Cyclopedic protein expression analysis of cultured canine mammary gland adenocarcinoma cells from six tumours

Research in Veterinary Science 80 (2006) 317–323 www.elsevier.com/locate/rvsc

Cyclopedic protein expression analysis of cultured canine mammary gland adenocarcinoma cells from six tumours T. Nakagawa a, M. Watanabe b, E. Ohashi a, R. Uyama a, S. Takauji a, M. Mochizuki a, R. Nishimura a, H. Ogawa a, S. Sugano b, N. Sasaki a,* a

Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan b Laboratory of Functional Genomics, Department of Medical Genome sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo 108-8639, Japan Accepted 19 July 2005

Abstract We characterised cultured canine mammary gland adenocarcinoma cells by exhaustive step protein expression analysis to identify factors associated with tumour progression or metastasis of canine mammary gland tumour. Cultured adenocarcinoma cells derived from a total of 3 primary and 3 metastatic lesions from 3 dogs (CHMp/m, CIPp/m and CNMp/m, where CHM, CIP, and CNM indicate the 3 animals) were used in this study. The expression of 24 proteins reported to be related to tumourigenesis or malignancy of human breast cancers were examined by Western blot analysis using 24 antibodies. The expression of sialyl Lewis X [sLe(x)] was only observed in CHMm cells, which were derived from pleural effusion. This expression was further confirmed by immunohistochemistry. The levels of some factors, such as 14-3-3r, cyclinD1 and Rb, differed among cells or between the primary and metastatic cells in the pair. Though the difference in their expression was not consistent within the cells from primary and metastatic origin, this characterisation should provide useful information for further molecular analysis of these cultured cells. Since some of the factors, such as sLe(x), 14-3-3r, cyclinD1 and Rb, showed different levels of expression in the pair, these cultured cells might be meaningful tools for clarification of distant metastasis in canine mammary gland tumours. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Mammary gland adenocarcinoma; Dog; Protein expression; Sialyl lewis X[sLe(x)]; 14-3-3r

1. Introduction Canine mammary gland tumour (CMGT) is one of the most common neoplasias, because it accounts for approximately 50% of all tumours in female dogs (Brodey et al., 1983; Dorn et al., 1968; MacEwen and Withrow, 1996). Histologically, about 50–60% of CMGTs are considered malignant (Brodey et al., 1983; Dorn et al., 1968; MacEwen and Withrow, 1996). Tumour invasion to the surrounding tissues and metastasis are the most significant prognostic factors in malignant tu*

Corresponding author. Tel.: +81 3 5841 5420; fax: +81 3 5841 8996. E-mail address: [email protected] (N. Sasaki).

0034-5288/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2005.07.011

mours (Hahn et al., 1992; Kurzman and Gilbertson, 1986; Shofer et al., 1989). Through in vitro and in vivo studies on cancers, several factors, including cell adhesion molecules (Phillips et al., 1990; Vleminckx et al., 1991), cell cycle and growth regulators (Howe and Brown, 2004; Narita et al., 1993a; Sherr, 1996), apoptosis-related molecules (Sawan et al., 1992), scatter folding factors (Dubois et al., 1997) and hormone receptors (Issemann and Green, 1990; Slamon et al., 1987), have been found to be related to tumour invasion and metastasis. The identification and analysis of these factors has helped to clarify the mechanisms of tumourigenesis and malignant formation in human cancers, and some of them have

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been applied to clinical diagnosis and treatment. For example, NCC-ST-439 and CA15-3 have been used as markers to diagnosis and evaluate the prognosis of human breast cancers (Gion et al., 1999; Narita et al., 1993b). In addition, anti c-erbB2 antibody (HerceptinÒ; Genentech, Inc., San Francisco, CA, USA) has been used for antibody therapy in human breast cancer patients with c-erbB2 (HER2/neu)-positive tumours (Ross and Fletcher, 1998). However, basic information on the expression of these factors in CMGTs has been limited. In this study, we characterised cultured canine mammary adenocarcinoma cells derived from primary and metastatic tumours by exhaustive step protein expression analysis to identify factors associated with the tumour progression or metastasis of CMGTs.

sialyl Lewis X [sLe(x)], E-cadherin, Rb, proliferating cell nuclear antigen (PCNA), glycogen synthase kinase-3b (GSK-3b), b-catenin and protein phosphatase 2A (PP2A)(BD Transduction Laboratories, Lexington, KY, USA), vimentin (Nichirei, Tokyo, Japan), cyclinD1, p53 and peroxisome proliferator-activated receptor-c (PPAR-c) (Santa Cruz Biotech, CA, USA), 14-3-3a, 14-3-3b, 14-3-3c, 14-3-3e, 14-3-3f, 14-3-3g, 14-3-3r and 14-3-3s (Immuno-Biochemical Laboratories, Gunma, Japan), c-erbB2 (DAKO, Glostrup, Denmark), prolactin receptor (Affinity BioReagents, Golden, CO, USA), NCC-ST-439 (Nihonkayaku, Tokyo, Japan), a-tubulin and b-actin (NeoMarker, Fremont, CA, USA) (internal controls). 2.3. Western blot analysis

2. Materials and methods 2.1. Cell cultures Cultured canine mammary gland adenocarcinoma cells from 6 tumours were used in this study. These cultured cells were derived from both primary and metastatic lesions of each of 3 patients and the letters CHM, CIP, and CNM indicate the same dogs, and the letters ‘‘p’’ and ‘‘m’’ indicate a primary or metastatic lesion. In the case of CHM, metastatic cells were collected from pleural effusion (see Table 1). The breed, age, source of cell cultures, method of collection, TNM classification and clinical staging (Owen, 1980) of these animals are shown in Table 1. Cultured cells were maintained in RPMI1640 medium supplemented with 10% foetal bovine serum, 5 mg/L gentamicin sulfate and 6 mg/L fungizone, and incubated at 37 °C in a humidified atmosphere of 5% CO2. Doubling times of CHMp/m, CIPp/m and CNMp/m cells were 24.7/23.1, 24.6/20.7 and 50.1/25.6 h, respectively. The cell passage numbers of CHMp/m, CIPp/m and CNMp/m cells were 52/52, 71/74 and 46/82, respectively. 2.2. Antibodies The primary antibodies used for the Western blot evaluation were obtained from the following sources:

Sub-confluent cultured cells were lysed in radio immuno precipitation assay (RIPA) buffer (10 mM Tris– HCl, 1% NP40, 0.1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 1 mM EDTA, 10 lg/ml aprotinin, 0.1 mM sodium molybdate, 2 mM sodium orthovanadate, 1 mM phenylmethansulfonyl fluoride, 10 mM sodium fluoride, 25 mM sodium b-glycerophosphate, 10 mM sodium pyrophosphoric acid and 1 mM EGTA). Protein concentrations were measured using the bicinchoninic acid (BCA) protein assay reagent (Pierce, Rockford, IL, USA). Cell lysates were boiled for 5 min in 2X SDS sample buffer and resolved by SDS–PAGE at about 10 lg per lane in a gel containing an appropriate concentration of acrylamide. The concentration was dependent on the weight of proteins (5%, 7.5%, 10% and 12%). Lysates of other species known to stain positive with each primary antibody were used as a positive control (data not shown). After electrophoresis, separated proteins were blotted onto a polyvinylidene difluoride (PVDF) membrane (Bio-rad, Hercules, CA, USA). The blots were then incubated for 16 h at 4 °C with 0.05% Tween 20 phosphate-buffered saline buffer (PBS-T) containing 5% non-fat milk. The blots were incubated with appropriate primary antibodies for 2 h at room temperature, and subsequently incubated with horseradish peroxidase (HRP) conjugated antibodies against mouse Ig or rabbit Ig (Amersham Bioscience, Piscataway, NJ, USA) for 1 h at room temperature with

Table 1 The dogs with spontaneous mammary adenocarcinomas from which the cells were derived Cells

Breed

Age (years)

Source of cells

Method of collection

TNM classificationa

Clinical stagea

CHMp CHMm

Mixed

12

Primary mass Pleural effusion

Surgical specimen Thoracocentesis

T4N1(+)M1

IV

CIPp CIPm

Shih Tzu

10

Primary mass Metastased regional lymph node

Surgical specimen Surgical specimen

T1cN1(+)M1

IV

CNMp CNMm

Maltese

11

Primary mass Metastased regional lymph node

Surgical specimen Surgical specimen

T1cN1(+)M0

II

a

TNM classification and clinical stage was defined by the WHO clinical stage classification (Owen, 1980).

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vigorous agitation. Signals were visualized with diaminobenzidine (DAB)/hydrogen peroxide solution (WAKO, Osaka, Japan). 2.4. Measurement of signal intensity To evaluate differences in the expression of these factors accurately, the signal intensity was measured by using an ATTO light capture system (AE-6962; ATTO, Tokyo, Japan). The expression levels of each factor were normalised with that of a-tubulin. 2.5. Immunohistochemistry The expression of sLe(x) on cultured canine mammary adenocarcinoma cultured cells was analyzed by immunohistochemistry. Each cell was cultured on LabTek II chamber slides (Nalge Nunc International, Rochester, NY, USA) to sub-confluent growth and fixed in 10% buffered formalin. Immunohistochemical procedures were performed using a DAKO ENVISION+ kit/HRP (DAKO). Briefly, endogenous peroxidase activity was abolished by treatment with 0.03% hydrogen peroxide containing sodium azide. For the staining of sLe(x), samples were reacted with the primary antibody against sLe(x) at room temperature for 2 h, and incubated with polymer solution containing HRP conjugated antibody against mouse Ig at room temperature for 30 min. Sections were visualised with DAB/hydrogen peroxide solution and counterstained with haematoxylin before observation.

3. Results

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Expression of cyclinD1, a positive regulator of the cell cycle in G1 phase, varied among the samples. In the CIP pair, CIPp showed higher expression than CIPm, while the opposite result was obtained in other pairs. Rb, a negative regulator of the cell cycle in G1 phase, showed strong expression in CNMm cells, while there was little expression in the CHMp and CIP pair (Fig. 1(b)). Expression of p53, a tumour suppressor factor associated with DNA repair, cell cycle arrest and apoptosis, was observed in all the cells. In the CHM pair, CHMm showed higher expression than CHMp, while in the CIP and CNM pairs the expression was similar between cells of primary and metastatic origin (Fig. 1(c)). Among 14-3-3 protein and their 7 isoforms, which amplify or suppress the signaling pathway, 14-3-3r exhibited various levels of expression in cultured adenocarcinoma cells. In the CIP pair, CIPp showed lower expression than CIPm, while in the CNM pair CNMm showed lower expression (Fig. 1(d)). Expression of c-erbB2, one of the membrane proteins and the target of antibody therapy in human breast cancers, was observed in all the cells, but its expression varied randomly without any consistent tendency. NCC-ST-439, a carbohydrate antigen used in diagnosis and evaluation of human breast cancers, was also observed in all the cells. CHMm and CNMm cells expressed this antigen more strongly than other cells (Fig. 1(e)). PCNA, GSK-3b, b-catenin, PP2A, 14-3-3 protein and the 6 other isoforms of 14-3-3 proteins (b, c, e, f, g and s), prolactin receptor and PPAR-c were detected in all the cells investigated with almost similar intensity.

3.1. Western blot analysis 3.2. Measurement of signal intensities Among proteins analyzed, the level of expression of sLe(x), E-cadherin, vimentin, cyclinD1, Rb, p53, 14-33r, c-erbB2 and NCC-ST-439 differed either among the cultured adenocarcinoma cell samples or between cells of primary and metastatic origin in an individual animal. Strong expression of sLe(x), which serves as a ligand of E-selectin, was only shown in CHMm cells, while in other cells this was not detected. CNMm showed lower expression of E-cadherin than CNMp, while CHMm showed higher expression of E-cadherin than CHMp. In CIPp and CIPm cells, E-cadherin expression was almost similar. Vimentin is known to be a constituent part of cytoskeletal fibres and a marker of mesenchymal cells. In the present study, vimentin was expressed in all the cell samples, but at different levels (Fig. 1(a)); CHMm and CIPp showed very slight expression, whereas strong signals were observed in CIPm and CNMp.

The calculated values of each signal are shown in Fig. 2. The differences in the calculated values were comparable to those evaluated visually. Differences in the calculated values ranged within 0.9–57.3% of a-tubulin intensities where no differences were observed visually. 3.3. Immunohistochemistry Because Western blot analysis revealed specific expression of the carbohydrate antigen sLe(x) on CHMm cells, we measured the expression of sLe(x) by immunohistochemistry to confirm this result and to investigate sLe(x) localization in these cells. In CHMm cells, the cell surface was strongly stained and the cytoplasm was also stained (Fig. 3), while there was no positive reaction in the other 5 cells.

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Fig. 1. Western blotting analysis of canine mammary adenocarcinoma cultured cells for 24 oncological factors. Lysates (10 lg) of CHMp, CHMm, CIPp, CIPm, CNMp and CNMm were applied sequentially from the left. Factors analyzed were categorized as follows: cell adhesion and structure molecules (a), cell cycle factors and growth regulators (b), apoptosis-related molecule (c), scatter folding factors (d) and receptors and glycoproteins (e).

1.0

epsilon

1.0

vimentin

1.0

zeta

1.0

cyclinD1

1.0

eta

1.0

Rb

1.0

sigma

1.0

PCNA

1.0

tau

1.0

GSK-3β

1.0

c-erbB2

1.0

β -catenin

1.0

NCC-ST-439

1.0

PP2A

1.0

prolactin R

1.0

p53

1.0

PPAR-γ

1.0

14-3-3 all

1.0

beta

CNMm

E-cadherin

CNMp

1.0

CIPm

14-3-3 gamma

CIPp

1.0

CHMm

sLe(x)

CHMp

1.0

% of α-tublin

CNMm

CNMp

CIPm

CIPp

CHMm

CHMp

Fig. 2. Signal intensities measured by an ATTO light capture system.

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Fig. 3. Immunohistochemical reactions of canine mammary adenocarcinoma cultured cells with anti-sLe(x) antibody. CHMm (b) cells exhibited positive staining for sLe(x), while CHMp (a), CIPp (c), CIPm (d), CNMp (e) and CNMm (f) exhibited negative reactions.

4. Discussion In this study, we evaluated the expression of various factors associated with differentiation and tumourigenesis in cultured canine mammary adenocarcinoma cells from 6 tumour samples. Among these factors, sLe(x) showed the most interesting expression – namely, only CHMm was positive for sLe(x). Furthermore, immunohistochemical analysis showed strong staining on the cell surface of CHMm cells, whereas no positive signal was observed in the other 5 cells, which supported the results of Western blotting analysis. sLe(x) is a carbohydrate ligand which adheres to Eselectin (Phillips et al., 1990). This ligand is expressed on granulocytes and monocytes, and has been implicated in their adhesion to vascular endothelial cells in the acute inflammation process (Berg et al., 1991; Lowe et al., 1990). sLe(x) is also reported to be expressed on human cancer cells and is thought to play important roles in haematogenous metastasis of the cancer, in which it mediates the initial adhesional step of tumour

cells to the distal vascular endothelial cells by sLe(x)E-selectin adhesion (Fukushi et al., 1985; Fukushima et al., 1984; Turner, 1982). The degree of expression of the ligand at the surface of cancer cells has been shown to be well correlated with the frequency of haematogenous metastasis and poor prognostic outcome of patients with cancers (Nakagoe et al., 1993, 1998; Renkonen et al., 1997; Sugiyama et al., 1992). We previously reported the expression of sLe(x) in canine and feline mammary gland tumour tissues, in which about 60% of spontaneous tumours were positively stained (Nakagawa et al., 2002). In addition, its expression was not detected histologically in normal mammary gland tissues bearing MGT (Nakagawa et al., 2002). These results suggested that the expression of this ligand might be related to tumourigenesis of CMGT. In this study, only CHMm cells with strong expression of sLe(x) were derived from the distant metastatic lesion (thoracic effusion). Other cells were derived from the primary lesion (CHMp, CIPp and CNMp) or metastatic lesion of the lymph node (CIPm and CNMm). This result

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suggested that the acquisition of sLe(x) expression may be an important factor in haematogenous metastasis in CMGT and further studies will be needed to clarify the role of sLe(x) in the metastasis of CMGT. The expressions of E-cadherin, cyclinD1, Rb, p53, 14-3-3r, c-erbB2 and NCC-ST-439 were found to differ among cells or between the cells of primary and metastatic origin in each pair. E-cadherin plays a crucial role in epithelial cell-cell adhesion and in maintenance of tissue architecture (Vleminckx et al., 1991). CyclinD1 and Rb are major regulators of the cell cycle in G1 phase (Sherr, 1996). The p53 protein regulates the expression of a wide variety of genes involved in cell cycle arrest and apoptosis in response to genotoxic or cellular stress (Sawan et al., 1992). 14-3-3r is one of the 14-3-3 protein isoforms and functions as a negative regulator of the cell cycle in G2/M phase (Dubois et al., 1997). Changes in these factors have been reported to be associated with tumour malignancy, invasiveness, metastasis, and poor prognosis in human breast cancers (Barbareschi et al., 1997; Jares et al., 1997; Urano et al., 2002) and, some of them in CMGTs (Restucci et al., 1997; Lee et al., 2004). Overexpression of C-erbB2, a transmembrane protein with intrinsic tyrosine kinase activity, is observed in human breast cancers and used as a target of antibody therapy (Slamon et al., 1987, 1989). NCCST-439 has high sensitivity as a tumour marker for breast cancers, and its serum level is thought to reflect tumour progression or metastasis (Narita et al., 1993b). Though differences in the expression of these factors were not consistent among cells or within the cells of primary or metastatic origin, however, in some of the cultured cell pairs, different expression within pairs was observed, suggesting the potential usefulness of these cells in analyzing these factors. Vimentin is known to be a constituent part of cytoskeletal fibres and a marker of mesenchymal cells. Half of CMGT tumours have been reported to consist of both epithelial and mesenchymal cells (Hellmen and Lindgren, 1989; Jones et al., 1997). Histologically they are diagnosed as complex or mixed tumours (Misdorp et al., 1999). In this study, we detected the signal of vimentin in all the cells with the various level of expression. The reason why it was detected in cultured adenocarcinoma cells was not clear and further investigation on the expression of both vimentin and cytokeratin, a marker of epithelial cells (Moll, 1994), may be needed to clarify the character and origin of these cells. The expression of PPAR-c which is an isotype of peroxisome proliferator-activated receptors (Issemann and Green, 1990), was observed in this study. This receptor indicates the level of differentiation of tumour cells (Kitamura et al., 1999; Mueller et al., 1998). Troglitazone, a selective ligand for PPAR-c, has antitumour activity in various cancer models and is thought to be effective in the treatment of cancers as an adjuvant ther-

apy (Keshamouni et al., 2004; Motomura et al., 2004; Yoshimura et al., 2003). Though the expression of this molecule in canine mammary adenocarcinoma cells varied, the detection of PPAR-c in these cells suggested the possibility of the application of PPAR-c target therapy for CMGTs. In this study, we measured the expression of various factors related to malignancy in cultured canine mammary adenocarcinoma cells, although differences in their expression were not consistent within the cells of primary or metastatic origin. However, the findings using the markers should provide useful information on the characteristics of cells derived from the same animal since they may have the same genomic background.

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