Sea urchin embryo, DNA-damaged cell cycle checkpoint and the mechanisms initiating cancer development

Cell division is an essential process for heredity, maintenance and evolution of the whole living kingdom. Sea urchin early development represents an excellent experimental model for the analysis of cell cycle checkpoint mechanisms since embryonic cells contain a functional DNA-damage checkpoint and since the whole sea urchin genome is sequenced. The DNA-damaged checkpoint is responsible for an arrest in the cell cycle when DNA is damaged or incorrectly replicated, for activation of the DNA repair mechanism, and for commitment to cell death by apoptosis in the case of failure to repair. New insights in cancer biology lead to two fundamental concepts about the very first origin of cancerogenesis. Cancers result from dysfunction of DNA-damaged checkpoints and cancers appear as a result of normal stem cell (NCS) transformation into a cancer stem cell (CSC). The second aspect suggests a new definition of "cancer", since CSC can be detected well before any clinical evidence. Since early development starts from the zygote, which is a primary stem cell, sea urchin early development allows analysis of the early steps of the cancerization process. Although sea urchins do not develop cancers, the model is alternative and complementary to stem cells which are not easy to isolate, do not divide in a short time and do not divide synchronously. In the field of toxicology and incidence on human health, the sea urchin experimental model allows assessment of cancer risk from single or combined molecules long before any epidemiologic evidence is available. Sea urchin embryos were used to test the worldwide used pesticide Roundup that contains glyphosate as the active herbicide agent; it was shown to activate the DNA-damage checkpoint of the first cell cycle of development. The model therefore allows considerable increase in risk evaluation of new products in the field of cancer and offers a tool for the discovery of molecular markers for early diagnostic in cancer biology. Prevention and early diagnosis are two decisive elements of human cancer therapy.     

Roundup disrupts male reproductive functions by triggering calcium-mediated cell death in rat testis and Sertoli cells

Glyphosate is the primary active constituent of the commercial pesticide Roundup. The present results show that acute Roundup exposure at low doses (36 ppm, 0.036 g/L) for 30 min induces oxidative stress and activates multiple stress-response pathways leading to Sertoli cell death in prepubertal rat testis. The pesticide increased intracellular Ca²⁺ concentration by opening L-type voltage-dependent Ca²⁺ channels as well as endoplasmic reticulum IP₃ and ryanodine receptors, leading to Ca²⁺ overload within the cells, which set off oxidative stress and necrotic cell death. Similarly, 30 min incubation of testis with glyphosate alone (36 ppm) also increased ⁴⁵Ca²⁺ uptake. These events were prevented by the antioxidants Trolox and ascorbic acid. Activated protein kinase C, phosphatidylinositol 3-kinase, and the mitogen-activated protein kinases such as ERK1/2 and p38MAPK play a role in eliciting Ca²⁺ influx and cell death. Roundup decreased the levels of reduced glutathione (GSH) and increased the amounts of thiobarbituric acid-reactive species (TBARS) and protein carbonyls. Also, exposure to glyphosate–Roundup stimulated the activity of glutathione peroxidase, glutathione reductase, glutathione S-transferase, γ-glutamyltransferase, catalase, superoxide dismutase, and glucose-6-phosphate dehydrogenase, supporting downregulated GSH levels. Glyphosate has been described as an endocrine disruptor affecting the male reproductive system; however, the molecular basis of its toxicity remains to be clarified. We propose that Roundup toxicity, implicated in Ca²⁺ overload, cell signaling misregulation, stress response of the endoplasmic reticulum, and/or depleted antioxidant defenses, could contribute to Sertoli cell disruption in spermatogenesis that could have an impact on male fertility.     

Doxorubicin and menadione decrease cell proliferation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> by different mechanisms

The aim of this study was to examine the effect of doxorubicin and menadione on cell proliferation, cell cycle, glutathione concentration, the expression of ribonucleotide reductase, and the Yap1 dependent redox-sensitive pathway in Saccharomyces cerevisiae as a eukaryote cell model. Our data showed that menadione induced cell-cycle arrest in the G₁ phase, decreased intracellular GSH and GSSG concentrations, dose-dependently increased expression of ribonucleotide reductase and the activity of Yap1 pathway. Doxorubicin induced the cell-cycle arrest in G₁ and S phases, increased the GSH and GSSG concentration and the expression of ribonucleotide reductase, and modulated the Yap-dependent pathway activity.     

Inhibition of ultraviolet B (UVB) induced apoptosis in A431 cells by mimosine is not dependent on cell cycle arrest

Ultraviolet (UV) radiation is a strong apoptotic trigger in many cell types. We have previously reported that a plant amino acid, mimosine (beta [N-(3-hydroxy-4-pyridone)]-alpha-aminopropionic acid), with a well-known reversible G1 cell cycle arrest activity can inhibit apoptosis induced by UV irradiation and RNA polymerase II blockage in human A431 cells. Here, apoptosis was measured with a fluorimetric caspase activation assay. Interestingly, the protective state was effective up to 24 h following removal of mimosine from the culture medium while cells were progressing in the cell cycle. Our results demonstrate that the protective effect of mimosine against UV-induced apoptosis can be dissociated from its G1 cell-cycle arrest activity.     

Gossypin induces G2/M arrest in human malignant glioma U251 cells by the activation of Chk1/Cdc25C pathway

Gossypin is a flavone that was originally isolated from Hibiscus vitifolius and has traditionally been used for the treatment of diabetes, jaundice, and inflammation. Recently, gossypin was found to have potent anticancer properties; however, its effect on human gliomas still remain unknown. To investigate the potential anticancer effects of gossypin on malignant gliomas and analyze the associated molecular mechanisms, we treated human glioma U251 cells with gossypin. Our study showed that the treatment of U251 cells with gossypin inhibited cell proliferation in a dose- and time-dependent manner and was observed to be minimally toxic to normal human astrocytes. Gossypin's effect on cell cycle distribution was observed, and we found that it induced G2/M-phase arrest in U251 cells. An analysis of cell-cycle regulatory proteins indicated that the arresting effect of gossypin on the cell cycle at G2/M phase was involved in the phosphorylation of cell division cycle 25C (Cdc25C) tyrosine phosphatase via the activation of checkpoint kinase 1 (Chk1). These findings indicate that gossypin is a potential treatment of gliomas because of gossypin's potential to regulate the proliferation of U251 cells via the cell-cycle regulatory proteins Chk1 and Cdc25C.     

MicroRNA destabilization enables dynamic regulation of the miR-16 family in response to cell-cycle changes

The miR-16 family, which targets genes important for the G1-S transition, is a known modulator of the cell cycle, and members of this family are often deleted or downregulated in many types of cancers. Here, we report the reciprocal relationship-that of the cell cycle controlling the miR-16 family. Levels of this family increase rapidly as cells are arrested in G0. Conversely, as cells are released from G0 arrest, levels of the miR-16 family rapidly decrease. Such rapid changes are made possible by the unusual instabilities of several family members. The repression mediated by the miR-16 family is sensitive to these cell-cycle changes, which suggests that the rapid upregulation of the miR-16 family reinforces cell-cycle arrest in G0. Upon cell-cycle re-entry, the rapid decay of several members allows levels of the family to decrease, alleviating repression of target genes and allowing proper resumption of the cell cycle.     

D609 对Neuro-2a 脑神经瘤细胞增殖和细胞周期的影响

目的:探讨小分子化合物D609对脑神经瘤细胞Neuro-2a的生长抑制及诱导细胞周期阻滞的效应,并初步研究其机制。方法:采用CCK-8法检测D609对Neuro-2a细胞的生长抑制作用;利用流式细胞术(FACS)检测D609处理对细胞周期进程的影响;利用免疫印迹实验(Western blot)检测不同浓度的D609处理后,细胞裂解液中细胞周期蛋白抑制因子p27的表达水平。结果:CCK-8的实验结果显示,加入150μmol/L D609处理72小时后,细胞生长受到明显地抑制,且伴有剂量依赖效应;流式细胞术的结果表明,D609处理使细胞周期阻滞在G0/G1期;免疫印迹的结果表明药物处理提升了p27的表达,且随药物浓度升高其表达亦增强。结论:D609可以有效地抑制Neuro-2a细胞的生长;进一步研究表明药物处理可以提升p27的表达水平并可以诱导将细胞阻滞在G0/G1期。因此,此研究将为脑神经瘤的治疗提供借鉴。     

Application of bromodeoxyuridine incorporation measurements to the determination of cell distribution within the S phase of the cell cycle

Flow cytofluorimetric measurement of incorporated bromodeoxyuridine, using a double-stained cell population, allows the determination of the distribution of cells along the cell cycle. We have developed a simple computer program for the direct treatment of 64 x 64 channel histograms. This analysis appears to provide interesting data about the distribution of cells in the various phases of the cell cycle, namely the S phase. Two examples have been chosen to illustrate possible fields for the application of such a program. Comparison of two cell lines such as friend murine erythroleukemia cells (MELC) and fibroblasts FR3T3 cells has shown that this analysis can be used for cell-cycle characterization of a given cell line. The program also allows the differential analysis of cell distribution along the cell cycle as a function of a given parameter. This possibility has been applied to study the variation of cell-cycle parameters as a function of the time of induced differentiation of MELC and reveals changes in the distribution of the cells along the various phases of the cell cycle, namely in the S phase.     

DNA replicatiion and cell-cycle progresssion of cultured mouse FM3A cells after treatment with 8-methoxypsoralen plus near-UV radiation

To investigate the response of cells to one type of DNA damage — namely DNA crosslinks — cell-cycle progression and macromolecular synthesis were studied with cultured mouse FM3A cells. Treatment of the cells with low doses of 8-methoxypsoralen (8-MOP) plus near-UV radiation (0.1 μg/ml plus 5 kJ/m² or 1.0 μg/ml plus 1–2.5 kJ/m²)_halted the progression of cells through the cell cycle temporarily for the first several hours. Then the cells resumed progression through the cell cycle, and most of the cells reached, and were finally arrested at, the G2 phase of the cycle. There was a rapid decrease of incorporation of [³H]thymidine into cellular DNA immediately after the treatment. Then, after 8 h of incubation, the incorporation of [³H]thymidine recovered to some extent depending on the dose of 8-MOP plus near-UV radiation. Thus the decrease and recovery of the incorporation of [³]Hthymidine were correlated with the halt and resumption in the cell-cycle process.Synthesis of RNA and protein was measured by determination of the amounts in the cells or by the incorporation of radioactive precursors after treatment. RNA and protein synthesis were stimulated by low doses of 8-MOP plus near-UV radiation, but inhibited severely by high doses.     

Periostin induces proliferation of differentiated cardiomyocytes and promotes cardiac repair

Adult mammalian hearts respond to injury with scar formation and not with cardiomyocyte proliferation, the cellular basis of regeneration. Although cardiogenic progenitor cells may maintain myocardial turnover, they do not give rise to a robust regenerative response. Here we show that extracellular periostin induced reentry of differentiated mammalian cardiomyocytes into the cell cycle. Periostin stimulated mononucleated cardiomyocytes to go through the full mitotic cell cycle. Periostin activated alphaV, beta1, beta3 and beta5 integrins located in the cardiomyocyte cell membrane. Activation of phosphatidylinositol-3-OH kinase was required for periostin-induced reentry of cardiomyocytes into the cell cycle and was sufficient for cell-cycle reentry in the absence of periostin. After myocardial infarction, periostin-induced cardiomyocyte cell-cycle reentry and mitosis were associated with improved ventricular remodeling and myocardial function, reduced fibrosis and infarct size, and increased angiogenesis. Thus, periostin and the pathway that it regulates may provide a target for innovative strategies to treat heart failure.     

Cyclin A but not cyclin D1 is essential for c-myc-modulated cell-cycle progression

The proto-oncogene c-myc is a key player in cell-cycle regulation and is deregulated in a broad range of human cancers and cell proliferation disorders. Here we reported that overexpression of c-myc in human embryonic lung fibroblasts (HEL) that have low endogenous c-myc enriched S phase cells with increased expression of cyclin D3, E, A, Cdk2, and Cdk4, and decreased expression of p21 and p27. To the opposite, using RNAi to downregulate c-myc expression in A549 cells that have high endogenous c-myc enriched G1 phase cells with decreased expression of cyclin D3, E, A, Cdk2, Cdk4, and increased expression of p21 and p27. We found that cyclin A expression was the most susceptive to changes in c-myc levels and essential in c-myc-modulated cell cycle pathway via co-transfection, however, cyclin D1 showed no change between treated and control groups in either HEL or A549 cells. Our results indicated that upregulation of c-myc expression promotes cell cycling in HEL cells, whereas downregulation of c-myc expression causes G1 phase arrest in A549 cells, and the c-myc-mediated cell-cycle regulation pathway was dependent on cyclin A and involved cyclin D3, E, Cdk2, Cdk4, p21, and p27, but not cyclin D1.     

Resveratrol inhibits proliferation and survival of Epstein Barr virus-infected Burkitt's lymphoma cells depending on viral latency program

Resveratrol (3,4',5-trihydroxy-trans-stilbene), a polyphenolic natural product, shows chemopreventive properties against several cancers, heart diseases, inflammation, and viral infections. Epstein Barr virus (EBV), a γ-herpesvirus, contributes to the development of several human cancers including Burkitt's lymphoma (BL). In this study, we asked whether treatment with resveratrol would affect the viability of EBV-positive BL cells displaying different forms of latency. We report here that resveratrol, regardless of EBV status, induces caspase-dependent apoptosis by arresting cell-cycle progression in G(1) phase. However, resveratrol strongly induced apoptosis in EBV(-) and latency I EBV(+) cells, whereas latency II and latency III EBV(+) BL cells showed a survival advantage that increased with the extent of the pattern of viral gene expression. Resveratrol-induced cell-cycle arrest and apoptosis occurred in association with induction of p38 MAPK phosphorylation and suppression of ERK1/2 signaling pathway. Moreover, NF-κB DNA-binding activity was inhibited in all BL lines except EBV(+) latency III cells. LMP1 oncogene, which is expressed in latency III phenotype, is involved with the higher resistance to the antiproliferative effect of resveratrol because siRNA-mediated inhibition of LMP1 greatly increased the sensitivity of latency III BL cells as well as that of lymphoblastoid cell lines to the polyphenol. We propose that a combined resveratrol/siRNA strategy may be a novel approach for the treatment of EBV-associated B-cell malignancies in which the viral pattern of gene expression has been defined.     

Activation of the RhoB Signaling Pathway by Thyroid Hormone Receptor β in Thyroid Cancer Cells

Thyroid hormone receptor (TR) mediates the crucial effects of the thyroid hormone (T3) on cellular growth, development, and differentiation. Decreased expression or inactivating somatic mutations of TRs have been found in human cancers of the liver, breast, lung, and thyroid. The mechanisms of TR-associated carcinogenesis are still not clear. To establish the function of TRβ in thyroid cancer cell proliferation, we constructed a recombinant adenovirus vector, AdTRβ, which expresses human TRβ1 cDNA. Thyroid cancer cell lines in which TRβ protein levels were significantly decreased as compared to intact thyroid tissues were infected with AdTRβ and the function of TRβ on cell proliferation and migration was analyzed. Ligand-bound TRβ induced HDAC1 and HDAC3 dissociation from, and histone acetylation associated with the RhoB promoter and enhanced the expression of RhoB mRNA and protein. In AdTRβ-infected cells, T3 and farnesyl transferase inhibitor (FTI)-treatment induced the distribution of RhoB on the cell membrane and enhanced the abundance of active GTP-bound RhoB. This RhoB protein led to p21-associated cell-cycle arrest in the G0/G1 phase, following inhibition of cell proliferation and invasion. Conversely, lowering cellular RhoB by small interfering RNA knockdown in AdTRβ-infected cells led to downregulation of p21 and inhibited cell-cycle arrest. The growth of BHP18-21v tumor xenografts in vivo was significantly inhibited by AdTRβ injection with FTIs-treatment, as compared to control virus-injected tumors. This novel signaling pathway triggered by ligand-bound TRβ provides insight into possible mechanisms of proliferation and invasion of thyroid cancer and may provide new therapeutic targets for thyroid cancers.     

Induction of autophagy by dimethyl cardamonin is associated with proliferative arrest in human colorectal carcinoma HCT116 and LOVO cells

Dimethyl cardamonin (2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone; DMC) is a naturally occurring chalcone, and it is the major compound isolated from the leaves of Syzygium samarangense (Blume) Merr. & L.M. Perry (Myrtaceae). Experiments were conducted to determine the effects of DMC on cell proliferation, cell-cycle distribution, and programmed cell death in cultures of human colorectal carcinoma HCT116 and LOVO cells. Results showed that DMC inhibited HCT116 and LOVO cell proliferation and induced G(2) /M cell cycle arrest, which was associated with the conversion of microtubule associated protein light chain 3 (LC3)-I-LC3-II, an autophagosome marker, and the incorporation of monodansylcadaverine (MDC), a marker for the acidic compartment of autolysosomes or acidic vesicular organelles. The treatment of HCT116 and LOVO cells using a combination of DMC with an autophagy inhibitor, such as 3-methyladenine (3-MA), beclin 1 siRNA, or atg5 siRNA, suppressed the effect of DMC-mediated anti-proliferation. These results imply that DMC can suppress colorectal carcinoma HCT116 and LOVO cell proliferation through a G(2) /M phase cell-cycle delay, and can induce autophagy, the hallmark of Type II programmed cell death (PCD). Taken together, our results suggest that DMC may be an effective chemotherapeutic agent for HCT116 and LOVO colorectal carcinoma cells.     

Cytotoxicity of apigenin on leukemia cell lines: implications for prevention and therapy

Natural-food-based compounds show substantial promise for prevention and biotherapy of cancers including leukemia. In general, their mechanism of action remains unclear, hampering rational use of these compounds. Herein we show that the common dietary flavonoid apigenin has anticancer activity, but also may decrease chemotherapy sensitivity, depending on the cell type. We analyzed the molecular consequences of apigenin treatment in two types of leukemia, the myeloid and erythroid subtypes. Apigenin blocked proliferation in both lineages through cell-cycle arrest in G₂/M phase for myeloid HL60 and G₀/G₁ phase for erythroid TF1 cells. In both cell lines the JAK/STAT pathway was one of major targets of apigenin. Apigenin inhibited PI3K/PKB pathway in HL60 and induced caspase-dependent apoptosis. In contrast, no apoptosis was detected in TF1 cells, but initiation of autophagy was observed. The block in cell cycle and induction of autophagy observed in this erythroleukemia cell line resulted in a reduced susceptibility toward the commonly used therapeutic agent vincristine. Thus, this study shows that although apigenin is a potential chemopreventive agent due to the induction of leukemia cell-cycle arrest, caution in dietary intake of apigenin should be taken during disease as it potentially interferes with cancer treatment.     

Dynamic Proteomics of Human Protein Level and Localization across the Cell Cycle

Regulation of proteins across the cell cycle is a basic process in cell biology. It has been difficult to study this globally in human cells due to lack of methods to accurately follow protein levels and localizations over time. Estimates based on global mRNA measurements suggest that only a few percent of human genes have cell-cycle dependent mRNA levels. Here, we used dynamic proteomics to study the cell-cycle dependence of proteins. We used 495 clones of a human cell line, each with a different protein tagged fluorescently at its endogenous locus. Protein level and localization was quantified in individual cells over 24h of growth using time-lapse microscopy. Instead of standard chemical or mechanical methods for cell synchronization, we employed in-silico synchronization to place protein levels and localization on a time axis between two cell divisions. This non-perturbative synchronization approach, together with the high accuracy of the measurements, allowed a sensitive assay of cell-cycle dependence. We further developed a computational approach that uses texture features to evaluate changes in protein localizations. We find that 40% of the proteins showed cell cycle dependence, of which 11% showed changes in protein level and 35% in localization. This suggests that a broader range of cell-cycle dependent proteins exists in human cells than was previously appreciated. Most of the cell-cycle dependent proteins exhibit changes in cellular localization. Such changes can be a useful tool in the regulation of the cell-cycle being fast and efficient.