Cell Adhesion ; drug effects ; Cell Movement ; drug effects ; Focal Adhesion Kinase 1 ; genetics ; metabolism ; Focal Adhesion Protein-Tyrosine Kinases ; Neoplasms ; Phosphorylation

OBJECTIVE: To study the expressions of focal adhesion kinase (FAK) and phospho-FAK( p-FAK) during skin incised wound healing and the applicability of time-dependent expressions of FAK and p-FAK. METHODS: The expression of FAK and p-FAK in cutaneous incised wound in mouse were investigated by immunohistochmeistry and Western blotting. RESULTS: FAK and p-FAK expression were detected in polymorphonuclear cells (PMNs) in the wound and adjacent regions 3 hours post-injury. The expressions of FAK and p-FAK were detected in a large number of infiltrating PMNs and some of mononuclear cells (MNCs) from 6 to 24 hours after injury. The MNCs and fibroblastic cells (FBCs) accounted for most part of the FAK and p-FAK positive cells from 3 to 14 days after injury. The numbers of FAK-positive cells increased continuously, reaching a peak at post-injury day 3, and then started to decrease from post-injury day 5 to 14. The changes of p-FAK-positive cells were similar to that of the FAKs, and reached a peak at 12 hours after injury. CONCLUSION Both FAK and p-FAK displayed a time-dependent expression during skin incised wound healing in mouse, with p-FAK being superior to FAK. Both FAK and p-FAK may potentially be used as new markers for determination of the wound interval.
Animals ; Focal Adhesion Kinase 1/metabolism* ; Mice ; Phosphorylation ; Skin/injuries* ; Time Factors ; Wound Healing

Cell migration plays an important role in repair of injury, angiogenesis, cancer metastasis and so on. In this paper the effects of basic fibroblast growth factor (bFGF) of different concentrations on ECV-304 cell migration, and the dynamic changes in focal adhesion kinase (FAK) were observed. The relationship between FAK and cell migration induced by bFGF was studied. A ECV-304 cell scratch wound model was established and the images of cell migration were quantitatively measured using a computer-assisted videomicroscopic system. The dynamic changes in FAK content (Western blot), FAK activity (immunoprecipitation plus Western blot) and FAK mRNA (RT-PCR) were measured in vitro. The expression of integrin alpha3 was investigated using immunocytochemical staining (ABC method). The results showed that bFGF produced a dual-phase regulatory effect on ECV-304 migration when the cell confluent areas reached 90%-95% in culture. It was found that compared with the control group (0 ng/ml bFGF), the cell migration was stimulated (P<0.05), inhibited (P<0.05) and unchanged when the cultured cells were treated with bFGF at the concentrations of 5, 10 and 15 ng/ml, respectively. The content and activity of FAK protein were markedly up-regulated in 5 ng/ml bFGF group and down-regulated in 15 ng/ml bFGF group, respectively. FAK mRNA expression came to the peak in 5 ng/ml bFGF group after 6 h culture and there was a significant difference compared with the control group. In various experimental groups there were no significant differences in the expression of integrin alpha3 compared with the control group according to the immunocytochemical staining. The results mentioned above suggest that different concentrations of bFGF have a dual-phase effect on the migration of cultured ECV-304 cells, which correlates positively with FAK content, activity and mRNA in cultured ECV-304 cell scratch wound model. The FAK plays an important role in the signal transduction pathway of cell migration induced by bFGF, while bFGF can regulate the content of FAK in ECV-304 cells at gene transcription level.
Cell Movement ; physiology ; Cells, Cultured ; Endothelium, Vascular ; cytology ; Fibroblast Growth Factor 2 ; physiology ; Focal Adhesion Kinase 1 ; Focal Adhesion Protein-Tyrosine Kinases ; Humans ; Integrin alpha3 ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; RNA, Messenger ; metabolism ; Umbilical Veins ; cytology

Animals ; Colonic Neoplasms ; metabolism ; pathology ; Focal Adhesion Kinase 1 ; Focal Adhesion Protein-Tyrosine Kinases ; Humans ; Integrins ; metabolism ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Protein-Tyrosine Kinases ; metabolism ; Rectal Neoplasms ; metabolism ; pathology ; Signal Transduction

OBJECTIVESTo investigate the effects of Arg-Gly-Asp-Ser (RGDS) tetrapeptide on integrin signaling and apoptosis in fibronectin (FN) -stimulated hepatic stellate cells (HSCs).

METHODS3H-thymidine incorporation, annexin-V/propidium iodide double-labeled flow cytometry (FCM) and transmission electron microscopy were employed to estimate the influence of RGDS on the proliferation and apoptosis of HSCs. And the adhesion rates were observed by toluidine blue colorimetric assay. The expression of focal adhesion kinase (FAK) mRNA and protein in HSCs was detected using RT-PCR and western blotting analysis, respectively.

RESULTSRGDS tetrapeptide at the concentrations of 25 microg/ml, 50 microg/ml and 100 microg/ml inhibited the proliferation of HSCs and induced HSCs apoptosis in dose-dependent and time-dependent manners, with the apoptotic rates of 9.49%, 27.67%, 31.59%, and the necrotic rates of 3.47%, 5.38%, 9.10%, respectively. Both the rates were higher than those in FN group (apoptotic rate: 3.44%; necrotic rate: 2.39%), F=8.02, P<0.05. After adding RGDS tetrapeptide to HSCs for 2 hours, the adhesive inhibition rates were 8.82%, 29.41% and 45.59%, respectively, which were higher than that in FN group (F=20.58, P<0.01). After exposure of HSCs to RGDS tetrapeptide for 24 hours, FAK protein decreased, and FAK mRNA was down-regulated earlier, about 2 hours after exposure to RGDS tetrapeptide.

CONCLUSIONThese results suggest that RGDS tetrapeptide may induce apoptosis of HSC in both dose-dependent and time-dependent manners in vitro, which may be related to the disruption of cell matrix adhesion and down-regulation of FAK expression.

Apoptosis ; drug effects ; Fibronectins ; pharmacology ; Focal Adhesion Kinase 1 ; Focal Adhesion Protein-Tyrosine Kinases ; Hepatocytes ; cytology ; physiology ; Humans ; Integrins ; metabolism ; physiology ; Liver ; cytology ; physiology ; Oligopeptides ; pharmacology ; Platelet Aggregation Inhibitors ; pharmacology ; Protein-Tyrosine Kinases ; biosynthesis ; genetics ; metabolism ; Signal Transduction

This study was aimed to investigate the effects of focal adhesion kinase (FAK) gene silence on leukemia cell growth, leukemogenesis and efficacy of chemotherapy drug. Vector containing lentiviral-FAK-shRNA was constructed and transfected into BCR/ABL-BaF3 leukemic cells, the cell growth and apoptosis were detected in vitro. The effect of FAK shRNA on leukemogenesis was studied in a murine model with leukemia. The apoptosis of leukemia cells and survival of leukemic mice treated by FAK shRNA combined with drug STI571 were monitored. The results showed that FAK gene expression was knocked down by lentiviral-FAK-shRNA. FAK gene silencing inhibited leukemia cell growth in vitro. The apoptosis test results showed that the percentages of Annexin V(+) cells in vector control group and FAK shRNA group were (3.46 ± 0.56)% and (7.3 ± 0.79)%, respectively, and the difference was statistically significant (p < 0.05). The mice in vector control group died at day 21 to 27, while the mice in FAK shRNA group died between day 52 and 60, and the difference was statistically significant (p < 0.05). Moreover, FAK gene silence combined with drug STI571 could enhance the apoptosis of leukemia cells and prolong survival time of leukemic mice. It is concluded that FAK gene silence inhibits leukemogenesis and promotes efficacy of chemotherapy drug on leukemia cells, indicating FAK gene silence may be considered as a new therapeutic strategy for leukemia.
Animals ; Focal Adhesion Kinase 1 ; genetics ; Gene Silencing ; Genetic Vectors ; Leukemia, Experimental ; genetics ; therapy ; Male ; Mice ; Mice, Inbred BALB C ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo investigate the effect of the wild type phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor-suppressor gene on the proliferation and apoptosis of human chronic myeloid leukemia (CML) cells line (K562) in vitro and explore the influence of PTEN-FAK signaling pathway on invasion and metastasis of leukemia cells.

METHODSThe recombinant Ad-PTEN gene containing green fluorescent protein gene (Ad-PTEN-GFP) or the empty vector (Ad-GFP) was transfected into K562 cells and fresh leukemia cells from CML patients in blast crisis. The growth of K562 cells was assayed by MTT assay; the apoptosis rate was assessed by flow cytometry (FCM). PTEN and FAK mRNA levels were detected by real-time fluorescent relative- quantification reverse transcriptional PCR (FQ-PCR) and its protein levels by Western blot. The metastasis and invasive ability was examined by transwell chamber assay.

RESULTSThe growth of K562 cells was suppressed markedly when Ad-PTEN-GFP was transfected into K562 cells at the 200 multiplicity of infection (MOI). The maximum growth inhibition rate was 35.2%. Transwell results showed the number of cells entered the lower chamber in Ad-GFP group was 9.1 fold more than that in Ad-PTEN-GFP group;The ability of metastasis and invasion of fresh leukemia cells was also suppressed after transfection with Ad-PTEN-GFP. FAK and p-FAK proteins were down-regulated by 0.72 and 0.16 fold lower after transfected with Ad-PTEN-GFP compared with Ad-GFP group.

CONCLUSIONSPTEN gene might inhibit the proliferation, metastasis and invasive ability of leukemia cells via down-regulating FAK expression.

Apoptosis ; Cell Movement ; Cell Proliferation ; Focal Adhesion Kinase 1 ; genetics ; metabolism ; Genetic Vectors ; Humans ; K562 Cells ; Leukemic Infiltration ; PTEN Phosphohydrolase ; genetics ; metabolism ; Signal Transduction ; Transfection

This study was purposed to investigate the effect of phycocyanin at different concentration on proliferation of K562 cells, to detect the changes of integrin beta1 expression and intracellular focal adhesion kinase (FAK) gene expression on the surface K562 cells treated with phycocyanin, and to explore the possible mechanism of integrin beta1 effect on phycocyanin inhibiting proliferation of K562 cells. The expression level of integrin beta1 on the surface of K562 cells was evaluated by flow cytometry (FCM); the growth of K562 cells treated with phycocyanin was measured by MTT assay; the expression level of FAK mRNA was analyzed by relatively quantitative RT-PCR after four-day culture of K562 cells with phycocyanin of 40 microg/ml, 80 microg/ml and 160 microg/ml, respectively. The results showed that integrin beta1 expression on the surface of K562 cells was significantly higher than that in bone marrow mononuclear cells (BMMNC) from normal subjects. Phycocyanin could not change the level of integrin beta1 expression. Phycocyanin could increase the expression of FAK gene on K562 cells and inhibit the proliferation of K562 cells. It is concluded that phycocyanin can inhibit the proliferation of K562 cells through enhancing the conjunction of cell stroma with integrin beta1 on K562 cell surface, up-regulating the expression level of FAK gene in K562 cells, restoring the signaling pathway of proliferation inhibition mediated by integrin beta1. The possible mechanism of phycocyanin in the proliferation inhibition of K562 cells is to increase the expression of FAK gene. The phycocyanin may be considered as a potential agent for inhibition of cancer cell proliferation.
Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Proliferation ; drug effects ; Focal Adhesion Kinase 1 ; biosynthesis ; genetics ; Humans ; Integrin beta1 ; biosynthesis ; genetics ; K562 Cells ; Phycocyanin ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics

The aim of this study was to investigate the effects of tyrosine kinase inhibitor PTK787 on cell proliferation, cell cycle and the expression of fak mRNA of human chronic myeloid leukemia (CML) cell line K562, and to explore the mechanism of PTK787 against acute myeloid leukemia. The MTT method was used to detect the effects of PTK787 in various concentrations and at different time points on proliferation of K562 cells; the flow cytometry was used to determine the effects of PTK787 in different concentrations on cell cycle of K562 cells; the RT-PCR was used to assay the expression of fak mRNA in K562 cells treated with PTK787 for 48 hours. The results showed that along with increasing of the concentration and prolonging of time, the inhibitory rate of PTK787 on K562 proliferation was gradually enhanced. The comparison between various concentration groups at same time or comparison between various time groups in same concentration showed significant differences (p < 0.05), in which the effect of 320 micromol/L PTK787 on cells was strongest, while the continuous increase of PTK787 concentration or prolong of action time did not enhance the inhibitory rate on K562 proliferation. With increasing of drug concentration, the cell proportion in G(1) phase gradually increased, the cell proportion in S phase gradually decreased, the comparison between various groups revealed significant differences (p < 0.05), however the continuous increase of drug concentration from 160 micromol/L did not obviously change the cell proportion in phases of cell cycle. With increasing of drug concentration, the expression of fak mRNA in K562 cells gradually reduced with significant differences between various groups (p < 0.05), but with continuous increase of drug concentration from 160 micromol/L, the effect of PTK787 on the expression of fak mRNA in K562 cells also did not obviously change. It is concluded that the PTK787 shows effect of anti-leukemia cells through inhibiting transformation of the K562 cells from G(1) phase into S phase and decreasing the expression of fak mRNA in cells.
Cell Cycle ; Cell Proliferation ; drug effects ; Focal Adhesion Kinase 1 ; genetics ; Gene Expression Regulation, Leukemic ; Humans ; K562 Cells ; Phthalazines ; pharmacology ; Pyridines ; pharmacology ; RNA, Messenger ; genetics

OBJECTIVETo study the mechanism of integrin in hypertrophic scar.

METHODSFibroblasts from 10 samples of human hypertrophic scars was cultured, FQ-PCR assay was applied to detect mRNA expression of alpha-SMA in hypertrophic scar fibroblasts after integrin and FAK antibody blocking.

RESULTSmRNA of alpha-SMA in fibroblasts expressed obviously lower after integrin and FAK antibody blocking than that of control groups ( P < 0.05).

CONCLUSIONThrough accelerating the synthesis of alpha-SMA, integrin and FAK play an important role in contracture of hypertrophic scar.

Actins ; biosynthesis ; Adult ; Cells, Cultured ; Cicatrix, Hypertrophic ; metabolism ; pathology ; Contracture ; metabolism ; pathology ; Fibroblasts ; metabolism ; Focal Adhesion Kinase 1 ; biosynthesis ; Humans ; Integrins ; biosynthesis ; RNA, Messenger ; metabolism ; Young Adult