5-parameter (5P), and the 6-parameter (6P) model. The tissue kinematics is fitted to the models by using the Levenberg-Marquardt algorithm implemented in IDL (RSI Inc.) programming language to minimize the weighted sum of squares. The relevant tissue physiologic parameters extracted from the models are the renal blood flow (RBF), glomerular filtration rate (GFR), fractional plasma volume, fractional tubular volumes and urine formation rates. The experimental results indicate that the deviation of the tissue physiologic parameters is within the limits required for tracking disease physiology in vivo and thus small animal functional X-ray CT would be able to determine changes in tissue physiology in vivo.

Mechanisms regulating the constitutive activation of the extracellular signal-regulated kinase (ERK) signaling pathway in ovarian cancer and the effect of ribonucleic acid interference for ERK1/2 on cancer cell proliferation.

The ERK1/2 MAPK pathway is a critical signaling system that mediates ligand-stimulated signals for the induction of cell proliferation, differentiation, and cell survival. Studies have shown that this pathway is constitutively active in several human malignancies and may be involved in the pathogenesis of these tumors. In the present study we examined the ERK1/2 pathway in cell lines derived from epithelial and granulosa cell tumors, two distinct forms of ovarian cancer. We show that ERK1 and ERK2 are constitutively active and that this activation results from both MAPK kinase-dependent and independent mechanisms and is correlated with elevated BRAF expression. MAPK phosphatase 1 (MKP-1) expression, which is involved in ERK1/2 deactivation, is down-regulated in the cancer cells, thus further contributing to ERK hyperactivity in these cells. Treatment of these cancer cell lines with the proteasome inhibitor ZLLF-CHO increased MKP-1 but not MKP-2 expression and decreased ERK1/2 phosphorylation. More importantly, silencing of ERK1/2 protein expression using RNA interference led to the complete suppression of tumor cell proliferation. These results provide evidence that the ERK pathway plays a major role in ovarian cancer pathogenesis and that down-regulation of this master signaling pathway is highly effective for the inhibition of ovarian tumor growth.

Premature thelarche and granulosa cell tumors: a search for FSH receptor and G5alpha activating mutations.

Activating mutations of the Gsalpha gene are responsible for McCune-Albright syndrome and have also been identified in sporadic tumors of the pituitary and thyroid. When associated with malignancy, activating Gsalpha mutations are known as gsp-oncogenes. We hypothesized that similar activating mutations might also account for some cases of premature thelarche and/ or granulosa cell tumors. Polymerase chain reaction and DNA sequencing was used to screen for activating mutations of Gsalpha genes in children with premature thelarche and in pathologic specimens from juvenile and adult granulosa cell tumors. Because these disorders involve over-activity of the FSH-signaling pathway, we also screened for activating mutations of the FSH receptor. No mutations were detected in either the Gsalpha or the FSHR fragment studied. Previously reported polymorphisms (Ser680Asn and Ala307Thr) of the FSHR were detected in 25/27 tumor samples and 9/9 premature thelarche samples. We conclude that activating mutations in previously identified mutation 'hot-spots' in the Gsalpha and FSH receptor genes are probably not a major cause of premature thelarche or granulosa cell tumors. In contrast, polymorphisms of the FSH receptor are common.

No activating mutations of FSH receptor in four children with ovarian juvenile granulosa cell tumors and the association of these tumors with central precocious puberty.

STUDY OBJECTIVE: The stimulation of the follicle-stimulating hormone receptor (FSHR) by circulating FSH or some activating mutations of the FSHR may play a causal role in the development of granulosa cell tumors of ovaries. STUDY DESIGN: We evaluated four patients with ovarian juvenile granulosa cell tumors (age range, 2.4 to 7.2; median, 2.9 years) and five healthy pubertal girls (age range, 16 to 18.5; median, 16.8 years) for activating mutations in exon 10 of the FSHR. The patients were followed and evaluated clinically. Genomic DNA was extracted from the peripheral blood. Exon10 of the FSHR was evaluated for mutations. RESULTS: All four patients presented with signs of precocious puberty. One patient, who had markedly accelerated growth velocity and advanced bone age, developed central precocious puberty after the removal of her tumor. Another patient was diagnosed to have a left ovarian cyst without tumor recurrence approximately 3.3 years after the removal of the tumor. Activating mutations were not found, but previously reported polymorphisms (Ser680Asn and Ala307Thr) of the FSHR were detected in three of four patients and in three of five controls. The follow-up period of these four patients ranged from 4.5 to 8.8 years, with a median value of 6.7 years. CONCLUSIONS: We did not find any activating mutation in exon 10 of the FSHR in our patients, and one patient developed precocious puberty after removal of her tumor. The development of ovarian tumors in these patients may have been caused by mutations at other exons of the FSHR and G protein subunits, so the association noted between central precocious puberty and granulosa cell tumors might not be coincidental.

Lysophosphatidic acid stimulates cell migration, invasion, and colony formation as well as tumorigenesis/metastasis of mouse ovarian cancer in immunocompetent mice.

We have already established human xenographic models for the effect of lysophosphatidic acid (LPA) on tumor metastasis in vivo. The purpose of this work is to establish a preclinical LPA effect model in immunocompetent mice. We first characterized the mouse epithelial ovarian cancer (EOC) cell line ID8 for its responsiveness to LPA in cell proliferation, migration, and invasion and compared these properties with those of human EOC. The signaling pathways related to cell migration were further investigated using pharmacologic and genetic approaches. The effects of LPA on the tumorigenesis of ID8 cells and mouse survival were then examined using two different mouse models (i.p. and orthotopic injections). LPA stimulated cell proliferation, migration, and invasion of mouse EOC ID8 cells in a manner closely resembling its activity in human EOC cells. The signaling pathways involved in LPA-induced cell migration in ID8 cells were also similar to those identified in human EOC cells. We have identified cyclooxygenase-1 and 15-lipoxygenase as two new signaling molecules involved in LPA-induced cell migration in both human and mouse EOC cells. In addition, LPA enhanced the tumorigenesis/metastasis of ID8 cell in vivo as assessed by increased tumor size, early onset of ascites formation, and reduced animal survival. We have established the first LPA-EOC preclinical model in immunocompetent mice. Because ID8 cells respond to LPA similar to human EOC cells, this model is very valuable in developing and testing therapeutic reagents targeting LPA in EOC.

Abnormalities in osteoclastogenesis and decreased tumorigenesis in mice deficient for ovarian cancer G protein-coupled receptor 1.

Ovarian cancer G protein-coupled receptor 1 (OGR1) has been shown to be a proton sensing receptor in vitro. We have shown that OGR1 functions as a tumor metastasis suppressor gene when it is over-expressed in human prostate cancer cells in vivo. To examine the physiological functions of OGR1, we generated conditional OGR1 deficient mice by homologous recombination. OGR1 deficient mice were viable and upon gross-inspection appeared normal. Consistent with in vitro studies showing that OGR1 is involved in osteoclastogenesis, reduced osteoclasts were detected in OGR1 deficient mice. A pH-dependent osteoclasts survival effect was also observed. However, overall abnormality in the bones of these animals was not observed. In addition, melanoma cell tumorigenesis was significantly inhibited in OGR1 deficient mice. OGR1 deficient mice in the mixed background produced significantly less peritoneal macrophages when stimulated with thioglycolate. These macrophages also showed altered extracellular signal-regulated kinases (ERK) activation and nitric oxide (NO) production in response to lipopolysaccharide. OGR1-dependent pH responses assessed by cAMP production and cell survival in macrophages or brown fat cells were not observed, presumably due to the presence of other proton sensing receptors in these cells. Our results indicate that OGR1's role in osteoclastogenesis is not strong enough to affect overall bone development and its role in tumorigenesis warrants further investigation. The mice generated can be potentially used for several disease models, including cancers or osteoclast-related diseases.

Ovarian cancer G protein-coupled receptor 1, a new metastasis suppressor gene in prostate cancer.

BACKGROUND: Metastasis is a process by which tumors spread from primary organs to other sites in the body and is the major cause of death for cancer patients. The ovarian cancer G protein-coupled receptor 1 (OGR1) gene has been shown to be expressed at lower levels in metastatic compared with primary prostate cancer tissues. METHODS: We used an orthotopic mouse metastasis model, in which we injected PC3 metastatic human prostate cancer cells stably transfected with empty vector (vector-PC3) or OGR1-expressing vector (OGR1-PC3) into the prostate lobes of athymic or NOD/SCID mice (n = 3-8 mice per group). Migration of PC3 cells transiently transfected with vector control or with OGR1- or GPR4 (a G protein-coupled receptor with the highest homology to OGR1)-expressing vectors was measured in vitro by Boyden chamber assays. G protein alpha-inhibitory subunit 1 (G alpha(i1)) expression after treatment with pertussis toxin (PTX) was measured using immunoblotting analysis. The inhibitory factor present in the conditioned medium was extracted using organic solvents and analyzed by mass spectrometry. RESULTS: In vivo, all 26 mice carrying tumors that were derived from vector-PC3 cells developed prostate cancer metastases (mean = 100%, 95% confidence interval [CI] = 83.97% to 100%) but few (4 of 32) mice carrying tumors derived from OGR1-expressing PC3 cells (mean = 12.50%, 95% CI = 4.08% to 29.93%) developed metastases. However, exogenous OGR1 overexpression had no effect on primary prostate tumor growth in vivo. In vitro, expression of OGR1, but not GPR4, inhibited cell migration (mean percentage of cells migrated, 30.2% versus 100%, difference = 69.8%, 95% CI = 63.0% to 75.9%; P<.001) via increased expression of G alpha(i1) and the secretion of a chloroform/methanol-extractable heat-insensitive factor into the conditioned medium through a PTX-sensitive pathway. CONCLUSION: OGR1 is a novel metastasis suppressor gene for prostate cancer. OGR1's constitutive activity via G alpha(i) contributes to its inhibitory effect on cell migration in vitro.

Imaging the Progression of Intra-tumor Heterogeneity in Prostate and Ovarian Xenografts Using Dynamic Contrast-Enhanced CT.

The purpose of this study is to measure the progressive intra-tumor heterogeneous physiological states for prostate (CWRrv) and ovarian (SKOV3x) xenograft mouse models. Dynamic contrast-enhanced CT was used to measure the change in a tumor's physiological state when transitioning from stage I (<7 mm diameter) to stage II (7-20 mm diameter). Images from stage I tumors are in the initial stages of angiogenesis: neovasculature growth. A radial heterogeneity begins to form with signs of angiogenic activity throughout the tumor. Stage II tumors develop both a saccular heterogeneity and a strong radial heterogeneity between periphery and core consistent with the effects of inflammatory and maturation processes. Imaging intra-tumor heterogeneity has the potential to track tumor stage and to be used as predictive factor in determining mortality rates.

Is McCune-Albright syndrome overlooked in subjects with fibrous dysplasia of bone?

OBJECTIVE: McCune-Albright syndrome (MAS) is characterized by a clinical triad of endocrinopathies, café au lait pigmentation, and polyostotic fibrous dysplasia of bone. We hypothesized that children diagnosed with fibrous dysplasia are not routinely being evaluated for coexisting endocrine dysfunction or MAS. Our objective was to prospectively screen subjects with fibrous dysplasia for endocrine disease and G(s)alpha gene (GNAS1 )-activating mutations. STUDY DESIGN: Nine subjects who presented with fibrous dysplasia and were followed in orthopedic clinics were evaluated for other manifestations of MAS. Genomic DNA was isolated from blood, and mutation analysis of GNAS1 was performed. RESULTS: On physical examination, 5 of 9 subjects were found to have café au lait pigmentation. Three of 9 subjects had TSH levels below the normal range. One of these subjects was found to have hyperthyroidism and was treated by total thyroidectomy. GNAS1 mutations were identified in 5 of 9 subjects with either monostotic or polyostotic fibrous dysplasia of bone. CONCLUSIONS: We conclude that a substantial proportion of children being followed for fibrous dysplasia of bone have unrecognized clinical and laboratory features of MAS. These children are at risk for endocrinopathy and should be screened accordingly.

Differential expression of GRK isoforms in nonmalignant and malignant human granulosa cells.

Granulosa cell tumors are serious ovarian neoplasms that can occur in women of all ages. While there have been numerous attempts to understand the cause of these malignancies, the pathogenesis of granulosa cell tumors (GCTs) still remains largely unknown. G-protein coupled receptor kinases (GRKs) are important regulators of signal transduction through the process of receptor desensitization and internalization. Receptors that are regulated by GRKs are members of the large family of seven-transmembrane receptors and include the follicle stimulating hormone receptor (FSHR). In granulosa cells, the FSH signaling system is responsible for cell proliferation, differentiation, and steroidogenesis. In the studies presented, we examined GRK mRNA and protein expression in nonmalignant human granulosa cells, in KGN cells, a human GCT cell line, and in a panel of human GCT samples. The KGN tumor cells express significantly less GRK4 alpha/beta protein and higher levels of GRK2 and GRK4 gamma/delta protein as compared to nonmalignant human granulosa cells. In human GCT samples, GRK4 alpha/beta protein was detected in 3 of the 13 tumor samples, whereas gamma/delta proteins expression was detected in all samples. These findings suggest that GRK protein expression is altered in GCTs and may be involved in the pathogenesis of these tumors.

Determination of trace isoflavone phytoestrogens in biological materials by capillary electrochromatography.

Capillary electrochromatography using a specialty monolithic matrix was utilized in developing a rapid and highly efficient separation of isoflavones in biological materials. Without a preconcentration technique, it is relatively easy to reach ppm-ppb concentrations of these compounds in soy-based foods and verify them structurally using a photodiode array detector. With on-column preconcentration, we were able to measure low-ppb levels in human serum. Using blood samples from human volunteers, whose diet was supplemented by a soy-based product, the method has been validated for high-throughput screening of isoflavones in clinical studies.