Multiple Micronuclei Research Articles

P-262 Clinical viability of oocytes with micronuclei at fertilisation

Abstract Study question Does the presence of micronuclei at fertilisation impact on embryo potential? Summary answer Embryos displaying micronuclei at fertilisation exhibit decreased utilisation rates and lower euploidy rates, however are capable of resulting in clinical pregnancies. What is known already The appearance of two pronuclei defines normal fertilisation. With advances in imaging techniques, a range of pronuclei phenotypes can now be identified in more detail, including additional micronuclei. Micronuclei are smaller satellite structures appearing around the pronuclei. Previous studies have determined that embryos containing two primary pronuclei and one micronuclei are generally diploid and can develop to blastocyst stage. However, these studies were limited in sample size, did not have consistent imaging techniques and assessed only the presence or absence of micronuclei, not their size or number. Study design, size, duration Retrospective cohort analysis in a single, large IVF centre including all oocytes entering culture between January 2022 and December 2023 (n = 33,237). All embryos were cultured in Embryoscope+ using Vitrolife sequential media at 6% CO2 and 5% O2. ICSI and naturally inseminated oocytes were included. All embryos were cultured to day 5/6 before determining suitability for transfer, freezing and/or genetic testing. Participants/materials, setting, methods During routine daily annotation, embryologists assessed fertilised oocytes for the presence of micronuclei. This study re-examined all embryos annotated as displaying micronuclei and further assessed: number of micronuclei, their position (‘apposed’ or ‘distant’ from the pronuclei, and in the case of > 1 micronuclei ‘clustered’ or ‘spread’) along with the area (μm2) of the two largest micronuclei and area (μm2) of primary pronuclei using Embryoscope+ software. Clinical pregnancy was defined as presence of a gestational sac. Main results and the role of chance Micronuclei were observed in 0.45% (n = 149/33,237) of all oocytes assessed. The majority of these presented as one additional nuclei 92.60% (n = 138), 7.38% (n = 11) showed two additional nuclei and 0.67%(n = 1) had multiple (more than 2) micronuclei. All micronuclei appeared apposing a primary pronucleus. In the event of multiple micronuclei, 82% were clustered whereas 9% was spread throughout the around the primary pronuclei. The average area of the largest micronuclei in each oocyte was 80.33μm2 ± 26.81.The mean area of all micronuclei was 76.80μm2, approximately 16% of the size of primary pronuclei measured, 465.09μm2. The utilisation rates of embryos derived from micronuclei fertilisation were 28.18% (N = 42) compared to 49%, n = 4118 of 2PN derived embryos. All but one of these presented two primary pronuclei and one additional micronuclei, with the remaining embryo displaying two micronuclei. Twelve of these embryos were transferred (in a fresh stimulated cycle (n = 5) or a subsequent frozen embryo transfer cycle (n = 7), resulting in four clinical pregnancies, however only one ongoing clinical pregnancy. PGT testing was performed on 19 embryos, with only two, 6.67% returning a euploid results, 68.42% resulting an aneuploid, of the remaining four embryos, 2 achieved a mosaic result and 2 received a no result. Limitations, reasons for caution As the occurrence of micronuclei is rare, only a limited number of embryos were available for analysis. Furthermore, this study was conducted in a large single laboratory and can not account for differences in culture conditions between laboratories. Inter-operator variability among initial fertilisation annotations could not be excluded. Wider implications of the findings By creating a definition of the micronuclei measurement, it provides safeguard for micronuclei to be assessed in culture. The data presented here can help guide clinical decisions where micronuclei are present. Trial registration number not applicable

Intratumor Heterogeneity and Treatment Resistance of Solid Tumors with a Focus on Polyploid/Senescent Giant Cancer Cells (PGCCs).

Single cell biology has revealed that solid tumors and tumor-derived cell lines typically contain subpopulations of cancer cells that are readily distinguishable from the bulk of cancer cells by virtue of their enormous size. Such cells with a highly enlarged nucleus, multiple nuclei, and/or multiple micronuclei are often referred to as polyploid giant cancer cells (PGCCs), and may exhibit features of senescence. PGCCs may enter a dormant phase (active sleep) after they are formed, but a subset remain viable, secrete growth promoting factors, and can give rise to therapy resistant and tumor repopulating progeny. Here we will briefly discuss the prevalence and prognostic value of PGCCs across different cancer types, the current understanding of the mechanisms of their formation and fate, and possible reasons why these tumor repopulating "monsters" continue to be ignored in most cancer therapy-related preclinical studies. In addition to PGCCs, other subpopulations of cancer cells within a solid tumor (such as oncogenic caspase 3-activated cancer cells and drug-tolerant persister cancer cells) can also contribute to therapy resistance and pose major challenges to the delivery of cancer therapy.

Morphologic and molecular characterization of Apertospathula pilata n. sp., a novel freshwater spathidiid (Ciliophora, Litostomatea) from Idaho, USA

Order Spathidiida Foissner and Foissner, 1988 comprises a large group of morphologically diverse, primarily predatory, free living ciliates, the phylogeny of which has remained stubbornly unresolved. Families Arcuospathidiidae and Apertospathulidae are two morphologically similar groups established on the basis of differences in the morphology of the oral bulge and circumoral kinety. While Arcuospathidiidae is non-monophyletic in 18S rRNA gene analyses, the Apertospathulidae has been represented by only a single Apertospathula sequence in public databases. In this report, a novel freshwater species, Apertospathula pilata n. sp. is described on the basis of living observation, silver impregnation, and scanning electron microscopy. The phylogeny of the new species is assessed based on the rRNA cistron. The main features distinguishing A. pilata n. sp. from all congeners are: the oral bulge extrusomes (filiform, up to 25 µm long), the combination of body size (130–193 µm) and shape (spatulate), the extensive oral bulge length (41% of the cell length after protargol impregnation), and multiple micronuclei (one to five, two on average). The monophyly of Apertospathulidae Foissner, Xu and Kreutz, 2005 is rejected.

Selective ATM inhibition augments radiation-induced inflammatory signaling and cancer cell death

Over half of all cancer patients undergo radiation therapy but there is an unmet need for more efficacious combination strategies with molecular targeted drugs. DNA damage response has emerged as an important intervention point for improving anti-tumor effects of radiation and several inhibitors are currently in development. Ataxia telangiectasia mutated (ATM) kinase is a key regulator of cellular response to DNA double strand breaks and a potential target for radiosensitization. We recently reported two new potent and selective ATM inhibitors, M3541 and M4076, that effectively sensitize cancer cells to radiation and regress human xenografts in clinically relevant animal models. Here, we dive deeper into the cellular events in irradiated cancer cells exposed to ATM inhibitors. Suppression of ATM activity inhibited radiation-induced ATM signaling and abrogated G1 checkpoint activation resulting in enhanced cell death. Our data indicated that entry into mitosis with gross structural abnormalities in multiple chromosomes is the main mechanism behind the increased cell killing. Misalignment and mis-segregation led to formation of multiple micronuclei and robust activation of the interferon response and inflammatory signaling via the cGAS/STING/TBK1 pathway. Cancer cells exposed to radiation in the presence of M3541 were more susceptible to killing in co-culture with NK cells from healthy donors. In addition, strong upregulation of PD-L1 expression was observed in the surviving irradiated cancer cells exposed to M3541. Simultaneous activation of the STING pathway and PD-L1 suggested that combination of radiation, ATM inhibitors and PD-L1 targeted therapy may offer a novel approach to radio-immunotherapy of locally advanced tumors.

Characterization of radiation-induced micronuclei associated with premature senescence, and their selective removal by senolytic drug, ABT-263

Radiotherapy is well-recognized as an efficient non-invasive remedy for cancer treatment. Since 10 Gy, a weekly total dose for conventional radiotherapy, was proven to create unreparable and residual DNA double-strand breaks (DSBs), they were found to give rise to mitotic failure, such as mitotic catastrophe, which resulted in multiple micronuclei associated with premature senescence. We demonstrated that pulverization of micronuclear DNA was caspase-dependent and triggered not ATM-dependent but DNA-PK-dependent DNA damage response, including phosphorylation of histone H2AX. Pulverization of micronuclear DNA and senescence-associated secretory phenotype (SASP) worsen tumor microenvironment after radiotherapy, so that senolytic drug was applied to eliminate senescent cancer cells. Prematurely senescent cancer cells with micronuclei caused by 10 Gy of γ-irradiation were subjected to 5 μM of ABT-263, a Bcl-2 family inhibitor, and selective cancer cell death by apoptosis was observed, while ABT-263 had little effect on growing cancer cells. Western blot analysis showed augmented expression of both apoptotic and anti-apoptotic proteins in senescent cells, indicating that increased apoptotic factors are essential for selective apoptotic cell death in combination with ABT-263. Our results suggested that selective elimination of senescent cells alleviates SASP and micronuclei-mediated the cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) activation, both of which lead to unfavorable adverse effects caused by radiotherapy.

Breaking malignant nuclei as a non-mitotic mechanism of taxol/paclitaxel.

Discovered in a large-scale screening of natural plant chemicals, Taxol/paclitaxel and the taxane family of compounds are surprisingly successful anti-cancer drugs, used in treatment of the majority of solid tumors, and especially suitable for metastatic and recurrent cancer. Paclitaxel is often used in combination with platinum agents and is administrated in a dose dense regimen to treat recurrent cancer.The enthusiasm and clinical development were prompted by the discovery that Taxol binds beta-tubulins specifically found within microtubules and stabilizes the filaments, and consequently inhibits mitosis. However, questions on how paclitaxel suppresses cancer persist, as other specific mitotic inhibitors are impressive in pre-clinical studies but fail to achieve significant clinical activity. Thus, additional mechanisms, such as promoting mitotic catastrophe and impacting non-mitotic targets, have been proposed and studied. A good understanding of how paclitaxel, and additional new microtubule stabilizing agents, kill cancer cells will advance the clinical application of these common chemotherapeutic agents.A recent study provides a potential non-mitotic mechanism of paclitaxel action, that paclitaxel-induced rigid microtubules act to break malleable cancer nuclei into multiple micronuclei. Previous studies have established that cancer cells have a less sturdy, more pliable nuclear envelope due to the loss or reduction of lamin A/C proteins. Such changes in nuclear structure provide a selectivity for paclitaxel to break the nuclear membrane and kill cancer cells over non-neoplastic cells that have a sturdier nuclear envelope.The formation of multiple micronuclei appears to be an important aspect of paclitaxel in the killing of cancer cells, either by a mitotic or non-mitotic mechanism. Additionally, by binding to microtubule, paclitaxel is readily sequestered and concentrated within cells.This unique pharmacokinetic property allows the impact of paclitaxel on cells to persist for several days, even though the circulating drug level is much reduced following drug administration/infusion. The retention of paclitaxel within cells likely is another factor contributing to the efficacy of the drugs.Overall, the new understanding of Taxol/paclitaxel killing mechanism—rigid microtubule-induced multiple micronucleation—will likely provide new strategies to overcome drug resistance and for rational drug combination.

Micronuclei Elimination in MCF-7 Human Breast Adenocarcinoma Cells

t—Recent literature data have demonstrated that cells are able to eliminate micronuclei. Induction of micronuclei with antitubulin agents is widely used in antitumor therapy and elimination of micronuclei presumably facilitates the survival of tumor cells during therapy. In this regard, the investigation of micronuclei elimination has both fundamental and practical significance. In the present study, we show presence of two subpopulations of micronuclei (small single and large multiple micronuclei) in MCF-7 cells (human breast adenocarcinoma cell line, p53+) both in control and after paclitaxel treatment. Each of these subpopulations obviously has different mechanisms of formation. It has been shown that the lysosomal compartment can be involved in the elimination of micronuclei (for small micronuclei). It has been found that elimination of a micronucleus is a rare phenomenon. Most micronuclei were preserved, but the micronuclei could have morphological defects. We described different defects in a micronuclear envelope: lack of peripheral heterochromatin, swelling of the perinuclear space, micronuclear membrane breaks, and complete or partial absence of a nuclear lamina. We found the presence of p53-negative micronuclei. Lack of p53 activation is more characteristic for small single micronuclei. The obtained data allow us to conclude that the elimination of micronuclei cannot have a significant impact on the results of therapy. However, there is a danger of micronuclear cells progressing through the cell cycle (primarily for cells with small single micronuclei).

Chromothripsis in Hepatocarcinogenesis: The Role of a Micronuclear Aberration and Polyploidy

In this study, we used the mouse model of chemically induced hepatocarcinogenesis to investigate the chromosomal aberrations in hepatic cells. The model was obtained by combined treatment of mice with Dipin (radiomimetic drug) followed by partial hepatectomy. Cytological analysis of isolated liver cells treated with Dipin has demonstrated a number of hepatocytes with structural nuclear abnormalities and multiple micronuclei. Karyotype analysis of polyploid hepatocytes has shown numerous chromosomal aberrations including alleged morphological manifestations of chromothripsis, a special type of genomic reorganization characterized by the local disintegration of chromosomes. Micronuclei with chromosomal fragments have developed as a result of double-strand DNA breaks and might serve as the initial substrate for chromothripsis. The emergence of micronuclei containing chromosomal fragments is the most important result of the treatment employed. Therefore, the presented model of liver cancer (hepatocarcinogenesis) can be used to study the process of chromothripsis in the future.

Creutzfeldt astrocytes may be seen in IDH-wildtype glioblastoma and retain expression of DNA repair and chromatin binding proteins.

Astrocytes with multiple micronuclei ("Creutzfeldt cells") in a brain biopsy are classically associated with demyelinating disease. However, glioblastoma may also have prominent Creutzfeldt astrocytes, along with granular mitoses. Therefore, Creutzfeldt cells may raise the diagnostic dilemma of high-grade glioma vs tumefactive demyelination. While cases of glioblastoma (GBM) with Creutzfeldt astrocytes have been reported, their clinicopathologic spectrum and genetic features are not understood. Studies have proposed that micronuclei in Creutzfeldt cells are a consequence of DNA damage, or may be susceptible to DNA damage and chromothripsis, but their biology in the context of glioblastoma remains unclear. Based on a challenging index case of GBM with mild hypercellularity, Creutzfeldt astrocytes, and granular mitoses on biopsy, we searched our archives for additional cases with similar histopathologic features. We identified 13 cases, reviewed their clinico-radiologic and pathologic features, and examined them for recurrent genetic alterations via NGS (9 cases) and for evidence of DNA damage by immunohistochemistry for DNA repair and chromatin remodeling proteins. We found that Creutzfeldt cell-rich GBMs were IDH-wildtype with no recurring genetic alterations. To test our hypothesis that micronuclei demonstrate loss of DNA repair or chromatin remodeling proteins, we examined the expression of various proteins (MDM2, p53, MLH1, MSH2, PMS2, MSH6, ATRX, INI1, SATB2, Ki67, pHH3) in Creutzfeldt cell rich-GBM. There was intact expression of DNA repair and chromatin remodeling proteins, with accumulation of p53 and reduced MDM2 expression within micronuclei. In contrast, granular mitoses showed pHH3 expression, confirming these cells are undergoing mitotic division, with no accumulation of p53 and reduced expression of DNA repair proteins. Our results emphasize that Creutzfeldt cells are part of the morphologic spectrum of IDH-wildtype glioblastoma. We did not find a role for DNA damage in the generation of Creutzfeldt cells, as both DNA repair and chromatin remodeling protein expression was retained in these cells.

Time-lapse observation and transcriptome analysis of a case with repeated multiple pronuclei after IVF/ICSI.

The purpose of this study was to investigate the cause of repeated multipronucleus (MPN) formation in zygotes in a patient after both in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). This is a case study. A patient had unexplained primary infertility with recurring total MPN zygotes after IVF and ICSI cycles. Time-lapse monitoring of pronucleus formation was carried out. Embryos developed from MPN zygotes were analyzed by fluorescence in situ hybridization (FISH). Single-cell RNA-seq analysis was used to identify gene expression profiles of the patient's oocyte and zygote, and these were compared to the data from oocytes and zygotes from donors with normal fertilization (patient, n=1; donors, n=4). Oocyte-specific genes with differential expression were selected by the Amazonia! From time-lapse analysis, we observed the formation of multiple micronuclei near the site of the second polar body extrusion. These micronuclei migrated, expanded, and juxtaposed with the male pronucleus leading to a multipronucleus. None of these MPN zygotes could develop to the blastocyst stage, and FISH analysis revealed a chaotic chromosomal complement in the arrested embryos. RNA-seq analysis showed 113 differentially expressed genes (DEGs) between the patient and the donor oocytes and zygotes. Moreover, 25 of the 113 DEGs were unique or highly expressed in oocytes and early embryos. From 25 DEGs, three genes, DYNC2LI1, NEK2, and CCNH, which are involved in meiosis and the chromosome separation process, were further validated by real-time PCR. We identified several candidate genes affecting pronucleus formation as a new cause of infertility.

Chelidonine induces mitotic slippage and apoptotic-like death in SGC-7901 human gastric carcinoma cells.

The aim of the present study was to investigate the effect of chelidonine on mitotic slippage and apoptotic-like death in SGC-7901 human gastric cancer cells. The MTT assay was performed to detect the antiproliferative effect of chelidonine. Following treatment with chelidonine (10 µmol/l), the ultrastructure changes in SGC-7901, MCF-7 and HepG2 cells were observed by transmission electron microscopy. The effects of chelidonine on G2/M phase arrest and apoptosis of SGC-7901 cells were determined by flow cytometry. Indirect immunofluorescence assay and laser scanning confocal microscopy (LSCM) were used to detect the phosphorylation level of histone H3 (Ser10) and microtubule formation was detected using LSCM following immunofluorescent labeling. Subsequent to treatment with chelidonine (10 µmol/l), expression levels of mitotic slippage-associated proteins, including BUB1 mitotic checkpoint serine/threonine kinase B (BubR1), cyclin-dependent kinase 1 (Cdk1) and cyclin B1, and apoptosis-associated protein, caspase-3 were examined by western blotting at 24, 48 and 72 h. The half maximal inhibitory concentration of chelidonine was 23.13 µmol/l over 48 h and chelidonine induced G2/M phase arrest of cells. The phosphorylation of histone H3 at Ser10 was significantly increased following treatment with chelidonine for 24 h, indicating that chelidonine arrested the SGC-7901 cells in the M phase. Chelidonine inhibited microtubule polymerization, destroyed microtubule structures and induced cell cycle arrest in the M phase. Giant cells were observed with multiple micronuclei of varying sizes, which indicated that following a prolonged arrest in the M phase, the cells underwent mitotic catastrophe. Western blotting demonstrated that the protein expression levels of BubR1, cyclin B1 and Cdk1 decreased significantly between 48 and 72 h. Low expression levels of BubR1 and inactivation of the cyclin B1-Cdk1 complex results in the cells being arrested at mitosis and leads to mitotic slippage. In addition, apoptotic morphological changes in multinucleated cells were observed, the apoptosis rates increased gradually with administration of chelidonine in a time-dependent manner and the protein levels of caspase-3 increased significantly between 24 and 72 h. Thus, chelidonine induces mitotic slippage, and apoptotic-like death occurs in SGC-7901 cells undergoing mitotic catastrophe. Gastric cancer is a common malignancy, and ranks second in overall cancer-associated mortalities worldwide. The present study demonstrated that chelidonine induces M phase arrest and mitotic slippage of SGC-7901 human gastric carcinoma cells via downregulating the expression of BubR1, Cdk1 and cyclin B1 proteins. With the prolongation of chelidonine treatment, the giant cells with multiple micronuclei underwent mitotic slippage and were maintained in the G1 phase and did not survive. A number of multinucleated cells underwent apoptosis via a caspase-dependent signaling pathway. The current study proposes that chelidonine induces mitotic slippage and apoptotic-like death of SGC-7901 cells.

VE-821, an ATR inhibitor, causes radiosensitization in human tumor cells irradiated with high LET radiation.

BackgroundHigh linear energy transfer (LET) radiation such as carbon ion particles is successfully used for treatment of solid tumors. The reason why high LET radiation accomplishes greater tumor-killing than X-rays is still not completely understood. One factor would be the clustered or complex-type DNA damages. We previously reported that complex DNA double-strand breaks produced by high LET radiation enhanced DNA end resection, and this could lead to higher kinase activity of ATR protein recruited to RPA-coated single-stranded DNA. Although the effect of ATR inhibition on cells exposed to low LET gamma-rays has recently been reported, little is known regarding the effect of ATR inhibitor on cells treated with high LET radiation. The purpose of this study is to investigate the effects of the ATR inhibitor VE-821 in human tumor and normal cells irradiated with high LET carbon ions.FindingsHeLa, U2OS, and 1BR-hTERT (normal) cells were pre-treated with 1 μM VE-821 for 1 hour and irradiated with either high LET carbon ions or X-rays. Cell survival, cell cycle distribution, cell growth, and micronuclei formation were evaluated. VE-821 caused abrogation of G2/M checkpoint and forced irradiated cells to divide into daughter cells. We also found that carbon ions caused a higher number of multiple micronuclei than X-rays, leading to decreased cell survival in tumor cells when treated with VE-821, while the survival of irradiated normal cells were not significantly affected by this inhibitor.ConclusionsATR inhibitor would be an effective tumor radiosensitizer with carbon ion irradiation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13014-015-0464-y) contains supplementary material, which is available to authorized users.

HBx transfection limits proliferative capacity of podocytes through cell cycle regulation.

Our previous studies have shown that podocyte number is significantly decreased in glomeruli of children with hepatitis B virus (HBV)-associated glomerulonephritis. In this study, we aimed to explore whether exogenous expression of HBx protein could directly inhibit podocyte proliferation in vitro, and to investigate its role in cell cycle regulation. HBx gene was delivered into cultured mouse podocytes through an adenovirus-based vector. Cell morphology was evaluated with Wright-Giemsa staining. Cell growth and proliferation were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5,6-carboxyfluorescein diacetate, succinimidyl ester (CFSE)-based proliferation assays. Cell cycle phase was analyzed by flow cytometry, and the expression of cell cycle regulatory proteins was examined by western blot analysis. It was found that the aberrant nuclear changes like double and multiple micronuclei, which reflect mitotic catastrophe, accumulated in podocytes after 5 days post-infection. MTT assay showed that Ad.HBx-infected podocytes grew much more slowly than controls at day 4 post-infection and thereafter. Furthermore, CFSE-based proliferation assay also showed that the proliferation of HBx-expressing podocytes was significantly inhibited than that of controls at 3-day post-infection, and that the difference became much more obvious at day 5 post-infection. Cell cycle analysis showed that the transfection of HBx resulted in significant up-regulation of both cyclin B1 and CDK-inhibitor p21 expression and G2/M phase arrest, and slight down-regulation of cyclin A expression. These results demonstrated that exogenous expression of HBx might limit the proliferative capacity of podocytes through cell cycle regulation, thus suggesting that HBx may play a role in podocyte injuries in HBV-associated glomerulonephritis.

Abstract 1335: Lack of casein kinase 1 delta induces DNA damage, inhibition of mTORC1 signaling and nucleophagy

Abstract Casein kinase 1 delta (CK1δ) is a conserved serine/threonine protein kinase that regulates diverse cellular processes including vesicle trafficking, cell cycle progression, chromosomal segregation, DNA repair, circadian rhythm, neurite outgrowth and ciliogenesis. CK1δ has been reported to phosphorylate Mdm2, the E3 ubiquitin ligase for p53, resulting in Mdm2 ubiquitination/degradation and concomitant accumulation of p53, the latter functioning as a mediator of cell cycle checkpoints and DNA repair. Mice that lack Csnk1d exhibit a perinatal lethal phenotype and typically weigh 30-50% less than their wild type littermates. The exact causes of death and small size are unknown. We hypothesized that the absence of CK1δ initiates cellular stresses that adversely affect cell survival. To examine this idea, we collected mouse embryonic fibroblasts (MEFs) from mice homozygous for a Csnk1d floxed allele, and generated MEF Csnk1d null cells by infection with adenovirus expressing Cre. Endogenous p53 protein level was significantly lower in MEF Csnk1d null cells relative to MEF Ctl cells infected with adenovirus-GFP. Substantial DNA damage also was detected in MEF Csnk1d null cells, as measured by γ-H2AX staining. Flow cytometric analysis revealed that early passage (P3) MEF Csnk1d null cells exhibited a larger sub-G0 fraction (64%) than MEF Ctl cells (34%). The sub-G0 fraction decreased sharply with increasing passage number, becoming negligible by P10. In addition to the decline in sub-G0 fraction, we observed a transient population of MEF Csnk1d null cells with multiple DAPI-stained micronuclei containing both γ-H2AX and membranes positive for the autophagosomal marker LC3 and lysosomal marker LAMP1. These features were preceded by and coincided with mTORC1 pathway inactivation, as indicated by low concentrations of phospho-Akt, phospho-mTOR and phospho-S6K, consistent with the view that mTORC1 inhibition facilitates the induction of autophagy. Subsequently, these differences between MEF Csnk1d null and MEF Ctl cells subsided. Our findings demonstrate that loss of CK1δ expression in proliferating MEF cells results in DNA damage and initially a large sub-G0 peak, followed by a wave of nucleophagy and ultimately cell survival in culture. Ongoing investigation will explore whether similar processes occur in embryonic tissues from Csnk1d null mice and contribute to their pathologic phenotype. Citation Format: Yoshimi Endo Greer, Bo Gao, Yingzi Yang, Jeffrey S. Rubin. Lack of casein kinase 1 delta induces DNA damage, inhibition of mTORC1 signaling and nucleophagy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1335. doi:10.1158/1538-7445.AM2014-1335

Dynamic Assembly of Brambleberry Mediates Nuclear Envelope Fusion during Early Development

To accommodate the large cells following zygote formation, early blastomeres employ modified cell divisions. Karyomeres are one such modification, mitotic intermediates wherein individual chromatin masses are surrounded by nuclear envelope; the karyomeres then fuse to form a single mononucleus. We identified brambleberry, a maternal-effect zebrafish mutant that disrupts karyomere fusion, resulting in formation of multiple micronuclei. As karyomeres form, Brambleberry protein localizes to the nuclear envelope, with prominent puncta evident near karyomere-karyomere interfaces corresponding to membrane fusion sites. brambleberry corresponds to an unannotated gene with similarity to Kar5p, a protein that participates in nuclear fusion in yeast. We also demonstrate that Brambleberry is required for pronuclear fusion following fertilization in zebrafish. Our studies provide insight into the machinery required for karyomere fusion and suggest that specialized proteins are necessary for proper nuclear division in large dividing blastomeres.

Retraction Note: Role of senescence and mitotic catastrophe in cancer therapy

Senescence and mitotic catastrophe (MC) are two distinct crucial non-apoptotic mechanisms, often triggered in cancer cells and tissues in response to anti-cancer drugs. Chemotherapeuticals and myriad other factors induce cell eradication via these routes. While senescence drives the cells to a state of quiescence, MC drives the cells towards death during the course of mitosis. The senescent phenotype distinguishes tumor cells that survived drug exposure but lost the ability to form colonies from those that recover and proliferate after treatment. Although senescent cells do not proliferate, they are metabolically active and may secrete proteins with potential tumor-promoting activities. The other anti-proliferative response of tumor cells is MC that is a form of cell death that results from abnormal mitosis and leads to the formation of interphase cells with multiple micronuclei. Different classes of cytotoxic agents induce MC, but the pathways of abnormal mitosis differ depending on the nature of the inducer and the status of cell-cycle checkpoints. In this review, we compare the two pathways and mention that they are activated to curb the growth of tumors. Altogether, we have highlighted the possibilities of the use of senescence targeting drugs, mitotic kinases and anti-mitotic agents in fabricating novel strategies in cancer control.

A p27Kip1 mutant that does not inhibit CDK activity promotes centrosome amplification and micronucleation

Mitotic catastrophe occurs when cells enter mitosis with damaged DNA or excess centrosomes. Cells overexpressing the centrosome protein CP110 or depleted of cyclin F, which targets CP110 for destruction, have more than two centrosomes and undergo mitotic catastrophe. Our studies show centrosome reduplication and mitotic catastrophe in osteosarcoma cells inducibly expressing a p27Kip1 mutant (termed p27K) that binds cyclins but not cyclin-dependent kinases (CDKs). p27K inhibited cell proliferation but not CDK activity or cell cycle progression. It did not induce apoptosis; however, cells expressing p27K had more than two centrosomes and, indicative of mitotic catastrophe, irregularly shaped nuclei or multiple micronuclei. p27K interacted with cyclin F in vivo (as did endogenous p27Kip1) and displaced cyclin F from CP110. Depletion of CP110 rescued p27K-expressing cells from centrosome reduplication and mitotic catastrophe. Collectively, our data show that p27Kip1 can perturb mitosis and suggest that it does so by sequestering cyclin F, which prevents its interaction with and the subsequent degradation of CP110, ultimately resulting in centrosome reduplication, mitotic catastrophe and abrogation of cell proliferation.

Inhibition of Radiation-Induced DNA Damage by Jamun, Syzygium cumini, in the Cultured Splenocytes of Mice Exposed to Different Doses of γ-Radiation

The radioprotective property of 50 mg/kg body weight jamun (Syzygium cumini) extract was studied in the cultured splenocytes of mice exposed to 0, 0.5, 1, 2, 3, or 4 Gy of γ-radiation. The spleens of irradiated mice were removed aseptically and the splenocytes were extracted from the individual spleens and cultured. The micronuclei were prepared 72 hours after irradiation in binucleate splenocytes by blocking cytokinesis with cytochalasin-B. Irradiation of mice resulted in a dose-dependent elevation in the micronucleated splenocytes. The exposure of mice not only elevated splenocytes bearing one micronucleus but also cells bearing 2 and multiple (>2) micronuclei indicating induction of complex DNA damage after irradiation. Oral treatment of mice with 50 mg/kg body weight of jamun leaf extract protected against the radiation-induced micronuclei formation. Jamun extract also protected against the formation of 2 and multiple micronuclei indicating repair or inhibition of complex DNA damage. The assessment of lipid peroxidation in mice brain homogenate has indicated a concentration dependent inhibition of lipid peroxidation by jamun extract. Studies in a cell free system revealed that jamun extract inhibited the formation of OH, O(2)-, DPPH, and ABTS(+) free radicals in a concentration dependent manner. Our study demonstrates that jamun extract protected mice against the radiation-induced DNA damage and inhibition of radiation-induced free radical formation may be one of the mechanisms of radioprotection.

2-(Naphthalene-1-yl)-6-pyrrolidinyl-4-quinazolinone Inhibits Skin Cancer M21 Cell Proliferation through Aberrant Expression of Microtubules and the Cell Cycle

Microtubules are a proven target for anticancer drug development because they are critical for mitotic spindle formation and the separation of chromosomes at mitosis. 2-(Naphthalene-1-yl)-6-pyrrolidinyl-4-quinazolinone (HL66) induced cell death with the large cells and multiple micronuclei in M21 skin cancer cells. We demonstrated that HL66-induced cell death is caspase-independent and accompanied by the failure of cell cycle progression. Therefore, HL66-induced cell death may be a mitotic catastrophe. HL66 inhibits the dephosphorylation on Thr14 or Tyr15 of cyclin-dependent kinase (Cdk) 1 and the formation of Cdk1/cyclin B1 complex, which might be associated with cell cycle arrest at the S and G(2)/M phases. HL66 is an antimicrotubule agent by molecular modeling on the basis of ligand binding to tubulin molecule. Furthermore, we also demonstrated that HL66, like vinblastine, is a tubulin-destabilizing agent via microtubule disruption in M21 cells. These results describe a novel pharmacological property of HL66 as a microtubule inhibitor, which may make it an attractive new agent for the treatment of skin cancer.

Mitotic Catastrophe in Malignant Epithelial Tumors: The Pathologist’s Viewpoint

Mitotic catastrophe is a common phenomenon occurring in tumor cells with impaired p53 function exposed to various cytotoxic and genotoxic agents. The defective p53 checkpoint causes improper segregation of chromosomes, resulting in aberrant mitosis, multiple micronuclei, multinucleate giant cells, and eventual necrosis-like death and centrosome aberration. Although various descriptions explaining mitotic catastrophe exist, there is still no generally accepted definition of this phenomenon. However, the syndrome of mitotic catastrophe may be a unifying morphological concept of particular interest to cancer research, as it integrally links cell death to checkpoints of the cell cycle. Morphological findings compatible with mitotic catastrophe may be found in pleomorphic, giant cell carcinomas—neoplasms characterized by a poor prognosis. The inclusion of mitotic catastrophe as part of the microscopic evaluation of tumors will add further insight to the pathobiology of tumor progression and in novel therapeutic designs. Finally, the possibility of assimilating mitotic catastrophe into a prognostic score is discussed.