Effects of oriented substrates on cell morphology, the cell cycle, and the cytoskeleton in Ros 17/2.8 cells

Absence of gravity or microgravity influences the cellular functions of bone forming osteoblasts. The underlying mechanism, however, of cellular sensing and responding to the gravity vector is poorly understood. This work quantified the impact of vector-directional gravity on the biological responses of Ros 17/2.8 cells grown on upward-, downward- or edge-on-oriented substrates. Cell morphology and nuclear translocation, cell proliferation and the cell cycle, and cytoskeletal reorganization were found to vary significantly in the three orientations. All of the responses were duration-dependent. These results provide a new insight into understanding how osteoblasts respond to static vector-directional gravity.

potential mechanisms involved in resistant phenotype of MCF-7breast carcinoma cells to ionizing radiation induced apoptosis

In the present study, we investigated the mechanisms of apoptosis resistance and the roles of the phosphorylation of BRCA1, p21, the Bax/Bcl-2 protein ratio and cell cycle arrest in IR-induced apoptosis in MCF-7 cells. X-irradiation, in particular at low dose (1 Gy), but not carbon ion irradiation, had a significant antiproliferative effect on the growth of MCF-7 cells. 1 Gy X-irradiation resulted in G1 and G2 phase arrest, but 4 Gy induced a significant G1 block. In contrast, carbon ion irradiation resulted in a significant accumulation in the G2 phase. Concomitant with the phosphorylation of H2AX induced by DNA damage,carbon ion irradiation resulted in an approximately 1.9–2.8-fold increase in the phosphorylation of BRCA1 on serine residue 1524, significantly greater than that detected for X-irradiation. Carbon ion irradiation caused a dramatic increase in p21 expression and drastic decrease in Bax expression compared with X-irradiation. The data implicated that phosphorylation of BRCA1 on serine residue 1524 might,at least partially, induce p21 expression but repress Bax expression. Together, our results suggested that the phosphorylation of BRCA1 at Ser-1524 might contribute to the G2 phase arrest and might be an upstream signal involved in preventing apoptosis signal via upregulation of p21 and downregulation of the Bax/Bcl-2 ratio.

Cell cycle and apoptosis alteration of human hepatocarcinoma cells by subclinical-dose C-12(6+)-beam irradiation

Objective The purpose of this study is to investigate the effect of subdinical-dose C-12(6+)-beam irradiation on cell cycle and cell apoptosis in hepatocarcinoma cells. Materials and methods The HepG(2) cells were exposed to 0-2.0 Gy of either the C-12(6+) beam or a gamma-ray. Cell survival was detected by clonogenic assay. Cell cycle was determined by flow-cytometry analysis. The apoptosis was monitored by fluorescence microscope with DAPI staining. p53 and p21 expression were detected by Western blot. Results The G(0)/G(1) cells in the irradiated groups were significantly more than those in the control (P<0.05). The C-12(6+)-ion irradiation had a greater effect on the cell cycle of HepG(2) cells (including promoting G(1)-phase and G(2)-phase arrest) than gamma-ray irradiation. The apoptotic cells induced by C-12(6+) beam were significantly more numerous than those induced by gamma-ray (P<0.05). The carbon ions had a stronger effect on p53 and p21 expression than the gamma-ray irradiation. The survival fractions for cells irradiated by C-12(6+) beam were significantly smaller than those irradiated by gamma-ray (P<0.05).

Study on the apoptosis and Bcl-2/Bax expression of human hepatoma SMMC-7721 cells after exposure to heavy-ion and X-ray irradiations

This study is aimed at observing the apoptosis and Bcl-2/Bax gene expression of mammalian cells following heavy-ion and X-ray irradiations. Exponentially growing human hepatoma SMMC-7721 cells cultured in vitro were irradiated with a C-12 ion beam of 50 MeV/u (corresponding to a LET value of 44.56 keV/mu m) from Heavy Ion Research Facility in Lanzhou (HIRFL) at doses varying from 0 to 3 Gy. The X-ray irradiation (8 MV) was performed in the therapy unit of the General Hospital of the Lanzhou Military Area. Survival fractions of irradiated cells at various doses were measured by means of MTT assay. Apoptotic cells after irradiation were analyzed with fluorescence microscope and flow cytometer (FCM). Immuno-histological assay were applied to detect the expression of Bcl-2/Bax genes in the irradiated cells. The survival fraction of SMMC-7721 cells decreased gradually (vs. control p<0.05) with increasing the dose of the carbon ion beam more obviously than X-ray irradiation, and the carbon ion irradiation efficiently induced cell apoptosis and significantly promoted the expression of Bax gene while Bcl-2 gene expression was restrained. High-LET heavy ion beam would induce cell apoptosis effectively than low-LET X-ray, and the apoptosis rate is correlated with the transcription of Bcl-2/Bax and the ratio of Bcl-2/Bax in human hepatoma SMMC-7721 cells after irradiation to heavy ion beam.

Survivin表达在高LET射线诱导细胞凋亡中作用机理的初步研究；A Preliminary Study on Action Mechanisms of Survivin Expression in Cell Apoptosis Induced by High-LET Radiation

先前的研究表明,肿瘤细胞中survivin的高表达与细胞对高传能线密度(LET)射线的辐射抗性相关。研究了survivin表达在高LET射线诱导的细胞凋亡中的作用,发现抑制survivin表达对高LETC离子辐射诱导的Bcl-2和Bax表达没有明显的影响。在高LET射线辐照中,survivin可能通过抑制caspase-3和-9活性的途径,抑制了细胞凋亡。

Hemizygosity for Atm and Brca1 influence the balance between cell transformation and apoptosis

Background: In recent years data from both mouse models and human tumors suggest that loss of one allele of genes involved in DNA repair pathways may play a central role in genomic instability and carcinogenesis. Additionally several examples in mouse models confirmed that loss of one allele of two functionally related genes may have an additive effect on tumor development. To understand some of the mechanisms involved, we examined the role of monoallelic loss or Atm and Brca1 on cell transformation and apoptosis induced by radiation. Methods: Cell transformation and apoptosis were measured in mouse embryo fibroblasts (MEF) and thymocytes respectively. Combinations of wild type and hemizygous genotypes for ATM and BRCA1 were tested in various comparisons. Results: Haploinsufficiency of either ATM or BRCA1 resulted in an increase in the incidence of radiation-induced transformation of MEF and a corresponding decrease in the proportion of thymocytes dying an apoptotic death, compared with cells from wild-type animals. Combined haploinsufficiency for both genes resulted in an even larger effect on apoptosis. Conclusions: Under stress, the efficiency and capacity for DNA repair mediated by the ATM/BRCA1 cell signalling network depends on the expression levels of both proteins.

Targeted RNA interference of cyclin A(2) mediated by functionalized single-walled carbon nanotubes induces proliferation arrest and apoptosis in chronic myelogenous leukemia K562 cells

Cyclin A(2) plays critical role in DNA replication, transcription, and cell cycle regulation. Its overexpression has been detected and related to many types of cancers including leukemia, suggesting that suppression of cyclin A(2) would be an attractive strategy to prevent tumor progression. Herein, we apply functionalized single wall carbon nanotubes (f-SWNTs) to carry small interfering RNA (siRNA) into K562 cells and determine whether inhibition of cyclin A(2) would be a potential therapeutic target for chronic myelogenous leukemia.

Knocking-Down Cyclin A(2) by siRNA Suppresses Apoptosis and Switches Differentiation Pathways in K562 Cells upon Administration with Doxorubicin

Cyclin A(2) is critical for the initiation of DNA replication, transcription and cell cycle regulation. Cumulative evidences indicate that the deregulation of cyclin A(2) is tightly linked to the chromosomal instability, neoplastic transformation and tumor proliferation. Here we report that treatment of chronic myelogenous leukaemia K562 cells with doxorubicin results in an accumulation of cyclin A(2) and follows by induction of apoptotic cell death. To investigate the potential preclinical relevance, K562 cells were transiently transfected with the siRNA targeting cyclin A(2) by functionalized single wall carbon nanotubes. Knocking down the expression of cyclin A(2) in K562 cells suppressed doxorubicin-induced growth arrest and cell apoptosis. Upon administration with doxorubicin, K562 cells with reduced cyclin A(2) showed a significant decrease in erythroid differentiation, and a small fraction of cells were differentiated along megakaryocytic and monocyte-macrophage pathways. The results demonstrate the pro-apoptotic role of cyclin A(2) and suggest that cyclin A(2) is a key regulator of cell differentiation.

Molecular coordinated regulation of gene expression during ovarian development in the penaeid shrimp

To understand the molecular events of ovarian development in penaeid shrimp, RNA arbitrarily primed polymerase chain reaction (RAP-PCR) was used to identify differentially expressed genes during ovarian maturation in Metapenaeus ensis. From a screening of 700 clones in a cDNA library of the shrimp ovary by the products of RAP-PCR of different maturation stages, 91 fragments with differentially expressed pattern as revealed by dot-blot hybridization were isolated and sequenced. Forty-two of these fragments show significant sequence similarity to known gene products and the differentially expressed pattern of 10 putative genes were further characterized via Northern hybridization. Putative glyceraldehyde-3-phosphate dehydrogenase and arginine kinase are related to provision of energy for active cellular function in oocyte development. Translationally controlled tumor protein, actin, and keratin are related to the organization of cytoskeleton to accomplish growth and development of oocytes. High mobility group protein DSP1, heat shock protein 70, and nucleoside diphosphate kinase may act as repressors before the onset of ovarian maturation. Peptidyl-prolyl cis-trans isomerase and glutathione peroxidase are related to the stabilization of proteins and oocytes. This study provides new insights on the molecular events in the ovarian development in the shrimp.

Rhein induces apoptosis and cell cycle arrest in human hepatocellular carcinoma BEL-7402 cells

Rhein, an anthraquinone derivative of rhubarb, inhibits the proliferation of various human cancer cells. In this paper, we focused on studying the effects of rhein on human hepatocelluar carcinoma BEL-7402 cells and further understanding the underlying molecular mechanism in an effort to make the potential development of rhein in the treatment of cancers. Using MTT assay and flow cytometry, we demonstrate a critical role of rhein in the suppression of BEL-7402 cell proliferation in a concentration- and time-dependent manner. The increase of apoptosis rate was observed after incubation of BEL-7402 cells with rhein at 50-200 mu M for 48 hours, and the cells exhibit typical apoptotic features including cellular morphological change and chromatin condensation. Moreover, rhein-induced cell cycle S-phase arrest. Additionally, after rhein treatment, expression levels of c-Myc gene were decreased, while those of caspase-3 gene were increased in a dose-dependent manner by using real-time PCR assay. The results demonstrate for the first time that cell cycle S-phase arrest is one of the mechanisms of rhein in inhibition of BEL-7402 cells. Rhein plays its role by inducing cell cycle arrest via downregulation of oncogene c-Myc and apoptosis through the caspase-dependent pathway. It is expected that rhein will be effective and useful as a new agent in hepatocelluar carcinoma treatment in the future.

Emodin-induced apoptosis in human breast cancer BCap-37 cells through the mitochondrial signaling pathway

Emodin, a natural anthraquinone compound isolated from the rhizome of rhubarb, is reported to suppress the growth of tumor in many clinical situations. In this study, we focused on the effect of emodin in human breast cancer BCap-37 cells and further understand the underlying molecular mechanism in treating breast cancer. Using MTT assay and flow cytometry, we demonstrated the critical role of emodin in the suppression of the proliferation of BCap-37 cells based on a concentration- and time-dependent manner. The increase of apoptotic rate was also observed after incubation of BCap-37 cells on emodin at 20 mu M and 50 mu M for 48 h. The cells exhibited typical apoptotic features including cellular morphological change, chromatin condensation and membrane blebbing. The results of the study further showed that Bcl-2 level decreased, while Bax and cytosolic cytochrome c levels in sample cells increased after the emodin treatment by using Western blot. The decline in the Bcl-2/Bax ratio and the increase of cytosolic cytochrome c concentration were consistent with the increase of the apoptotic ratio. The results strongly suggest that the disruption of the mitochondrial signaling pathway was involved in emodin-induced apoptosis in BCap-37 cells.

Endothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis

Background: Endothelial cells (EC) shed endothelial microparticles (EMP) in activation and apoptosis. Objectives: We compared the antigenic expression of EMP species released during activation as compared to apoptosis, in three cell lines. Methods: EC from renal and brain microvascular (MiVEC) and coronary macrovascular (MaVEC) origin were incubated with TNF-alpha to induce activation, or deprived of growth factors to induce apoptosis. Antigens expressed on EMP and EC were assayed flow cytometrically and included constitutive markers (CD31, CD51/61, CD105), inducible markers (CD54, CD62E and CD106), and annexin V binding. Results: It was found that in apoptosis, constitutive markers in EMP were markedly increased (CD31>CD105), with a concomitant decrease in expression in EC. Annexin V EC surface binding and annexin V+ EMP were more sharply increased in apoptosis than in activation. In contrast, in activation, inducible markers in EMP were markedly increased in both EMP and EC (CD62E>CD54>CD 106). Coronary MaVEC released significantly less EMP than MiVEC. Conclusion: EC release qualitatively and quantitatively distinct EMP during activation compared to apoptosis. Analysis of EMP phenotypic signatures may provide clinically useful information on the status of the endothelium. (C) 2003 Elsevier Science Ltd. All rights reserved.

Microfluidic devices for the analysis of apoptosis

Apoptosis is the outcome of a metabolic cascade that results in cell death in a controlled manner. Due to its important role in maintaining balance in organisms, in mechanisms of diseases, and tissue homeostasis, apoptosis is of great interest in the emerging fields of systems biology. Research into cell death regulation and efforts to model apoptosis processes have become powerful drivers for new technologies to acquire ever more comprehensive information from cells and cell populations. The microfluidic technology promises to integrate and miniaturize many bioanalytical processes, which offers an alternative platform for the analysis of apoptosis. This review aims to highlight the recent developments of microfluidic devices in measuring the hallmarks as well as the dynamic process of cellular apoptosis. The potential capability and an outlook of microfluidic devices for the study of apoptosis are addressed.

Pancreatic and adrenal development and function in an ovine model of polycystic ovary syndrome.

Polycystic Ovary Syndrome (PCOS) is a complex disorder encompassing reproductive and metabolic dysfunction. Ovarian hyperandrogenism is an endocrine hallmark of human PCOS. In animal models, PCOS-like abnormalities can be recreated by in utero over-exposure to androgenic steroid hormones. This thesis investigated pancreatic and adrenal development and function in a unique model of PCOS. Fetal sheep were directly exposed (day 62 and day 82 of gestation) to steroidal excesses - androgen excess (testosterone propionate - TP), estrogen excess (diethylstilbestrol - DES) or glucocorticoid excess (dexamethasone - DEX). At d90 gestation there was elevated expression of genes involved in β- cell development and function: PDX-1 (P<0.001), and INS (P<0.05), INSR (P<0.05) driven by androgenic excess only in the female fetal pancreas. β- cell numbers (P<0.001) and in vitro insulin secretion (P<0.05) were also elevated in androgen exposed female fetuses. There was a significant increase in insulin secreting β-cell numbers (P<0.001) and in vivo insulin secretion (glucose stimulated) (P<0.01) in adult female offspring, specifically associated with prenatal androgen excess. At d90 gestation, female fetal adrenal gene expression was perturbed by fetal estrogenic exposure. Male fetal adrenal gene expression was altered more dramatically by fetal glucocorticoid exposure. In female adult offspring from androgen exposed pregnancies there was increased adrenal steroidogenic gene expression and in vivo testosterone secretion (P<0.01). This highlights that the adrenal glands may contribute towards excess androgen secretion in PCOS, but such effects might be secondary to other metabolic alterations driven by prenatal androgen exposure, such as excess insulin secretion Thus there may be dialogue between the pancreas and adrenal gland, programmed during early life, with implications for adult health Given both hyperinsulinaemia and hyperandrogenism are common features in PCOS, we suggest that their origins may be at least partially due to altered fetal steroidal environments, specifically excess androgenic stimulation

Quality control autophagy: a joint effort of ubiquitin, protein deacetylase and actin cytoskeleton.

Autophagy has been predominantly studied as a nonselective self-digestion process that recycles macromolecules and produces energy in response to starvation. However, autophagy independent of nutrient status has long been known to exist. Recent evidence suggests that this form of autophagy enforces intracellular quality control by selectively disposing of aberrant protein aggregates and damaged organelles--common denominators in various forms of neurodegenerative diseases. By definition, this form of autophagy, termed quality-control (QC) autophagy, must be different from nutrient-regulated autophagy in substrate selectivity, regulation and function. We have recently identified the ubiquitin-binding deacetylase, HDAC6, as a key component that establishes QC. HDAC6 is not required for autophagy activation per se; rather, it is recruited to ubiquitinated autophagic substrates where it stimulates autophagosome-lysosome fusion by promoting F-actin remodeling in a cortactin-dependent manner. Remarkably, HDAC6 and cortactin are dispensable for starvation-induced autophagy. These findings reveal that autophagosomes associated with QC are molecularly and biochemically distinct from those associated with starvation autophagy, thereby providing a new molecular framework to understand the emerging complexity of autophagy and therapeutic potential of this unique machinery.