Isolation and characterization of a canine mammary cell line prepared for proteomics analysis

Tissue and Cell 45 (2013) 183–190

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Isolation and characterization of a canine mammary cell line prepared for proteomics analysis Mohamad Zamani-Ahmadmahmudi a , Seyed Mahdi Nassiri a,∗ , Issa Jahanzad b , Dariush Shirani c , Reza Rahbarghazi a , Babak Yazdani d a

Department of Clinical Pathology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran Department of Pathology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran c Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran d Department of Oncology, Osvah Pharmaceutical Company, Tehran, Iran b

a r t i c l e

i n f o

Article history: Received 28 June 2012 Received in revised form 28 October 2012 Accepted 29 November 2012 Available online 29 January 2013 Keywords: Canine Mammary tumor Cell line Characterization Proteomics

a b s t r a c t Mammary cancer is the most common tumor in female dogs. Canine mammary tumor serves as an excellent model for human breast cancer biology. Cancer cell lines are routinely used as the source of protein for proteomics studies because antigen homogeneity is essential for protein profiling of tumors. In this study, we sought to isolate and characterize a canine mammary cell line that was subject to protein profiling analysis through 2-dimensional electrophoresis (2-DE) method. Mammary tumor was collected from a 6-year-old terrier dog. Tumor fragments were treated with collagenase, and dissociated cells were cultured. The cell line was subcultured over 50 times. Characterization profile included population doubling time, colony forming assay, spheroid formation/migration potency, immunocytochemistry for steroid receptors and intermediate filaments, karyotyping, RT-PCR for cytokeratins 8, 14, and 18, and 2-DE pattern. The cell line revealed three growth phases including normal, dormant, and immortal phase. Immunocytochemistry showed that the cell line was positive for estrogen receptor, pancytokeratin, cytokeratin-low and vimentin, and negative for progesterone receptor, cytokeratin-high. RT-PCR supported the immunocytochemistry results. 2-DE pattern and proteome analysis of the cell line revealed that protein composition was stable, indicating the cell line as an appropriate source of protein for canine mammary proteomics studies. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction Mammary cancer is the most common tumor in female dogs (Dorn et al., 1968). Reportedly, approximately 30% of the surgically removed canine mammary tumors are malignant (Misdorp, 2002). These tumors, which are reported to occur almost exclusively in female dogs, have histopathologic features, biologic behavior and metastatic pattern similar to those seen in humans (MacEwen, 1990). Proteomics is an effective technique that is used for the identification of changes of protein patterns in a wide variety of diseases, especially neoplastic disorders (McCaw et al., 2007). Proteomics techniques allow generation of tumor-specific proteomic profile, which will not only yield novel biomarkers with predictive value to improve diagnostic and prognostic information, but also provide

∗ Corresponding author at: University of Tehran, Faculty of Veterinary Medicine, Qareeb St., Azadi Ave., PO Box 14155-6453, Tehran, Iran. Tel.: +98 2161117128; fax: +98 2166438327. E-mail addresses: [email protected], [email protected] (S.M. Nassiri). 0040-8166/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tice.2012.11.002

an avenue to bolster understanding of tumor pathogenesis (Srinivas et al., 2001, 2002). Although these techniques are being used routinely in human cancers, they are rarely used for canine cancer investigation (McCaw et al., 2007) which serves as an excellent model for human breast cancer biology (MacEwen, 1990). Various sources of protein lysate including cell lines, tissues or biological fluids are used in 2-dimensional electrophoresis (2-DE) (Hanash, 2003). Solid tumor samples have a heterogeneous nature because they consist of many elements such as epithelial cell, vascular and stromal structures, nerves, inflammatory components, etc. (Hondermarck, 2003; Johann et al., 2011). This heterogeneity provides a major problem for analyzing protein profiling using 2DE and subsequent mass spectrometry (Johann et al., 2011). Hence, proteomics researchers are eager to use cell lines instead of whole tissue specimens (Huber et al., 2004; Li et al., 2006; Hamsher et al., 2007; Toillon et al., 2007; Hamrita et al., 2008; Ou et al., 2008). Protein lysate homogeneity is extremely critical for all proteomics techniques. To improve the consistency and reproducibility of results of the proteomics techniques, antigen homogeneity is essential. There are several established and well-characterized human breast cell lines that are commercially available from

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different companies, e.g., American Type Culture Collection (ATCC). However, only three unestablished and not fully characterized canine mammary cell lines have been provided so far by ATCC (www.atcc.org). No comparable cell line is available in European Collection of Cell Cultures (ECACC) (www.hpacultures.org), Japanese Collection of Research Bioresources (JCRB) and Korean Cell Line Collection (KCLB) (Lee et al., 2010). A certain number of canine mammary tumor cell lines have been already established and the authors performed some detailed characterization (Hellmén, 1992; Lee et al., 2010; van der Burg et al., 1989), however, the proteomic profiling of these cell lines, which must be quite useful for proteomics analysis, has not been performed. Indeed, there is not a standard canine mammary tumor cell line that might be appropriate for applying proteomic studies. The present study was conducted to isolate and characterize a canine mammary cell line, which was subject to protein profiling analysis through 2-DE.

2.4. Soft agar colony forming assay

2. Materials and methods

The cells at passage 51 were used for invasion assay. Spheroid formation and invasion assay were performed according to the standard protocol with minor modification (Duca et al., 2004; Nowicki et al., 2008). Upon 80–90% confluence, cells were trypsinzed and counted. 20 ␮L of cell culture media containing 45 × 103 cells was placed on the lids of 10 cm-cell culture dishes. The lids were then inverted on dishes containing 10 mL DMEM and the hanging drops were incubated at 37 ◦ C for 48 h. The cells in the drops formed mammospheres after 48 h of incubation. To determine the invasiveness of epithelial tumor cells, a three-dimensional sphere invasion assay was developed. Briefly, neutralized collagen (BD Biosciences) was mixed with 0.5% agarose (Gibco-BRL) in a 2:1 ratio in 0.5X DMEM–2% FBS. The mammospheres were sandwiched between two layers of collagen/agarose matrix in 24-well culture plates and incubated at 37 ◦ C for 8 days. The migration of cells in the matrix was monitored daily.

2.1. Tumor sample Mammary gland tumor was collected from a 6-year-old terrier dog that underwent surgical tumor resection at the Department of Surgery of the University of Tehran Veterinary Teaching Hospital. Complete history, physical examination, laboratory and imaging findings and tumor dimensions were recorded. Then, tumor fragments were prepared under aseptic conditions for cell culture. Thereafter, the tumor was fixed in 10% buffered formalin, and then slices were embedded in paraffin, cut into 7 ␮m sections, and stained with hematoxylin-eosin (H&E) for histopathologic examination. 2.2. Cell culture The aseptically excised tumor was cut into small fragments (approximately 1 mm) with a razor blade. Tumor fragments were dissociated after 3 h treatment with collagenase (Sigma, USA). The larger fragments were discarded. Small fragments and dissociated cells were then pelleted by centrifugation at 5000 rpm for 5 min and resuspended in DMEM F12 medium (Gibco-BRL, Grand Island, NY, USA) supplemented with heat inactivated 15% fetal bovine serum (FBS) (Gibco-BRL). Cells were then introduced into a 25-cm2 tissue culture flask and incubated with 95% air and 5% CO2 at 37 ◦ C. Medium was changed twice per week. Outgrown cells were passaged when the culture reach 80% confluence. Cell morphology at different passages was recorded using a camera attached to an inverted microscope (Nikon, Japan). 2.3. Growth rate and population doubling time (PDT) Growth rate and population doubling time (PDT) were assayed using a standard method (Freshney, 2010). Cell suspensions were diluted to 1 × 104 , 3 × 104 , and 7 × 104 cells/ml of culture medium. Each dilution was seeded in triplicate in 12-well plates. The plates were kept in a CO2 incubator. Every 24 h, the cells (three wells for each concentration) were trypsinzed and counted. The PDT was calculated using the following equation (Ahmadbeigi et al., 2011):

DT =

h LN(N2 /N1 )/LN(2)

where N1 is the number of seeded cells and N2 is the number of counted cells at time h.

The cloning efficiency was assayed as descried previously with some minor modifications (Hellmén, 1992). Briefly, a 0.8% base agar layer was prepared in double strength DMEM supplemented with 20% FBS, then the mixture was poured into 6-well culture plates. The base agar layer was allowed to stand for 15 min at 4 ◦ C. Then, a 0.7% top agarose solution was prepared in double strength DMEM supplemented with 20% FBS and mixed equally with cell suspension. The prepared suspension was then added to the base agar layer. Each well contained 2000 cells. The cells were incubated at 37 ◦ C for 10 days in a CO2 incubator, and fed every 2–3 days. After 10 days, the number of colonies formed was counted after staining with 0.005% Crystal Violet. Cloning efficiency was assayed as the percentage of single cells seeded that formed colonies. 2.5. Spheroid formation/cell invasion (migration assay)

2.6. Karyotype analysis Karyotype analysis was performed as previously described (Hellmén, 1992). Cells at the growth phase were treated with 0.1 ␮g/ml colchicine for 3 h. Then adherent cells were dissociated by trypsin–EDTA (0.25%) (Gibco-BRL), and collected cells were resuspended in a hypotonic KCL (0.075 M). The cell suspension was then fixed in methanol:acetic acid (3:1) and added dropwise to clean, cold slides. Finally, prepared slides were stained with Giemsa stain, and chromosome numbers per cell were counted for 50 metaphase cells. 2.7. RT-PCR assay Total RNA extraction was performed using Tripure isolation reagent (Roche, Germany) according to the manufacturer’s protocol. Briefly, Tripure isolation reagent was added directly to the culture flask and cell lysate was prepared after pipetting several times. Chloroform (Merck, Germany) was used to separate the solution into three phases. To recover RNA, the colorless aqueous phase was used. The RNA was precipitated with isopropanol (Merck), followed by washing with 75% ethanol (Merck). Finally, the RNA pellet was dissolved in RNase-free water. cDNA was synthesized using Maxime RT PreMix Kit (Intron Biotechnology, Korea) according to the manufacturer’s instructions. The cDNA synthesis reaction was run at 45 ◦ C for 60 min, followed by 95 ◦ C for 5 min. Then, the PCR reaction was carried out using specific primers for CK8, CK14, CK18 and HPRT (Supplementary Table 1) at 94 ◦ C for 30 s, 52 ◦ C for 45 s, and 72 ◦ C for 1 min for 30 cycles. HPRT was used as internal control.

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Supplementary material related to this article found, in the online version, at http://dx.doi.org/10.1016/j.tice.2012.11.002. 2.8. Immunocytochemistry (ICC) Immunocytochemical study was performed using primary antibodies against steroid receptors (estrogen receptor (ER), Clone 1D5; progesterone receptor (PR), polyclonal antibody), pancytokeratin (Clone MNF116), cytokeratin high molecular weight (CK-high, Clone 34␤E1), cytokeratin low molecular weight (CK-low, Clone 35␤h11), and vimentin (Clone Vim 3B4) according to the manufacturer’s instructions. All antibodies were purchased from Dako Company (Glostrup, Denmark) and prepared at a dilution of 1:100. For ICC, which was performed on adherent cultured cells, paraformaldehyde 4% (Sigma, USA) was used to fix the cells. After washing three times with PBS, the cell membrane was permeabilized with 0.4% Triton X-100 (Sigma). The cells were then rinsed three times in PBS, and proteins blocked in 1% bovine serum albumin. Thereafter, primary antibodies (diluted in Triton X-100 0.04%) were applied overnight at 4 ◦ C. Then, cells were washed three times with PBS–Tween 0.1%, incubated with HRP-conjugated secondary antibodies (Dako, Denmark) for 3 h at room temperature, and then washed with PBS–Tween 0.1% three times. Signals were detected using 3,3 -diaminobenzidine (DAB) (Sigma) as chromogen. 2.9. Protein extraction and 2-DE Cultured cells were washed in washing buffer containing 10 mM Tris stock solution and 250 mM sucrose (Merck). 3 × 106 cells were lysed using a lysis buffer containing 7 M urea (Bio-Rad, NY), 2 M thiourea (Merck), 4% CHAPS (MP biomedical, USA), Tris stock solution (40 mM), and 0.2% carrier ampholyte (pH 3–10) (Sigma). After centrifugation (13,000 × g for 15 min at 4 ◦ C), the supernatant was collected and kept in −80 ◦ C. 2-DE analysis was conducted using Bio-Rad system (USA) as described previously (Hamrita et al., 2008). Briefly, proteins (300 ␮g) were subject to isoelectric focusing on 7 cm linear IPG strips (pH 3–10) at 250 V for 20 min, 4000 V for 2 h (linear mode), and 4000 V for 5 h (rapid mode). The first-dimension gels were equilibrated in a buffer containing 6 M urea, 2% SDS, 1.5 M Tris–HCL, and 17.4% glycerol. The equilibrated gel strip was placed on the top of a 12% SDS gel slab. The SDS-PAGE was performed at 80 V for 2 h. Gel slabs were fixed with fixation solution (40% ethanol and 10% acetic acid) for at least 60 min. Protein pattern was visualized by Coomassie-blue G-250 staining method. Gel slabs were destained with 1% acetic acid. 2-DE patterns from cell passages 10, 35, and 51 were analyzed using Melanie 7.0 software (Geneva Bioinformatics [GeneBio] S.A., Geneva, Switzerland). The following parameters were considered for detecting and matching spots: smooth = 2, saliency = 1, and min area = 5. 2.10. Spot picking, in-gel digestion, and protein identification by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometer (MALDI-TOF/TOF-MS) After matching spots, five spots that were common between the three 2-DE gels from the cell line at passages 10, 35, and 51 were selected and excised from the gel using a new scalpel blade for each spot (totally 15 spots). Gel pieces were transferred to the labeled 1.5 mL Eppendorf tubes. After washing gel pieces in 50 mM ammonium bicarbonate solution, they were shrunken in 50% acetonitrile, 25 mM ammonium bicarbonate solution for 15 min. The gel pieces were dried under vacuum for 5 min. Then, gel fragments were dehydrated and alkylated by 50 mM ammonium bicarbonate–10 mM DTT and 50 mM ammonium bicarbonate–100 mM iodoacetamide, respectively. Again, gel

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pieces were washed in 50 mM ammonium bicarbonate solution for 10 min and shrunken in 50% acetonitrile buffer. Tryptic digestion was then performed by adding 28 ␮L of the enzyme solution containing porcine trypsin (Promega, V5111) prepared in 50 mM ammonium bicarbonate to each tube. After adding 10 ␮L of 25 mM ammonium bicarbonate to each tube, the digestion was performed at room temperature for at least 3 h. Finally, peptides were extracted by incubating the samples in 50 ␮L of 5% formic acid–50% acetonitrile for 20 min. Peptides were identified by a UltraflexIIITM MALDI-TOF/TOF-MS (Bruker Daltonics, Bremen, Germany) operating in the reflectron mode. ␣-Cyano-4-hydroxycinnamic acid (Sigma, USA) was used as matrix. MALDI-TOF/TOF-MS were performed as described previously (Buckley et al., 2009). The resulted query masses were compared with masses database using engine MASCOT (Matrix Science, London, UK). Then the identified queries were searched against the NCBInr database. The search was only restricted to the canine sequences database. 3. Results 3.1. Tumor histopathology The tumor measured 2 cm × 1 cm. The dog was of clinical stage IIa according to the TNM-based clinical staging system. H&E stained tumor tissue was then reviewed. The tumor was classified as simple carcinoma according to the WHO-AFIP classification of canine mammary tumors. The tumor had a tubulopapillary pattern consisted of branching tubules and a complex of papillae lined by bland flat, cuboidal, and polygonal cells (Fig. 1A and B). Malignancy criteria included marked pleomorphism, anisokaryosis, hyperchromatism with a moderate mitotic index (10/hpf), and infiltration into surrounding tissues and vessels. In addition, the papillary pattern limited the stromal component of the tissue. The tumor grade was II on the basis of a three-grade system as described by Misdorp (2002), in which tubule formation, hyperchromatism and mitosis, and irregular size and shape of nuclei are used for tumor grading. 3.2. Isolation of the cell line After 24 h, the cultured cells reached 90% confluence. The PDT at passage 9 was 18.2 h. Until passage 10, cells proliferated rapidly and appeared spindle shaped (Fig. 2A: passage 3). Cell morphology changed gradually into large polygonal or spindle-shaped cells, and PDT increased to 48 h. Moreover, the number of cells with large nuclei and multiple nucleoli increased (Fig. 2B: passage 15). Cells at passage 20 changed the morphology from large polygonal to small spindle-shaped cells (Fig. 2C). Proliferation rate gradually increased with further passages (PDT 25 h at passage 23). The cell line was cultured over 50 passages. 3.3. Characterization of the cell line The cell line was able to form multiple colonies within agar after 10 days (Fig. 3). Cloning efficiency was 2.4 ± 0.08%. The cells efficiently produced spheroids in the hanging drops 48 h after incubation (Fig. 4A). The harvested mammospheres cultured on the collagen matrix showed low invasion potency because minimum cells migration was observed after 4 days (Fig. 4B). In addition, 8 days after culture, the tumor cells did not migrate to any appreciable extent (Fig. 4C). Karyotype analysis revealed a mean chromosome number of 72. Hypodiploidy was stable at passage 20 with a mean chromosome number of 73. The cells at passages 9 and 35 were used for the ICC assay. Pancytokeratin, CK low and vimentin showed a strong positive reactivity with a diffuse cytoplasmic pattern in 100% of the cells

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Fig. 1. The histological section of the excised mammy tumor (H&E staining). The histopathology shows a predominant tubulopapillary growth pattern with a mixture of papillary structures lined by flat to polygonal cells (left panel). The boxed area in the left panel is magnified in the right panel. A marked pleomorphism, anisokaryosis, hyperchromatism, and infiltration into surrounding tissues are observed (right panel). The tumor corresponds to a simple carcinoma: tubular type and grade II.

Fig. 2. Morphology of the isolated cell line at different passages. Morphological changes were observed during long-term cell culture. Dense bundles of the spindle-shaped cells proliferated rapidly at early passages (passage 3) (PDT: 18.2 h) (A). The cell morphology gradually became large polygonal (arrows) (passage 15) (B). Again at immortal phase, cell proliferation rate increased (PDT: 25 h) and morphology changed to small spindle-shaped cells (passage 23) (C).

(Fig. 5A–C). Cells did not stain with the antibody against CK high (Fig. 5D). The staining pattern of all markers remained unchanged during cell passages (in passages 9 and 35). The cell line was also evaluated for steroid receptors at the passage 51. The cells

Fig. 3. Clonogenic assay. Cells cultured in double strength DMEM/agarose mixture formed a lot of colonies 10 days post-culture.

expressed estrogen receptor with a diffuse staining of the nuclei, but they were negative for progesterone receptor (Fig. 5E and F). To support the ICC results, RT-PCR reaction was performed to assess the expression of CK8 (as high molecular weight cytokeratin) and CK14, and 18 (as low molecular weight cytokeratin) genes. The cells at passage 35 were used for RT-PCR assay. RT-PCR results were consistent with immunocytochemistry findings. A strong expression of CK18 mRNA was detected, whereas there was no visible band for CK8 or for CK14 mRNA (Fig. 6). Results of 2-DE affirmed this cell line as a suitable source of protein for various proteomics studies of canine mammary breast tumor. A consistent and reproducible 2-DE pattern was achieved from different passages after staining the gels (Fig. 7A and B). 2-DE patterns from different cell passages (10, 35, and 51) were analyzed by Melanie software. 169, 163, and 170 spots were detected on the Coomassie-blue-stained 2-DE gels at passages 10, 35, and 51, respectively. Gel matching analysis revealed 150 common spots between patterns (Fig. 7C and D). We also analyzed five common spots through MALDITOF/TOF-MS. All of these five spots corresponded to similar proteins at different cell passages. Cytokeratins family (1, 2, and 10), manganese superoxide dismutase (Mn-SOD), and elongation factor-like protein (EF-Tu) were detected using MALDI-TOF/TOF technique (Table 1).

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Fig. 4. Spheroid formation and invasion assay. The cells aggregated to form spheroids 48 h after incubation at 37 ◦ C in hanging drops. Spheroids cultured in double-layer collagen at day 1 (A), day 4 (B) and day 8 (C). The arrow indicates the migratory zone. The tumor cells show low migration ability in the collagen matrix.

4. Discussion Canine mammary cell line in this study was isolated from fresh tissue sample because freezing process probably alters the results of cell differentiation (Hellmén, 1992). In a study, to increase the purity of the isolated cell line, the authors treated fetal calf serum (FCS) with dithiothreitol (DTT) and idoacetamide (IAA) followed by heating the serum at 56 ◦ C and then treatment twice with dextrancoated charcoal at 45 ◦ C to inactivate some growth factors such as platelet-derived growth factor and transforming growth factor ␤ in order to suppress fibroblast outgrowth (van der Burg et al., 1989). However, growth factors, which are necessary for normal cell growth, are sensitive molecules and manipulations on FBS or FCS may destroy a variety of them. In agreement with other studies (Hellmén, 1992; Lee et al., 2010), the present study demonstrated that a simple treatment to FBS (heating at 56 ◦ C for 5 min) yield a pure cell line after a few passages. In line with previous studies, it seems that fibroblast cell growth stops after passaging cells for several times. The ICC findings for CK high and pancytokeratine intermediate filaments support this hypothesis. Three phases can be considered for isolated cell line growth: normal, dormant, and immortal phase. In normal phase, cells proliferated with PDT 18.2 h and cell morphology was spindle-shaped. Gradually, the cells entered the dormant phase where cell size and PDT increased. Multiple nuclei and large size of cultured cells were notified in the dormant phase. The most important mechanism for these changes was reported to be nuclear division without cytokinesis (Edgar and Orr-Weaver, 2001). Other possible mechanisms include culture conditions such as cultured media and culture container (Ahmadbeigi et al., 2011). Finally, the cells entered their immortalized phase in which cell proliferation rate gradually increased and cell morphology changed to small spindle-shaped.

Epithelial characteristics were shown for cultured cells by using various methods. The cell line uniformly expressed pancytokeratin, CK low, and vimentin. The same results were reported by some authors (Hellmén and Lindgren, 1989; Raymond and Leong, 1989; Hellmén, 1992; Destexhe et al., 1993; Griffey et al., 1993), whereas some others indicated a variable staining pattern (weak, strong, or negative) of these intermediate filaments over time (Hellmén, 1992). The most malignant mammary gland tumors such as ductular and lobular carcinoma (Complex type, Scirrhous type, and Comedo type) stain positively for both vimentin and cytokeratin in both humans and dogs (Hellmén and Lindgren, 1989). Luminal epithelial cells express CK8, 13, 16, 18, and 19, but myoepithelial cells express CK4, 14, and 17 (Bartek et al., 1985; Guelstein et al., 1988; Hellmén, 1992). ER and PR are positive in 40–60% of canine benign and malignant mammary tumors (Misdorp, 2002). The more metastatic and malignant tumors show less steroid receptors expression (Donnay et al., 1993; Rutteman et al., 1990). The previously isolated canine ER-negative mammary cell line showed a high malignancy phenotype (van der Burg et al., 1989), however the isolated cell line in this study had a significant expression of ER with low invasion potency. The mRNA expression analysis using RT-PCR method revealed that the cell line has probably a luminal origin since the CK14 mRNA expression was negative. A limited number of primary canine mammary tumors or isolated cell lines have been studied cytogenetically (Hellmén, 1992; van der Burg et al., 1989). Aneuploidy is frequently indicated in malignant mammary tumors (Rutteman et al., 1988; Misdorp, 2002). The isolated cell line in this study was found to have a stable hypodiploidy. In dogs, chromosome hypodiploidy was not only reported in mammary tumors, but also recognized in lymphosarcoma, fibrosarcoma, and osteosarcoma (Hellmén, 1992). To investigate the protein composition stability during passages a 2-DE gel analysis software (Melanie 7.0) was employed

Table 1 Analysis of five spots of the canine mammary cell line by MALDI-TOF/TOF-MS. Spot

Protein name

Accession number

Mass score

Number of queriesa

Molecular weight (kDa)/pI

1 2 3 4 5

Cytokeratin 10 Cytokeratin 2 Cytokeratin 1 Elongation factor-like protein (EF-Tu) Manganese superoxide dismutase (Mn-SOD)

NP 001013443.1 NP 001003386.1 NP 001003392.1 XP 536924.3 XP 533463.3

135 198 57 433 127

10 10 7 10 10

39.832/4.72 66.111/8.07 66.149/8.16 43.889/5.78 20.963/7.74

a

Number of queries that were subjected to engine MASCOT for searching in Matrix Science database.

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Fig. 5. A strong diffuse reaction of the cells with anti pancytokeratin (A), CK-low (B) and vimentin antibodies (C). The cells are negative for CK-high (D). The nuclei are stained positive for ER (E), and negative for PR (F).

Fig. 6. Gene expression of cytokeratin subtypes 8, 14, and 18. The cells expressed CK18 mRNA. HPRT gene was used as internal control.

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Fig. 7. Protein profiling pattern of the isolated cell line using 2-dimensional electrophoresis (2-DE). The protein lysate was separated by 2-DE and stained with Coomassieblue staining method. A consistent 2-DE pattern was obtained (A: passage 10, B: passage 35). The common excised spots were showed (arrow). The 2-DE gel analysis from passage 10 (C) and passage 35 (D) cells using Melanie software. The red lines determine the zone of each spot. The spots were compared between the two gels: common spots are shown with blue dots. Blue rods in panel C display the corresponding spots in panel D with minor displacement. More than 90% of the spots are common between the gels. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

for detecting and matching common spots. Almost 90% of the detected spots were matched between analyzed gels. In addition, mass spectrometry of five common spots from the three different gels identified similar proteins. They included cytokertins family, Mn-SOD, and elongation factor-like protein (EF-Tu). The cytokeratins are types I and II intermediate filaments, which are acidic and basic proteins, respectively. The cytokeratins family has similar structural, biochemical, and immunological properties. These proteins are considered as tumor marker especially in epithelial carcinoma. Although they are non-specific tumor markers, they can be used for predicting disease status (Barak et al., 2004). Moreover, some cytokeratins subtypes can elicit humoral response in a variety of malignancies including breast cancer, hepatocelluar carcinoma, and gastric cancer (Barak et al., 2004; Hamrita et al., 2008). Manganese superoxide dismutase (Mn-SOD) is a mitochondrial enzyme that plays an important role in protecting the cell against oxidant agents. Expression of Mn-SOD in neoplastic tissues has been investigated by some authors (Holley et al., 2012). Overexpression of this enzyme was detected in colorectal, lung, gastric, and breast cancers, hepatocelluar carcinoma, and autoimmune hepatitis (Holley et al., 2012; Takashima et al., 2006); its overexpression has been suggested to possess tumor suppression properties because some less malignant tumors showed higher

Mn-SOD expression level than more invasive tumors (Holley et al., 2012). Elongation factor EF-Tu is a member of the GTPase superfamily. EF-Tu involves in transferring aminoacyl-tRNA on the ribosome, signal transduction, cell proliferation, and chaperon activities. Autoantibodies against EF-Tu were found in a majority of human patients with infiltrating ductal breast carcinoma, and in twothird of patients with melanoma, which candidate this factor as an antigen that raises a humoral response during neoplastic transformation, with appreciable importance as tumor marker for early detection of neoplastic disease (Forgber et al., 2009; Hamrita et al., 2011). EF-Tu has an important effect in activating the proteins involved in tumorigenesis and cell growth (Hamrita et al., 2011). Interestingly, the expression of this protein was also detected in the canine mammary tumor cell line isolated in this study, indicating the similarity of breast cancer protein profile in dogs with that in human beings. A number of human breast cell lines have been used for proteomics studies, most of which aimed to detect novel biomarkers for breast cancer. In particular, immunoproteomic approaches are implemented to identify biomarkers for early tumor detection, diagnosis, and prognosis. For example, by using MCF-7 or SUM-44 cell line lysates as the source of protein, a number of tumor

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