Multipolar mitosis and aneuploidy after chrysotile treatment: a consequence of abscission failure and cytokinesis regression

Chrysotile, like other types of asbestos, has been associated with mesothelioma, lung cancer and asbestosis. However, the cellular abnormalities induced by these fibers involved in cancer development have not been elucidated yet. Previous works show that chrysotile fibers induce features of cancer cells, such as aneuploidy, multinucleation and multipolar mitosis. In the present study, normal and cancer derived human cell lines were treated with chrysotile and the cellular and molecular mechanisms related to generation of aneuploid cells was elucidated. The first alteration observed was cytokinesis regression, the main cause of multinucleated cells formation and centrosome amplification. The multinucleated cells formed after cytokinesis regression were able to progress through cell cycle and generated aneuploid cells after abnormal mitosis. To understand the process of cytokinesis regression, localization of cytokinetic proteins was investigated. It was observed mislocalization of Anillin, Aurora B, Septin 9 and Alix in the intercellular bridge, and no determination of secondary constriction and abscission sites. Fiber treatment also led to overexpression of genes related to cancer, cytokinesis and cell cycle. The results show that chrysotile fibers induce cellular and molecular alterations in normal and tumor cells that have been related to cancer initiation and progression, and that tetraploidization and aneuploid cell formation are striking events after fiber internalization, which could generate a favorable context to cancer development

The multiple facets of drug resistance: one history, different approaches

Abstract\ud Some cancers like melanoma and pancreatic and ovarian cancers, for example, commonly display resistance to chemotherapy, and this is the major obstacle to a better prognosis of patients. Frequently, literature presents studies in monolayer cell cultures, 3D cell cultures or in vivo studies, but rarely the same work compares results of drug resistance in different models. Several of these works are presented in this review and show that usually cells in 3D culture are more resistant to drugs than monolayer cultured cells due to different mechanisms. Searching for new strategies to sensitize different tumors to chemotherapy, many methods have been studied to understand the mechanisms whereby cancer cells acquire drug resistance. These methods have been strongly advanced along the years and therapies using different drugs have been increasingly proposed to induce cell death in resistant cells of different cancers. Recently, cancer stem cells (CSCs) have been extensively studied because they would be the only cells capable of sustaining tumorigenesis. It is believed that the resistance of CSCs to currently used chemotherapeutics is a major contributing factor in cancer recurrence and later metastasis development. This review aims to appraise the experimental progress in the study of acquired drug resistance of cancer cells in different models as well as to understand the role of CSCs as the major contributing factor in cancer recurrence and metastasis development, describing how CSCs can be identified and isolated.We thank Roberdo Cabado for helping with the Figure 1 final art. The works\ud of our laboratory are supported by FAPESP, CNPq and CAPES

The Fate of Chrysotile-Induced Multipolar Mitosis and Aneuploid Population in Cultured Lung Cancer Cells

Chrysotile is one of the six types of asbestos, and it is the only one that can still be commercialized in many countries. Exposure to other types of asbestos has been associated with serious diseases, such as lung carcinomas and pleural mesotheliomas. The association of chrysotile exposure with disease is controversial. However, in vitro studies show the mutagenic potential of chrysotile, which can induce DNA and cell damage. The present work aimed to analyze alterations in lung small cell carcinoma cultures after 48 h of chrysotile exposure, followed by 2, 4 and 8 days of recovery in fiber-free culture medium. Some alterations, such as aneuploid cell formation, increased number of cells in G2/M phase and cells in multipolar mitosis were observed even after 8 days of recovery. The presence of chrysotile fibers in the cell cultures was detected and cell morphology was observed by laser scanning confocal microscopy. After 4 and 8 days of recovery, only a few chrysotile fragments were present in some cells, and the cellular morphology was similar to that of control cells. Cells transfected with the GFP-tagged α-tubulin plasmid were treated with chrysotile for 24 or 48 h and cells in multipolar mitosis were observed by time-lapse microscopy. Fates of these cells were established: retention in metaphase, cell death, progression through M phase generating more than two daughter cells or cell fusion during telophase or cytokinesis. Some of them were related to the formation of aneuploid cells and cells with abnormal number of centrosomes

Níveis disfuncionais de ansiedade relacionada ao Coronavírus em estudantes de medicina: Dysfunctional levels of Coronavirus-related anxiety in medical students

As preocupações com a saúde mental das pessoas afetadas pela pandemia de coronavírus não foram abordadas adequadamente. Isso é surpreendente, uma vez que tragédias em massa, particularmente aquelas que envolvem doenças infecciosas, muitas vezes desencadeiam ondas de medo e ansiedade elevados que são conhecidos por causar perturbações maciças no comportamento e no bem-estar psicológico de muitos na população. Assim, o objetivo desse trabalho é demonstrar os níveis disfuncionais de ansiedade relacionada ao coronavírus em estudantes de medicina. Para isso, foi realizado uma revisão sistemática sobre a temática

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

Chrysotile interaction with human lung carcinoma cell culture: interference on mitosis using report genes and real time microscopy and the study of genotoxic potential

Asbesto é um nome geral dado a seis tipos de fibras minerais encontradas naturalmente na crosta terrestre. Estas fibras vêm sendo exploradas industrialmente desde 1970, porém diversos trabalhadores expostos às fibras apresentaram patologias no trato respiratório, como fibroses e carcinomas. Alguns tipos de fibra foram banidos do mercado, porém o tipo de asbesto crisotila ainda pode ser comercializado na maioria dos países. Estudos in vivo e in vitro tentam elucidar as alterações causadas pela exposição à asbesto nos tecidos e nas células que possam estar relacionadas ao aparecimento de doenças, e foi verificado que a exposição às fibras leva a quebras na dupla fita de DNA, estresse oxidativo, formação de células micronucleadas e células aneuploides. O presente estudo teve como objetivo verificar a presença de alterações causadas em células em cultura expostas à crisotila por 48 h e recuperadas em meio livre de fibras por 48 h, 4 dias e 8 dias, além de observar por microscopia em tempo real divisões aberrantes após a exposição as fibras por 24 e 48h. Foram verificadas alterações que permaneceram na cultura mesmo após 8 dias de recuperação, quando não foram mais observadas fibras na cultura, como formação de células aneuploides, diminuição de frequência de células em G0/G1, aumento de células em G2/M e aumento relativo de células em metáfase quanto à porcentagem de células em fases mais tardias da fase M do ciclo. Já aumento da frequência de células micronucleadas ocorreu apenas nos períodos quando foram observadas fibras na cultura. Para análise da formação de mitoses multipolares e destinos destas células foram construídos vetores para expressão de tubulinas fusionadas a proteínas fluorescentes RFP e GFP, padronizadas as condições de transfecção e de aquisição de imagens para que as células tratadas com crisotila fossem observadas por time-lapse. Alguns destinos de mitoses multipolares causadas pelo tratamento com crisotila foram observados, como morte em metáfase, divisão gerando duas ou três células filhas, fusão de células durante a telófase e retenção em metáfase. Os dados sugerem também a indução da amplificação centrossômica, que parece ocorrer inicialmente em células interfásicas, e também devido à fusão de células.Asbestos is a general name given to six different fibrous silicate minerals found naturally in the earth\'s crust. These fibers are being exploited industrially since 1970, but several workers exposed to the fibers developed diseases in the respiratory tract, such as fibrosis and carcinomas. Some types of fiber were banished from the market, but the type of asbestos chrysotile can still be marketed in most countries. Studies in vivo and in vitro are trying to elucidate the asbestos effects in tissues and cells that could be related to the development of diseases, and these studies verified that asbestos exposure lead to DNA double strand breaks, oxidative stress, multinucleated and aneuploid cell formation. The present work aimed to verify the alterations in culture cells exposed to chrysotile for 48 h and recovered in fiber-free medium for 48 h, 4 days and 8 days, and also observe aberrant mitosis using time-lapse microscopy after 24 h and 48 h of chrysotile exposure. Some alterations were observed and remained in cell culture even after 8 days of recovery when chrysotile fibers were no longer observed - such as aneuploid cell formation, increased frequencies of G2/M cell, decreased frequencies of G1 cells, and increased frequencies of cells in early M phases as metaphase. The induction of micronuclei occurred only during the periods that fibers were observed in cell culture. For the analysis of multipolar mitosis formation and destinies of these cells after chrysotile treatment, DNA vectors for the expression of tubulins fused to fluorescent proteins (GFP and RFP) were constructed, and the conditions for cells transfection and image acquisition for time-lapse microscopy were established. The fate of some multipolar metaphases was observed: cell retention on metaphase, cell cycle progression generating two or three daughter cells, cell fusion during cytokinesis or during telophase after a multipolar anaphase, and cell death. The centrosome amplification was not observed during the M phase of cell cycle, and may occur in interphase, and also despite cell fusion

Different approaches to understand aneuploidy: interfering with mitosis using chrysotile and vincristine

A aneuploidia é uma característica dos tumores sólidos. Ela pode ser resultado de diferentes erros durante a mitose, como a amplificação centrossômica, mitoses multipolares, e anormalidades durante a citocinese. Hoje se sabe que a aneuploidia pode estar relacionada à supressão ou progressão tumoral dependendo do grau da aneuploidia e do contexto genético das células, e assim esforços vem sendo feitos a fim de elucidar quais erros durante a mitose estão relacionados à formação de células aneuploides viáveis e inviáveis. Estudos prévios do nosso grupo mostraram que tratamentos de células em cultura com fibras de crisotila e com vincristina levam a formação de células aneuploides. Agora direcionados nossos esforços para elucidar os mecanismos envolvidos na formação dessas células, investigando alterações nos centrossomos, número de cromossomos, e origens e destinos de mitoses multipolares após o tratamento com crisotila e com vincristina. As fibras de crisotila, em linhagens de células tumorais e normais, levaram a padrões de localização alterados de proteínas relacionadas à abscisão durante a citocinese, e ocorreu a regressão deste processo e consequente formação de apenas uma célula-filha com o dobro do conteúdo de cromossomos e de centrossomos. Nas duas linhagens estudadas essas células tetraploides progrediram no ciclo celular, gerando mitoses multipolares e consequente formação de células aneuploides. O tratamento com vincristina levou a respostas similares e também distintas em células normais e tumorais. Durante a retenção em metáfase ocorreu a fragmentação da matriz pericentriolar, e as células foram encaminhadas à morte celular ou à saída da mitose sem a ocorrência de divisão celular. Entretanto, células de origem normal tetraploides não progrediram no ciclo celular e não formaram mitoses multipolares, enquanto na linhagem tumoral as células apresentaram aumento da expressão de Aurora A e células com conteúdo cromossômico aumentado e aneuploide em mitoses multipolares. As mitoses multipolares formaram uma, duas ou três células e apresentaram diversas anormalidades no processo de divisão. As alterações observadas no número e composição dos centrossomos após o tratamento com as duas linhagens indicaram que processos de amplificação centrossômica ocorreram após o tratamento. Os dados foram compatíveis com a reduplicação dos centrossomos e com a formação de centríolos a partir do aumento da matriz pericentriolar. Os dados reunidos mostram que apenas células tumorais foram capazes de proliferar mesmo após diferentes erros mitóticos, enquanto células normais puderam apenas superar os erros ocasionados pelas fibras de crisotilaAneuploidy is a feature of solid tumors. Aneuploid cells result from errors during mitosis, such as centrosome amplification, multipolar mitosis and cytokinesis abnormalities. The capability of aneuploidy to promote and to suppress tumorigenesis has driven the efforts to characterize mitotic errors that form viable and not viable aneuploid cells. We have previously shown that chrysotile, an asbestos fiber, and vincristine, a chemotherapeutic agent, are able to induce aneuploidy. Now we directed our focus to discover possible mechanisms involved in aneuploid cell formation. Herein we evaluated centrosome morphology, chromosome number, and origins and fates of multipolar mitosis after chrysotile and vincristine treatment. Chrysotile fibers, in normal and cancer cells, led to mislocalization of proteins involved in abscission, which resulted in cytokinesis regression and tetraploid cells. These cells were able to enter cell cycle, giving rise to multipolar mitosis and aneuploid cells. Vincristine treatment led to specific and common responses in normal and cancer cells. During metaphase arrest, pericentrosomal matrix was fragmented, and the cells could be conducted to mitotic slippage in both lineages. However, normal tetraploid cells could not progress through cell cycle and neither to form multipolar mitosis, while cancer tetraploid cells showed Aurora A overexpression, structural and numerical centrosome abnormalities, multipolar mitosis and high levels of aneuploidy. The results showed that cancer cells could proliferate even after several mitotic errors, while normal cells could only overcome errors induced by chrysotile treatmen

Percentage of aneuploid nuclei after chrysotile exposure and recovery.

<p>Nuclear DNA content of control and chrysotile-treated (for 48 h) HK2 cells and allowed to recover in fiber-free medium for 2, 4 or 8 days was quantified, and nuclei with DNA content >5.1 C were considered aneuploid. Nuclei from mono-, bi- and multi-nucleated cells were quantified independently, and the aneuploid nuclei were identified. After 2 days of recovery, aneuploid nuclei were mainly in multinucleated cells, different to control cells and after long recovery periods – 4 and 8 days -, the aneuploid nuclei were predominantly in mononucleated cells. (P≤0.01: a x b, d x e, e x f, d x f; P<0.001: g x h, g x i; P = 0.1: h x i; P = 0.02: b x c).</p

Alterations in the morphology of HK2 cells after 24 h or 48 h of chrysotile treatment.

<p>Cells were processed by immunofluorescence to visualize nuclei, actin filaments and microtubules, and chrysotile fibers were observed by their autofluorescence. A) Confocal images of HK2 cells treated with chrysotile for 24 h or 48 h showing long, thick fibers interacting with the cells and multipolar mitosis; B) the alterations in cell morphology were analyzed, and after 24 h or 48 h of chrysotile treatment, the number of binucleated and multinucleated cells increased, as well the number of micronucleated cells, apoptotic cells and cells in multipolar mitosis (P<0.001).</p