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.

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.

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.