Changes In F-actin Cytoskeleton Research Articles

Gamma oscillations in the pedunculopontine nucleus are regulated by F-actin: neuroepigenetic implications.

The pedunculopontine nucleus (PPN) is part of the reticular activating system (RAS) in charge of arousal and rapid eye movement sleep. The presence of high-frequency membrane oscillations in the gamma-band range in the PPN has been extensively demonstrated both in vivo and in vitro. Our group previously described histone deacetylation (HDAC) inhibition in vitro induced protein changes in F-actin cytoskeleton and intracellular Ca2+ concentration regulation proteins in the PPN. Here, we present evidence that supports the presence of a fine balance between HDAC function and calcium calmodulin kinase II-F-actin interactions in the PPN. We modified F-actin polymerization in vitro by using jasplakinolide (1 μM, a promoter of F-actin stabilization), or latrunculin-B (1 μM, an inhibitor of actin polymerization). Our results showed that shifting the balance in either direction significantly reduced PPN gamma oscillation as well as voltage-dependent calcium currents.

Homeobox B7 accelerates the cancer progression of gastric carcinoma cells by promoting epithelial-mesenchymal transition (EMT) and activating Src-FAK pathway.

Aim: To study the carcinogenetic mechanism of HOXB7 in gastric cancer (GC) remains.Methods: Two human GC cell lines — SGC7901 and SNU1 — were used for this study. SGC7901 cells were transfected with siRNA-HOXB7 (siHOXB7) to knock down HOXB7 expression, whereas, SNU1 cells were transduced with pCDNA3.1-HOXB7 to overexpress HOXB7. After transfection, cancer progression was assessed by determining cell proliferation, wound-healing process, cell cycle, apoptosis, invasion, and migration. The effect of HOXB7 on epithelial–mesenchymal transition (EMT) was measured by observing changes in F-actin cytoskeleton and evaluating the expression of EMT markers. p-Scr and p-FAK were evaluated to assess the mechanism.Results: Knockdown of HOXB7 suppressed cell proliferation, alleviated the wound-healing process, inhibited cell migration and invasion, and arrested the cell cycle while promoting cell apoptosis, suggesting the tumor-suppressive effect of siHOXB7 in human GC cells. On the contrary, HOXB7 overexpression showed a tumor-promoting effect on human GC cells. Moreover, we confirmed an inhibitory effect of siHOXB7 on the EMT process by preventing epithelial cells from acquiring a mesenchymal phenotype and downregulating mesenchymal markers (vimentin, β-catenin, N-cadherin, Twist) while upregulating epithelial markers (E-cadherin). Our data revealed that HOXB7 was associated with Src/FAK and favored the activation of the Src–FAK pathway in human GC cells.Conclusion: HOXB7 accelerated the malignancy of GC, by facilitating EMT and regulating the Scr–FAK pathway.

C-Myc activation promotes cofilin-mediated F-actin cytoskeleton remodeling and telomere homeostasis as a response to oxidant-based DNA damage in medulloblastoma cells

Medulloblastoma (MB) is a common and highly aggressive pediatric brain tumor of a heterogeneous nature. According to transcriptome-based profiling, four molecular subgroups of MB have been revealed, namely WNT, SHH, Group 3 and Group 4. High MYC mRNA expression and MYC gene amplification in MB have been considered as indicators of poor prognosis. However, the role of c-Myc in MB biology is still not well established. In the present study, the effects of c-Myc activation in UW228-MycER MB cell line were investigated using 4-hydroxytamoxifen (4-OHT) induction system. Upon 4-OHT stimulation, an increase in metabolic activity, large-cell/anaplastic (LC/A) phenotype and oxidative stress-mediated DNA damage were observed. However, 53BP1 foci were not implicated in DNA damage response. Instead, cofilin nuclear translocation, changes in F-actin cytoskeleton and the levels of cytoskeletal proteins were shown. Moreover, the telomere length was found to be unaffected that may be associated with the upregulation of TRF proteins. Transcription of nascent RNA (synthesis of new rRNA) and the expression of RNA polymerase I-specific transcription initiation factor RRN3/TIF-IA were also elevated. Moreover, increased levels of DNMT2, a modulator of stress responses, were observed. A small fraction of cells responded differently as oncogene-induced senescence was also noticed. We postulate that c-Myc-mediated modulation of genetic stability of MB cells may trigger cellular heterogeneity and affect adaptive responses to changing environment.

BCL-2 inhibitor combined with low dose cytarabine in the treatment of relapsed acute myeloid leukemia after allogeneic hematopoietic stem cell transplantation: a case report and literature review

目的探索GTP结合蛋白氯苄乙胺2（Septin2）在霍奇金淋巴瘤细胞株L428细胞中的表达及其在H/RS（Hodgkin/Reed-Sternberg）细胞向B细胞再分化中的作用。方法采用实时荧光定量PCR（RT-qPCR）、Western blot、激光共聚焦显微镜、免疫细胞化学法检测霍奇金淋巴瘤细胞株L428细胞过表达CD99前后Septin2在mRNA和蛋白水平的表达；采用RT-qPCR和Western blot检测L428细胞转染Septin2干扰片段（Septin2-siRNA）后Septin2的表达；采用激光共聚焦显微镜、CCK8法和流式细胞术检测干扰Septin2基因对L428细胞骨架蛋白、细胞增殖活性和免疫表型的影响。结果与L428细胞空白对照组比较，L428细胞Septin2 mRNA表达水平在CD99过表达后（0.329±0.019对1.000，P=0.001）和转染Septin2-siRNA后（0.276±0.025对1.000，P=0.000）均下降，蛋白表达水平也下降。与转染空载体组比较，转染Septin2-siRNA后的L428细胞较前者胞体缩小，细胞增殖活性下降（F=204.927，P<0.001)，细胞中的微丝骨架发生了重建，CD30和CD15表达下降，CD19、CD10、CD38表达增高。结论Septin2在L428细胞中高表达，干扰Septin2可促进H/RS细胞向B细胞方向再分化。

Lobaplatin-induced changes of F-actin and α-tubulin cytoskeleton in cholangiocarcinoma cell line RBE

Objective To investigate the F-actin and α- tubulin cytoskeleton changes in cholangiocarcinoma cell line RBE after treatment with Lobaplatin. Methods The RBE cells were cultured in vitro, followed by treatment with various concentrations of lobaplatin for 24 h. The changes of F-actin cytoskeleton were scanned by confocal laser scanning microscopy(CLSM)after staining with phalloidin-fluoresceine isothiocyanate(FITC)and DAPI. Immunohistochemistry and real-time polymerase chain reaction(Real-time PCR)were used to observe α-tubulin cytoskeleton changes and α-tubulin mRNA transcript levels respectively. Results CLSM and immunohistochemistry showed that both the F-actin and the α- tubulin cytoskeleton of RBE cells had disorganization and rearrangement after the treatment with lobaplatin. The staining of α - tubulin was getting deep(P<0.05),and the Real- time PCR determinedα- tubulin mRNA/β- actin ratio in 5.0 mg/L and 25.0 mg/L lobaplatin- treated groups was 2.08±0.13 and 1.91±0.46 respectively (P<0.05). Conclusion The changes of microfilaments and microtubules in RBE cells play a positive role in the apoptosis induced by lobaplatin. Key words: Cholangiocarcinoma; Lobaplatin; Cytoskeleton; F-actin; α-tubulin

Effect of simulated microgravity on osteocytes responding to fluid shear stress

Osteocytes, as most abundant cells and major mechanical sensor in bone, play an important role in the mechanism of microgravity-induced bone loss. The response of osteocytes to fluid flow stress under simulated microgravity was investigated in this study. MLO-Y4, an osteocyte-like cell line, was cultured under simulated microgravity condition for 5 days. Then cells were sheared at 15dyn/cm2 in flow chamber. After 15min shear, nitric oxide (NO) was examined by Griess Reagent and prostaglandin E2 (PGE2) by ELISA. After 6h shear, alkaline phosphatase (ALP) was examined by PNPP, osteocalcin (OC) and procollagen type I N propeptide (PINP) by ELISA. Cells were divided into four groups: CON (1G with no shear), CON-S (1G with shear), SM (simulated microgravity with no shear) and SM-S (simulated microgravity with shear). The results showed that (1) NO, ALP activity, OC and PINP increased significantly while PGE2 showed no change in SM compared with CON. (2) NO, PGE2, ALP activity and PINP increased significantly while OC decreased significantly in CON-S compared with CON. (3) NO in SM-S had no significant difference compared to SM, PGE2 and OC increased while ALP activity and PINP decreased significantly in SM-S compared with SM. (4) The increasing amplitude of PGE2 and OC, the decreasing amplitude of ALP activity in SM-S to SM was lower than that in CON-S to CON. In addition, some changes of F-actin cytoskeleton were observed by confocal microscopy. All results indicated that the response induced by fluid shear in osteocytes could be inhibited by simulated microgravity, namely the mechanosensibility of osteocytes decreased under simulated microgravity. This may partly contribute to the mechanism of microgravity-induced osteoporosis and will be helpful to find out effective description.

The effect of 3‐(5‐nitro‐2‐thienyl)‐9‐chloro‐5‐morpholin‐4‐yl[1,2,4]triazolo[4,3‐c]quinazoline on cell growth, cell cycle, induction of DNA fragmentation, and activity of caspase 3 in murine leukemia L1210 cells and fibroblast NIH‐3T3 cells

Quinazolines are multitarget agents, which have broad spectrum of biological activity, and some of them are now in cancer clinical testing. 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline is a new synthetically prepared derivative, which in our previous study showed cytotoxic effects on cancer cell lines HeLa and B16. Quinazoline, at micromolar concentrations, induced morphological changes and necrosis of B16 cells, and at nanomolar concentrations it produced changes of F-actin cytoskeleton. It did not cause changes in the cell cycle, did not induce apoptotic cell death in B16 cells, did not have a mutagenic effect, and did not even behave as a typical intercalating agent. Little significant reduction of tumor volume in intramuscular transplanted B16 cells was observed. The aim of the present study was to examine the cytotoxic effect of 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline on murine leukemia L1210 cells and fibroblast NIH-3T3 cells. Induction of cell morphology and cell cycle changes, induction of apoptosis and caspase 3 activity were studied. Quinazoline acted cytotoxically on both cell lines. The sensitivity of leukemia L1210 cells to the quinazoline was higher than that of fibroblast NIH-3T3. The IC(100) was 12 microM for L1210 cells and 24 microM for NIH-3T3 cells. No effect of quinazoline on the cell cycle profile of L1210 and NIH-3T3 was detected, however, quinazoline induced an increase of the sub-G(0) cell fraction, apoptotic DNA fragmentation, and apoptotic morphological changes at a concentration of 12 microM. This quinazoline concentration induced caspase 3 activity. Our results demonstrated that induction of apoptotic cell death via activation of caspase 3 contributed to the cytotoxic effects of 3-(5-nitro-2-thienyl)-9-chloro-5-morpholin-4-yl[1,2,4]triazolo[4,3-c]quinazoline in murine leukemia L1210 cells.

Contact of Chlamydophila pneumoniae with type II cell triggers activation of calcium-mediated NF-κB pathway

Nuclear factor-κB (NF-κB) plays an important role in inflammation, proliferation and regulation of apoptosis. The purpose of the present study on type II cells was to investigate whether Chlamydophila pneumoniae contact induces (I) a Ca 2+ release, that (II) disrupts F-actin/β-tubulin cytoskeletal association with NF-κB/IκBα, leading to (III) a subsequent NF-κB activation. Incubation of rat type II pneumocytes with C. pneumoniae caused an intracellular calcium release within seconds. Confocal laser scanning microscopy (CLSM) revealed that bacterial contact with cell surface leads to a disappearance of the microvilli and disturbs the co-localization between F-actin and NF-κB (p65). Using semi-quantitative CLSM, we show that at 10–30 min IκBα was decreased and p65 or p50 was simultaneously translocated from cytoplasm to the nucleus, resulting in a 19-fold and 17-fold increase versus control cells. During this time no bacteria were internalized into type II cells. The pre-treatment of cells with BAPTA-AM inhibited C. pneumoniae-mediated calcium release. BAPTA-AM or SN50 prevented the C. pneumoniae-induced changes in F-actin cytoskeleton and inhibited NF-κB activation. Paclitaxel reduced C. pneumoniae-mediated changes of β-tubulin cytoskeleton and activation of NF-κB. These results suggest that calcium-mediated cytoskeleton reorganization is involved in C. pneumoniae-induced NF-κB activation in type II cells.