MCP Sign the guestbook
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/mcp.T600051-MCP200 on February 10, 2007.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Supplemental Data
Right arrow All Versions of this Article:
T600051-MCP200v1
6/5/939    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Glossary
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zanardi, A.
Right arrow Articles by Carbone, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zanardi, A.
Right arrow Articles by Carbone, R.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Molecular & Cellular Proteomics 6:939-947, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Technology

Immunocell-array for Molecular Dissection of Multiple Signaling Pathways in Mammalian Cells*,S

Andrea Zanardi{ddagger}, Luca Giorgetti§,¶, Oronza A. Botrugno§,||, Saverio Minucci§, Paolo Milani, Pier Giuseppe Pelicci§,** and Roberta Carbone{ddagger},{ddagger}{ddagger}

From {ddagger} Tethis S.r.l., Via Russoli 3, 20143 Milan, Italy, § European Institute of Oncology (EIO), Via Ripamonti 435, 20141 Milan, Italy, CIMAINA, Centro Interdisciplinare Materiali e Interfacce Nanostrutturati, Via Celoria 16, 20133 Milan, Italy, and ** IFOM, Istituto Fondazione Italiana per la Ricerca sul Cancro (FIRC) di Oncologia Molecolare, Via Adamello 16, 20139 Milan, Italy


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The knowledge of signaling pathways that are triggered by physiological and pathological conditions or drug treatment is essential for the comprehension of the biological events that regulate cellular responses. Recently novel platforms based on "reverse-phase protein arrays" have proven to be useful in the study of different pathways, but they still lack the possibility to detect events in the complexity of a cellular context. We developed an "immunocell-array" of cells on chip where, upon cell plating, growing, drug treatment, and fixation, by spotting specific antibodies we can detect the localization and state of hundreds of proteins involved in specific signaling pathways. By applying this technology to mammalian cells we analyzed signaling proteins involved in the response to DNA damage and identified a chromatin remodeling pathway following bleomycin treatment. We propose our technology as a new tool for the array-based multiplexed analysis of signaling pathways in drug response screening, for the proteomics of profiling patient cells, and ultimately for the high throughput screening of antibodies for immunofluorescence applications.

The misregulation or hyperactivity of intracellular and extracellular signaling cascades may represent the molecular signature of human diseases (1). Thus, the capability of monitoring the complexity of all biochemical networks involved in specific pathologies is essential for the realization of patient-tailored therapies and for the characterization of drug response (2, 3). Signaling pathways are complex, and they are regulated by specific spatiotemporal dynamics taking place in a cellular contest. To deal with this complexity, approaches are required that can describe "when and where" (4, 5) a single event takes place. New genomics and proteomics technologies have recently been developed (6–8) allowing the simultaneous analysis of multiple events and the characterization of complex samples. Among them, reverse-phase protein microarrays (9) represent the most valid approach to probe tissue or cell lysates with a large number of antibodies or patient sera to allow the identification of new biomarkers, the analysis of protein expression profiles, and the efficacy and toxicity of drug candidates. These novel protein array platforms have proven to be very powerful in the study of different molecular pathways; however, although this technology represents an important tool for proteomics studies, it does not allow the detection of multiple events in the "cellular" context of compartments and organelles where phenomena have to be analyzed for the comprehension of biological networks.

We developed an "immunocell-array" of cells on glass slides where, upon fixation, we can detect simultaneously, with specific antibodies, the localization and state of hundreds of proteins involved in different signaling pathways, minimizing reagents and cell consumption and obtaining high quality images by high resolution microscopy. Using this tool we analyzed the kinetic response (protein post-transcriptional modification and profiling) of different target proteins upon DNA damage and identified a chromatin remodeling pathway in mammalian cells. We expect that this new cell-based technology will offer a powerful tool in proteomics studies for drug discovery and will introduce a new immunofluorescence methodology in life science applications.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and Culture Conditions—
All cell lines were grown in tissue culture plates (Falcon, BD Bioscience) at 37 °C in a humidified incubator with 5% CO2. Human osteosarcoma U2OS (American Type Culture Collection (ATCC)) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS,1 1% L-glutamine, and 1% P/S. Human fibroblast Tig3-hTert (Tig3 fibroblasts immortalized with human telomerase reverse transcriptase, kindly provided by Dr. C. Moroni IFOM-IEO Campus, Milan, Italy) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS (origin, United States), 1% L-glutamine, and 1% P/S.

Primary human melanocytes were isolated in our laboratory according to the protocol developed by Meenhard Herlyn.2 Cells were cultured in McCoy’s 5A medium (Invitrogen) supplemented with 2% FBS (United States origin), 5 µg/ml recombinant human insulin (Roche Applied Science), 5 µg/ml human holotransferrin (Sigma), 0.5 µg/ml hydrocortisone (Sigma), 20 pM cholera toxin from Vibrio cholerae (Sigma), 16 nM phorbol 12-myristate 13-acetate (Sigma), 10 nM endothelin 1 human, porcine (Sigma), 10 ng/ml recombinant human stem cell factor (Peprotech), and 1 ng/ml recombinant human fibroblast growth factor basic (Peprotech). NIH3T3 cells (ATCC) were cultured in 10% calf serum (Colorado Serum Co. CS1334), 1% L-glutamine, and 1% P/S. For the immunocell-array experiment cells were plated (typically 2 x 105–6 x 105) on 0.2% gelatin (Sigma type B from bovine skin)-coated glass slides (ArrayIt® SuperClean2, TeleChem International) and placed in Vivascience® Vivadish plates (microbiological grade). For the immunofluorescence staining typically 8 x 104–105 cells were plated on coverslips in 6 wells/plate. In the DNA damage experiments cells were treated with 25 µg/ml bleomycin (Sigma) and then processed after 8, 24, or 48 h as specifically indicated for the different experimental approaches.

Immunocell-array—
Cells were fixed with a solution of 4% paraformaldehyde in Pipes buffer (80 mM Pipes, pH 6.8, 5 mM EGTA pH 8, 2 mM MgCl2) for 15 min. After fixation slides were washed twice with 1x PBS; at this point slides can be stored for several days at 4 °C. After washing, cells were permeabilized for 10 min with 0.1% Triton in a solution of 1x PBS + 0.2% BSA, washed twice with 1x PBS, and blocked for 1 h with 2% BSA. After blocking, the slides were washed twice with 1x PBS, and finally slides were drained to remove excess buffer and placed in the slide holder. The antibodies were dispensed from a 96-multiwell plate (40 µl of antibody dilution in each well) using a Biodot® BioJet Plus spotter with Axsys software (BioDot Inc.). Usually drops of 30 nl were spotted for each antibody with a pitch of 1–2 mm, keeping uniform humidity at 65% in the spotting chamber during the experiment. Primary antibodies were spotted and subsequently incubated on the slide for 45 min at 4 °C in a small chamber with an NaCl-saturated water solution in 75% humidity. After incubation, slides were washed twice with 1x PBS + 0.1% Tween and four times in 1x PBS, drained as above, and repositioned in the spotting chamber where secondary antibodies were spotted. Secondary antibodies were incubated as above for 30 min. After washing twice in 1x PBS + 0.1% Tween and four times in 1xPBS, cells were stained with DAPI for 5 min, washed with 1x PBS and H2O, and mounted with Mowiol.

Immunofluorescence Staining—
For conventional immunofluorescence staining we plated cells on glass coverslips and, at appropriate time points, processed them for 4% paraformaldehyde fixation, 0.1% Triton permeabilization, and 2% BSA blocking. Coverslips were further incubated with primary and secondary antibodies at the appropriate dilutions (as described under "Immunocell-array") and finally mounted with Mowiol. Image acquisition and analysis were performed as described below under "Image Acquisition" and "Image Analysis."

Primary and Secondary Antibodies—
Primary antibodies included: monoclonal anti-AP-1{gamma} (Sigma, clone 100/3), monoclonal anti-AP-2{alpha} (Affinity BioReagents, MA1-064), Monoclonal anti-Caveolin (homemade supernatant), monoclonal anti-clathrin (Affinity BioReagents, MA1-065), monoclonal anti-E3B1 (homemade), monoclonal anti-Eps8 (BD Transduction Laboratories, 610143), monoclonal anti-Eps15 (homemade), monoclonal anti-paxillin (Zymed Laboratories Inc., 03-6100), monoclonal anti-p-tyrosine (Upstate, clone 4G10), monoclonal anti-VASP (BD Transduction Laboratories, 610447), monoclonal anti-vinculin (Sigma, h-VIN1), polyclonal anti-{alpha}-actinin (homemade), polyclonal anti-giantin (Babco, PRB-114C), polyclonal anti-profilin (homemade), monoclonal anti-p53 (Santa Cruz Biotechnology, DO-1), monoclonal anti-cortactin (Upstate, 05-180), monoclonal anti-PML (homemade), monoclonal anti-NPM (homemade), monoclonal anti-{gamma}-H2AX (Upstate, 05-636), monoclonal anti-Chk1 (Santa Cruz Biotechnology, catalog number sc-8408), monoclonal anti-BMI1 (homemade), monoclonal anti-small ubiquitin-like modifier (Zymed Laboratories Inc., catalog number 33-2400), monoclonal anti-SC35 (Sigma, clone SC35), monoclonal T25 (homemade) (10), polyclonal anti-p-H3 (phospho-histone H3 Ser-28, mitosis marker) (Upstate, 06-570), polyclonal anti-DNA-dependent protein kinase (Santa Cruz Biotechnology, sc-1551), monoclonal T52 (homemade) (10), monoclonal anti-EZH2 (homemade), polyclonal anti-p-ATM (Rockland, 600-401-400), polyclonal anti-CASP3 (caspase 3) (Cell Signaling Technology, 5A1), polyclonal anti-DAXX (Santa Cruz Biotechnology, sc-7152), monoclonal anti-HDAC1 (homemade), polyclonal anti-CBP (Santa Cruz Biotechnology, sc-369), and polyclonal anti-Sirt6 (homemade). Secondary antibodies included: goat anti-mouse and anti-rabbit Alexa Fluor 488 (Molecular Probes-Invitrogen) and goat anti-mouse and anti-rabbit Cy3 (Jackson ImmunoResearch Laboratories). For control staining mouse IgG1 isotype (BD Biosciences) was used at 1 µg/ml.

Image Acquisition—
Imaging of immunocell-arrays and immunostained coverslips was performed with an automated fluorescence microscope (Olympus IX81 Scan–R microscope-based imaging platform for screening, Olympus Europa, Hamburg, Germany) equipped with a digital camera (ORCA-ER C4742-80, Hamamatsu) using 10x, 20x, 40x, and 60x UPlanApo magnification objectives. For the immunocell-arrays analyzed in Figs. 4 and 6, each antibody was spotted in a 10x replicate, and one 20x field per spot (containing ~35 cells) was acquired; population to be compared thus contained ~350 cells. For the immunofluorescence staining of the experiment shown in Fig. 7A, 25 images were acquired with a 20x magnification objective for each antibody at each time point.


Figure 4
View larger version (19K):
[in this window]
[in a new window]

  		FIG. 4. Immunocell-arrays assessing the interslide consistency by using anti-lamin-B as reference standard. a, response of lamin-B, p53, p-ATM, and {gamma}-H2AX at different times after bleomycin treatment (0 h corresponds to untreated). The colors are proportional to the K-S statistics for the empirical cumulative distributions at 8 and 24 h against the untreated (0 h) condition, and they reflect the extent of protein up-regulation (green) or down-regulation (red) with respect to the untreated population as detected by a K-S test with significance threshold p < 0.001. b, empirical cumulative distributions of the fluorescence intensities of the antibodies used in the immunocell-array experiment from which the K-S statistics of a were calculated. The fraction of cells over the total population is plotted as a function of its maximum nuclear mean fluorescence intensity (arbitrary units). Dotted line, untreated cells; dashed line, 8-h treatment; solid line, 24-h treatment.

 

Figure 6
View larger version (26K):
[in this window]
[in a new window]

  		FIG. 6. Immunocell-arrays detect a chromatin remodeling pathway in response to DNA damage in U2OS cells. A, heat map showing the response of the 22 proteins (and BSA as control) that were stained in the immunocell-array of Fig. 5. Colors are proportional to the K-S statistics of the empirical cumulative distributions at 8 and 24 h against the untreated (0 h) condition and reflect the extent of protein up-regulation (green) or down-regulation (red) as detected by a K-S test with p < 0.001. B, empirical cumulative distributions of fluorescence intensities of all the antibodies used in the immunocell-array experiment from which K-S statistics of a were calculated. The fraction of cells over the total population is plotted as a function of its maximum nuclear mean fluorescence intensity (arbitrary units). Dotted lines, untreated population (0 h); dashed lines, population 8 h after treatment with bleomycin; solid lines, population 24 h after treatment with bleomycin. Distributions shifted to the right correspond to increased fluorescence signals and vice versa. pTYR, phosphotyrosine; SUMO, small ubiquitin-like modifier; DNAPK, DNA-dependent protein kinase.

 

Figure 7
View larger version (43K):
[in this window]
[in a new window]

  		FIG. 7. Validation of down-regulation of chromatin acetylation upon DNA damage with bleomycin by conventional immunofluorescence staining, Western Blot, and immunoprecipitation. A, panel of representative images for p53, {gamma}-H2AX, T25, and T52 staining at different time points (0 h corresponding to untreated and 8 and 24 h after bleomycin treatment). Scale bar, 20 µm. B, heat map showing the response of the four proteins whose staining is reported in A. Colors are proportional to the K-S statistics of the empirical cumulative distributions at 8 and 24 h against the untreated (0 h) condition with p < 0.001. C, empirical cumulative distributions of fluorescence intensities of the antibodies used in the experiment from which the K-S statistics of B were calculated. Dotted lines, untreated population (0 h); dashed lines, population 8 h after treatment with bleomycin; solid lines, population 24 h after treatment with bleomycin. D, Western blot detection of untreated (NT) and bleomycin-treated U2OS cells at 24 and 48 h with T52 and T25 antibodies; H3 was used as loading control. Lower panel, silver staining of SDS-PAGE gel of the same U2OS lysates immunoprecipitated with T52 shows acetyl-H3 (Ac-H3) and acetyl-H4 (Ac-H4) levels. IP, immunoprecipitate.

 
Image Analysis—
Images were processed and analyzed with automated routines written in Matlab programming language with Image Processing Toolbox. Background was removed by subtracting from each image its morphological opening with a circle of radius 20 pixels. After background subtraction, a binary mask was generated identifying nuclear regions by applying a threshold segmentation on DAPI fluorescence images; Cy3 nuclear mean intensity was then calculated in each of these regions, and single cell data were stored for further analysis. For cytoplasmic antibodies, Cy3 intensity was calculated in a perinuclear cytoplasmic region of thickness 4 pixels around nuclei. For each antibody {alpha} and each time point t (t = 0, 8, and 24 h), the empirical cumulative distribution F({alpha},t,I) of fluorescence intensity I was calculated. Given the small size of samples and the non-gaussian distribution of fluorescence over the populations we used the two-sample Kolmogorov-Smirnov test, relying on the comparison of empirical cumulative distributions of fluorescence distributions (11, 12). For each population (cells belonging to the 10 spots or to the images of coverslips that were stained against a given antibody at a given time point) the Cy3 mean intensity was measured cell-by-cell after DAPI-based identification of nuclei. For each antibody {alpha}, the Kolmogorov-Smirnov (K-S) statistics for the distributions at times t > 0 against the untreated distributions at t = 0 were then calculated as D({alpha},t) = maxI({nmid}F({alpha},t,I) – F({alpha},0,I){nmid}). The distribution at t > 0 was held to differ from the one at t = 0 if the two-sided p value for the null hypothesis (overlapping distributions), as calculated from the corresponding D({alpha},t), was smaller than 0.001. The measure plotted in the heat maps in Figs. 4a, 6A, and 7B is the K-S statistics with sign, defined by KS({alpha},t) = D({alpha},t) sign(F({alpha},t,IM) – F({alpha},0,IM)) where IM is the fluorescence intensity where {nmid}F({alpha},t,I) – F({alpha},0,I){nmid} is maximum.

Immunoprecipitation and Western Blot—
Lysates from untreated and bleomycin-treated U2OS cells were prepared in 50 mM Hepes, pH 7.5, 0.5% Nonidet P-40, 250 mM NaCl, 1 mM sodium orthovanadate, 10 mM NaF, anti-proteases and subjected to mild sonication. After clarification by centrifugation, 1.5 mg of lysates were incubated with protein G-agarose beads coated with the T52 antibody. Immunoprecipitation was carried out for 4 h at 4 °C with constant rotation. Immunoprecipitates were then washed five times in lysis buffer and three times in PBS. Immunoprecipitates were fractionated by SDS-PAGE and silver-stained as described elsewhere (13). For the Western blot procedure 15 µg of lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Primary antibodies were detected with the ECL detection system (Amersham Biosciences). Control of protein loading was carried out with polyclonal anti-histone H3 (Abcam).


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Setup of an Immunocell-array Protocol on Glass Slides—
We set up a novel method for simultaneous staining with multiple antibodies spotted in array format on fixed cells grown directly on glass slide as described in Fig. 1. Tig3-hTert fibroblasts were plated on microarray-grade glass slides coated with gelatin, grown at the desired confluency, and fixed by 4% paraformaldehyde for 10 min. Cells were subsequently permeabilized, blocked by BSA incubation, and further processed for primary and AlexaFluor488-conjugated secondary antibody spotting. Antibodies were spotted by means of a non-contact spotter; the antibody dilutions were prepared in 96-well plates. For primary antibody spotting, fixed slides were gently washed and, after draining the excess washing solution, placed on the slide holder in 65% humidity.


Figure 1
View larger version (50K):
[in this window]
[in a new window]

  		FIG. 1. Schematic depiction of the immunocell-array technology. Cells are cultured directly on slides and processed at the appropriate time for immunostaining. After fixation, permeabilization, and blocking, primary and secondary antibodies are spotted on top of the cell layer. Slides are further counterstained by DAPI and mounted with Mowiol, and images are acquired with an automated fluorescence microscope and analyzed by imaging software.

 
To verify the specificity of staining in localized areas, we spotted a monoclonal antibody against PML, a nuclear protein localized in nuclear dots (PML bodies) (14), and a mouse IgG1 isotype in 2% BSA in alternate fashion in a 7 x 7 array format so that we could easily score eventual PML staining in adjacent IgG1 spots due to antibody cross-reactivity. After primary antibody spotting, the slide was incubated to allow antibody-antigen recognition at 4 °C, washed with PBS to remove the unbound antibodies, and placed on the spotter sample holder for the secondary antibody spotting. After incubation at 4 °C, slides were washed with PBS, stained with DAPI to allow the identification of nuclei, then mounted with Mowiol, and processed for image acquisition with an automated platform for fluorescence microscopy. The results are summarized in Fig. 2 and show that PML staining was specifically localized in the nucleus of cells belonging to confined regions without the occurrence of cross-contamination of cells in IgG1 adjacent spots. We confirmed the PML nuclear localization and the IgG1 staining of the immunocell-array (Fig. 2, E and F, respectively) by conventional immunofluorescence on coverslips (Fig 2, G and H, respectively). We also tested cross-reactivity by spotting in alternate fashion anti-PML antibody and 2% BSA in PBS; also in this case we did not observe cross-contamination among spots (data not shown).


Figure 2
View larger version (71K):
[in this window]
[in a new window]

  		FIG. 2. Setup of the immunocell-array technology. Tig3-hTert fibroblasts were fixed, permeabilized, and stained with a monoclonal antibody against PML and mouse IgG1 isotype in an alternate 7 x 7 array format. A, the entire array is show at 20x magnification. Green, PML staining; blue, DAPI staining. Scale bar, 500 µm. B, image magnification of an area of A; no cross-contaminations among adjacent areas can be observed. Scale bar, 250 µm. C, same as in A but showing only PML staining. D, same as in B but showing only PML staining to exclude possible cross-contaminations among adjacent regions. E and G, 60x magnification of cells of, respectively, one spot of the immunocell-array and one field of a coverslip stained by conventional immunofluorescence (PML staining). F and H, 60x magnification of cells of, respectively, one spot of the immunocell-array and one field of a coverslip stained by conventional immunofluorescence (IgG1 staining). Scale bar, 10 µm.

 
Adult Primary Melanocytes and NIH3T3 Cells Are Suitable Cellular Model Systems for Targeting Cytoplasmic and Nuclear Proteins by Immunocell-array—
To further explore the potential of this technology, we decided to perform immunocell-array experiments on different cellular types targeting several nuclear, cytoplasmic, and membrane proteins. We thus tested NIH3T3 cells and adult human primary melanocytes. NIH3T3 cells represent a suitable model for cytoskeleton-associated signaling pathways. In Fig. 3A we show a 7 x 19 immunocell-array of NIH3T3 cells grown on slide for 3 days, fixed, and stained with a panel of different antibodies (17 different antibodies and two lanes of BSA as negative control; see Table I for the antibody list); Cy3-conjugated antibodies were used as secondary antibodies. In Fig. 3B higher resolution images show detailed staining of all the tested antibodies for which we obtained specific staining. In particular we detected actin-associated structures by anti-{alpha}-actinin (15), -cortactin (16), -Eps8 (17), -Abi1 (18), -VASP (19); phosphorylated proteins by anti-p-tyrosine; Golgi staining by anti-giantin (20); focal adhesions by anti-paxillin (21) and anti-vinculin (21); vesicles by clathrin (22) and caveolin (23) staining; and cytoplasmic and vesicle-associated Eps15 staining (24) and NPM nucleolar staining (25). Primary melanocytes represent important tools for the biological studies related to diseases like pigmentation disorders, tumors (melanoma), or other genetic abnormalities; the possibility to perform high throughput signaling studies on patient-derived samples, where the availability of cells is one of the major constraints, certainly represents a valuable resource. We performed a 10 x 20 immunocell-array (Fig. 3C) by staining with 18 different antibodies and BSA as negative control (see Table I); AlexaFluor488-conjugated antibodies were used as secondary antibodies. Fig. 3D shows images at 40x magnification of all the tested antibodies; we obtained specific staining for all the targets confirming the reproducibility of our technology and the capability of employing different cellular models.


Figure 3
View larger version (57K):
[in this window]
[in a new window]

  		FIG. 3. Immunocell-arrays on NIH3T3 cells and primary adult melanocytes. A, 7 x 19 immunocell-array of NIH3T3 cells stained with 17 different antibodies (BSA was spotted as control in the first and last lanes). Blue, DAPI staining; red, Cy3-conjugated secondary antibodies. Scale bar, 500 µm. B, magnification of small areas of different spots is shown to evaluate in detail specific protein localization. Scale bar, 20 µm. C, 10 x 20 immunocell-array of adult primary melanocytes stained with 18 different antibodies (BSA was spotted as control in the first and last lanes). Blue, DAPI staining; green, AlexaFluor488-conjugated secondary antibodies. Scale bar, 500 µm. D, magnifications of small areas of different spots are shown to evaluate in detail specific protein localization. Scale bar, 25 µm. Images in A and C were acquired at 20x magnification; those in B and D at were acquired at 40x magnification. p-TYR, phosphotyrosine.

 

View this table:
[in this window]
[in a new window]

  		TABLE I List of antigens tested in the immunocell-array with U2OS cells, NIH3T3 cells, and primary human adult melanocytes

SUMO, small ubiquitin-like modifier; DNA-PK, DNA-dependent protein kinase; p-TYR, phosphotyrosine.

 
Immunocell-arrays Detect Deacetylation of Chromatin in Bleomycin-treated U2OS Cells—
To evaluate the capability of immunocell-arrays in dissecting molecular pathways, we studied the DNA damage response following bleomycin treatment of U2OS cells by analyzing a panel of selected antibodies (see Table I for the antibody list). To perform a quantitative analysis of signal intensity of the different antibodies among different slides, we performed an immunocell-array experiment by plating U2OS cells on three slides, treating them with bleomycin for 8 and 24 h, and comparing them with an untreated slide. We performed on each slide an immunostaining for lamin-B, p-ATM, p53, and {gamma}-H2AX in a 10 x 4 array format. p-ATM, p53, and {gamma}-H2AX are well known targets of DNA damage response and become activated or up-regulated upon treatment. Because lamin-B, a nuclear envelope structural protein, is not expected to undergo changes in expression/regulation upon bleomycin treatment (26), we considered it as a control over interslide staining consistency. As expected, no significant changes in lamin-B staining were detected, whereas we observed an increase in expression of p-ATM, p53, and {gamma}-H2AX (p < 0.001). These results are summarized in Fig. 4 with the heat map reporting the K-S statistics with sign (see "Experimental Procedures" for image analysis) for the various antibodies. Red color corresponds to increasing levels with respect to the untreated population, green corresponds to decreasing levels, and black corresponds to constant levels; the magnitude of the shift equals the K-S statistics against the untreated population (see "Experimental Procedures"). The cumulative distribution functions for each antibody at the different time points, from which the K-S statistics were driven, are reported in Fig. 4b.

For the DNA damage experiment, as shown in Supplemental Fig. 1, three slides were plated with U2OS cells; two of them were treated with bleomycin for 8 and 24 h (a, untreated; b, 8 h; c, 24 h). The three samples were processed for staining with 22 different antibodies (plus two lanes of BSA samples as background control) in a 24 x 10 array format and analyzed through automated fluorescence microscopy. Image magnifications (40x magnification) for all the tested antibodies at time 0 and 24 h of bleomycin treatment are reported in Fig. 5. Fig. 6 summarizes the results concerning the 22 antibodies used in the experiment. The heat map in Fig. 6A reflects the time course behavior of the various antibodies. Cumulative distribution functions for each antibody at the different time points are reported in Fig. 6B.


Figure 5
View larger version (34K):
[in this window]
[in a new window]

  		FIG. 5. 8 x 24 immunocell-arrays of U2OS cells treated with bleomycin. 40x magnifications of small areas of spots representing all the immunostained proteins in untreated (0 h) and 24-h bleomycin-treated slides are shown. p-TYR, phosphotyrosine; SUMO, small ubiquitin-like modifier; GIANT, giantin; DNA-PK, DNA-dependent protein kinase.

 
We observed and confirmed that known targets of DNA damage response, such as p-ATM (27), {gamma}-H2AX (28), and p53 (27), were activated or up-regulated upon treatment. Moreover Ezh2, which is a putative target of p53 (29), was down-regulated. Interestingly another protein of the polycomb group, BMI1, appeared to be down-regulated upon damage, suggesting its possible involvement in the cell cycle response through functional interaction with pathways of cell cycle block (30). Other proteins like Sirt6 (31), the deacetylase HDAC1 (32), the proapoptotic DAXX (33), and the transcriptional coactivator CBP (34) did not show significant alteration. Interestingly T52 and T25 antibodies revealed down-regulation of respective targets (10) (acetyl-lysine and acetyl-histone H4) suggesting that DNA damage is inducing a global down-regulation of chromatin acetylation at long term.

We confirmed our data of down-regulation of acetylation at long term by conventional immunostaining on coverslips, Western blot, and immunoprecipitation. Fig. 7, A, B, and C, shows the results obtained by performing immunofluorescence on coverslips: we observed up-regulation of expected targets of DNA damage (p53 and {gamma}-H2AX) and a significant decrease in T25 and T52 staining at long term upon bleomycin treatment. We then performed Western blot with T25 and T52 antibodies of U2OS cells that were not treated or treated with bleomycin for 24 and 48 h; as shown in Fig. 7D a significant down-regulation of histone acetylation (both H3 and H4) was observed at 48 h. We further confirmed these data by performing immunoprecipitation with T52 on the same lysates followed by silver staining of the SDS-PAGE gel (see "Experimental Procedures"); we observed specific down-regulation of acetyl-H3 and acetyl-H4 at the earlier time point (24 h), in accordance with our immunocell-array detection, probably due to the intrinsic higher sensitivity of the method. Previous work done in yeast (35) and recently in mammalian cells (36) has underlined a mechanism of localized chromatin remodeling activity triggered by the double strand DNA repair pathway. Our results, obtained by immunocell-array technology and validated by conventional immunofluorescence, Western blot, and immunoprecipitation, further strengthen the role of chromatin state in DNA repair and genomic stability but mainly characterize our technology as a novel tool for protein profiling, in the context of cellular complexity, in the field of drug discovery.


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We set up a new cell-array-based method for multiplexed immunofluorescence analysis of target proteins in different cellular models that provides many advantages over existing immunofluorescence-based methods. 1) It decreases the cost of the assay in terms of antibodies because only 1 µl of the primary or secondary antibody is required for producing up to 1000 spots in one experiment so that only a few nanoliters of reagents can be used. Rare patient cells can be processed for profiling studies, opening interesting perspectives in the monitoring of human disease. 2) It provides a flexible "high content" approach "on chip" for multiple targets and different kinetics in the context of whole-cell analysis where multiparametric data can be obtained, 3) Data can be acquired by high resolution automated microscopy and other commercial automated platform for image acquisition and analysis. 4) Finally the technology ensures a high degree of reproducibility and no well-to-well variability.

We propose the immunocell-array technology as a powerful tool for the molecular dissection of signaling pathways (kinase response, post-translational modifications, protein localization, and protein profiling) upon drug response in normal and disease states, for the characterization of cancer signatures (protein overexpression and mislocalization) on patient cell samples, and ultimately for the screening of monoclonal and polyclonal antibodies for immunofluorescence applications. In particular, by using immunocell-array, we identified a recently described pathway of deacetylation in mammalian cells upon DNA damage (36) that probably involves specific chromatin-modifying enzymes. We validated this finding by conventional immunofluorescence, Western blot, and immunoprecipitation approaches. A further detailed biochemical analysis of this pathway, however, is beyond the scope of our work, which is mainly directed toward the development and validation of a new technological tool for protein profiling. Finally with the increased availability of highly specific and selective antibodies to cancer-relevant targets, the present technology could accelerate the drug development process and offer new tools to patient monitoring during disease therapy.


   ACKNOWLEDGMENTS
 
We thank Giorgio Scita, Emanuela Colombo, and Susanna Chiocca for the gift of many primary antibodies, the Monoclonal Facility of the Department of Experimental Oncology of European Institute of Oncology (EIO) for providing many custom-made antibodies, and Ida Marangi for help. We are grateful to Giuseppina Giardina and Cristina Spinelli for providing melanocytes. We especially thank Mario Faretta and Dario Parazzoli for precious help in automated image acquisition.


  FOOTNOTES
 
Received, September 14, 2006, and in revised form, January 29, 2007.

Published, MCP Papers in Press, February 10, 2007, DOI 10.1074/mcp.T600051-MCP200

1 The abbreviations used are: FBS, fetal bovine serum; P/S, penicillin/streptomycin; DAPI, 4',6-diamidino-2-phenylindole; p-, phospho-; VASP, vasodilator-stimulated phosphoprotein; PML, promyelocytic leukemia; ATM, ataxia-telangiectasia mutated; DAXX, death domain-associated protein 6; HDAC, histone deacetylase; CBP, cAMP-response element-binding protein (CREB)-binding protein; K-S, Kolmogorov-Smirnov. Back

2 M. Herlyn, unpublished data. Back

* This work was supported in part by Associazione Italiana per la Ricerca sul Cancro under Oncogenomic Grant (OGCG) "Development and integration of high-throughput technologies for the functional genomics of cancer" and by the European Union under "INTACT" (Identification of Novel Targets for Cancer Therapy) Grant LSHC-CT-2003-506803. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

|| Supported by a FIRC fellowship. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Tethis S.r.l., Via Russoli 3, 20143 Milan, Italy. Tel.: 39-0236568349; Mobile: 39-392-9774561; Fax: 39-0236569183; E-mail: roberta.carbone{at}tethis-lab.com


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Buckhaults, P. (2006) Gene expression determinants of clinical outcome. Curr. Opin. Oncol. 18, 57 –61[Medline]

  2. Liotta, L. A., Kohn, E. C., and Petricoin, E. F. (2001) Clinical proteomics: personalized molecular medicine. J. Am. Med. Assoc. 286, 2211 –2214[Free Full Text]

  3. Petricoin, E. F., III, Bichsel, V. E., Calvert, V. S., Espina, V., Winters, M., Young, L., Belluco, C., Trock, B. J., Lippman, M., Fishman, D. A., Sgroi, D. C., Munson, P. J., Esserman, L. J., and Liotta, L. A. (2005) Mapping molecular networks using proteomics: a vision for patient-tailored combination therapy. J. Clin. Oncol. 23, 3614 –3621[Abstract/Free Full Text]

  4. Cadigan, K. M., and Liu, Y. I. (2006) Wnt signaling: complexity at the surface. J. Cell Sci. 119, 395 –402[Abstract/Free Full Text]

  5. Fink, L., Kwapiszewska, G., Wilhelm, J., and Bohle, R. M. (2006) Laser-microdissection for cell type- and compartment-specific analyses on genomic and proteomic level. Exp. Toxicol. Pathol. 57, Suppl. 2,25 –29[CrossRef][Medline]

  6. Carr, K. M., Rosenblatt, K., Petricoin, E. F., and Liotta, L. A. (2004) Genomic and proteomic approaches for studying human cancer: prospects for true patient-tailored therapy. Hum. Genomics 1, 134 –140[Medline]

  7. Kingsmore, S. F. (2006) Multiplexed protein measurement: technologies and applications of protein and antibody arrays. Nat. Rev. Drug Discov. 5, 310 –321[CrossRef][Medline]

  8. Chen, H., Kovar, J., Sissons, S., Cox, K., Matter, W., Chadwell, F., Luan, P., Vlahos, C. J., Schutz-Geschwender, A., and Olive, D. M. (2005) A cell-based immunocytochemical assay for monitoring kinase signaling pathways and drug efficacy. Anal. Biochem. 338, 136 –142[CrossRef][Medline]

  9. Chan, S. M., Ermann, J., Su, L., Fathman, C. G., and Utz, P. J. (2004) Protein microarrays for multiplex analysis of signal transduction pathways. Nat. Med. 10, 1390 –1396[CrossRef][Medline]

  10. Ronzoni, S., Faretta, M., Ballarini, M., Pelicci, P., and Minucci, S. (2005) New method to detect histone acetylation levels by flow cytometry. Cytometry A 66, 52 –61[Medline]

  11. Press, W. H., Teukolsky, S. A., Vetterling, W. T., and B. P., F. (2002) Numerical Recipes in C++: the Art of Scientific Computing, 2nd Ed., pp.623 –628, Cambridge University Press, Cambridge

  12. Perlman, Z. E., Slack, M. D., Feng, Y., Mitchison, T. J., Wu, L. F., and Altschuler, S. J. (2004) Multidimensional drug profiling by automated microscopy. Science 306, 1194 –1198[Abstract/Free Full Text]

  13. Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850 –858[Medline]

  14. Eskiw, C. H., Dellaire, G., Mymryk, J. S., and Bazett-Jones, D. P. (2003) Size, position and dynamic behavior of PML nuclear bodies following cell stress as a paradigm for supramolecular trafficking and assembly. J. Cell Sci. 116, 4455 –4466[Abstract/Free Full Text]

  15. Lazarides, E., and Burridge, K. (1975) {alpha}-Actinin: immunofluorescent localization of a muscle structural protein in nonmuscle cells. Cell 6, 289 –298[CrossRef][Medline]

  16. McNiven, M. A., Kim, L., Krueger, E. W., Orth, J. D., Cao, H., and Wong, T. W. (2000) Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape. J. Cell Biol. 151, 187 –198[Abstract/Free Full Text]

  17. Provenzano, C., Gallo, R., Carbone, R., Di Fiore, P. P., Falcone, G., Castellani, L., and Alema, S. (1998) Eps8, a tyrosine kinase substrate, is recruited to the cell cortex and dynamic F-actin upon cytoskeleton remodeling. Exp. Cell Res. 242, 186 –200[CrossRef][Medline]

  18. Stradal, T., Courtney, K. D., Rottner, K., Hahne, P., Small, J. V., and Pendergast, A. M. (2001) The Abl interactor proteins localize to sites of actin polymerization at the tips of lamellipodia and filopodia. Curr. Biol. 11, 891 –895[CrossRef][Medline]

  19. Quinlan, M. P. (2004) Vinculin, VASP, and profilin are coordinately regulated during actin remodeling in epithelial cells, which requires de novo protein synthesis and protein kinase signal transduction pathways. J. Cell. Physiol. 200, 277 –290[CrossRef][Medline]

  20. Linstedt, A. D., and Hauri, H. P. (1993) Giantin, a novel conserved Golgi membrane protein containing a cytoplasmic domain of at least 350 kDa. Mol. Biol. Cell 4, 679 –693[Abstract]

  21. Turner, C. E., Glenney, J. R., Jr., and Burridge, K. (1990) Paxillin: a new vinculin-binding protein present in focal adhesions. J. Cell Biol. 111, 1059 –1068[Abstract/Free Full Text]

  22. Anderson, R. G., Vasile, E., Mello, R. J., Brown, M. S., and Goldstein, J. L. (1978) Immunocytochemical visualization of coated pits and vesicles in human fibroblasts: relation to low density lipoprotein receptor distribution. Cell 15, 919 –933[CrossRef][Medline]

  23. Smart, E. J., Ying, Y. S., Conrad, P. A., and Anderson, R. G. (1994) Caveolin moves from caveolae to the Golgi apparatus in response to cholesterol oxidation. J. Cell Biol. 127, 1185 –1197[Abstract/Free Full Text]

  24. Tebar, F., Sorkina, T., Sorkin, A., Ericsson, M., and Kirchhausen, T. (1996) Eps15 is a component of clathrin-coated pits and vesicles and is located at the rim of coated pits. J. Biol. Chem. 271, 28727 –28730[Abstract/Free Full Text]

  25. Chan, P. K., and Chan, F. Y. (1999) A study of correlation between NPM-translocation and apoptosis in cells induced by daunomycin. Biochem. Pharmacol. 57, 1265 –1273[CrossRef][Medline]

  26. von Kobbe, C., Harrigan, J. A., May, A., Opresko, P. L., Dawut, L., Cheng, W. H., and Bohr, V. A. (2003) Central role for the Werner syndrome protein/poly(ADP-ribose) polymerase 1 complex in the poly(ADP-ribosyl)ation pathway after DNA damage. Mol. Cell. Biol. 23, 8601 –8613[Abstract/Free Full Text]

  27. Turenne, G. A., Paul, P., Laflair, L., and Price, B. D. (2001) Activation of p53 transcriptional activity requires ATM’s kinase domain and multiple N-terminal serine residues of p53. Oncogene 20, 5100 –5110[CrossRef][Medline]

  28. Tomilin, N. V., Solovjeva, L. V., Svetlova, M. P., Pleskach, N. M., Zalenskaya, I. A., Yau, P. M., and Bradbury, E. M. (2001) Visualization of focal nuclear sites of DNA repair synthesis induced by bleomycin in human cells. Radiat. Res. 156, 347 –354[CrossRef][Medline]

  29. Tang, X., Milyavsky, M., Shats, I., Erez, N., Goldfinger, N., and Rotter, V. (2004) Activated p53 suppresses the histone methyltransferase EZH2 gene. Oncogene 23, 5759 –5769[CrossRef][Medline]

  30. Nowak, K., Kerl, K., Fehr, D., Kramps, C., Gessner, C., Killmer, K., Samans, B., Berwanger, B., Christiansen, H., and Lutz, W. (2006) BMI1 is a target gene of E2F-1 and is strongly expressed in primary neuroblastomas. Nucleic Acids Res. 34, 1745 –1754[Abstract/Free Full Text]

  31. Liszt, G., Ford, E., Kurtev, M., and Guarente, L. (2005) Mouse Sir2 homolog SIRT6 is a nuclear ADP-ribosyltransferase. J. Biol. Chem. 280, 21313 –21320[Abstract/Free Full Text]

  32. Minucci, S., and Pelicci, P. G. (2006) Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat. Rev. Cancer 6, 38 –51[CrossRef][Medline]

  33. Salomoni, P., and Khelifi, A. F. (2006) Daxx: death or survival protein. Trends Cell Biol. 16, 97 –104[CrossRef][Medline]

  34. Grossman, S. R. (2001) p300/CBP/p53 interaction and regulation of the p53 response. Eur. J. Biochem. 268, 2773 –2778[Medline]

  35. Tamburini, B. A., and Tyler, J. K. (2005) Localized histone acetylation and deacetylation triggered by the homologous recombination pathway of double-strand DNA repair. Mol. Cell. Biol. 25, 4903 –4913[Abstract/Free Full Text]

  36. Murr, R., Loizou, J. I., Yang, Y. G., Cuenin, C., Li, H., Wang, Z. Q., and Herceg, Z. (2006) Histone acetylation by Trrap-Tip60 modulates loading of repair proteins and repair of DNA double-strand breaks. Nat. Cell Biol. 8, 91 –99[CrossRef][Medline]


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow Supplemental Data
Right arrow All Versions of this Article:
T600051-MCP200v1
6/5/939    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Glossary
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zanardi, A.
Right arrow Articles by Carbone, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zanardi, A.
Right arrow Articles by Carbone, R.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Journal of Biological Chemistry 
 Journal of Lipid Research   ASBMB Today