Truncation, Deamidation, and Oxidation of Histone H2B in Cells

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Truncation, Deamidation, and Oxidation of Histone H2B in Cells Cultured with Nickel(II) Aldona A. Karaczyn, Filip Golebiowski, and Kazimierz S. Kasprzak* Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Frederick, Maryland 21702 Received May 4, 2005

Molecular mechanisms of nickel-induced carcinogenesis include interactions of Ni(II) cations with histones. Previously, we demonstrated in vitro and in cells that Ni(II) cleaved off the -SHHKAKGK C-terminal motif of histone H2A. In the present study, Western blotting of histones isolated from rat and human cell lines, cultured for 3-5 days with 0.05-0.5 mM Ni(II), revealed time- and dose-dependent appearance of a new band of histone H2B. This effect was also induced by Co(II), but not by Cu(II), Cd(II), and Zn(II). Mass spectrometry and amino acid sequencing of proteins from the new band allowed for identification of two derivatives of the major variant of histone H2B. The larger protein was histone H2B lacking 16 N-terminal amino acids. The smaller one was histone H2B which, in addition to being shortened at the N-terminus, had nine amino acids deleted from its C-terminus. At both termini, the truncation occurred between lysine and alanine in the two identical -KAVTK- repeats of histone H2B. Also, the truncated H2B proteins had their Q22 residues deamidated and M59 and M62 residues oxidized to sulfoxides, a signature of oxidative stress. The truncation did not concur with apoptosis. Its mechanism involved activation by Ni(II) treatment of specific nuclear proteolytic enzymes belonging to the calpain family. The terminal tails of core histones participate in structuring chromatin and regulating gene expression. Therefore, the observed truncation and other modifications of histone H2B may assist in Ni(II) carcinogenesis through epigenetic mechanisms.

Introduction Occupational exposure to nickel compounds is associated with respiratory tract toxicity and carcinogenesis (reviewed in refs 1, 2). Mechanistic concepts proposed for nickel carcinogenesis include genotoxic effects such as promutagenic DNA damage (2, 3) and epigenetic effects associated with the impairment of DNA repair mechanisms (4, 5), interference with Ca(II) signaling (6), or induction of the hypoxic response (7, 8). Some of these effects may result from Ni(II) attack on chromatin mediated through binding to histones (9). Previously, we have found that Ni(II) can bind to the -CAIH- motif of histone H3 (10) and the -TESHHK- motif of histone H2A (11). Others have later implicated the -AKRHRKmotif of histone H4 (12) and the -ELAKHA- motif of histone H2B (13) as possible Ni(II)-binding sites. As we have also found, Ni(II) bound to histone H2A assists in hydrolysis of the -TESHHK- motif between the glutamic acid and serine residues in both test tube and cultured cells experiments that leads to the emergence of a new truncated version of histone H2A (q-H2A)1 (14, 15). Investigating the identity of q-H2A in cellular histone * To whom correspondence should be addressed: Kazimierz S. Kasprzak, Bldg 538, Room 205E, NCI at Frederick, Frederick, MD 21702-1201. Tel: 301 846-5738. Fax: 301 846-6946. E-mail: kasprkaz@ mail.ncifcrf.gov. 1 Abbreviations: AK295, Z-L-aminobutyric acid-CONH(CH ) -mor2 3 pholine; MALDI-TOF/TOF MS, matrix-assisted laser desorption/ ionization and tandem mass spectrometry; Ni(II), divalent nickel; PBS, phosphate-buffered normal saline, pH 7.4; PD 150606, 3-(4-iodophenyl)2-mercapto-(Z)-2 propenoic acid; PVDF, polyvinylidene fluoride; q-H2B, truncated histone H2B.

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extracts, we noticed that in one-dimensional gels the q-H2A band was not homogeneous (15). The present study has been, therefore, aimed at identification of the other protein(s) comigrating with q-H2A. Mass spectral analysis of tryptic digests of the q-H2A band revealed the presence of peptides originating not only from histone H2A, but also from H2B, thus indicating the possibility that the latter, like H2A, might undergo modification which, judging by the electrophoretic mobility, looked like truncation. Test tube experiments, that we performed first, showed a complete resistance of commercial (human recombinant) histone H2B to Ni(II)-mediated hydrolysis under conditions which would facilitate H2A truncation. So, the suspected H2B degradation appeared to be a strictly cellular phenomenon. To investigate it more closely, in the present study, rat kidney epithelial (NRK52E) cells and human lung (HPL1D and 1HAEo-) cells were cultured in media containing noncytotoxic concentrations of Ni(II) or, for a comparison, other transition metals, Co(II), Cu(II), Zn(II), and Cd(II). The histones were isolated from cell nuclei and analyzed by gel electrophoresis, Western blotting, LC/MS, and amino acid sequencing. As we found, Ni(II) and to a lesser extent also Co(II) were indeed capable of inducing histone H2B truncation. The scission occurred between lysine and alanine residues at two identical amino acid sequences, -KAVTK-, present at both the N- and C-terminal sides of this histone. This suggested the involvement of a specific proteolytic enzyme which was identified, using specific inhibitors, as belonging to the calpain family. In addition, the truncated H2B was found to be oxidized at

This article not subject to U.S. Copyright. Published 2005 by the American Chemical Society Published on Web 10/21/2005

Ni(II)-Induced Modifications of Histone H2B

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Figure 1. Percent of adherent cells excluding Trypan blue. Means ( SD (n ) 4) for 3 days or range (n ) 2) for 5 days. Numbers under the bars indicate metal concentrations (µM). For this assay, cells were seeded at day -1 in a 96-well 12.5 cm × 85-cm plastic plate and allowed to grow for 1 day in metal-free media, the same as those used for the flask cultures (see Materials and Methods). The metals were added on day 0 into two wells per each metal concentration/time point. The 5-day cultures had their metal-containing or metal-free (untreated controls) media changed at day 3. The harvested wells were washed twice with PBS, and the adherent cells were collected with the help of trypsin, stained in PBS with 1:1 0.4% Trypan blue for 1 min, and counted in a hemocytometer. Asterisks denote treatments resulting in histone H2B truncation and other modifications.

its two methionine residues and deamidated at two glutamine residues, a very likely result of Ni(II)-induced oxidative stress. Mass spectral analysis suggested the presence of other modifications, as well.

Materials and Methods Cell Culture. In the experiments, we used normal rat kidney epithelial-like cells (NRK-52E; ATCC-CRL 1571), human pulmonary cells (HPL1D; a generous gift of Dr. T. Takahashi, Nagoya, Japan), and human airway epithelial cells [1HAEo-; obtained from Dr. D. C. Gruenert, University of California, San Francisco, CA (16)]. The cells were cultured in 162 cm2 flasks at 37 °C, under 5% CO2-containing air, for 3 or 5 days, in DMEM or EMEM media (NRK-52E and 1HAEo- cells, respectively), and F-12 medium (HPL1D cells), containing 5% (DMEM) or 10% (EMEM and F-12) fetal bovine serum, to which Ni(II) acetate (J. T. Baker Chemical Co., Phillipsburg, NJ) was added, starting at approximately 70% of cell confluence, to make 0.05-0.5 mM Ni(II) final concentrations. For the 5-day cultures, media were changed after 3 days. The other metals were tested as chlorides (from Aldrich Chemical, Milwaukee, WI) at concentrations of similar toxicity: 0.05-0.2 mM for Co(II) and Zn(II), 0.01-0.04 mM for Cu(II), and 0.001-0.01 mM for Cd(II). At termination of exposure, the media were discarded, the cultures were rinsed twice with ice-cold PBS, pH 7.4, and only cells attached to the bottom of the flasks were collected for histone analysis. The untreated control cells were harvested when the cultures approached 95% confluence. Although Ni(II) and the other metals gradually halted the cell proliferation in a dose-dependent manner, as found by the Trypan blue exclusion method, the adherent NRK-52E cells remained viable in over 95% for the lowest to 75-90% for the highest metal ion concentrations tested, depending on the metal. The slower growing 1HAEo-

and, especially, HPL1D cells were somewhat more sensitive to the metals, with the viability of the adherent cells dropping to 50% at single cases of the highest metal concentrations (Figure 1 and Supporting Information). Extraction of Histones and Gel Electrophoresis. Histones were isolated from cell nuclei by acid extraction with 0.5 M HCl as described previously (17), and separated by onedimensional (1-D) gel electrophoresis using a 10% or 4-12% gradient NuPage SDS Bis-Tris gels (Invitrogen, Carlsbad, CA) under reducing conditions provided by 10% v/v β-mercaptoethanol. Total protein concentration in the extracts was determined using the Bradford method with albumin calibration (Pierce, Rockford, IL). Amounts ranging from 10 to 20 µg of total protein were loaded on the gels and separated in 2-(-Nmorpholino)ethano-sulfonic acid (MES) running buffer at 70-150 V for 1.5 h. Two-dimensional (2-D) electrophoresis was used for fine separation of H2B fractions for mass spectral analysis. This was performed as described earlier for the modified acetic acid-urea-triton system (AUT) (18). Acetic acid-urea minislab 15% polyacrylamide gels were used for the first dimension, and 4-12% Bis-Tris gels were used for the second dimension. Histone proteins (50 µg) were dissolved in loading buffer (8 M urea, 1 M acetic acid, and 1 M β-mercaptoethanol) and incubated for 2-3 h at 37 °C. The protein bands in gels were visualized with Simple Blue staining (Invitrogen). Western Blotting. Proteins separated by 1-D or 2-D gel electrophoresis were transferred at 4 °C onto nitrocellulose membranes (Amersham Pharmacia Biotech, Piscataway, NJ) in 25 mM 3-[(1,1-dimethyl-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (AMPSO) buffer, pH 9.5, at 35 V for 1.5 h. The histone H2B bands were visualized with the use of a series of primary rabbit anti-H2B antibodies from Upstate Biotechnology (Lake Placid, NY) or Santa Cruz Biotechnology (Santa Cruz, CA). Anti-rabbit antibodies labeled with horseradish

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peroxidase (Cell Signaling Technology, Beverly, MA) served as the secondary antibodies together with Super Signal (Pierce) reagents to detect the chemiluminescent signal. LC/MS Analysis. Histone extracts and commercial histone standards were separated by 1-D and 2-D gel electrophoresis and bands were visualized by Simple Blue staining (Invitrogen). Protein bands were excised from the gel and destained in aqueous solution of 50% methanol and 10% acetic acid for 4-6 h at 25 °C with gentle vortexing. After rinsing with deionized water, the bands were dried and then extracted with 30 µL of formic acid/water/2-propanol (1:3:2, v/v/v) (19). The resulting mixture was vortexed for 4 h at room temperature and centrifuged through 30 kDa filters (Millipore Corp., Bedford, MA) at 2000g for 15 min. The HP 1100 Series LC/MSD instrument (Hewlett-Packard Co., Palo Alto, CA) was used for on-line LC/MS analysis of the protein fractions. For the LC separation, a reverse phase column, ZORBAX 300SB-C3 (Agilent Technologies, Palo Alto, CA) was used with linear gradient of 5% v/v aqueous acetic acid to 100% acetonitrile (0-100% acetonitrile in 30 min). The protein fractions were eluted at a flow rate of 0.2 mL/min, detected at 280 nm, and introduced on-line to the ESI source. Mass-spectral sample analysis was performed using software provided by the manufacturer, Agilent Technologies. MALDI-MS Analysis. Protein spots were excised from gels, destained in 10% acetic acid, and macerated with a scalpel. Ingel trypsin (Promega, Madison, WI) digestion was performed as previously described (20). Briefly, the gel pieces were transferred to a microfuge tube and washed with 50% acetonitrile/25 mM ammonium bicarbonate. Protein gel spots were digested overnight with 100 ng of trypsin at 37 °C, and the resulting peptides were eluted with 50% acetonitrile/5% formic acid in deionized water. After concentration in a Speed Vac Concentrator (Savant, Holbrook, NY), portions (typically 1/20) of the unseparated tryptic digests were cocrystallized in a matrix of R-cyano-4-hydroxycinnamic acid and analyzed using a PerSeptive Biosystems DE-PRO mass spectrometer (Foster City, CA) equipped with delayed extraction operated in the reflector mode. Spectra were internally calibrated using trypsin autoproteolysis peaks, and the accuracy of mass measurements of all peptides was in the range of (0.05 Da. The MS/MS data were acquired as described before (19) on a matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) mass spectrometer (4700 Proteomics Analyzer, Applied Biosystems, Foster City, CA). MS and MS/MS spectra were interpreted to yield protein identities using the MS-Fit and MS-Tag programs (http://prospector.ucsf.edu) (21). Amino Acid Sequencing. The proteins were transferred onto PVDF membranes (Pall Gelman Laboratory, Ann Arbor, MI) and stained with Simple Blue. N-terminal sequence determination by Edman degradation was carried out in an Applied Biosystems (Foster City, CA) model 494 LLC Procise sequenator. Phenylthiohydantoin derivatives of amino acids were analyzed on-line with an Applied Biosystems model 785A/140C/610A analyzer. Tests for Apoptosis. To check possible association of the observed truncation of histone H2B with apoptosis, we tested the cells for signs of apoptosis under conditions inducing the truncation, using the Annexin-V binding assay in combination with 7-AAD (7-amino-actinomycin D) or PI (propidium iodide; BD Biosciences, San Diego, CA), and the caspase 3-mediated PARP [poly(ADP ribose) polymerase] protein cleavage assay (Trevigen, Gaithersburg, MD). The positive control cells from these assays, treated with staurosporine (Upstate) or adriamycin (Calbiochem, San Diego, CA), respectively, were also tested for the presence of q-H2B by gel electrophoresis/Western blotting, as described above. For the Annexin-V assay, cells grown in the absence and presence of 0.1-0.5 mM Ni(II) for 3 and 5 days, attached to the bottom of the flasks, were collected with trypsin and washed twice with ice-cold PBS. Supernatants were discarded, and cells were resuspended in 0.3 mL of ice-cold binding buffer to obtain a suspension of 106 cells/mL. Positive controls were prepared

Karaczyn et al. from cells treated with 0.5-10 µM staurosporine for 6 h and collected as above. One microliter of Annexin-V labeled with a fluorescent marker phycoerythrin (PE) or fluorescein isothiocyanate (FITC), and 2 µL of 7-AAD or PI, respectively, were added to each 0.3-mL sample of the cell suspension. Additionally, samples with only Annexin-V and only 7-AAD or PI were prepared as binding controls. Tubes were incubated for 10 min at room temperature without exposure to light prior to analysis by flow cytometry, using the BD LSR1 instrument (BectonDickinson, San Diego, CA). Twenty thousand cells were analyzed per sample. For the PARP protein cleavage assay, cells were grown without or with 0.05-0.5 mM Ni(II) for 5 days. The positive control cells were cultured with 0.3 µM adriamycin for 5 h. Cultures were harvested by scraping, and cellular proteins were extracted with RIPA buffer (0.05M Tris-HCl, pH 7.4, 0.15 M NaCl, 0.25% deoxycholic acid, 1% NP-40, and 1 mM EDTA). Samples containing 40 µg of total protein were analyzed by Western blotting, using an antibody that recognized both the intact (∼116 kDa) and cleaved (∼85 kDa) forms of PARP protein (Upstate). Search for the Enzyme Causing Histone H2B Truncation. Since the tests for apoptosis were negative, our search for proteolytic enzymes involved in the truncation was focused on proteinases whose major function is to modify rather than fully degrade proteins. A literature search for such enzymes with a specific affinity for the -KAVTK- sequence in histone H2B, found in our mass-spectrometric and sequencing experiments (see Results), pointed at calpains as the most likely culprits (22, 23). Therefore, we tested the effects of specific calpain inhibitors, PD150606 and AK295 (EMD Biosciences, San Diego, CA) on the truncation of histone H2B by nickel. In these experiments, cells were first grown with 0.01 mM PD150606 or 0.005 and 0.05 mM AK295 (or without the inhibitors to obtain the Ni(II)only controls) for 6 h, then Ni(II) was added to make 0.25 or 0.5 mM concentration, and the cells were cultured for 18 h. This cycle was repeated for 5 days. Untreated control cells had their media replaced every 24 h. Cell harvest was followed by histone extraction, gel separation, and Western blotting analysis, as above.

Results Histone Separation and Western Blotting. As shown in Figure 2, Western blotting of the histone separation gels revealed that treatment of cells with Ni(II) resulted in the advent of a new protein band (q-H2B) reactive with anti-histone H2B antibodies and having higher electrophoretic mobility than that of the wild-type H2B (wt-H2B). Three days of incubation revealed the formation of q-H2B in HPL1D and NRK-52E cells exposed to 0.5 mM Ni(II), whereas after 5 days, this band was present in cells of all three lines grown with 0.5 mM Ni(II) and in HPL1D and NRK cells also after culture with lower Ni(II) concentrations. Thus, the emergence of q-H2B depended on cell type, Ni(II) concentration, and time of exposure. Figure 3 shows the q-H2B band separated on a 2-D gel. Among the other metals tested, only Co(II) caused the appearance of the q-H2B band in all three cell lines, whereas Cu(II), Zn(II), and Cd(II) did not produce this effect (Figure 4). Mass Spectral Analysis and N-Terminal Sequencing. To further characterize the q-H2B protein(s), various mass spectral and sequencing techniques were used. The LC/MS analysis of the q-H2B band excised from 1-D and 2-D gels revealed the presence of two comigrating proteins with molecular masses of 12 206 and 11 324 Da. The band was next analyzed by tryptic digestion and mass spectrometry. As shown in Table 1, the MALDIMS analysis yielded six peptides with molecular masses


Figure 2. Nickel(II) exposure causes histone H2B truncation. Human pulmonary HPL1D (A) and 1HAEo- (B) cells, and rat renal epithelial NRK-52E cells (C) were cultured with 0.1-0.5 mM Ni(II) for 3 or 5 days. Histones were extracted with acid from cell nuclei and separated by 1-D gel electrophoresis followed by Western blotting with anti-histone H2B antibodies and visualization with secondary antibodies labeled to produce a chemiluminescent signal, as described under Materials and Methods. The truncated histone H2B band (q-H2B) is visible beneath the wild-type histone H2B band. Ctrl, cells cultured without Ni(II).

Figure 3. The truncated histones H2B (q-H2B) migrate in a single band in 2-D electrophoresis. For fine separation of histones extracted from cell nuclei, acetic acid-urea minislab 15% polyacrylamide gels were used for the first dimension (AU), and 4-12% Bis-Tris gels were used for the second dimension (SDS-PAGE). The protein bands from NRK-52E cells were visualized by Western blotting as described in Figure 1 and under Materials and Methods. Ctrl, cells cultured without Ni(II).

ranging from 901.5 to 1775.7 Da, all representing the highly conserved amino acid sequences unique for all major variants of mammalian histone H2B. The identities of the 953.6, 1265.6, and 1775.7 Da peptides were confirmed by MALDI-MS/MS. The latter peptide appeared to be oxidized on both methionine residues. The N-terminal sequencing of the q-H2B proteins isolated from NRK-52E and 1HAEo- cells revealed the following amino acid sequence: AVTKAEKKDGKK-, denoting that both proteins were derived from wt-H2B by deletion of the first 16 amino acids from its N-terminal tail (compare the sequences in Table 1). Very interestingly, the highly evolutionarily conserved glutamine residue Q22, present solely in wt-H2B extracts from untreated cells and predominantly in Ni(II)-exposed cells, was fully deamidated to glutamic acid in histone q-H2B (E6 in the shorter N-terminal tail of q-H2B; Table 1). Although the C-terminal sequences of the two individual q-H2B proteins could not be analyzed due to technical difficulties (comigration and low abundance of the smaller protein), these proteins could be identified based on the combination of the results of N-terminal sequencing and mass spectral analysis of the whole proteins and their tryptic digests. Thus, considering the published amino acid sequence of rat histone H2B (see footnote to Table 1) and knowing the N-terminal amino acids and the modifications of some residues of q-H2B, found in the


present experiment (Q22 deamidation; M59 and M62 oxidation), we can calculate the molecular mass of the larger q-H2B protein as 12 205 Da, that is, very close to that found in the present experiment. To get the exact mass of 12 206 Da, as measured, we had to assume deamidation of one more glutamine residue, perhaps in the inner peptide, -SREIQTAVR-, which did not show up in the tryptic digests. The smaller 11 324-Da q-H2B protein could be derived from the larger q-H2B by removal of the last nine amino acid residues, -AVTKYTSSK, from its C-terminal end, and acetylation of two lysine residues. It is important to notice that the -KAVTK- motif in which the second cutting is postulated to occur is identical with that at the N-terminus where the cutting was demonstrated by N-terminal sequencing. Tests for Apoptosis. The results of the flow cytometric assays did not reveal any significant enhancement of the level of apoptosis in Ni(II)-treated cells, including those with q-H2B, over the level of apoptosis observed in the respective control cells (Figure 5A and Supporting Information). Likewise negative was the endogenous PARP protein truncation test that would reveal PARP cleavage by caspase 3 if the latter were activated by apoptosis, as was the case for our positive control with adriamycin (Figure 5B). Most importantly, q-H2B was not detected in the positive control cell cultures containing between 12 and 18% apoptotic cells (Figure 5C and Supporting Information). Effect of Specific Calpain Inhibitors, PD150606 and AK295. As presented in Figure 6, pretreatment of cells with PD150606 or AK295 prevented the Ni(II)mediated truncation of histone H2B, thus, pointing at calpains as the most likely culprit.

Discussion Nickel, a well-established human carcinogen (1) has been known to interact with and chemically modify (damage) the major moieties of chromatin, DNA, and histones (reviewed in ref 2). In our previous study investigating the interaction of Ni(II) with histone H2A in cells, we noticed that the new electrophoretic band of this histone resulting from such interaction contained not only H2A damaged by truncation (q-H2A) but also other proteins which were absent in untreated cells (15). Therefore, in the present study, we continued experiments to identify those proteins. A preliminary tryptic digest analysis of the q-H2A band revealed the presence of peptides typical for histone H2A and, in addition, also for H2B. To further confirm the identity of the latter, we first improved the 1-D gel electrophoresis conditions and used 2-D electrophoresis to better separate the q-H2A band from comigrating proteins. Then, employing the Western blotting technique, we found that, indeed, one of the new protein bands, migrating faster than wildtype histone H2B (wt-H2B), was reactive with antihistone H2B antibodies. The final identification steps included LC/MS analysis and N-terminal sequencing which revealed that this band (q-H2B) contained two proteins derived from wt-H2B by cutting off the first 16 amino acids from its N-terminal tail (both proteins) and the last 9 amino acids from its C-terminal part (the smaller of these two). These two proteins could not be separated in our gels despite a significant difference in molecular masses (882 Da) and were distinguished only by mass spectrometry. The reasons for the comigration

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Figure 4. (A) Cobalt(II) exposure causes histone H2B truncation. Rat renal epithelial NRK-52E cells (NRK), human pulmonary HPL1D (HPL), and 1HAEo- (HAE) cells were cultured with 0.1 or 0.2 mM Co(II) for 3 and 5 days. (B) Exposure of NRK cells to Zn(II), Cu(II), or Cd(II) at the given concentrations for 3 and 5 days does not result in histone H2B truncation. Histones were extracted with acid from cell nuclei and separated by 1-D gel electrophoresis followed by Western blotting with anti-histone H2B antibodies and visualization with secondary antibodies labeled to produce a chemiluminescent signal, as described under Materials and Methods. The truncated histone H2B band (q-H2B), if any, is visible beneath the wild-type histone H2B band. Ctrl, cells cultured without metals. Table 1. Peptides Found in Tryptic Digests and N-Terminus Sequencing of Histone q-H2B peptidea,b LAHYNKR LLLPGELAK ESYSVYVYK KESYSVYVYK VLKQVHPDTGISSK AoxMGIoxMNSFVNDIFER

molecular mass (Da)c 901.5 953.6d 1 137.5 1265.6d 1508.8 1775.7d

N-terminus sequencing qH2B: AVTKAEKKDGKKe,f wt-H2B: PEPAKSAPAP KKG SKKAVTK AQKKDGe,f a NRK-52 cells cultured with 0.5 mM Ni(II) for 5 days. b Amino acid sequences corresponding to the above peptides in the rat histone H2B, PubMed Accession No. 0506206A, in bolde: PEPAKSAPAP KKGSKKAVTK AQKKDGKKRK RSRKESYSVY VYKVLKQVHP DTGISSKAMG IMNSFVNDIF ERIAGEASRL AHYNKRSTIT SREIQTAVRL LLPGELAKHA VSEGTKAVTK YTSSK. c Obtained by MALDI-MS. d Confirmed by MALDIMS/MS. e Q22 deamidation site underlined. f NRK-52 and 1HAEocells. Wt-H2B isolated from untreated cells. In wt-H2B from Ni(II)exposed cells, Q and E were both found at site of Q22.

are not clear. Perhaps, the expected increase in mobility of the smaller protein due to mass reduction was prevented by a simultaneous loss of electric charges owing to truncation of two lysines in the cutoff C-terminal -AVTKYTSSK sequence and modification of other residues postulated to account for the molecular mass found (see below). Most interestingly, the q-H2B histone showed clear indications of oxidative damage such as oxidation of its two methionine residues, M59 and M62, to sulfoxides, and deamidation of glutamine residues (Q22 found; deamidation of one more glutamine, perhaps Q95, is indicated by mass spectrometric analysis). Mediation of oxidation of

cellular proteins and peptides by transition metals via various mechanisms is a known phenomenon (24-28). Nickel(II) has been reported to enhance oxidative degradation of certain histone- and protamine-derived peptides, but only in test tube experiments (29). Oxidative histone damage at specific sites in cells exposed to this metal is reported here for the first time. Although artifactual oxidation of methionines during the analytical procedure was unlikely (not found in untreated cells), the observed deamidation of Q22 could be eventually produced by hydrolysis in the strong acids used for histone isolation from cells and gels. To test this, we sequenced wt-H2B histone obtained from untreated cells and prepared for amino acid sequencing under the same conditions as those used for q-H2B obtained from Ni(II)-exposed cells. No detectable E22 was found. Likewise negative were the results of similar tests for artifactual histone deamidation performed by others (30). Also, glutamine residues in proteins are much more resistant to nonenzymatic deamidation than are asparagine residues (31), yet no asparagine deamidation was detected in q-H2B. The exact molecular basis for the observed deamidation and its biological relevance deserves further studies. Oxidation of the methionine residues found in the same samples may be suggestive of the involvement of reactive oxygen also in the deamidation mechanism. Indeed, reactive oxygen species have been already implicated in deamidation of isolated proteins and peptides (32) or in cellular proteins (33), and Ni(II) is known to generate oxidative stress in cells and tissues resulting in oxidative damage to chromatin components (9) (reviewed in ref 2). Deamidation of peptides has also been found after incubation with nitric oxide donors (Li Kong et al., unpublished work). Thus, Ni(II), which does enhance the



Figure 6. Specific calpain inhibitors, PD150606 and AK295, prevent Ni(II)-induced truncation of histone H2B. 1HAEo- cells were grown with Ni(II) for 5 days in the presence and absence of the inhibitors (Inh.), PD150606 (A) and AK295 (B), under conditions closely resembling those in which Ni(II) alone produced detectable histone H2B truncation, as specified in Materials and Methods. The cells were tested for the presence of the truncated histone H2B (q-H2B) by Western blotting with anti-histone H2B antibodies.

Figure 5. Ni(II)-induced histone H2B truncation is not temporally associated with apoptosis. (A) NRK-52 cells were grown with Ni(II) under conditions producing histone H2B truncation, for 3 days (light bars) or 5 days (dark bars), and analyzed for apoptosis using the Annexin-V/7-AAD flow cytometric assay as described under Materials and Methods. For a positive control (gray bar), cells were prepared by incubation with 0.5 µM staurosporine for 6 h and analyzed as above. (B) NRK-52 cells were grown with Ni(II) for 5 days and analyzed for apoptosis using the PARP assay based on Western blotting with antiPARP antibodies as described under Materials and Methods. Unlike in the positive control cells cultured for 5 h with 0.3 µM adriamycin (Adriam.), truncated PARP (q-PARP) protein indicative of apoptosis was not found in untreated (Ctrl) and Ni(II)treated cells. (C) As revealed by Western blotting with antihistone H2B antibodies, q-H2B was absent in NRK-52E cells containing up to 18% of adherent early apoptotic cells following exposure to 0.5-10 µM staurosporine for 6 h.

expression of inducible nitric oxide synthase (iNOS) (34), could possibly mediate the deamidation through nitric oxide and its products, for example, nitrite, with site specificity of this effect depending on target accessibility. The deamidation might well be due to activation by Ni(II) of specific amide hydrolases, a result that would resemble the observed activation of another family of enzymes, the calpains, discussed later on. In such a case, the most likely candidates could be transglutaminases (35, 36). Transglutaminases’ activity depends on Ca(II) and may be further regulated by calpains (37) and nitric oxide (36). The major function of this family of enzymes is catalysis of protein cross-linking (38), but they may, as well, cause deamidation of glutamine residues in proteins (35, 39). Most significantly, Q22 and Q95 in histone H2B have already been identified as targets for nuclear transglutaminase TG2 in transamination reaction with a polyamine, but deamidation was not studied (39, 40). No matter what the actual mechanism of the

glutamine deamidation found in the present experiment is, the conversion of glutamine to glutamic acid may have regulatory (signaling) and/or pathogenic consequences as does the conversion of asparagine to aspartic acid isomers (30). As we have found before, the truncation of histone H2A is a direct result of Ni(II) binding and assistance in hydrolysis of a peptide bond at the truncation site (14, 15). The mechanism of the observed truncation of histone H2B is clearly different. Although isolated histone H2B was shown recently to have some binding capacity for Ni(II) (13), the binding site at the -ELAKHA- (residues 105-110) sequence, distant from the truncation sites at 16/17 and 116/117 -KA- residues, and lack of any detectable in vitro hydrolysis, observed in the present study, both indicated an indirect mechanism involving a specific proteolytic enzyme. Since Ni(II) can induce apoptosis (41), we first suspected proteinases activated by apoptosis as possible mediators of the truncation. However, the frequency of apoptosis in cells harvested after Ni(II) exposure, showing the presence of q-H2B, was low (approximately 1-2%) and not markedly different from that in cells cultured without the metal. And vice versa, the apoptosis-positive control cultures, which contained up to 10 times more apoptotic cells, did not produce q-H2B. This, along with the apparent site specificity of the truncation, brought us to calpains, whose role in cell is thought to be predominantly regulatory, through limited and specific post-translational modification of proteins rather than destructive, through extensive proteolysis. The sequential and conformational determinants of substrate recognition by calpains are not fully understood (22). To date, observations on over a 100 known calpain substrates suggest that the cleavage depends on both higher order structural factors and complex amino acid preferences at and around the cleaved bond that allows for some shifts in site specificity of substrate recognition. Among many other proteins, calpains have already been described by Sakai et al. to in vitro hydrolyze histones H2A, H2B, and H3, with H2B being more susceptible than H2A and H3 (42). Thus, calf thymus histone H2B was cleaved in 14 different sites at

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the Ser, Lys, Tyr, Gly, Ala, Thr, Leu, and Glu residues. Most importantly, in concordance with our results, one of them was at Lys-116 in the -116KAVTK- sequence. However, the second cleavage site at Lys-16 found in the present study was not reported previously. This discrepancy is a likely result of the above-mentioned shifts in cleavage sites, depending on higher order structure of the target protein. Deamidation of 22Q near the cleavage site might also play a role. And, obviously, the conformation of histone H2B attacked by calpains in chromatin in our cells was different from that in a test tube where the multiple cleavage sites were determined by Sakai et al. (42). Although hydrolysis of cellular histones by proteinases of various types in living cells has been reported before (43-46), the exceptional specificity of the two inhibitors used by us (22, 47, 48) argues very strongly against possible involvement of enzymes other than calpains in the observed Ni(II)-induced truncation of histone H2B. Calpains are intracellular neutral cysteine proteinases activated by either micromolar (µ-calpains) or nearly millimolar (m-calpains) concentrations of Ca(II). They form a family distinct from other cysteine proteinases, such as papain, cathepsins, or caspases (49). Important for this discussion is also the fact that, in addition to that in cytosol and other subcellular organelles, calpain activity was observed in cell nuclei (50, 51). Calcium is coordinated to the calpain molecule through a calmodulin-like Ca(II)-binding domain (22). In the absence of Ca(II), several calmodulin-dependent enzymes were found to be activated in a hormetic way by Ni(II) and certain other metal cations (52, 53). This would offer a mechanistic explanation of the activation of calpains observed in the present experiment. However, Ni(II) exposure is known to elevate Ca(II) level in cultured cells (6), and in the presence of Ca(II), Ni(II) inhibits the calmodulinregulated enzymes (52, 53). Such inhibition was also demonstrated for isolated calpains, although at high millimolar levels of Ca(II) and Ni(II) and in the absence of calpastatin, the cellular calpain inhibitor (54). This makes it difficult to be sure about the inhibition in cells. So, the exact mechanism of the observed calpain activation is not clear and requires further investigations, including testing Ca(II) exposures for the truncation effect and identification of the enzyme isoform (µ-calpain, m-calpain, other) and the activating metal involved plus its possible interaction with calpastatin. There are reports that only µ-calpain, but not m-calpain is transported into the cell nucleus (22, 55, 56). In chromatin, the core histone tails regulate its overall structure and accessibility of DNA to gene transcription factors, DNA repair machinery, or apoptotic enzymes (57, 58). The regulation is achieved through post-transcriptional modifications of the tails. It is, therefore, tempting to speculate that their truncation, that by itself may neither be lethal nor detrimental to cell division, as found in yeast (59) and slime mold (60), has the potential to affect those modifications and eventually gene expression and/or DNA repair. A preferential site-specific proteolysis of histone H2B has been found in human lymphocytes exposed to sulfur mustard that damages DNA and causes G2/M arrest. In this case, the histone was cleaved only at its N-terminal tail’s Val-18 by a chymotrypsin-like proteinase, and the cleavage was thought to facilitate DNA excision repair (58). Interestingly, Ni(II) action on cells also includes DNA damage leading to G2/M arrest

Karaczyn et al.

(41) and, as found in this experiment, to proteolytic scission of histone H2B at a site very close to the Val-18 residue. This indicates that, perhaps, the N-terminal -KAVTKA- sequence in H2B serves as a safety sensor to stop cell division when DNA and/or the histones are damaged (oxidation, deamidation) and call for repair. The significance of the observed cleavage in the second, C-terminal -KAVTKA- sequence remains to be investigated. The reported site-specific truncation, oxidation, and deamidation of histone H2B, concurrent with the truncation of histone H2A described by us previously (14, 15), in Ni(II)-exposed cells, provide further examples supporting the hypothesis of epigenetic nature of nickelinduced cell transformation and carcinogenesis formulated by Lee et al. (61). These effects may, indeed, dysregulate the histone code and alter gene expression. Moreover, the enzymes involved in producing of some of the effects are capable of attacking other nuclear proteins as well. For example, the p53 tumor suppressor protein also is a substrate for calpains (51). Their action enhanced by Ni(II) may, therefore, distort the functions of both histone H2B and p53 protein and assist in cell transformation to a malignant phenotype.

Acknowledgment. The Authors wish to thank Drs. A. Byrd and S. Tarasov, Structural Biophysics Laboratory, National Cancer Institute at Frederick, for valuable technical advice and making the LC/MS instrument available to us, and R. J. Fisher and Young Kim, Protein Chemistry Laboratory, SAIC Frederick, for help in amino acid sequencing. Critical comments of our laboratory colleagues Drs. G. Buzard and K. Salnikow on this study and editorial help of Ms. Kathy Breeze are also gratefully acknowledged. This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. Supporting Information Available: Figures showing cell proliferation rates (Figure 1S) and FACS profiles of NRK-52E cells (Figure 2S). This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) IARC (1990) Nickel, Chromium, and Welding, in International Agency for Research on Cancer (IARC) Monographs on the Evaluation of Carcinogenic Risk to Humans (IARC, Ed.) pp 257445, IARC, Lyon, France. (2) Kasprzak, K. S., Sunderman, F. W., Jr., and Salnikow, K. (2003) Nickel carcinogenesis. Mutat. Res. 533, 67-97. (3) Kasprzak, K. S. (1995) Possible role of oxidative damage in metalinduced carcinogenesis. Cancer Invest. 13, 411-430. (4) Dally, H., and Hartwig, A. (1997) Induction and repair inhibition of oxidative DNA damage by nickel(II) and cadmium(II) in mammalian cells. Carcinogenesis 18, 1021-1026. (5) Hartmann, M., and Hartwig, A. (1998) Disturbance of DNA damage recognition after UV-irradiation by nickel(II) and cadmium(II) in mammalian cells. Carcinogenesis 19, 617-621. (6) Zhou, D., Salnikow, K., and Costa, M. (1998) Cap43, a novel gene specifically induced by Ni2+ compounds. Cancer Res. 58, 21822189. (7) Maxwell, P., and Salnikow, K. (2004) HIF-1: an oxygen and metal responsive transcription factor. Cancer Biol. Ther. 3, 29-35. (8) Salnikow, K., Donald, S. P., Bruick, R. K., Zhitkovich, A., Phang, J. M., and Kasprzak, K. S. (2004) Depletion of intracellular ascorbate by the carcinogenic metals nickel and cobalt results in the induction of hypoxic stress. J. Biol. Chem. 279, 40337-40344. (9) Huang, X., Kitahara, J., Zhitkovich, A., Dowjat, K., and Costa, M. (1995) Heterochromatic proteins specifically enhance nickelinduced 8-oxo-dG formation. Carcinogenesis 16, 1753-1759. (10) Bal, W., Lukszo, J., Jezowska-Bojczuk, M., and Kasprzak, K. S. (1995) Interactions of nickel(II) with histones. Stability and


(11)

(12)

(13)

(14)

(15)

(16)

(17)

(18)

(19)

(20)

(21)

(22)

(23)

(24)

(25)

(26)

(27)

(28)

(29)

(30)

(31)

solution structure of complexes with CH3CO-Cys-Ala-Ile-HisNH2, a putative metal binding sequence of histone H3. Chem. Res. Toxicol. 8, 683-692. Bal, W., Lukszo, J., Bialkowski, K., and Kasprzak, K. S. (1998) Interactions of Nickel(II) with histones: interactions of Nickel(II) with CH3CO-Thr-Glu-Ser-His-His-Lys-NH2, a peptide modeling the potential metal binding site in the “C-Tail” region of histone H2A. Chem. Res. Toxicol. 11, 1014-1023. Zoroddu, M. A., Schinocca, L., Kowalik-Jankowska, T., Kozlowski, H., Salnikow, K., and Costa, M. (2002) Molecular mechanisms in nickel carcinogenesis: modeling Ni(II) binding site in histone H4. Environ. Health Perspect. 110 (Suppl. 5), 719-723. Karavelas, T., Mylonas, M., Malandrinos, G., Plakatouras, J. C., Hadjiliadis, N., Mlynarz, P., and Kozlowski, H. (2005) Coordination properties of Cu(II) and Ni(II) ions towards the C-terminal peptide fragment -ELAKHA- of histone H2B. J. Inorg. Biochem. 99, 606-615. Bal, W., Liang, R., Lukszo, J., Lee, S. H., Dizdaroglu, M., and Kasprzak, K. S. (2000) Ni(II) specifically cleaves the C-terminal tail of the major variant of histone H2A and forms an oxidative damage-mediating complex with the cleaved-off octapeptide. Chem. Res. Toxicol. 13, 616-624. Karaczyn, A. A., Bal, W., North, S. L., Bare, R. M., Hoang, V. M., Fisher, R. J., and Kasprzak, K. S. (2003) The octapeptidic end of the C-terminal tail of histone H2A is cleaved off in cells exposed to carcinogenic nickel(II). Chem. Res. Toxicol. 16, 15551559. Gruenert, D. C., Finkbeiner, W. E., and Widdicombe, J. H. (1995) Culture and transformation of human airway epithelial cells. Am. J. Physiol. 268, L347-360. Bonner, W. M., West, M. H., and Stedman, J. D. (1980) Twodimensional gel analysis of histones in acid extracts of nuclei, cells, and tissues. Eur. J. Biochem. 109, 17-23. Dimitrov, S. I., and Wolffe, A. P. (1997) Fine resolution of histones by two-dimensional polyacrylamide gel electrophoresis: developmental implications. Methods 12, 57-61. Cohen, S. L., and Chait, B. T. (1997) Mass spectrometry of whole proteins eluted from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Anal. Biochem. 247, 257-267. Matsui, N. M., Smith, D. M., Clauser, K. R., Fichmann, J., Andrews, L. E., Sullivan, C. M., Burlingame, A. L., and Epstein, L. B. (1997) Immobilized pH gradient two-dimensional gel electrophoresis and mass spectrometric identification of cytokineregulated proteins in ME-180 cervical carcinoma cells. Electrophoresis 18, 409-417. Clauser, K. R., Baker, P., and Burlingame, A. L. (1999) Role of accurate mass measurement ((10 ppm) in protein identification strategies employing MS or MS/MS and database searching. Anal. Chem. 71, 2871-2882. Sorimachi, H., Ishiura, S., and Suzuki, K. (1997) Structure and physiological function of calpains. Biochem. J. 328 (Pt. 3), 721732. Tompa, P., Buzder-Lantos, P., Tantos, A., Farkas, A., Szilagyi, A., Banoczi, Z., Hudecz, F., and Friedrich, P. (2004) On the sequential determinants of calpain cleavage. J. Biol. Chem. 279, 20775-20785. Stadtman, E. R., and Oliver, C. N. (1991) Metal-catalyzed oxidation of proteins. Physiological consequences. J. Biol. Chem. 266, 2005-2008. Stadtman, E. R. (1993) Oxidation of free amino acids and amino acid residues in proteins by radiolysis and by metal-catalyzed reactions. Annu. Rev. Biochem. 62, 797-821. Levine, R. L., Mosoni, L., Berlett, B. S., and Stadtman, E. R. (1996) Methionine residues as endogenous antioxidants in proteins. Proc. Natl. Acad. Sci. U.S.A. 93, 15036-15040. Berlett, B. S., and Stadtman, E. R. (1997) Protein oxidation in aging, disease, and oxidative stress. J. Biol. Chem. 272, 2031320316. Stadtman, E. R., and Levine, R. L. (2003) Free radical-mediated oxidation of free amino acids and amino acid residues in proteins. Amino Acids 25, 207-218. Bal, W., Lukszo, J., and Kasprzak, K. S. (1997) Mediation of oxidative DNA damage by nickel(II) and copper(II) complexes with the N-terminal sequence of human protamine HP2. Chem. Res. Toxicol. 10, 915-921. Lindner, H., Sarg, B., Hoertnagl, B., and Helliger, W. (1998) The microheterogeneity of the mammalian H1(0) histone. Evidence for an age-dependent deamidation. J. Biol. Chem. 273, 1332413330. Robinson, N. E., and Robinson, A. B. (2001) Deamidation of human proteins. Proc. Natl. Acad. Sci. U.S.A. 98, 1240912413.

Chem. Res. Toxicol., Vol. 18, No. 12, 2005 1941 (32) Robinson, A. B., Irving, K., and McCrea, M. (1973) Acceleration of the rate of deamidation of GlyArgAsnArgGly and of human transferrin by addition of L-ascorbic acid. Proc. Natl. Acad. Sci. U.S.A. 70, 2122-2123. (33) D’Angelo, S., Ingrosso, D., Perfetto, B., Baroni, A., Zappia, M., Lobianco, L. L., Tufano, M. A., and Galletti, P. (2001) UVA irradiation induces L-isoaspartyl formation in melanoma cell proteins. Free Radical Biol. Med. 31, 1-9. (34) Cruz, M. T., Goncalo, M., Figueiredo, A., Carvalho, A. P., Duarte, C. B., and Lopes, M. C. (2004) Contact sensitizer nickel sulfate activates the transcription factors NF-kB and AP-1 and increases the expression of nitric oxide synthase in a skin dendritic cell line. Exp. Dermatol. 13, 18-26. (35) Mycek, M. J., and Waelsch, H. (1960) The enzymatic deamidation of proteins. J. Biol. Chem. 235, 3513-3517. (36) Beninati, S., and Piacentini, M. (2004) The transglutaminase family: an overview: minireview article. Amino Acids 26, 367372. (37) Ando, Y., Imamura, S., Murachi, T., and Kannagi, R. (1988) Calpain activates two transglutaminases from porcine skin. Arch. Dermatol. Res. 280, 380-384. (38) Kim, J. H., Nam, K. H., Kwon, O. S., Kim, I. G., Bustin, M., Choy, H. E., and Park, S. C. (2002) Histone cross-linking by transglutaminase. Biochem. Biophys. Res. Commun. 293, 1453-1457. (39) Esposito, C., and Caputo, I. (2005) Mammalian transglutaminases. Identification of substrates as a key to physiological function and physiopathological relevance. FEBS J. 272, 615631. (40) Ballestar, E., Abad, C., Franco, L., Lopez-Camacho, C., Salgado, J., Lequerica, J. L., Madarro, A., and Polaina, J. (1996) Core histones are glutaminyl substrates for tissue transglutaminase. Amino acid substitutions enhancing thermostability of Bacillus polymyxa beta-glucosidase A. J. Biol. Chem. 271, 1881718824. (41) Shiao, Y. H., Lee, S. H., and Kasprzak, K. S. (1998) Cell cycle arrest, apoptosis and p53 expression in nickel(II) acetate-treated Chinese hamster ovary cells. Carcinogenesis 19, 1203-1207. (42) Sakai, K., Akanuma, H., Imahori, K., and Kawashima, S. (1987) A unique specificity of a calcium activated neutral protease indicated in histone hydrolysis. J. Biochem. (Tokyo) 101, 911918. (43) Bartley, J., and Chalkley, R. (1970) Further studies of a thymus nucleohistone-associated protease. J. Biol. Chem. 245, 4286-4292. (44) Dumuis-Kervabon, A., Encontre, I., Etienne, G., Jauregui-Adell, J., Mery, J., Mesnier, D., and Parello, J. (1986) A chromatin core particle obtained by selective cleavage of histones by clostripain. EMBO J. 5, 1735-1742. (45) Harvima, R. J., Yabe, K., Fraki, J. E., Fukuyama, K., and Epstein, W. L. (1988) Hydrolysis of histones by proteinases. Biochem. J. 250, 859-864. (46) Dino Rockel, T., and von Mikecz, A. (2002) Proteasome-dependent processing of nuclear proteins is correlated with their subnuclear localization. J. Struct. Biol. 140, 189-199. (47) Saatman, K. E., Murai, H., Bartus, R. T., Smith, D. H., Hayward, N. J., Perri, B. R., and McIntosh, T. K. (1996) Calpain inhibitor AK295 attenuates motor and cognitive deficits following experimental brain injury in the rat. Proc. Natl. Acad. Sci. U.S.A. 93, 3428-3433. (48) Wang, M. S., Davis, A. A., Culver, D. G., Wang, Q., Powers, J. C., and Glass, J. D. (2004) Calpain inhibition protects against Taxolinduced sensory neuropathy. Brain 127, 671-679. (49) Berti, P. J., and Storer, A. C. (1995) Alignment/phylogeny of the papain superfamily of cysteine proteases. J. Mol. Biol. 246, 273283. (50) Gopalakrishna, R., and Barsky, S. H. (1986) Hydrophobic association of calpains with subcellular organelles. Compartmentalization of calpains and the endogenous inhibitor calpastatin in tissues. J. Biol. Chem. 261, 13936-13942. (51) Kubbutat, M. H., and Vousden, K. H. (1997) Proteolytic cleavage of human p53 by calpain: a potential regulator of protein stability. Mol. Cell. Biol. 17, 460-468. (52) King, M. M., and Huang, C. Y. (1983) Activation of calcineurin by nickel ions. Biochem. Biophys. Res. Commun. 114, 955-961. (53) Weaver, J., Porasuphatana, S., Tsai, P., Cao, G. L., Budzichowski, T. A., Roman, L. J., and Rosen, G. M. (2004) The effect of divalent cations on neuronal nitric oxide synthase activity. Toxicol. Sci. 81, 325-331. (54) Gaitanaki, C., Papazafiri, P., and Beis, I. (2003) The calpaincalpastatin system and the calcium paradox in the isolated perfused pigeon heart. Cell. Physiol. Biochem. 13, 173-80. (55) Mellgren, R. L., and Lu, Q. (1994) Selective nuclear transport of mu-calpain. Biochem. Biophys. Res. Commun. 204, 544-550.

1942


(56) Zhang, W., Lu, Q., Xie, Z. J., and Mellgren, R. L. (1997) Inhibition of the growth of WI-38 fibroblasts by benzyloxycarbonyl-Leu-LeuTyr diazomethyl ketone: evidence that cleavage of p53 by a calpain-like protease is necessary for G1 to S-phase transition. Oncogene 14, 255-263. (57) Angelov, D., Vitolo, J. M., Mutskov, V., Dimitrov, S., and Hayes, J. J. (2001) Preferential interaction of the core histone tail domains with linker DNA. Proc. Natl. Acad. Sci. U.S.A. 98, 65996604. (58) Millard, C. B., Meier, H. L., and Broomfield, C. A. (1994) Exposure of human lymphocytes to bis-(2-chloroethyl)sulfide solubilizes truncated and intact core histones. Biochim. Biophys. Acta 1224, 389-394.

Karaczyn et al. (59) Schuster, T., Han, M., and Grunstein, M. (1986) Yeast histone H2A and H2B amino termini have interchangeable functions. Cell 45, 445-451. (60) Thiriet, C., and Hayes, J. J. (2001) A novel labeling technique reveals a function for histone H2A/H2B dimer tail domains in chromatin assembly in vivo. Genes Dev. 15, 2048-2053. (61) Lee, Y. W., Klein, C. B., Kargacin, B., Salnikow, K., Kitahara, J., Dowjat, K., Zhitkovich, A., Christie, N. T., and Costa, M. (1995) Carcinogenic nickel silences gene expression by chromatin condensation and DNA methylation: A new model for epigenetic carcinogens. Mol. Cell. Biol. 15, 2547-2557.

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Truncation, Deamidation, and Oxidation of Histone H2B in Cells

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