Results in Immunology 3 (2013) 95–103

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Results in Immunology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / r i n i m

Transcriptome analysis of neoplastic hemocytes in soft-shell clams ଝ Mya arenaria : Focus on cell cycle molecular mechanism

Ahmed Siah a,b,*, Patty McKenna b, Franck C.J. Berthe c, Luis O.B. Afonso d, Jean-Michel Danger e aBritish Columbia Centre for Aquatic Health Sciences, BC CAHS, 871A Island Highway, Campbell River, BC, Canada V9W 2C2 bDepartment of Pathology & Microbiology, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, Canada C1A 4P3 cAnimal Health and Welfare Unit, European Food Safety Authority (EFSA), Largo N, Palli 5IA, I-43100, Parma,Italy dSchool of Life & Environmental Sciences, Faculty of Science, Engineering and Built Environment, Deakin University, PO Box 423, Warrnambool, Victoria 3280, Australia eLaboratory of Ecotoxicology, University of Le Havre, 25 rue Philippe Lebon, BP540, 76058 Le Havre, France

a r t i c l e i n f o a b s t r a c t

Article history: In North America, a high mortality of soft-shell clams Mya arenaria was found to be related to the disease Received 24 August 2013 known as disseminated neoplasia (DN). Disseminated neoplasia is commonly recognized as a tetraploid disor-

Received in revised form 20 October 2013 der related to a disruption of the cell cycle. However, the molecular mechanisms by which hemocytes of clams Accepted 21 October 2013 are transformed in the course of DN remain by far unknown. This study aims at identifying the transcripts related to DN in soft shell clams ’ hemocytes using next generation of sequencing (Illumina HiSeq2000). This Keywords:

Transcriptomic study mainly focuses on transcripts and molecular mechanisms involved in cell cycle. Using Illumina next Neoplasia generation of sequencing, more than 95,399,159 reads count with an average length of 45 bp was generated Tetraploidy from three groups of hemocytes: (1) a healthy group with less than 10% of tetraploid cells; (2) an intermediate Mya arenaria group with tetraploid hemocytes ranging between 10% and 50% and (3) a diseased group with more than 50% Cell cycle of tetraploid cells. After the reads were cleaned by removing the adapters, de novo assembly was performed Molecular mechanisms on the sequences and more than 73,696 contigs were generated with a mean contig length estimated at 585 bp ranging from 189 bp to 14,773 bp. Once a Blastx search against NCBI Non Redundant database was performed and the duplicates removed, 18,378 annotated sequences matched known sequences, 3078 were hypothetical and 9002 were uncharacterized sequences. Fifty percent and 41% of known sequences match sequences from Mollusca and Gastropoda respectively. Among the bivalvia, 33%, 17%, 17% and 15% of the contigs match sequences from Ostreoida, Veneroida, Pectinoida and Mytiloida respectively. ontology analysis showed that metabolic, cellular, transport, cell communication and cell cycle represent 33%, 15%, 9%, 8.5% and 7% respectively of the total biological process. Approximately 70% of the component process is related to intracellular process and 15% is linked to and ribonucleoprotein complex. Catalytic activi- ties and binding molecular processes represent 39% and 33% of the total molecular functions. Interestingly, nucleic acid binding represents more than 18% of the total protein class. Transcripts involved in the molecular mechanisms of cell cycle are discussed providing new avenues for future investigations. c 2013 The Authors. Published by Elsevier B.V. All rights reserved.

1. Introduction exhibit higher DNA contents and mitosis rates than normal cells re- sulting in the formation of tetraploid cells [ 4 , 5 ]. During normal cell Disseminated neoplasia (DN) is defined as a leukemia-like dis- cycle, segregate only when an adequate kinetochore- ease affecting hemocytes of soft-shell clams. This disease is mainly microtubule attachment exists enabling cells to pass the spindle as- characterized by a high number of circulating hemocytes containing sembly checkpoint [ 6 ]. However, after prolonged arrest at the spindle pleomorphic nuclei with a high nucleus–cytoplasm ratio [ 1 , 2 ]. These assembly checkpoint that promotes cytokinesis failure, cells become abnormal circulating cells lose their function of phagocytosis [ 3 ] and tetraploid [ 7 ]. The etiology of DN in Mya arenaria remains unknown although several factors such as retrovirus infection or contamination seem

ଝ This is an open-access article distributed under the terms of the Creative Com- to contribute to the development of the disease. Retroviral infection mons Attribution-NonCommercial-No Derivative Works License, which permits non- was shown as the main factor [ 8 ] of DN development; however, other commercial use, distribution, and reproduction in any medium, provided the original studies did not succeed in detecting retroviral components in diseased author and source are credited. clams [ 9 ]. In addition, studies stipulate that environmental contami- * Corresponding author at: British Columbia Centre for Aquatic Health Sciences, BC

CAHS, 871A Island Highway, Campbell River, BCCanada V9W 2C2. Tel.: +1 250 286 nation by pesticides could be the main cause of DN in soft shell clams 6102; fax: +1 250 286 6103. collected from Prince Edward Island [ 10 , 11 ]. E-mail address: [email protected] (A. Siah).

2211-2839/ $ - see front matter c 2013 The Authors. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.rinim.2013.10.001 96 A. Siah et al. / Results in Immunology 3 (2013) 95–103

Fig. 1. Contigs length distribution after de novo assembly .

Many studies have investigated the molecular mechanisms in- volved in the development of the disease [ 12 –18 ]. For instance, some studies have shown that the proliferation of hemocytes in clams is due Fig. 2. Phylogenetic distribution of the best hit in animal kingdom (A) and eukaryota to the sequestration of by mortalin [ 13 , 14 ], whereas other studies (B) based on BlastX search in the NCBI nr database. demonstrated that p53’s pathways were disrupted by the activation of the Mouse Double Minute 2 (MDM2) proto-oncogene [ 15 ]. Our recent investigation using subtractive suppressive hybridization techniques suggested the involvement of proto-oncogene such as RAS like protein members, c- and c-jun in the development of DN [ 17 ]. It was hy- pothesized that the proto-oncogenes were regulated by the presence of transposase whose was increased in tetraploid hemocytes [ 16 ]. Briefly, although several studies have been initiated, the molecular mechanisms by which hemocytes become neoplastic are still to be unravelled. Unfortunately, little is known on the tran- scriptome of soft shell clams. Only few sequences are available in GenBank represented mainly by Map53 (Acc# AF253323.1 ), Map73 (Acc# AF253324.1 ), and mortalin (Acc # EF576660 ). The development of the new generation of sequencing technolo- gies represents a great opportunity to increase the number of tran- script sequences and would provide a basis platform for molecular mechanisms studies involved in DN in soft shell clams. Thus, this study aims at increasing the numbers of transcript se- quences in hemocytes of soft shell clams affected by DN. Our ap- proach focused specifically on sequencing mRNA isolated from dif- ferent groups of hemocytes characterized by their tetraploid status. The sequences generated in this study will constitute a baseline in un- ravelling the molecular pathway(s) involved in DN as well as a source of potential target transcripts which could be involved in hemocytes’ immune system of soft shell clams, M. arenaria .

2. Materials and methods

2.1. Sampling Fig. 3. Species distribution based on BlastX alignment in Bivalvia (A) and Mollusca (B) NCBI nr database. Approximately 5-cm-long specimens of M. arenaria were collected at low tide at a depth of 15–20 cm using a hand rake from May to November 2011 in North River (46 ◦15  10  N, 63 ◦10  42  W) (Char- 2.2. Flow cytometry lottetown, Prince Edward Island, Canada). Clams were washed with seawater and transported to the Atlantic Veterinary College at the Flow cytometric (FCM) analysis was used to assess the ploidy sta- University of Prince Edward Island for further analysis. tus of M. arenaria’s haemocytes according to the methods described A. Siah et al. / Results in Immunology 3 (2013) 95–103 97

Fig. 4. Percentage representation of mapping (A) biological processes, (B) cellular component and (C) molecular functions. by Delaporte et al. [ 5 ]. This technique enables the screening of cell 2.3. RNA extraction populations for DNA content. The protocol is based on the binding properties of propidium iodide (PI), which stains by intercalating into Based on their tetraploid status, 3 groups of clams were selected: DNA strands. The PI fluorescence intensity is proportional to the DNA (1) Group C (healthy clams considered as control) with a low percent- content in the cell. Briefly, hemolymph (500 μL) was withdrawn from age of tetraploid hemocytes ( < 10%); Group D (disease in develop- individual clams using a 3 mL syringe fitted with a 25-gauge needle. ment): individuals presenting a percentage of tetraploid cells ranging Haemocytes were fixed in 2.5 mL of cold absolute ethanol and stored between 10% and 50%; and Group E (established disease): clams with a at −20 ◦C for at least 30 min. Fixed cells were centrifuged (400 g for high percentage of tetraploid hemocytes ( > 50%). Hemolymph (2 mL) 10 min at room temperature), and the supernatant was discarded. was withdrawn from individual clams using a 3 mL syringe fitted with Haemocyte pellets were re-suspended in 0.01 M phosphate-buffered a 25-gauge needle. Hemolymph was centrifuged at 400 g for 15 min saline (PBS) and the cells were allowed to re-hydrate for 30 min at at 4 ◦C in order to isolate hemocytes from the serum. Total RNAs room temperature. After two washes in PBS (400 g for 10 min at room were extracted from hemocytes using RNeasy purification kit (Qia- temperature), cells were re-suspended in 380 μL of PBS solution and gen, USA), treated with DNaseI and the quality of RNA was analyzed transferred to flow cytometer tubes by filtering through an 80 μm using the bioanalyser Experion (Biorad, Canada). RNA concentrations nylon mesh. Propidium iodide (50 μg mL −1 ) and DNAse-Free RNase A were analyzed using a Nanodrop (ND 1000, USA) spectrophotometer. (50 μg mL −1 ) were added to each tube before the mixtures were incu- Samples with high quality (RNA quality index higher or equal at bated in the dark until optimal PI staining for 30 min. PI fluorescence, 8) and concentration higher than 100 ng mL −1 was selected. Based which is related to the DNA content of each cell, was detected on the on these criteria, 6 samples were pooled for the group C (0–10%), orange photomultiplicator of a FACSCalibur flow cytometer (BD Bio- 7 samples for the group D (10–50%) and 3 samples for the group E sciences) at a wavelength ranging between 550 and 600 nm. For each ( > 50%). sample, 10,000 particles were counted at low flow rate (15 μL min −1 ). For each cell event, a single pulse of PI fluorescence was represented 2.4. Library preparation and sequencing according to its area and width. The pulse width needs to be com- pared with the pulse area in order to discriminate the cells in the Library preparation and next generation of sequencing were car- phase G2 / M from doublets of G0 / G1 cells represented by the same ried out by Eurofin (Alabama, US). Three individual libraries (one from DNA quantity. To gate single haemocytes, PI fluorescence intensities each group) were prepared with NuGEN Ovation RNA-Seq system V2 were plotted as a FL2-area vs. FL2- width dot-plot. The region R1 was according to the manufacturer’s recommendations. NuGEN Ovation drawn in order to discriminate single cells from the doublets. The sin- RNA-Seq system V2 corresponds to three main steps: first strand gle cells gated in R1 were plotted on a FL2-area histogram and used to cDNA synthesis, second strand cDNA synthesis and purification and estimate the percentage of normal and tetraploid haemocytes in the SPIA amplification and purification. Briefly, First Strand Primer Mix analyzed cell population. Results are presented as the percentage of was mixed to RNA (100 ng). The mixture was warmed at 65 ◦ C for tetraploid haemocytes per clam. Three different classes of clams were 5 min. First Strand Master Mix (3 mL) was added to each tube and distinguished according to the percentage of tetraploid hemocytes. placed in a thermal cycler at 4 ◦ C for 1 min, 25 ◦C for 10 min, 42 ◦C for 10 min, and 70 ◦C for 15 min. Second Strand Master Mix (10 mL) was added to each First Strand reaction tube. The mixture was added and placed in a thermal cycler at 4 ◦C for 1 min, 25 ◦C for 10 min, 50 ◦C for 30 min, and 80 ◦C for 20 min. Second strand cDNA was purified using Agencourt RNAClean XP purification beads provided with a NuGEN 98 A. Siah et al. / Results in Immunology 3 (2013) 95–103

The emergence of the next generation of sequencing has dramat- ically increased the knowledge and information on bacteria, inverte- brate and vertebrate organisms’ transcriptome. Although the num- ber of Expressed Sequence Tags (ESTs) has increased during the last decade, the knowledge on mollusk bivalvia in particular M. arenaria is still limited. GenBank database contains only 248,489 nucleotides and 373,300 ESTs related to Bivalvia (4 / 15 / 2013). Ostreoida and Mytiloida represent the most representative groups with 223,017 (59.7%) and 71,328 (19.1%) ESTs respectively. Among the Ostreoida, Crassostrea is the main representative group with 222,785 (99.9%) ESTs and Mytilus have been mainly sequenced in the Mytiloida family with 67,990 (95.3%). Unfortunately, the number of ESTs related to M. arenaria is very poor with 100 entries represented mainly by cytochrome oxidase, microsatellite and ribosomal RNA. Among the 100 entries, only 10 transcripts have been recorded in GenBank (4 / 15 / 2013) rep- Fig. 5. Distribution of best hits annotated protein class based on the BlastX alignment and gene ontology mapping. resented by M. arenaria p53 tumor suppressor homolog (Myap53) mRNA, partial cds ( U45237.1 ); M. arenaria voltage-dependent sodium channel mRNA, partial cds ( AY847740.1 ); M. arenaria -like pro-

Ovation RNA-Seq system V2 kit. Finally, SPIA Master Mix (40 μL) was tein mRNA, complete cds (AF253324.1); M. arenaria E3 ubiquitin- added to each tube containing the double-stranded cDNA bound to protein ligase mRNA, complete cds (AF154109.2); M. arenaria aryl hy- the dried beads and placed in a thermo cycler at 4 ◦C for 1 min, 47 ◦C drocarbon -like protein mRNA, complete cds (AF261769.1); for 60 min, and 80 ◦C for 20 min. SPIA cDNA was purified using QI- M. arenaria p53 tumor suppressor-like protein mRNA, complete cds

AGEN MinElute Reaction Cleanup Kit (Qiagen, USA). SPIA cDNA was (AF253323.1); M. arenaria ribosomal protein S19 (S19) mRNA, com- sequenced on Illumina HiSeq2000 with 2 × 50 bp according to the plete cds (U63092.1); M. arenaria mitochondrial mortalin-2 precursor manufacturer’s recommendations. (Mot-2) mRNA, complete cds, nuclear gene for mitochondrial prod- uct ( AY326398.2 ); M. arenaria mitochondrial mortalin splice variant mRNA, complete cds, alternatively spliced, nuclear gene for mito-

2.5. Sequence analysis and bioinformatics chondrial product ( EF576660.1 ); and M. arenaria Rack1 gene for Re- ceptor of Activated Kinase C 1 ( AM404081.1 ). Most of the messengers

Reads generated from Illumina HiSeq2000 sequencing were as- cited above are related to the p53-like molecular mechanisms in- sembled using the CLC Genomics Workbench software v5.5. Adap- volved in DN in soft shell clams M. arenaria [ 12 , 23 , 24 ]. However, the tors and primer sequences were trimmed as well as low quality and number of sequences remains by far unrepresentative of M. arenaria ’s duplicates removed. De novo sequence assembly was performed us- transcriptome, which therefore makes it difficult to understand the ing de Bruijn graphs as described in [19–21]. The generated contigs molecular mechanisms by which hemocytes become neoplastic. This and isotigs were blasted against NCBI Non-Redundant database in- study attempts to populate M. arenaria ’s sequence database in partic- / cluding Swissprot, Metazoan Refseq and UniprotKB Trembl protein ular in the field of clams ’ pathology such as disseminated neoplasia. databases using the GenomeQuest bioinformatic platform (US) with These sequences could be used in studies interested in molecular −6 a cut-off of 10 . Gene ontology searches were performed using the mechanisms involved in disseminated neoplasia but also in ecotox-

Panther Pathway [22]. Sequences were submitted to BLAST compari- icogenomics where M. arenaria is used as a sentinel species for bio- son against the KEGG database to obtain KO (KEGG Orthology) surveillance programmes. assignments and to generate KEGG pathways. The threshold > 60 was applied to BLAST bit scores. Reads per kilobase per million mapped reads (RPKM) were quantified from the three groups. Ratio to the 3.2. Annotation healthy group C were calculated for group D and group E. Data are reported in a histogram representing Log2 RPKM ratio. A Blastx search was performed against NCBI Non Redundant database including Swissprot, Metazoan Refseq and UniprotKB / 3. Results and discussion Trembl protein databases. The GenomeQuest bioinformatic platform was used for the Blastx search and a cut-off of 10 −6 was applied for 3.1. De novo assembly the analysis. Once the duplicates were removed, 18,378 annotated sequences matched known sequences, 3078 were hypothetical and The tetraploid status of hemocytes has been screened using flow 9002 were uncharacterized sequences. Eighty-five percent of the an- cytometry. Three groups of hemocytes were determined: Group C, a notated sequences are among eukaryote identities ( Fig. 2 A) with 18% healthy group with less than 10% of tetraploid cells; Group D, an inter- and 5% linked to arthropoda and mollusca phylla ( Fig. 2 B). mediate group with tetraploid hemocytes ranging between 10% and Data showed that 50% and 41% of known sequences match with se- 50% and group E, a diseased group with more than 50% of tetraploid quences from Mollusca and Gastropoda respectively ( Fig. 3 A). Among cells. More than 95,399,159 reads count were generated from the the bivalvia, 33%, 17%, 17% and 15% of the contigs match sequences three groups with an average length of 45 bp. from Ostreoida, Veneroida, Pectinoida and Mytiloida respectively ( Fig. The bioinformatic analysis of the sequences was performed us- 3 B). ing the CLC Genomics Workbench software v5.5. The adaptors and primers were trimmed; in addition, duplicates and low quality se- quences were removed from the data set. Upon cleaning, de novo 3.3. Gene ontology assembly was performed on the sequences using de Bruijn graphs. More than 73,696 contigs were generated with a mean contig length Gene ontology analysis showed that metabolic, cellular, transport, estimated at 585 bp ranging from 189 bp to 14,773 bp. More than cell communication and cell cycle represent 33%, 15%, 9%, 8.5% and 90.3% of the contigs (66,612 sequences) had a length ranging from 7% of the total biological process respectively ( Fig. 4 A). The major- 189 to 1268 bp and less than 9.7% were longer than 1268 bp ( Fig. 1 ). ity (around 70%) of the component process is related to intracellular A. Siah et al. / Results in Immunology 3 (2013) 95–103 99

Fig. 6. Expression levels of gene identified in our sequence database during the phase G1–S. Histogram represents Log 2 RPKM ratio of group D (disease in development) and E (established disease) to group C (control). GSK3b: glycogen synthase kinase 3 beta; RBL1 (p107): retinoblastoma-like protein 1; / 5: E2F4 / 5; c-Myc: Myc proto-oncogene protein; Miz1: zinc finger and BTB domain-containing protein 17; SMAD 2 / 3: SMAD 2 / 3: mothers against decapentaplegic homolog 2 / 3; SMAD4: mothers against decapentaplegic homolog 4; CycD2: cyclin D2; CDK4: cyclin-dependent kinase 4; CycE: cyclin E; CDK2: cyclin-dependent kinase 2; SCF: S-phase kinase-associated protein 1; RBL2 (p107-p130): retinoblastoma-like protein 2; E2F4 / 5: transcription factor E2F4 / 5; and : transcription factor E2F3.

Fig. 7. Expression levels of gene identified in our sequence database during the phase S–G2. Histogram represents Log 2 RPKM ratio of group D (disease in development) and E (established disease) to group C (control). Cyc H: cyclin H; CycA cyclin A; CDC7 cell division control protein 7; CDC6: cell division control protein 6; ORC5: origin recognition complex subunit 5; ORC3: origin recognition complex subunit 3; ORC2: origin recognition complex subunit 2; ORC1: origin recognition complex subunit 1; MCM4: DNA replication licensing factor; MCM5: DNA replication licensing factor; MCM2: DNA replication licensing factor; MCM6: DNA replication licensing factor; MCM7: DNA replication licensing factor MCM7; MCM3: DNA replication licensing factor; Cdk7: cyclin-dependent kinase 7; CDK2: cyclin-dependent kinase 2; GADD45: growth arrest and DNA-damage-inducible protein; RBL2: retinoblastoma-like protein 2; RBL1: retinoblastoma-like protein 1; Rb: retinoblastoma-associated protein; MDM2: MDM2 Binding Protein; p300: E1A / CREB-binding protein; DNA-PK: DNA-dependent protein kinase catalytic subunit; ATM / ATR: ataxia telangiectasia mutated family protein; Chk1: serine / threonine-protein kinase; CDK1: cyclin-dependent kinase 1; and Wee: wee1-like protein kinase. 100 A. Siah et al. / Results in Immunology 3 (2013) 95–103

Fig. 8. Expression levels of gene identified in our sequence database during the phase G2–M. Histogram represents Log 2 RPKM ratio of group D (disease in development) and E (established disease) to group C (control). MPS1: serine / threonine-protein kinase; MAD1: mitotic spindle assembly checkpoint protein; MAD2: mitotic spindle assembly checkpoint protein; BUB3: cell cycle arrest protein; BUB1: checkpoint serine / threonine-protein kinase; BUB1 β/ BUBR1: mitotic checkpoint serine / threonine-protein kinase; 14–3–3 β−τ−ζ : 14–3–3 protein beta / theta / zeta; CycB: cyclin B; CDK1: cyclin-dependent kinase 1; Myt1: membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase; PLK1: polo-like kinase 1; SMC1: structural maintenance of 1; SMC3: structural maintenance of chromosome 3 (chondroitin sulfate proteoglycan 6); STAG1 / 2: cohesin complex subunit; Rad21: cohesin complex subunit SCC1; ESP1: separase; APC / C4: anaphase-promoting complex subunit 4; APC / C2: anaphase-promoting complex subunit 2; APC / C6: anaphase-promoting complex subunit 6; APC / C8: anaphase-promoting complex subunit 8; APC / C3: anaphase-promoting complex subunit 3; APC / C10: anaphase-promoting complex subunit 10; APC / C1: anaphase-promoting complex subunit 1; APC / C7: anaphase-promoting complex subunit 7; Cdh1 / CDC20: cell division cycle 20, cofactor of APC complex; and CDC 14: cell division cycle 14. processes and approximately 15% linked to protein and ribonucleo- to be related to centrosome amplification triggering aneuploidy [ 28 ]. protein complex respectively ( Fig. 4 B). Catalytic activities and bind- It was shown that cyclin E-CDK2 complex is a key regulator in cen- ing molecular processes represent 39% and 33% of the total molecular trosome duplication and DNA replication [ 29 ]. It was demonstrated functions respectively ( Fig. 4 C). Interestingly, nucleic acid binding that cyclin E can bind the centrosome using the centrosomal localiza- represents more than 18% of the total protein class ( Fig. 5 ). tion signal (CLS) and then initiate the S-phase of the cell cycle [ 30 ]. In addition, CLS domain allows cyclin E to bind the minimichrosome 3.4. Gene expression and cell cycle maintenance 5 (MCM 5) recognised as a DNA pre-replication complex factor [ 31 ]. It was shown that the expression of MCM5 inhibits cen- Tetraploid cells with double DNA quantity can lead to tumorgene- trosome amplification in CHO cells arrested in S-phase corroborating sis under a non-functional p53 gene [ 25 ]. Several studies have shown the essential role of MCM5 on the centrosome regulation [ 31 ]. How- the link between the increase of tetraploid tumor cells and the failure ever, in our study, MCM5 transcript level was similar to the control. of p53 or downstream gene such as p21 in mammalians [ 26 ]. The same Furthermore, our data showed an overexpression of c-myc in groups observations were described in soft shell clams M. arenaria where p53 D and E hemocytes ( Fig. 6 ) corroborating our previous findings [ 17 ]. function was affected by high level of mortalin in the cytoplasm or high expression of MDM2 [ 13 , 15 ]. The formation of tetraploid cells 3.4.2. Transcripts involved in S–G2 phase of cell cycle is a consequence of cell fusion, endoreduplication, cytokinesis fail- In late G1 phase, chromosome duplication is triggered by a spe- ure or mitotic slippage [ 26 ]. During the cell cycle, the chromosome cific cdk protein known as S-phase promoting cdks. DNA replication segregation is under control of mitotic checkpoint genes . is initiated by the formation of the pre-replicative complex (pre-RC). This complex activity is dependent on the association of Cdc6 with 3.4.1. Transcripts involved in G1– S phase of cell cycle the Origin Recognition Complex (ORC) [ 32 ]. Protein kinase such as At the G1 phase also known as the growth phase which corre- Dbf4-Cdc7 and Clb5 / 6(B-cyclin)-Cdc28 phosphorylate and activate sponds to a high rate of biosynthesis, the CDK4,6 like were proteins within the pre-RC initiating then the DNA replication in identified in our database. CDK4,6 linked to cyclin D is the first serine- phase S [ 33 ]. Cdc7 is a serine-threonine kinase which phosphorylates threonine kinases activated during the cell cycle. CDK4,6-cyclin D MCM2. MCM2 is a key component of the DNA replicative helicase, complex under proliferative signal activation allow the cell to enter which plays a central role in genome duplication. The transcript level the cell cycle at its first G1 phase [ 27 ]. Once activated, CDK4,6-Cyclin of Cdc7 in hemocytes from group D (disease in development) is 19.1 D initiates a sequential activation of downstream of cyclin E-CDK2, times higher than the control, whereas this level is 5 times higher in which regulates the G1–S transition and centrosome duplication. In diseased hemocytes with a tetraploidy status higher than 50% ( Fig. 7 ). our study, cyclin D2 and cyclin E transcripts were 4.6 and 3.8 times Interestingly, Cdc7 gene and protein are highly expressed in tumor respectively higher than the control during the development phase of cells including leukemia [ 34 , 35 ]. Several investigations targeted Cdc7 the disease ( Fig. 6 ). Once the disease was established, the expression as a cancer therapy by inhibiting Cdc7 expression in cancer cells [ 36 ]. level of cyclin D2 and E was 2.3 and 2.5 higher than the control respec- In G1–S transition phase, Cdc6 is a key component in DNA replication. tively. The regulation of centrosome duplication is key in the regula- Cdc 6 is an ATPase belonging to the AAA family with chaperone like tion of chromosome segregation. Several types of cancer were found activities as well as a role in transcriptional regulation [ 37 ]. As shown A. Siah et al. / Results in Immunology 3 (2013) 95–103 101

Fig. 9. KEGG mapping for cell cycle pathway. The green boxes represent the annotated protein found in our library. The white boxes are the unidentified proteins in our database sequences. for Cdc7, the level of Cdc6 transcript is 8.8 and 3.5 higher in groups D overexpression of cyclin B seems to coincide with a high expression and E respectively ( Fig. 7 ). In cancer cells, overexpression of Cdc6 in- of c-myc ( Fig. 6 ). It was shown that cyclin B1 is a c-myc target whose hibits expression of p16 INKa / p15 INKb bot activator of retinoblastoma overexpression induces chromosomal activity and tetraploidy [ 45 ]. pathway and a p53 stimulator, ARF [ 38 ]. In addition, it was shown In the M phase, the anaphase promoting complex / cyclosome that the overexpression of Cdc6 has a synergistic effect with mutant (APC / C) is an E3 ubiquitin ligase complex that targets the degradation p53 in tumor progression and chromosome instability [ 39 ]. of protein involved in the cell cycle during the M phase. APC / C’s ac- The growth arrest and DNA damage inducible gene (GADD45) is tivation depends on two WD-40 domain proteins, CDC20 and CDH1. a group of genes that are activated by genomic damage compounds In our study, APC / C family members identified in our database and in such as UV radiations, chemotherapeutic agents and growth factors particular APC / C sub-unit 7 showed a high expression level in hemo- [ 40 ]. Under activation by p53, GADD45 interacts with Cdc2 which is cyte with tetraploid status ranging between 10% and 50% as well as in cleaved from Cyclin B1 and the complex GADD45 / Cdc2 acts on the diseased organisms with more than 50% tetraploid hemocytes ( Fig. 8 ). cell cycle by arresting the division at the checkpoint G2 / M [ 41 ]. The Similarly, Cdh1 / Cdc20 is also higher in both groups D and E ( Fig. 8 ). In high expression of GADD45 recorded in group D (hemocytes with yeast mitosis, CDC20 and CDH1 bind to the APC / C, which is activated tetraploidy status ranging between 10% and 50%). during the mitosis process [ 46 ]. Cdc20 activates APC / C during early mitosis, while Cdh1 plays a key role and controls several phases from late mitosis to G1 / S transition [ 47 ]. It was shown that the deregu- 3.4.3. Transcripts involved in G2–M phase of cell cycle lation of Cdh1 impaired centrosome formation, cytokinesis and DNA / / G2 phase is mainly controlled by CycA CDK1 and CycB CDK1. Cy- re-replication [ 48 ]. Overall, the disruption of APC / Cdh1 activity may clin B1 is expressed at very low levels and its expression increases lead to cell cycle instability and thus the development of neoplastic at the G2–M phase [ 42 ]. However, in tumor cells, cyclin B1 is mainly cells [ 49 ]. localized in the cytoplasm and overexpressed throughout the cell cy- On the other hand, the expression level of separin (Esp1) is high cle due to the inactive state of p53 [ 43 ]. It was shown that cyclin A (Log2 ratio Med / Neg = 3.58) in hemocytes with a tetraploidy rang- is key in the progression of male germ cells through meiosis [ 44 ]. ing from 10% to 50% ( Fig. 8 ). Separin is among the protein complexes Over-expression of cyclin A was found in leukemic cells in more than known as cohesin. This complex maintains the cohesion between 50% of acute myeloid leukemia patients [ 44 ]. In our study, both cyclin the sister chromatid which is important for equal segregation of sis- A ( Fig. 7 ) and B ( Fig. 8 ) as well as CDK1 ( Fig. 8 ) are highly expressed in ter chromatids in anaphase [ 50 ]. Separin related proteins have been hemocytes with a tetraploid status ranging between 10% and 15%. The 102 A. Siah et al. / Results in Immunology 3 (2013) 95–103 identified in yeast [ 51 ], Aspergillus nidulans [ 52 ], Xenopus, and human expression levels in haemocytes of soft-shell clams ( Mya arenaria ). Ecotoxicol- [ 53 ] and now in M. arenaria (this study) suggesting that the mech- ogy 2011;20:1765–72 . [11] Muttray A, Reinisch C, Miller J, Ernst W, Gillis P, Losier M. Haemocytic leukemia anism of chromatid segregation is evolutionarily conserved among in Prince Edward Island (PEI) soft shell clam ( Mya arenaria ): spatial distribution organisms. in agriculturally impacted estuaries. Sci Total Environ 2012;424:130–42 . [12] Kelley ML, Winge P, Heaney JD, Stephens RE, Farell JH, Van Beneden RJ. Ex- pression of homologues for p53 and p73 in the softshell clam ( Mya arenaria ), a 3.5. Cell cycle molecular pathway in hemocytes of M. arenaria naturally-occurring model for human cancer. Oncogene 2001;20:748–58 . [13] Walker CW, B ottger¨ SA. A naturally occurring cancer with molecular connectiv-

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