Up-regulation of Tumor Susceptibility Gene 101 Protein in Ovarian Carcinomas Revealed by Proteomics Analyses
2006; Elsevier BV; Volume: 6; Issue: 2 Linguagem: Inglês
10.1074/mcp.m600305-mcp200
ISSN1535-9484
AutoresTravis W. Young, Fang Mei, Daniel Rosen, Gong Yang, Nan Li, Jinsong Liu, Xiaodong Cheng,
Tópico(s)RNA modifications and cancer
ResumoSmall GTPase RAS plays a critical role in cellular signaling and oncogenic transformation. Proteomics analysis of genetically defined human ovarian cancer models identified the tumor susceptibility gene 101 (TSG101) as a downstream target of RAS oncogene. Mechanistic studies revealed a novel post-translational regulation of TSG101 through the RAS/RAF/MEK/MAPK signaling pathway and downstream molecules p14ARF/HDM2. Immunoanalysis using ovarian cancer samples and microtissue array revealed elevated TSG101 levels in human ovarian carcinomas. Silencing of TSG101 by short interfering RNA in ovarian cancer cells led to growth inhibition and cell death. Concurrent with the apparent growth-inhibitory effect, the levels of the CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2) and hypoxia-inducible factor 1α (HIF-1α), as well as its cellular activity, were markedly reduced after TSG101 knockdown. These results demonstrate that TSG101 is important for CITED2- and HIF-1α-mediated cellular regulation in ovarian carcinomas. Small GTPase RAS plays a critical role in cellular signaling and oncogenic transformation. Proteomics analysis of genetically defined human ovarian cancer models identified the tumor susceptibility gene 101 (TSG101) as a downstream target of RAS oncogene. Mechanistic studies revealed a novel post-translational regulation of TSG101 through the RAS/RAF/MEK/MAPK signaling pathway and downstream molecules p14ARF/HDM2. Immunoanalysis using ovarian cancer samples and microtissue array revealed elevated TSG101 levels in human ovarian carcinomas. Silencing of TSG101 by short interfering RNA in ovarian cancer cells led to growth inhibition and cell death. Concurrent with the apparent growth-inhibitory effect, the levels of the CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2) and hypoxia-inducible factor 1α (HIF-1α), as well as its cellular activity, were markedly reduced after TSG101 knockdown. These results demonstrate that TSG101 is important for CITED2- and HIF-1α-mediated cellular regulation in ovarian carcinomas. Oncogenic transformation is an intricate process involving alterations of multiple genetic elements and signaling cascades. One critical signaling molecule that contributes directly to transformation is the small G-protein RAS. Activation of the K-RAS or H-RAS signaling pathways plays an important role in ovarian tumorigenesis. Mutations in K-RAS and its downstream effector B-RAF are involved in 60–70% of low grade serous ovarian cancers (1Singer G. Oldt III, R. Cohen Y. Wang B.G. Sidransky D. Kurman R.J. Shih I. Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma..J. Natl. Cancer Inst. 2003; 95: 484-486Crossref PubMed Google Scholar, 2Sieben N.L. Macropoulos P. Roemen G.M. Kolkman-Uljee S.M. Jan F.G. Houmadi R. Diss T. Warren B. Al Adnani M. De Goeij A.P. Krausz T. Flanagan A.M. In ovarian neoplasms, BRAF, but not KRAS, mutations are restricted to low-grade serous tumours..J. Pathol. 2004; 202: 336-340Crossref PubMed Scopus (204) Google Scholar). Although activating mutations of H-RAS are present in only about 6% of ovarian cancers (3Varras M.N. Sourvinos G. Diakomanolis E. Koumantakis E. Flouris G.A. Lekka-Katsouli J. Michalas S. Spandidos D.A. Detection and clinical correlations of ras gene mutations in human ovarian tumors..Oncology. 1999; 56: 89-96Crossref PubMed Scopus (34) Google Scholar), activation of H-RAS upstream and downstream effector pathways often occurs in the absence of an H-RAS mutation (4Patton S.E. Martin M.L. Nelsen L.L. Fang X. 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RAS functions as an intracellular molecular switch, cycling between the GDP-bound inactive state and the GTP-bound active state in response to external stimuli leading from cell surface receptor tyrosine kinases to nuclear transcription factors (7Marshall C.J. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation..Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4220) Google Scholar, 8Berchuck A. Carney M. Human ovarian cancer of the surface epithelium..Biochem. Pharmacol. 1997; 54: 541-544Crossref PubMed Scopus (74) Google Scholar). RAS-associated cell signaling is involved in many important cellular processes, such as cell growth, differentiation, and survival under physiological conditions. Several well known intracellular signaling cascades including the RAF/MEK 1The abbreviations used are: MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; 2-DE, two-dimensional electrophoresis; CITED2, CBP/p300-interacting transactivator with ED-rich tail 2; GFP, green fluorescent protein; HMD2, human homolog of MDM2; HIF, hypoxia-inducible factor; MDM2, mouse double minute 2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; TSG101, tumor susceptibility gene 101; siRNA, short interfering RNA; MAPK, mitogen-activated protein kinase; CBP, cAMP-response element-binding protein (CREB)-binding protein; ERK, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HRE, HIF-1α response element. 1The abbreviations used are: MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; 2-DE, two-dimensional electrophoresis; CITED2, CBP/p300-interacting transactivator with ED-rich tail 2; GFP, green fluorescent protein; HMD2, human homolog of MDM2; HIF, hypoxia-inducible factor; MDM2, mouse double minute 2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; TSG101, tumor susceptibility gene 101; siRNA, short interfering RNA; MAPK, mitogen-activated protein kinase; CBP, cAMP-response element-binding protein (CREB)-binding protein; ERK, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HRE, HIF-1α response element./ERK pathway, the phosphatidylinositol 3-kinase pathway, and the RAL-guanine nucleotide dissociation stimulator pathway have been identified as mediators of RAS downstream effects (9Vavvas D. 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TSG101 was originally identified as a potential tumor suppressor from a controlled homozygous functional knock-out screen. Inactivation of TSG101 in NIH3T3 mouse fibroblasts leads to focus formation in monolayer cell cultures, anchorage-independent growth in soft agar, and in vivo tumor formation in nude mice (15Li L. Cohen S.N. Tsg101: a novel tumor susceptibility gene isolated by controlled homozygous functional knockout of allelic loci in mammalian cells..Cell. 1996; 85: 319-329Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). Initial studies suggested that TSG101 was often mutated in human breast cancers (16Li L. Li X. Francke U. Cohen S.N. The TSG101 tumor susceptibility gene is located in chromosome 11 band p15 and is mutated in human breast cancer..Cell. 1997; 88: 143-154Abstract Full Text PDF PubMed Google Scholar), and its aberrant splice variants were frequently detected in different tumor types (17Li L. Francke U. Cohen S.N. Retraction. The TSG101 tumor susceptibility gene is located in chromosome 11 band p15 and is mutated in human breast cancer..Cell. 1998; 93 (following 660)Abstract Full Text Full Text PDF Google Scholar, 18Lee M.P. Feinberg A.P. Aberrant splicing but not mutations of TSG101 in human breast cancer..Cancer Res. 1997; 57: 3131-3134PubMed Google Scholar, 19Gayther S.A. Barski P. Batley S.J. Li L. de Foy K.A. Cohen S.N. Ponder B.A. Caldas C. Aberrant splicing of the TSG101 and FHIT genes occurs frequently in multiple malignancies and in normal tissues and mimics alterations previously described in tumours..Oncogene. 1997; 15: 2119-2126Crossref PubMed Google Scholar, 20Wang Q. Driouch K. Courtois S. Champeme M.H. Bieche I. Treilleux I. Briffod M. Rimokh R. Magaud J.P. Curmi P. Lidereau R. Puisieux A. Low frequency of TSG101/CC2 gene alterations in invasive human breast cancers..Oncogene. 1998; 16: 677-679Crossref PubMed Google Scholar, 21Steiner P. Barnes D.M. Harris W.H. Weinberg R.A. 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Chem. 2004; 279: 35984-35994Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), the role of TSG101 in tumor formation and development has proven to be complex and remains controversial. TSG101 was initially described as a potential tumor suppressor, and the expression of TSG101 has been shown to be decreased in certain cancer samples (28Bennett N.A. Pattillo R.A. Lin R.S. Hsieh C.Y. Murphy T. Lyn D. TSG101 expression in gynecological tumors: relationship to cyclin D1, cyclin E, p53 and p16 proteins..Cell. Mol. Biol. (Noisy-le-grand). 2001; 47: 1187-1193PubMed Google Scholar). However, more recent studies suggest that TSG101 levels are elevated in human cancers, including thyroid (29Liu R.T. Huang C.C. You H.L. Chou F.F. Hu C.C. Chao F.P. Chen C.M. Cheng J.T. Overexpression of tumor susceptibility gene TSG101 in human papillary thyroid carcinomas..Oncogene. 2002; 21: 4830-4837Crossref PubMed Scopus (61) Google Scholar) and gastrointestinal tumors (30Koon N. Schneider-Stock R. Sarlomo-Rikala M. Lasota J. Smolkin M. Petroni G. Zaika A. Boltze C. Meyer F. Andersson L. Knuutila S. Miettinen M. El Rifai W. Molecular targets for tumour progression in gastrointestinal stromal tumours..Gut. 2004; 53: 235-240Crossref PubMed Scopus (109) Google Scholar). Furthermore overexpression of TSG101 can also lead to neoplastic transformation (15Li L. Cohen S.N. Tsg101: a novel tumor susceptibility gene isolated by controlled homozygous functional knockout of allelic loci in mammalian cells..Cell. 1996; 85: 319-329Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). Gene silencing of TSG101 leads to growth arrest and cell death in breast and prostate cancer cells (31Zhu G. Gilchrist R. Borley N. Chng H.W. Morgan M. Marshall J.F. Camplejohn R.S. Muir G.H. Hart I.R. Reduction of TSG101 protein has a negative impact on tumor cell growth..Int. J. Cancer. 2004; 109: 541-547Crossref PubMed Scopus (49) Google Scholar) instead of growth promotion as would be expected for the loss of a true tumor suppressor. We propose that TSG101 is an important factor for maintaining normal cellular homeostasis and that elevated TSG101 expression contributes to oncogenic transformation. This hypothesis is consistent with the fact that steady-state TSG101 levels are tightly controlled in normal cells, primarily at the post-translational level, keeping protein concentrations within a narrow range (32Feng G.H. Lih C.J. Cohen S.N. TSG101 protein steady-state level is regulated posttranslationally by an evolutionarily conserved COOH-terminal sequence..Cancer Res. 2000; 60: 1736-1741PubMed Google Scholar). At the present time, the mechanism of TSG101 post-translational regulation is not clear. Understanding the cellular regulation of TSG101 is important for further elucidating the function of TSG101 under physiological and neoplastic conditions. In this study, we identified TSG101 as a RAS downstream target in human ovarian epithelial cells transformed by either oncogenic H-RASV12 or K-RASV12 using a two-dimensional electrophoresis (2-DE)-based proteomics analysis. Immunohistochemical analysis of ovarian cancer tissue array revealed that TSG101 is up-regulated in more than 70% of human ovarian carcinomas. Gene silencing using TSG101-specific siRNA inhibited the growth and tumorigenicity of SKOV-3 cells, a naturally occurring ovarian cancer cell line with elevated RAS activity. Our study for the first time provides a direct link between oncogenic RAS and TSG101 and demonstrates a potential role for TSG101 in RAS-mediated oncogenic transformation through regulation of the CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2) and hypoxia-inducible factor 1α (HIF-1α). T29, T29H, and T29K cells were generated through retroviral transfection as described previously (33Liu J. Yang G. Thompson-Lanza J.A. Glassman A. Hayes K. Patterson A. Marquez R.T. Auersperg N. Yu Y. Hahn W.C. Mills G.B. Bast Jr., R.C. A genetically defined model for human ovarian cancer..Cancer Res. 2004; 64: 1655-1663Crossref PubMed Scopus (230) Google Scholar) and grown in Medium 199/MCDB105 medium (1:1) (Sigma) containing 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen). SKOV-3 cells were maintained in RPMI 1640 medium containing 5% fetal bovine serum (Invitrogen) and 1% penicillin/streptomycin (Invitrogen). 2-DE proteomics analysis was performed as described previously (34Young T.W. Mei F.C. Yang G. Thompson-Lanza J.A. Liu J. Cheng X. Activation of antioxidant pathways in ras-mediated oncogenic transformation of human surface ovarian epithelial cells revealed by functional proteomics and mass spectrometry..Cancer Res. 2004; 64: 4577-4584Crossref PubMed Scopus (106) Google Scholar, 35Young T. Mei F. Liu J. Bast Jr., R.C. Kurosky A. Cheng X. Proteomics analysis of H-RAS-mediated oncogenic transformation in a genetically defined human ovarian cancer model..Oncogene. 2005; 24: 6174-6184Crossref PubMed Scopus (25) Google Scholar). Briefly cells were trypsinized, washed in PBS, and lysed in buffer containing the following: 7 m urea, 2 m thiourea, 4% CHAPS, 1 mm EDTA, 1 mm EGTA, 60 mm DTT, 1 mm PMSF, 25 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mm benzamidine, 1 mm sodium orthovanadate, and 1 mm microcystin. Total protein concentration was determined using the Bradford assay (Bio-Rad), and 500 μg was loaded onto 18-cm Immobiline pH gradient strips (GE Healthcare). After focusing for 56,000 V-h, strips were loaded onto 10% SDS-Tricine gels and electrophoresed for 20 h at 140 V. Gels for analysis and for protein excision were stained with silver as described previously (36Blum H. Beier H. Gross H.L. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels..Electrophoresis. 1987; 8: 93-99Crossref Scopus (3728) Google Scholar, 37Shevchenko A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels..Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7763) Google Scholar). Images were analyzed using Phoretix 2-D software (Nonlinear Dynamics). Spots of interest were excised and in-gel digested with trypsin as described previously (34Young T.W. Mei F.C. Yang G. Thompson-Lanza J.A. Liu J. Cheng X. Activation of antioxidant pathways in ras-mediated oncogenic transformation of human surface ovarian epithelial cells revealed by functional proteomics and mass spectrometry..Cancer Res. 2004; 64: 4577-4584Crossref PubMed Scopus (106) Google Scholar). Proteins were identified by MALDI-TOF analysis by comparison of tryptic fragment profiles with the National Center for Biotechnology Information (NCBI) database for theoretical peptide cleavage patterns. The protein concentration of cell lysates was assayed with the Bio-Rad protein assay reagent. Equal amounts of protein were loaded onto 12% SDS-polyacrylamide minigels (Bio-Rad) or 10% Tricine-SDS gels and transferred to PVDF membranes. PVDF blots and the remaining polyacrylamide gels were stained with Ponceau S and Coomassie Blue, respectively, to ensure equal loading and even transfer of the samples. After being blocked overnight in 5% milk in TBS-Tween, blots were incubated with corresponding primary antibodies for 1.5 h followed by horseradish peroxidase-conjugated secondary antibody (1:4000, Bio-Rad) for 45 min. Antigen-antibody complexes were detected by enhanced chemiluminescence (Pierce). Total RNA was isolated from cells using TRIzol reagent (Invitrogen), and the concentration was determined by absorbance at 260 nm. Real time PCR analysis was carried out using fluorescent TSG101 primers on an Applied Biosystems Prism 7000 sequence detection system. RT-PCR was carried out for TSG101 and HIF-1α using 1 μg of isolated total RNA under the following parameters: 94 °C for 1 min, 57 °C for 1 min, and 72 °C for 1.5 min; 35 cycles. Primers used for RT-PCR were TSG101 forward (5′-TCCAGTCTTCTCTCGTCCTATTTC-3′) and reverse (5′-TTTCCTCC TTCATCCGCCATCTC-3′) and HIF-1α forward (5′-CCTGCACTCAATCAAGAATTGC-3′) and reverse (5′-TTCCTGCTCTGTTTGGTGAGGCT-3′). For chemical inhibitor experiments, cells were grown to 50–60% confluence in 6-well plates and treated with DMSO vehicle or the following for 24 h: 20 μm U0126 (MEK inhibitor), 10 μm LY294002 (PI3K inhibitor), or 10 μm FTI-277 (H-RAS farnesylation inhibitor). Following treatments, cells were lysed, and the levels of total and cytoplasmic TSG101 were examined by immunoblotting using specific anti-TSG101 antibody (1:1000, Novus). SKOV-3 and T29H cells were transfected with two retrovirus-mediated H-RAS siRNA vectors (designated H1 and H2) that have been described previously (38Yang G. Thompson J.A. Fang B. Liu J. Silencing of H-ras gene expression by retrovirus-mediated siRNA decreases transformation efficiency and tumor growth in a model of human ovarian cancer..Oncogene. 2003; 22: 5694-5701Crossref PubMed Scopus (105) Google Scholar). Briefly H1 selectively silences mutant H-RASV12, whereas H2 suppresses both the H-RASV12 mutant and wild-type H-RAS expression. SKOV-3 and T29H cells grown to 50% confluence in 10-cm plates were transfected with H1 and H2 retroviral supernatants generated from Phoenix viral packaging cells. Following transfection, cells were selected for 7–10 days in 0.7 mg/ml G418 to establish stable cell lines. RAS-GTP binding assays and Western blotting using anti-H-RAS (1:2000, Santa Cruz Biotechnology) were used to measure the expression and activation levels of RAS proteins in these siRNA cell lines. The tissue microarray slides were subjected to immunohistochemical staining as follows. After initial deparaffinization, endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide. Deparaffinized sections were microwaved in 10 mm citrate buffer (pH 6.0) to unmask the epitopes. The slides were then incubated at 4 °C overnight against TSG101 (1:100, Clone 4A10, Novus Biologicals), next with biotin-labeled secondary antibody for 20 min, and finally with a 1:40 solution of streptavidin:peroxidase for 20 min. Tissues were then stained for 5 min with 0.05% 3′,3-diaminobenzidine tetrahydrochloride that had been freshly prepared in 0.05 m Tris buffer at pH 7.6 containing 0.024% H2O2, then counterstained with hematoxylin, dehydrated, and mounted. All of the dilutions of antibody, biotin-labeled secondary antibody, and streptavidin-peroxidase were made in phosphate-buffered saline (pH 7.4) containing 1% bovine serum albumin. Negative controls were made by replacing the primary antibody with phosphate-buffered saline. All controls gave satisfactory results. Immunostaining for TSG101 was analyzed by computerized automated image analysis (Ariol SL-50, Applied Imaging, San Jose, CA). Quantitation was done on the whole core tissue at 20× considering only tumor epithelial cells by appropriate training of the computerized system. Immunostaining for TSG101 was measured as the total integrated optical density and expressed in arbitrary optical density units (ΣOD). These units are expressed in a range from 0 to 255. An optical density unit closer to 0 corresponds to darker pixels, whereas a value closer to 255 corresponds to lighter pixels. Therefore, a ΣOD low value translates as a higher expression, and a high ΣOD value translates as a lower expression for the marker. For statistical analysis, all cases displaying total integrated optical density (mean ± S.E.) were then grouped together on a 0–3 scale. Negative staining (score 0) was defined as the total absence of marker (brown color). The mean of the results from the two replicate core samples from each tumor specimen was considered for each case. Counting criteria and software settings were identical for all slides. Quantitation was done blinded to clinicopathologic information. Normal ovarian epithelial cells were used as a comparison for intensity and pattern of staining. Freshly plated T29H cells (2 × 105 cells/plate in 6-cm plates) and SKOV-3 cells at 80% confluence were transfected with HDM2 vector (1 μg) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells were harvested at 24 and 48 h post-transfection or further selected for the generation of stable lines using 0.7 mg/ml G418. Whole cell lysates were processed as described above, and Western blots were probed using anti-HDM2 (1:1000, Santa Cruz Biotechnology). Cells were plated at 1 × 105 in 3.5-cm wells and grown overnight at 37 °C. The following day cells were transfected with either control siRNA duplex or TSG101 siRNA duplex at 375 ng/well (Dharmacon) using Lipofectamine 2000. Cells were then passaged at 48 h post-transfection and used for subsequent experiments. The HIF-1α response element (HRE)-GFP reporter (a generous gift from Mark Dewhirst) was transfected into cells 48 h after transfection with siRNA reagents, and cells were subsequently plated onto microscope coverslips. On the 5th day following siRNA transfection, cells on coverslips were rinsed in PBS and fixed in 2% paraformaldehyde for 15 min. Nuclear staining was carried out with 4′,6-diamidino-2-phenylindole (5 ng/ml) for 5 min after which slides were mounted and observed using a fluorescence microscope (Olympus BX51) equipped with a Hamamatsu digital camera (C4742-95). SKOV-3 cells were trypsinized 48 h post-transfection with siRNAs and plated at 2 × 103 cells/well in 96-well plates with five wells for each treatment. On subsequent days, medium was removed, and 100 μl/well fresh medium was added along with 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to a final concentration of 500 μg/ml. Cells were incubated for 3 h at 37 °C and then lysed by adding 200 μl/well DMSO and incubating at 37 °C for 1 h. Cell viability was measured by reduction of MTT, which registered absorbance at 595 nm on a Molecular Devices microplate reader. SKOV-3 cells were trypsinized 3 days following transfection with either control or TSG101 siRNA and subsequently washed and resuspended in phosphate-buffered saline at 5 × 106 cells/ml. 200 μl (1 × 106 cells) of control or TSG101-transfected cells were injected subcutaneously into the right and left flanks (respectively) of 4–6-week-old BALB/c athymic nude mice (The Jackson Laboratory, Bar Harbor, ME). Mice were examined every 5 days until visible tumors appeared. Subsequently tumor volume measurements were taken every 3–5 days, mice were sacrificed 6 weeks after initial injections, and a final measurement of tumor volume was taken. To systematically investigate the molecular mechanisms of oncogene RAS-mediated transformation and to explore the important signaling events associated with transformation of human ovarian epithelial cells, we compared the total protein expression profiles of three genetically engineered cell lines, T29, T29H, and T29K, derived from human ovarian surface epithelial cells. Although T29 cells stably transfected with SV40 T/t antigens and human telomerase reverse transcriptase are fully immortalized, only the addition of oncogenic H-RAS (in T29H) or K-RAS (in T29K) leads to the malignant transformation of T29 cells (33Liu J. Yang G. Thompson-Lanza J.A. Glassman A. Hayes K. Patterson A. Marquez R.T. Auersperg N. Yu Y. Hahn W.C. Mills G.B. Bast Jr., R.C. A genetically defined model for human ovarian cancer..Cancer Res. 2004; 64: 1655-1663Crossref PubMed Scopus (230) Google Scholar). To eliminate clonal variability, pools of early passages of T29, T29H, and T29K were used in this study. Total cell lysates from each cell line were analyzed by 2-DE using Immobiline dry strips (pH 4–7) and 10% Tricine-SDS-polyacrylamide gels. For each cell line, at least two independent cell lysates were prepared from cultures at different early passages, and four or more well resolved gels from six different runs were analyzed. Fig. 1 shows representative silver-stained 2-DE images of T29, T29H, and T29K. Approximately 2200 distinct protein spots were resolved within each gel. The intensity of each protein spot was determined, normalized to the sum of intensities of all spots on the gel, and quantified as a percentage of volume in each gel using Phoretix 2-D analysis software (Nonlinear Dynamics). Each individual protein spot was then matched with the identical protein spot from each replicate gel. Data for these matched spots were then averaged over replicate gels for each cell line. The average normalized volume of each spot in the transformed T29H or T29K cells was then compared with that of the matched spot in immortalized T29 cells. To determine what would constitute significant changes between transformed and untransformed cells, the intrinsic variance of each protein spot was determined for each cell line. The average variance for individual spots in the replicate gels for the T29, T29H, and T29K were 34, 29, and 30%, respectively. Based on these observed values, we selected spots whose average normalized volume increased or decreased by at least 1.5-fold between th
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