The SAM and HD domain containing protein 1 (Sterile alpha motif domain and HD domain-containing protein 1, SAMHD1) is a putative negative regulator of the antiviral innate immune response. It can significantly increase the antiviral immune response, mediates the interferon-induced inflammatory response involved in the host foreign-virus defense system. The early studies have focused on its gene mutations associated with Aicardi-Goutières syndrome (AGS), the latest study found that SAMHD1 as a potent dGTP-stimulated triphosphohydrolase restricts HIV-1 replication by hydrolyzing the majority of cellular dNTPs, thus inhibiting reverse transcription and viral complementary DNA (cDNA) synthesis. Auxiliary gene of HIV-2 and simian immunodeficiency virus (SIVsm / mac) encoding the Vpx protein can eliminate HIV-1 restriction. In recent years, the research on SAMHD1, mores forward rapidly this paper overviews the recent research progression related to the above fields.
Animals ; Cell Line ; HIV ; metabolism ; physiology ; Humans ; Monomeric GTP-Binding Proteins ; genetics ; metabolism ; SAM Domain and HD Domain-Containing Protein 1 ; Viral Regulatory and Accessory Proteins ; metabolism

mTOR (mammalian target of rapamycin) is the center for cellular activities. It controls many cell activities via inhibiting apoptosis and promoting cell growth. Rheb can activate mTOR signaling pathway and participate in genesis and development of multiple cancers. This study was purposed to explore the underlying role of Rheb in human myeloid leukemia by using the myeloid leukemia cell lines. Two myeloid leukemia cell lines HL-60 and K562 overexpressing Rheb were established with retrovirus containing Rheb. The mRNA and protein expressions of Rheb were determined by Real-Time PCR and Western blot respectively. Cell proliferation rate was examined by CCK-8 assay and apoptosis rate was analyzed using Annexin V and 7-AAD double-staining. The results showed that Rheb was overexpressed in both HL-60 and K562 cell lines. The Rheb overexpression cell lines were successfully established. It is found that overexpression of Rheb could promote cell growth. Furthermore, the overexpression of Rheb could accelerate cells entering into G2/M phase (P < 0.01), while did not affect the apoptosis. It is concluded that Rheb overexpression promotes myeloid leukemia cell proliferation through accelerating cell cycle progression.
Cell Cycle ; Cell Proliferation ; HL-60 Cells ; Humans ; K562 Cells ; Monomeric GTP-Binding Proteins ; metabolism ; Neuropeptides ; metabolism ; Ras Homolog Enriched in Brain Protein ; Signal Transduction

OBJECTIVETo transfect nm23-H1 into the BcaCD885 cell lines in order to get safe high-efficiency and low-toxicity, and to find out whether nm23-H1 could affect the invasion and metastases ability of BcaCD885 cell lines.

METHODSLipofect was used to transfect nm23-H1 into BcaCD885 cell lines; immunohistochemistry was used to detect the difference expression of nm23-H1 between transfected and non-transfected cell lines; then transwell-room and wash way were used to detect the difference of invasion and metastases ability between transfected and non-transfected cell lines.

RESULTSPCMV-NEO-BAM system gave the stability expression of nm23-H1; there was significant different NDPKA expression between transfected and non-transfected BcaCD885 cell lines; the invasion and metastases ability of transfected BcaCD885 cell lines decreased obviously.

CONCLUSIONnm23-H1 can inhibit the metastases of BcaCD885 cell lines significantly.

Cell Adhesion ; physiology ; Cell Movement ; physiology ; Genetic Vectors ; genetics ; Humans ; Monomeric GTP-Binding Proteins ; genetics ; metabolism ; Mouth Neoplasms ; genetics ; pathology ; physiopathology ; NM23 Nucleoside Diphosphate Kinases ; Nucleoside-Diphosphate Kinase ; Transcription Factors ; genetics ; metabolism ; Transfection ; Tumor Cells, Cultured

Human monocyte is an important cell type which is involved in various complex human diseases. To better understand the biology of human monocytes and facilitate further studies, we developed the first comprehensive proteome knowledge base specifically for human monocytes by integrating both in vivo and in vitro datasets. The top 2000 expressed genes from in vitro datasets and 779 genes from in vivo experiments were integrated into this study. Altogether, a total of 2237 unique monocyte-expressed genes were cataloged. Biological functions of these monocyte-expressed genes were annotated and classified via Gene Ontology (GO) analysis. Furthermore, by extracting the overlapped genes from in vivo and in vitro datasets, a core gene list including 541 unique genes was generated. Based on the core gene list, further gene-disease associations, pathway and network analyses were performed. Data analyses based on multiple bioinformatics tools produced a large body of biologically meaningful information, and revealed a number of genes such as SAMHD1, G6PD, GPD2 and ENO1, which have been reported to be related to immune response, blood biology, bone remodeling, and cancer respectively. As a unique resource, this study can serve as a reference map for future in-depth research on monocytes biology and monocyte-involved human diseases.
Aged ; Biomarkers, Tumor ; metabolism ; DNA-Binding Proteins ; metabolism ; Female ; Glucosephosphate Dehydrogenase ; metabolism ; Humans ; Mass Spectrometry ; methods ; Middle Aged ; Monocytes ; metabolism ; Monomeric GTP-Binding Proteins ; metabolism ; Phosphopyruvate Hydratase ; metabolism ; Proteomics ; methods ; SAM Domain and HD Domain-Containing Protein 1 ; Tumor Suppressor Proteins ; metabolism

The release of neurotransmitter is regulated in the processes of membrane docking and membrane fusion between synaptic vesicles and presynaptic plasma membranes. Synaptic vesicles contain a diverse set of proteins that participate in these processes. Small GTP-binding proteins exist in the synaptic vesicles and are suggested to play roles for the regulation of neurotransmitter release. We have examined a possible role of GTP-binding proteins in the regulation of protein phosphorylation in the synaptic vesicles. GTPgammaS stimulated the phosphorylation of 46 kappa Da protein (p46) with pI value of 5.0-5.2, but GDPbetaS did not. The p46 was identified as protein interacting with C-kinase 1 (PICK-1) by MALDI-TOF mass spectroscopy analysis, and anti-PICK-1 antibody recognized the p46 spot on 2-dimensional gel electrophoresis. Rab guanine nucleotide dissociation inhibitor (RabGDI), which dissociates Rab proteins from SVs, did not affect phosphorylation of p46. Ca2+/ calmodulin (CaM), which causes the small GTP- binding proteins like Rab3A and RalA to dissociate from the membranes and stimulates CaM- dependnet protein kinase(s) and phosphatase, strongly stimulate the phosphorylation of p46 in the presence of cyclosporin A and cyclophylin. However, RhoGDI, which dissociates Rho proteins from membranes, reduced the phosphorylation of p46 to the extent of about 50%. These results support that p46 was PICK-1, and its phosphorylation was stimulated by GTP and Ca2+/CaM directly or indirectly through GTP-binding protein(s) and Ca2+/CaM effector protein(s). The phosphorylation of p46 (PICK-1) by GTP and Ca2+/CaM may be important for the regulation of transporters and neurosecretion.
Animals ; Calcium/*metabolism ; Calmodulin/*metabolism ; Carrier Proteins/*chemistry/*metabolism ; Guanine Nucleotide Dissociation Inhibitors/metabolism ; Guanosine Triphosphate/metabolism/*pharmacology ; Molecular Weight ; Monomeric GTP-Binding Proteins/metabolism ; Phosphorylation/drug effects ; Rats ; Recombinant Fusion Proteins/*chemistry/*metabolism ; Synaptic Membranes/chemistry/drug effects/*metabolism ; Synaptic Vesicles/chemistry/drug effects/*metabolism

OBJECTIVETo construct recombinant lentiviral vectors carrying Rheb gene and its mutant Rheb'D60K gene, and examine their expression in human liver cancer cells.

METHODSRheb gene was amplified by PCR to construct the recombinant plasmid LV31-Rheb-WT and LV31-Rheb-D60K. HEK-293 FT cells were contransfected with the recombinant lentiviral vector together with a lentiviral package plasmid to produce the lentiviral particles. The expression of PS6 protein was detected in the lentivirus-infected MCF-7 cells. The apoptosis of SK-HEP-1 cells transfected with LV31-Rheb-WT or LV31-Rheb-D60K was observed.

RESULTSThe recombinant LV31-Rheb-WT and LV31-Rheb-D60K vectors were confirmed by PCR and DNA sequencing. Western blotting showed that PS6 protein expression was increased in LV31-Rheb-WT-transfected cells while decreased in LV31-Rheb-D60K-transfected cells. LV31-Rheb-D60K-transfected SK-HEP-1 cells showed more obvious apoptosis after starvation than LV31-Rheb-WT-transfected cells.

CONCLUSIONLentiviral vectors carrying Rheb gene and its mutant has been successfully constructed, which can be useful in further investigation of the role of Rheb gene in cancer cells.

Apoptosis ; genetics ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Genetic Vectors ; genetics ; HEK293 Cells ; Humans ; Lentivirus ; genetics ; metabolism ; Liver Neoplasms ; metabolism ; pathology ; MCF-7 Cells ; Monomeric GTP-Binding Proteins ; biosynthesis ; genetics ; Mutant Proteins ; genetics ; Neuropeptides ; biosynthesis ; genetics ; Ras Homolog Enriched in Brain Protein ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection

OBJECTIVETo investigate the role of Rheb (mTOR activator) in AML development by measuring Rheb expression in bone marrow of adult AML patients and in AML cell line HL-60.

METHODSReal-time PCR assay was used to measure the Rheb mRNA expression in 27 AML patients and 29 ITP patients as control. The relationship between Rheb mRNA expression and age, AML subtype, fusion gene, splenomegaly, hepatomegaly and survival of AML patients was analyzed and compared. In addition, HL-60 cell line over-expressing Rheb was established, and the HL-60 cells and HL-60 cells with overexpression of Rheb were treated with Ara-C of different concentrations, the proliferation level was detected by CCK-8 method, and the IC50 was calculated.

RESULTSThe mRNA level of Rheb in AML patients was similar to that in ITP patients (control). Interestingly, higher expression of Rheb was associated with better survival and was sensitive to Ara-C treatment. However, the expression level of Rheb was not associated with age, AML subtype, fusion gene, and hepatomegaly of patients. Lower expression level of Rheb was associated with splenomegaly. In vitro analysis of HL-60 line indicated that overexpression of Rheb could increased the cell sensitivity to Ara-C treatment (IC50=0.54 µmol/L) and caused HL-60 cell apoptosis.

CONCLUSIONThe lower Rheb expression is a poor prognostic indicator for AML patients, which is associated with AML splenomegaly, the patients and HL-60 cells with low expression of Rheb are insensitive to Ara-C treatment.

Adult ; Apoptosis ; Bone Marrow ; metabolism ; Cytarabine ; pharmacology ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; pathology ; Monomeric GTP-Binding Proteins ; genetics ; metabolism ; Neuropeptides ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Ras Homolog Enriched in Brain Protein ; Real-Time Polymerase Chain Reaction ; Spleen ; pathology

OBJECTIVETo understanding the molecular mechanisms in invasion and metastasis of the ovarian carcinoma, we investigate a novel candidate metastasis-associated gene (MTA1) and nm23H1 mRNA expression and mutation in ovarian carcinoma.

METHODSTwenty primary ovarian carcinoma specimens, 20 corresponding lymph nodes and 8 normal ovarian was examined for mRNA expression and mutation of MTA1 and nm23H1 genes by reverse-transcription polymerase chain reaction (RT-PCR) and RT-PCR-SSCP analysis. The level of the expression was determined by the relative optic density (ROD) of the PCR products.

RESULTSThe frequency of MAT1 overexpression was 100% (7/7) in primary ovarian carcinoma with metastasis but only 38.5% (5/13) in those without metastasis (P=0.0103). Overexpression of MAT1 was observed in 87.5% (6/7) of lymph nodes with metastasis but only 23% (3/13) of lymph nodes without metastasis (P=0.0118). In contrast with MAT1, low expression of nm23H1 mRNA was seen in 7 of 7 ovarian carcinoma with metastasis but only in 4 of 13 (30%) of those without metastasis (P=0.0043). Low nm23H1 expression was also seen in 7 of 7 lymph nodes with metastasis but only in 5 of 13 (38.5%) nonmetastatic lymph nodes (P=0.0102). The ROD ratio of MAT1 to nm23H1 increased with the development of metastasis. No mutation of MAT1 and nm23H1 genes was found by SSCP analysis.

CONCLUSIONThe mRNA expression of MTA1 and nm23H1 is positively and negatively correlated with lymph node metastasis, respectively. Expression abnormalities but not mutation of the two genes are frequent events related to lymph node metastasis of ovarian cancer.

Female ; Histone Deacetylases ; Humans ; Lymphatic Metastasis ; genetics ; Monomeric GTP-Binding Proteins ; biosynthesis ; genetics ; Mutation ; NM23 Nucleoside Diphosphate Kinases ; Neoplasm Invasiveness ; Neoplasm Proteins ; biosynthesis ; genetics ; Nucleoside-Diphosphate Kinase ; Ovarian Neoplasms ; genetics ; metabolism ; pathology ; RNA, Messenger ; genetics ; Repressor Proteins ; Transcription Factors ; biosynthesis ; genetics

OBJECTIVETo study angiogenesis and the expression of nm23-H(1) tumor metastatic suppressor gene in primary breast carcinoma and their relationship with axillary lymph node metastasis.

METHODSIntratumoral vascularization in 80 cases of breast cancer was observed preoperatively by power doppler imaging (PDI) and analysed with computer-assisted quantative assessment, and the expression of microvessel density (MVD) and nm23 protein was determined by immunohistochemical technique.

RESULTSBlood flow signals and blood vessels postitive area within masses were more in axillary node positive (LN+) group than in axillary node negative (LN-) group (t = 7.07, P < 0.01), MVD expression were higher in the LN+ group than in the LN- group (t = 6.34, P < 0.01). Meanwhile the expression of nm23-H(1) protein was lower in the LN+ group than in the LN- group, and significant correlation was found between angiogenesis and the expression of nm23-H(1) protein.

CONCLUSIONSAngiogenesis and the expression of nm23-H(1) protein may play an important role in the lymphatic metastasis process of breast cancer. Furthermore, angiogenesis may correlates with the expression of nm23-H(1) protein in that progress.

Adult ; Aged ; Aged, 80 and over ; Axilla ; Biomarkers, Tumor ; biosynthesis ; Breast Neoplasms ; diagnosis ; metabolism ; pathology ; physiopathology ; Female ; Genes, Tumor Suppressor ; Humans ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; diagnosis ; Middle Aged ; Monomeric GTP-Binding Proteins ; biosynthesis ; NM23 Nucleoside Diphosphate Kinases ; Neovascularization, Pathologic ; Nucleoside-Diphosphate Kinase ; Transcription Factors ; biosynthesis