Proto-Oncogene Proteins c-akt ; TOR Serine-Threonine Kinases ; GTP-Binding Protein alpha Subunits, Gi-Go/metabolism*

Humans ; Brain Neoplasms/metabolism* ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Glioma/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Signal Transduction ; ATPases Associated with Diverse Cellular Activities/metabolism* ; Mitochondrial Proteins/metabolism* ; GTP-Binding Protein alpha Subunits/metabolism*

BACKGROUNDThe addition of anti-human epidermal growth factor receptor 2 (HER2)-targeted drugs, such as trastuzumab, lapatinib, and trastuzumab emtansine (T-DM1), to chemotherapy significantly improved prognosis of HER2-positive breast cancer patients. However, it was confused that metastatic patients vary in the response of targeted drug. Therefore, methods of accurately predicting drug response were really needed. To overcome the spatial and temporal limitations of biopsies, we aimed to develop a more sensitive and less invasive method of detecting mutations associated with anti-HER2 therapeutic response through circulating-free DNA (cfDNA).

METHODSFrom March 6, 2014 to December 10, 2014, 24 plasma samples from 20 patients with HER2-positive metastatic breast cancer who received systemic therapy were eligible. We used a panel for detection of hot-spot mutations from 50 oncogenes and tumor suppressor genes, and then used targeted next-generation sequencing (NGS) to identify somatic mutation of these samples in those 50 genes. Samples taken before their first trastuzumab administration and subsequently proven with clinical benefit were grouped into sensitive group. The others were collected after disease progression of the trastuzumab-based therapy and were grouped into the resistant group.

RESULTSA total of 486 single-nucleotide variants from 46 genes were detected. Of these 46 genes, phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), proto-oncogene c-Kit (KIT), and tumor protein p53 (TP53) were the most common mutated genes. Seven genes, including epidermal growth factor receptor (EGFR), G protein subunit alpha S (GNAS), HRas proto-oncogene (HRAS), mutL homolog 1 (MLH1), cadherin 1 (CDH1), neuroblastoma RAS viral oncogene homolog (NRAS), and NOTCH1, that only occurred m utations in the resistant group were associated with the resistance of targeted therapy. In addition, we detected a HER2 S855I mutation in two patients who had persistent benefits from anti-HER2 therapy.

CONCLUSIONTargeted NGS of cfDNA has potential clinical utility to detect biomarkers from HER2-targeted therapies.

Adolescent ; Adult ; Aged ; Biomarkers, Tumor ; genetics ; Breast Neoplasms ; genetics ; metabolism ; Cadherins ; genetics ; Chromogranins ; genetics ; Class I Phosphatidylinositol 3-Kinases ; Drug Resistance, Neoplasm ; genetics ; Female ; GTP-Binding Protein alpha Subunits, Gs ; genetics ; Humans ; Male ; Middle Aged ; Mutation ; genetics ; Phosphatidylinositol 3-Kinases ; genetics ; Proto-Oncogene Proteins c-kit ; genetics ; Receptor, ErbB-2 ; metabolism ; Receptor, Notch1 ; genetics ; Tumor Suppressor Protein p53 ; genetics ; Young Adult

PURPOSE: Apoptosis of vascular endothelial cells is a type of endothelial damage that is associated with the pathogenesis of cardiovascular diseases such as atherosclerosis. Heterotrimeric GTP-binding proteins (G proteins), including the alpha 12 subunit of G protein (Galpha12), have been found to modulate cellular proliferation, differentiation, and apoptosis of numerous cell types. However, the role of Galpha12 in the regulation of apoptosis of vascular cells has not been elucidated. We investigated the role of Galpha12 in serum withdrawal-induced apoptosis of human umbilical vein endothelial cells (HUVECs) and its underlying mechanisms. MATERIALS AND METHODS: HUVECs were transfected with Galpha12 small-interfering RNA (siRNA) to knockdown the endogenous Galpha12 expression and were serum-deprived for 6 h to induce apoptosis. The apoptosis of HUVECs were assessed by Western blotting and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The expressions of microRNAs were analyzed by quantitative real-time PCR. RESULTS: Knockdown of Galpha12 with siRNA augmented the serum withdrawal-induced apoptosis of HUVECs and markedly repressed the expression of microRNA-155 (miR-155). Serum withdrawal-induced apoptosis of HUVECs was inhibited by the overexpression of miR-155 and increased significantly due to the inhibition of miR-155. Notably, the elevation of miR-155 expression prevented increased apoptosis of Galpha12-deficient HUVECs. CONCLUSION: From these results, we conclude that Galpha12 protects HUVECs from serum withdrawal-induced apoptosis by retaining miR-155 expression. This suggests that Galpha12 might play a protective role in vascular endothelial cells by regulating the expression of microRNAs.
*Apoptosis ; Atherosclerosis/*blood/genetics/immunology ; Cell Proliferation ; Endothelial Cells/*metabolism ; GTP-Binding Protein alpha Subunits, G12-G13/*genetics ; Gene Expression Profiling ; Gene Expression Regulation ; Human Umbilical Vein Endothelial Cells/cytology ; Humans ; MicroRNAs/*metabolism ; Protective Agents ; *RNA, Small Interfering ; Real-Time Polymerase Chain Reaction ; *Transfection

The Cre/LoxP system is a well-established approach to spatially and temporally control genetic inactivation. The calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIalpha) promoter limits expression to specific regions of the forebrain and thus has been utilized for the brain-specific inactivation of the genes. Here, we show that CaMKIIalpha-Cre can be utilized for simultaneous inactivation of genes in the adult brain and in male germ cells. Double transgenic Rosa26(+/stop-lacZ)::CaMKIIalpha-Cre(+/Cre) mice generated by crossing CaMKIIalpha-Cre(+/Cre) mice with floxed ROSA26 lacZ reporter (Rosa26(+/stop-lacZ)) mice exhibited lacZ expression in the brain and testis. When these mice were mated to wild-type females, about 27% of the offspring were whole body blue by X-gal staining without inheriting the Cre transgene. These results indicate that recombination can occur in the germ cells of male Rosa26(+/stop-lacZ)::CaMKIIalpha-Cre(+/Cre) mice. Similarly, when double transgenic Gnao(+/f)::CaMKIIalpha-Cre(+/Cre) mice carrying a floxed Go-alpha gene (Gnao(f/f)) were backcrossed to wild-type females, approximately 22% of the offspring carried the disrupted allele (Gnao(Delta)) without inheriting the Cre transgene. The Gnao(Delta/Delta) mice closely resembled conventional Go-alpha knockout mice (Gnao(-/-)) with respect to impairment of their behavior. Thus, we conclude that CaMKIIalpha-Cre mice afford recombination for both tissue- and time-controlled inactivation of floxed target genes in the brain and for their permanent disruption. This work also emphasizes that extra caution should be exercised in utilizing CaMKIIalpha-Cre mice as breeding pairs.
Animals ; Brain/*metabolism ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics ; Female ; GTP-Binding Protein alpha Subunits, Gi-Go/*genetics ; *Gene Deletion ; Gene Knockout Techniques/*methods ; Male ; Mice ; RNA, Untranslated/genetics ; Recombination, Genetic ; Spermatozoa/*metabolism

The aim of the present study is to explore the mechanism of estrogen on regulating cardiac function disorder by adjusting the stimulating adenylate cyclase G &alpha; protein (G&alpha;s)-cycle adenosine monophosphate (cAMP) signal pathway. Adult female rats were randomly divided into five groups: sham group, ovariectomized group (OVX), OVX and 17β-estradiol given group (OVX+E₂), OVX and isoprenaline injected group (OVX+ISO), OVX and 17β-estradiol, isoprenaline injected group (OVX+E₂+ISO). Rats were ovariectomized, and two weeks later, OVX+E₂group was injected with E₂, OVX+ISO group was injected with ISO, OVX+E₂+ISO group was injected with E₂and ISO. Another four weeks later, the hemodynamic parameters were monitored by carotid artery intubation: left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), maximal differentials of left ventricular developed pressure (+dp/dt(max)), and minimal differentials of left ventricular developed pressure (-dp/dt(max)). Brain natriuretic peptide (BNP) and cAMP concentration in plasma were determined; G&alpha;(s) protein expression in myocardium was determined. The results showed that the hemodynamic parameters, the concentration of BNP and cAMP in plasma had no significant changes after ovariectomy compared with sham group. But after isoprenaline injection in ovariectomized rats, LVSP and +dp/dt(max) declined (P < 0.01), LVEDP and -dp/dt(max) elevated (P < 0.01); plasma BNP concentration increased (P < 0.01); plasma cAMP concentration decreased (P < 0.01), compared with OVX group. Further estrogen supplements improved the heart function treated by isoprenaline: LVSP and +dp/dt(max) elevated (P < 0.01), LVEDP and -dp/dtmax declined (P < 0.05, P < 0.01); the plasma BNP concentration decreased (P < 0.01); the plasma cAMP concentration increased (P < 0.01). Estrogen had no significant influence on G&alpha;s protein expression. The results suggest that estrogen can alleviate myocardial injury and regulate cardiac function disorder by increasing cAMP level, finally improved the excessive suppression of myocardium.
Animals ; Cyclic AMP ; blood ; Estradiol ; pharmacology ; Estrogens ; pharmacology ; Female ; GTP-Binding Protein alpha Subunits, Gs ; metabolism ; Hemodynamics ; Isoproterenol ; adverse effects ; Myocardium ; pathology ; Natriuretic Peptide, Brain ; blood ; Ovariectomy ; Rats ; Signal Transduction

The worldwide prevalence of obesity is steadily increasing, nearly doubling between 1980 and 2008. Obesity is often associated with insulin resistance, a major risk factor for type 2 diabetes mellitus (T2DM): a costly chronic disease and serious public health problem. The underlying cause of T2DM is a failure of the beta cells of the pancreas to continue to produce enough insulin to counteract insulin resistance. Most current T2DM therapeutics do not prevent continued loss of insulin secretion capacity, and those that do have the potential to preserve beta cell mass and function are not effective in all patients. Therefore, developing new methods for preventing and treating obesity and T2DM is very timely and of great significance. There is now considerable literature demonstrating a link between inhibitory guanine nucleotide-binding protein (G protein) and G protein-coupled receptor (GPCR) signaling in insulin-responsive tissues and the pathogenesis of obesity and T2DM. These studies are suggesting new and emerging therapeutic targets for these conditions. In this review, we will discuss inhibitory G proteins and GPCRs that have primary actions in the beta cell and other peripheral sites as therapeutic targets for obesity and T2DM, improving satiety, insulin resistance and/or beta cell biology.
Animals ; Diabetes Mellitus, Type 2/drug therapy/*metabolism ; GTP-Binding Protein alpha Subunits/genetics/*metabolism ; Humans ; Insulin-Secreting Cells/metabolism ; Obesity/drug therapy/*metabolism ; Receptor, Melatonin, MT2/genetics/*metabolism ; Receptors, Adrenergic, alpha-1/genetics/*metabolism ; Receptors, Prostaglandin/genetics/*metabolism

OBJECTIVETo analyze the association between T393C single nucleotide polymorphism (SNP) of GNAS1 gene and non-valvular atrial fibrillation (AF) in Chinese Han patients.

METHODSNinety patients with non-valvular AF and 90 healthy subjects were examined for T393C SNP of GNAS1 gene using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The allele genotypes and the distribution of allele frequencies were analyzed and compared between the two groups. The relationship between allele frequency distribution characteristics and the heart rate variability (HRV) were also studied for analysis of the association between T393C SNP of GNAS1 gene and the autonomic nervous activation in non-valvular AF.

RESULTSThe two groups showed a significant difference in the frequencies of genotypes of T393C SNP of GNAS1 gene and allele frequencies (P<0.01). CC genotype and T393C allele frequency were significantly increased in the case group. pNN50, LF, or LF/HF showed no significant difference between different genotypes (P<0.05).

CONCLUTIONSThe T393C SNP of GNAS1 gene is closely associated with non-valvular AF in Chinese Han patients.

Adult ; Aged ; Aged, 80 and over ; Alleles ; Asian Continental Ancestry Group ; genetics ; Atrial Fibrillation ; genetics ; metabolism ; physiopathology ; Chromogranins ; Female ; GTP-Binding Protein alpha Subunits, Gs ; genetics ; Gene Frequency ; Genotype ; Heart Rate ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide ; Risk Factors

Helicobacter pylori (H. pylori) is an important risk factor for chronic gastritis, peptic ulcer, and gastric cancer. Proteinase-activated receptor 2 (PAR2), subgroup of G-protein coupled receptor family, is highly expressed in gastric cancer, and chronic expression of cyclooxygenase-2 (COX-2) plays an important role in H. pylori-associated gastric carcinogenesis and inflammation. We previously demonstrated that H. pylori induced the expression of PAR2 and COX-2 in gastric epithelial cells. Present study aims to investigate whether COX-2 expression induced by H. pylori in Korean isolates is mediated by PAR2 via activation of Gi protein and Src kinase in gastric epithelial AGS cells. Results showed that H. pylori-induced COX-2 expression was inhibited in the cells transfected with antisense oligonucleotide for PAR2 or treated with Gi protein blocker pertussis toxin, Src kinase inhibitor herbimycin A and soybean trypsin inbitor, indicating that COX-2 expression is mediated by PAR2 through activation of Gi protein and Src kinase in gastric epithelial cells infected with H. pylori in Korean isolates. Thus, targeting the activation of PAR2 may be beneficial for prevention or treatment of gastric inflammation and carcinogenesis associated with H. pylori infection.
Benzoquinones/*pharmacology ; Cell Line, Tumor ; Cyclooxygenase 2/genetics/*metabolism ; Epithelial Cells/enzymology/metabolism/microbiology ; GTP-Binding Protein alpha Subunits, Gi-Go/metabolism ; Gastric Mucosa/enzymology/metabolism/*microbiology ; *Helicobacter pylori ; Humans ; Lactams, Macrocyclic/*pharmacology ; Oligonucleotides, Antisense ; Pertussis Toxin/*pharmacology ; RNA, Messenger/metabolism ; Receptor, PAR-2/*physiology ; src-Family Kinases/metabolism

OBJECTIVETo study the significance of c-myc, p53 and p16 protein expression in fibrous dysplasia, to detect the GNAS1 gene mutation in fibrous dysplasia, and to explore the property of fibrous dysplasia.

METHODSThe expression of c-myc, p53 and p16 protein was evaluated by immunohistochemistry SP method in 35 cases of fibrous dysplasia including 1 FD with malignancy, 1 Mazabraud syndrome and 20 control cases (10 cases of bony callus, 10 cases of osteosarcoma). Genomic DNA extraction, PCR amplification and gene sequencing were used to detect GNAS1 gene mutation in 35 cases of fibrous dysplasia.

RESULTSC-myc protein immunoreactivity was detected in 91 percentage of FD (P = 0.001). Compared with the negative control group, the difference was significant. P16 positive was detected in 34 FD cases (P = 0.001). The difference was significant as compared with the positive control group. Positive p53 protein expression was detected in the only 1 case of fibrous dysplasia with malignant transformation. PCR amplification was successful in 12 of 35 FD cases. Two of the 12 FD cases were detected to have GNAS1 gene mutation, in which 1 case was FD of Mazabraud syndrome, 1 case was a monostotic lesion.

CONCLUSIONSC-myc could be another protooncogene in addition to c-fos in the fibrous dysplasia disease. P53 protein overexpression could be useful in the diagnosis of FD malignancy and in the prediction of the prognosis of FD. The abnormal expression of the gene p16 might play an important role in the formation of FD. The GNAS1 mutation exist in FD. All of the results indicate that FD could be a neoplasia disease, caused by multiple factors leading to a dysfunction of bone development.

Adolescent ; Adult ; Child ; Chromogranins ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Female ; Fibrous Dysplasia of Bone ; genetics ; metabolism ; pathology ; GTP-Binding Protein alpha Subunits, Gs ; genetics ; Humans ; Male ; Middle Aged ; Mutation ; Osteosarcoma ; genetics ; metabolism ; pathology ; Proto-Oncogene Proteins c-myc ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; Young Adult