The adapter protein Lamellipodin (Lpd) plays an important role in cell migration. In particular, Lpd mediates lamellipodia formation by regulating actin dynamics via interacting with Ena/VASP proteins. Its RA-PH tandem domain configuration suggests that like its paralog RIAM, Lpd may also mediate particular Ras GTPase signaling. We determined the crystal structures of the Lpd RA-PH domains alone and with an N-terminal coiled-coil region (cc-RA-PH). These structures reveal that apart from the anticipated coiled-coil interaction, Lpd may also oligomerize through a second intermolecular contact site. We then validated both oligomerization interfaces in solution by mutagenesis. A fluorescence-polarization study demonstrated that Lpd binds phosphoinositol with low affinity. Based on our crystallographic and biochemical data, we propose that Lpd and RIAM serve diverse functions: Lpd plays a predominant role in regulating actin polymerization, and its function in mediating Ras GTPase signaling is largely suppressed compared to RIAM.
Actins ; metabolism ; Amino Acid Sequence ; Binding Sites ; Carrier Proteins ; chemistry ; genetics ; metabolism ; Crystallography, X-Ray ; Humans ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Molecular Sequence Data ; Mutagenesis ; Phosphatidylinositols ; metabolism ; Polymerization ; Protein Binding ; Protein Structure, Tertiary ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics ; Signal Transduction ; ras Proteins ; 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 study the expression of RAS protein in human glioma tissues and its influence on tumor growth.

METHODSRAS protein expression in glioma tissues was determined by immunohistochemical (IHC) staining. Subsequently, MTT cell proliferation assay, flow cytometry and Western blotting were used to assay U251 cells with reduced RAS expression.

RESULTSThe expression of RAS in glioma was increased and strongly correlated with pathological grade. Downregulation of RAS resulted in glioma cells growth suppression and increased apoptosis.

CONCLUSIONThe expression level of RAS protein in human glioma was increased. Downregulation of RAS can inhibit glioblastoma cell growth through the RAS signal pathway.

Brain Neoplasms ; genetics ; metabolism ; pathology ; Cell Growth Processes ; genetics ; Cell Line, Tumor ; Down-Regulation ; Glioma ; genetics ; pathology ; Humans ; Immunohistochemistry ; ras Proteins ; biosynthesis ; genetics

BACKGROUND/AIMS: Mutations of the epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene (KRAS) are important in the pathogenesis of lung cancer, and recent reports have revealed racial and geographical differences in mutation expression. METHODS: This study was conducted to investigate the prevalence of EGFR and KRAS mutations and their correlation with clinical variables in Korean patients with adenocarcinoma of the lung. Formalin-fixed adenocarcinoma specimens from 104 randomly selected patients diagnosed at Kosin University Gospel Hospital from October 1996 to January 2005 were used for the study. RESULTS: We found a high prevalence of EGFR mutations and a low prevalence of KRAS mutations. EGFR mutations were present in 24% (25 of 104) of the samples: one mutation in exon 18, 13 in exon 19, one in exon 20, and 10 in exon 21. The presence of an EGFR mutation was not associated with gender, smoking history, histological grade, age, bronchioalveolar components, or cancer stage in patients with adenocarcinoma of the lung. CONCLUSIONS: Mutations of KRAS were present in 9.6% (9 of 94) of the samples: eight in codon 12 and one in codon 13. EGFR mutations were never found in tumors with KRAS mutations, suggesting a mutually exclusive relationship.
Adenocarcinoma/*genetics/metabolism/mortality ; Adult ; Aged ; DNA, Neoplasm/*genetics ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Korea/epidemiology ; Lung Neoplasms/*genetics/metabolism/mortality ; Male ; Middle Aged ; *Mutation ; Polymerase Chain Reaction ; Prognosis ; Proto-Oncogene Proteins/biosynthesis/*genetics ; Receptor, Epidermal Growth Factor/biosynthesis/*genetics ; Retrospective Studies ; Survival Rate ; ras Proteins/biosynthesis/*genetics

OBJECTIVETo investigate the effect of Abnormal Savda Munziq (ASMq) flavonoids on proliferation, apoptosis and apoptosis-related gene expression in human hepatoma (HepG2) cells in vitro and to probe the mechanism.

METHODThe effects of ASMq flavonoids on proliferation, apoptosis and apoptosis-related gene expression of HepG2 cells were investigated respectively by MTT assay, gel electrophoresis, flow cytometry and RT-PCR.

RESULTASMq flavonoids significantly inhibited growth of HepG2 cells in vitro, arrested HepG2 in the sub-G, phase, induced cell apoptosis and significantly down-regulated expression level of Bcl-2 mRNA, and up-regulated expression of p53, p21, Bax gene mRNA expressions.

CONCLUSIONASMq flavonoids has significantly regulative action on growth, apoptosis and apoptosis-related gene expression of cancer cells in vitro, which possibly are the important way to excert anticancer effect, and flavonoids are possibly a main active component of ASMq for exerting the anticancer effect.

Antineoplastic Agents, Phytogenic ; isolation & purification ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drug Combinations ; Flavonoids ; isolation & purification ; pharmacology ; Flow Cytometry ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Plants, Medicinal ; chemistry ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Proto-Oncogene Proteins p21(ras) ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics ; bcl-2-Associated X Protein ; biosynthesis ; genetics

Axin is a negative regulator of the Wnt/beta-catenin pathway and is involved in the regulation of axis formation and proliferation. Involvement of Axin in the regulation of other signaling pathways is poorly understood. In this study, we investigated the involvement of Akt in growth regulation by Axin in L929 fibroblasts stimulated by EGF. Akt activity was increased by EGF treatment and Ras activation, respectively. Both the EGF- and Ras-induced Akt activations were abolished by Axin induction, as revealed by both Western blot and immunocytochemical analyses. The proliferation and Akt activation induced by EGF were decreased by Axin induction, and the effects of EGF were abolished by treatment of an Akt-specific inhibitor. Therefore, Axin inhibits EGF-induced proliferation of L929 fibroblasts by blocking Akt activation.
Animals ; Cell Line ; Cell Nucleus/metabolism ; Cell Proliferation ; Epidermal Growth Factor/*pharmacology ; Fibroblasts/drug effects/physiology ; Mice ; Proto-Oncogene Proteins c-akt/antagonists & inhibitors/*metabolism ; Repressor Proteins/genetics/*physiology ; Signal Transduction ; ras Proteins/biosynthesis/genetics

OBJECTIVETo study the function of c-myc and K-ras in tumorigenesis of ovarian cancer.

METHODSK-ras and/or c-myc cDNAs were introduced into mouse ovarian surface epithelium cells (MOSE) using recombinant Moloney retroviral vectors. The resulting MOSE cells were studied by cell proliferation assays, the ability to form colonies in soft agarose, matrigel invasion assays and tumorigenicity assays in nude mice.

RESULTSK-ras and c-myc can be easily delivered to the normal MOSE cells by recombinant retroviruses. mRNA and protein of the target genes can be detected by RT-PCR and Western blot. Cell proliferation assays showed that MOSE-Ras cells and MOSE-RM cells (MOSE-Ras/Myc) grew more rapidly than parental cells (MOSE) and MOSE-Myc cells (P <0.01). In addtition, MOSE-RM cells grew more rapidly than MOSE-Ras cells (P <0. 05). Cell colony formation assays showed that while MOSE-Ras and MOSE-RM cells can form colonies in soft-agarose, the MOSE-Myc and MOSE cells did not. Matrigel invasion assays showed that MOSE-Ras and MOSE-RM cells have invasion ability, but not MOSE-Myc ascites and the control MOSE cells. Xenograft experiments showed that MOSE-Ras and MOSE-RM cells were able to form tumors in nude mice following intraperitoneal injection. Tumors were not observed in animals injected with either MOSE-Myc or MOSE cells.

CONCLUSIONThe recombinant Moloney retroviral system is a highly efficient and convenient method for introducing and expressing foreign genes in murine surface epithelial cell cultures. In this model, expression of K-ras alone is sufficient to generate tumorigenic MOSE, however expression of c-myc in conjunction with K-ras results in cells with a higher index of malignancy. Based on the assays described in this report, expression of c-myc alone can not transform MOSE cultures although it does play a role in cooperation with K-ras.

Animals ; Blotting, Western ; Cell Movement ; Cell Proliferation ; Cell Transformation, Neoplastic ; Cells, Cultured ; Epithelial Cells ; cytology ; metabolism ; Female ; Immunohistochemistry ; Mice ; Mice, Nude ; Mice, Transgenic ; Neoplasms, Experimental ; genetics ; metabolism ; pathology ; Oncogene Protein p21(ras) ; biosynthesis ; genetics ; Ovarian Neoplasms ; genetics ; metabolism ; pathology ; Ovary ; cytology ; Proto-Oncogene Proteins c-myc ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; Retroviridae ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVE: To investigate the expression of P21 in renal interstitial fibrosis rats and the effect of enalapril on it. METHODS: Sprague Dawley rats were randomly divided into 3 groups: a sham operation group,a unilateral urethral obstruction group, and an enalapril treatment group. The expression of P21 in renal tubular epithelial cells on the process was detected by immunohistochemistry at different time spots (7, 14, 21 d after UUO, sham-surgery or enalapril treatment). The expression of p21 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Seven days after the surgery, significant differences were found in P21 expression between UUO and SOR renal tubular cells. With degree of interstitial fibrosis aggravating, P21 expression increased. Enalapril can inhibit its expression. CONCLUSION In the kidney of UUO rats, P21 expression increased and enalapril possessed significant inhibitory effects on the procedure. P21 may participate in the pathogenesis of renal tubule-interstitial fibrosis.
Angiotensin-Converting Enzyme Inhibitors ; pharmacology ; therapeutic use ; Animals ; Enalapril ; pharmacology ; therapeutic use ; Kidney Tubules ; metabolism ; Male ; Nephrosclerosis ; drug therapy ; metabolism ; Proto-Oncogene Proteins p21(ras) ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Ureteral Obstruction ; complications

OBJECTIVETo investigate the role of Ras antisense oligoribonucleotide (ASODN) in multidrug resistance (MDR) of pancreatic carcinoma Pc-2 cells.

METHODSRas and P-gp expression was suppressed by Ras ASODN. Sensitivity of Pc-2 cells to chemotherapy was determined by the MTT assay. MDR-1 mRNA level was detected by fluorogenic probe quantitative reverse transcription polymerase chain (RT-PCR) method. Flow cytometry (FCM) was used to detect the accumulative concentration of adriamycin (ADR) in the cells.

RESULTSRas ASODN significantly inhibited the Ras and P-gp expression (P < 0.05), increased the sensitivity of Pc-2 cells to chemotherapeutic agents (P < 0.05), decreased MDR-1 gene level in Pc-2 cells (P < 0.05), and increased the intracellular intake of ADR in Pc-2 cells (P < 0.05).

CONCLUSIONRas ASODN may enhance the sensitivity of multidrug-resistant pancreatic cancer Pc-2 cells to chemotherapeutic agents by regulating MDR-1 gene level.

ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; Cell Line, Tumor ; Down-Regulation ; Doxorubicin ; metabolism ; Drug Resistance, Multiple ; drug effects ; genetics ; Drug Resistance, Neoplasm ; drug effects ; genetics ; Genes, MDR ; drug effects ; genetics ; Humans ; Oligonucleotides, Antisense ; pharmacology ; Pancreatic Neoplasms ; genetics ; metabolism ; pathology ; ras Proteins ; biosynthesis ; genetics

OBJECTIVE: To investigate the effects of long-term estrogen replacement treatment (ERT) on the expression of bcl-2 and H-ras in rat endometrium. METHODS: Thirty 5-month-old SD female rats were randomly divided into 3 groups: Control group ( sham operated and vehicle injected, n 10) , OVX group (OVX operated and vehicle injected, n = 10) , and ERT group (OVX operated and 17 beta-estradiol injected, n = 10). The rats were killed in the 13th week and the uteri were isolated and weighed, pathologically analyzed, and we measured the thickness of the endometrium. Immunochemistry and in situ hybridization analysis were used to examine the changes of bcl-2 and H-ras mRNA and Bcl and H-ras protein expression in the endometrium of the rats. RESULTS: Uterine weight and endometrial thickness of OVX decreased much more than those of the control (P <0.01 ) and ERT rats (P < 0.01). One simple hyperplasia and one squamous metaplasia of endometrium were found in ERT rats. Quantitatively, bcl-2 and H-ras mRNA and Bcl and H-ras protein level of ERT were higher than those of OVX rats (P < 0.01 ), and there were no statistical significances between the ERT group and the control rats. CONCLUSION Long-term estrogen replacement can keep the endometrium from atrophy, and lead to the genesis of simple hyperplasia and squamous metaplasia of the endometriun, which can increase the risk of endometrial carcinomas. Estrogen may inhibit apoptosis and promote the proliferation of endometrial cells through increasing the expression of bcl-2 and H-ras mRNA and Bcl-H-ras proteins.
Animals ; Endometrium ; metabolism ; Estradiol ; pharmacology ; Estrogen Replacement Therapy ; Female ; Genes, bcl-2 ; Genes, ras ; Ovariectomy ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Time Factors ; ras Proteins ; biosynthesis ; genetics