Proteomic Identification of Insulin-like Growth Factor-binding Protein-6 Induced by Sublethal H2O2 Stress from Human Diploid Fibroblasts

Fibroblasts are the most ubiquitous cell types within our body. They produce various factors to maintain the texture and structure of a particular organ or tissue. To identify protein factors secreted by fibroblasts and alteration of these protein factors upon oxidative stress, HCA3 human skin diploid fibroblasts were exposed to a sublethal dose of H2O2, which induces a prematurely senescent phenotype. Conditioned media from prematurely senescent cells versus control cells were analyzed for proteins using an LC-MS/MS-based proteomic technique. Collagen α1(VI), collagen α2(I), fibronectin, lumican, and matrix metalloproteinase 2 were among the proteins consistently detected from control and H2O2-treated cells. Insulin-like growth factor-binding protein-6 (IGFBP-6) consistently showed up in the conditioned medium of H2O2-treated cells but not from untreated cells. Increased IGFBP-6 production due to H2O2 treatment was confirmed by RT-PCR and Western blot analyses. While H2O2 induced a dose-dependent elevation of IGFBP-6 mRNA, Western blot analyses detected elevated levels of IGFBP-6 protein in the conditioned medium of H2O2-treated cells. In comparison, fibronectin or matrix metalloproteinase 2 did not show changes at the mRNA level in cell lysates or at the protein level in the conditioned medium by H2O2 treatment. Using several types of toxins at sublethal doses, including cis-platin, hydroxyurea, colchicine, l-mimosine, rhodamine, dithiothreitol, or N-ethylmaleimide, we found that these agents induced increases of IGFBP-6 at mRNA and protein levels. An increased level of IGFBP-6 protein was detected in the plasma of aging mice and of young mice treated with doxorubicin. These data suggest that IGFBP-6 may serve as a sensitive biomarker of cell degeneration or injury in vitro and in vivo. Fibroblasts are the most ubiquitous cell types within our body. They produce various factors to maintain the texture and structure of a particular organ or tissue. To identify protein factors secreted by fibroblasts and alteration of these protein factors upon oxidative stress, HCA3 human skin diploid fibroblasts were exposed to a sublethal dose of H2O2, which induces a prematurely senescent phenotype. Conditioned media from prematurely senescent cells versus control cells were analyzed for proteins using an LC-MS/MS-based proteomic technique. Collagen α1(VI), collagen α2(I), fibronectin, lumican, and matrix metalloproteinase 2 were among the proteins consistently detected from control and H2O2-treated cells. Insulin-like growth factor-binding protein-6 (IGFBP-6) consistently showed up in the conditioned medium of H2O2-treated cells but not from untreated cells. Increased IGFBP-6 production due to H2O2 treatment was confirmed by RT-PCR and Western blot analyses. While H2O2 induced a dose-dependent elevation of IGFBP-6 mRNA, Western blot analyses detected elevated levels of IGFBP-6 protein in the conditioned medium of H2O2-treated cells. In comparison, fibronectin or matrix metalloproteinase 2 did not show changes at the mRNA level in cell lysates or at the protein level in the conditioned medium by H2O2 treatment. Using several types of toxins at sublethal doses, including cis-platin, hydroxyurea, colchicine, l-mimosine, rhodamine, dithiothreitol, or N-ethylmaleimide, we found that these agents induced increases of IGFBP-6 at mRNA and protein levels. An increased level of IGFBP-6 protein was detected in the plasma of aging mice and of young mice treated with doxorubicin. These data suggest that IGFBP-6 may serve as a sensitive biomarker of cell degeneration or injury in vitro and in vivo. Fibroblasts reside in the stromal layer of the skin and actively maintain the integrity and architecture of the tissue by secreting matrix proteases and depositing extracellular matrix proteins. During the process of aging, it is thought that such cells show changes in biochemistry and gene expression patterns. Some of these changes, such as alterations in the secretion of degradative enzymes, inflammatory cytokines, and growth factors, are related to the senescent phenotype of normal diploid fibroblasts in culture (1Campisi J. Aging and cancer: the double-edged sword of replicative senescence.J. Am. Geriatr. Soc. 1997; 45: 482-488Google Scholar, 2Campisi J. The role of cellular senescence in skin aging.J. Investig. Dermatol. Symp. Proc. 1998; 3: 1-5Google Scholar, 3Saad M.J. Velloso L.A. Carvalho C.R. Angiotensin II induces tyrosine phosphorylation of insulin receptor substrate 1 and its association with phosphatidylinositol 3-kinase in rat heart.Biochem. 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With human diploid fibroblasts (HDFs) 1The abbreviations used are: HDF, human diploid fibroblast; MMP, matrix metalloproteinase; IGF, insulin-like growth factor; IGFBP, insulin-like growth factor-binding protein; PDL, population doubling level; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Dox, doxorubicin; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin. in culture, the senescent phenotype is typically achieved by serial passage (11Smith J.R. Pereira S.O. Replicative senescence: implications for in vivo aging and tumor suppression.Science. 1996; 273: 63-67Google Scholar, 12Cristofalo V.J. Pignolo R.J. Replicative senescence of human fibroblast-like cells in culture.Physiol. Rev. 1993; 73: 617-638Google Scholar). However, recent evidence suggests that early passage HDFs respond to a defined dose range of oxidants by entering a state of arrested growth and altered phenotype resembling replicative senescence (13Chen Q. 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Proteins secreted by senescent-like fibroblasts appear to exhibit the ability to promote the growth and colony formation of initiated keratinocytes (23Dilley T.K. Bowden G.T. Chen Q.M. Novel mechanisms of sublethal oxidant toxicity: induction of premature senescence in human fibroblasts confers tumor promoter activity.Exp. Cell Res. 2003; 290: 38-48Google Scholar). Uncovering the nature of the proteins secreted by prematurely senescent cells becomes important in understanding the interplay between oxidative stress, aging, and aging-associated diseases. Recent advancement in available genomic sequence information has provided an infrastructure for the emerging field of proteomics (24Yates III, J.R. Mass spectrometry and the age of the proteome.J. Mass Spectrom. 1998; 33: 1-19Google Scholar, 25Liebler D.C. Proteomic approaches to characterize protein modifications: new tools to study the effects of environmental exposures.Environ. 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A "shotgun" approach based on the separation capacity of liquid chromatography instrumentation becomes possible if the number of proteins is not overwhelmingly large, such as from a defined subproteome (29McDonald W.H. Yates III, J.R. Shotgun proteomics: integrating technologies to answer biological questions.Curr. Opin. Mol. Ther. 2003; 5: 302-309Google Scholar, 30Link A.J. Eng J. Schieltz D.M. Carmack E. Mize G.J. Morris D.R. Garvik B.M. Yates III, J.R. Direct analysis of protein complexes using mass spectrometry.Nat. Biotechnol. 1999; 17: 676-682Google Scholar). Compared with cell lysates, the subproteome of secreted proteins is less complex and allows meaningful identification of proteins using the shotgun approach. Based on the fact that a mass spectrometer measures a molecule based on its abundance given a mixture of different molecules, methods have been developed not only to identify the nature of proteins in a mixture but also to compare relative levels of a protein between different samples (24Yates III, J.R. Mass spectrometry and the age of the proteome.J. Mass Spectrom. 1998; 33: 1-19Google Scholar, 27Yates III, J.R. Mass spectrometry. From genomics to proteomics.Trends Genet. 2000; 16: 5-8Google Scholar, 28Yates III, J.R. Mass spectral analysis in proteomics.Annu. Rev. Biophys. Biomol. Struct. 2004; 33: 297-316Google Scholar, 31Pasa-Tolic L. Harkewicz R. Anderson G.A. Tolic N. Shen Y. Zhao R. Thrall B. Masselon C. Smith R.D. Increased proteome coverage for quantitative peptide abundance measurements based upon high performance separations and DREAMS FTICR mass spectrometry.J. Am. Soc. Mass Spectrom. 2002; 13: 954-963Google Scholar, 32Yates III, J.R. Link A.J. Schieltz D. Direct analysis of proteins in mixtures. Application to protein complexes.Methods Mol. Biol. 2000; 146: 17-26Google Scholar, 33Gygi S.P. Rist B. Aebersold R. Measuring gene expression by quantitative proteome analysis.Curr. Opin. Biotechnol. 2000; 11: 396-401Google Scholar). Protease digestion of the proteins from conditioned media followed by analysis of the resulting peptides using ESI-LC-MS/MS allowed us to measure the alteration of secreted protein factors following oxidative stress. Chemicals were purchased from Sigma unless otherwise indicated. Stabilized H2O2 (H-1009, Sigma) was used, and the concentration of the stock was verified by absorbency at 240 nm. HCA3 human dermal fibroblasts at the population doubling level (PDL) 20 were obtained from Dr. Olivia Periera-Smith. These cells typically reach replicative senescence after PDL 80 and were used for this study at PDL 26–40. HCA3 cells were subcultured weekly in 10 ml of Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal bovine serum, 50 units/ml penicillin, and 50 μg/ml streptomycin (Invitrogen) at a seeding density of 1 × 106 cells/100-mm Falcon dish. Under these conditions, the cells reached confluence 6–7 days after subculture. HCA3 cells were seeded at a density of 2 × 106/100-mm dish 5 days before treatment. At the time of H2O2 treatment, the cells had reached confluence, and the density of cells was 10.48 ± 0.85 × 106. Confluent cells were treated with 600 μm H2O2 in a 100-mm dish containing 10 ml of medium. This dose is equivalent to ∼0.6 pmol of H2O2/cell. The dose less than 0.85 pmol/cell has been shown to be non-lethal and induce premature senescence in early passage HDFs (13Chen Q. Ames B.N. Senescence-like growth arrest induced by hydrogen peroxide in human diploid fibroblast F65 cells.Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4130-4134Google Scholar). For the dose-response experiments, cells were treated with H2O2 from 150 to 600 μm. After 2-h incubation in the presence of H2O2, cells were placed in fresh DMEM containing 10% (v/v) FBS and were allowed to develop a senescent phenotype in 3 days. cis-Platin (50 μm), hydroxyurea (50 mm), colchicine (100 μm), l-mimosine (750 μm), rhodamine (1 μm), dithiothreitol (1 mm), N-ethylmaleimide (8 μm), and H2O2 (250 μm) were used to treat confluent cultures of HCA3 cells for 4 h. The cells were then placed in fresh DMEM containing 10% (v/v) FBS for 3 days of culture. For treatment with retinoic acid (1 μm), confluent cells were incubated in the medium containing this drug for 3 days without medium change. To collect conditioned media of HDFs for proteomic analysis, culture media for HCA3 cells in 100-mm dishes were removed 3 days after H2O2 or other toxicant treatment. The cells were rinsed two times in DMEM and were placed in 6 ml of fresh DMEM containing 0% FBS for 3 days of culture. The serum-free conditioned media were collected, filtered through a 0.45-μm filter to remove cell debris, dialyzed against 0.01 n NH4HCO3, and concentrated 100 times down in volume using a speed vacuum concentrator. Protein concentration in the concentrated medium was determined by the Bradford method according to the manufacturer's instructions (Bio-Rad). The concentrated media were digested overnight with trypsin at a 50:1 ratio, i.e. 1 μg of trypsin/50 μg of protein (34Shevchenko A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.Anal. Chem. 1996; 68: 850-858Google Scholar). The resulting peptides were analyzed by a ThermoFinnigan (San Jose, CA) LCQ Classic quadrupole ion trap mass spectrometer equipped with a Michrom (Auburn, CA) MAGIC2002 HPLC instrument and a nanospray ion source (University of Washington). A mixture of peptides equivalent to 7 μg of proteins was loaded onto a 10-cm-long capillary column with a diameter of 365 μm (outer diameter) or 100 μm (inner diameter). The capillary column was generated using a P2000 capillary puller (Sutter Instrument Co., Novato, CA) and was packed with 5–6 cm of Vydac C18 material. Samples were eluted at a flow rate of 200–300 nl/min into a mass spectrometer using reversed phase solvent conditions. Tandem MS spectra of peptides were analyzed with the Turbo SEQUEST software that assigns peptide sequences to the spectra (28Yates III, J.R. Mass spectral analysis in proteomics.Annu. Rev. Biophys. Biomol. Struct. 2004; 33: 297-316Google Scholar). The software was used to search for known human proteins in the non-redundant database from the National Center for Biotechnology Information (NCBI). Proteins from conditioned media were separated by SDS-polyacrylamide gel electrophoresis using a mini-Protean II electrophoresis apparatus (Bio-Rad) run at 90 V. The separated proteins were transferred to PVDF membranes (Millipore, Bedford, MA) by electrophoresis. The membrane was incubated with antibodies against IGFBP-6 (1:200 dilution; polyclonal, H-70, Santa Cruz Biotechnology, Inc.), fibronectin (1:2000 dilution; polyclonal, F-3648, Sigma), or matrix metalloproteinase 2 (MMP-2) (1:2000 dilution; polyclonal, AB19015, Chemicon International). The bound antibody was detected using a secondary antibody conjugated with horseradish peroxidase (1:8000; Zymed Laboratories Inc.) for an ECL reaction. Total RNA was extracted from cells with TRIzol (Invitrogen) and was used as a template for RT-PCR. Following the RT reaction using 2 μg of total RNA from each sample, 3 μl of the 35-μl RT reaction mixture was used for each PCR. PCR for IGFBP-6 was carried out in 28 cycles with the primer pair 5′-GAATCCAGGCACCTCTACCA-3′ and 5′-GGTAGAAGCCTCGATGGTCA-3′ at 94 °C for 30 s, 62 °C for 30 s, and 72 °C for 30 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene and as an internal control. PCR for GAPDH was carried out using the primers 5′-CGTCTTCACCATGGAGA-3′ and 5′-CGGCCATACGCCCACAGTTT-3′ at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s for 30 cycles. The PCR products were detected by agarose gel electrophoresis and ethidium bromide staining. Blood was collected from BL6 × 129SF1 J mice at 5–7 weeks (young) or 16–18 months (old) of age from the abdominal vena cava. Total blood (200–300 μl) from an animal was centrifuged at 2000 rpm at 4 °C to remove blood cells and sediments. The remaining plasma (40–45% of total blood) is clear and transparent. For doxorubicin (Dox) treatment, 5–7-week-old male (18–22 g) mice were treated with Dox via intraperitoneal injection at the dose of 4 mg/kg (10 ml/kg of body weight) according to the protocol described by Sun et al. (35Sun X. Zhou Z. Kang Y.J. Attenuation of doxorubicin chronic toxicity in metallothionein-overexpressing transgenic mouse heart.Cancer Res. 2001; 61: 3382-3387Google Scholar). The animals were injected twice a week for a total of 10 injections. Control animals were injected with saline at the same volume. The animals were not treated for 2 weeks between the first four injections and the last six injections to allow for recovery of bone marrow depression. The blood from the animals was collected 2 weeks after the last injection. Early passage HCA3 human skin fibroblasts were treated with H2O2 and were allowed to develop a stable senescence-like phenotype. To determine the effect of H2O2 treatment on protein factors secreted, we compared the profiles of proteins in the conditioned medium from control versus H2O2-treated cells. Serum-free conditioned media were collected for concentration and protease digestion. The resulting peptide mixtures were injected into the ESI-LC-MS/MS instrument. A representative total ion current chromatogram from the conditioned medium of control or H2O2-treated cells is shown in Fig. 1. The mass spectrometer was operated in a data-dependent MS to MS/MS switching mode so that precursor peptide ions detected in a MS survey scan trigger an ion fragmentation for obtaining MS/MS spectra for each of the precursor peptide ions. MS/MS spectra indicate primarily fragment ions originating from either the C terminus (y ion series) or N terminus (b ion series) of a peptide and were searched against a human protein sequence database using the Turbo SEQUEST software. This software searches the entries against all peptide sequences in the database and assigns correlation scores for the probability of matches. The judgment of a confident match is largely based on two parameters: "Xcorr" and "Ions." Xcorr represents the cross-correlation value computed from the experimental MS/MS spectrum when compared with the theoretical candidate peptide MS/MS spectrum, while Ions stands for the number of matched ions in the experimental MS/MS spectrum with the total number of possible sequence ions theoretically predicated for the peptide sequence. Based on the recommendation of the SEQUEST software, selection criteria for a confident protein identification include Xcorr ≥ 1.8 for +1 ions, Xcorr ≥ 2.5 for +2 ions, and Xcorr ≥ 3.5 for +3 ions. In all cases, the value of Ions must be greater than 50%. Table I lists proteins identified by the criteria described above in the conditioned media collected from three independent experiments. The reproducibility of the analytical method is 80–90% between different runs with the same sample. Several proteins consistently showed up in all three experiments in both control and H2O2-treated groups (Table I). These proteins include collagen α1(IV) chain, collagen α2(I) chain, lumican, fibronectin, and MMP-2 (gelatinase A or 72-kDa type IV collagenase). Table II summarizes the scores of Xcorr and Ions and the number of peptides identified for these proteins. IGFBP-6 appeared in the conditioned medium of H2O2-treated cells from all three experiments (Table II). The MS/MS spectra and SEQUEST Flicka protein information output on IGFBP-6 identified from three experiments are shown in Fig. 2, A–D. Each MS/MS spectrum has a high ion-matching ratio. Despite the fact that only one peptide was identified in each experiment for IGFBP-6, the MS/MS spectra and Xcorr and Ions scores all provide high confidence in the detection. This suggests that IGFBP-6 may appear only in the conditioned medium of H2O2-treated cells or that H2O2-treated cells produce more IGFBP-6 than control untreated cells.Table IList of proteins identified from all three experimentsControl fibroblastsH2O2-treated fibroblastsExp. 1Collagen α1(I) chainCollagen α1(I) chainCollagen α1(VI) chainCollagen α1(VI) chainCollagen α2(I) chainCollagen α2(I) chainCollagen α2(V) chainFibronectinFibronectinFibulin 1LumicanLumicanMMP-2MMP-2Plasma protease C1 inhibitor precursorTIMP-1β2-MicroglobulinTIMP-2IGFBP-6Protein kinase C, μ typeβ2-MicroglobulinExp. 2Collagen α1(I) chainCollagen α1(VI) chainCollagen α1(VI) chainCollagen α2(I) chainCollagen α2(I) chainFibronectinComplement C1r componentFibulin 1FibronectinLumicanFibulin 1MMP-1LumicanMMP-2MMP-2TIMP-1TIMP-1IGFBP-6Quiescin Q6Quiescin Q6Superoxide dismutase 3ClusterinTetranectin (plasminogen-binding protein)Plasminogen activator inhibitorHuman complement C1s proteaseProcollagen C-endopeptidase enhancerZinc finger protein 335Decorin isoform a preproproteinDecorin isoform a preproproteinComplement component 1, s subcomponentVimentinExp.3Collagen α1(I) chainCollagen α1(I) chainCollagen α1(VI) chainCollagen α1(VI) chainCollagen α2(I) chainCollagen α2(I) chainFibronectinFibronectinFibulin 1Fibulin 1LumicanLumicanMMP-1MMP-1MMP-2MMP-2TIMP-1TIMP-1IGFBP-4TIMP-2IGFBP-7IGFBP-6Procollagen C-endopeptidase enhancerQuiescin Q6Complement C1r component precursorProcollagen C-endopeptidase enhancerAutotaxinComplement C1r component precursorGlia-derived nexin precursorPigment epithelium-derived factor precursorVimentinVimentin Open table in a new tab Table IISummary of proteins consistently appearing in all three experimentsNameExp.GroupXcorrIonsNo. of peptides%Proteins appearing in both control and H2O2 groups Collagen α1(VI) chain1Ctr2.6667.71H2O22.6450.02Ctr3.04 ± 0.5156.9 ± 6.85H2O24.30 ± 1.0960.1 ± 1.533Ctr3.21 ± 0.5759.4 ± 7.43H2O24.19 ± 0.8868.1 ± 9.73 Collagen α2(I) chain1Ctr4.01 ± 0.8063.7 ± 7.741H2O23.63 ± 0.4061.2 ± 8.9102Ctr3.54 ± 0.8256.7 ± 4.214H2O23.66 ± 0.9156.7 ± 8.273Ctr3.38 ± 0.5158.3 ± 6.36H2O23.72 ± 0.8257.2 ± 5.712 Fibronectin1Ctr3.7757.141H2O23.24 ± 0.6361.5 ± 11.832Ctr3.07 ± 0.4460.0 ± 10.95H2O23.53 ± 0.3163.1 ± 15.153Ctr3.30 ± 0.5161.7 ± 8.17H2O23.17 ± 0.1861.2 ± 10.09 Lumican1Ctr3.17 ± 0.1271.4 ± 19.32H2O23.257212Ctr3.61 ± 0.8667.1 ± 12.15H2O23.49 ± 0.7066.0 ± 9.443Ctr3.70 ± 1.0372.0 ± 8.53H2O23.49 ± 0.7372.5 ± 9.05 MMP-21Ctr3.1752.81H2O22.97 ± 0.1069.0 ± 19.022Ctr3.37 ± 0.6567.9 ± 9.53H2O23.17 ± 0.5762.8 ± 4.833Ctr3.54 ± 0.6765.0 ± 7.45H2O23.17 ± 0.3466.1 ± 10.68Proteins appearing in H2O2 group alone IGFBP-61H2O22.759412H2O25.137913H2O24.63751 Open table in a new tab Fig. 2MS/MS spectra of IGFBP-6 peptide and SEQUEST Flicka output for detected IGFBP-6 peptides. MS/MS spectra of the IGFBP-6 peptide detected in three independent experiments are shown in A–C. The bold letters indicate the detected b and y ions matching the predicted ion mass in the database. In D, the SEQUEST Flicka protein information page for IGFBP-6 shows the fragments detected.View Large Image Figure ViewerDownload (PPT) The LC-MS/MS-based shotgun proteomic method led to the identification of IGFBP-6 from H2O2-treated cells and several proteins, including fibronectin and MMP-2, from both control and H2O2-treated cells. To verify these results, we performed Western blot analyses using conditioned media collected from control or H2O2-treated cells. The data indicate that there is no significant difference in the level of MMP-2 and fibronectin in the conditioned media between control and H2O2-treated cells (Fig. 3). With cell lysates, a minor elevation of fibronectin was detected with H2O2 treatment. MMP-2 was not detected with cell lysates, suggesting that MMP-2 is a secreted protein. IGFBP-6 is known to be O-glycosylated at 5 amino acid residues (Thr126, Ser144, Thr145, Thr146, and Ser152) (36Bach L.A. Insulin-like growth factor binding protein-6: the "forgotten" binding protein?.Horm. Metab. Res. 1999; 31: 226-234Google Scholar, 37Bach L.A. Thotakura N.R. Rechler M.M. Human insulin-like growth factor binding protein-6 is O-glycosylated.Biochem. Biophys. Res. Commun. 1992; 186: 301-307Google Scholar). Western blot analyses of conditioned media showed two bands of IGFBP-6 (Fig. 3). Presumably the lower molecular weight band represents the non-glycosylated form, and the higher molecular weight band represents the glycosylated form of IGFBP-6. With either form, IGFBP-6 protein showed an elevation in the conditioned medium of H2O2-treated cells (Fig. 3). IGFBP-6 protein from cell lysates showed a molecular weight between the two forms present in the conditioned medium (Fig. 3), suggesting that the protein is partially glycosylated. The level of IGFBP-6 protein from cell lysates did not show a dramatic increase with H2O2 treatment. These data demonstrate that we are able to verify the data on IGFBP-6 obtained by LC-MS/MS with Western blot analyses. To characterize the induction of IGFBP-6 by H2O2 treatment, we performed dose-response studies to measure levels of IGFBP-6 mRNA in cell lysates and to determine levels of IGFBP-6 protein in the conditioned medium. HCA3 cells were treated with 0, 150, 300, 450, or 600 μm H2O2. RNA and conditioned media were collected in parallel from the same set of samples. Western blot analyses showed a dose-dependent increase of IGFBP-6 protein, both glycosylated and non-glycosylated forms, in the conditioned medium of H2O2-treated cells (Fig. 4A). In comparison, no significant changes of fibronectin and MMP-2 at the protein level were detected in the conditioned media of HCA3 cells treated with various doses of H2O2. Consistent with the protein measurement data, semiquantitative RT-PCR showed a dose-dependent increase of IGFBP-6 mRNA with H2O2 treatment (Fig. 4B). We extended our study by asking whether or not IGFBP-6 can serve as a biomarker of oxidative injury. HCA3 cells were treated with a variety of chemicals, including a DNA-damaging agent (cis-platin), DNA polymerase inhibitor (hydroxyurea), mi