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Proteomics-based analysis of a pair of glioma cell lines with different tumor forming characteristics
Neuroscience Letters 401 (2006) 59–64
Proteomics-based analysis of a pair of glioma cell lines with different tumor forming characteristics Le Zhou a,b , Yan Wang a , Ying-tao Zhang a , Yi-ping Geng a , Lu-sheng Si a , Yi-li Wang a,∗ a
Institute for Cancer Research, The Key Laboratory of Biomedical Information Engineering of Chinese Education Ministry, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710061, China b Department of Neurosurgery, The Second Hospital of Xi’an Jiaotong University, Xi’an 710004, China Received 18 September 2005; received in revised form 1 January 2006; accepted 27 February 2006
Abstract Glioma is the most common malignant disease in the brain, and recurrence is the main cause of death from this disease. Tumor recurrence involves multiple steps, and requires the accumulation of the altered expression of many different proteins. Identification of the recurrence associated protein profile in glioma cell lines will be helpful in clarifying the molecular mechanisms underlying glioma recurrence. In this report, two glioma cell lines with distinct tumor forming ability in vitro and in vivo were chosen and the different protein expression patterns were analyzed by proteomics method. To confirm the utility of this method, we validated the differential expression of one protein, cathepsin D, by immunohistochemistry analysis. Forty-six proteins appeared differently between two cell lines and 18 of them were identified. These 18 are involved in cell proliferation, DNA replication, protein synthesis, invasion, angiogenesis and neurotrophic factor. All of these molecules are important in tumor growth, and a subset of them may be related to glioma recurrence. These findings may contribute to the discovery of new diagnostic markers and therapeutic targets of glioma. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Glioma; Recurrence; Proteomics
Gliomas are the most common primary brain tumors in adults [33]. Despite the tremendous advances in neuroimaging, neurosurgery, radiation and medical oncology, a complete surgical resection of infiltrating tumor cells in the brain parenchyma surrounding the main tumor mass is never achieved at the time of surgery and more than 90% of the tumors will recur within 2 cm of the primary tumor location. This makes the prognosis of the disease very poor [1,25]. Tumor recurrence involves multiple steps, and requires the accumulation of the altered expression of many different proteins. Recent studies on the tumorigenesis indicate that only a small subpopulation of tumor cells with stem cell character is capable of initiating tumor formation and maintaining the nonstop growth [27]. Independent analysis of any single protein would be insufficient to understand all of the aspects of glioma recurrence. Systematical analysis of the tumor cells with differ-
∗
Corresponding author. Tel.: +86 29 82655429 808; fax: +86 29 82655499. E-mail address: [email protected] (Y.-l. Wang).
0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.02.080
ent biological behaviors may be helpful to elucidate the nature of the tumor recurrence. Currently, the application of the proteomic approach has made it possible to characterize global alterations in cell protein expression. This promises new insight into the cellular mechanisms involved in tumor recurrence and is likely to result in the discovery of novel diagnostic markers and new therapeutic opportunities. In the present study, proteomics approach was used to analyze of a pair of human glioma cell lines (U251 and A172) which are widely used in laboratory. The biological behaviors of the two cell lines have been extensively investigated. More importantly the two cell lines are demonstrated different tumor forming potentiality. Cell line U251 can form subcutaneous tumor mass in nude mice but A172 cannot [2,13,17]. It is considered that the highly invasive nature of the tumor cell are associated with the frequent recurrence, and the ability of colony forming in vitro and tumor forming in nude mice reflects the malignant potentiality to certain extent. Although the two cell lines are derived from different patients, the distinct growth patterns make it a meaningful model for investigation of glioma forming. Com-
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L. Zhou et al. / Neuroscience Letters 401 (2006) 59–64
parative study on the protein expression profile may disclose the differential functional proteins and provide useful information. By two-Dimensional gel Electrophoresis (2-DE) and MALDI-TOF MS analysis, several differentially expressed proteins were identified, and the utility of this proteomics method was reconfirmed by immunocytochemistry. A subset of these differentially expressed proteins may be relevant to glioma recurrence. U251 and A172 cells were purchased from Cell Bank of Chinese Academic of Science (Shanghai, China). The cells were cultured in DMEM (Gibco BRL, USA) supplemented with 15% fetal calf serum at 37 ◦ C in an atmosphere of 5% CO2 in air. To confirm the colony forming ability of the two cell lines, anchorage independent growth in soft agar was performed. A certain number of tumor cells were seeded onto per dish and cultured at 37 ◦ C in an atmosphere of 5% CO2 in air for 3 weeks. The numbers of colonies that contain more than 50 cells were counted. Statistical analysis of the significance of differences between the two cell lines was determined by Student’s t-test, and p-values less than 0.05 were considered significant. Sample preparation for 2-DE was performed as follows. The cells were harvested by trypsinization at the exponential growth phase. After washing in Hanks’ solution and icecold phosphatebuffered saline, the cells were counted and centrifuged in 2.0 ml microtubes. The cell pellets were dissolved in lysis buffer (9 M urea, 4% CHAPS, 40 mM Tris-base, 40 mM DTT) and centrifuged at 40,000 × g for 1 h at 4 ◦ C. Protein concentration was determined by the Bradford protein assay method. All samples were stored in aliquots at −80 ◦ C before analyzing. The first dimensional isoelectric focusing (IEF) was carried out on an IPGphor system (Amersham Biosciences, Sweden)
using 13 cm strips and 80 g protein in 250 l sample solution. Focusing was started at 200 V and the voltage was gradually increased to 8000 V, and then focusing at 8000 V until total volt-hours reaching 26000 V h at 20 ◦ C. After IEF, the focused strips were then equilibrated in buffer I (6 M urea, 30% glycerol, 2% SDS, 1% DTT) and then buffer II (DTT was replaced with 2.5% IAA) each for 15 min with gentle shaking. The second dimensional separation was carried out on 12.5% SDS-polyacrylamide gels using Hoefer SE 600 vertical chambers. After 2-DE, the gels were stained with silver nitrate. The stained gels were captured by transmission scanning and were analyzed with Image Master5.0. Following analysis, selected protein spots were manually excised from gels, destained, and dried. Proteins were then digested with TPCKtrypsin (Sigma, USA) overnight at 37 ◦ C. The peptides were extracted and peptide mass finger printings (PMF) were generated by MALDI-TOF MS. PMFs were matched to the theoretical tryptic digests of proteins in database NCBInr by Mascot software (http://www.matrixscience.com). The selected taxonomy category was Homo sapien (human). Max Missed Cut limited in 0-1. Peptide mass tolerance was settled at 100 ppm. Modifications were allowed for carbamidomethyl of cysteine and oxidation of methionine. To confirm the validity of this proteomics method, we selected one of identified proteins (spot 25, which was identified as cathepsin D and expression level is 2.46-fold higher in U251 than in A172) and validated the differential expression using immunohistochemistry analysis. Antibody for cathepsin D (1:100 dilution) was obtained from Chemicon, USA. Immunohistochemistry was performed according to standard techniques [22].
Fig. 1. The 2-DE gel images of A172 (A) and U251 (B) cells. The Arabic numerals show the differential expressed proteins.
L. Zhou et al. / Neuroscience Letters 401 (2006) 59–64
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Fig. 2. The PMF of spot 25 obtained by MALDI-TOF MS after trypsin digestion. The protein was identified as gi|30582659, cathepsin D, at 90 score. (Protein scores greater than 64 are significant (p < 0.05)).
The results of anchorage independent growth in soft agar showed that U251 cells formed 202 ± 15 colonies per dish, whereas A172 cells formed 104 ± 15 colonies per dish. Statistical analysis showed there was a significant difference between two cell lines in colony formation ability. This result was consistent with the character of tumor formation in nude mice of the two cell lines. The 2-DE gels results of the protein profile expressed by U251 and A172 cells were presented in Fig. 1. Good reproducibility and resolution were achieved on silver stained gels for each cell line. Following comparison with software Image Master5.0, 46 protein spots, which were marked as numbers in the Fig. 1, were found more than two-fold changes in amount. Among them, 18 protein spots (number 20–37) increased while nineteen protein spots (number 1–19) decreased in U251 cells; five protein spots (number 42–46) were detected only in U251; and four protein spots (number 38–41) were exclusive in A172. The PMF spectrums of the proteins were obtained by MALDI-TOF-MS analysis following in-gel digestion. Fig. 2 is a MALDI-TOF mass spectrum of peptides derived from protein spot 25. The protein was identified as cathepsin D by PMF searching in NCBInr database. Cathepsin D was observed in
both cell lines by immunohistochemistry stain. It expressed almost in all U251 cells, but only in part of A172 cells (Fig. 3). This is consistent with the result of 2-DE and MALDI-TOF MS analysis. It demonstrates that proteomics analysis is a reliable method for this research. A summary of the identification data is show in Table 1. We compared the protein profiles of two human glioma cell lines that possess different tumor forming potential by proteomics technologies. The results indicate that the tumor forming potential of tumor cells may be regulated by the different expression of several specific proteins. These proteins involved in cell proliferation, DNA replication, protein synthesis, invasion, angiogenesis, and neurotrophic factor. It is interesting that the proteins up-regulated in U251 cells usually have positive relationship to tumor proliferation. In contrast, the proteins with higher expression levels in A172 cells usually have a negative relationship to tumor proliferation. Transketolase, a thiamine-dependent enzyme, links the pentose phosphate pathway with the glycolytic pathway and is an important enzyme in nucleotide metabolism. This protein plays a crucial role in tumor cell nucleic acid synthesis [8]. Moreover, over expression of transketolase was detected in most nervous
Fig. 3. Cell A172 (A) and U251 (B) immuno-labeled for Cathepsin D (×200).
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Table 1 The identified protein spots in cell line A172 and U251 No.
NCBI no.
Peptides matched
Theoretical MW/pI
Sequence coverage (%)
Protein name
1 4 5 7 11 12 13 18 20 21 23 25 26 30 34 35 36 40
15029910 32891807 31543380 4502599 505104 4505773 21411003 1731411 14141166 51895760 2228737 30582659 34147630 6983940 37267 3392940 21739410 50949567
5/26 9/28 6/14 5/16 6/19 9/29 9/20 9/32 5/16 4/11 5/23 6/15 9/40 5/17 10/48 4/11 7/19 8/29
26.34/4.3 22.22/7.1 20.05/6.3 30.64/8.6 45.30/6.2 29.86/5.6 36.71/9.2 55.47/9.0 38.60/6.3 18.23/7.7 53.28/8.9 45.04/6.1 50.19/7.3 44.90/9.2 68.44/7.9 55.38/7.2 80.38/5.2 21.12/9.0
17 71 32 25 17 55 25 21 16 35 26 14 22 22 27 10 17 21
PR48 proteina Biliverdin reductase Ba DJ-1 proteina Carbonyl reductase 1a KIAA0071a Prohibitina Poly(ADP-ribose) polymerase (PARP)a Zinc finger protein 135a Poly(rc)-binding protein 2 (PCBP2)b Peptidylprolyl isomerase A (cyclophilin A)b Glial cell line-derived neurotrophic factor receptor (Grf) Cathepsin Db Tu translation elongation factor, mitochondrial (TUFM)b p47-phoxb Transketolaseb AIREb Hypothetical proteinb Hypothetical proteinc
a b c
Expressed higher in A172. Expressed higher in U251. Expressed only in A172.
system tumors as compared with cerebral cortex [7]. PR48 is the regulatory subunit of protein phosphatase 2A (PP2A), one of initiators of DNA replication in eukaryotes. Over-expression of PR48 in human cells causes a G1 arrest [31]. Considering that DNA replication is the basis of cell proliferation, higher expression of transketolase and low expression of PR48 may enhance U251 cell proliferation by promoting DNA replication. Prohibitin has been shown to be a potential tumor suppressor protein, which can bind to the Rb protein as well as E2F. This binding is necessary to suppress cell proliferation [19]. Recently, a notable under-expression of prohibitin was observed in Grade III glioma compared with control tissues [6]. Thus, decreased expression of prohibitin may be the other reason for promoting U251 cell proliferation. Poly(ADP-ribose) polymerase (PARP), expressed higher in U251, can catalyze the transfer of multiple ADP-ribose units from NAD to substrate proteins [23], and plays a role in DNA repair and the recovery of cells from DNA damage [14]. Inhibition of PARP may increase the efficacy of temozolomide (a DNA-methylating agent used in the treatment of gliomas) in the treatment of gliomas, particularly in tumors deficient in DNA mismatch repair [4]. These findings suggest that higher expression of PARP is important for U251 growth. Wild type AIRE protein acted as a strong transcriptional activator in mammalian cells [3]. KIAA0071 can bind to the RE1 silencing transcription factor in vitro and vivo and exhibits transcriptional corepressor activity [16]. Zinc finger protein (ZFP) is a large class of transcriptional regulators. Engineered ZFP transcription factors are shown to be potent regulators of gene expression with therapeutic promise in the treatment of glioma [29]. Poly(rc)-binding protein 2 (PCBP2) binds the major stemloop structure (stem-loop IV) in the internal ribosome entry site and is essential for translation initiation. At the same time, PCBP2 is able to stimulate the activity of the proto-oncogene
c-myc [9]. TUFM is highly conserved throughout evolution. It can deliver amino-acylated tRNAs to the ribosome during the elongation step of translation. Absence of the TUFM is lethal in S. pombe [5]. AIRE, PCBP2 and TUFM, which can promote protein synthesis, are increasingly expressed in U251, but expression level of KIAA0071 and ZFP135 which can inhibit protein synthesis are down-regulated. Predominance in protein synthesis may be one of reasons for U251 tumor forming. Invasion is an important characteristic in glioma. Cathepsin D and Carbonyl reductase (CBR) are both involved in tumor cell invasion. Cathepsin D, a lysosomal aspartyl endoproteinase, takes part in degradation of the extracellular matrix and is strongly expressed by invading glioblastoma cells at the infiltrative margin. Several reports have indicated that Cathepsin D can stimulate cancer cell proliferation and increase metastatic potential in vivo [21]. Antibody to Cathepsin D was shown to inhibit the invasion of U251 cell line in a dose-dependent manner [28]. Fukuda et al. even took cathepsin D as a potential serum marker for the prediction of aggressive nature of human gliomas [12]. CBR can catalyze prostaglandins which have been associated with more aggressive tumor invasion and metastases. Therefore, modulation of prostaglandin levels by CBR was suggested as a possible mechanism of restraining the ability of cancer cell invasion [11]. Higher level of cathepsin D and lower level of CBR in U251 imply that invasion associated molecules may play an important role in tumor forming. The process of angiogenesis plays a key role in development and progression of solid tumors. Cyclophilin A (CypA) is a modulator of endothelial cell functions in vascular disease. It can increase endothelial cell proliferation, migration, invasive capacity, and tubulogenesis at low concentrations [20]. Yang et al. demonstrates that CypA has a mitogenic effect on human aorta smooth muscle cells and human lung microvascular endothelial cells [32]. The expression of CypA will increase
L. Zhou et al. / Neuroscience Letters 401 (2006) 59–64
with the help of reactive oxygen species (ROS) [18]. Protein P47-phox can improve ROS generation. Interestingly, CypA and P47-phox are both over-expressed in the U251 cell line, which might make U251 cells possess higher angiogenesis ability in tumor formation. Glial cell line-derived neurotrophic factor receptor (Grf) is a receptor of Glial cell line-derived neurotrophic factor (GDNF). Immunohistochemical analysis revealed that Grf expression levels were significantly higher in proliferative diabetic retinopathy subjects [15]. Therefore, Grf may favor tumor formation. DJ-1 protein is strongly and homogenously expressed in all CNS regions. P53 protein is known as an apoptotic factor for glioma cells [24]. DJ-1 can bind to p53 and positively regulates p53 in vitro and in vivo [26]. Low expression of DJ-1 may help U251 cell escape from P53 inducing apoptosis. Few reports have focused on the role of biliverdin reductase in cancer pathogenesis and progression. Only Fang reported it might be helpful in Heme oxygenase-1 antitumor effect. During this process, biliverdin reductase catalyzes biliverdin to bilirubin [10]. This is consistent with higher level of biliverdin reductase in A172 than in U251 and their biological characteristics. Unfortunately, there are still some proteins with unclear function (hypothetical protein) or proteins cannot be identified. This will be resolved following the improvement of mass spectrometry technology and enrichment of the protein database. Recently Vogel et al. [30] have applied 2-DE gel analysis on glioblastoma multiforme (GBM) and glioma cell lines (including U251 and A172). This study focused on different protein profiles between primary GBMs and the glioma cell-lines that are likely due to the selection pressures in vitro culture. They identified a number of differentially expressed proteins that are involved in cytoskeletal movement or scaffolding, secretion of matrix metalloproteinases, tumor suppressor, DNA transcription and cellular metabolism. In our study, we took the advantage of the two cell lines with distinct growth patterns, to disclose the discrepancy of their protein profile in order to demonstrate any of the clues associated with their biological property. We did not find any overlapping proteins between their findings and our results. Furthermore, Vogel et al. found large quantities of proteins are shared proteins while the number of unique proteins for individual cell lines was small (5–10 proteins). We also found that differently expressed proteins are a small part of total proteins between U251 and A172 cell line. On the basis of these consistent results, it is considered that in vitro cultured cell lines have the similarity in protein profile under the same selection pressures in vitro culture microenvironment, but each individual cell line still maintains its unique character. That is also explainable why the established tumor cell lines have been extensively used in research, and the comparison between cell lines in vitro could reflect the different characteristics of cell lines itself. In summary, we use proteomic methods to compare protein profiles between two cell lines with different tumor forming characteristics. This comparative analysis revealed the differential expression of several proteins involved in cell proliferation, DNA replication, protein synthesis, invasion, angiogenesis and neurotrophic factors. Additional studies are under way to
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determine sensitivity, specificity, and prognostic value of these proteins in a larger spectrum of human gliomas, which may permit their use as diagnositic and prognostic markers, to predict response to therapy, or to identify new molecular targets for therapy. Acknowledgements This work was supported by the Institute for Cancer research of Xi’an Jiaotong University, China. References [1] L. Bello, C. Giussani, G. Carrabba, M. Pluderi, V. Lucini, M. Pannacci, D. Caronzolo, G. Tomei, R. Villani, F. Scaglione, R.S. Carroll, A. Bikfalvi, Suppression of malignant glioma recurrence in a newly developed animal model by endogenous inhibitors, Clin. Cancer Res. 8 (2002) 3539–3548. [2] D.D. Bigner, S.H. Bigner, J. Ponten, B. Westermark, M.S. Mahaley, E. Ruoslahti, H. Herschman, L.F. Eng, C.J. Wikstrand, Heterogeneity of genotypic and phenotypic characteristics of fifteen permanent cell lines derived from human gliomas, J. Neuropathol. Exp. Neurol. 40 (1981) 201–229. [3] P. Bjorses, M. Halonen, J.J. Palvimo, M. Kolmer, J. Aaltonen, P. Ellonen, J. Perheentupa, I. Ulmanen, L. Peltonen, Mutations in the AIRE gene: effects on subcellular location and transactivation function of the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy protein, Am. J. Hum. Genet. 66 (2000) 378–392. [4] C.L. Cheng, S.P. Johnson, S.T. Keir, J.A. Quinn, O.F. Ali, C. Szabo, H. Li, A.L. Salzman, M.E. Dolan, P. Modrich, D.D. Bigner, H.S. Friedman, Poly(ADP-ribose) polymerase-1 inhibition reverses temozolomide resistance in a DNA mismatch repair-deficient malignant glioma xenograft, Mol. Cancer Ther. 4 (2005) 1364–1368. [5] S. Chiron, A. Suleau, N. Bonnefoy, Mitochondrial translation: elongation factor tu is essential in fission yeast and depends on an exchange factor conserved in humans but not in budding yeast, Genetics 169 (2005) 1891–1901. [6] V.C. Chumbalkar, C. Subhashini, V.M. Dhople, C.S. Sundaram, M.V. Jagannadham, K.N. Kumar, P.N. Srinivas, R. Mythili, M.K. Rao, M.J. Kulkarni, S. Hegde, A.S. Hegde, C. Samual, V. Santosh, L. Singh, R. Sirdeshmukh, Differential protein expression in human gliomas and molecular insights, Proteomics 5 (2005) 1167–1177. [7] M.T. Coleman, N. Allen, Activities of enzymes of the hexose monophosphate pathway in nervous system tumors induced by ethylnitrosourea, Acta Neuropathol. (Berl.) 41 (1978) 223–227. [8] M.X. Du, J. Sim, L. Fang, Z. Yin, S. Koh, J. Stratton, J. Pons, J.J. Wang, B. Carte, Identification of novel small-molecule inhibitors for human transketolase by high-throughput screening with fluorescent intensity (FLINT) assay, J. Biomol. Screen. 9 (2004) 427–433. [9] J.R. Evans, S.A. Mitchell, K.A. Spriggs, J. Ostrowski, K. Bomsztyk, D. Ostarek, A.E. Willis, Members of the poly (rC) binding protein family stimulate the activity of the c-myc internal ribosome entry segment in vitro and in vivo, Oncogene 22 (2003) 8012–8020. [10] J. Fang, T. Akaike, H. Maeda, Antiapoptotic role of heme oxygenase (HO) and the potential of HO as a target in anticancer treatment, Apoptosis 9 (2004) 27–35. [11] G.L. Forrest, B. Gonzalez, Carbonyl reductase, Chem. Biol. Interact. 129 (2000) 21–40. [12] M.E. Fukuda, Y. Iwadate, T. Machida, T. Hiwasa, Y. Nimura, Y. Nagai, M. Takiguchi, H. Tanzawa, A. Yamaura, N. Seki, Cathepsin D is a potential serum marker for poor prognosis in glioma patients, Cancer Res. 65 (2005) 5190–5194. [13] D.J. Giard, S.A. Aaronson, G.J. Todaro, P. Arnstein, J.H. Kersey, H. Dosik, W.P. Parks, In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors, J. Natl. Cancer Inst. 51 (1973) 1417–1423.
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Proteomics-based analysis of a pair of glioma cell lines with different tumor forming characteristics Le Zhou a,b , Yan Wang a , Ying-tao Zhang a , Yi-ping Geng a , Lu-sheng Si a , Yi-li Wang a,∗ a
Institute for Cancer Research, The Key Laboratory of Biomedical Information Engineering of Chinese Education Ministry, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710061, China b Department of Neurosurgery, The Second Hospital of Xi’an Jiaotong University, Xi’an 710004, China Received 18 September 2005; received in revised form 1 January 2006; accepted 27 February 2006
Abstract Glioma is the most common malignant disease in the brain, and recurrence is the main cause of death from this disease. Tumor recurrence involves multiple steps, and requires the accumulation of the altered expression of many different proteins. Identification of the recurrence associated protein profile in glioma cell lines will be helpful in clarifying the molecular mechanisms underlying glioma recurrence. In this report, two glioma cell lines with distinct tumor forming ability in vitro and in vivo were chosen and the different protein expression patterns were analyzed by proteomics method. To confirm the utility of this method, we validated the differential expression of one protein, cathepsin D, by immunohistochemistry analysis. Forty-six proteins appeared differently between two cell lines and 18 of them were identified. These 18 are involved in cell proliferation, DNA replication, protein synthesis, invasion, angiogenesis and neurotrophic factor. All of these molecules are important in tumor growth, and a subset of them may be related to glioma recurrence. These findings may contribute to the discovery of new diagnostic markers and therapeutic targets of glioma. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Glioma; Recurrence; Proteomics
Gliomas are the most common primary brain tumors in adults [33]. Despite the tremendous advances in neuroimaging, neurosurgery, radiation and medical oncology, a complete surgical resection of infiltrating tumor cells in the brain parenchyma surrounding the main tumor mass is never achieved at the time of surgery and more than 90% of the tumors will recur within 2 cm of the primary tumor location. This makes the prognosis of the disease very poor [1,25]. Tumor recurrence involves multiple steps, and requires the accumulation of the altered expression of many different proteins. Recent studies on the tumorigenesis indicate that only a small subpopulation of tumor cells with stem cell character is capable of initiating tumor formation and maintaining the nonstop growth [27]. Independent analysis of any single protein would be insufficient to understand all of the aspects of glioma recurrence. Systematical analysis of the tumor cells with differ-
∗
Corresponding author. Tel.: +86 29 82655429 808; fax: +86 29 82655499. E-mail address: [email protected] (Y.-l. Wang).
0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.02.080
ent biological behaviors may be helpful to elucidate the nature of the tumor recurrence. Currently, the application of the proteomic approach has made it possible to characterize global alterations in cell protein expression. This promises new insight into the cellular mechanisms involved in tumor recurrence and is likely to result in the discovery of novel diagnostic markers and new therapeutic opportunities. In the present study, proteomics approach was used to analyze of a pair of human glioma cell lines (U251 and A172) which are widely used in laboratory. The biological behaviors of the two cell lines have been extensively investigated. More importantly the two cell lines are demonstrated different tumor forming potentiality. Cell line U251 can form subcutaneous tumor mass in nude mice but A172 cannot [2,13,17]. It is considered that the highly invasive nature of the tumor cell are associated with the frequent recurrence, and the ability of colony forming in vitro and tumor forming in nude mice reflects the malignant potentiality to certain extent. Although the two cell lines are derived from different patients, the distinct growth patterns make it a meaningful model for investigation of glioma forming. Com-
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parative study on the protein expression profile may disclose the differential functional proteins and provide useful information. By two-Dimensional gel Electrophoresis (2-DE) and MALDI-TOF MS analysis, several differentially expressed proteins were identified, and the utility of this proteomics method was reconfirmed by immunocytochemistry. A subset of these differentially expressed proteins may be relevant to glioma recurrence. U251 and A172 cells were purchased from Cell Bank of Chinese Academic of Science (Shanghai, China). The cells were cultured in DMEM (Gibco BRL, USA) supplemented with 15% fetal calf serum at 37 ◦ C in an atmosphere of 5% CO2 in air. To confirm the colony forming ability of the two cell lines, anchorage independent growth in soft agar was performed. A certain number of tumor cells were seeded onto per dish and cultured at 37 ◦ C in an atmosphere of 5% CO2 in air for 3 weeks. The numbers of colonies that contain more than 50 cells were counted. Statistical analysis of the significance of differences between the two cell lines was determined by Student’s t-test, and p-values less than 0.05 were considered significant. Sample preparation for 2-DE was performed as follows. The cells were harvested by trypsinization at the exponential growth phase. After washing in Hanks’ solution and icecold phosphatebuffered saline, the cells were counted and centrifuged in 2.0 ml microtubes. The cell pellets were dissolved in lysis buffer (9 M urea, 4% CHAPS, 40 mM Tris-base, 40 mM DTT) and centrifuged at 40,000 × g for 1 h at 4 ◦ C. Protein concentration was determined by the Bradford protein assay method. All samples were stored in aliquots at −80 ◦ C before analyzing. The first dimensional isoelectric focusing (IEF) was carried out on an IPGphor system (Amersham Biosciences, Sweden)
using 13 cm strips and 80 g protein in 250 l sample solution. Focusing was started at 200 V and the voltage was gradually increased to 8000 V, and then focusing at 8000 V until total volt-hours reaching 26000 V h at 20 ◦ C. After IEF, the focused strips were then equilibrated in buffer I (6 M urea, 30% glycerol, 2% SDS, 1% DTT) and then buffer II (DTT was replaced with 2.5% IAA) each for 15 min with gentle shaking. The second dimensional separation was carried out on 12.5% SDS-polyacrylamide gels using Hoefer SE 600 vertical chambers. After 2-DE, the gels were stained with silver nitrate. The stained gels were captured by transmission scanning and were analyzed with Image Master5.0. Following analysis, selected protein spots were manually excised from gels, destained, and dried. Proteins were then digested with TPCKtrypsin (Sigma, USA) overnight at 37 ◦ C. The peptides were extracted and peptide mass finger printings (PMF) were generated by MALDI-TOF MS. PMFs were matched to the theoretical tryptic digests of proteins in database NCBInr by Mascot software (http://www.matrixscience.com). The selected taxonomy category was Homo sapien (human). Max Missed Cut limited in 0-1. Peptide mass tolerance was settled at 100 ppm. Modifications were allowed for carbamidomethyl of cysteine and oxidation of methionine. To confirm the validity of this proteomics method, we selected one of identified proteins (spot 25, which was identified as cathepsin D and expression level is 2.46-fold higher in U251 than in A172) and validated the differential expression using immunohistochemistry analysis. Antibody for cathepsin D (1:100 dilution) was obtained from Chemicon, USA. Immunohistochemistry was performed according to standard techniques [22].
Fig. 1. The 2-DE gel images of A172 (A) and U251 (B) cells. The Arabic numerals show the differential expressed proteins.
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Fig. 2. The PMF of spot 25 obtained by MALDI-TOF MS after trypsin digestion. The protein was identified as gi|30582659, cathepsin D, at 90 score. (Protein scores greater than 64 are significant (p < 0.05)).
The results of anchorage independent growth in soft agar showed that U251 cells formed 202 ± 15 colonies per dish, whereas A172 cells formed 104 ± 15 colonies per dish. Statistical analysis showed there was a significant difference between two cell lines in colony formation ability. This result was consistent with the character of tumor formation in nude mice of the two cell lines. The 2-DE gels results of the protein profile expressed by U251 and A172 cells were presented in Fig. 1. Good reproducibility and resolution were achieved on silver stained gels for each cell line. Following comparison with software Image Master5.0, 46 protein spots, which were marked as numbers in the Fig. 1, were found more than two-fold changes in amount. Among them, 18 protein spots (number 20–37) increased while nineteen protein spots (number 1–19) decreased in U251 cells; five protein spots (number 42–46) were detected only in U251; and four protein spots (number 38–41) were exclusive in A172. The PMF spectrums of the proteins were obtained by MALDI-TOF-MS analysis following in-gel digestion. Fig. 2 is a MALDI-TOF mass spectrum of peptides derived from protein spot 25. The protein was identified as cathepsin D by PMF searching in NCBInr database. Cathepsin D was observed in
both cell lines by immunohistochemistry stain. It expressed almost in all U251 cells, but only in part of A172 cells (Fig. 3). This is consistent with the result of 2-DE and MALDI-TOF MS analysis. It demonstrates that proteomics analysis is a reliable method for this research. A summary of the identification data is show in Table 1. We compared the protein profiles of two human glioma cell lines that possess different tumor forming potential by proteomics technologies. The results indicate that the tumor forming potential of tumor cells may be regulated by the different expression of several specific proteins. These proteins involved in cell proliferation, DNA replication, protein synthesis, invasion, angiogenesis, and neurotrophic factor. It is interesting that the proteins up-regulated in U251 cells usually have positive relationship to tumor proliferation. In contrast, the proteins with higher expression levels in A172 cells usually have a negative relationship to tumor proliferation. Transketolase, a thiamine-dependent enzyme, links the pentose phosphate pathway with the glycolytic pathway and is an important enzyme in nucleotide metabolism. This protein plays a crucial role in tumor cell nucleic acid synthesis [8]. Moreover, over expression of transketolase was detected in most nervous
Fig. 3. Cell A172 (A) and U251 (B) immuno-labeled for Cathepsin D (×200).
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Table 1 The identified protein spots in cell line A172 and U251 No.
NCBI no.
Peptides matched
Theoretical MW/pI
Sequence coverage (%)
Protein name
1 4 5 7 11 12 13 18 20 21 23 25 26 30 34 35 36 40
15029910 32891807 31543380 4502599 505104 4505773 21411003 1731411 14141166 51895760 2228737 30582659 34147630 6983940 37267 3392940 21739410 50949567
5/26 9/28 6/14 5/16 6/19 9/29 9/20 9/32 5/16 4/11 5/23 6/15 9/40 5/17 10/48 4/11 7/19 8/29
26.34/4.3 22.22/7.1 20.05/6.3 30.64/8.6 45.30/6.2 29.86/5.6 36.71/9.2 55.47/9.0 38.60/6.3 18.23/7.7 53.28/8.9 45.04/6.1 50.19/7.3 44.90/9.2 68.44/7.9 55.38/7.2 80.38/5.2 21.12/9.0
17 71 32 25 17 55 25 21 16 35 26 14 22 22 27 10 17 21
PR48 proteina Biliverdin reductase Ba DJ-1 proteina Carbonyl reductase 1a KIAA0071a Prohibitina Poly(ADP-ribose) polymerase (PARP)a Zinc finger protein 135a Poly(rc)-binding protein 2 (PCBP2)b Peptidylprolyl isomerase A (cyclophilin A)b Glial cell line-derived neurotrophic factor receptor (Grf) Cathepsin Db Tu translation elongation factor, mitochondrial (TUFM)b p47-phoxb Transketolaseb AIREb Hypothetical proteinb Hypothetical proteinc
a b c
Expressed higher in A172. Expressed higher in U251. Expressed only in A172.
system tumors as compared with cerebral cortex [7]. PR48 is the regulatory subunit of protein phosphatase 2A (PP2A), one of initiators of DNA replication in eukaryotes. Over-expression of PR48 in human cells causes a G1 arrest [31]. Considering that DNA replication is the basis of cell proliferation, higher expression of transketolase and low expression of PR48 may enhance U251 cell proliferation by promoting DNA replication. Prohibitin has been shown to be a potential tumor suppressor protein, which can bind to the Rb protein as well as E2F. This binding is necessary to suppress cell proliferation [19]. Recently, a notable under-expression of prohibitin was observed in Grade III glioma compared with control tissues [6]. Thus, decreased expression of prohibitin may be the other reason for promoting U251 cell proliferation. Poly(ADP-ribose) polymerase (PARP), expressed higher in U251, can catalyze the transfer of multiple ADP-ribose units from NAD to substrate proteins [23], and plays a role in DNA repair and the recovery of cells from DNA damage [14]. Inhibition of PARP may increase the efficacy of temozolomide (a DNA-methylating agent used in the treatment of gliomas) in the treatment of gliomas, particularly in tumors deficient in DNA mismatch repair [4]. These findings suggest that higher expression of PARP is important for U251 growth. Wild type AIRE protein acted as a strong transcriptional activator in mammalian cells [3]. KIAA0071 can bind to the RE1 silencing transcription factor in vitro and vivo and exhibits transcriptional corepressor activity [16]. Zinc finger protein (ZFP) is a large class of transcriptional regulators. Engineered ZFP transcription factors are shown to be potent regulators of gene expression with therapeutic promise in the treatment of glioma [29]. Poly(rc)-binding protein 2 (PCBP2) binds the major stemloop structure (stem-loop IV) in the internal ribosome entry site and is essential for translation initiation. At the same time, PCBP2 is able to stimulate the activity of the proto-oncogene
c-myc [9]. TUFM is highly conserved throughout evolution. It can deliver amino-acylated tRNAs to the ribosome during the elongation step of translation. Absence of the TUFM is lethal in S. pombe [5]. AIRE, PCBP2 and TUFM, which can promote protein synthesis, are increasingly expressed in U251, but expression level of KIAA0071 and ZFP135 which can inhibit protein synthesis are down-regulated. Predominance in protein synthesis may be one of reasons for U251 tumor forming. Invasion is an important characteristic in glioma. Cathepsin D and Carbonyl reductase (CBR) are both involved in tumor cell invasion. Cathepsin D, a lysosomal aspartyl endoproteinase, takes part in degradation of the extracellular matrix and is strongly expressed by invading glioblastoma cells at the infiltrative margin. Several reports have indicated that Cathepsin D can stimulate cancer cell proliferation and increase metastatic potential in vivo [21]. Antibody to Cathepsin D was shown to inhibit the invasion of U251 cell line in a dose-dependent manner [28]. Fukuda et al. even took cathepsin D as a potential serum marker for the prediction of aggressive nature of human gliomas [12]. CBR can catalyze prostaglandins which have been associated with more aggressive tumor invasion and metastases. Therefore, modulation of prostaglandin levels by CBR was suggested as a possible mechanism of restraining the ability of cancer cell invasion [11]. Higher level of cathepsin D and lower level of CBR in U251 imply that invasion associated molecules may play an important role in tumor forming. The process of angiogenesis plays a key role in development and progression of solid tumors. Cyclophilin A (CypA) is a modulator of endothelial cell functions in vascular disease. It can increase endothelial cell proliferation, migration, invasive capacity, and tubulogenesis at low concentrations [20]. Yang et al. demonstrates that CypA has a mitogenic effect on human aorta smooth muscle cells and human lung microvascular endothelial cells [32]. The expression of CypA will increase
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with the help of reactive oxygen species (ROS) [18]. Protein P47-phox can improve ROS generation. Interestingly, CypA and P47-phox are both over-expressed in the U251 cell line, which might make U251 cells possess higher angiogenesis ability in tumor formation. Glial cell line-derived neurotrophic factor receptor (Grf) is a receptor of Glial cell line-derived neurotrophic factor (GDNF). Immunohistochemical analysis revealed that Grf expression levels were significantly higher in proliferative diabetic retinopathy subjects [15]. Therefore, Grf may favor tumor formation. DJ-1 protein is strongly and homogenously expressed in all CNS regions. P53 protein is known as an apoptotic factor for glioma cells [24]. DJ-1 can bind to p53 and positively regulates p53 in vitro and in vivo [26]. Low expression of DJ-1 may help U251 cell escape from P53 inducing apoptosis. Few reports have focused on the role of biliverdin reductase in cancer pathogenesis and progression. Only Fang reported it might be helpful in Heme oxygenase-1 antitumor effect. During this process, biliverdin reductase catalyzes biliverdin to bilirubin [10]. This is consistent with higher level of biliverdin reductase in A172 than in U251 and their biological characteristics. Unfortunately, there are still some proteins with unclear function (hypothetical protein) or proteins cannot be identified. This will be resolved following the improvement of mass spectrometry technology and enrichment of the protein database. Recently Vogel et al. [30] have applied 2-DE gel analysis on glioblastoma multiforme (GBM) and glioma cell lines (including U251 and A172). This study focused on different protein profiles between primary GBMs and the glioma cell-lines that are likely due to the selection pressures in vitro culture. They identified a number of differentially expressed proteins that are involved in cytoskeletal movement or scaffolding, secretion of matrix metalloproteinases, tumor suppressor, DNA transcription and cellular metabolism. In our study, we took the advantage of the two cell lines with distinct growth patterns, to disclose the discrepancy of their protein profile in order to demonstrate any of the clues associated with their biological property. We did not find any overlapping proteins between their findings and our results. Furthermore, Vogel et al. found large quantities of proteins are shared proteins while the number of unique proteins for individual cell lines was small (5–10 proteins). We also found that differently expressed proteins are a small part of total proteins between U251 and A172 cell line. On the basis of these consistent results, it is considered that in vitro cultured cell lines have the similarity in protein profile under the same selection pressures in vitro culture microenvironment, but each individual cell line still maintains its unique character. That is also explainable why the established tumor cell lines have been extensively used in research, and the comparison between cell lines in vitro could reflect the different characteristics of cell lines itself. In summary, we use proteomic methods to compare protein profiles between two cell lines with different tumor forming characteristics. This comparative analysis revealed the differential expression of several proteins involved in cell proliferation, DNA replication, protein synthesis, invasion, angiogenesis and neurotrophic factors. Additional studies are under way to
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