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Clusterin is associated with spontaneous breast cancer in TA2 mice
FEBS Letters 581 (2007) 3277–3282
Clusterin is associated with spontaneous breast cancer in TA2 mice Baocun Suna,b,c,*,1, Shiwu Zhanga,c,1, Danfang Zhanga,c,1, Yanqing Liua, Yan Lia, Zhe Ronga, Yue Zhua, Xinghong Jiad a
Department of Pathology, Tianjin Cancer Hospital, Tianjin Medical University, Tianjin 300060, PR China b Department of Pathology, Tianjin Medical University, Tianjin 300070, China c The Key Laboratory, Breast Cancer Prevention and Treatment, The Ministry of Education, PR China d The Experimental Animal Center, Tianjin Medical University, Tianjin 300070, China Received 17 May 2007; revised 28 May 2007; accepted 12 June 2007 Available online 21 June 2007 Edited by Lukas Huber
Abstract Two-dimensional electrophoresis and Matrix-assisted laser desorption ionization-time of flight-time of mass spectrometry were used to detect the differentially expressed proteins in serum of tientsin albinao 2 mice with spontaneous breast cancer, normal tientsin albinao 2 mice and tientsin albinao 1 mice. Only nuclear clusterin (n-CLU) was expressed in tientsin albinao 1. Immunohistochemistry and western blot validated that n-CLU was present in normal tientsin albinao 2 and tientsin albinao 1 mammary epithelium, and secretory clusterin expressed in the cytoplasm of normal tientsin albinao 2 mammary epithelium and spontaneous breast cancer. n-CLU may play an important role in tientsin albinao 2 spontaneous breast cancer initiation and development. Ó 2007 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
ing that F1 hybrids have the same histocompatibility genes. TA2 mice have a high incidence of spontaneous breast cancer without any chemical stimulus [6,7]. TA1 and TA2 mice have similar genetic backgrounds. In this study, the serum total protein extracts from TA1 mice, TA2 mice and TA2 mice that had spontaneous breast cancer were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with immobilized pH gradients followed by mass spectrometry to detect proteins that are absent or expressed in the serum of normal TA1 mice [8]. Immunohistochemical staining and Western blot were performed to validate the expression of proteins.
2. Materials and methods Keywords: Two-dimensional electrophoresis; Electrospray ion trap mass spectrometry; n-Clusterin; Spontaneous breast cancer
2.1. Mice There were four TA2 mice that had spontaneous breast cancer. Two of these mice have bred three times, and the others have bred four times. The average time it takes for is about 278 days. Four normal female TA1 mice coming from the same mother were selected, and so as the 4 normal female TA2 mice. The age of these particular TA1 and TA2 mice was almost the same, about 7–8 weeks.
1. Introduction With breast cancer morbidity increasing worldwide [1], breast cancer has become a significant threat to woman life. Many studies of breast cancer have focused on the levels of DNA and mRNA, but the function of a gene is actually fulfilled by the expression of its protein [2–4]. Mice are frequently used in genetic studies, as the genetic background of many particular strains has been established. After selection of many years, Tianjin Medical University has established two spontaneous breast cancer models: the TA1 strain has a low incidence of spontaneous breast cancer, while the TA2 strain has a high incidence [5]. Studies have indicated that skin transplantation is successful between F1 hybrids of TA1 and TA2, demonstrat* Corresponding author. Address: Department of Pathology, Tianjin Cancer Hospital, Tianjin Medical University, Tianjin 300060, PR China. Fax: +86 022 23537796. E-mail address: [email protected] (S. Zhang). 1
These authors contributed equally to this work.
Abbreviations: CLU, clusterin; s-CLU, secretory clusterin; n-CLU, nuclear clusterin; NLSs, nuclear localization signals; MALDI-TOFMS, Matrix-assisted laser desorption ionization-time of flight-time of mass spectrometry
2.2. Two-dimensional electrophoresis and mass spectrometric detection Blood was collected from the epicanthic venous plexus of the mice and the serum was separated from the other blood components. Two-dimensional electrophoresis with immobilized pH gradients was performed according to the methods of Gorg et al. [9] and the Manual of Operations for the IPGphor isoelectric focusing system. The serum sample (15 ll) was loaded into the sample cup, ensuring that the IPG strip was covered with 2–3 ml of mineral oil. Isoelectric focusing was conducted with the immobilized pH 3–10 non-linear gradient strips (Amersham Biosciences) at 0–500 V for an hour, 500 V for 5 h, 500– 3500 V for 5 h and 3500 V for 12 h. After IEF two SDS–PAGE gels (12%) were made to perform the second dimension. Electrophoresis was stopped when the dye front was approximately 1 mm from the bottom of the gel. After electrophoresis, the gel was removed from the gel cassette and stained [10]. 2.3. Analysis of the gel image ImageMaster 2D 5.0, Imagescanner and LabScan image collection software were used to capture, store, evaluate, and present information contained in the gel. All of the data were analyzed in SPSS10.0 and Excel. The spots absent in one sample and the spots which quantity increase or decrease three-fold were defined as the differential proteins. 2.4. Mass spectrometric detection The differentially expressed protein spots were excised and destained. Enough digestion buffer was added to just cover the gel slices and make them swell, and then the digestion buffer was removed and
0014-5793/$32.00 Ó 2007 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. doi:10.1016/j.febslet.2007.06.021
3278 replaced with 20 ll of 50 mM NH4HCO3. The digestion proceeded overnight at 37 °C. The peptides were extracted twice with 35 ll 0.1% trifluoroacetic acid (TFA) and 50% acetonitrile (ACN). All of the extracts were pooled and dried with nitrogen. The dry extracts were then re-dissolved in 3 ll of 0.1% TFA and 50% ACN. 0.3 ll samples and 0.3 ll CHCA solution were mined and spot on the plate. The mass spectrometer was operated under 18 kV accelerating voltage in the reflectron mode and a m/z range of 800–4000. Peak lists (S/N > 10) obtained from MALDI-TOF-MS were extracted from the raw data and were analyzed by DPS ExplorerTM software (Version 3.5, Applied Biosystems, USA). They were interpreted with MASCOT (Version 1.9.01, Matrix Science, Boston, MA, USA) and the proteins with over 95% CI were identified. The tandem mass spectra from the MS/MS experiments were searched against the non-redundant protein sequence database downloaded from NCBI (www.ncbi.nlm.nil. gov060915). Searches against the NCBI database revealed individual ion scores above 33 for 95% peptide identity significance level, and above 40 for the 95% level. The minimum criteria for protein identification were set to two unique peptides above the 99% level. If the protein was identified twice only the best peptide coverage was reported.
3. Immunohistochemistry The normal mammary gland tissue and the breast cancer tissue of the above 12 mice were fixed with formalin and then processed and embedded in paraffin. Five micrometer-thick paraffin-embedded tissue sections were cut and stained immunohistochemically (SP method). The primary antibody (mouse monoclonal antihuman Clusterin-a/b, clone: H-330:sc-8354, Santa Cruz Biotechnology) was diluted to 1:200. The sections were pretreated with citrate buffer (0.01 M citric acid, pH 6.0) for 20 min at 100 °C in a microwave oven. The slides were incubated overnight at 4 °C, washed with PBS, and incubated with the biotinylated secondary antibody and preformed avidin-biotinylated peroxidase complex by turns. The color was developed with DAB. Finally, all of the sections were counterstained with hematoxylin.
4. Western blot The normal breast tissue and breast cancer tissue were lysated with the buffer (1% SDS, 10 Mm Tris–Hcl, pH 7.6, 20 lg/ml aptotenin, 20 lg/ml leupeptin and 1 mM AEBSF). The protein concentrations were determined using Bradford method. Five micrograms of protein were separated on 12% of SDS–PAGE gels and transferred to PVDF membranes. After blocked with 10% non-fat milk, the membranes were
B. Sun et al. / FEBS Letters 581 (2007) 3277–3282
incubated with anti-clusterin polyclonal antibody (Clusterina/b, clone: H-330:sc-8354, Santa Cruz Biotechnology Inc., 1:200 dilution) at 4 °C overnight. After washing for three times, the membranes were incubated with goat anti-rabbit IgG at room temperature for 30 min. The signals were developed with the ECL kit (Beijing Zhongshan Biotechnology Inc.) and using anti b-actin antibody (sc-1616, Santa Cruz Biotechnology Inc.) as an internal control.
5. Results 5.1. Pathological examination The location of each spontaneous tumor in the mice is different: two of the four were located in the mammary gland near the forward limb, while the other two were located in the fourth mammary gland near the hind limb. Under the light microscope, the tumor cells formed many nests or tubules (Fig. 1A and B). The spontaneous breast cancer cells were negative for ER, PR, and Her-2. 5.2. Analysis of the 2D electrophoresis maps Serum protein from mice with spontaneous breast cancer, normal TA1 mice and TA2 mice was separated by IPG2DE-SDS–PAGE, with every sample tested three times. According to analysis by ImageMaster 2D 5.0, every sample had good reproducibility. The gel was silver-stained after 2D electrophoresis, and 2DE maps with good resolution and reproducibility were obtained. Silver staining was preferred over Coomassie blue staining because the resolution is higher with silver staining. Isoelectric focusing, SDS–PAGE and staining were performed identically for these six maps. The molecular mass of the proteins ranged between 10 and 100 kD, the isoelectric point was between 4 and 9, and the number of acidic points exceeded the number of basic points. Comparing the six maps, the main differentially expressed proteins were located between PI 4.5 and 6.5. Sorting of the protein spots from the three groups can be seen in Table 1. 5.3. Analysis of the differentially expressed proteins in mice with spontaneous breast cancer, normal TA1 and TA2 mice Analysis with ImageMaster 2D 5.0 software indicated that the main differentially expressed proteins were located in the range between PI 4.5 and 6.5 and had a molecular mass between 25 and 97 kD. A difference more than 10 times was
Fig. 1. A TA2 mouse with spontaneous breast cancer. (A) The tumor is in the second mammary gland of the forward limb. (B) H&E stain of a tumor section shows the tumor cells formed many nests or tubules.
B. Sun et al. / FEBS Letters 581 (2007) 3277–3282
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Table 1 Number of protein points matched across 2D gels for each mouse type TA1 TA2 TA2 with spontaneous breast cancer
TA1
TA2
TA2 with spontaneous breast cancer
305 (100%) 271 (81.14%) 295 (84.53%)
271 (88.85%) 334 (100%) 303 (86.82%)
298 (97.7%) 303 (90.72%) 349 (100%)
Fig. 2. 2D electrophoresis maps of serum protein from (A) normal TA2 mice, (B) normal TA1 mice, and (C) TA2 mice with spontaneous breast cancer.
Table 2 Proteins expressed differentially among the three groups of mice ID
TA2 mice with spontaneous breast cancer
Normal TA2 mice
Normal TA1 mice
Identified by mass spectrometry
0204 0205 2208 6614 2627 2207 6610 1101 1201 4101 5202
Expressed Expressed Expressed Expressed Downregulated Expressed Expressed Expressed Expressed Downregulated Expressed
Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Expressed Downregulated
Absent Absent Absent Absent Expressed Absent Downregulated Absent Absent Downregulated Absent
Haptoglobin ATP-binding protein Hypothetical protein MtrDRAFT_AC140721g10v1 Modification methylase HemK Clusterin precursor Paraoxonase Pregnancy zone protein Putative Transaldolase AGL175Wp SAP Peptide transporter-like protein
Fig. 3. Number: 2627 serum electrophoresis maps of (A) normal TA1 mice, (B) normal TA2 mice, and (C) TA2 mice with spontaneous breast cancer.
used as the cutoff to determine if a certain protein was differentially expressed. Differential protein points were searched and compared in the three groups (Fig. 2). Eleven differential protein points were found, and the expression of these 11 proteins can be seen in Table 2.
5.4. Mass spectrographic analysis of the differentially expressed proteins and database searches The differentially expressed proteins were identified by mass spectrometry on the basis of their peptide mass fingerprints (PMF) (Fig. 3A–C, Table 3). One differentially expressed
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Table 3 Differential proteins expressed among the three groups of mice Accession number
Protein name
MW (Da)
Protein PI
Sequence coverage (%)
Ion score
Ion score C.I.%
gij8248739 gij95108443 gij92870298 gij77965817 gij729152 gij1628624 gij34785996 gij52005360 gij44985692 gij297547 gij34393815
Haptoglobin ATP-binding protein Hypothetical protein MtrDRAFT_AC140721g10v1 Modification methylase HemK Clusterin precursor Paraoxonase Pregnancy zone protein Putative Transaldolase AGL175Wp SAP Peptide transporter-like protein
38749.51172 70630.64844 18858.36914 29695.49023 51655.71000 39540.32031 165775.8125 24397.60938 43013.06641 26230.26953 33643.53906
6.08 5.59 8.52 4.86 5.46 5.07 6.24 6.75 5.62 5.98 10.99
56 47 76 55 46 49 66 77 59 68 54
88.86000061 50.40000153 41.479999542 53.98999977 86.5552847 74.31999969 47.95999908 54.00999832 46.49000168 101.7900009 66.06999969
99.9997123 97.32645933 99.68657546 98.5432135 98.541879 99.99137276 99.567824 97.07588288 95.0347847 100 96.5428713
Fig. 4. Mass spectrometry on the basis of the peptide mass fingerprints validates that the molecular mass of protein No. 2627 is 51655.71 D and the protein was found to be n-CLU by searching the public database.
Fig. 5. n-CLU expression (A) n-CLU is highly expressed in the normal mammary gland of TA1 mice. Positive grains can be seen in breast ductal epithelial cell nuclei. (B) In TA2 mice positive grains are located in both the cytoplasm and nucleus of breast ductal epithelial cells. (C) TA2 spontaneous breast cancer tissue is n-CLU negative.
protein was identified by PMF and tandem mass spectrometry. Its molecular mass is 51655.71 (ID number: 2627.3033), and the protein was identified as the clusterin precursor (n-CLU) by searching the database (Fig. 4). Most of the other differentially expressed proteins are highly abundant serum proteins, and have no relation to tumorigenesis.
5.5. Immunohistochemical staining and Western blot results Clusterin-a/b (clone: H-330) can detect the secretory clusterin (s-CLU) and n-CLU. As seen in the immunohistochemical staining, positive grains can be seen in the nucleus of breast ductal epithelial cells, which implied n-CLU is expressed in the normal mammary gland of TA1 mice. In TA2 mice positive
B. Sun et al. / FEBS Letters 581 (2007) 3277–3282
Fig. 6. The results of western blot also indicates that clusterin in mammary epithelium of TA1 mice is 55 kDa isoform, both of 40 kDa and 55 kDa isoforms are detected in TA2 mice mammary gland and 40 kDa clusterin isoform is present in TA2 spontaneous breast cancer.
grains were located in both the cytoplasm and nucleus. There were no positive grains in the nucleolus of the tumor cells. However, the cytoplasm of tumor cells showed strongly expression for clusterin (Fig. 5A–C). Western blot analyzed clusterin proteins expressed in TA1 and TA2 normal breast tissue and TA2 spontaneous breast cancer. The results indicated that clusterin in mammary epithelium of TA1 mice was 55 kDa isoform , both of 40 kDa and 55 kDa isoforms were detected in TA2 mice mammary gland and the expression of 40 kDa clusterin isoform was present in TA2 spontaneous breast cancer (Fig. 6).
6. Discussion The mouse model is the best subject for many genetic studies, as its genetic background is well known. The TA1 and TA2 mouse strains have a low and a high morbidity of breast cancer, respectively [5]. After being inbred for 160 consecutive generations, TA1 and TA2 mice have highly consistent genotypes, and they can reproduce healthy offspring. The morbidity of spontaneous breast cancer in parous female mice is 84.1% within an average of 280 days after birth, and the morbidity of virginal mice is 41.4% within an average of 400 days after birth. The morbidity in TA1 mice is only 1% [6]. The difference in morbidity between TA1 and TA2 mice may be because there are some mutations in the chromosomal structure of the TA2 strain. The mutations of genes may be result in different proteins expression. In this study, we used two-dimensional electrophoresis and mass spectrometry to analyze the serum of mice with spontaneous breast cancer, normal TA1 mice and TA2 mice. Using TA1 mice as the control, we screened serum protein samples for proteins that are expressed or absent in the TA2 strain. Decreased expression of clusterin precursor protein can be seen in mice with spontaneous breast cancer and in normal TA2 mice, while it is expressed in the TA1 strain. Immunohistochemistry also indicated n-CLU, located in the nucleus, was expressed in TA1 mice. Clusterin, also known as apolipoprotein J (APOJ), testosterone-repressed prostate message 2 (TRPM2), sulfated glycoprotein 2 (SGP2), complement-associated protein SP-40, and complement lysis inhibitor (CLI), is a secreted heterodimeric disulfide-linked glycoprotein with a molecular mass of approximately 70 kD [11]. Clusterin maps to 8q21 and encodes a protein composed of 448 amino acids. Clusterin, first isolated
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from testicular tissue in 1983, is a prostaglandin synthetase inhibitor. It is expressed in virtually all tissues and is found in all human body fluids [12]. Clusterin is involved in numerous physiological processes important for carcinogenesis and tumor growth [13,14], including apoptotic cell death [6,15], cell cycle regulation, DNA repair [16], cell adhesion, tissue remodeling, lipid transport, membrane recycling, and immune system regulation [17]. Clusterin can also act as a molecular chaperone to eliminate the metaproteins produced when cells are under stress [18]. There are two known clusterin protein isoforms generated in human cells: the secreted form (s-CLU) and the nuclear form (n-clusterin) [19]. s-CLU expression is initiated by translation from the first AUG codon of the full-length clusterin mRNA. This precursor protein, with a molecular mass of about 60 kD, is directed to the endoplasmic reticulum and then it is transported toward the Golgi apparatus where it is heavily glycosylated and the a and b subunits that are linked by disulfide bonds are cleaved. Under physiological conditions, the mature 70 kD protein that is secreted appears as a smear at 37–40 kD on a reducing SDS–PAGE immunoblot. Nuclear clusterin (nCLU), with a molecular weight of about 55 kD, is synthesized from a second in-frame AUG codon using an alternatively spliced n-CLU mRNA. Unlike s-CLU, n-CLU does not undergo subunit cleavage, nor does this protein appear to be extensively glycosylated. Expression of n-CLU results in a pro-death signal that inhibits cell growth and survival, so it is also called a cell death protein [20]. Expression of n-CLU is induced by cytotoxic stress and it can lead to cell death [21]. n-CLU’s C-terminal coiled-coil domain can associate with the DNA double stranded break repair protein Ku70. This domain of n-CLU is the minimal region required for Ku70 binding and apoptosis. The binding of n-CLU and Ku70 can lead to the formation of Ku70/Ku80 dimers that can affect DNA non-homologous repair and eventually lead to genomic instability and cell death. In normal cells, the inactive of nuclear clusterin precursor (pnCLU) exists in the cytoplasm of normal cells and has two targets of nuclear localization signals (NLSs). Under damage, pnCLU can be decorated and form the 55 kDa nCLU. With the help of NLSs, nCLU transferred into the nucleus and induce the cell apoptosis [21]. It is indicated that clusterin is highly associated with cell apoptosis and survival [15,22]. The relationship between clusterin and the pathogenesis and progression of cancer is determined by the presence of the different isoforms, the intracellular location of the protein and the glycosylational modifications [23–26]. It is reported that clusterin takes part in the pathogenesis and progression of cancer only after glycosylation and cellular relocalization [27–29]. The immature precursor and n-CLU exist together in the nucleus of tumor cells, while the mature clusterin localizes in the cytoplasm. Unlike glycosylated mature clusterin which can promote the pathogenesis and progression of cancer [30], n-CLU may be proapoptotic [31,32] and is capable of eliminating abnormally replicating cells to avert tumor progression [33,34]. The antibody we used in this study to detect clusterin a/b is a polyclonal antibody. It can detect both cytoplasmic clusterin and n-CLU and can therefore distinguish the glycosylated mature clusterin from n-CLU depending on the location of the positive grains. Results from our study show that clusterin in breast epithelial cells in TA1 mice is located in the cell nucleus, suggesting that n-CLU is present in the breast epithe-
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lial cell nuclei where it plays a part in inhibition of malignant transformation. Acknowledgements: We thank Valerie Dunmire for her expert editorial assistance with this manuscript. This work was partially supported by Tianjin Key Science and Technology Committee Foundation of China (043115211-1).
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Clusterin is associated with spontaneous breast cancer in TA2 mice Baocun Suna,b,c,*,1, Shiwu Zhanga,c,1, Danfang Zhanga,c,1, Yanqing Liua, Yan Lia, Zhe Ronga, Yue Zhua, Xinghong Jiad a
Department of Pathology, Tianjin Cancer Hospital, Tianjin Medical University, Tianjin 300060, PR China b Department of Pathology, Tianjin Medical University, Tianjin 300070, China c The Key Laboratory, Breast Cancer Prevention and Treatment, The Ministry of Education, PR China d The Experimental Animal Center, Tianjin Medical University, Tianjin 300070, China Received 17 May 2007; revised 28 May 2007; accepted 12 June 2007 Available online 21 June 2007 Edited by Lukas Huber
Abstract Two-dimensional electrophoresis and Matrix-assisted laser desorption ionization-time of flight-time of mass spectrometry were used to detect the differentially expressed proteins in serum of tientsin albinao 2 mice with spontaneous breast cancer, normal tientsin albinao 2 mice and tientsin albinao 1 mice. Only nuclear clusterin (n-CLU) was expressed in tientsin albinao 1. Immunohistochemistry and western blot validated that n-CLU was present in normal tientsin albinao 2 and tientsin albinao 1 mammary epithelium, and secretory clusterin expressed in the cytoplasm of normal tientsin albinao 2 mammary epithelium and spontaneous breast cancer. n-CLU may play an important role in tientsin albinao 2 spontaneous breast cancer initiation and development. Ó 2007 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
ing that F1 hybrids have the same histocompatibility genes. TA2 mice have a high incidence of spontaneous breast cancer without any chemical stimulus [6,7]. TA1 and TA2 mice have similar genetic backgrounds. In this study, the serum total protein extracts from TA1 mice, TA2 mice and TA2 mice that had spontaneous breast cancer were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with immobilized pH gradients followed by mass spectrometry to detect proteins that are absent or expressed in the serum of normal TA1 mice [8]. Immunohistochemical staining and Western blot were performed to validate the expression of proteins.
2. Materials and methods Keywords: Two-dimensional electrophoresis; Electrospray ion trap mass spectrometry; n-Clusterin; Spontaneous breast cancer
2.1. Mice There were four TA2 mice that had spontaneous breast cancer. Two of these mice have bred three times, and the others have bred four times. The average time it takes for is about 278 days. Four normal female TA1 mice coming from the same mother were selected, and so as the 4 normal female TA2 mice. The age of these particular TA1 and TA2 mice was almost the same, about 7–8 weeks.
1. Introduction With breast cancer morbidity increasing worldwide [1], breast cancer has become a significant threat to woman life. Many studies of breast cancer have focused on the levels of DNA and mRNA, but the function of a gene is actually fulfilled by the expression of its protein [2–4]. Mice are frequently used in genetic studies, as the genetic background of many particular strains has been established. After selection of many years, Tianjin Medical University has established two spontaneous breast cancer models: the TA1 strain has a low incidence of spontaneous breast cancer, while the TA2 strain has a high incidence [5]. Studies have indicated that skin transplantation is successful between F1 hybrids of TA1 and TA2, demonstrat* Corresponding author. Address: Department of Pathology, Tianjin Cancer Hospital, Tianjin Medical University, Tianjin 300060, PR China. Fax: +86 022 23537796. E-mail address: [email protected] (S. Zhang). 1
These authors contributed equally to this work.
Abbreviations: CLU, clusterin; s-CLU, secretory clusterin; n-CLU, nuclear clusterin; NLSs, nuclear localization signals; MALDI-TOFMS, Matrix-assisted laser desorption ionization-time of flight-time of mass spectrometry
2.2. Two-dimensional electrophoresis and mass spectrometric detection Blood was collected from the epicanthic venous plexus of the mice and the serum was separated from the other blood components. Two-dimensional electrophoresis with immobilized pH gradients was performed according to the methods of Gorg et al. [9] and the Manual of Operations for the IPGphor isoelectric focusing system. The serum sample (15 ll) was loaded into the sample cup, ensuring that the IPG strip was covered with 2–3 ml of mineral oil. Isoelectric focusing was conducted with the immobilized pH 3–10 non-linear gradient strips (Amersham Biosciences) at 0–500 V for an hour, 500 V for 5 h, 500– 3500 V for 5 h and 3500 V for 12 h. After IEF two SDS–PAGE gels (12%) were made to perform the second dimension. Electrophoresis was stopped when the dye front was approximately 1 mm from the bottom of the gel. After electrophoresis, the gel was removed from the gel cassette and stained [10]. 2.3. Analysis of the gel image ImageMaster 2D 5.0, Imagescanner and LabScan image collection software were used to capture, store, evaluate, and present information contained in the gel. All of the data were analyzed in SPSS10.0 and Excel. The spots absent in one sample and the spots which quantity increase or decrease three-fold were defined as the differential proteins. 2.4. Mass spectrometric detection The differentially expressed protein spots were excised and destained. Enough digestion buffer was added to just cover the gel slices and make them swell, and then the digestion buffer was removed and
0014-5793/$32.00 Ó 2007 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. doi:10.1016/j.febslet.2007.06.021
3278 replaced with 20 ll of 50 mM NH4HCO3. The digestion proceeded overnight at 37 °C. The peptides were extracted twice with 35 ll 0.1% trifluoroacetic acid (TFA) and 50% acetonitrile (ACN). All of the extracts were pooled and dried with nitrogen. The dry extracts were then re-dissolved in 3 ll of 0.1% TFA and 50% ACN. 0.3 ll samples and 0.3 ll CHCA solution were mined and spot on the plate. The mass spectrometer was operated under 18 kV accelerating voltage in the reflectron mode and a m/z range of 800–4000. Peak lists (S/N > 10) obtained from MALDI-TOF-MS were extracted from the raw data and were analyzed by DPS ExplorerTM software (Version 3.5, Applied Biosystems, USA). They were interpreted with MASCOT (Version 1.9.01, Matrix Science, Boston, MA, USA) and the proteins with over 95% CI were identified. The tandem mass spectra from the MS/MS experiments were searched against the non-redundant protein sequence database downloaded from NCBI (www.ncbi.nlm.nil. gov060915). Searches against the NCBI database revealed individual ion scores above 33 for 95% peptide identity significance level, and above 40 for the 95% level. The minimum criteria for protein identification were set to two unique peptides above the 99% level. If the protein was identified twice only the best peptide coverage was reported.
3. Immunohistochemistry The normal mammary gland tissue and the breast cancer tissue of the above 12 mice were fixed with formalin and then processed and embedded in paraffin. Five micrometer-thick paraffin-embedded tissue sections were cut and stained immunohistochemically (SP method). The primary antibody (mouse monoclonal antihuman Clusterin-a/b, clone: H-330:sc-8354, Santa Cruz Biotechnology) was diluted to 1:200. The sections were pretreated with citrate buffer (0.01 M citric acid, pH 6.0) for 20 min at 100 °C in a microwave oven. The slides were incubated overnight at 4 °C, washed with PBS, and incubated with the biotinylated secondary antibody and preformed avidin-biotinylated peroxidase complex by turns. The color was developed with DAB. Finally, all of the sections were counterstained with hematoxylin.
4. Western blot The normal breast tissue and breast cancer tissue were lysated with the buffer (1% SDS, 10 Mm Tris–Hcl, pH 7.6, 20 lg/ml aptotenin, 20 lg/ml leupeptin and 1 mM AEBSF). The protein concentrations were determined using Bradford method. Five micrograms of protein were separated on 12% of SDS–PAGE gels and transferred to PVDF membranes. After blocked with 10% non-fat milk, the membranes were
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incubated with anti-clusterin polyclonal antibody (Clusterina/b, clone: H-330:sc-8354, Santa Cruz Biotechnology Inc., 1:200 dilution) at 4 °C overnight. After washing for three times, the membranes were incubated with goat anti-rabbit IgG at room temperature for 30 min. The signals were developed with the ECL kit (Beijing Zhongshan Biotechnology Inc.) and using anti b-actin antibody (sc-1616, Santa Cruz Biotechnology Inc.) as an internal control.
5. Results 5.1. Pathological examination The location of each spontaneous tumor in the mice is different: two of the four were located in the mammary gland near the forward limb, while the other two were located in the fourth mammary gland near the hind limb. Under the light microscope, the tumor cells formed many nests or tubules (Fig. 1A and B). The spontaneous breast cancer cells were negative for ER, PR, and Her-2. 5.2. Analysis of the 2D electrophoresis maps Serum protein from mice with spontaneous breast cancer, normal TA1 mice and TA2 mice was separated by IPG2DE-SDS–PAGE, with every sample tested three times. According to analysis by ImageMaster 2D 5.0, every sample had good reproducibility. The gel was silver-stained after 2D electrophoresis, and 2DE maps with good resolution and reproducibility were obtained. Silver staining was preferred over Coomassie blue staining because the resolution is higher with silver staining. Isoelectric focusing, SDS–PAGE and staining were performed identically for these six maps. The molecular mass of the proteins ranged between 10 and 100 kD, the isoelectric point was between 4 and 9, and the number of acidic points exceeded the number of basic points. Comparing the six maps, the main differentially expressed proteins were located between PI 4.5 and 6.5. Sorting of the protein spots from the three groups can be seen in Table 1. 5.3. Analysis of the differentially expressed proteins in mice with spontaneous breast cancer, normal TA1 and TA2 mice Analysis with ImageMaster 2D 5.0 software indicated that the main differentially expressed proteins were located in the range between PI 4.5 and 6.5 and had a molecular mass between 25 and 97 kD. A difference more than 10 times was
Fig. 1. A TA2 mouse with spontaneous breast cancer. (A) The tumor is in the second mammary gland of the forward limb. (B) H&E stain of a tumor section shows the tumor cells formed many nests or tubules.
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Table 1 Number of protein points matched across 2D gels for each mouse type TA1 TA2 TA2 with spontaneous breast cancer
TA1
TA2
TA2 with spontaneous breast cancer
305 (100%) 271 (81.14%) 295 (84.53%)
271 (88.85%) 334 (100%) 303 (86.82%)
298 (97.7%) 303 (90.72%) 349 (100%)
Fig. 2. 2D electrophoresis maps of serum protein from (A) normal TA2 mice, (B) normal TA1 mice, and (C) TA2 mice with spontaneous breast cancer.
Table 2 Proteins expressed differentially among the three groups of mice ID
TA2 mice with spontaneous breast cancer
Normal TA2 mice
Normal TA1 mice
Identified by mass spectrometry
0204 0205 2208 6614 2627 2207 6610 1101 1201 4101 5202
Expressed Expressed Expressed Expressed Downregulated Expressed Expressed Expressed Expressed Downregulated Expressed
Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Downregulated Expressed Downregulated
Absent Absent Absent Absent Expressed Absent Downregulated Absent Absent Downregulated Absent
Haptoglobin ATP-binding protein Hypothetical protein MtrDRAFT_AC140721g10v1 Modification methylase HemK Clusterin precursor Paraoxonase Pregnancy zone protein Putative Transaldolase AGL175Wp SAP Peptide transporter-like protein
Fig. 3. Number: 2627 serum electrophoresis maps of (A) normal TA1 mice, (B) normal TA2 mice, and (C) TA2 mice with spontaneous breast cancer.
used as the cutoff to determine if a certain protein was differentially expressed. Differential protein points were searched and compared in the three groups (Fig. 2). Eleven differential protein points were found, and the expression of these 11 proteins can be seen in Table 2.
5.4. Mass spectrographic analysis of the differentially expressed proteins and database searches The differentially expressed proteins were identified by mass spectrometry on the basis of their peptide mass fingerprints (PMF) (Fig. 3A–C, Table 3). One differentially expressed
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Table 3 Differential proteins expressed among the three groups of mice Accession number
Protein name
MW (Da)
Protein PI
Sequence coverage (%)
Ion score
Ion score C.I.%
gij8248739 gij95108443 gij92870298 gij77965817 gij729152 gij1628624 gij34785996 gij52005360 gij44985692 gij297547 gij34393815
Haptoglobin ATP-binding protein Hypothetical protein MtrDRAFT_AC140721g10v1 Modification methylase HemK Clusterin precursor Paraoxonase Pregnancy zone protein Putative Transaldolase AGL175Wp SAP Peptide transporter-like protein
38749.51172 70630.64844 18858.36914 29695.49023 51655.71000 39540.32031 165775.8125 24397.60938 43013.06641 26230.26953 33643.53906
6.08 5.59 8.52 4.86 5.46 5.07 6.24 6.75 5.62 5.98 10.99
56 47 76 55 46 49 66 77 59 68 54
88.86000061 50.40000153 41.479999542 53.98999977 86.5552847 74.31999969 47.95999908 54.00999832 46.49000168 101.7900009 66.06999969
99.9997123 97.32645933 99.68657546 98.5432135 98.541879 99.99137276 99.567824 97.07588288 95.0347847 100 96.5428713
Fig. 4. Mass spectrometry on the basis of the peptide mass fingerprints validates that the molecular mass of protein No. 2627 is 51655.71 D and the protein was found to be n-CLU by searching the public database.
Fig. 5. n-CLU expression (A) n-CLU is highly expressed in the normal mammary gland of TA1 mice. Positive grains can be seen in breast ductal epithelial cell nuclei. (B) In TA2 mice positive grains are located in both the cytoplasm and nucleus of breast ductal epithelial cells. (C) TA2 spontaneous breast cancer tissue is n-CLU negative.
protein was identified by PMF and tandem mass spectrometry. Its molecular mass is 51655.71 (ID number: 2627.3033), and the protein was identified as the clusterin precursor (n-CLU) by searching the database (Fig. 4). Most of the other differentially expressed proteins are highly abundant serum proteins, and have no relation to tumorigenesis.
5.5. Immunohistochemical staining and Western blot results Clusterin-a/b (clone: H-330) can detect the secretory clusterin (s-CLU) and n-CLU. As seen in the immunohistochemical staining, positive grains can be seen in the nucleus of breast ductal epithelial cells, which implied n-CLU is expressed in the normal mammary gland of TA1 mice. In TA2 mice positive
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Fig. 6. The results of western blot also indicates that clusterin in mammary epithelium of TA1 mice is 55 kDa isoform, both of 40 kDa and 55 kDa isoforms are detected in TA2 mice mammary gland and 40 kDa clusterin isoform is present in TA2 spontaneous breast cancer.
grains were located in both the cytoplasm and nucleus. There were no positive grains in the nucleolus of the tumor cells. However, the cytoplasm of tumor cells showed strongly expression for clusterin (Fig. 5A–C). Western blot analyzed clusterin proteins expressed in TA1 and TA2 normal breast tissue and TA2 spontaneous breast cancer. The results indicated that clusterin in mammary epithelium of TA1 mice was 55 kDa isoform , both of 40 kDa and 55 kDa isoforms were detected in TA2 mice mammary gland and the expression of 40 kDa clusterin isoform was present in TA2 spontaneous breast cancer (Fig. 6).
6. Discussion The mouse model is the best subject for many genetic studies, as its genetic background is well known. The TA1 and TA2 mouse strains have a low and a high morbidity of breast cancer, respectively [5]. After being inbred for 160 consecutive generations, TA1 and TA2 mice have highly consistent genotypes, and they can reproduce healthy offspring. The morbidity of spontaneous breast cancer in parous female mice is 84.1% within an average of 280 days after birth, and the morbidity of virginal mice is 41.4% within an average of 400 days after birth. The morbidity in TA1 mice is only 1% [6]. The difference in morbidity between TA1 and TA2 mice may be because there are some mutations in the chromosomal structure of the TA2 strain. The mutations of genes may be result in different proteins expression. In this study, we used two-dimensional electrophoresis and mass spectrometry to analyze the serum of mice with spontaneous breast cancer, normal TA1 mice and TA2 mice. Using TA1 mice as the control, we screened serum protein samples for proteins that are expressed or absent in the TA2 strain. Decreased expression of clusterin precursor protein can be seen in mice with spontaneous breast cancer and in normal TA2 mice, while it is expressed in the TA1 strain. Immunohistochemistry also indicated n-CLU, located in the nucleus, was expressed in TA1 mice. Clusterin, also known as apolipoprotein J (APOJ), testosterone-repressed prostate message 2 (TRPM2), sulfated glycoprotein 2 (SGP2), complement-associated protein SP-40, and complement lysis inhibitor (CLI), is a secreted heterodimeric disulfide-linked glycoprotein with a molecular mass of approximately 70 kD [11]. Clusterin maps to 8q21 and encodes a protein composed of 448 amino acids. Clusterin, first isolated
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from testicular tissue in 1983, is a prostaglandin synthetase inhibitor. It is expressed in virtually all tissues and is found in all human body fluids [12]. Clusterin is involved in numerous physiological processes important for carcinogenesis and tumor growth [13,14], including apoptotic cell death [6,15], cell cycle regulation, DNA repair [16], cell adhesion, tissue remodeling, lipid transport, membrane recycling, and immune system regulation [17]. Clusterin can also act as a molecular chaperone to eliminate the metaproteins produced when cells are under stress [18]. There are two known clusterin protein isoforms generated in human cells: the secreted form (s-CLU) and the nuclear form (n-clusterin) [19]. s-CLU expression is initiated by translation from the first AUG codon of the full-length clusterin mRNA. This precursor protein, with a molecular mass of about 60 kD, is directed to the endoplasmic reticulum and then it is transported toward the Golgi apparatus where it is heavily glycosylated and the a and b subunits that are linked by disulfide bonds are cleaved. Under physiological conditions, the mature 70 kD protein that is secreted appears as a smear at 37–40 kD on a reducing SDS–PAGE immunoblot. Nuclear clusterin (nCLU), with a molecular weight of about 55 kD, is synthesized from a second in-frame AUG codon using an alternatively spliced n-CLU mRNA. Unlike s-CLU, n-CLU does not undergo subunit cleavage, nor does this protein appear to be extensively glycosylated. Expression of n-CLU results in a pro-death signal that inhibits cell growth and survival, so it is also called a cell death protein [20]. Expression of n-CLU is induced by cytotoxic stress and it can lead to cell death [21]. n-CLU’s C-terminal coiled-coil domain can associate with the DNA double stranded break repair protein Ku70. This domain of n-CLU is the minimal region required for Ku70 binding and apoptosis. The binding of n-CLU and Ku70 can lead to the formation of Ku70/Ku80 dimers that can affect DNA non-homologous repair and eventually lead to genomic instability and cell death. In normal cells, the inactive of nuclear clusterin precursor (pnCLU) exists in the cytoplasm of normal cells and has two targets of nuclear localization signals (NLSs). Under damage, pnCLU can be decorated and form the 55 kDa nCLU. With the help of NLSs, nCLU transferred into the nucleus and induce the cell apoptosis [21]. It is indicated that clusterin is highly associated with cell apoptosis and survival [15,22]. The relationship between clusterin and the pathogenesis and progression of cancer is determined by the presence of the different isoforms, the intracellular location of the protein and the glycosylational modifications [23–26]. It is reported that clusterin takes part in the pathogenesis and progression of cancer only after glycosylation and cellular relocalization [27–29]. The immature precursor and n-CLU exist together in the nucleus of tumor cells, while the mature clusterin localizes in the cytoplasm. Unlike glycosylated mature clusterin which can promote the pathogenesis and progression of cancer [30], n-CLU may be proapoptotic [31,32] and is capable of eliminating abnormally replicating cells to avert tumor progression [33,34]. The antibody we used in this study to detect clusterin a/b is a polyclonal antibody. It can detect both cytoplasmic clusterin and n-CLU and can therefore distinguish the glycosylated mature clusterin from n-CLU depending on the location of the positive grains. Results from our study show that clusterin in breast epithelial cells in TA1 mice is located in the cell nucleus, suggesting that n-CLU is present in the breast epithe-
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lial cell nuclei where it plays a part in inhibition of malignant transformation. Acknowledgements: We thank Valerie Dunmire for her expert editorial assistance with this manuscript. This work was partially supported by Tianjin Key Science and Technology Committee Foundation of China (043115211-1).
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