Combretastatin CA-4 and combretastatin derivative induce mitotic catastrophe dependent on spindle checkpoint and caspase-3 activation in non-small cell lung cancer cells

Combretastatin A-4 (CA-4), a natural stilbenoid isolated from Combretum caffrum, is a new vascular targeting agent (VTA) known for its antitumor activity due to its anti-tubulin properties. We investigated the molecular mechanisms leading to cell death in non-small cell lung cancer H460 cells induced by natural (CA-4) and synthetic stilbenoids (ST2151) structurally related to CA-4. We found that both compounds induced depolymerization and rearrangement of spindle microtubules, as well as an increasingly aberrant organization of metaphase chromosomes in a dose- and time-dependent manner. Prolonged exposition to ST2151 led cells to organize multiple sites of tubulin repolymerization, whereas tubulin repolymerization was observed only after CA-4 washout. H460 cells were arrested at a pro-metaphase stage, with condensed chromosomes and a triggered spindle assembly checkpoint, as evaluated by kinetochore localization of Bub1 and Mad1 antibodies. Persistent checkpoint activation led to mitochondrial membrane permeabilization (MMP) alterations, cytochrome c release, activation of caspase-9 and -3, PARP cleavage and DNA fragmentation. On the other hand, caspase-2, and -8 were not activated by the drug treatment. The ability of cells to reassemble tubulin in the presence of an activated checkpoint may be responsible for ST2151-induced multinucleation, a recognized sign of mitotic catastrophe. In conclusion, we believe that discovery of new agents able to trigger mitotic catastrophe cell death as a result of mitotic block and prolonged spindle checkpoint activation is particularly worthwhile, considering that tumor cells have a high proliferative rate and mitotic failure occurs irrespective of p53 status. Electronic Supplementary Material Supplementary material is available in the online version of this article at . Ilio Vitale and Antonio Antoccia contribuited equally to this work.     

New alternative phosphorylation sites on the cyclin dependent kinase 1/cyclin a complex in p53-deficient human cells treated with etoposide: possible association with etoposide-induced apoptosis

Cell cycle arrest is a major cellular response to DNA damage preceding the decision to repair or die. Many malignant cells have non-functional p53 rendering them more “aggressive” in nature. Arrest in p53-negative cells occurs at the G2M cell cycle checkpoint. Failure of DNA damaged cells to arrest at G2 results in entry into mitosis and potential death through aberrant mitosis and/or apoptosis. The pivotal kinase regulating the G2M checkpoint is Cdk1/cyclin B whose activity is controlled by phosphorylation. The p53-negative myeloid leukemia cell lines K562 and HL-60 were used to determine Cdk1 phosphorylation status during etoposide treatment. Cdk1 tyrosine 15 phosphorylation was associated with G2M arrest, but not with cell death. Cdk1 tyrosine 15 phosphorylation also led to suppression of nuclear cyclin B-associated Cdk1 kinase activity. However cell death, associated with broader tyrosine phosphorylation of Cdk1 was not attributed to tyrosine 15 alone. This broader phosphoryl isoform of Cdk1 was associated with cyclin A and not cyclin B. Alternative phosphorylations sites were predicted as tyrosines 4, 99 and 237 by computer analysis. No similar pattern was found on Cdk2. These findings suggest novel Cdk1 phosphorylation sites, which appear to be associated with p53-independent cell death following etoposide treatment.     

NoDe: a fast error-correction algorithm for pyrosequencing amplicon reads

Background

The popularity of new sequencing technologies has led to an explosion of possible applications, including new approaches in biodiversity studies. However each of these sequencing technologies suffers from sequencing errors originating from different factors. For 16S rRNA metagenomics studies, the 454 pyrosequencing technology is one of the most frequently used platforms, but sequencing errors still lead to important data analysis issues (e.g. in clustering in taxonomic units and biodiversity estimation). Moreover, retaining a higher portion of the sequencing data by preserving as much of the read length as possible while maintaining the error rate within an acceptable range, will have important consequences at the level of taxonomic precision.

Results

The new error correction algorithm proposed in this work - NoDe (Noise Detector) - is trained to identify those positions in 454 sequencing reads that are likely to have an error, and subsequently clusters those error-prone reads with correct reads resulting in error-free representative read. A benchmarking study with other denoising algorithms shows that NoDe can detect up to 75% more errors in a large scale mock community dataset, and this with a low computational cost compared to the second best algorithm considered in this study. The positive effect of NoDe in 16S rRNA studies was confirmed by the beneficial effect on the precision of the clustering of pyrosequencing reads in operational taxonomic units.

Conclusions

NoDe was shown to be a computational efficient denoising algorithm for pyrosequencing reads, producing the lowest error rates in an extensive benchmarking study with other denoising algorithms.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-015-0520-5) contains supplementary material, which is available to authorized users.     

Cutting edge: Chk1 directs senescence and mitotic catastrophe in recovery from G2 checkpoint arrest

Besides the well‐understood DNA damage response via establishment of G₂ checkpoint arrest, novel studies focus on the recovery from arrest by checkpoint override to monitor cell cycle re‐entry. The aim of this study was to investigate the role of Chk1 in the recovery from G₂ checkpoint arrest in HCT116 (human colorectal cancer) wt, p53^–/– and p21^–/– cell lines following H₂O₂ treatment. Firstly, DNA damage caused G₂ checkpoint activation via Chk1. Secondly, overriding G₂ checkpoint led to (i) mitotic slippage, cell cycle re‐entry in G₁ and subsequent G₁ arrest associated with senescence or (ii) premature mitotic entry in the absence of p53/p21^WAF1 causing mitotic catastrophe. We revealed subtle differences in the initial Chk1‐involved G₂ arrest with respect to p53/p21^WAF1: absence of either protein led to late G₂ arrest instead of the classic G₂ arrest during checkpoint initiation, and this impacted the release back into the cell cycle. Thus, G₂ arrest correlated with downstream senescence, but late G₂ arrest led to mitotic catastrophe, although both cell cycle re‐entries were linked to upstream Chk1 signalling. Chk1 knockdown deciphered that Chk1 defines long‐term DNA damage responses causing cell cycle re‐entry. We propose that recovery from oxidative DNA damage‐induced G₂ arrest requires Chk1. It works as cutting edge and navigates cells to senescence or mitotic catastrophe. The decision, however, seems to depend on p53/p21^WAF1. The general relevance of Chk1 as an important determinant of recovery from G₂ checkpoint arrest was verified in HT29 colorectal cancer cells.     

Cdc2 and Cdk2 play critical roles in low dose doxorubicin-induced cell death through mitotic catastrophe but not in high dose doxorubicin-induced apoptosis

In Huh-7 hepatoma cells, low dose (LD) doxorubicin treatment induces cell death through mitotic catastrophe accompanying the formation of large cells with multiple micronuclei, whereas high dose (HD) doxorubicin induces apoptosis. In this study, we investigated the role of Cdc2 and Cdk2 kinase in the regulation of the two modes of cell death induced by doxorubicin. During HD doxorubicin-induced apoptosis, the histone H1-associated activities of Cdc2 and Cdk2 both progressively declined in parallel with reductions in cyclin A and cyclin B protein levels. In contrast, during LD doxorubicin-induced cell death through mitotic catastrophe, the Cdc2 and Cdk2 kinases were transiently activated 1 day post-treatment, with similar changes seen in the protein levels of cyclin A, cyclin B, and Cdc2. Treatment with roscovitine, a specific inhibitor of Cdc2 and Cdk2, significantly blocked LD doxorubicin-induced mitotic catastrophe and cell death, but did not affect HD doxorubicin-induced apoptosis in Huh-7, SNU-398, and SNU-449 hepatoma cell lines. Our results demonstrate that differential regulation of Cdc2 and Cdk2 activity by different doses of doxorubicin may contribute to the induction of two distinct modes of cell death in hepatoma cells, either apoptosis or cell death through mitotic catastrophe.     

Error threshold in RNA quasispecies models with complementation

A general assumption of quasispecies models of replicons dynamics is that the fitness of a genotype is entirely determined by its sequence. However, a more biologically plausible situation is that fitness depends on the proteins that catalyze metabolic reactions, including replication. In a stirred population of replicons, such as viruses replicating and accumulating within the same cell, the association between a given genome and the proteins it encodes is not tight as it can be replicated by proteins translated from other genomes. We have investigated how this complementation phenomenon affects the error threshold in simple quasispecies mean field models. We first studied a model in which the master and the mutant genomes code for wild-type and mutant replicases, respectively. We assume that the mutant replicase has a reduced activity and that the wild-type replicase does not have increased affinity for the master genome. The whole pool of replicases can bind and replicate both genomes. We then analyze a different model considering a more extreme case of mutant genomes, the defective interfering particles (DIPs) described in many cases of viral infection. DIPs, with a higher replication rate owed to their shorter genomes, do not code for replicase, but they are able of using the replicase translated from the master genome. Our models allow to study how the probability of interaction between the genomes and the whole pool of replicases affects the error threshold. In both systems we characterize the scenario of coexistence between master and mutant genomes, providing the critical values of mutation rate, μ_c, and the critical interaction rate between master genomes and replicases, γ_c, at which the quasispecies enters into error catastrophe, a situation in which the mutant genomes dominate the population. In both cases, we showed that the error-threshold transition is given by transcritical-like bifurcations, suggesting a continuous phase transition. We have also found that the region in the parameter space (μ,γ) in which the master sequence survives is reduced when DIPs are introduced into the system.     

切尔诺贝利核事故以来隔离区动物生态的研究概况

综述了切尔诺贝利核事故以来乌克兰Schmalhausen动物研究所(SIZ)对核工厂周围隔离区(exclusionzone ,EZ)的野生动物所进行的长达20年的放射生态学调查研究。基于乌克兰以往鸟类迁移的观察资料,评估了137Cs和 90Sr在隔离区候鸟体内的含量。而且还选择一些动物物种作为环境状况的标准指示生物,目的是为了阐明1 37Cs在隔离区脊椎动物体内的污染情况;整个第聂伯河流域、基辅行政区和隔离区软体动物贝壳内 90Sr的β活性。结果表明不同物种相对放射性核累积、迁移和累积因素呈有规律的季节性和长期性变化趋势,这些参数的运用可以大大地减少数据的波动和复杂性。直接辐射毁坏森林后,营养链的崩溃和病死树昆虫害虫的爆发导致了其次级生态变化。99 %的隔离区并不直接受辐射的影响,人员撤离、农业和森林管理停止以及大规模排除污染是这些区域生态变化的主要因素。在初始变化之后,由于自然资源、捕食者和偷猎者等的限制,隔离区的动物密度和分布达到一个稳定的极限值。数年前成功地在隔离区引入了一群蒙古野马,该群体保持了稳定增长。重新评估了以前划定的若干自然保护位点目前的保护状况,并提出了建议扩大这些自然保护区的范围等保护措施。     

Variable selection for logistic regression using a prediction-focused information criterion

In biostatistical practice, it is common to use information criteria as a guide for model selection. We propose new versions of the focused information criterion (FIC) for variable selection in logistic regression. The FIC gives, depending on the quantity to be estimated, possibly different sets of selected variables. The standard version of the FIC measures the mean squared error of the estimator of the quantity of interest in the selected model. In this article, we propose more general versions of the FIC, allowing other risk measures such as the one based on L(p) error. When prediction of an event is important, as is often the case in medical applications, we construct an FIC using the error rate as a natural risk measure. The advantages of using an information criterion which depends on both the quantity of interest and the selected risk measure are illustrated by means of a simulation study and application to a study on diabetic retinopathy.     

Detection of competitive enzyme inhibition with end point progress curve data

A model for a dimensionless factor, the inhibition detection limit (IDL), which describes the limit of detection of competitive inhibition for end point assays as a function of the proportion of substrate converted into product, has been developed. For a given end point enzymatic assay, the IDL function has a maximum that is dependent on the error structure parameters (four parameters) of the assay, the value of [S]o/K(ms), and the extent of product inhibition (K(ms)/K(mp)). Accordingly, the substrate conversion level that maximized the ability to detect samples with high Ki/[I] ratios was predicted for each member of a population of simulated assays. Furthermore, we identified a consensus substrate conversion level where the probability of a near-optimal robustness and detection limit for all the members of the assay population is maximal. Unlike the optimal substrate conversion level for individual assays, this consensus substrate conversion level was dependent only on [S]o/K(m), K(ms)/K(mp), and whether the signal increases or decreases during the course of the reaction. Consensus substrate conversion levels were beyond the initial velocity range for almost all the analyzed assay populations. It was shown that the IDL factor was a more informative indicator of assay quality than the popular Z' factor.     

On the synthesis of DNA error correcting codes

DNA error correcting codes over the edit metric consist of embeddable markers for sequencing projects that are tolerant of sequencing errors. When a genetic library has multiple sources for its sequences, use of embedded markers permit tracking of sequence origin. This study compares different methods for synthesizing DNA error correcting codes. A new code-finding technique called the salmon algorithm is introduced and used to improve the size of best known codes in five difficult cases of the problem, including the most studied case: length six, distance three codes. An updated table of the best known code sizes with 36 improved values, resulting from three different algorithms, is presented. Mathematical background results for the problem from multiple sources are summarized. A discussion of practical details that arise in application, including biological design and decoding, is also given in this study.