Mitotic Phase Research Articles

Formation of Gefitinib-Resistant Lung Cancer Cell Lines and their Changes in Epidermal Growth Factor Receptor Signaling Pathway

Lung cancer is one of the top ten malignant tumors, seriously endangering people’s lives and health, with morbidity and mortality ranking first. Molecular targeted therapy overcomes the shortcomings of traditional chemotherapy. In recent years, the application of tumor therapy has become wider and wider. Gefitinib is the most successful molecular targeted drug for the treatment of lung cancer in recent years. The purpose of this article is to explore the establishment method of gefitinib-resistant lung cancer cell lines and the mechanism of epidermal growth factor receptor signaling pathway change. The method of gradually increasing the concentration of gefitinib was used to successfully establish NC-H1985 gefitinibresistant cells. The strains were also analyzed for epidermal growth factor receptor, messenger ribonucleic acid expression at different times using the anti-tubercular treatment method. The results show that this cell line has a certain difference compared to the parental NC-H1985 cell. With the extension of the culture time, the ribonucleic acid concentration decreased by 51 % and the 18S ribosomal ribonucleic acid, cycle threshold value increased by 39 %. In addition, the higher the relative expression of messenger ribonucleic acid in gefitinib-resistant lung cancer cells, the epidermal growth factor receptor signaling pathway is likely to change. From 24 h to 72 h, the relative expression of messenger ribonucleic acid has increased by 37.5 %. After gefitinib resistance culture of NC-H1985 gefitinib-resistant cells were 44.1 % in resting phase, 36.9 % in growth 1 phase, 11.4 % in synthesis phase, 4.3 % in growth 2 phase and 3.3 % in mitotic phase.

Assessment of Cytogenotoxicity Induced by Lead Acetate on Root Tip Cells of Onion (Allium cepa L.)

The aim of present study was to assess the cytotoxic and genotoxic potency of lead acetate using an Allium root tip bioassay. Root tips of Allium cepa L. were treated with different concentrations of lead acetate viz., 0.03, 0.06, and 0.09 mg mL-1 of lead acetate for 48 h. Mitotic index (MI), phase index (PI), and total chromosomal abnormalities (TA) were calculated. The effect of lead acetate was found to be dose-dependent. The frequency of MI ranged from 8.4 to 5.7%, PI of prophase from 4.2 to 3.4%, PI of metaphase from 1.14 to 1.11%, PI of anaphase from 1.8 to 0.6%, and PI of telophase from 1.1 to 0.4% The total chromosomal abnormality (TA) ranged from 0.42 to 1.39% The differences between the averages of all the studied properties were significantly affected by the heavy metal lead acetate concentration variant applied. Mitotic observation showed that increasing of concentration variants doses decrease mitotic index and increased chromosomal abnormality percentage. Various types of physiological and clastogenic (laggard, stickiness, abnormal metaphase, chromatin bridge, and delayed anaphase were observed in Allium cepa root tip cells. Moreover, the A. cepa root tip bioassay method gives critical data for evaluating an agent's action mechanisms and impacts on genetic material.

The Effect of Water Boiled in Aluminum Cookwares on Genomic Abnormalities in the Meristematic Cells of Onion Root

Introduction: One of the ways of human exposure to aluminum (Al) is Al food packaging materials and cookwares. Although many studies have examined the biotic influence of nanoparticles or ionic form of heavy metals, there are limited studies conducted on the possible health risks of metals in the form of alloy used for making utensils. In this study, the effect of water boiled in Al cookwares with defined concentrations of Al on the genomic abnormalities and cell division of meristematic cells of onion root was evaluated using Allium cepa assay. Materials and Methods: The onion roots were treated with water boiled in Al utensils (three pots) with different concentrations of Al (5 and 10 mg/l) for 42 to 43 hours and then analyzed for mitotic index (MI) and mitotic phase aberrations (MPA). Results: The percent of MI in the study groups treated with 5 mg/l of Al from pot 1 and 10 mg/l from all pots increased significantly compared to the control group (P < 0.05). Also, the frequency of total MPA in all Al treated groups significantly increased compared to the control group (P < 0.05). The most significant results were derived by sticky chromosomes, anaphase bridge, going ahead chromosome and disturbed mitosis, respectively. Conclusion: The result of this study confirmed the genotoxic effect of water boiled in Al cookwares containing the examined range of Al concentrations on the meristematic cells of onion root.

Function-oriented synthesis of fluorescent chemosensor for selective detection of Al3+ in neat aqueous solution: Paperstrip detection & DNA bioimaging

Fluorescence based sensors have gained popularity in quick response devices for detection of different analytes selectively. Most of them being organic conjugated scaffolds, often lack solubility in water that limits their application. Herein we report a completely water-soluble sulfonated derivative of salicylaldehyde, designed for selective detection of Aluminium ions in neat aqueous solution. The photophysical properties of S1 & a π-conjugated sulfanilinic acid analogue SA1 was studied and they were screened for their selectivity, sensitivity, binding modes & applications in spot paper tests as well as bioimaging. Both the sensor molecules exhibited high degree of selectivity towards Al3+ in preference to other metal ions. They displayed large Kb values of nearly 104 with a stoichiometry of 3:1 as evidenced by B-H & quantitation plots which was subsequently validated by ESI-MS. Exceptional fluorescent responses were observed with increasing Al3+ concentration exhibiting detection limits of 0.827 μM & 1.67 μM for S1 &SA1 respectively. These values are far lower than the permissible limits of Al(III) in potable water according to WHO guidelines. The binding modes and stoichiometry were confirmed by DFT studies on optimised geometries of the sensors and their complexes using B3LYP hybrid functional which correlates well with experimental findings. Excellent water solubility and a resultant biomembrane permeability facilitated its application in bioimaging of DNA. We have demonstrated fluorescence microscopic imaging of onion root tip cells for different mitotic phases. Furthermore, the sensor has also been applied in qualitative visual detection of Al(III) ions in aqueous solution by using paper strip test.

Abstract P170: SpliceCore® a platform for identifying aberrant alternative splicing in triple negative breast cancer for novel therapeutic development

Abstract Splicing dysregulation is a major hallmark of cancer, affecting tumor progression, metastasis, and therapy resistance. Multiple studies have demonstrated the oncogenic activity of specific cis splicing errors and trans-acting splicing factor misregulation in patient tumors. As such, cancer-associated splicing dysregulation is a novel source of clinically actionable biomarkers and therapeutic targets, particularly for treatment insensitive cancers such as TNBC. Envisagenics has developed SpliceCore, an innovative cloud-based software platform that integrates machine learning (ML) algorithms with high performance computing to analyze large RNA-seq datasets to predict biologically relevant, novel, and highly prevalent tumor specific alternative splicing (AS) changes. Using SpliceCore, we have analyzed >2500 RNAseq samples from different breast cancer subtypes as well as normal breast tissue and identified several AS targets with a potential to translate into therapeutic candidates for TNBC. Here, we will discuss one of our most promising AS targets; it is present in 60.5% of TNBC patients and correlates with poor overall survival. Using SpliceCore, we predicted and designed an optimal set of splice switching oligos (SSO) that can efficiently switch the skipping isoform to an inclusion isoform in TNBC cells. Detailed mechanism of action studies of isoform switching by SSO-0205 have demonstrated the critical role of the isoform in a TGFβ dependent tumor progression mechanism. Pretreatment of the TNBC cells with SSO-0205 24 hours before TGFβ pathway activation reversed the cell proliferative response associated with it. This resulted in a strong inhibition of p21 gene expression, accompanied by a 50% decrease on the number of cells in G2, the mitotic phase of the cell cycle, and 40% decrease on cell viability. Additionally, migratory response induced by TGFβ in TNBC cells was also significantly inhibited by SSO-0205 pretreatment, which downregulated ANGPTL4 gene expression followed by a 55% decrease in cell migration. Together, we provide experimental proof of concept to demonstrate SpliceCore’s ability to discover novel disease specific AS targets and design splice correcting oligonucleotides for subsequent correction and therapeutic development. Using this approach, we were able to uncover a novel therapeutic target for TNBC, whose aberrant splicing contributes to TNBC pathogenesis by promoting an overactivation of the TGFβ pathway. Reversal of this aberrant TNBC specific splicing using SSOs represent a new and promising therapeutic approach that will have a significant impact on TNBC treatment and clinical care. Citation Format: Miguel A. Manzanares, Dhingra Priyanka, Kendall Anderson, Vanessa Frederick, Adam Geier, Alyssa Casill, Martin Akerman, Gayatri Arun. SpliceCore® a platform for identifying aberrant alternative splicing in triple negative breast cancer for novel therapeutic development [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P170.

Human breast tumor derived endothelial cells exhibit distinct biological properties.

Excessive angiogenesis characterized by leaky, tortuous, and chaotic vasculature is one of the hallmarks of cancers and is significantly correlated to poor prognosis. Disorganized angiogenesis leads to poor perfusion of anti-cancer drugs and limits access to immune cells. Hence, impeding angiogenesis is one of the attractive therapeutic targets to inhibit progression and metastasis in several solid tumors including breast. We have developed a robust and reproducible method for isolating and ex vivo culture of endothelial cells (EC) derived from non-malignant (Endo-N) and malignant (Endo-T) part from clinically characterized human breast tumors. RT-PCR and immunoblotting analysis indicated that these cells exhibited expression of endothelial specific genes such as PECAM-1 (CD31), Endoglin (CD105), eNOS, VE-cadherin, VCAM1, and MCAM. Vasculogenic mimicry and contamination of progenitor EC recruited in tumors was ruled out by absence of CD133 expression and normal karyotype. Both the cell types showed stable expression of CD31 and CD105 up to seven passages. Furthermore, compared to Endo-N cells, Endo-T cells showed (a) constitutively increased proliferation marked by nearly 36% of cells in mitotic phase, (b) requirement of glutamine for cell survival, (c) pro-migratory phenotype, (d) produced increased number of sprouts in 3D cultures, and (e) resistance to sorafenib. Tumor derived EC showed distinct biological properties compared to normal breast EC. Our method for isolating endothelial cell types from human breast tumors may be explored to (a) understand cellular and molecular mechanisms, (b) screen anti-angiogenic molecules, and (c) formulate organoid cultures to develop personalized medicine facilitating better clinical management of breast cancers.

Identification and Validation of Afatinib Potential Drug Resistance Gene BIRC5 in Non-Small Cell Lung Cancer.

IntroductionResistance to second-generation epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), afatinib, is the most significant challenge in the clinical management of non-small cell lung cancer (NSCLC), and the underlying mechanisms remain unclear.MethodsGenomic signatures that may confer afatinib resistance in NSCLC were identified via data mining of public databases and integrative bioinformatic analyses. Furthermore, acquired afatinib-resistant lung adenocarcinoma cell lines (HCC827 AR) were established by long-term exposure under afatinib in vitro for stepwise escalation. The expression of baculovirus IAP repeat protein 5 (BIRC5) was detected by western blot, and cellular viability of HCC827 AR was determined by CCK8.ResultsThrough integrative bioinformatic analyses of public datasets, overexpression of baculovirus IAP repeat protein 5 (BIRC5) was identified in both afatinib-resistant NSCLC cells and tissues, and BIRC5 overexpression was positively correlated with lymph node metastasis as well as pathological stage in NSCLC. Furthermore, NSCLC patients with BIRC5 overexpression showed poor survival outcomes. Immune infiltration analysis suggested that BIRC5 expression was significantly inversely correlated with tumor-infiltrating cell numbers and immune biomarker expression in NSCLC. The functions of genes co-expressed with BIRC5 were mainly enriched in cell cycle mitotic phase transition, double-strand break repair, and negative regulation of the cell cycle process signaling pathway. In addition, overexpression of BIRC5 protein was detected in afatinib-resistant cells by western blot, while BIRC5-expressing cells treated with BIRC5 inhibitor, YM155, were sensitive to afatinib.ConclusionsIn this study, we showed that overexpression of BIRC5 resulted in resistance to afatinib in NSCLC and BIRC5-specific inhibitors may overcome the resistant phenotype, indicating that dysregulation of the apoptotic cell death pathway may be the key mechanism underlying TKI resistance in the development of NSCLC.

Cell-cycle dependent GATA2 subcellular localization in mouse 2-cell embryos

GATA factors are essential transcription factors for embryonic development that broadly control the transcription of other genes. This study aimed to examine GATA2 protein localization in mouse embryos at the 2-cell stage, when drastic transformation in gene expression occurs for subsequent development in early embryos. We first analyzed GATA2 localization in 2-cell embryos at the interphase and mitotic phases by immunofluorescence analysis. In the interphase, GATA2 protein was localized in the nucleus, as a common transcription factor. In the mitotic phase, GATA2 protein was observed as a focally-aggregated spot around the nucleus of each blastomere. To explore the relationship between GATA2 protein localization and cell cycle progression in mouse 2-cell stage embryos, GFP-labeled GATA2 protein was overexpressed in the blastomere of 2-cell embryos. Overexpression of GFP-labeled GATA2 protein arrested cellular mitosis, focally aggregated GATA2 protein expression was not observed. This mitotic arrest by GATA2 overexpression was not accompanied with the upregulation of a 2-cell stage specific gene, murine endogenous retrovirus-L. These results suggest that GATA2 protein localization changes dynamically depending on cell cycle progression in mouse 2-cell embryos; in particular, focally aggregated localization of GATA2 in the mitotic phase requires appropriate cell cycle progression.

Phosphorylation at Ser68 facilitates DCAF11-mediated ubiquitination and degradation of CENP-A during the cell cycle.

CENP-A (centromeric protein A), a histone H3 variant, specifies centromere identity and is essential to centromere maintenance. Little is known about how protein levels of CENP-A are controlled in mammalian cells. Here, we report that the phosphorylation of CENP-A Ser68 primes the ubiquitin-proteasome-mediated proteolysis of CENP-A during mitotic phase in human cultured cells. We identify two major polyubiquitination sites that are responsible for this phosphorylation-dependent degradation. Substituting the two residues, Lys49 and Lys124, with arginines abrogates proper CENP-A degradation and results in CENP-A mislocalization to non-centromeric regions. Furthermore, we find that DCAF11 (DDB1 and CUL4 associated factor 11/WDR23) is the E3 ligase that specifically mediates the observed polyubiquitination. Deletion of DCAF11 hampers CENP-A degradation and causes its mislocalization. We conclude that the Ser68 phosphorylation plays an important role in regulating cellular CENP-A homeostasis via DCAF11-mediated degradation to prevent ectopic localization of CENP-A during the cell cycle.

Mitotic Index Determination on Live Cells From Label-Free Acquired Quantitative Phase Images Using a Supervised Autoencoder.

This interdisciplinary work focuses on the interest of a new auto-encoder for supervised classification of live cell populations growing in a thermostated imaging station and acquired by a Quantitative Phase Imaging (QPI) camera. This type of camera produces interferograms that have to be processed to extract features derived from quantitative linear retardance and birefringence measurements. QPI is performed on living populations without any manipulation or treatment of the cells. We use the efficient new autoencoder classification method instead of the classical Douglas-Rachford method. Using this new supervised autoencoder, we show that the accuracy of the classification of the cells present in the mitotic phase of the cell cycle is very high using QPI features. This is a very important finding since we demonstrate that it is now possible to very precisely follow cell growth in a non-invasive manner, without any bias. No dye or any kind of markers are necessary for this live monitoring. Any studies requiring analysis of cell growth or cellular response to any treatment could benefit from this new approach by simply monitoring the proportion of cells entering mitosis in the studied cell population.

Modulation of osteoclastogenesis through adrenomedullin receptors on osteoclast precursors: initiation of differentiation by asymmetric cell division

Adrenomedullin (ADM), a member of the calcitonin family of peptides, is a potent vasodilator and was shown to have the ability to modulate bone metabolism. We have previously found a unique cell surface antigen (Kat1 antigen) expressed in rat osteoclasts, which is involved in the functional regulation of the calcitonin receptor (CTR). Cross-linking of cell surface Kat1 antigen with anti-Kat1 antigen monoclonal antibody (mAbKat1) stimulated osteoclast formation only under conditions suppressed by calcitonin. Here, we found that ADM provoked a significant stimulation in osteoclastogenesis only in the presence of calcitonin; a similar biological effect was seen with mAbKat1 in the bone marrow culture system. This stimulatory effect on osteoclastogenesis mediated by ADM was abolished by the addition of mAbKat1. 125I-labeled rat ADM (125I-ADM)-binding experiments involving micro-autoradiographic studies demonstrated that mononuclear precursors of osteoclasts abundantly expressed ADM receptors, and the specific binding of 125I-ADM was markedly inhibited by the addition of mAbKat1, suggesting a close relationship between the Kat1 antigen and the functional ADM receptors expressed on cells in the osteoclast lineage. ADM receptors were also detected in the osteoclast progenitor cells in the late mitotic phase, in which only one daughter cell of the dividing cell express ADM receptors, suggesting the semiconservative cell division of the osteoclast progenitors in the initiation of osteoclastogenesis. Messenger RNAs for the receptor activity-modifying-protein 1 (RAMP1) and calcitonin receptor-like receptor (CRLR) were expressed in cells in the osteoclast lineage; however, the expression of RAMP2 or RAMP3 was not detected in these cells. It is suggested that the Kat1 antigen is involved in the functional ADM receptor distinct from the general ADM receptor, consisting of CRLR and RAMP2 or RAMP3. Modulation of osteoclastogenesis through functional ADM receptors abundantly expressed on mononuclear osteoclast precursors is supposed to be important in the fine regulation of osteoclast differentiation in a specific osteotrophic hormonal condition with a high level of calcitonin in blood.Adrenomedullin (ADM) significantly stimulates osteoclastogenesis only in the presence of calcitonin. Functional ADM receptor partly composed of osteoclast-specific cell surface Kat1 antigen was detected in cells in the osteoclast-lineage. Expression of ADM receptor was firstly detected in proliferating osteoclast progenitors performing asymmetric cell division and is then expressed in mononuclear osteoclast precursors. Specific regulation of ADM receptors expressed on mononuclear osteoclast precursors could provide an alternative way to modulate bone remodeling therapeutically.

RNF20 affects porcine adipocyte differentiation via regulation of mitotic clonal expansion.

ObjectivesRNF20 is recognized as a main E3 ligase for monoubiquitination of histone H2B at lysine 120 (H2Bub). The critical role of RNF20 and H2Bub in various molecular events, such as DNA replication, RNA transcription, and DNA damage response, has been widely investigated and documented. However, its role in porcine adipogenesis remains unknown. In this study, we aimed to clarify the effect of RNF20 on porcine preadipocyte differentiation.Materials and MethodsBackfat tissues from fat‐type pigs (Bama and Meishan) and lean‐type pigs (Yorkshire and Landrace) were collected to detect the expression level of RNF20. Preadipocytes were isolated from Bama piglets and induced to differentiation. Small interfering RNAs were applied to deplete RNF20. Oil Red O staining, quantitative real‐time PCR, RNA‐seq, Western blot analysis, and EdU assays were performed to study the regulatory mechanism of RNF20 during adipogenesis.ResultsWe found that the expression levels of RNF20 and H2Bub were significantly higher in backfat tissues from fat‐type pigs than in those from lean‐type pigs. Consistently, the significantly induced expression of RNF20 and H2Bub was also observed in porcine differentiated adipocytes. In addition, knockdown of RNF20 greatly inhibited porcine adipogenesis, as evidenced by dramatically decreased lipid droplet formation and lower expression levels of adipogenic transcription masters in RNF20 knockdown cells. Mechanistically, the depletion of RNF20 decreases the cell proliferation and the level of p‐C/EBPβ via the Ras‐Raf‐MEK1/2‐ERK1/2 cascade pathway at the mitotic clonal expansion phase and therefore suppresses cell differentiation.ConclusionsOur results demonstrate that RNF20 is required for porcine preadipocyte differentiation.

Dynamics of mitochondrial distribution during development and asymmetric division of rice zygotes.

Mitochondria change their distribution from nuclear peripheral to uniformly distributed in cytoplasm during zygotic development of rice, and the mitochondria re-distribute around nucleus for even segregation into daughter cells. Mitochondria are highly dynamic organelles that actively move and change their localization along with actin filaments during the cell cycle. Studies of mitochondrial dynamics and distribution in plant cells have mainly been conducted on somatic cells, and our understanding about these aspects during the formation and development of zygotes remains limited. In this study, mitochondrial nucleoids of rice egg cells and zygotes were successfully stained by using N-aryl pyrido cyanine 3 (PC3), and their intracellular localization and distribution were demonstrated. Mitochondria in rice egg cells were small and coccoid in shape and were primarily distributed around the nucleus. Upon gamete fusion, the resulting zygotes showed mitochondrial dispersion and accumulation equivalent to those in rice egg cells until 8h after fusion (HAF). Around 12 HAF, the mitochondria started to disperse throughout the cytoplasm of the zygotes, and this dispersive distribution pattern continued until the zygotes entered the mitotic phase. At early prophase, the mitochondria redistributed from dispersive to densely accumulated around the nucleus, and during the metaphase and anaphase, the mitochondria were depleted from possible mitotic spindle region. Thereafter, during cell plate formation between daughter nuclei, the mitochondria distributed along the phragmoplast, where the new cell wall was formed. Finally, relatively equivalent amounts of mitochondria were detected in the apical and basal cells which were produced through asymmetric division of the zygotes. Further observation by treating the egg cell with latrunculin B revealed that the accumulation of mitochondria around the nuclear periphery in egg cells and early zygotes depended on the actin meshwork converging toward the egg or zygote nucleus.

Хромосомні аберації у лімфоцитах людини за зовнішнього та/або внутрішнього опромінення зразків крові 137Cs у модельних експериментах in vitro

The linear dependence of the yield of unstable chromosomal aberrations in human lymphocytes on the dose of prolonged external and/or internal irradiation of peripheral blood samples by 137Cs in the dose range of 0.09 - 0.54 Gy in vitro is presented. The higher cytogenetic efficiency of internal 137Cs irradiation is shown in comparison with external. It is shown that the cytogenetic efficiency of 137Cs in blood contamination is caused by influence on cells not only in the mitotic rest phase but during proliferation, within intracellular radioactivity through their cultivation. It is proposed to use the presented dose dependences of the yield of chromosomal aberrations in lymphocytes in vitro for human biological dosimetry during radiation accidents associated with prolonged external exposure or 137Cs incorporation to the body as a supplement to the results of physical dosimetry.

Genotoxic effect of two commonly used textile dyes Reactive Blue 19 and Reactive Black 5 using Allium cepa L. as an indicator

In this study, the cytotoxic and genotoxic effects of Reactive Blue 19 and Reactive Black 5 were investigated using the Allium test and comet assay. These chemicals are fabric dyes used in textile industries in various parts of Turkey. Bulbs with roots of Allium cepa L. were treated with different concentrations (25, 50 and 100 ppm) of these textile dyes for 24 h. and 48 h. The root tips were processed for cytological studies by the aseto-orcein squash procedure. Distilled water and methyl methane sulfonate (MMS, 10 ppm) were used as an negative and positive control, respectively. Exposure of Reactive Blue 19 and Reactive Black 5 significantly decreased mitotic index values. Additionally, all treatments changed the frequency of mitotic phases when compared with the control groups. These dyes increased chromosome aberrations in test material. Among these abnormalities were anaphase bridges, c-mitosis, laggards, micronuclei and stickiness. A significant increase in DNA damage was also observed at all concentrations of both Reactive Blue 19 and Reactive Black 5 examined by comet assay.

Metabolomics and Physiological Approach to Understand Allelopathic Effect of Horseradish Extract on Onion Root and Lettuce Seed as Model Organism.

In the present study, we assessed the allelopathic effects of various concentrations (0%, 0.1%, 0.2%, and 0.3%) of horseradish root extract (HRE) on onion root. The average growth of onion root tips during the 0% HRE treatment (deionized water treatment) was 0.9 cm/day, which was the highest among the growth rates obtained with all HRE treatments. Moreover, the average growth during 0.3% HRE treatment was 0.1 cm/day. During cell cycle analysis, the mitotic phase fraction of the control (deionized water treatment) cells was 6.5% of all dividing cells, with this percentage being the highest among the values obtained for all treatment groups. In the control group, all cell cycle phases were identified; however, in the 0.1%, 0.2%, and 0.3% treatment groups, telophase was not identified. The ROS accumulation area of the onion root decreased, as the HRE treatment concentration increased. In the control root, the area of dead tissue was 0%; however, in the 0.1% and 0.2% HRE treatment roots, the ratio was 5% and 50%, respectively. These findings indicate that the allelopathic effect of HRE depends on the concentration of HRE applied to the onion root.

Single-cell adhesion strength and contact density drops in the M phase of cancer cells

The high throughput, cost effective and sensitive quantification of cell adhesion strength at the single-cell level is still a challenging task. The adhesion force between tissue cells and their environment is crucial in all multicellular organisms. Integrins transmit force between the intracellular cytoskeleton and the extracellular matrix. This force is not only a mechanical interaction but a way of signal transduction as well. For instance, adhesion-dependent cells switch to an apoptotic mode in the lack of adhesion forces. Adhesion of tumor cells is a potential therapeutic target, as it is actively modulated during tissue invasion and cell release to the bloodstream resulting in metastasis. We investigated the integrin-mediated adhesion between cancer cells and their RGD (Arg-Gly-Asp) motif displaying biomimetic substratum using the HeLa cell line transfected by the Fucci fluorescent cell cycle reporter construct. We employed a computer-controlled micropipette and a high spatial resolution label-free resonant waveguide grating-based optical sensor calibrated to adhesion force and energy at the single-cell level. We found that the overall adhesion strength of single cancer cells is approximately constant in all phases except the mitotic (M) phase with a significantly lower adhesion. Single-cell evanescent field based biosensor measurements revealed that at the mitotic phase the cell material mass per unit area inside the cell-substratum contact zone is significantly less, too. Importantly, the weaker mitotic adhesion is not simply a direct consequence of the measured smaller contact area. Our results highlight these differences in the mitotic reticular adhesions and confirm that cell adhesion is a promising target of selective cancer drugs as the vast majority of normal, differentiated tissue cells do not enter the M phase and do not divide.

The Mitotic Index of Cajanus cajan from Kisar Island, in the Southwest of Maluku

The mitotic index of the roots of pigeon pea can be the basis for determining the growth of pigeon pea. The purpose of this research was to determine the time of root cell division, to observe the mitotic phases, and to determine the mitotic index of pigeon pea root cells. The preparation of the pigeon pea was carried out for 4 days to grow the roots. The roots were cut off at 08.00, 08.15, and 08.30 WIT (Eastern Indonesian Time). The roots were cut 0.5-1cm. Carnoy’s solution was used as the fixative solution using the Squash technique. The prepared roots were then observed using an Olympus cx-22 microscope and an OptiLab camera with a magnification of 100x40. The data were descriptively analyzed to describe the images of mitotic phases and the mitotic index presentation in the root cells of pigeon pea. The results of this research showed that the cell division of the pigeon pea roots began at 08.00 WIT, which was marked by the presence of a lot of prophase. The next phases that appeared were prometaphase, metaphase, and anaphase which occurred from 08.15 to 08.30 with different numbers. The highest mitotic index occurred at 08.15, when most of the root cells underwent metaphase. This study succeeded in revealing that the optimum time for pigeon pea root cell division is 08.15 WIT. In the future, this research can help pigeon pea farmers in Southwest of Maluku to carry out vegetative reproduction which is closely related to this mitotic study.

Mutagenic and Cytotoxic Activity of Insecticide Napoleon 4EC in Allium cepa and Ames Test

The objective of this study was to explore the mutagenic and cytotoxic effects of Napoleon 4EC pesticide used in Turkey to control insect pest by using two standard assays. The Allium cepa test was used for determined the cytotoxic effects of this pesticide. For this test, onion seeds were exposed to Napoleon 4EC (100, 200, and 400 ppm) for 24, 48, and 72 hours. For each test group root tip cells were stained with Feulgen and five slides were prepared for each concentration and counted microscopically. The concentrations Napoleon 4EC was compared with the value for the negative control using Dunnet-t test, 2 sided. The results indicated that mitotic index was clearly decreased with increasing the concentration of Napoleon 4EC in each treatment group as compared to the controls. The percentage of mitotic phases has been markedly impacted. Five different doses of the pesticide (50, 100, 200, 400, 800 μg/plate) were examined with Ames test using Salmonella typhimurium strains TA98 and TA100 with and without S9 metabolic activation for mutagenic activity. Ames test results showed a dose dependent effect, but not twice the negative control for S. typhimurium TA98 and TA100, with or without S9 mix except 800 μg/plate doses. In 800 μg/plate doses, colony numbers are two-fold increase according to colony number of control group. So, this places the this compound as a weak mutagen according to the parameters.

Curcumin-like structure (CCA-1.1) induces permanent mitotic arrest (Senescence) on Triple-negative breast cancer (TNBC) cells, 4T1

Triple-negative breast cancer (TNBC) remains as the deadliest cancer type due to the lack of treatment options. Hence, several attempts have been made to develop new anticancer for TNBC therapy. This study intended to challenge curcumin analog (CCA)-1.1, which is derived from pentagamavunone-1 structure, against the 4T1 cell line and TNBC cell model, covering the cytotoxic activity in correlation with cell cycle progression, apoptosis induction, reactive oxygen species (ROS) generation, and senescence evidence. The cell viability, cell cycle profile, apoptosis induction, intracellular ROS level, and senescence induction were determined in vitro using trypan blue exclusion, propidium iodide (PI) staining, Annexin-PI staining, dichlorofluorescein diacetate staining, and senescence-associated-β-gal method. CCA-1.1 showed cytotoxic activity on 4T1 cells, giving half maximal inhibitory concentration value of 3M, but was less toxic on non-cancerous 3T3-L1 cells. CCA-1.1 induced rapid cell death and inhibited cell cycle progression at the mitotic phase. Instead, of causing apoptosis, CCA-1.1 induced mitotic catastrophe. Furthermore, CCA-1.1 itself increased the intracellular ROS level and induced senescence, possibly through catastrophic cell death. Altogether, our preliminary study strengthens the potency of CCA-1.1 for its anticancer activities against TNBC cells and prospective to be pharmaceutically developed as a novel candidate for cancer therapy.