Abstract

Background: Arsenic trioxide (ATO) is an effective drug used in acute promyelocytic leukemia (AML). Many reports suggest that ATO can also be applied as an anticancer agent for solid tumors in the future. The influence of arsenic trioxide on the expression of different cell cycle regulators is poorly recognized. The purpose of the current study is to investigate how arsenic trioxide affects cyclin A expression and localization in the A549 cell line.Materials and methods: Morphological and ultrastructural changes in A549 cells were observed using light and transmission electron microscopes. Cyclin A localization was determined by immunofluorescence. Image-based cytometry was applied to evaluate the effect of arsenic trioxide on apoptosis and the cell cycle. Expression of cyclin A mRNA was quantified by real-time PCR.Results: After treatment with arsenic trioxide, increased numbers of cells with cytoplasmic localization of cyclin A were observed. The doses of 10 and 15 μM ATO slightly reduced expression of cyclin A mRNA. The apoptotic phenotype of cells was poorly represented, and the Tali imagebased cytometry analysis showed low percentages of apoptotic cells. The A549 population displayed an enriched fraction of cells in G0/G1 phase in the presence of 5μM ATO, whereas starting from the higher concentrations of the drug, i.e. 10 and 15 μM ATO, the G2/M fraction was on the increase.Discussion: Low expression of cyclin A in the A549 cell line may constitute a potential factor determining arsenic trioxide resistance. It could be hypothesized that the observed alterations in cyclin A expression/distribution may correlate well with changes in cell cycle regulation in our model, which in turn determines the outcome of the treatment.

Introduction

Arsenic trioxide (ATO) is an effective drug for acute promyelocytic leukemia (APL). The mechanism of action of As2 O3 in APL mainly involves interaction with the fusion protein PML-RARα, which is characteristic of APL [19]. Scientific data indicate that arsenic trioxide can be an efficient therapeutic agent for the treatment of solid tumors. Apart from its impact on PML-RARα, arsenic trioxide affects a lot of molecular pathways, the activation or downregulation of which can induce apoptosis. In addition, ATO can induce cell cycle arrest in G0/G1 and G2/M phases. Triggering another type of cell death, autophagy, has also been reported [3,20,28]. Despite the efforts to investigate arsenic trioxide’s mechanism of action, specific interactions between ATO and its molecular targets remain to be elucidated. The influence of arsenic trioxide on cell cycle regulators is poorly investigated. Cyclins are proteins that are responsible for cell cycle transition in a time-dependent coordinated manner. Each cyclin phosphorylates its respective cyclin-dependent kinase (CDK), which in turn induces a cascade of events and progression through the cell cycle. During S phase, cyclin A binds to CDK2 and determines the accuracy of the replication process. On the turn of G2 and M phases, cyclin A activates CDK1 and with participation of MPF complex (cyclin B/ CDK1/p34) controls the G2/M transition and mitosis initiation. Cyclin A localizes predominantly in the nucleus during S phase, where it phosphorylates CDC6, which results in its inactivation and degradation, thus preventing interruption of the replication [4]. At the G2/M transition, cyclin A is responsible for accumulation and activation of CDK1/cyclin B complex [14]. Additionally, cyclin A takes part in chromatin condensation and nuclear envelope breakdown [10-11]. Cyclin A controls transition from prophase to metaphase, affecting kinetochore microtubules stabilization, and assures optimal time of prophase duration for faithful chromosome segregation [17]. The cyclin A/CDK1 complex phosphorylates APC protein at early M phase, which is indispensable for proper orientation of the mitotic spindle [2]. Overexpression or depleted levels of cyclin A have been found in several types of human tumors. It has been proven that arsenic trioxide is able to change the expression profile of cell cycle and apoptosis-related proteins in the A549 cell line; however, its impact on cyclin expression is poorly known [26]. The A549 cell line used in the current study was initiated in 1972 through explant culture of lung carcinomatous tissue from a 58-year-old Caucasian male. A549 cells could synthesize lecithin with a high percentage of desaturated fatty acids utilizing the cytidine diphosphocholine pathway. The cells are positive for keratin in immunoperoxidase staining [9,21]. The A549 cells originate from type II alveolar epithelium and display expression of several cytochrome P450 (CYP) enzymes, which undergo differential regulation in response to xenobiotics. This non-small cell lung carcinoma cell line is therefore believed to constitute a perfect model for studies concerning the pulmonary CYP system [7,15]. In the current study we evaluated the influence of arsenic trioxide on cyclin A expression and localization in the A549 cell line. Moreover, we determined morphological and ultrastructural changes in A549 cells after ATO treatment. We also investigated ATO-induced apoptosis and cell cycle arrest. Our results suggest that the A549 cell line may display a low level of cyclin A expression, which however may be differentially regulated depending on the cell cycle arrest resulting from the ATO dose used for the treatment.

MaterialsAndMethods

Cell culture

The human non-small cell lung carcinoma cell line A549 was kindly provided by P. Kopiński, Ph.D. (Department of Gene Therapy, Ludwik Rydygier Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Poland). The cells were cultured in monolayers at 37°C in a CO2 incubator in Dulbecco’s Modified Eagle Medium (DMEM) (Lonza, Verviers, Belgium) with the addition of 10% fetal bovine serum (FBS; Gibco/Life Technologies, Carlsbad, CA, USA) and 50 μg/ml of gentamycin (SigmaAldrich, St. Louis, MO, USA). Arsenic trioxide (Adriblastin PFS; Pharmacia Italia S.p.A., Pfizer Group) was diluted in sterile water and was added to the cell culture at appropriate doses. Twenty-four hours after seeding, the cells were exposed to ATO (5, 10 and 15 μM) for 24 h.

Light microscopy

The light microscope was applied to investigate morphological alternations in the A549 cell line. Cells were fixed in 4% paraformaldehyde, then washed three times for 5 minutes with PBS and five times for 30 seconds with distilled water. The cells were stained with Mayer’s hematoxylin, then washed three times with tap water for 5 minutes. The cells were incubated for 10 minutes with PBS and then mounted with Aqua-Poly/Mount (Polysciences Warrington, PA, USA). The preparations were analyzed in an Eclipse E800 microscope (Nikon) with the NIS-Elements image analysis system and a CCD camera (DS-5Mc-U1; Nikon).

Transmission electron microscopy

Conventional electron microscopy was used to visualize A549 cell morphology at the ultrastructural level. The cells were washed with sodium cacodylate buffer and fixed in 3.6% glutaraldehyde (pH 7.2, Polysciences, Warrington, PA, USA) (30 min, room temperature [RT]). After washing in 0.1 M sodium cacodylate buffer (pH 7.4; Roth, Karlsruhe, Germany), the cells were postfixed in 1% buffered OsO4 (Serva, Heidelberg, Germany) for 1 h, dehydrated in ethanol (30–90%) and acetone (90-100%) and embedded in Epon E812 (Roth, Karlsruhe, Germany) polymerized for 24 h at 37°C and 7 days at 65°C. Semithin sections were stained with 1% toluidine blue and used for targeting the cells. Ultrathin sections (40 nm thick) were cut with a diamond knife (Diatome, Bienne, Switzerland) on a Reichert Om U3 ultramicrotome (Leica Microsystems, Vienna, Austria) on copper grids (SigmaAldrich; St. Louis, MO, USA) and stained with 1% uranyl acetate (Ted Pella, Inc., Redding, CA, USA) and lead citrate (Ted Pella, Inc., Redding, CA, USA). The material was examined using a JEM 100 CX transmission electron microscope (JEOL, Tokyo, Japan) operating at 60 kV on IMAGO – EM23 film (NDT System, Warsaw, Poland).

Annexin V/propidium iodide (PI) binding assay

To assess the extent of cell death, the Tali Apoptosis kit – Annexin V Alexa Fluor 488 and Propidium Iodide (Invitrogen) was used according to the manufacturer’s instructions. In short, after the arsenic trioxide treatment, the cells were collected from 6-well plates using trypsin-EDTA solution, centrifuged at 300 × g for 8 min, resuspended in ABB (annexin binding buffer) and incubated with Annexin V Alexa Fluor 488 at RT in the dark for 20 minutes. Following the centrifugation at 300 × g for 5 min, the cells were again resuspended in ABB and incubated with propidium iodide at RT in the dark for 5 minutes. The cells were analyzed using a Tali ImageBased Cytometer (Invitrogen). The data were quantified by FCS Express Research Edition software (version 4.03; De Novo Software, New Jersey, NJ, USA) and expressed as the percentage of cells in each population (viable Annexin V-/PI-; early apoptotic Annexin V+/PI-; late apoptotic Annexin V+/PI+; necrotic Annexin V-/PI+).

DNA content analysis

For DNA content analysis, the Tali Cell Cycle Kit (Invitrogen) was used according to the manufacturer’s instructions. Briefly, the treated cells were harvested from 6-well plates by trypsinization, rinsed with PBS, fixed in ice-cold 70% ethanol at 4°C, and left at -25°C overnight. The cells were then centrifuged at 1000 × g for 5 min at 4°C and washed with PBS the second day. After centrifugation at 500 × g for 10 min at 4°C, the cells were resuspended in Tali Cell Cycle Solution containing propidium iodide (PI), RNase A, and Triton X-100. Following 30-min incubation at RT in the dark, the cells were analyzed using a Tali Image-Based Cytometer (Invitrogen) and the percentage of cells in each phase of the cell cycle was determined using FCS Express Research Edition software (version 4.03; De Novo Software, New Jersey, NJ, USA).

Flow cytometric analysis for cyclin A expression

Cells cultured on 6-well plates were trypsinized, washed and then suspended in PBS. Cells were centrifuged (5  min, 300 x g). The obtained pellet was fixed with Cytofix/Cytoperm Fixation Permeabilization Solution (BD Pharmingen, San Diego, CA, USA), for 30 minutes. The cells were washed with Perm/Wash Buffer (BD Pharmingen, San Diego, CA, USA). Following centrifugation (5 min, 300 x g), the cells were permeabilized with Cytofix/CytopermPlus Permeabilization Buffer (BD Pharmingen, San Diego, CA, USA), and incubated for 10 minutes on ice. The cells were washed with Perm/ Wash Buffer, then centrifuged (5 min, 300 x g) and resuspended in Cytofix/Cytoperm Fixation Permeabilization Solution. After 5 minutes, the cells were washed with Perm/Wash Buffer, centrifuged (5 min, 300 x g), incubated for 10 minutes with 0.5% BSA/PBS and again centrifuged. For intracellular staining, the cell suspensions were transferred into flow cytometric tubes containing 20 μl of FITC conjugated mouse anti-human cyclin A (BD Pharmingen, San Diego, CA, USA) and 200 μl of 0.5% BSA. Following a 45-min incubation (4°C, in the dark) and washing with BSA, the cells were centrifuged (5 min, 300 x g) to wash off excess antibody and resuspended in 200 μl of BSA for flow cytometric analysis on a FACScan instrument (Becton-Dickinson, San Jose, CA, USA), with BD CellQuest Pro software Version 5.2.1 (BD Biosciences, San Jose, CA, USA) and with FlowJo 7.5.5 (Tree Star Inc., Ashland, OR, USA).

Quantitative real-time PCR analysis

To determine the expression level of cyclin A, SYBR green-based quantitative real-time PCR was performed using LightCycler 2.0 Instrument (Roche Applied Science; Mannheim, Germany) and LightCycler Software Version 4.0. Total RNA from the A549 cells was prepared by using the Total RNA kit (A&A biotechnology; Gdynia, Poland) according to the manufacturer’s protocol. The reverse transcription and quantitative PCR reactions were performed in a single 20 μl LightCycler capillary (Roche Applied Science; Mannheim, Germany) as a onestep real-time qRT-PCR with TranScriba reverse transcriptase and Master Mix SYBR (TranScriba-qPCR Master Mix SYBR kit; A&A biotechnology; Gdynia, Poland) as described by the manufacturer. The total reaction mixture (20 µl) contained 100 ng of RNA and 1 μM of each primer in addition to the TranScriba-qPCR Master Mix SYBR kit components. The primers used were as follows: forward, 5’-GTCACATGCTCATCATTTACA-3’; reverse, 5’-GGTACTGAAGTCCGGGAACC-3’. One cycle of reverse transcription was carried out for 10 min at 50°C, one cycle of denaturation for 3 min at 95°C, and 40 cycles of denaturation for 15 s at 95°C, followed by annealing and elongation for 30 s at 60°C. Relative cyclin A mRNA expression levels were quantified using the comparative threshold cycle (CT) method [22] and the results were normalized to the expression of the housekeeping gene TBP (TATA-binding protein) and presented as a fold difference relative to a calibrator sample (untreated cells).

Fluorescence microscopy

For observations using fluorescence microscopy, A549 cells were briefly washed with PBS, fixed in 4% paraformaldehyde (15 min, RT) and then washed with PBS (3 x 5 min). After that, the cells were incubated in permeabilization solution (0.25% Triton X-100 in PBS) and blocked with 1% BSA. After permeabilization, the cells were incubated with mouse monoclonal anti-cyclin A antibody (Sigma-Aldrich, St. Louis, MO, USA) (60 min, RT), washed three times with PBS and incubated with Alexa Fluor 488 goat anti-mouse IgG (Invitrogen/Life Technologies, Carlsbad, CA, USA) (45 min, RT, in the dark). Nuclear staining was performed with DAPI (Sigma-Aldrich, St. Louis, MO, USA). After incubation, the cells were washed with PBS and then mounted on slides in Aqua Poly/ Mount (Polysciences, Warrington, PA, USA). Both cyclin A and DAPI staining were examined using an Eclipse E800 microscope with a Y-FL fluorescence attachment (Nikon), NIS-Elements 3.30 image analysis system and CCD camera (DS-5Mc-U1; Nikon).

Results

Control cells exhibited typical epithelial-like morphology with kidney-shaped or oval nuclei. The cells maintained their adherence to each other. An increasing number of morphological alternations was observed in a dose-dependent manner. After ATO treatment the cells became enlarged and rounded. Vacuolization, chromatin condensation, nucleus shrinkage and loss of adhesion between cells were also characteristic of ATOtreated cells (Fig. 1). At the electron microscope level, alterations in nuclei structure and shape, swollen mitochondria and lysosome-like structures were seen. Giant flattened cells with one big nucleus – which suggested mitotic catastrophe – were observed as well (Fig. 2). Fluorescence microscopic analysis of cyclin A revealed low expression of this protein in the control and ATOtreated cells. In the control cells and cells incubated with 5 μM of ATO, cyclin A expression was observed mainly in the nucleus. With increasing doses of arsenic trioxide, numbers of cells with cytoplasmic expression of cyclin A increased simultaneously, with the protein often localized in the form of cytoplasmic foci (Fig. 3).

Real-time PCR analysis showed slightly decreased levels of cyclin A mRNA after treatment with 10 µM and 15 µM of ATO. In turn, the lowest dose of drug did not affect the expression level of cyclin A mRNA (Fig. 4). Expression of cyclin A in A549 cells was too low for flow cytometric analysis (data not shown). Our results showed that arsenic trioxide affected distribution of the cell cycle after 24 h incubation. A statistically significant increase in the percentage of G0/G1 cells was observed after incubation with 5 μM arsenic as compared to the control population, which was followed by decreases in higher doses of the drug. Concomitantly, an inverted tendency resulted from the treatment as regards the G2/M fraction of cells, i.e. decreased percentages at 5 μM ATO were followed by the enriched population at higher concentrations of the agent. The dose of 5 μM also featured a compromised population of S-phase cells. Although the subG1 fraction of cells was reduced as a result of the treatment (Fig. 5), a more in-depth image-based cytometric analysis of apoptosis using annexin V/PI showed rather high resistance of A549 cells to arsenic trioxide. The drug caused only a slight decrease in the number of viable cells, with early and late apoptotic cells constituting only a small percentage of cells. The predominant type of cell death induced in this study was necrosis (Fig. 6).

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