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Methods in Molecular Biology TM VOLUME 218 Cancer Cell Signaling Methods and Protocols Edited by David M. Terrian HUMANA PRESS Antimitogenic Activity of Tumor Suppression 3 1 Functional Analysis of the Antimitogenic Activity of Tumor Suppressors Erik S. Knudsen and Steven P. Angus Abstract Loss of tumor suppressors contributes to numerous cancer types. Many, but not all, proteins encoded by tumor suppressor genes have antiproliferative activity and halt cell-cycle progression. In this chapter, we present three methods that have been utilized to monitor the antimitogenic action exerted by tumor suppressors. Tumor suppressor function can be demonstrated by colony formation assays and acquisition of the flat-cell phenotype. Because of the antiproliferative action of these agents, we also present two transient assays that monitor the effect of tumor suppressors on cell-cycle progression. One is based on BrdU incorporation (i.e., DNA replication) and the other on flow cytometry. Together, this triad of techniques is sufficient to determine the action of tumor suppressors and other antiproliferative agents. Key Words: Tumor suppressor; green fluorescent protein; bromo-deoxyuridine; retinoblastoma; cell cycle; cyclin; flow cytometry; mitogen; fluorescence microscopy. 1. Introduction The discovery of tumor suppressor genes, whose loss predisposes to tumor development, has revolutionized the molecular analysis of cancer (1–3). By definition, tumor suppressor genes are genetically linked to a cancer. For example, the retinoblastoma (RB) tumor suppressor was first identified as a gene that was specifically lost in familial RB (4–6). The majority of tumor suppressors From: Methods in Molecular Biology, vol. 218: Cancer Cell Signaling: Methods and Protocols Edited by: D. M. Terrian © Humana Press Inc., Totowa, NJ 3 4 Knudsen and Angus has been identified based on linkage analysis and subsequent epidemiological studies, however, initial understanding of their mode of action was relatively limited. As the number of tumor suppressors has increased, understanding the mechanism through which tumor suppressors function has become an important aspect of cancer biology. In general, tumors exhibit uncontrolled proliferation. This phenotype can arise from loss of tumor suppressors that regulate progression through the cell cycle (e.g., RB or p16ink4a) or upstream mitogenic signaling (e.g., NF1 or PTEN) cascades (1,3,7–9). Thus, specific tumor suppressors can function to suppress proliferation. However, not all tumor suppressors act in this manner. For example, mismatch repair factors (e.g., MSH2 or MLH-1) lost in hereditary nonpolyposis colorectal cancer (HNPCC) function not to inhibit proliferation, but to prevent further mutations (10–12). Additionally, other tumor suppressors have multiple functions, for example, p53 can function to either induce cell death or halt cell-cycle progression (9,13). Functional analysis of tumor suppressors relies on a host of methods to determine how or if they inhibit proliferation. Later, we will focus on methods that have been used to assess the antimitogenic potential of the RB-pathway (2,3,7, 14). However, these same approaches are amenable to any tumor suppressor or antimitogenic molecule. Assays used to evaluate antimitogenic activity are based either on the halt of proliferation or cell-cycle progression. Cell proliferation assays, as described later, have been extensively utilized to demonstrate the antiproliferative effect of tumor suppressors (15–20). However, these assays do not illuminate whether the observed effects are attributable to cell-cycle arrest or apoptosis. Additionally, because of the antiproliferative action of many tumor suppressors, it is difficult to obtain sufficient populations of cells for analysis. This obstacle can be surmounted through the use of transient assays to monitor cell-cycle effects (16,19,21–25). Two different transient approaches to analyze tumor suppressor action on the cell cycle are also described. 2. Materials 2.1. Cell Culture and Transfection of Antimitogen/Tumor Suppressor 1. SAOS-2 human osteosarcoma cell line (ATCC #HTB-85). 2. Dulbecco’s modification of Eagle’s medium (DMEM, Cellgro, cat #10-017-CV) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Atlanta Biologicals, cat #S12450), 100 U/mL penicillin-streptomycin and 2 mM L-glutamine (Gibco-BRL). Antimitogenic Activity of Tumor Suppression 5 3. Dulbecco’s phosphate-buffered saline (PBS), tissue culture grade, without calcium and magnesium (Cellgro, cat #21-031-CV). 4. 1X Trypsin-EDTA solution (Cellgro, cat #25-052-CI). 5. 60-mm tissue-culture dishes. 6. Six-well tissue-culture dishes. 7. 12-mm circular glass cover slips (Fisher), sterilized. 8. Mammalian expression system (e.g., pcDNA3.1, Invitrogen). 9. Relevant cDNAs: RB, Histone 2B (H2B)-GFP [from G. Wahl, The Salk Institute, La Jolla, CA (26)], pBABE-puro [puromycin resistance plasmid, (27)]. 10. 0.25M CaCl2: dissolve in ddH2O; filter (0.2 µm) sterilize and store in aliquots at −20ºC. 11. 2X BES-buffered solution (2X BBS): 50 mM N,N-bis (2-hydroxyethyl)-2-aminoethanesulfonic acid, 280 mM NaCl, 1.5 mM Na2HPO4, adjust pH to 6.95 in ddH2O, filter (0.2 µm) sterilize and store in aliquots at −20ºC. 12. Inverted fluorescence microscope (Zeiss). 2.2. Inhibition of BrdU Incorporation in Transiently-Transfected Cells 1. Transfected SAOS-2 cells. 2. Cell proliferation-labeling reagent, BrdU/FdU (Amersham Pharmacia, cat# RPN201). 3. PBS: 136 mM NaCl, 2.6 mM KCl, 10mM Na2HPO4, 2.7 mM KH2PO4 in ddH2O; pH to 7.4 with HCl; sterilize in autoclave. 4. 3.7% (v/v) formaldehyde in PBS: dilute fresh from 37% w/w stock solution (Fisher). 5. 0.3% (v/v) Triton X-100 (Fisher) in PBS. 6. Immunofluorescence (IF) buffer: 0.5% v/v Nonidet P-40 (Fisher) and 5 mg/mL (w/v) bovine serum albumin (Sigma) in PBS; store at 4ºC. 7. 1M MgCl2. 8. DNase I, RNase-free (10 U/µL) (Roche, cat# 776 785). 9. Monoclonal rat anti-BrdU antibody (Accurate Scientific, cat #YSRTOBT-0030). 10. Donkey anti-rat IgG, Red X-conjugated (Jackson Immunoresearch, cat #712-295153). 11. 1 mg/mL (w/v) Hoechst 33258 (Sigma, cat #B2883). 12. Microscope slides. 13. Gel/Mount (Biomeda Corp., cat #MØ1) 14. Inverted fluorescence microscope (Zeiss). 2.3. Cell-Cycle Analysis of Transiently-Transfected Cells 1. Transfected SAOS-2 cells. 2. PBS. 3. 1X Trypsin-ethylene diamine tetraacetic acid (EDTA) solution (Cellgro, cat #25052-CI). 4. Clinical centrifuge. 6 Knudsen and Angus 5. 100% ethanol stored at −20ºC. 6. 40 mg/mL (w/v) RNase A (Sigma, cat #R-4875): Dissolve in sterile double-distilled (dd)H2O at 100ºC, 15 min; aliquot and store at −20ºC. 7. 100X propidium iodide (PI) solution: 20 mg/mL (w/v) propidium iodide (Sigma, cat #P-4170) in PBS; cover with foil to protect from light and store at 4ºC. 8. 5-mL polystyrene round-bottom tubes (Becton Dickinson, cat #35-2058). 9. Coulter Epics XL flow cytometer. 10. FlowJo data analysis software (Treestar). 11. ModFit cell-cycle analysis software (Verity). 2.4. Flat-Cell Assay and Colony Inhibition in Stably-Transfected Cells 1. 2. 3. 4. Transfected SAOS-2 cells. 2.5 mg/mL puromycin (w/v) (Sigma, cat #P-7255). 1% crystal violet (w/v) (Fisher, cat #C581-25)/20% ethanol solution. Inverted microscope with camera. 3. Methods 3.1. Cell Culture and Transfection of Antimitogen/Tumor Suppressor 3.1.1. Cell Culture 1. Seed approx 1 × 105 cells per well of a six-well plate or 3 × 105 cells per 60-mm dish in DMEM supplemented with 10% FBS and penicillin-streptomycin. 2. SAOS-2 cells should attach to the tissue culture dish within 4–6 h. 3.1.2. Cell Transfection 1. Prepare purified plasmid DNA stocks at 1 mg/mL concentration in TE buffer. 2. Add DNA to 1.5-mL Eppendorf tube (4.25 µg per well of a six-well plate, 8.5 µg total per 60-mm dish). 3. Add 0.25M CaCl2 to DNA and mix by pipeting. 4. Add 2X BBS solution and mix by inverting. 5. Incubate tubes at room temperature for 20 min. 6. Add DNA/CaCl2/BBS solution to cells dropwise. 7. Inspect the cells for the presence of precipitate using an inverted microscope (20× power is sufficient) (see Note 1). 8. Return cells to tissue culture incubator (37ºC, 5% CO2). 9. 16 h postaddition of precipitate, wash cells three times briefly with PBS. 10. Inspect dishes to ensure removal of precipitate. 11. Add fresh media to cells. Antimitogenic Activity of Tumor Suppression 7 3.1.3. Confirmation of Transfection/ Determining Transfection Efficiency 1. Take live plates of cells transfected 16 h prior with H2B-GFP and either vector or antimitogen/tumor suppressor out of the incubator. 2. Aspirate media. 3. Replace with PBS. 4. Visualize transfected cells by GFP fluorescence using an inverted fluorescent microscope (20X power is sufficient). 5. Using the GFP fluorescence and phase contrast, determine the percentage of GFPpositive cells by counting random fields of cells. 6. Compare the relative transfection efficiencies between vector control and antimitogen/tumor suppressor. 3.2. Inhibition of BrdU Incorporation in Transiently Transfected Cells 3.2.1. Cell Culture 1. Culture cells at 60% confluence (approx 1 × 105 cells/well) on coverslips in a sixwell plate (four cover slips per well). 3.2.2. Cell Transfection 1. Use 4 µg of CMV-vector or CMV-RB and 0.25 µg of CMV-H2B-GFP. 2. Use 0.125 mL CaCl2 and 0.125 mL 2X BBS. 3.2.3. BrdU Labeling 1. 36–48 h after adding fresh media to transfected cells, add cell proliferation-labeling reagent directly to media in wells (1:1000 dilution) (see Note 2). 2. Return six-well dish to tissue-culture incubator for 16 h. 3.2.4. Fixation 1. 2. 3. 4. 5. 6. Aspirate media from wells. Wash cells gently with PBS. Fix cells at room temperature with 3.7% formaldehyde in PBS for 15 min. Aspirate formaldehyde. Add PBS to wells. Cover slips in PBS may be stored in dark at 4ºC. 3.2.5. BrdU Staining 1. 2. 3. 4. Aspirate PBS. Add 0.3% Triton X-100 in PBS to wells to permeabilize the cells (see Note 3). Incubate dish at room temperature for 15 min. Aspirate 0.3% Triton X-100 and replace with PBS. 8 Knudsen and Angus Fig. 1. Diagram of BrdU staining in a humidified chamber of fixed and permeabilized cells grown on glass cover slips. 5. Prepare primary antibody solution by diluting the following in IF buffer: a. 1:50 1M MgCl2. b. 1:500 Rat anti-BrdU. c. 1:500 DNase I (see Note 4). 6. Pipet 35 µL primary antibody solution onto each cover slip. 7. Incubate cover slips in a humidified chamber at 37ºC for 45 min (see Fig. 1). 8. Wash cover slips in PBS in six-well dish for 5 min with 2–3 changes. 9. Prepare secondary antibody solution by diluting the following in IF buffer: a. 1:100 Donkey anti-rat Red-X. b. 1:100 Hoechst (10 µg/mL final conc.). 10. Pipet 35 µL secondary antibody solution onto each cover slip. 11. Incubate cover slips in humidified chamber at 37ºC for 45 min. 12. Wash cover slips in PBS in six-well dish for 5 min with 2–3 changes. 13. Mount cover slips on slides using Gel/Mount. 14. Examine cover slips using an inverted fluorescence microscope. 15. Inhibition determined by counting. Antimitogenic Activity of Tumor Suppression 9 Fig. 2. SAOS-2 cells were cotransfected with H2B-GFP and either CMV-vector or CMV-RB. Cells were pulse-labeled with BrdU for 16 h. Fixation, permeabilization, and immunostaining were performed as described. Photomicrographs of immunofluorescent cells were taken at equal magnification. Arrows indicate transfected cells. Quantification of this approach is presented in refs. (19,21–23). 3.2.6. Quantitation and Documentation 1. Quantitation of BrdU inhibition. a. Count the number of transfected (i.e., GFP-positive) cells in a random field). b. Without changing fields, count the number of GFP-positive cells that are also BrdU-positive (i.e., Red-X-positive). c. Repeat steps a and b until 150–200 GFP-positive cells have been counted. d. Calculate the percent BrdU-positive (BrdU-positive/GFP-positive). e. As a control, determine the percentage of BrdU-positive cells from untransfected (GFP-negative) cells on the same cover slips. f. Compare the effect of antimitogen expression vs vector expression on BrdU incorporation (see Fig. 2). 2. Documentation a. Take representative photomicrographs of selected fields. b. Use blue (Hoechst), green (H2B-GFP), and red (Red-X) channels to obtain photomicrographs of the same field. 3.3. Cell-Cycle Arrest in Transiently-Transfected Cells 3.3.1. Cell Culture 1. Culture cells in 60-mm dishes at 60% confluence. 2. Include a dish that will not be transfected. 3.3.2. Cell Transfection 1. Use 8 µg of CMV-vector or CMV-RB and 0.5 µg of CMV-H2B-GFP (see Note 5). 2. Use 0.25 mL CaCl2 and 0.25 mL 2X BBS. 10 Knudsen and Angus 3.3.3. Cell Harvesting and Fixation 1. 36–48 h after adding fresh media to transfected cells, add trysin (approx 0.75 mL) to dishes. 2. Confirm that cells have detached after 1–2 min using inverted microscope. 3. Inactivate trypsin by adding an equal volume of media. 4. Transfer suspended cells to 15-mL conical tubes. 5. Pellet cells in a clinical centrifuge at 1000 rpm, 2–3 min. 6. Aspirate media. 7. Add 2–3 mL PBS to wash cell pellet. 8. Repeat centrifugation. 9. Aspirate PBS. 10. Resuspend cell pellet in 200 µL PBS. 11. Slowly add 1 mL ice-cold 100% ethanol while vortexing gently. 12. Tubes may be stored in the dark at 4ºC for 1–2 wk. 3.3.4. Propidium Iodide Staining 1. 2. 3. 4. 5. 6. 7. Prepare 1X PI by diluting 100X PI stock solution in PBS (see Note 6). Add RNase A to 1X PI at a 1:1000 dilution (final concentration = 40 µg/mL). Pellet fixed cells at 200g, 2–3 min. Aspirate ethanol. Resuspend cell pellet in approx 1 mL 1X PI containing RNase A. Transfer resuspended cells to 5-mL polystyrene round-bottom tubes. Incubate tubes in the dark at room temperature for at least 15 min prior to analysis (see Note 7). 3.3.5. FACS 1. Run untransfected control to set background levels of GFP signal and to establish PI parameters. 2. Gate H2B-GFP-positive cells (either positive or negative) (see Fig. 3 and Note 8). 3. Analyze PI staining in GFP-positive cells. 4. Perform ModFit analysis on PI histograms (see Fig. 3). 3.4. Flat-Cell Assay/Colony Inhibition in Stably Transfected Cells 3.4.1. Cell Culture 1. Culture 1 × 105 cells in 60-mm dishes. 2. Include a control plate that will not be transfected. 3.4.2. Cell Transfection 1. Use 8 µg of CMV-vector or CMV-RB and 0.5 µg of pBABE-puro. 2. Use 0.25 mL CaCl2 and 0.25 mL 2X BBS. Antimitogenic Activity of Tumor Suppression 11 Fig. 3. SAOS-2 cells either untransfected (left column) or transfected with H2B-GFP and either CMV-vector (middle column), or RB (right column) were fixed in ethanol and stained with propidium iodide (PI). Cells were subsequently analyzed by FACS. Top row, Cells were gated to distinguish the GFP-negative population from the GFPpositive population. Hatched line indicates gate position (GFP-positive cells above line, GFP-negative cells below). Middle row, GFP-negative cells were analyzed for DNA content (PI) and ModFit analysis was performed to quantitate cell cycle distribution (% phase) as indicated. Bottom row, GFP-positive cells were analyzed for DNA content (PI) and ModFit analysis was performed to quantitate cell cycle distribution (% phase) as indicated. 3.4.3. Puromycin Selection and Staining 1. 24 h after adding fresh media to transfected cells, add puromycin to media at a 1:1000 dilution (final concentration = 2.5 µg/mL puromycin). 2. Confirm puromycin selection by visual analysis of untransfected cells.